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WO2025081042A1 - Nickase-retron template-based precision editing system and methods of use - Google Patents

Nickase-retron template-based precision editing system and methods of use Download PDF

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Publication number
WO2025081042A1
WO2025081042A1 PCT/US2024/051048 US2024051048W WO2025081042A1 WO 2025081042 A1 WO2025081042 A1 WO 2025081042A1 US 2024051048 W US2024051048 W US 2024051048W WO 2025081042 A1 WO2025081042 A1 WO 2025081042A1
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ncrna
variant
seq
region
msd
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Hari JAYARAM
Jacob LAYER
Kyong-Rim KIEFFER-KWON
Zhongxia YI
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Renagade Therapeutics Management Inc
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Renagade Therapeutics Management Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • the present disclosure generally relates to systems, methods and pharmaceutical compositions used for precise genome editing, including nucleic acid edits, insertions, replacements, and deletions at targeted and precise genome sites, wherein said systems, methods, and compositions are based on a chimeric editing system comprising one or more components of a prime editor and one or more components of a retron editor.
  • Gene editing tools encompass a diverse set of technologies that can make many types of genetic alterations in various contexts. These technologies have evolved over the last couple of decades to provide a range of user-programmable editing tools that include ZFN (zinc finger) nuclease editing systems, meganuclease editing systems, and TALENS (transcription activator-like effector nucleases).
  • ZFN zinc finger
  • meganuclease editing systems and TALENS (transcription activator-like effector nucleases).
  • CRISPR clustered regularly interspaced short palindromic repeats
  • CRISPR-Cas9 CRISPR-associated proteins
  • Jinek et al. “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,” Science, Vol.337 (6096), pp.816-821, meganuclease editors, for example, as described, Boissel et al., “megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering,” Nucleic Acids Research 42: pp.2591-2601, and bacterial retron systems, RNG043-WO1 PCT Application for example, as described, in Schubert et al., “High-throughput functional variant screens via in vivo production of single-stranded DNA,” PNAS, April 27, 2021,
  • CRISPR-Cas9 has been derivatized in numerous ways to expand upon its guide RNA- based programmable double-strand cutting activity to form systems ranging from finding alternative CRISPR Cas nuclease enzymes having different PAM requirements and cutting properties (e.g., Cas12a, Cas12f, Cas13a, and Cas13b) forto base editing, for example, as described in Komor et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage,” Nature, May 19, 2016, 533 (7603); pp.420-424 [cytosine base editors or CBEs] and Gaudelli et al., “Programmable base editing of A-T to G-C in genomic DNA without DNA cleavage,” Nature, Vol.551, pp.464-471 [adenine base editors or ABEs]) to prime editing, for example, as described in Anzalone et al., “Search-and-replace genome editing without
  • the present disclosure describes a chimeric gene editing system that comprises one or more components of site-specific gene editing system (e.g., a prime editing system or a prime editor-like system) with one or more components of a retron editing system, producing an entirely new gene editing system with distinct properties, capabilities and advantages.
  • site-specific gene editing system e.g., a prime editing system or a prime editor-like system
  • the chimeric gene editing system comprises (a) a nickase component, (b) a guide RNA that complexes with the nickase component and directs it to a target DNA sequence, (c) a polymerase component, (d) and a polymerase template sequence, wherein the polymerase template sequence is provided by a modified retron ncRNA comprising the polymerase template sequence (herein referred to as a “templated ncRNA” or “tncRNA”).
  • a modified retron ncRNA comprising the polymerase template sequence
  • the tncRNA comprises an altered structural configuration relative to a wildtype retron ncRNA wherein the alterations include (i) a linker joining the 5’ end of the ncRNA a1 region to the 3’ end of the ncRNA a2 region, (ii) a deletion of the wildtype msd region or portion thereof, or (iii) installation of a single-strand RNA sequence comprising the polymerase template in place of the deleted msd sequence.
  • the chimeric gene editing system may also optionally comprise a retron reverse transcriptase (RT) to convert the templated ncRNA to a cognate msDNA, which is referred to herein as the “templated msDNA” or “tmsDNA”.
  • RT retron reverse transcriptase
  • the tncRNA comprises an altered structural configuration relative to a wildtype retron ncRNA wherein sthe alterations include (i) a linker joining the 5’ end of the ncRNA a1 region to the 3’ end of the ncRNA a2 region, (ii) a deletion of the wildtype msd region or portion thereof, or (iii) installation of a single-strand RNA sequence comprising the polymerase template in place of the deleted msd sequence.
  • the chimeric gene editing system may also optionally comprise a retron RT (or a nucleic acid sequence encoding a retron RT) to convert the templated ncRNA to a cognate msDNA, which is referred to herein as the “templated msDNA” or “tmsDNA”.
  • a prime editor which includes (a) a reverse transcriptase component fused to a (b) CRISPR Cas9 nickase component, and which is complexed with (c) a prime editor guide RNA (pegRNA) (i.e., the PE:pegRNA complex).
  • pegRNA prime editor guide RNA
  • the pegRNA comprises a (i) primer binding site (PBS) and (ii) a template sequence.
  • PBS primer binding site
  • the mechanism of prime editing is described in the art.
  • the PE:pegRNA complex binds to a target DNA at a site specified by the spacer sequence of the pegRNA.
  • the Cas9 nickase then nicks one strand, generating a 3’-ended flap of endogenous DNA (“the 3’ flap”).
  • RNG043-WO1 PCT Application located on the pegRNA, binds to the 3’ flap and the edited RNA sequence is reverse transcribed from the 3’ flap against the template portion of the pegRNA by the reverse transcriptase of the PE:pegRNA complex.
  • the reverse transcription product effectively extends from the endogenous 3’ flap to form a newly synthesized single strand of DNA (i.e., the RT product or edited strand).
  • the edited strand displaces the endogenous strand sitting immediately downstream of the nick on the nicked strand, forming a 5’ flap of displaced endogenous DNA, which is removed by cell enzymes.
  • the edited DNA is permanently installed onto both strands as a result of endogenous DNA repair enzymes and a round of DNA replication. This process can be enhanced by using an additional normal-length guide RNA that is programmed to install (by again complexing with the PE) a second nick into the unedited strand.
  • Retrons encode and transcribe as a single RNA, which comprises a non-coding RNA (ncRNA) portion and a portion encoding a specialized reverse transcriptase (RT) (see Panel 1).
  • ncRNA non-coding RNA
  • RT reverse transcriptase
  • the translated RT typically recognizes certain secondary structures in the ncRNA, and binds the RNA template downstream from the msd region.
  • the RT initiates reverse transcription of the RNA towards its 5 ⁇ end, starting from the 2’-end of a conserved guanosine (G) residue found immediately after a double-stranded RNA structure (the a1/a2 region) within the ncRNA.
  • G conserved guanosine
  • a portion of the ncRNA serves as a template for reverse transcription, and reverse transcription terminates before reaching the msr locus.
  • cellular RNase H degrades the segment of the ncRNA that serves as template, but not other parts of the ncRNA.
  • the present disclosure describes a chimeric gene editing system comprising one or more components of a prime editor system and one or more components of a retron editor.
  • the resulting chimeric editing system is referred to herein as a “nickase/retron template precision editing system.”
  • RNG043-WO1 PCT Application [0012] The present disclosure contemplates a variety of configurations and interchangeable components that may constitute different embodiments envisioned for the nickase/retron template precision editing system described herein.
  • the nickase/retron template precision editing system described herein may comprise: (1) a nickase or a nucleic acid sequence encoding a nickase (e.g., a mRNA encoding a nickase); (2) a guide RNA that complexes with the nickase and directs the nickase to a target DNA sequence; (3) a polymerase or a nucleic acid sequence encoding a polymerase (e.g., a mRNA encoding a polymerase); and (4) a polymerase template sequence for synthesis of an edit strand to be incorporated into a target site, wherein the polymerase template sequence is provided by a templated ncRNA.
  • a nickase or a nucleic acid sequence encoding a nickase e.g., a mRNA encoding a nickase
  • a guide RNA that complexes with the nicka
  • the tncRNA comprises an altered structural configuration relative to a wildtype retron ncRNA wherein the alterations include, but are not limited to, (i) a linker joining the 5’ end of the ncRNA a1 region to the 3’ end of the ncRNA a2 region, (ii) a deletion of the wildtype msd region or a portion thereof, or/and (iii) installation of a single- strand RNA sequence comprising the polymerase template in place of the deleted msd sequence.
  • the polymerase template may also comprise a primer binding site which anneals to an endogenous 3’ flap produced as a result of introducing a nick at the target site by the nickase.
  • the annealing of the primer binding site to the endogenous 3’ flap creates a polymerization initiation site for synthesis of a new strand of single-stranded DNA (“the edited strand” or “the edited flap”) from the 3’ end of the endogenous flap and using the polymerase template as instructions.
  • the edited strand displaces the endogenous strand sitting immediately downstream of the nick on the nicked strand, forming a 5’ flap of displaced endogenous DNA, which is removed by cell enzymes.
  • the edited flap is permanently installed onto both strands as a result of endogenous DNA repair enzymes and DNA replication.
  • This process can be enhanced by using an additional normal-length guide RNA that is programmed to install (by again complexing with the nickase) a second nick into the unedited strand.
  • This particular DNA lesion is recognized by the cells, compelling the cell to repair the unedited strand to match as the complement of the edited strand.
  • This process leaves both strands in the edited configuration, thereby permanently installing the edit.
  • the chimeric gene editing system shown in FIG.3 may also optionally comprise a retron RT (or a nucleic acid sequence encoding a retron RT) to convert the templated ncRNA to a cognate msDNA, which is referred to herein as the “templated msDNA” or “tmsDNA”.
  • the polymerase template may be provided directly by a templated ncRNA (e.g., as shown in FIG.6).
  • the polymerase template of the nickase/retron template precision editing system described herein may be provided by a templated ncRNA and a retron RT which converts the ncRNA to a templated msDNA, which then effectivley provides the templating function for the polymerase to synthesize and edited strand for incorporation.
  • a templated ncRNA and a retron RT which converts the ncRNA to a templated msDNA, which then effectivley provides the templating function for the polymerase to synthesize and edited strand for incorporation.
  • FIG.5 the nickase/retron template precision editing system described herein comprises a templated ncRNA and/or a templated msDNA, as illustrated in FIG.4. Referring to panel A, a non-folded wildtype ncRNA is depicted.
  • the molecule comprises in the 5’ to 3’ direction an a1 region, an msr region, an msd region, and an a2 region.
  • the msr region forms 1 or more stem loops, as well as in the msd region.
  • FIG.2 for a detailed schematic on how the ncRNA is conventionally converted to an msRNA by a retron RT.
  • panels C and D are components of the nickase/retron template precision editing system described herein.
  • Panel C illustrates an embodiment of a templated ncRNA.
  • the tncRNA comprises an altered structural configuration relative to a wildtype retron ncRNA (e.g., as shown in Panel B) wherein said alterations include, but are not limited to, (i) a linker joining the 5’ end of the ncRNA a1 region to the 3’ end of the ncRNA a2 region, (ii) a deletion of the wildtype msd region or a portion thereof, and (iii) installation of a single-strand RNA sequence comprising the polymerase template ((i) or (ii)) in place of the deleted msd sequence.
  • alterations include, but are not limited to, (i) a linker joining the 5’ end of the ncRNA a1 region to the 3’ end of the ncRNA a2 region, (ii) a deletion of the wildtype msd region or a portion thereof, and (iii) installation of a single-strand RNA sequence comprising the polymerase template ((i) or (i
  • a complex comprising a fusion protein comprising a nickase and a polymerase and a guide RNA binds to and cuts an unedited target DNA to introduce a nick in the DNA (“nicked DNA”).
  • the templated msDNA (comprising a polymerase template RNG043-WO1 PCT Application with a PBS) associates with the nicked DNA by annealing the PBS to the endogenous 3’ flap immediately upstream of the nick on the nicked strand.
  • the guide RNA and the ncRNA can be delivered separately and do not need to be joined, as shown in FIG.8B.
  • the structure of the ncRNA may be further minimized by removing all or a portion of the a1/a2 stem/loop.
  • the guide RNA may be fused to the ncRNA or unfused. Other configurations on delivering ncRNAs and guide RNAs are depicted in FIG.8E.
  • FIG.10A and FIG.10B depict yet another embodiment which is differs from the system of FIG.9 in that the ncRNAs each have a first and second PBS.
  • a first ncRNA comprising a polymerase template with flanking PBS sites (PBS1 and PBS2).
  • Panel (2) depicts that as a first step the RT (e.g., retron RT) of a first fusion protein (the first fusion protein comprises a nickase (dotted black circle) and RT) synthesizes DNA against the first RTT from the 3’ of the endogenous 3’ flap, extending over the entire RTT and the PBS2 downstream of the RTT.
  • the RT e.g., retron RT
  • the PBS2 is encoded into the resulting first edited DNA flap a counterpart PBS2’ DNA sequence, which anneals to a second 3’ endogenous flap formed at a second nick site as shown in Panel 3.
  • DNA is again synthesized by a second editing fusion protein (e.g. by a DNA-dependent DNA polymerase, which in theory could be an engineered retron RT or another DNA-dependent DNA polymerase).
  • the second synthesis uses the first synthesis product as a template.
  • the double stranded DNA duplex is then incorporated into the DNA thereby installing the edited strands.
  • each of the components of the nickase/retron template precision editing system may comprises various modifications resulting from optimization, engineering, mutagenesis, substitution of orthologs, and other engineering processes to change, alter, or improve editing functions, stability, and processivity, and/or other characteristics.
  • the disclosure provides pharmaceutical compositions, such as lipid nanoparticles (LNPs) comprising an appropriate set of cargo elements that constitute the components of the nickase/retron template precision editing system.
  • LNPs lipid nanoparticles
  • Any delivery system may be used to delivery the nickase/retron template precision editing systems as proteins, nucleic acids, or protein-nucleic acid complexes, etc.
  • the nickase/retron template precision editing system may be delivered in an all-RNA configuration comprising one or more mRNAs encoding a nickase, a polymerase, and an optionally a retron RT (i.e., where the polymerase template is an msDNA such that it is converted from a ncRNA), as well as function RNA components such as targeting guide RNAs, second nicking guide RNAs, and ncRNAs.
  • the present disclosure further provides nucleic acid molecules encoding the gene editing system components (e.g., a recombinant ncRNA and/or a RNG043-WO1 PCT Application recombinant retron RT).
  • the present disclosure provides genome editing systems comprising recombinant retron components (e.g., recombinant ncRNA and/or recombinant RT), programmable nickases, and guide RNAs.
  • recombinant retron components e.g., recombinant ncRNA and/or recombinant RT
  • programmable nickases e.g., telomeres
  • guide RNAs e.g., telomerecombinant RNA and/or recombinant RT
  • nucleic acid molecules encoding the described genome editing systems and said components thereof, as well as polypeptides making up the components of said genome editing systems.
  • the disclosure provides vectors for transferring and/or expressing said genome editing systems, e.g., under in vitro, ex vivo, and in vivo conditions.
  • the disclosure provides cell-delivery compositions and methods, including compositions for passive and/or active transport to cells (e.g., plasmids), delivery by virus- based recombinant vectors (e.g., AAV and/or lentivirus vectors), delivery by non-virus-based systems (e.g., liposomes and LNPs), and delivery by virus-like particles.
  • cells e.g., plasmids
  • virus-based recombinant vectors e.g., AAV and/or lentivirus vectors
  • non-virus-based systems e.g., liposomes and LNPs
  • the genome editing systems described herein may be delivered in the form of DNA (e.g., plasmids or DNA-based virus vectors), RNA (e.g., ncRNA and mRNA delivered by LNPs), a mixture of DNA and RNA, protein (e.g., virus-like particles), and ribonucleoprotein (RNP) complexes.
  • DNA e.g., plasmids or DNA-based virus vectors
  • RNA e.g., ncRNA and mRNA delivered by LNPs
  • protein e.g., virus-like particles
  • RNP ribonucleoprotein
  • the disclosure provides formulations comprising any of the aforementioned components for delivery to cells and/or tissues, including in vitro, in vivo, and ex vivo delivery, recombinant cells and/or tissues modified by the recombinant retron-based genome modification systems and methods described herein, and methods of modifying cells by conducting genome editing and related DNA donor-dependent methods, such as recombineering, or cell recording, using the herein disclosed retron-based genome modification systems.
  • the disclosure also provides methods of making the recombinant retrons, retron-based genome modification systems, vectors, compositions, and formulations described herein, as well as to pharmaceutical compositions and kits for modifying cells under in vitro, in vivo, and ex vivo conditions that comprise the herein disclosed genome editing and/or modification systems.
  • this disclosure or the inventions herein provide a gene editing system comprising one or more delivery vehicles, wherein: the delivery vehicle(s) comprise RNA cargo; the RNA cargo comprises (a) at least one mRNA molecule encoding (i) a nucleic acid programmable nuclease (e.g., a nickase) and (ii) a polymerase (e.g., a DNA-dependent DNA polymerase, an RNA-dependent DNA polymerase, e.g., a retron reverse transcriptase), (b) an engineered templated retron ncRNA, and (c) guide RNA for the programmable nuclease; and each delivery vehicle contains (a)(i) and/or (a)(ii) and/or (b) and/or (c); whereby one delivery vehicle or more than one delivery vehicle delivers (a)(i), (a)(ii), (b), and (c).
  • a nucleic acid programmable nuclease e
  • (a)(i) and (a)(ii) comprise a single mRNA molecule encoding the nucleic acid programmable nuclease and the polymerase (e.g, retron reverse transcriptase).
  • the polymerase e.g, retron reverse transcriptase
  • (a)(i) and (a)(ii) are encoded and expressed as a fusion protein.
  • the fusion protein comprises the C-terminal end of the nucleic acid programmable nuclease fused to the N-terminal end of the polymerase (e.g. retron reverse transcriptase (nuclease:polymerase)); or the fusion protein comprises the N-terminal end of the nucleic acid programmable nuclease fused to the C-terminal end of the retron reverse transcriptase (polymerase:nuclease fusion).
  • the polymerase e.g. retron reverse transcriptase (nuclease:polymerase)
  • the fusion protein comprises the N-terminal end of the nucleic acid programmable nuclease fused to the C-terminal end of the retron reverse transcriptase (polymerase:nuclease fusion).
  • (a)(i) and (a)(ii) comprise a first mRNA molecule encoding the nucleic acid programmable nuclease and a second mRNA molecule encoding the polymerase (e.g., retron reverse transcriptase).
  • (c) is separate from (a)(i), (a)(ii) and (b) or is provided in trans.
  • the engineered retron ncRNA, and (c) the guide RNA are fused or are provided in cis.
  • the engineered retron ncRNA, and (c) the guide RNA are fused or are provided in cis and the guide RNA is fused to the 5’ end of the retron ncRNA.
  • the engineered retron ncRNA, and (c) the guide RNA are fused or are provided in cis and the guide RNA is fused to the 3’ end of the retron ncRNA.
  • the engineered retron ncRNA, and (c) the guide RNA are fused or are provided in cis and the engineered ncRNA comprises a first RNG043-WO1 PCT Application guide RNA fused to the 5’ end of the retron ncRNA, and a second guide RNA fused to the 3’ end of the retron ncRNA, and the first and second guide RNAs target different sequences.
  • guide RNA for the programmable nuclease can comprise one or more guides that target the same or different target sequences.
  • Such guide RNA(s) in an embodiment can be single guide RNA(s) or sgRNA(s); for instance, when the nucleic acid programmable nuclease comprises a Cas9.
  • the one or more delivery vehicles comprise a liposome or a lipid nanoparticle (LNP).
  • LNP lipid nanoparticle
  • the at least one mRNA molecule encoding i) the nucleic acid programmable nuclease and (ii) the polymerase (e.g., retron reverse transcriptase), and (b) the engineered retron ncRNA, are in the same delivery vehicle.
  • the at least one mRNA molecule encoding (i) the nucleic acid programmable nuclease and (ii) the polymerase (e.g., retron reverse transcriptase), and (b) the engineered retron ncRNA, are in separate delivery vehicles.
  • the nucleic acid programmable nuclease and the polymerase e.g., retron reverse transcriptase
  • the polymerase e.g., retron reverse transcriptase
  • the editing systems can be used in an animal cell, or a mammalian cell (e.g., a primate, a non-human primate, or a domesticated mammal such as a cat or dog or horse) or a human cell; for instance to correct, address, treat, mitigate a genetic condition in the animal, mammal, domesticated mammal, cat, dog, horse or human.
  • a mammalian cell e.g., a primate, a non-human primate, or a domesticated mammal such as a cat or dog or horse
  • a human cell for instance to correct, address, treat, mitigate a genetic condition in the animal, mammal, domesticated mammal, cat, dog, horse or human.
  • Such can be done in plant cells to introduce mutations that give rise to favorable phenotypic characteristics such as disease resistance or other favorable plant trait(s).
  • the nucleic acid programmable nuclease comprises a Cas9 nuclease, a TnpB nuclease, or a Cas12a nuclease.
  • the nuclease is a nickase such that only one of the two strands in the target region is cut.
  • the templated ncRNA may be derived from a baseline or wildtype ncRNA, such as any of those ncRNAs disclosed in Table B or a nucleotide sequence of Table B of U.S. Application No.18/087,673, or International Application No.
  • PCT/US2023/061038, or International Application No. PCT/US2023/072872 (each are incorporated herein by reference in their entireties, including their Sequence Listings), or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with a sequence from Table B of any of the aforementioned applications.
  • the polymerase template can be heterologous to a cell. Alternatively, the polymerase template can be endogenous to the cell.
  • the cell can contain a sequence that is typical for those in a population having a disease state and the donor polynucleotide can be a sequence that is typical for those in the population not having a non-disease state (e.g., the donor can be for a genetic correction or repair of a cell to modify the cell from having a mutation or modification that gives rise to a disease state to having a sequence typical of not having the disease state).
  • the gene editing system can comprise any combination(s) of the foregoing embodiments of the gene editing system.
  • this disclosure or the inventions herein provide a cell, such as an isolated cell comprising the gene editing system disclosed herein, such as in any of the foregoing paragraphs.
  • the cell e.g., isolated cell
  • the cell can be a eukaryotic cell.
  • the eukaryotic cell can be a plant cell or an animal cell or a mammalian cell, e.g., an isolated plant cell or an isolated animal cell or an isolated mammalian cell.
  • the mammalian cell e.g., an isolated mammalian cell
  • the cell can be a prokaryotic cell, e.g., a bacterial cell.
  • the donor polynucleotide can code for antibiotic susceptibility; and thus, the present disclosure and any inventions disclosed herein can involve a means for addressing antibiotic resistant bacteria by rendering such bacteria susceptible to antibiotics (and a subject to whom the gene editing system is administered can also then receive antibiotics to which the bacteria are rendered susceptible by the gene editing system).
  • this disclosure or the inventions herein provide a composition comprising: a) the gene editing system disclosed herein, such as in any of the foregoing paragraphs; and b)a pharmaceutically or veterinarily acceptable carrier.
  • the delivery vehicle in the composition can comprise a lipid nanoparticle comprising: a) one or RNG043-WO1 PCT Application more ionizable lipids; b) one or more structural lipids; c) one or more PEGylated lipids; and d) one or more phospholipids.
  • the one or more ionizable lipids comprises an ionizable lipid set forth in Table 2 of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038.
  • this disclosure or the inventions herein provide uses of the gene editing system embodiments and/or the compositions disclosed herein, such as in any of the foregoing paragraphs; for instance, use in modifying a cell or genetically modifying a cell, e.g., a eukaryotic or a prokaryotic cell and/or an animal cell and/or a mammalian and/or a human cell and/or a bacterial cell and/or a plant cell, in vivo, in vitro or ex vivo (e.g., any cell discussed herein wherein the cell comprises an isolated cell).
  • a cell or genetically modifying a cell e.g., a eukaryotic or a prokaryotic cell and/or an animal cell and/or a mammalian and/or a human cell and/or a bacterial cell and/or a plant cell, in vivo, in vitro or ex vivo (e.g., any cell discussed herein wherein the cell comprises an isolated cell
  • this disclosure or the inventions herein provide uses of the gene editing system embodiments and/or the compositions disclosed herein, such as in any of the foregoing paragraphs; for instance, use in treating or addressing a genetic condition of a subject, [0050]
  • this disclosure or the inventions herein provide methods of genetically modifying a cell comprising: contacting a gene editing system as herein discussed, such as in any of the foregoing paragraphs, or a composition as herein discussed, such as in any of the foregoing paragraphs (which comprises a gene editing system as herein discussed, such as in any of the foregoing paragraphs), advantageously a gene editing system that includes a sequence of interest encoding a donor polynucleotide comprising an intended edit to be integrated at a target sequence in a cell, said method comprising contacting the composition or the gene editing system with the cell, thereby delivering the RNA cargo to the cell, wherein: the nucleic acid programmable nuclease forms a complex with the
  • the cell can be a eukaryotic or a prokaryotic cell or an animal cell or a mammalian cell or a human cell or a bacterial cell or a plant cell.
  • a non-coding RNA (ncRNA) variant comprising a reference retron ncRNA with one or more modifications, wherein the reference retron ncRNA comprises from 5’ to 3’ direction: a1 region, first branching guanosine, msr, msd, and a2 region, and the one or more modifications comprises (i) linkage of the a1 region and the a2 region, (ii) deletion of at least a portion of the msr; (iii) deletion of at least a portion RNG043-WO1 PCT Application of the msd, (iv) addition of a single-stranded RNA comprising a polymerase template, or (v) addition of an RNA motif.
  • the one or more modifications comprise the linkage of the a1 region and the a2 region, wherein the linkage of the a1 region and the a2 region is formed by directly joining the 5’ end of the a1 region to the 3’ end of the a2 region without a linker.
  • the a1 region and the a2 region are partially or completely complementary to each other and the a1 region and the a2 region form a stem-loop structure after the linkage.
  • the ncRNA variant further comprises a second branching guanosine.
  • the one or more modifications further comprises a breakage between the msd and the a2 region, thereby generating the ncRNA variant comprising from 5’ to 3’ direction: the a2 region, the linkage between the a1 and the a2 region, the a1 region, the first branching guanosine, the msr, and the msd.
  • the one or more modifications comprises the deletion of at least a portion of the msr.
  • the portion deleted from the msr comprises a spacer between two stem-loops in the msr or a portion of the spacer.
  • the portion deleted from the msr comprises a portion of the spacer, whereby the deletion preserves a remaining 1-15 base pairs between the two stem-loops in the msr, as compared to the reference retron ncRNA.
  • the ncRNA variant comprises a sequence selected from RTX3_6342_msr_stem_var1 (SEQ ID NO: 19644), RTX3_6342_msr_stem_var20 (SEQ ID NO: 19663), RTX3_6342_msr_stem_var5 (SEQ ID NO: 19648), RTX3_6342_a1a2_var6 (SEQ ID NO: 19548), RTX3_6342_a1a2_var10 (SEQ ID NO: 19552), RTX3_6342_a1a2_var15 (SEQ ID NO: 19557), RTX3_6342_a1a2_var16 (SEQ ID NO: 19558), RTX3_6342_a1a2_var19 (SEQ ID NO: 19561), RTX3_6342_a1a2_var20 (SEQ ID NO: 19562), RTX3_6342_a1a2_var19 (SEQ ID NO
  • the ncRNA variant comprises a sequence selected from msR Spacer Del-1 (SEQ ID NO: 19939), msD Spacer Del- 1 (SEQ ID NO: 19940), msD Spacer Del-2 (SEQ ID NO: 19941), and msR Spacer Del-1/msD Spacer Del-2 (SEQ ID NO: 19942).
  • the ncRNA variant comprises one or more modifications in msR Spacer Del-1 (SEQ ID NO: 19939), msD Spacer Del-1 (SEQ ID NO: 19940), msD Spacer Del-2 (SEQ ID NO: 19941), or msR Spacer Del-1/msD Spacer Del-2 (SEQ ID NO: 19942) compared to WT R6342 ncRNA (SEQ ID NO: 19734).
  • the ncRNA variant comprises a sequence selected from Alt1 msR Spacer Del- (SEQ ID NO: 19947)1, Alt1 msD Spacer Del-1 (SEQ ID NO: 19948), Alt1 msD Spacer Del-2 (SEQ ID NO: 19949), Alt1 msR Spacer Del-1/msD Spacer Del-2 (SEQ ID NO: 19950), Alt1 msR Spacer Del-2/msD Del-3 (SEQ ID NO: 19951), Alt1 msR Spacer Del-2/msD Del-3 MS2 (SEQ ID NO: 19952), and Alt1 msR Spacer Del-2/msD Del-3 U7 (SEQ ID NO: 19953).
  • the ncRNA variant comprises one or more modifications in Alt1 msR Spacer Del- (SEQ ID NO: 19947)1, Alt1 msD Spacer Del-1 (SEQ ID NO: 19948), Alt1 msD Spacer Del-2 (SEQ ID NO: 19949), Alt1 msR Spacer Del-1/msD Spacer Del-2 (SEQ ID NO: 19950), Alt1 msR Spacer Del-2/msD Del-3 (SEQ ID NO: 19951), Alt1 msR Spacer Del- 2/msD Del-3 MS2 (SEQ ID NO: 19952), or Alt1 msR Spacer Del-2/msD Del-3 U7 (SEQ ID NO: 19953) compared to WT R6342 ncRNA (SEQ ID NO: 19734).
  • the ncRNA variant comprises a sequence selected from msR spacer del_30HA (SEQ ID NO: 19955), msR spacer del_45HA (SEQ ID NO: 19956), WT PAM mt, (SEQ ID NO: 19957) Alt del1+del2 PAM mt (SEQ ID NO: 19958), or Alt del1+del2+U7 PAM mt (SEQ ID NO: 19959), and Alt del1+del2 25bp ins (SEQ ID NO: 19960).
  • the ncRNA variant comprises one or more modifications in msR spacer del_30HA (SEQ ID NO: 19955), msR spacer del_45HA (SEQ ID NO: 19956), WT PAM mt, (SEQ ID NO: 19957) Alt del1+del2 PAM mt (SEQ ID NO: 19958), or Alt del1+del2+U7 PAM mt (SEQ ID NO: RNG043-WO1 PCT Application 19959), or Alt del1+del225bp ins (SEQ ID NO: 19960)compared to WT R6342 ncRNA (SEQ ID NO: 19734).
  • the one or more modifications further comprises addition of 2’O methyl or phosphorothioate bond at the 5’ or 3’ end of the ncRNA variant. In an embodiment, the one or more modifications further comprises the addition of the 2’O methyl or the phosphorothioate bond at both the 5’ and 3’ ends of the ncRNA variant.
  • the present disclosure provides a retron variant comprising the ncRNA variant of any one of the preceding embodiments and an RNA encoding a first polymerase, optionally downstream of the msd of the ncRNA variant.
  • the first polymerase is a reverse transcriptase, optionally wherein the reverse transcriptase is originated from a naturally occurring retron or retron-like sequence.
  • the first polymerase is a reverse transcriptase selected from: EcoI-RT, Efe1-RT, Mva1-RT, Cex1-RT, Eco8-RT, Vap1- RT, and Vro1-RT.
  • the reverse transcriptase is originated from a same naturally occurring retron as the reference retron ncRNA.
  • the present disclosure provides a chimeric gene editing composition
  • a chimeric gene editing composition comprising: (a) the ncRNA variant of any one of the preceding embodiments, or the retron variant of any one of the preceding embodiments, (b) a nuclease or a first mRNA encoding the nuclease, (c) a guide RNA (gRNA) associated with the nuclease, and (d) optionally, a second polymerase or a second mRNA encoding the second polymerase.
  • the chimeric gene editing composition comprises the second polymerase or the second mRNA encoding the second polymerase, wherein the second polymerase is a reverse transcriptase.
  • the chimeric gene editing composition comprises the second polymerase or the second mRNA encoding the second polymerase, wherein the second polymerase is a DNA polymerase.
  • the nuclease is a nickase.
  • the nickase comprises a Cas9 nuclease, a Cas9(D10A) nuclease, a TnpB nuclease, or a Cas12a nuclease.
  • the ncRNA variant or the retron variant is linked to the gRNA directly or indirectly.
  • the chimeric gene editing composition further comprises delivery vehicles.
  • the delivery vehicles encapsulate one or more components selected from: (a) the ncRNA variant or the retron variant, (b) the nuclease or the first mRNA encoding the nuclease, (c) the gRNA associated with the nuclease, and (d) optionally, the second polymerase or the second mRNA encoding the second polymerase.
  • the one or more polynucleotides further comprise a coding sequence of the first polymerase. [0086] In an embodiment of any one of the preceding embodiments, the one or more polynucleotides further comprise a coding sequence of a second polymerase.
  • the one or more polynucleotides further comprise one or more promoters, wherein each of the one or more promoters is operably linked to (i) the coding sequence of the ncRNA variant or the retron variant, (ii) a coding sequence of a nuclease, (iii) a coding sequence of a gRNA, (iv) a coding sequence of the first polymerase, or (v) a coding sequence of a second polymerase.
  • the present disclosure provides a vector comprising the one or more polynucleotides of any one of the preceding embodiments.
  • the ncRNA is linked to the gRNA directly or indirectly.
  • the ncRNA, the gRNA, and the first mRNA encoding the nickase are linked directly or indirectly.
  • FIG.6 illustrates the process of editing with a gene editing system described herein that utilizes a ncRNA and a retron RT to use the ncRNA as the polymerase template to edit the target DNA.
  • FIG.7A-7D illustrates the mechanism of editing with the gene editing system described herein.
  • FIG.8A-8C illustrates various configurations envisioned for the guide RNA and ncRNA components of the gene editing systems described herein.
  • FIG.8D illustrates various modifications that may be introduced in the templated ncRNAs described herein.
  • FIG.8E illustrates various configurations envisioned for the guide RNA and ncRNA components of the gene editing systems described herein.
  • FIG.9 illustrates an dual-editor embodiment of the gene editing system described herein.
  • FIG.10A-10B illustrates an dual-editor embodiment of the gene editing system described herein.
  • FIG.11 is a schematic illustrating various modifications of the gene editing system described herein.
  • FIG.12 is a schematic of an LNP formulation comprising RNA cargo for delivery the gene editing system of the disclosure to a cell, tissue, or patient under in vitro, in vivo, or ex vivo conditions.
  • FIG.13 is a scatter plot from a pooled screen assay, where a1:a2 variants were identified that showed higher performance than WT retron R6342.
  • FIG.17 is a set of graphs and a graphic showing the effects of altering the a1a2 stem on editing efficiency. These results suggest that the a1:a2 stem structure, instead of sequence, is the important feature for the editing output for ncRNA.
  • FIG.18 is a set of graphs and a graphic showing the effects of deleting the msD and msR spacer on editing efficiency. Deleting msR spacer alone did not affect the gene editing outcome while msD deletion variants exhibited lower editing efficiency, partially due to the decrease in indel rate in these cell lines.
  • FIG.19 is a set of graphs and a graphic showing the effects of a fused a1a2 ncRNA configuration on editing efficiency.
  • a fused a1a2 design (ALT) with both long and short R6342 retron ncRNA (R6342L and R6342S, respectively) was generated.
  • the alternative design with the fused a1a2 loop did not impact editing efficiency for R6342L ncRNA and slightly reduced the editing efficiency of R6342S ncRNA.
  • FIG.20 is a set of graphs and a graphic showing the effects of deletion of the msR and msD spacers with a fused a1a2 ncRNA configuration on editing efficiency. Deletion of the spacer sequences generally reduced editing efficiencyas compared to WT, but overall the modified constructs still demonstrated appreciable activity.
  • FIG.21 is a set of graphs and a graphic showing the effects of deletion of the msD stem loop in combination with the msD and msR spacers.
  • ncRNA with an msD stem- loop deletion with a further shortening of the msD spacer and a complete removal of the msR spacer, with the fused a1a2 stem (Alt1 msR Spacer Del-2/msD Del-3) only suffered a small negative effect on gene editing.
  • Incorporation of a MS2 stem-loop at 3’ end of the ncRNA significantly reduced the editing efficiency of such ncRNA (Alt1 msR Spacer Del-2/msD Del- 3 MS2), while the 3’ tail from a snRNA, U7, maintained similar editing efficiency (Alt1 msR Spacer Del-2/msD Del-3 MS2).
  • FIG.22 is a set of graphs and a graphic showing that the U7 snRNA sequence strongly boosted ssDNA production in the short ncRNA. ssDNA production with short ncRNA with deletions of msD, and msR stem-loop was measured. It was found that such ncRNA variants result in a 6-fold reduction in ssDNA production.
  • FIG.23 is a set of graphs showing that a minimization variant ncRNA with msr spacer deletion allowed up to 20% editing in human hematopoietic stem cells (HSCs) for M (ATG)>T (ACC) mutation installation in the UBA1 gene at 2.5 fold higher efficiency than WT version.
  • HSCs human hematopoietic stem cells
  • FIG.24 is a set of graphs showing ncRNA with msr/msd spacer deletion and alternative configuration (as presented in FIG.19) allowed up to 40% editing in human hematopoietic stem cells (HSC) for PAM mutation installation or 25 bp insertion in AAVS1 gene, at 2.5 fold higher efficiency than WT version.
  • HSC human hematopoietic stem cells
  • the bulge can be symmetrical (viz., the two unbase-paired single-stranded regions have the same number of nucleotides), or asymmetrical (viz., the unbase-paired single stranded region(s) have different or unequal numbers of nucleotides), or there is only one unbase-paired nucleotide on one strand.
  • a bulge can be described as A/B (such as a “2/2 bulge,” or a “1/0 bulge”) wherein A represents the number of unpaired nucleotides on the upstream strand of the RNG043-WO1 PCT Application stem, and B represents the number of unpaired nucleotides on the downstream strand of the stem.
  • cDNA refers to a strand of DNA copied from an RNA template, e.g., by a reverse transcriptase.
  • Cognate refers to two biomolecules that normally interact or co-exist in nature.
  • sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable or hybridizable. Moreover, a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a bulge, a loop structure or hairpin structure, etc.).
  • DNA-guided nuclease or “DNA-guided endonuclease” refers to a nuclease that associates covalently or non-covalently with a guide RNA thereby forming a complex between the guide RNA and the DNA-guided nuclease.
  • the guide RNA comprises a spacer sequence which comprises a nucleotide sequence having complementarity with a strand of a target DNA sequence.
  • the DNA-guided nuclease is indirectly guided or programmed to localize to a specific site in a DNA molecule through its association with the guide RNA, which directly binds or anneals to a strand of the target DNA through its complementarity region via Watson- Crick base-pairing.
  • DNA regulatory sequences can be used interchangeably herein to refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and/or regulate transcription of a non-coding sequence (e.g., guide RNA) or a coding sequence and/or regulate translation of a mRNA into an encoded polypeptide.
  • a non-coding sequence e.g., guide RNA
  • a coding sequence e.g., coding sequence and/or regulate translation of a mRNA into an encoded polypeptide.
  • the donor polynucleotide can contain sufficient homology to a genomic sequence at the target site, e.g.70%, 80%, 85%, 90%, 95%, or 100% homology with the nucleotide sequences flanking the target site, e.g., within about 200 bases or less of the target site, e.g., within about 190 bases or less of the target site, e.g., within about 180 bases or less of the target site, e.g., within about 170 bases or less of the target site, e.g., within about 160 bases or less of the target site, e.g., within about 150 bases or less of the target site, e.g., within about 140 bases or less of the target site, e.g., within about 130 bases or less of the target site, e.g., within about 120 bases or less of the target site, e.g., within about 110 bases or less of the target site, e.g., within about 100 bases or less of the target site, e.
  • the retron DNA may encode the ncRNA loci (which includes the msr and msd regions) as well as a retron RT.
  • Engineered retron [00140] As used herein, the term “engineered retron” or equivalently, “recombinant retron” or “retron variant” refers to a retron that does not occur in nature.
  • engineered retrons can include wildtype or naturally-occurring retrons that are modified to contain at least one modification, including a single nucleotide substitution, insertion, or deletion, or a substitution, insertion, or deletion of more than one nucleotide, i.e., up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, RNG043-WO1 PCT Application 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,
  • nucleotide of a starting point retron e.g., a wildtype retron
  • the nucleotides may be contiguous or non- contiguous.
  • an engineered retron as a whole is not naturally-occurring, it may include components such as nucleotide sequences that do occur in nature.
  • an engineered retron can have nucleotide sequences from different organisms (e.g., from different bacteria species), or from completely synthetic / artificial / recombinant nucleic acid sequences.
  • an engineered retron can have a bacterial nucleotide sequence, a human nucleotide sequence, a viral nucleotide sequence, and/or a synthetic / artificial / recombinant nucleotide sequence, and/or combinations of such sequences.
  • modifications of the recombinant retrons disclosed herein include the insertion of a heterologous nucleic acid sequence in a retron, for example, inserted into the ncRNA locus, such as in the msr or the msd loci.
  • exosomes are generally released into an extracellular environment from host/progenitor cells post fusion of multivesicular bodies the cellular plasma membrane.
  • exosomes can include components of the progenitor membrane in addition to designed components (e.g. engineered retron).
  • Exosome membranes are generally lamellar, composed of a bilayer of lipids, with an aqueous inter-nanoparticle space.
  • expression vector or “expression construct” refers to a vector that includes one or more expression control sequences, and an “expression control sequence” is a DNA sequence that controls and regulates the transcription and/or translation of another DNA sequence.
  • the present disclosure comprehends recombinant vectors that may include viral vectors, bacterial vectors, protozoan vectors, DNA vectors, or recombinants thereof.
  • Guide RNA The RNA molecule that binds to a programmable nuclease of a retron-based gene editing systems and which targets the nuclease to a specific location within the targeted polynucleotide sequence is referred to herein as the “guide RNA” or “guide RNA polynucleotide” (also referred to herein as a “guide RNA” or “gRNA” or “crRNA”).
  • a guide RNA comprises two segments, a “DNA-targeting segment” and a “protein-binding segment.”
  • segment it is meant a segment/section/region of a molecule, e.g., a contiguous stretch of nucleotides in an RNA.
  • a protein-binding segment of a guide RNA can comprise base pairs 5-20 of the RNA molecule that is 40 base pairs in length; and the DNA-targeting segment can comprise base pairs 21-40 of the RNA molecule that is 40 base pairs in length.
  • the definition of “segment,” unless otherwise specifically defined in a particular context, is not limited to a specific number of total base pairs, is not limited to any particular number of base pairs from a given RNA molecule, is not limited to a particular number of separate molecules within a complex, and may include regions of RNA molecules that are of any total length and may or may not include regions with complementarity to other molecules.
  • the DNA-targeting segment (or “DNA-targeting sequence”) comprises a nucleotide sequence that is complementary to a specific sequence within a targeted polynucleotide sequence (the complementary strand of the targeted polynucleotide sequence) designated the “protospacer-like” sequence herein.
  • the protein-binding segment interacts with a site-directed modifying polypeptide.
  • site-directed modifying polypeptide is a CRISPR nuclease
  • site-specific cleavage of the targeted polynucleotide sequence may occur at locations determined by both (i) base-pairing complementarity between the guide RNA and the targeted polynucleotide sequence; and (ii) a short motif (referred to as the protospacer adjacent motif (PAM)) in the targeted polynucleotide sequence.
  • PAM protospacer adjacent motif
  • heterologous nucleic acid refers to a genotypically distinct entity from that of the rest of the entity to which it is compared or into which it is introduced or incorporated.
  • a polynucleotide introduced by genetic engineering techniques into a different cell type is a heterologous polynucleotide (e.g., DNA or RNA) and, if expressed, can encode a heterologous polypeptide.
  • a cellular sequence e.g., a gene or portion thereof
  • Heterologous nucleic acid sequences introduced into retrons can including without limitation guide RNA sequences, HDR donor templates, protein-encoding genes, or non-coding functional RNA elements (e.g., stem-loops, hairpins, and bulges).
  • Homology-directed repair refers to the specialized form DNA repair that takes place, for example, during repair of double-strand breaks in cells. This process requires nucleotide sequence homology, uses a “donor” molecule to template repair of a “target” molecule (i.e., the one that experienced the double-strand break), and leads to the transfer of genetic information from the donor to the target.
  • Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, solvents, the base composition of the hybridizing species, length of the complementary regions, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art.
  • One having ordinary skill in the art knows how to vary these parameters to achieve a particular stringency of hybridization.
  • LNP Lipid nanoparticle
  • the term “lipid nanoparticle” or LNP refers to a type of lipid particle delivery system formed of small solid or semi-solid particles possessing an exterior lipid layer with a hydrophilic exterior surface that is exposed to the non-LNP environment, an interior space which may aqueous (vesicle like) or non-aqueous (micelle like), and at least one hydrophobic inter-membrane space.
  • LNP membranes may be lamellar or non-lamellar and may be comprised of 1, 2, 3, 4, 5 or more layers.
  • LNPs may comprise a nucleic acid (e.g.
  • Liposomes are a versatile carrier platform in that they are capable of transporting hydrophobic or hydrophilic molecules, including small molecules, proteins, and nucleic acids into cells. They were the earliest developed generation of nanoscale medicine delivery platform. Numerous liposomal drug formulations have been approved for human medicines, e.g., Doxil, a lipid nanoparticle formulation of the antitumor agent doxorubicin.
  • the liposomes can include those described in Tenchov et al., “Lipid Nanoparticles – From Liposomes to mRNA Vaccine Delivery, a Landscape of Diversity and Advancement,” ACS Nano, 2021, 15, pp.16982-17015 (the contents of which are incorporated by reference).
  • the term may also encompass oligonucleotides containing known analogues of natural nucleotides.
  • the term also may also encompass nucleic acid-like structures with synthetic backbones, such as, for example, in Eckstein, 1991; Baserga et al., 1992; Milligan, 1993; WO 97/03211; WO 96/39154; Mata, 1997; Strauss-Soukup, 1997; and Straus, 1996.
  • the term encompasses both ribonucleic acid (RNA) and DNA, including cDNA (including RT DNA), genomic DNA, synthetic, synthesized (e.g., chemically synthesized) DNA, and/or DNA (or RNA) containing nucleic acid analogs.
  • nucleic acid-guided nuclease or “nucleic acid-guided endonuclease” refers to a nuclease that associates covalently or non-covalently with a guide nucleic acid (e.g., a guide RNA or a guide DNA) thereby forming a complex between the guide nucleic acid and the nucleic acid-guided nuclease.
  • the guide nucleic acid comprises a spacer sequence which comprises a nucleotide sequence having complementarity with a strand of a target DNA sequence.
  • nucleic acid-guided nuclease is programmed by associating with a RNG043-WO1 PCT Application guide RNA molecule and in such cases the nuclease may be called “RNA-guided nuclease.” When programmed by a guide DNA, the nuclease may be called a “DNA-guided nuclease.”
  • Nucleic acid-guided, RNA-guided, or DNA-guided nucleases may also be referred to as “programmable nucleases,” which also include other classes of programmable nucleases which associate with specific DNA sequences through amino acid / nucleotide sequence recognition (e.g., zinc fingers nucleases (ZFN) and transcription activator like effector nucleases (TALEN)) rather than through guide RNAs.
  • ZFN zinc fingers nucleases
  • TALEN transcription activator like effector nucleases
  • a nuclease that has been modified to remove, inactivate, or otherwise eliminate at least one nuclease activity but which still retains at least one nuclease activity may be referred to as a “nickase” nuclease.
  • a nickase nuclease cuts one strand of a double-stranded DNA molecule, but not both strands.
  • a CRISPR Cas9 naturally comprises two distinct nuclease activity domains, namely, the HNH domain and the RuvC domain. The HNH domain cuts the strand of DNA bound to the guide RNA and the RuvC domain cuts the protospacer strand.
  • Programmable nuclease is meant to refer to a polypeptide that has the property of selective localization to a specific desired nucleotide sequence target in a nucleic acid molecule (e.g., to a specific gene target) due to one or more RNG043-WO1 PCT Application targeting functions.
  • targeting functions can include one or more DNA-binding domains, such as zinc finger domains characteristic of many different types of DNA binding proteins or TALE domains characteristic of TALEN proteins.
  • Such targeting function may also include the ability to associate and/or form a complex with a guide RNA, which then localizes to a specific site on the DNA which bears a sequence that is complementary to a portion of the guide RNA (i.e., the spacer of the guide RNA).
  • the programmable nuclease may be a single protein which comprises both a domain that binds directly (e.g., a ZF protein) or indirectly (e.g., an RNA-guided protein) to a target DNA site, as well as a nuclease domain.
  • a “recombinant nucleic acid” or “recombinant nucleotide” refers to a molecule that is constructed by joining nucleic acid molecules, which optionally may self-replicate in a live cell.
  • sequence identity refers to the overall relatedness between polymeric molecules, e.g., between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
  • the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller as described in CABIOS, 1989, 4:11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna CMP matrix. Methods commonly employed to determine percent identity between sequences can include, but are not limited to those disclosed in Carillo, H. and Lipman, D., SIAM J Applied Math., 48:1073 (1988); incorporated herein by reference.
  • Exemplary computer software to determine homology between two sequences can include, but are not limited to, GCG program package as described in Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984), BLASTP, BLASTN, and FASTA as described in Altschul, S. F. et al., J. Molec. Biol., 215, 403 (1990).
  • RNG043-WO1 PCT Application [00167] It is noted that when this disclosure speaks to a polypeptide (including anywhere in this specification, including in Table A of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No.
  • ⁇ -helical recognition lobe REC
  • NUC nuclease lobe
  • WED Wedge
  • REC ⁇ -helical recognition lobe
  • PI PAM-interacting
  • RuvC nuclease Bridge Helix
  • BH Bridge Helix
  • the term“subject” refers to an individual organism, for example, an individual mammal. In some embodiments, the subject is a human.
  • the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, a goat, a cattle, a cat, or a dog. In some embodiments, the subject is a vertebrate, an amphibian, a reptile, a fish, an insect, a fly, or a nematode. In some embodiments, the subject is a research animal. In some embodiments, the subject is genetically engineered, e.g., a genetically engineered non-human subject. The subject may be of either sex and at any stage of development.
  • stem refers to two or more base pairs, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more base pairs, formed by inverted repeat sequences connected at a “tip,” where the more 5 ⁇ or “upstream” strand of the stem bends to allows the more 3 ⁇ or “downstream” strand to base-pair with the upstream strand.
  • the number of base pairs in a stem is the “length” of the stem.
  • the tip of the stem is typically at least 3 nucleotides, but can be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more nucleotides.
  • An otherwise continuous stem may be interrupted by one or more bulges as defined herein.
  • the number of unpaired nucleotides in the bulge(s) are not included in the length of the stem.
  • the position of a bulge closest to the tip can be described by the number of base pairs between the bulge and the tip (e.g., the bulge is 4 bps from the tip).
  • the position of the other bulges (if any) further away from the tip can be described by the number of base pairs in the stem between the bulge in question and the tip, excluding any unpaired bases of other bulges in between.
  • Synthetic or artificial nucleic acid refers nucleic acids that are non-naturally occurring sequences. Such sequences do not originate from, or are not known to be present in RNG043-WO1 PCT Application any living organism (e.g., based on sequence search in existing sequence databases). Recombinant nucleic acids and synthetic nucleic acids also include those molecules that result from the replication of either of the foregoing.
  • a target sequence is the sequence to which the guide sequence of a guide nucleic acid (e.g., guide RNA) will hybridize.
  • a guide nucleic acid e.g., guide RNA
  • the target site (or target sequence) 5 ⁇ - GTCAATGGACC-3 ⁇ (SEQ ID NO:19933) within a target nucleic acid is targeted by (or is bound by, or hybridizes with, or is complementary to) the sequence 5 ⁇ -GGTCCATTGAC-3 ⁇ (SEQ ID NO:19934).
  • Suitable hybridization conditions include physiological conditions normally present in a cell.
  • the strand of the target nucleic acid that is complementary to and hybridizes with the guide RNA is referred to as the “complementary strand” or “target strand”; while the strand of the target nucleic acid that is complementary to the “target strand” (and is therefore not complementary to the guide RNA) is referred to as the “non-target strand” or “non-complementary strand.”
  • Treatment refers to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof, as described herein.
  • treatment may be administered after one or more symptoms have developed and/or after a disease has been diagnosed.
  • treatment may be administered in the absence of symptoms, e.g., to prevent or delay onset of a symptom or inhibit onset or progression of a disease.
  • treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their recurrence.
  • upstream and downstream RNG043-WO1 PCT Application are terms of relativity that define the linear position of at least two elements located in a nucleic acid molecule (whether single or double-stranded) that is orientated in a 5 ⁇ -to-3 ⁇ direction.
  • a first element is said to be upstream of a second element in a nucleic acid molecule where the first element is positioned somewhere that is 5 ⁇ to the second element.
  • a first element is downstream of a second element in a nucleic acid molecule where the first element is positioned somewhere that is 3 ⁇ to the second element.
  • variant retron RT is retron RT comprising one or more changes in amino acid residues as compared to a wild type retron RT amino acid sequence.
  • variant retron RT encompasses homologous proteins having at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% percent identity with a reference sequence and having the same or substantially the same functional activity or activities as the reference sequence.
  • Wild type is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene, protein, or characteristic as it occurs in nature as distinguished from mutant or variant forms.
  • DETAILED DESCRIPTION [00177] The present disclosure provides systems, methods and compositions used for precise genome editing, including installing nucleic acid insertions, replacements, and deletions at targeted and precise genome sites, wherein said systems, methods, and compositions are based on novel and/or modified retrons or components thereof, such as modified versions of the retron RTs of Table X of U.S. Application No.18/087,673, or RNG043-WO1 PCT Application International Application No.
  • the present disclosure provides a non-coding RNA variant (ncRNA variant) comprising a reference retron non-coding RNA (ncRNA) with one or more modifications.
  • the reference retron ncRNA can comprise from 5’ to 3’ direction: a1 region, first branching guanosine, msr, msd, and a2 region, In some embodiments, the reference retron ncRNA further comprises second branching guanisine. In some embodiments, the one or more modifications comprises (i) linkage of the a1 region and the a2 region, (ii) deletion of at least a portion of the msr; (iii) deletion of at least a portion of the msd, (iv) addition of a single- stranded RNA comprising a polymerase template, or (v) addition of an RNA motif.
  • the ncRNA variant can be incorporated into a recombinant or engineered retron and provide improved functionality and/or properties compared to the reference retron.
  • the present disclosure provides recombinant retrons comprising one or more genetic modifications which improves the functionality and/or properties of a retron.
  • Such genetic modifications can include a mutation, insertion, deletion, inversion, replacement, substitution, or translocation of one or more contiguous or non- contiguous nucleobases in a nucleic acid molecule encoding a retron or a component of a retron, such as an ncRNA or a reverse transcriptase.
  • the retron that becomes modifed with the one or more genetic modifications is a naturally occurring retron or retron component (e.g., naturally occuring ncRNA of Table A of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872 or RT) with the ability to facilitate homology-dependent recombination (or HDR) in a cell, thereby resulting in a relative increase in the concentrations or amounts of msDNA comprising a DNA donor template.
  • a naturally occurring retron or retron component e.g., naturally occuring ncRNA of Table A of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872 or RT
  • HDR homology-dependent recombination
  • the recombinant retrons are based on and/or derived from a naturally-occurring retron, such as any retron- related sequence provided by Table X of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872 (the introduction of the one or more genetic modifications into a set of 7257 previously unknown retrons discovered through computational methods described herein (e.g., see RNG043-WO1 PCT Application Examples).
  • a naturally-occurring retron such as any retron- related sequence provided by Table X of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872 (the introduction of the one or more genetic modifications into a set of 7257 previously unknown retrons discovered through computational methods described herein (e.g., see RNG043-WO1 PCT Application Examples).
  • the recombinant retrons can be based on introducing the one or more genetic modifications into previously available retron sequences, as described in “Mestre et al., Systematic Prediction of Genes Functionally Associated with Bacterial Retrons and Classification of The Encoded Tripartite Systems, Nucleic Acids Research, Volume 48, Issue 22, 16 December 2020, Pages 12632-12647” (incorporated herein by reference)) to achieve recombinant retrons with the enhanced ability to produce increased concentrations or amounts of msDNA comprising a DNA donor template.
  • the present disclosure further provides nucleic acid molecules encoding the recombinant retrons and/or recombinant retron components (e.g., a recombinant ncRNA and/or a recombinant retron RT).
  • the present disclosure provides genome editing systems comprising recombinant retron components (e.g., recombinant ncRNA and/or recombinant RT), programmable nucleases (e.g., RNA-guided nucleases, such as CRISPR-Cas proteins, ZFPs, and TALENS), and guide RNAs (in the case where RNA-guide nucleases are used in said genome editing systems).
  • recombinant retron components e.g., recombinant ncRNA and/or recombinant RT
  • programmable nucleases e.g., RNA-guided nucleases, such as CRISPR-Cas proteins, ZFPs,
  • the disclosure provides nucleic acid molecules encoding the described genome editing systems and said components thereof, as well as polypeptides making up the components of said genome editing systems.
  • the disclosure provides vectors for transferring and/or expressing the genome editing systems, e.g., under in vitro, ex vivo, and in vivo conditions.
  • the retron-based genome editing systems described herein may be delivered in the form of DNA (e.g., plasmids or DNA-based virus vectors), RNA (e.g., ncRNA and mRNA delivered by LNPs), a mixture of DNA and RNA, protein (e.g., virus-like particles), and ribonucleoprotein (RNP) complexes.
  • DNA e.g., plasmids or DNA-based virus vectors
  • RNA e.g., ncRNA and mRNA delivered by LNPs
  • protein e.g., virus-like particles
  • RNP ribonucleoprotein
  • each of the components of the retron-based genome editing system is delivered by an all-RNA system, e.g., the delivery of one or more RNA molecules (e.g., mRNA and/or ncRNA) by one or more LNPs, wherein the one or more RNA molecules form the ncRNA and guide RNA (as needed) and/or are translated into the polypeptide components (e.g., the RT and a programmable nuclease).
  • an all-RNA system e.g., the delivery of one or more RNA molecules (e.g., mRNA and/or ncRNA) by one or more LNPs, wherein the one or more RNA molecules form the ncRNA and guide RNA (as needed) and/or are translated into the polypeptide components (e.g., the RT and a programmable nuclease).
  • the disclosure provides formulations comprising any of the aforementioned components for delivery to cells and/or tissues, including in vitro, in vivo, and ex vivo delivery, recombinant cells and/or tissues modified by the recombinant retron-based genome modification systems and methods described herein, and methods of modifying cells by conducting genome editing and related DNA donor-dependent methods, such as recombineering, or cell recording, using the herein disclosed retron-based genome modification systems.
  • the engineered retron may comprise a sequence modification (e.g., insertion, deletion, and/or substitution of one or more nucleotide(s)) in the msr locus and/or the msd locus that: i) modulates (e.g., enhances) reverse transcription, processivity, accuracy/fidelity, and/or production of the msDNA (e.g., in the mammalian cell); ii) modulates (e.g., reduces) immunogenicity of the ncRNA encoded by the engineered retron (e.g., encoded by the msr locus and/or the msd locus) in a host (e.g., a host comprising the mammalian cell); iii) comprises a nucleotide sequence that modulates (e.g., inhibits or antagonizes) a function of the msDNA; and/or iv) modulates (e.g., improves
  • a sequence modification
  • the engineered retron is an engineered nucleic acid construct comprising: a) a first polynucleotide encoding a non-coding RNA (ncRNA), the first polynucleotide comprising: i) an msr locus encoding the msr RNA portion of a multi-copy RNG043-WO1 PCT Application single-stranded DNA (msDNA); and ii) an msd locus encoding the msd RNA portion of the msDNA; and b) one or more heterologous nucleic acids inserted at or within a location selected from: the msd locus, upstream of the msr locus, upstream of the msd locus, and downstream of the msd locus.
  • ncRNA non-coding RNA
  • the engineered nucleic acid construct may further comprise a second polynucleotide encoding a reverse transcriptase (RT), or a portion thereof, wherein the encoded RT is capable of synthesizing a DNA copy of at least a portion of the msd locus encoding the msDNA.
  • the engineered retron of the present disclosure encodes a reverse transcriptase (RT) or a functional domain thereof, comprising: i) a polypeptide listed in Table A of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No.
  • PCT/US2023/072872 or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polypeptide listed in Table A of U.S. Application No. 18/087,673, or International Application No.
  • the engineered retron of the present disclosure encodes a reverse transcriptase (RT) or a functional domain thereof, comprising: i) a polynucleotide listed in Table A of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No.
  • RT reverse transcriptase
  • PCT/US2023/072872 or a polynucleotide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polynucleotide listed in Table A of U.S.
  • the polynucleotide encoding the RT does not comprise a polynucleotide of Table X of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No.
  • PCT/US2023/072872 or an ncRNA having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to an ncRNA in Table B of U.S. Application No.18/087,673, or International Application No.
  • PCT/US2023/072872 or an ncRNA having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to an ncRNA listed in Table B of U.S. Application No. 18/087,673, or International Application No.
  • the reverse transcriptase (RT) or functional domain thereof comprises: (A) i) a polypeptide listed in Table A of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No.
  • PCT/US2023/072872 or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least RNG043-WO1 PCT Application 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polypeptide listed in Table A of U.S.
  • the polynucleotide encoding the RT does not comprise a polynucleotide in Table X of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872.
  • Another aspect of the disclosure provides a vector system comprising a vector comprising the engineered retron described herein.
  • RNG043-WO1 PCT Application [00195] Another aspect of the disclosure provides an isolated host cell comprising the engineered retron described herein, or the vector system described herein.
  • Another aspect of the disclosure provides a pharmaceutical composition comprising the engineered retron described herein, or the vector system described herein.
  • Another aspect of the disclosure provides a delivery vehicle comprising the engineered retron described herein or the ncRNA encoded by the engineered retron described herein, the vector or vector system described herein, the host cell described herein, or the pharmaceutical composition described herein.
  • kits comprising the engineered retron described herein or the ncRNA encoded by the engineered retron described herein, and optionally instructions for genetically modifying a cell using the engineered retron described herein or the ncRNA encoded by the engineered retron described herein.
  • the target sequence is recognized by a suitable nuclease, such as a CRISPR/Cas effector enzyme, a ZFN, a TALEN, a meganuclease, TnpB, IscB, or a restriction endonuclease (RE), and a double- stranded break (DSB) is created by the nuclease to facilitate / promote the insertion of the part of the heterologous nucleic acid into the target sequence.
  • a suitable nuclease such as a CRISPR/Cas effector enzyme, a ZFN, a TALEN, a meganuclease, TnpB, IscB, or a restriction endonuclease (RE), and a double- stranded break (DSB) is created by the nuclease to facilitate / promote the insertion of the part of the heterologous nucleic acid into the target sequence.
  • Retrons encode and transcribe as a single RNA, which comprises a non-coding RNA (ncRNA) portion and a portion encoding a specialized reverse transcriptase (RT).
  • ncRNA non-coding RNA
  • RT reverse transcriptase
  • the retron ncRNA (msr and msd) is the precursor of the hybrid molecule that eventually forms, and it initially folds into a typical RNA secondary structure that is recognized by the accompanying RT.
  • the translated RT typically recognizes certain secondary structures in the ncRNA, and binds the RNA template downstream from the msd region.
  • the RT initiates reverse transcription of the RNA towards its 5 ⁇ end, starting from the 2’-end of a conserved guanosine (G) residue found immediately after a double-stranded RNA structure (the a1/a2 region) within the ncRNA.
  • G conserved guanosine
  • a portion of the ncRNA serves as a template for reverse transcription, and reverse transcription terminates before reaching the msr locus.
  • cellular RNase H degrades the segment of the ncRNA that serves as template, but not other parts of the ncRNA.
  • an engineered retron can have a bacterial nucleotide sequence, a human nucleotide sequence, a viral nucleotide sequence, and/or a synthetic / artificial / recombinant nucleotide sequence, and/or combinations of such sequences.
  • modifications of the recombinant retrons disclosed herein include the insertion of a heterologous nucleic acid sequence in a retron, for example, inserted into the ncRNA locus, such as in the msr or the msd loci.
  • Linking guide RNA molecules to the 5 ⁇ and/or 3 ⁇ ends also represents another modification contemplated by the recombinant retrons disclosed herein.
  • the guide RNA molecules may also be categorized or referred to more generally as types of heterologous nucleic acid sequences used to modify starting point retrons. Examples of such modifications are depicted in FIG.8A-8E.
  • the DNA encoding the RT may also be modified to obtain a recombinant RT.
  • Such modifications to the DNA encoding ncRNA and/or RT may modulate the function of the ncRNA and/or RT in various ways, including i) modulating (e.g., enhancing) reverse transcription, processivity, accuracy/fidelity, and/or production of the msDNA (e.g., in the mammalian cell); ii) modulating (e.g., reducing) immunogenicity of ncRNA (msr locus and msd locus) encoded by the engineered retron in a host (e.g., a host comprising the mammalian cell); iii) modulating (e.g., inhibits, either permanently or transiently) a function of the msDNA; and/or iv) modulating (e.g., improving) efficiency of targeted genome editing / engineering.
  • modulating e.g., enhancing
  • reverse transcription e.g., processivity, accuracy/fidelity, and/or production of the msDNA
  • the present disclosure provides recombinant retrons having the general structure of: a) an msr locus; b) an msd locus encoding the msd RNA portion of the msDNA; c) a sequence encoding a retron reverse transcriptase (RT) (optionally in trans to the ncRNA), wherein the msd RNA is capable of being reverse transcribed (e.g., in a host cell such as a mammalian cell) to form an msDNA by the retron reverse transcriptase (RT); and/or d) a polymerase template nucleic acid capable of being transcribed.
  • RT retron reverse transcriptase
  • the retrons have a general structure comprising from 5’ to 3’ direction: a1 region, first branching guanosine, msr, msd, and a2 region.
  • the engineered retrons of the present disclosure are optionally structurally further modified to include one or more heterologous nucleic acids.
  • the engineered retron may be further modified to provide various functional improvements, such as (without limitation), to enhance the production of msDNA in a cell (e.g., a mammalian cell, including a human cell).
  • the engineered retron of the present disclosure encodes a reverse transcriptase (RT) or a functional domain thereof, comprising: i) a polypeptide listed in aforementioned Table A, or ii) a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polypeptide listed in aforementioned Table A.
  • RT reverse transcriptase
  • the engineered retron of the present disclosure encodes a reverse transcriptase (RT) or a functional domain thereof, comprising: i) a polynucleotide RNG043-WO1 PCT Application listed in aforementioned Table A, or a polynucleotide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polynucleotide of Table A of U.S.
  • the templated ncRNA is derived from a ncRNA comprising: (I) an ncRNA listed in aforementioned Table B, or (II) an ncRNA having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to an ncRNA of aforementioned Table B.
  • the engineered retron of the present disclosure encodes an ncRNA and a reverse transcriptase (RT) or a functional domain thereof, wherein the ncRNA and the RT or functional domain thereof are as described above.
  • RT reverse transcriptase
  • the engineered retrons are engineered based on clades defined on retron/retron RTs, in which the retrons are associated with a tripartite system composed of the ncRNA, the RT and an additional protein or RT-fused domain with diverse enzymatic functions, such as, for example, as described in “Mestre et al., Systematic Prediction of Genes Functionally Associated with Bacterial Retrons and Classification of The Encoded Tripartite Systems, Nucleic Acids Research, Volume 48, Issue 22, 16 December 2020, Pages 12632-12647” (incorporated herein by reference).
  • sequence alignments between different retron sequences are based on software known to one of ordinary skill in the art.
  • the retron ncRNA sequences including msr and msd sequences within the same clade may be highly conserved at certain positions, while being less conserved at other positions.
  • the a1 and a2 regions are both single- stranded and substantially reverse complementary to each other, forming a stem with optional interruption by a symmetric or asymmetric bulge, with optional one or more 5 ⁇ and/or 3 ⁇ overhang/unpaired nucleotide(s), wherein the a1 region generally ends before (e.g., ends immediately 5 ⁇ to) the conserved branching guanosine (G) providing the 2’-OH for reverse transcription priming.
  • G conserved branching guanosine
  • the sequence change comprises a mutated, reduced, or eliminated bulge in the a1/a2 stem region, including sequence change(s) in one (i.e., a1 or a2) strand, or both a1 and a2 strands.
  • the sequence change comprises deleting nucleotides from a1, a2, or both a1 and a2, such that the size of the bulge is reduced, or a symmetrical bulge becomes asymmetrical or vice versa, or a bulge is eliminated.
  • the sequence change comprises replacing/substituting nucleotides in a1, a2, or both a1 and a2, such that previously unpaired bases in the bulge become base-paired.
  • the sequence change comprises replacing an unpaired purine base with one or more unpaired pyrimidine base(s).
  • RNG043-WO1 PCT Application [00229] In some embodiments, the sequence change comprises replacing an unpaired pyrimidine base with one or more unpaired purine base(s).
  • the sequence change comprises replacing one unpaired purine base (e.g., A or G) with another unpaired purine base (e.g., G or A, respectively).
  • the sequence change comprises replacing one unpaired pyrimidine base (e.g., T/U or C) with another unpaired pyrimidine base (e.g., C or T/U, respectively).
  • the sequence change comprises an extension or shortening of a1, a2, or both a1 and a2.
  • the length of a1 can be shortened by deleting 5 ⁇ overhang, deleting any upstream bulge nucleotides, deleting bases involved in base-pairing.
  • the length of a1 can be extended by adding 5 ⁇ overhang, adding any upstream bulge nucleotides, adding bases involved in base-pairing.
  • the length of a2 can be shortened by deleting 5 ⁇ overhang, deleting any downstream bulge nucleotides, deleting bases involved in base-pairing. Likewise, the length of a2 can be extended by adding 5 ⁇ overhang, adding any downstream bulge nucleotides, adding bases involved in base-pairing.
  • Retron and ncRNA variants [00235] The ncRNA variants provided herein comprise one or more modifications compared to a reference retron ncRNA.
  • the one or more modifications can comprise (i) linkage of the a1 region and the a2 region, (ii) deletion of at least a portion of the msr; (iii) deletion of at least a portion of the msd, (iv) addition of a single-stranded RNA comprising a polymerase template, and/or (v) addition of an RNA motif.
  • the ncRNAs disclosed herein may be modified by introducing additional RNA motifs into the ncRNAs, e.g., at the 5 ⁇ and 3 ⁇ termini of the ncRNAs, or even at positions therein between (e.g., in the msr or msd regions) to improve transcriptional production and/or stability and/or function (e.g., RT-DNA production).
  • Such structures may include, but are not limited to RNA hairpins, RNA step-loops, RNA quadruplexes, cap structures, and poly(A) tails, or ribozyme functions and the like.
  • ncRNAs could also be modified to include one or more nuclear localization sequences.
  • RNA motifs could also improve RT processivity of the ncRNA or enhance ncRNA activity by enhancing RT binding.
  • Addition of dimerization motifs - such as kissing loops or a GNRA tetraloop/tetraloop receptor pair - at the 5 ⁇ and 3 ⁇ termini of the ncRNA could also result in effective circularization of the ncRNA, improving stability.
  • RNG043-WO1 PCT Application it is envisioned that addition of these motifs could enable the physical separation of ncRNA components, e.g., separation of the msr and msd regions.
  • ncRNAs could be further improved via directed evolution, in an analogous fashion to how protein function can be improved. Directed evolution could enhance ncRNA recognition by RT and/or reduce off-site targeting and/or indels and/or improve precise editing efficiency.
  • the present disclosure contemplates any such ways to further improve the stability and/or functionality of the ncRNAs disclosed here.
  • the RNAs (including the guide RNAs and the ncRNAs) used in the compositions of the disclosure have undergone a chemical or biological modification to render them more stable.
  • Exemplary modifications to an RNA include the depletion of a base (e.g., by deletion or by the substitution of one nucleotide for another) or modification of a base, for example, the chemical modification of a base.
  • RNA modifications includes modifications which introduce chemistries which differ from those seen in naturally occurring RNA, for example, covalent modifications such as the introduction of modified nucleotides, (e.g., nucleotide analogs, or the inclusion of pendant groups which are not naturally found in such mRNA molecules).
  • RNAs used in the compositions of the disclosure include, but are not limited to, 4'- thio- modified bases: 4'-thio-adenosine, 4'-thio-guanosine, 4'-thio-cytidine, 4'-thio-uridine, 4'- thio- 5-methyl-cytidine, 4'-thio-pseudouridine, and 4'-thio-2-thiouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2- thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1- carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5- tau
  • modification also includes, for example, the incorporation of non-nucleotide linkages or modified nucleotides into the mRNA sequences of the present disclosure (e.g., modifications to one or both of the 3' and 5' ends of an mRNA molecule encoding a functional protein or enzyme).
  • modifications include the addition of bases to an mRNA sequence (e.g., the inclusion of a poly A tail or a longer poly A tail), the alteration of the 3' UTR or the 5' UTR, complexing the mRNA with an agent (e.g., a protein or a complementary nucleic acid molecule), and inclusion of elements which change the structure of an RNA molecule (e.g., which form secondary structures).
  • RNAs include a 5' cap structure.
  • a 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5'5'5 triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase.
  • GTP guanosine triphosphate
  • cap structures include, but are not limited to, m7G(5')ppp (5'(A,G(5')ppp(5')A and G(5')ppp(5')G.
  • Naturally occurring cap structures comprise a 7- methyl guanosine that is linked via a triphosphate bridge to the 5'-end of the first transcribed RNG043-WO1 PCT Application nucleotide, resulting in a dinucleotide cap of m7G(5')ppp(5')N, where N is any nucleoside.
  • the cap is added enzymatically; the cap is added in the nucleus and is catalyzed by the enzyme guanylyl transferase.
  • Additional cap analogs include, but are not limited to, a chemical structures selected from the group consisting of m7GpppG, m7GpppA, m7GpppC; unmethylated cap analogs (e.g., GpppG); dimethylated cap analog (e.g., m2,7GpppG), trimethylated cap analog (e.g., m2,2,7GpppG), dimethylated symmetrical cap analogs (e.g., m7Gpppm7G), or anti reverse cap analogs (e.g., ARCA; m7,2'OmeGpppG, m72'dGpppG, m7,3'OmeGpppG, m7,3'dGpppG and their tetraphosphate derivatives), for example, as described in Jemielity, J.
  • the length of a poly A or poly U tail can be at least about 10, 50, 100, 200, 300, 400 at least 500 nucleotides.
  • a poly-A tail on the 3' terminus of mRNA typically includes about 10 to 300 adenosine nucleotides (e.g., about 10 to 200 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 100 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60 adenosine nucleotides).
  • mRNAs include a 3' poly(C) tail structure.
  • a suitable poly-C tail on the 3' terminus of mRNA typically include about 10 to 200 cytosine nucleotides (e.g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides).
  • the RNG043-WO1 PCT Application poly-C tail may be added to the poly-A or poly U tail or may substitute the poly-A or poly U tail.
  • RNAs according to the present disclosure e.g., ncRNAs
  • the chimeric gene editing composition includes one or more polymerases, which can be an retron RT.
  • the retron RT in such embodiments would handle converting a template ncRNA to a cognate templated msDNA, as well as being the RNA-dependent DNA polymerase functioning on the target DNA at the nick site (see Figures for details mechanism of action).
  • RNG043-WO1 PCT Application [00249]
  • the retron RTs that may be used with the gene editing system described herein may be any retron RT described in the art, including any of those described in Table A of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No.
  • Reverse transcriptases are enzymes present in all three domains of life, which are DNA polymerase using RNA as a template. Reverse transcriptases of the present disclosure are used to reverse transcribe template msd RNA into single-stranded msDNA.
  • a heterologous nucleic acid inserted at or within a location selected from: the msd locus, upstream of the msr locus, upstream of the msd locus, and downstream of the msd locus; and c.
  • the RT does not comprise a polypeptide listed in the abovementioned Table X.
  • the disclosure relates to an engineered nucleic acid-enzyme construct comprising: a) a non-coding RNA (ncRNA) comprising: i) an msr locus encoding the msr RNA portion of a multi-copy single-stranded DNA (msDNA); and ii) an msd locus encoding the msd RNA portion of the msDNA, b) a heterologous nucleic acid inserted at or within a location selected from: the msd locus; upstream of the msr locus; upstream of the msd locus; and downstream of the msd locus; and c) a reverse transcriptase (RT), or a portion thereof, wherein the RT is capable of synthesizing a DNA copy of at least a portion of the msd
  • ncRNA non-coding RNA
  • the chimeric gene editing system comprises (a) a nickase component, (b) a guide RNA that complexes with the nickase component and directs it to a target DNA sequence, (c) a polymerase component, and/or (d) and a polymerase template sequence, wherein the polymerase template sequence is provided by a modified retron ncRNA comprising the polymerase template sequence (herein referred to as a “templated ncRNA” or RNG043-WO1 PCT Application “tncRNA”).
  • a modified retroncRNA comprising the polymerase template sequence
  • the tncRNA comprises an altered structural configuration relative to a wildtype retron ncRNA wherein the alterations include (i) a linker joining the 5’ end of the ncRNA a1 region to the 3’ end of the ncRNA a2 region, (ii) a deletion of the wildtype msd region or portion thereof, and/or (iii) installation of a single-strand RNA sequence comprising the polymerase template in place of the deleted msd sequence.
  • the chimeric gene editing system may also optionally comprise a retron RT to convert the templated ncRNA to a cognate msDNA, which is referred to herein as the “templated msDNA” or “tmsDNA”.
  • the nickase component of the chimeric editing system described herein may be any programmable CRISPR nickase, including nickases based on CRISPR system Class 1, Type I, II, or III Cas nucleases; Class 2, Type II nuclease (such as Cas9); a Class 2, Type V nuclease (such as Cpfl), or a Class 2, Type VI nuclease (such as C2c2).
  • Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9 (Csnl or Csxl2), CaslO, CaslOd, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Cs
  • a Class 1, type II CRISPR system Cas9 endonuclease is used.
  • Cas9 nucleases from any species, or biologically active fragments, variants, analogs, or derivatives thereof that retain Cas9 endonuclease activity i.e., catalyze site-directed cleavage of DNA to generate double-strand breaks
  • the Cas9 need not be physically derived from an organism but may be synthetically or recombinantly produced.
  • sequences or a variant thereof comprising a sequence having at least about 70- 100% sequence identity thereto, including any percent identity within this range, such as 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used for genome editing, as described herein and in some examples, as described in Fonfara et al. (2014) Nucleic Acids Res. 42(4):2577-90; Kapitonov et al. (2015) J.
  • Cpfl does not require a tracrRNA and only depends on a crRNA in its guide RNA, which provides the advantage that shorter guide RNAs can be used with Cpfl for targeting than Cas9.
  • Cpfl is capable of cleaving either DNA or RNA.
  • the PAM sites recognized by Cpfl have the sequences 5 ⁇ -YTN-3 ⁇ (where “Y” is a pyrimidine and “N” is any nucleobase) or 5 ⁇ -TTN-3 ⁇ , in contrast to the G-rich PAM site recognized by Cas9.
  • Cpfl cleavage of DNA produces double-stranded breaks with a sticky-ends having a 4 or 5 nucleotide overhang.
  • additional information on examples of Cpfl is disclosed in Ledford et al.
  • C2c1 (Cas12b) is another class II CRISPR/Cas system RNA-guided nuclease that may be used.
  • C2cl similarly to Cas9, depends on both a crRNA and tracrRNA for guidance to target sites, such as, for example, those disclosed in Shmakov et al. (2015) Mol Cell.60(3):385-397, Zhang et al. (2017) Front Plant Sci.8:177; herein incorporated by reference.
  • a nucleic acid sequence-programmable DNA binding domain can be associated with or complexed with at least one guide nucleic acid (e.g., guide RNA or a pegRNA), which localizes the DNA binding domain to a DNA sequence that comprises a DNA strand (i.e., a target strand) that is complementary to the guide nucleic acid, or a portion RNG043-WO1 PCT Application thereof (e.g., the spacer of a guide RNA which anneals to the protospacer of the DNA target).
  • the guide nucleic-acid “programs” the DNA binding domain (e.g., Cas9 or equivalent) to localize and bind to complementary sequence of the protospacer in the DNA.
  • nucleic acid sequence-programmable DNA binding domain may be any Class 2 CRISPR-Cas system, including any type II, type V, or type VI CRISPR-Cas enzyme.
  • CRISPR-Cas As a tool for genome editing, there have been constant developments in the nomenclature used to describe and/or identify CRISPR-Cas enzymes, such as Cas9 and Cas9 orthologs.
  • CRISPR-Cas nomenclature is discussed in Makarova et al., “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?,” The CRISPR Journal, Vol.1. No.5, 2018, the entire contents of which are incorporated herein by reference.
  • the mechanism of action of certain CRISPR Cas enzymes contemplated herein includes the step of forming an R-loop whereby the Cas protein induces the unwinding of a double-strand DNA target, thereby separating the strands in the region bound by the Cas protein.
  • the guide RNA spacer then hybridizes to the “target strand” at a region that is complementary to the protospacer sequence of the DNA.
  • the Cas protein may include one or more nuclease activities, which then cut the DNA leaving various types of lesions.
  • the Cas protein may comprises a nuclease activity that cuts the non-target strand at a first location, and/ or cuts the target strand at a second location.
  • the target DNA can be cut to form a “double-stranded break” whereby both strands are cut.
  • the target DNA can be cut at only a single site, i.e., the DNA is “nicked” on one strand.
  • Exemplary Cas proteins with different nuclease activities include “Cas9 nickase” (“nCas9”) and a deactivated Cas9 having no nuclease activities (“dead Cas9” or “dCas9”).
  • nCas9 Cas9 nickase
  • deactivated Cas9 having no nuclease activities deactivated Cas9 having no nuclease activities
  • d Cas9 deactivated Cas9 having no nuclease activities
  • the below description of various Cas proteins which can be used in connection with the presently disclosed LNP-delivered gene editing systems is not meant to be limiting in any way.
  • the gene editing systems may comprise the canonical SpCas9, or any ortholog Cas9 protein, or any variant Cas9 protein—including any naturally occurring variant, mutant, or otherwise engineered version of Cas9—that is known or which can be made or evolved through a directed evolutionary or otherwise mutagenic process.
  • the Cas9 nuclease sequences and structures can be those described in “Complete genome sequence of an M1 strain of Streptococcus pyogenes.” Ferretti et al., J.J., McShan W.M., Ajdic D.J., Savic D.J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A.N., Kenton S., Lai H.S., Lin S.P., Qian Y., Jia H.G., Najar F.Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S.W., Roe B.A., McLaughlin R.E., Proc.
  • the Cas9-derived nickase has one or more mutations in the REC Lobe domain. In one embodiment, the Cas9-derived nickase has a N497A, R661A, and/or Q695A mutation in the REC Lobe domain. In some embodiment, the Cas9-derived nickase has one or more mutations in the HNH domain. In one embodiment, the Cas9-derived nickase has H840A, N863A, and/or D839A in the HNH domain.
  • PCT/US2021/044924 filed August 6, 2021; International Application No. PCT/KR2021/009794, filed July 28, 2021; International Application No. PCT/US2021/031439, filed May 7, 2021; International Application No. PCT/US2021/034996, filed May 28, 2021; International Application No. PCT/KR2021/005244, filed April 26, 2021; International Application No. PCT/KR2021/005031, filed April 21, 2021; International Application No. PCT/US2020/023730, filed March 19, 2020; International Application No. PCT/US2020/023713, filed March 19, 2020; International Application No. PCT/EP2021/054228, filed February 19, 2021; International Application No.
  • PCT/US2020/023732 filed March 19, 2020; International Application No. PCT/US2020/023712, filed March 19, 2020; International Application No. PCT/US2020/023725, filed March 19, 2020; International Application No. PCT/US2020/023713, filed March 19, 2020; International Application No. PCT/US2020/023727, filed March 19, 2020; International Application No. PCT/US2020/023724, filed March 19, 2020; International Application No. PCT/US2020/023583, filed March 19, 2020; International Application No. PCT/US2020/023723, filed March 19, 2020; International Application No. PCT/CN2020/074218, filed February 3, 2020; U.S. Application No.15/616,756, filed June 7, 2017, now U.S.
  • the chimeric gene editing composition comprises a prime editing system or a polynucleotide encoding a prime editing system.
  • the cargo comprises a component of a prime editing system or a polynucleotide encoding a component of a prime editing system.
  • the prime editor comprises a catalytically impaired Cas protein fused to an engineered reverse transcriptase which can precisely and permanently edit one or more target nucleobases in a target polynucleotide.
  • the prime editor comprises an engineered Moloney murine leukemia virus (“M-MLV”) reverse transcriptase (“RT”) fused to a Cas-H840A nickase (called “PE2”).
  • M-MLV Moloney murine leukemia virus
  • RT Cas-H840A nickase
  • the prime editor comprises an engineered M-MLV RT fused to a Cas9-H840A nickase.
  • the prime editor comprises an engineered M-MLV RT fused to a Streptococcus pyogenes Cas9 (spCas9)-H840A nickase.
  • PE modifications include increased PAM flexibility to increase the utility of PE2 editing, expanding the coverage of targetable pathogenic variants in the ClinVar database that can now be prime edited to 94.4%.
  • the prime editing system further comprises a prime editing guide RNA (“pegRNA”).
  • the cargo comprises a pegRNA or a polynucleotide encoding a pegRNA.
  • the prime editing system further comprises a second guide RNA targeting the complementary strand, allowing the Cas9 nickase to also nick the non-edited strand (called “PE3”), which biases mismatch DNA repair in favor of the edited sequence.
  • the second guide RNA is designed to recognize the complementary strand of DNA only after the PE3 edit has occurred (called “PE3b”), which reduces indel formation.
  • the prime editing system comprises an uracil glycosylase inhibitor.
  • the prime editing system comprises a Cas9 protein fused to an uracil glycosylase inhibitor.
  • the cargo comprises an uracil glycosylase inhibitor or a polynucleotide encoding an uracil glycosylase inhibitor.
  • the cargo comprises a Cas9 protein fused to an uracil glycosylase inhibitor or a polynucleotide encoding a Cas9 protein fused to an uracil glycosylase inhibitor.
  • the present disclosure further provides guide RNAs for use in accordance with the disclosed nucleic acid programmable DNA binding proteins (e.g., Cas9) for use in methods of editing.
  • the disclosure provides guide RNAs that are designed to recognize target sequences.
  • Such gRNAs may be designed to have guide sequences (or “spacers”) having complementarity to a target sequence.
  • Such gRNAs may be designed to have not only a guide sequences having complementarity to a target sequence to be edited, but also to have a backbone sequence that interacts specifically with the nucleic acid programmable DNA binding protein.
  • the guide RNA may be 15-100 nucleotides in length and comprise a sequence of at least 10, at least 15, or at least 20 contiguous nucleotides that is complementary to a target nucleotide sequence.
  • the guide RNA may comprise a spacer sequence of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides that is complementary to a target nucleotide sequence.
  • the guide sequence has a length in a range of from 17-30 nucleotides (nt) (e.g., from 17-25, 17-22, 17-20, 19-30, 19-25, 19-22, 19-20, 20-30, 20-25, or 20-22 nt). In some cases, the guide sequence has a length in a range of from 17-25 nucleotides (nt) (e.g., from 17- 22, 17-20, 19-25, 19-22, 19-20, 20-25, or 20-22 nt).
  • the guide sequence has a length of 17 or more nt (e.g., 18 or more, 19 or more, 20 or more, 21 or more, or 22 or more nt; 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, etc.). In some cases, the guide sequence has a length of 19 or more nt (e.g., 20 or more, 21 or more, or 22 or more nt; 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, etc.). In some cases, the guide sequence has a length of 17 nt.
  • nt e.g., 18 or more, 19 or more, 20 or more, 21 or more, or 22 or more nt; 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, etc.
  • the guide sequence has a length of 18 nt. In some cases, the guide sequence has a length of 19 nt. In some cases, the guide sequence has a length of 20 nt. In some cases, the guide sequence has a length of 21 nt. In some cases, the guide sequence has a length of 22 nt. In some cases, the guide sequence has a length of 23 nt.
  • the spacer sequence has a length of from 15 to 50 nucleotides (e.g., from 15 nucleotides (nt) to 20 nt, from 20 nt to 25 nt, from 25 nt to 30 nt, from 30 nt to 35 nt, from 35 nt to 40 nt, from 40 nt to 45 nt, or from 45 nt to 50 nt).
  • 15 nucleotides (nt) to 20 nt from 20 nt to 25 nt, from 25 nt to 30 nt, from 30 nt to 35 nt, from 35 nt to 40 nt, from 40 nt to 45 nt, or from 45 nt to 50 nt.
  • a subject guide RNA can interact with a target nucleic acid (e.g., double stranded DNA (dsDNA), single stranded DNA (ssDNA), single stranded RNA (ssRNA), or double stranded RNA (dsRNA)) in a sequence-specific manner via hybridization (i.e., base pairing).
  • a target nucleic acid e.g., double stranded DNA (dsDNA), single stranded DNA (ssDNA), single stranded RNA (ssRNA), or double stranded RNA (dsRNA)
  • dsRNA double stranded DNA
  • the guide RNA can be modified to hybridize to any desired target sequence (e.g., while taking the PAM into account, e.g., when targeting a dsDNA target) within a target RNG043-WO1 PCT
  • Application nucleic acid e.g., a eukaryotic target nucleic acid such as genomic DNA.
  • the percent complementarity between the spacer sequence of the guide and the target site of the target nucleic acid is 60% or more (e.g., 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the spacer and the target site of the target nucleic acid is 80% or more (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%).
  • a guide RNA (including pegRNA) component comprises ribonucleotides in a region that binds to a target sequence and one or more deoxyribonucletides and/or nucleotide analogs in a region that binds to a nucleic acid programmable DNA binding protein (e.g., Cas9 nickase).
  • deoxyribonucleotides and/or nucleotide analogs are incorporated in engineered guide RNA (including pegRNA) component structures.
  • 3-5 nucleotides at either the 3’ or the 5’ end of a guide RNA (including pegRNA) component are chemically modified.
  • all of the phosphodiester bonds of a guide RNA (including pegRNA) component are substituted with phosphorothioates (PS) for enhancing levels of gene disruption.
  • more than five nucleotides at the 5’ and/or the 3’ end of the guide RNA (including pegRNA) component are chemically modified with 2’-0-Me, 2’-F or S-constrained ethyl(cEt).
  • Such chemically modified guide RNA (including pegRNA) component can mediate enhanced levels of gene disruption, such as, for example, those described in Ragdarm et al., 2015, PNAS, E7110-E7111.
  • Such chemically modified guide RNA (including pegRNA) component can be used to identify or enrich cells generically edited by a gene editing system described herein.
  • other guide RNA (including pegRNA) modifications are described in Kim, D.Y., Lee, J.M., Moon, S.B. et al. Efficient CRISPR editing with a hypercompact Cas12f1 and engineered guide RNAs delivered by adeno-associated virus. Nat Biotechnol 40, 94–102 (2022).
  • the guide RNA (including pegRNA) are modified in one or more locations within the molecule: MS1, an internal penta(uridinylate) (UUUUU) sequence in the tracrRNA; MS2, the 3′ terminus of the crRNA; MS3, the ‘stem 1’ region of the tracrRNA; MS4, the tracrRNA–crRNA complementary region; and MS5, the ‘stem 2’ region of the tracrRNA.
  • Various aspects of the present disclosure provide methods and compositions for improved guide RNA (including pegRNA) stability via chemical modifications, such as, for examples, the methods and compositions described in Braasch, D.
  • RNA targeting therapeutics molecular mechanisms of antisense oligonucleotides as a therapeutic platform.
  • Hendel et al. improved guide RNA stability by chemically modifying gRNA ends to reduce degradation by exonucleases and RNA nuclease. See Hendel, A., Bak, R. O., Clark, J. T., Kennedy, A. B., Ryan, D. E., Roy, S., et al. (2015a). Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat. Biotechnol.33, 985–989.
  • the genome editing system comprising a guide RNA (including pegRNA) and further comprises one or more chemical modifications selected from, but not limited to the modifications described in Allen et al.
  • chemical modifications to the guide RNA include modifications on the ribose rings and phosphate backbone of guide RNA (including pegRNA) and modifications at the 2′OH include 2′-O-Me, 2′-F, and 2′F- ANA. More extensive ribose modifications include 2′F-4′-C ⁇ -OMe and 2′,4′-di-C ⁇ -OMe combine modification at both the 2′ and 4′ carbons.
  • Phosphodiester modifications include sulfide-based Phosphorothioate (PS) or acetate-based phosphonoacetate alterations. Combinations of the ribose and phosphodiester modifications have given way to formulations such as 2′-O-methyl 3′phosphorothioate (MS), or 2′-O-methyl-3′-thioPACE (MSP), and 2′-O- methyl-3′-phosphonoacetate (MP) RNAs.
  • MS 2′-O-methyl 3′phosphorothioate
  • MSP 2′-O-methyl-3′-thioPACE
  • MP 2′-O- methyl-3′-phosphonoacetate
  • Locked and unlocked nucleotides such as locked nucleic acid (LNA), bridged nucleic acids (BNA), S-constrained ethyl (cEt), and unlocked nucleic acid (UNA) are examples of sterically hindered nucleotide modifications. Modifications to make a phosphodiester bond between the 2′ and 5′ carbons (2′,5′-RNA) of adjacent RNAs as well as a butane 4-carbon chain link between adjacent RNAs have been described. [00330] In certain embodiments, the ncRNA and the guide RNA can be delivered as a single molecule, i.e., with the guide RNA fused to the 5’ and/or 3’ end of the ncRNA.
  • the ncRNA may have guide RNAs located at both ends in some embodiments.
  • the guide RNA and ncRNA may be provided and/or delivered as separate components. Separation of the guide RNA from the ncRNA can result in increased editing efficiency.
  • a ncRNA-gRNA fusion may be co-delivered with a separate guide RNA.
  • the disclosure provides compositions for transferring and/or expressing the retron-based gene editing systems, e.g., under in vitro, ex vivo, and in RNG043-WO1 PCT Application vivo conditions.
  • the disclosure provides cell-delivery compositions and methods, including compositions for passive and/or active transport to cells (e.g., plasmids), delivery by virus-based recombinant vectors (e.g., AAV and/or lentivirus vectors), delivery by non-virus-based systems (e.g., liposomes and LNPs), and delivery by virus-like particles of the retron-based gene editing systems described herein.
  • cells e.g., plasmids
  • virus-based recombinant vectors e.g., AAV and/or lentivirus vectors
  • non-virus-based systems e.g., liposomes and LNPs
  • the retron-based gene editing systems described herein may be delivered in the form of DNA (e.g., plasmids or DNA-based virus vectors), RNA (e.g., guide RNA and mRNA delivered by LNPs), a mixture of DNA and RNA, protein (e.g., virus-like particles), a mixture of DNA or RNA and protein, and ribonucleoprotein (RNP) complexes.
  • DNA e.g., plasmids or DNA-based virus vectors
  • RNA e.g., guide RNA and mRNA delivered by LNPs
  • protein e.g., virus-like particles
  • RNP ribonucleoprotein
  • the retron-based gene editing systems and/or components thereof can be delivered by any known delivery system such as those described above, including (a) without vectors (e.g., electroporation), (b) viral delivery systems and (c) non-viral delivery systems.
  • Viral delivery systems include expression vectors, adeno-associated virus (AAV) vectors, retroviral vectors, lentiviral vectors, and the like.
  • An expression construct can be replicated in a living cell, or it can be made synthetically.
  • Non-viral delivery systems include without limitation lipid particles (e.g.
  • Lipid nanoparticles (LNPs)), non-lipid nanoparticles, exosomes, liposomes, micelles, viral particles, stable nucleic-acid-lipid particles (SNALPs), lipoplexes/polyplexes, DNA nanoclews, Gold nanoparticles, iTOP, Streptolysin O (SLO), multifunctional envelope-type nanodevice (MEND), lipid-coated mesoporous silica particles, inorganic nanoparticles, and polymeric delivery technology (e.g., polymer-based particles).
  • SNALPs stable nucleic-acid-lipid particles
  • SLO stable nucleic-acid-lipid particles
  • SLO stable nucleic-acid-lipid particles
  • SLO stable nucleic-acid-lipid particles
  • SLO stable nucleic-acid-lipid particles
  • SLO stable nucleic-acid-lipid particles
  • SLO stable nucleic-acid-lipid particles
  • SLO stable nucleic-
  • the engineered retron-based gene editing systems may be introduced into any type of cell, including any cell from a prokaryotic, eukaryotic, or archaeon organism, including bacteria, archaea, fungi, protists, plants (e.g., monocotyledonous and dicotyledonous plants); and animals (e.g., vertebrates and invertebrates).
  • animals e.g., vertebrates and invertebrates.
  • animals that may be transfected with an engineered retron-based gene editing systems include, without limitation, vertebrates such as fish, birds, mammals (e.g., human and non-human primates, farm animals, pets, and laboratory animals), reptiles, and amphibians.
  • the engineered retron-based gene editing systems can be introduced into a single cell or a population of cells.
  • Cells from tissues, organs, and biopsies, as well as recombinant cells, genetically modified cells, cells from cell lines cultured in vitro, and artificial cells (e.g., nanoparticles, liposomes, polymersomes, or microcapsules encapsulating nucleic acids) may all be transfected with the engineered retron-based gene editing systems.
  • the engineered retron-based gene editing systems can be introduced into cellular fragments, cell components, or organelles (e.g., mitochondria in animal and plant cells, plastids (e.g., chloroplasts) in plant cells and algae).
  • organelles e.g., mitochondria in animal and plant cells, plastids (e.g., chloroplasts) in plant cells and algae.
  • Cells may be cultured or expanded after transfection with the engineered retron- based gene editing systems.
  • Methods of introducing nucleic acids into a host cell are well known in the art.
  • Commonly used methods include chemically induced transformation, typically using divalent cations (e.g., CaCl 2 ), dextran-mediated transfection, polybrene mediated transfection, lipofectamine and LT-1 mediated transfection, electroporation, protoplast fusion, encapsulation of nucleic acids in liposomes, and direct microinjection of the nucleic acids comprising Cas12a editing systems into nuclei, such as, for example, the methods described in Sambrook et al. RNG043-WO1 PCT Application (2001) Molecular Cloning, a laboratory manual, 3rd edition, Cold Spring Harbor Laboratories, New York, Davis et al.
  • Plant cells may also be targeted by the retron-based gene editing systems (or components thereof) disclosed herein.
  • methods for genetic transformation of plant cells can be those described in in US2022/0145296, and U.S. Pat. Nos.8,575,425; 7,692,068; 8,802,934; 7,541,517; Rakoczy-Trojanowska, M. (2002) Cell Mol Biol Lett.7:849- 858; Jones et al. (2005) Plant Methods 1:5; Rivera et al.
  • the plant cells that have been transformed may be grown into a transgenic organism, such as a plant, in accordance with conventional methods for example, such as those described in McCormick et al. (1986) Plant Cell Reports 5:81-84.
  • Plant material that may be transformed with the retron-based gene editing systems (or components thereof) described herein includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like. Progeny, variants, and mutants of the regenerated plants are also included within the scope of the disclosure, provided that these parts comprise the genetic modification introduced by the retron-based gene editing systems.
  • retron-based gene editing systems described herein may be used to produce transgenic plants with desired phenotypes, including but not limited to, increased disease resistance (e.g., increased viral, bacterial of fungal resistance), increased insect resistance, increased drought resistance, increased yield, and altered fruit ripening characteristics, sugar and oil composition, and color.
  • the retron msr gene, msd gene, and/or ret gene can be expressed in vitro from a vector, such as in an in RNG043-WO1 PCT Application vitro transcription system.
  • the resulting ncRNA or msDNA can be isolated before being packaged and/or formulated for direct delivery into a host cell.
  • the isolated ncRNA or msDNA can be packaged/formulated in a delivery vehicle such as lipid nanoparticles as described in other sections.
  • the retron msr gene, msd gene, and/or ret gene are expressed in vivo from a vector within a cell.
  • the retron msr gene, msd gene, and/or ret gene can be introduced into a cell with a single vector or in multiple separate vectors to produce msDNA in a host subject.
  • the retron msr gene, msd gene, and/or ret gene, and any other components of the retron-based genome editing systems described herein may be expressed in vivo from RNA delivered to the cell.
  • the retron msr gene, msd gene, and/or ret gene can be introduced into a cell with a single vector or in multiple separate vectors to produce msDNA in a host subject.
  • Vectors and/or nucleic acid molecules encoding the recombinant retron-based genome editing system or components thereof can include control elements operably linked to the retron sequences, which allow for the production of msDNA either in vitro, or in vivo in the subject species.
  • the retron msr gene, msd gene, and/or ret gene can be operably linked to a promoter to allow expression of the retron reverse transcriptase and/or the msDNA product.
  • heterologous sequences encoding desired products of interest may be inserted in the msr gene and/or msd gene.
  • desired products of interest e.g., polynucleotide encoding polypeptide or regulatory RNA, donor polynucleotide for gene editing, or protospacer DNA for molecular recording
  • the retron-based gene editing systems are produced by a vector system comprising one or more vectors.
  • Numerous vectors are available for use in the vector or vector system, including but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • the retron-based gene editing systems (or components thereof) disclosed herein may be delivered in an “all-RNA” format.
  • all-RNA refers to the fact that each of the components of a retron editing system (e.g., the retron RT, the programmable nuclease, the sgRNA, and the ncRNA) are delivered and/or administered as RNA (e.g., coding RNA or non-coding RNA).
  • RNA e.g., coding RNA or non-coding RNA.
  • RNG043-WO1 PCT Application the RNA components may be delivered to cells and/or tissues by direct means, such as electroporation or transfection.
  • the RNA components may be delivered to cells and/or tissues by way of a delivery vehicle, such as an LNP or liposome.
  • the retron editing systems described herein may comprise a coding RNA (e.g., linear or circular mRNA) that encodes a retron reverse transcriptase (e.g., any RT from the abovementioned Table X or the abovementioned Table A), a coding RNA (e.g., linear or circular mRNA) that encodes a programmable nuclease (e.g., a Cas9, Cas12a, or TnpB nuclease), a retron ncRNA (e.g., a ncRNA from the abovementioned Table B), and a guide RNA for targeting the programmable nuclease to a particular desired target sequence.
  • a coding RNA e.g., linear or circular mRNA
  • a coding RNA e.g., linear or
  • RT and nuclease components may be encoded on the same coding RNA molecule.
  • the proteins may also be expressed from separate coding RNA molecules.
  • the RT and the nuclease components can be fused together as a singular fusion polypeptide having an RT domain and a nuclease domain optionally joined by a linker.
  • the ncRNA and the guide RNA may be fused together as a single RNA molecule.
  • the guide RNA may be located at the 5’ end of the ncRNA. In other embodiments, the guide RNA may be located at the 3’ end of the ncRNA.
  • the ncRNA may comprise a guide RNA at both the 3’ and the 5’ ends of the ncRNA.
  • the ncRNA and the guide RNA may be separate molecules, i.e., delivered separately.
  • the retron editing system may include both a ncRNA-guide RNA fusion and an additional guide RNA provided as a separate molecule.
  • the different RNA components of the all-RNA retron editing system can be combined and administered (e.g., directly or within a delivery vehicle) in different ratios. In some embodiments, the ratios of such RNA components or species can be expressed as molar ratios.
  • the molar ratio of RT coding RNA to nuclease coding RNA can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20.
  • Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 RNG043-WO1 PCT Application to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40.
  • the molar ratio of nuclease coding RNA to RT coding RNA can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20.
  • Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40.
  • Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40.
  • the molar ratio of separate guide RNA to ncRNA or ncRNA-guide RNA fusion can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20.
  • Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40.
  • the molar ratio of ncRNA to separate guide RNA can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20.
  • Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40.
  • the molar ratio of separate guide RNA to ncRNA can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20.
  • Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, RNG043-WO1 PCT Application from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40.
  • the molar ratio of a coding RNA (e.g., encoding RT and/or nuclease) to ncRNA or ncRNA-guide RNA fusion, as the case may be, can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20.
  • Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40.
  • the molar ratio of a coding RNA encoding a retron RT to ncRNA or ncRNA-guide RNA fusion can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20.
  • Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40.
  • the molar ratio of a coding RNA encoding a programmable nuclease to ncRNA or ncRNA-guide RNA fusion can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20.
  • Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40.
  • the molar ratio of a coding RNA encoding a retron RT or a nuclease to a separate guide RNA can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20.
  • Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40.
  • the RT mRNA: ncRNA-sgRNA ratio is about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20.
  • Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40.
  • multiple genetic loci are targeted hence the ncRNA-sgRNA includes a mixture of ncRNA-sgRNA species and the same ratios and ranges are applicable.
  • the retron-based gene editing systems (or components thereof) disclosed herein may be delivered in a composition comprising protein, DNA and/or RNAs.
  • a retron editing system e.g., the sgRNA, and the ncRNA
  • RNA and other components e.g., the retron RT, the programmable nuclease
  • the RNA or DNA components and the protein components are delivered to cells or tissues by the same means, such as electroporation or transfection, LNP, liposome, exosomes, etc.
  • the RNA or DNA components and the protein components are be delivered to cells or tissues by different types of delivery vehicles.
  • RNG043-WO1 PCT Application [00372] In some embodiments, the RNA or DNA components and the protein components are delivered concurrently. In some embodiments, the RNA or DNA components and the protein components are delivered separately, e.g., sequentially.
  • Viral vector delivery [00373] In various embodiments, the retron-based gene editing systems described herein may be delivered in viral vectors. [00374] Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus (AAV) vectors, retroviral vectors, lentiviral vectors, and the like.
  • AAV adeno-associated virus
  • an expression construct can be replicated in a living cell, or it can be made synthetically.
  • the nucleic acid comprising an retron-based gene editing system (or component thereof) is under transcriptional control of a promoter.
  • the promoter is competent for initiating transcription of an operably linked coding sequence by a RNA polymerase I, II, or III.
  • Exemplary promoters for mammalian cell expression include the SV40 early promoter, a CMV promoter such as the CMV immediate early promoter, such as, for example, as described in U.S.
  • Other nonviral promoters, such as a promoter derived from the murine metallothionein gene, will also find use for mammalian expression.
  • exemplary promoters for plant cell expression include the CaMV 35S promoter for example as described in Odell et al., 1985, Nature 313:810-812; the rice actin promoter for example as described in McElroy et al., 1990, Plant Cell 2:163-171; the ubiquitin promoter for example as described in Christensen et al., 1989, Plant Mol. Biol. 12:619-632; and Christensen et al., 1992, Plant Mol. Biol.18:675-689; the pEMU promoter for example as described in Last et al., 1991, Theor. Appl.
  • the retron-based vectors may also comprise tissue-specific promoters to start expression only after it is delivered into a specific tissue.
  • Non- limiting exemplary tissue-specific promoters include B29 promoter, CD14 promoter, CD43 promoter, CD45 promoter, CD68 promoter, desmin promoter, elastase- 1 promoter, endoglin promoter, fibronectin promoter, Flt-1 promoter, GFAP promoter, GPIIb promoter, ICAM- 2 RNG043-WO1 PCT Application promoter, INF-b promoter, Mb promoter, Nphsl promoter, OG-2 promoter, SP-B promoter, SYN1 promoter, and WASP promoter.
  • these and other promoters can be obtained from or incorporated into commercially available plasmids, using techniques as described in Sambrook et al., supra.
  • one or more enhancer elements is/are used in association with the promoter to increase expression levels of the constructs. Examples include the SV40 early gene enhancer, as described in Dijkema et al., EMBOJ (1985) 4:761, the enhancer/promoter derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus, as described in Gorman et al., Proc. Natl. Acad. Sci.
  • LTR long terminal repeat
  • an expression vector for expressing a retron-based gene editing system (or component thereof) comprises a promoter operably linked to a polynucleotide encoding the components.
  • the components of the retron-based gene editing system may be configured as individual gene transcripts or as fused constructs.
  • the nuclease component may be fused with the reverse transcriptase component.
  • the ncRNA component may be fused with the guide RNA component.
  • the nuclease component may be fused with the reverse transcriptase component, but the guide RNA and the ncRNA are separate.
  • the guide RNA and ncRNA components may be fused, but the reverse transcripase and nuclease components are separately provided. Any functional combinations of fused components and non-fused components are contemplated.
  • the vector or vector system also comprises a transcription terminator/polyadenylation signal. Examples of such sequences include, but are not limited to, those derived from SV40, as described in Sambrook et al., supra, as well as a bovine growth hormone terminator sequence, as described in U.S. Patent No.5,122,458.
  • 5 ⁇ - UTR sequences can be placed adjacent to the coding sequence to further enhance the expression.
  • Such sequences may include UTRs comprising an internal ribosome entry site (IRES).
  • IRES internal ribosome entry site
  • IRES permits the translation of one or more open reading frames from a vector.
  • the IRES element attracts a eukaryotic ribosomal translation initiation complex and promotes translation initiation, for example, as described in Kaufman et al., Nuc. Acids Res. (1991) 19:4485-4490; Gurtu et al., Biochem. RNG043-WO1 PCT Application Biophys. Res. Comm.
  • IRES sequences are known and include sequences derived from a wide variety of viruses, such as, for example, from leader sequences of picomaviruses such as the encephalomyocarditis virus (EMCV) UTR, for example, as described in Jang et al. Virol.
  • EMCV encephalomyocarditis virus
  • nonviral IRES sequences will also find use herein, including, but not limited to IRES sequences from yeast, as well as the human angiotensin II type 1 receptor IRES for example as described in Martin et al., Mol. Cell Endocrinol. (2003) 212:51-61, fibroblast growth factor IRESs (FGF-1 IRES and FGF-2 IRES, for example as described in Martineau et al. (2004) Mol. Cell. Biol.24(17): 7622-7635), vascular endothelial growth factor IRES for example as described in Baranick et al. (2008) Proc. Natl. Acad Sci.
  • IRES sequences from yeast as well as the human angiotensin II type 1 receptor IRES for example as described in Martin et al., Mol. Cell Endocrinol. (2003) 212:51-61, fibroblast growth factor IRESs (FGF-1 IRES and FGF-2 IRES, for example as described in Martineau et al. (2004) Mol.
  • IRES sequence may be included in a vector, for example, to express multiple bacteriophage recombination proteins for recombineering or an RNA-guided nuclease (e.g., Cas9) for HDR in combination with a retron reverse transcriptase from an expression cassette.
  • RNA-guided nuclease e.g., Cas9
  • a polynucleotide encoding a viral self-cleaving 2A peptide such as a T2A peptide
  • a viral self-cleaving 2A peptide can be used to allow production of multiple protein products (e.g., Cas9, bacteriophage recombination proteins, retron reverse transcriptase) from a single vector or a single transcription unit under one promoter.
  • One or more 2A linker peptides can be inserted between the coding sequences in the multicistronic construct.
  • the 2A peptide which is self-cleaving, allows co-expressed proteins from the multicistronic construct to be produced at equimolar levels.
  • 2A peptides from various viruses may be used, RNG043-WO1 PCT Application including, but not limited to 2A peptides derived from the foot-and-mouth disease virus, equine rhinitis A virus, Jhosea asigna virus and porcine teschovirus, such as those described in Kim et al. (2011) PLoS One 6(4): el8556, Trichas et al. (2008) BMC Biol.6:40, Provost et al. (2007) Genesis 45(10): 625-629, Furler et al. (2001) Gene Ther.8(11):864-873; herein incorporated by reference in their entireties.
  • bacterial plasmids may contain antibiotic selection markers (e.g., ampicillin, kanamycin, erythromycin, carbenicillin, streptomycin, or tetracycline resistance), a lacZ gene (b-galactosidase produces blue pigment from x-gal substrate), fluorescent markers (e.g., GFP. mCherry), or other markers for selection of transformed bacteria, such as, for example, those described in Sambrook et al., supra.
  • the expression construct comprises a plasmid suitable for transforming a yeast cell.
  • Yeast expression plasmids typically contain a yeast-specific origin of replication (ORI) and nutritional selection markers (e.g., HIS3, URA3, LYS2, LEU2, TRP1, METIS, ura4+, leul+, ade6+), antibiotic selection markers (e.g., kanamycin resistance), fluorescent markers (e.g., mCherry), or other markers for selection of transformed yeast cells.
  • the yeast plasmid may further contain components to allow shuttling between a bacterial host (e.g., E coli) and yeast cells.
  • yeast plasmids A number of different types are available including yeast integrating plasmids (Yip), which lack an ORI and are integrated into host chromosomes by homologous recombination; yeast replicating plasmids (YRp), which contain an autonomously replicating sequence (ARS) and can replicate independently; yeast centromere plasmids (YCp), which are low copy vectors containing a part of an ARS and part of a centromere sequence (CEN); and yeast episomal plasmids (YEp), which are high copy number plasmids comprising a fragment from a 2 micron circle (a natural yeast plasmid) that allows for 50 or more copies to be stably propagated per cell.
  • Yip yeast integrating plasmids
  • ARS autonomously replicating sequence
  • YCp yeast centromere plasmids
  • CEN yeast episomal plasmids
  • yeast episomal plasmids YEp
  • the expression construct does not comprise a plasmid suitable for transforming a yeast cell.
  • the expression construct comprises a virus or engineered construct derived from a viral genome.
  • a number of viral based systems have been developed for gene transfer into mammalian cells.
  • the viral based systems include RNG043-WO1 PCT Application adenoviruses, retroviruses (g-retroviruses and lentiviruses), poxviruses, adeno-associated viruses, baculoviruses, and herpes simplex viruses, such as, for example, those described in Wamock et al. (2011) Methods Mol.
  • retroviruses provide a convenient platform for gene delivery systems. Selected sequences can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo.
  • the retroviral system can be the one described in U.S. Pat. No.5,219,740; Miller and Rosman (1989) BioTechniques 7:980- 990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Bums et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop.3:102-109; and Ferry et al. (2011) Curr. Pharm. Des.17(24): 2516-2527).
  • Lentiviruses are a class of retroviruses that are particularly useful for delivering polynucleotides to mammalian cells because they are able to infect both dividing and nondividing cells, such as, for example, as described in Lois et al. (2002) Science 295:868- 872; Durand et al. (2011) Viruses 3(2): 132-159; herein incorporated by reference).
  • a number of adenoviral vectors have also been described. Unlike retroviruses which integrate into the host genome, adenoviruses persist extrachromosomally thus minimizing the risks associated with insertional mutagenesis.
  • AAV vectors can be readily constructed using techniques as described in U.S. Pat. Nos.5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 (published 23 January 1992) and WO 93/03769 (published 4 March 1993); Lebkowski et al., Molec. Cell. Biol. (1988) 8:3988-3996; Vincent et al., Vaccines 90 (1990) (Cold Spring Harbor LaboratoryPress); Carter, B. J. Current Opinion in Biotechnology (1992) 3:533-539; Muzyczka, N.
  • another vector system useful for delivering nucleic acids encoding the Cas12a editing system components is the enterically administered recombinant RNG043-WO1 PCT Application poxvirus vaccines as described by Small, Jr., P. A., et al. (U.S. Pat. No.5,676,950, issued Oct. 14, 1997, herein incorporated by reference).
  • viral vectors include those derived from the pox family of viruses, including vaccinia virus and avian poxvirus.
  • vaccinia virus recombinants expressing a nucleic acid molecule of interest can be constructed as follows. The DNA encoding the particular nucleic acid sequence is first inserted into an appropriate vector so that it is adjacent to a vaccinia promoter and flanking vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK). This vector is then used to transfect cells which are simultaneously infected with vaccinia.
  • TK thymidine kinase
  • avipoxviruses such as the fowlpox and canarypox viruses, can also be used to deliver the nucleic acid molecules of interest.
  • the use of an avipox vector is particularly desirable in human and other mammalian species since members of the avipox genus can only productively replicate in susceptible avian species and therefore are not infective in mammalian cells.
  • molecular conjugate vectors such as the adenovirus chimeric vectors described in Michael et al., J. Biol. Chem. (1993) 268:6866-6869 and Wagner et al., Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can also be used for gene delivery.
  • Sindbis-virus derived vectors can be those described in Dubensky et al. (1996) J. Virol.70:508-519; and International Publication Nos. WO 95/07995, WO 96/17072; as well as, Dubensky, Jr., T. W., et al., U.S. Pat.
  • chimeric alphavirus vectors comprised of sequences derived from Sindbis virus and Venezuelan equine encephalitis virus, such as, for example, those described in Perri et al. (2003) J. Virol.77: RNG043-WO1 PCT Application 10394-10403 and International Publication Nos. WO 02/099035, WO 02/080982, WO 01/81609, and WO 00/61772; each herein incorporated by reference in their entireties.
  • a vaccinia-based infection/transfection system can be conveniently used to provide for inducible, transient expression of the nucleic acids of interest (e.g., Cas12a editing system) in a host cell.
  • cells are first infected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase.
  • This polymerase displays extraordinar specificity in that it only transcribes templates bearing T7 promoters.
  • cells are transfected with the nucleic acid of interest, driven by a T7 promoter.
  • the polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA.
  • the method provides for high level, transient, cytoplasmic production of large quantities of RNA, such as, for example, as described in Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990) 87:6743- 6747; Fuerst et al., Proc. Natl. Acad. Sci. USA (1986) 83:8122-8126.
  • an amplification system can be used that will lead to high level expression following introduction into host cells.
  • T7 RNA polymerase promoter preceding the coding region for T7 RNA polymerase can be engineered. Translation of RNA derived from this template will generate T7 RNA polymerase which in turn will transcribe more templates. Concomitantly, there will be a cDNA whose expression is under the control of the T7 promoter. Thus, some of the T7 RNA polymerase generated from translation of the amplification template RNA will lead to transcription of the desired gene. Because some T7 RNA polymerase is required to initiate the amplification, T7 RNA polymerase can be introduced into cells along with the template(s) to prime the transcription reaction.
  • the polymerase can be introduced as a protein or on a plasmid encoding the RNA polymerase.
  • the T7 system can be those described in International Publication No. WO 94/26911; Studier and Moffatt, J. Mol. Biol. (1986) 189:113-130; Deng and Wolff, Gene (1994) 143:245-249; Gao et al., Biochem. Biophys. Res. Commun. (1994) 200:1201-1206; Gao and Huang, Nuc. Acids Res. (1993) 21:2867-2872; Chen et al., Nuc. Acids Res. (1994) 22:2114-2120; and U.S. Pat. No.5,135,855.
  • Insect cell expression systems such as baculovirus systems
  • baculovirus systems can also be used, such as, for example those described in Baculovirus and Insect Cell Expression Protocols (Methods in Molecular Biology, D.W. Murhammer ed., Humana Press, 2nd edition, 2007) and L. King The Baculovirus Expression System: A laboratory guide (Springer, 1992).
  • Materials and methods for baculovirus/insect cell expression systems are commercially available in kit RNG043-WO1 PCT Application form from, inter alia, Thermo Fisher Scientific (Waltham, MA) and Clontech (Mountain View, CA).
  • Plant expression systems can also be used for transforming plant cells.
  • such systems use virus-based vectors to transfect plant cells with heterologous genes.
  • such systems can include those described in Porta et al., Mol. Biotech. (1996) 5:209-221; and hackland et al., Arch. Virol. (1994) 139:1-22.
  • the expression constructs and/or RNA components must be delivered into a cell. This delivery may be accomplished in vitro, as in laboratory procedures for transforming cells lines, or in vivo or ex vivo, as in the treatment of certain disease states.
  • One mechanism for delivery is via viral infection where the expression construct is encapsulated in an infectious viral particle.
  • Non-viral delivery methods [00403] Several non-viral methods for the transfer of expression constructs are available for delivering the retron-based editing systems or components thereof into cells also are contemplated. These include the use of calcium phosphate precipitation, DEAE-dextran, electroporation, direct microinjection, DNA-loaded liposomes, lipofectamine-DNA complexes, cell sonication, gene bombardment using high velocity microprojectiles, and receptor-mediated transfection.
  • the non-viral methods can include those described in Graham and Van Der Eb (1973) Virology 52:456-467; Chen and Okayama (1987) Mol. Cell Biol.7:2745- 2752; Rippe et al. (1990) Mol.
  • nucleic acid molecules encoding the retron-based editing systems or components thereof may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation).
  • the nucleic acid may be stably maintained in the RNG043-WO1 PCT Application cell as a separate, episomal segment of DNA.
  • Such nucleic acid segments or episomes encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.
  • expression constructs encoding the retron-based editing systems or components thereof may simply consist of naked recombinant DNA or plasmids comprising nucleotide sequences encoding said retron-based editing systems or components thereof.
  • Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well.
  • Dubensky et al. Proc. Natl. Acad. Sci. USA (1984) 81:7529-7533
  • polyomavirus DNA in the form of calcium phosphate precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection.
  • Benvenisty & Neshif Proc. Natl. Acad. Sci.
  • DNA expression constructs encoding the retron- based editing systems or components thereof may be transferred into cells by particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them, such as, for example, as described in Klein et al. (1987) Nature 327:70-73.
  • Liposomes [00407]
  • constructs encoding the retron-based editing systems or components thereof may be delivered using liposomes. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium.
  • the liposome may be complexed with a hemagglutinating virus (HVJ).
  • HVJ hemagglutinating virus
  • the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-I), for example, described in Kato et al. (1991) J. Biol. Chem.266(6):3361-3364.
  • HMG-I nuclear non-histone chromosomal proteins
  • the liposome may be complexed or employed in conjunction with both HVJ and HMG-I.
  • a bacterial promoter is employed in the DNA construct, it also will be desirable to include within the liposome an appropriate bacterial polymerase.
  • Liposomes can be made from several different types of lipids, e.g., phospholipids.
  • a liposome may comprise natural phospholipids and lipids such as l,2- distearoryl-sn-glycero-3 -phosphatidyl choline (DSPC), sphingomyelin, egg phosphatidylcholines, monosialoganglioside, or any combination thereof.
  • DSPC distearoryl-sn-glycero-3 -phosphatidyl choline
  • sphingomyelin sphingomyelin
  • egg phosphatidylcholines monosialoganglioside, or any combination thereof.
  • monosialoganglioside monosialoganglioside
  • the transport polymer is a Histidine-Lysine co-polymer (HKP) used to package and deliver mRNA and other cargo, for example, as described in U.S. Patent Nos., 7,163,695, and 7,772,201, incorporated herein by reference in their entireties, [00413]
  • the lipid particles may be stable nucleic acid lipid particles (SNALPs).
  • SNALPs may comprise an ionizable lipid (DLinDMA) (e.g., cationic at low pH), a RNG043-WO1 PCT Application neutral helper lipid, cholesterol, a diffusible polyethylene glycol (PEG)-lipid, or any combination thereof.
  • SNALPs may comprise synthetic cholesterol, dipalmitoylphosphatidylcholine, 3-N-[(w-methoxy polyethylene glycol)2000)carbamoyl]-l,2- dimyrestyloxypropylamine, and cationic l,2-dilinoleyloxy-3-N,Ndimethylaminopropane.
  • SNALPs may comprise synthetic cholesterol, l,2-distearoyl-sn-glycero-3- phosphocholine, PEG- eDMA, and 1,2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane (DLinDMA).
  • Polymer based vehicles [00414]
  • the retron-based editing systems or components thereof may be encapsulated by delivery vehicles that comprise polymer-based particles (e.g., nanoparticles).
  • the polymer-based particles may mimic a viral mechanism of membrane fusion.
  • the polymer-based particles may be a synthetic copy of Influenza virus machinery and form transfection complexes with various types of nucleic acids ((siRNA, miRNA, plasmid DNA or snucleic acid component, mRNA) that cells take up via the endocytosis pathway, a process that involves the formation of an acidic compartment.
  • nucleic acids (siRNA, miRNA, plasmid DNA or snucleic acid component, mRNA) that cells take up via the endocytosis pathway, a process that involves the formation of an acidic compartment.
  • the low pH in late endosomes acts as a chemical switch that renders the particle surface hydrophobic and facilitates membrane crossing. Once in the cytosol, the particle releases its payload for cellular action.
  • This Active Endosome Escape technology is safe and maximizes transfection efficiency as it is using a natural uptake pathway.
  • the polymer-based particles may comprise alkylated and carboxyalkylated branched polyethylenimine.
  • the polymer-based particles are VIROMER, e.g., VIROMER RNAi, VIROMER RED, VIROMER mRNA.
  • methods of delivering the systems and compositions herein include those described in Bawage SS et al., Synthetic mRNA expressed Cast 3a mitigates RNA virus infections, biorxiv.org/content/10.1101/370460vl. full doi: doi.org/10.1101/370460, Viromer® RED, a powerful tool for transfection of keratinocytes.
  • the delivery vehicles may comprise exosomes.
  • Exosomes include membrane bound extracellular vesicles, which can be used to contain and delivery various types of biomolecules, such as proteins, carbohydrates, lipids, and nucleic acids, and complexes thereof (e.g., RNPs).
  • the exosomes can include those described in Schroeder A, et al., J Intern Med.2010 Jan;267(l):9-21; El-Andaloussi S, et al., Nat Protoc.2012 Dec;7(12):2112-26; Uno Y, et al., Hum Gene Ther.2011 Jun;22(6):711-9; Zou W, et al., Hum RNG043-WO1 PCT Application Gene Then 2011 Apr;22(4):465-75.
  • Exemplary exosomes can be generated from 293F cells, with mRNA-loaded exosomes driving higher mRNA expression than mRNA loaded LNPs in some instances, for example, as described in J. Biol. Chem.
  • the exosome may form a complex (e.g., by binding directly or indirectly) to one or more components of the cargo.
  • a molecule of an exosome may be fused with first adapter protein and a component of the cargo may be fused with a second adapter protein.
  • the first and the second adapter protein may specifically bind each other, thus associating the cargo with the exosome. Examples of such exosomes can include those described in Ye Y, et al., Biomater Sci.2020 Apr 28. doi: 10.1039/d0bm00427h.
  • Receptor-mediated delivery [00417] Other expression constructs encoding the retron-based editing systems or components thereof are receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific, such as, for example, as described in Wu and Wu (1993) Adv. Drug Delivery Rev.12:159- 167). Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA-binding agent. Several ligands have been used for receptor-mediated gene transfer.
  • the ligands can include asialoorosomucoid (ASOR) and transferrin, as described in Wu and Wu (1987), supra; Wagner et al. (1990) Proc. Natl. Acad. Sci. USA 87(9):3410- 3414).
  • the ligands can include synthetic neoglycoprotein, which recognizes the same receptor as ASOR, which can be used as a gene delivery vehicle, as described in Ferkol et al. (1993) FASEB J.7:1081-1091; Perales et al. (1994) Proc. Natl. Acad. Sci. USA 91(9):4086-4090.
  • the ligands can include epidermal growth factor (EGF) has also been used to deliver genes to squamous carcinoma cells, for example, as described in Myers, EPO 0273085.
  • delivery vehicle comprising one or more expression constructs encoding the retron-based editing systems or components thereof may comprise a ligand and a liposome, such as, for example, those described in Nicolau et al. (Methods Enzymol. (1987) 149:157-176), employed lactosy 1-ceramide, a galactose-terminal asialoganglioside, incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes.
  • a nucleic acid encoding a particular gene also may be specifically delivered into a cell by any number of receptor-ligand systems with or without liposomes.
  • antibodies to surface antigens on cells can similarly be used as targeting moieties.
  • the promoters that may be used in the retron-based editing systems or components thereof described herein may be constitutive, inducible, or tissue-specific. In some embodiments, the promoters may be a constitutive promoters.
  • Non- limiting exemplary constitutive promoters include cytomegalovirus immediate early promoter (CMV), simian virus (SV40) promoter, adenovirus major late (MLP) promoter, Rous sarcoma virus (RSV) promoter, mouse mammary tumor virus (MMTV) promoter, phosphoglycerate kinase (PGK) promoter, elongation factor-alpha (EFla) promoter, ubiquitin promoters, actin promoters, tubulin promoters, immunoglobulin promoters, a functional fragment thereof, or a combination of any of the foregoing.
  • the promoter may be a CMV promoter.
  • the promoter may be a truncated CMV promoter. In other embodiments, the promoter may be an EFla promoter. In some embodiments, the promoter may be an inducible promoter. Non-limiting exemplary inducible promoters include those inducible by heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohol. In some embodiments, the inducible promoter may be one that has a low basal (non-induced) expression level, such as, e.g., the Tet-On® promoter (Clontech). In some embodiments, the promoter may be a tissue-specific promoter. In some embodiments, the tissue-specific promoter is exclusively or predominantly expressed in liver tissue.
  • Non-limiting exemplary tissue-specific promoters include B29 promoter, CD14 promoter, CD43 promoter, CD45 promoter, CD68 promoter, desmin promoter, elastase- 1 promoter, endoglin promoter, fibronectin promoter, Flt-1 promoter, GFAP promoter, GPIIb promoter, ICAM- 2 promoter, INF-b promoter, Mb promoter, Nphsl promoter, OG-2 promoter, SP-B promoter, SYN1 promoter, and WASP promoter.
  • the present disclosure further provides delivery systems for delivery of a therapeutic payload (e.g., the RNA payloads described herein which may encode a polypeptide of interest, e.g., a nucleobase editing system or a therapeutic protein) disclosed herein.
  • a delivery system suitable for delivery of the therapeutic payload disclosed herein comprises a lipid nanoparticle (LNP) formulation.
  • LNP lipid nanoparticle
  • an LNP of the present disclosure comprises an ionizable lipid, a structural lipid, a PEGylated lipid (aka PEG lipid), and a phospholipid.
  • an LNP comprises an ionizable lipid, a structural lipid, a PEGylated lipid (aka PEG lipid), and a zwitterionic amino acid lipid.
  • an LNP further comprises a 5th lipid, besides any of the aforementioned lipid components.
  • the LNP encapsulates one or more elements of the active agent of the present RNG043-WO1 PCT Application disclosure.
  • an LNP further comprises a targeting moiety covalently or non-covalently bound to the outer surface of the LNP.
  • the targeting moiety is a targeting moiety that binds to, or otherwise facilitates uptake by, cells of a particular organ system.
  • an LNP has a diameter of at least about 20nm, 30 nm, 40nm, 50nm, 60nm, 70nm, 80nm, or 90nm. In some embodiments, an LNP has a diameter of less than about 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, or 160nm. In some embodiments, an LNP has a diameter of less than about 100nm. In some embodiments, an LNP has a diameter of less than about 90nm. In some embodiments, an LNP has a diameter of less than about 80nm. In some embodiments, an LNP has a diameter of about 60-100nm.
  • an LNP has a diameter of about 75-80nm.
  • the lipid nanoparticle compositions of the present disclosure are described according to the respective molar ratios of the component lipids in the formulation.
  • the mol-% of the ionizable lipid may be from about 10 mol-% to about 80 mol-%.
  • the mol-% of the ionizable lipid may be from about 20 mol-% to about 70 mol-%.
  • the mol-% of the ionizable lipid may be from about 30 mol-% to about 60 mol-%.
  • the mol-% of the ionizable lipid may be from about 35 mol-% to about 55 mol-%. As a non- limiting example, the mol-% of the ionizable lipid may be from about 40 mol-% to about 50 mol-%. [00424] In some embodiments, the mol-% of the phospholipid may be from about 1 mol-% to about 50 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 2 mol-% to about 45 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 3 mol-% to about 40 mol-%.
  • the mol-% of the phospholipid may be from about 4 mol-% to about 35 mol-%. In some embodiments, the mol- % of the phospholipid may be from about 5 mol-% to about 30 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 10 mol-% to about 20 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 5 mol-% to about 20 mol-%. [00425] In some embodiments, the mol-% of the structural lipid may be from about 10 mol-% to about 80 mol-%.
  • the mol-% of the structural lipid may be from about 20 mol-% to about 70 mol-%. In some embodiments, the mol-% of the structural lipid may be from about 30 mol-% to about 60 mol-%. In some embodiments, the mol-% of the structural lipid may be from about 35 mol-% to about 55 mol-%. In some embodiments, the mol-% of the structural lipid may be from about 40 mol-% to about 50 mol-%. RNG043-WO1 PCT Application [00426] In some embodiments, the mol-% of the PEG lipid may be from about 0.1 mol- % to about 10 mol-%.
  • the mol-% of the PEG lipid may be from about 0.2 mol-% to about 5 mol-%. In some embodiments, the mol-% of the PEG lipid may be from about 0.5 mol-% to about 3 mol-%. In some embodiments, the mol-% of the PEG lipid may be from about 1 mol-% to about 2 mol-%. In some embodiments, the mol-% of the PEG lipid may be about 1.5 mol-%. In some embodiments, the mol-% of the PEG lipid may be about 2.5 mol- %.
  • an LNP disclosed herein comprises an ionizable lipid.
  • an LNP comprises two or more ionizable lipids.
  • an LNP of the present disclosure comprises an ionizable lipid, such as those disclosed in US 2023/0053437; US 2019/0240354; US 2010/0130588; US 2021/0087135; WO 2021/204179; US 2021/0128488; US 2020/0121809; US 2017/0119904; US 2013/0108685; US 2013/0195920; US 2015/0005363; US 2014/0308304; US 2013/0053572; WO 2019/232095A1; WO 2021/077067; WO 2019/152557; US 2017/0210697; or WO 2019/089828A1, each of which is incorporated by reference herein in their entirety.
  • an LNP described herein comprises an ionizable lipid, such as those disclosed in PCT Publications WO2023044343A1, WO2023044333A1, WO2023122752A1, WO2024044728A1 and WO2023196931A1 and PCT Application PCT/US2024/019990, each of which is incorporated by reference herein, in its entirety.
  • an LNP described herein comprises a lipid, e.g., an ionizable lipid, such as those disclosed in US Application publication US2017/0119904, which is incorporated by reference herein, in its entirety.
  • an LNP described herein comprises a lipid, e.g., an ionizable lipid, such as those disclosed in PCT Application publication WO2021/204179, which is incorporated by reference herein, in its entirety.
  • an LNP described herein comprises a lipid, e.g., an ionizable lipid, such as those disclosed in PCT Application WO2022/251665A1, which is incorporated by reference herein, in its entirety.
  • an LNP of the present disclosure comprises an ionizable lipid, such as those disclosed in PCT Application Publication WO2023044343A1, which is incorporated by reference herein, in its entirety.
  • an LNP of the present disclosure comprises an ionizable lipid, such as those disclosed in PCT Application Publication WO2023044333A1, which is incorporated by reference herein, in its entirety.
  • an LNP of the present disclosure comprises an ionizable lipid, such as those disclosed in PCT Publication WO2023122752A1, which is incorporated by reference herein, in its entirety.
  • an LNP of the present disclosure comprises an ionizable lipid, such as those disclosed in PCT Application PCT/US2023/065477, which is incorporated by reference herein, in its entirety.
  • an LNP comprises a structural lipid.
  • Structural lipids can be selected from the group consisting of, but are not limited to, cholesterol, fecosterol, fucosterol, beta sitosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, cholic acid, sitostanol, litocholic acid, tomatine, ursolic acid, alpha-tocopherol, Vitamin D3, Vitamin D2, Calcipotriol, botulin, lupeol, oleanolic acid, beta-sitosterol-acetate and mixtures thereof.
  • the structural lipid is cholesterol.
  • the structural lipid is a cholesterol analogue, such as the one disclosed by Patel, et al., Nat Commun., 11, 983 (2020), which is incorporated herein by reference in its entirety.
  • the structural lipid includes cholesterol and a corticosteroid (such as prednisolone, dexamethasone, prednisone, and hydrocortisone), or any combinations thereof.
  • a structural lipid can be a structural lipid as described in international patent application WO2019152557A1, which is incorporated herein by reference in its entirety. [00439]
  • a structural lipid is a cholesterol analog.
  • a structural lipid is a phytosterol.
  • Using a phytosterol may enhance endosomal escape, for example, as described in Herrera et al., Illuminating endosomal escape of polymorphic lipid nanoparticles that boost mRNA delivery, Biomaterials Science (2020), which is incorporated herein by reference.
  • a structural lipid contains plant sterol mimetics for enhanced endosomal release.
  • PEGylated lipids [00442]
  • a PEGylated lipid is a lipid modified with polyethylene glycol.
  • PEGylated lipid and PEG lipid are used interchangeably to refer to the same concept.
  • an LNP comprises one, two or more PEGylated lipid or PEG-modified lipid.
  • a PEGylated lipid may be selected from the non-limiting group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG- modified dialkylglycerols, and mixtures thereof.
  • a PEG lipid may be PEG-c- DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.
  • the PEGylated lipid is selected from (R)-2,3- bis(octadecyloxy)propyl-1-(methoxypoly(ethyleneglycol)2000)propylcarbamate, PEG-S-DSG, PEG-S-DMG, PEG-PE, PEG-PAA, PEG-OH DSPE C18, PEG-DSPE, PEG-DSG, PEG-DPG, PEG-DOMG, PEG-DMPE Na, PEG-DMPE, PEG-DMG2000, PEG-DMG C14, PEG-DMG 2000, PEG-DMG, PEG-DMA, PEG-Ceramide C16, PEG-C-DOMG, PEG-c-DMOG, PEG-c- DMA, PEG-cDMA, PEGA, PEG750-C-DMA, PEG400, PEG2k-DMG, PEG2k-C11, PEG2000-PE, PEG2000
  • the LNP comprises a PEGylated lipid, such as, for example, a PEGylated lipid as disclosed in one of US 2019/0240354; US 2010/0130588; US 2021/0087135; WO 2021/204179; US 2021/0128488; US 2020/0121809; US 2017/0119904; US 2013/0108685; US 2013/0195920; US 2015/0005363; US 2014/0308304; US 2013/0053572; WO 2019/232095A1; WO 2021/077067; WO 2019/152557; US 2015/0203446; US 2017/0210697; US 2014/0200257; or WO 2019/089828A1, each of which is incorporated by reference herein in their entirety.
  • a PEGylated lipid such as, for example, a PEGylated lipid as disclosed in one of US 2019/0240354; US 2010/0130588; US 2021/0087135;
  • an LNP includes DSPC. In certain embodiments, an LNP includes DOPE. In some embodiments, an LNP includes both DSPC and DOPE. [00450] In some embodiments, an LNP comprises a phospholipid selected from 1- pentadecanoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-myristoyl-2-palmitoyl-sn- glycero-3-phosphocholine, 1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine, 1- palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-stearoyl-sn-glycero-3- phosphocholine, 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine, 1-palmitoyl-2-linoleoyl- sn-glycero-3-phosphocholine, 1-palmito
  • the lipid nanoparticle further comprises a targeting moiety.
  • the targeting moiety may be an antibody or a fragment thereof.
  • the targeting moiety may be capable of binding to a target antigen.
  • the pharmaceutical composition comprises a targeting moiety that is operably connected to a lipid nanoparticle.
  • the targeting moiety is capable of binding to a target antigen.
  • the target antigen is expressed in a target organ. In some embodiments, the target antigen is expressed more in the target organ than it is in the liver.
  • an LNP comprises a zwitterionic lipid.
  • an LNP comprising a zwitterionic lipid does not comprise a phospholipid.
  • Zwitterionic amino lipids have been shown to be able to self-assemble into LNPs without phospholipids to load, stabilize, and release mRNAs intracellularly, for example, as described in U.S. Patent Publication 20210121411, which is incorporated herein by reference in its entirety.
  • zwitterionic, ionizable cationic and permanently cationic helper lipids can enable tissue-selective mRNA delivery and CRISPR-Cas9 gene editing in spleen, liver and lungs as described in Liu et al., Membrane-destablizing ionizable phospholipids for organ-selective mRNA delivery and CRISPR-Cas gene editing, Nat Mater. (2021), which is incorporated herein by reference in its entirety.
  • the zwitterionic lipids may have head groups containing a cationic amine and an anionic carboxylate, such as the ones described in Walsh et al., Synthesis, Characterization and Evaluation of Ionizable Lysine-Based Lipids for siRNA Delivery, Bioconjug Chem. (2013), which is incorporated herein by reference in its entirety.
  • Ionizable lysine-based lipids containing a lysine head group linked to a long-chain dialkylamine through an amide linkage at the lysine ⁇ -amine may reduce immunogenicity, for example, as described in Walsh et al., Synthesis, Characterization and Evaluation of Ionizable Lysine-Based Lipids for siRNA Delivery, Bioconjug Chem. (2013). viii. Additional lipid components [00460] In some embodiments, the LNP compositions of the present disclosure further comprise one or more additional lipid components capable of influencing the tropism of the LNP.
  • the LNP further comprises at least one lipid selected from DDAB, EPC, 14PA, 18BMP, DODAP, DOTAP, and C12-200, such as described in Cheng, et al. Nat Nanotechnol.2020 April; 15(4): 313–320.; Dillard, et al. PNAS 2021 Vol.118 No.52.
  • the LNP compositions of the present disclosure comprise, or further comprise one or more lipids selected from 1,2-di-O-octadecenyl-sn- glycero-3-phosphocholine (18:0 Diether PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine RNG043-WO1 PCT Application (18:3 PC), Acylcarnosine (AC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), N- oleoyl-sphingomyelin (SPM) (C18:l), N-lignoceryl SPM (C24:0), N-nervonoylshphingomyelin (C24:l), Cardiolipin (CL), l,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC8- 9PC),
  • a nanoparticle includes an ionizable lipid, a phospholipid, a PEG lipid, and a structural lipid.
  • the lipid component of the nanoparticle composition includes about 30 mol % to about 60 mol % ionizable lipid, about 0 mol % to about 30 mol % phospholipid, about 18.5 mol % to about 48.5 mol % structural lipid, and about 0 mol% to about 10 mol% of PEG lipid, provided that the total mol % does not exceed 100%.
  • the lipid component of the nanoparticle composition includes about 35 mol % to about 55 mol % ionizable lipid, about 5 mol % to about 25 mol % phospholipid, about 30 mol % to about 40 mol % structural lipid, and about 0 mol % to about 10 mol % of PEG lipid.
  • the lipid component includes about 50 mol % ionizable lipid, about 10 mol % phospholipid, about 38.5 mol % structural lipid, and about 1.5 mol% of PEG lipid.
  • the lipid component includes about 40 mol % ionizable lipid, about 20 mol % phospholipid, about 38.5 mol % structural lipid, and about 1.5 mol % of PEG lipid. In another particular embodiment, the lipid component includes about 48.5 mol % ionizable lipid, about 10 mol % phospholipid, about 40 mol % structural lipid, and about 1.5 mol % of PEG lipid. In another particular embodiment, the lipid component includes about 48.5 mol % ionizable lipid, about 10 mol % phospholipid, about 39 mol % structural lipid, and about 2.5 mol % of PEG lipid.
  • the phospholipid may be DOPE or DSPC.
  • the PEG lipid may be PEG-DMG and/or the structural lipid may be cholesterol.
  • the amount of active agent in a nanoparticle composition may depend on the size, composition, desired target and/or application, or other properties of the nanoparticle composition as well as on the properties of the active agent.
  • the amount of active agent useful in a nanoparticle composition may depend on the size, sequence, and other characteristics of the active agent.
  • the relative amounts of active agent and other elements (e.g., lipids) in a nanoparticle composition may also vary.
  • the wt/wt ratio of the lipid component to an enzyme in a nanoparticle composition may be from about 5:1 to about 60:1, such as 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, and 60:1.
  • the amount of a enzyme in a nanoparticle composition may, for example, be measured using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy).
  • the lipid component of the nanoparticle comprises: (a) about 1 mol% to about 2 mol% of PEG lipid; (b) about 25 mol% to about 40 mol% structural lipid; (c) about 20 mol% to about 45 mol% phospholipid, non-ionizable lipid or zwitterionic lipid; and (d) about 30 mol% to about 60 mol% of an ionizable lipid.
  • the lipid component of the nanoparticle comprises: (a) about 2 mol% of PEG lipid; (b) about 25 mol% structural lipid; (c) about 40 mol% phospholipid, non-ionizable lipid or zwitterionic lipid; and (d) about 33 mol% of an ionizable lipid.
  • the lipid component of the nanoparticle comprises: (a) about 2.5 mol% of PEG lipid; (b) about 39 mol% structural lipid; (c) about 10 mol% phospholipid, non-ionizable lipid or zwitterionic lipid; and (d) about 48.5 mol% of an ionizable lipid.
  • the lipid component of the nanoparticle comprises: (a) about 1.5 mol% of PEG lipid; (b) about 40 mol% structural lipid; (c) about 10 mol% phospholipid, non-ionizable lipid or zwitterionic lipid; and (d) about 48.5 mol% of an ionizable lipid.
  • the lipid component of the nanoparticle composition comprises about 30 mol % to about 60 mol % ionizable lipid, about 0 mol % to about 30 mol % phospholipid, about 18.5 mol % to about 48.5 mol % structural lipid, and about 0 mol% to about 10 mol% of PEG lipid, provided that the total mol % does not exceed 100%.
  • the lipid component of the nanoparticle composition comprises about 20 mol % to about 45 mol % ionizable lipid, about 30 mol % to about 60 mol % phospholipid, about 10 RNG043-WO1 PCT Application mol % to about 30 mol % structural lipid, and about 0 mol% to about 10 mol% of PEG lipid, provided that the total mol % does not exceed 100%.
  • the lipid component of the nanoparticle composition comprises about 35 mol % to about 55 mol % ionizable lipid, about 5 mol % to about 25 mol % phospholipid, about 30 mol % to about 40 mol % structural lipid, and about 0 mol % to about 10 mol % of PEG lipid, provided that the total mol % does not exceed 100%.
  • the lipid component of the nanoparticle composition comprises about 30 mol % to about 40 mol % ionizable lipid, about 35 mol % to about 45 mol % phospholipid, about 20 mol % to about 30 mol % structural lipid, and about 0.5 mol % to about 5 mol % of PEG lipid, provided that the total mol % does not exceed 100%.
  • the lipid component of the nanoparticle composition comprises about 25 mol % to about 45 mol % ionizable lipid, about 35 mol % to about 50 mol % phospholipid, about 10 mol % to about 25 mol % structural lipid, and about 1 mol% to about 5 mol% of PEG lipid, provided that the total mol % does not exceed 100%.
  • the lipid component comprises about 50 mol % ionizable lipid, about 10 mol % phospholipid, about 38.5 mol % structural lipid, and about 1.5 mol% of PEG lipid.
  • the lipid component comprises about 40 mol % ionizable lipid, about 20 mol % phospholipid, about 38.5 mol % structural lipid, and about 1.5 mol % of PEG lipid. In another particular embodiment, the lipid component comprises about 48.5 mol % ionizable lipid, about 10 mol % phospholipid, about 40 mol % structural lipid, and about 1.5 mol % of PEG lipid. In another particular embodiment, the lipid component comprises about 48.5 mol % ionizable lipid, about 10 mol % phospholipid, about 39 mol % structural lipid, and about 2.5 mol % of PEG lipid.
  • the lipid component comprises about 33 mol % ionizable lipid, about 40 mol % phospholipid, about 25 mol % structural lipid, and about 2 mol % of PEG lipid.
  • the phospholipid is DOPE or DSPC.
  • the phospholipid is DSPC.
  • the phospholipid is a sphingolipid.
  • the phospholipid is a sphingomyelin.
  • the PEG lipid is PEG-DMG (eg. PEG2K-DMG).
  • the PEG lipid is PEG-DSPE (eg. PEG2K-DSPE).
  • the PEG lipid is PEG- DMPE (eg. PEG2K-DMPE).
  • the structural lipid is cholesterol.
  • the PEG lipid is PEG-DMG and/or the structural lipid is cholesterol.
  • the PEG lipids is PEG2K-DMG, the structural lipid is cholesterol, and the phospholipid is DSPC.
  • the PEG lipids is PEG2K-DMG, the structural lipid is cholesterol, and the phospholipid is sphingomyelin.
  • the PEG lipids is PEG-DMG, the structural lipid is cholesterol, and the phospholipid is a mixture of RNG043-WO1 PCT Application DSPC and sphingomyelin.
  • the LNP comprises about 33mol% ionizable lipid (eg. at least one ionizable lipid of a Formula described herein), about 40mol% of a sphingolipid, about 25mol% cholesterol and about 2mol% PEG2K-DMG.
  • the PEG lipids is PEG2K-DSPE, the structural lipid is cholesterol, and the phospholipid is DSPC.
  • the PEG lipids is PEG2K-DSPE, the structural lipid is cholesterol, and the phospholipid is sphingomyelin. In some embodiments, the PEG lipids is PEG-DSPE, the structural lipid is cholesterol, and the phospholipid is a mixture of DSPC and sphingomyelin. In some embodiments, the PEG lipids is PEG2K-DMG, the structural lipid is cholesterol, and the phospholipid is DOPE. In some embodiments, the PEG lipids is PEG2K-DMG, the structural lipid is cholesterol, and the phospholipid is DOPC.
  • the PEG lipids is PEG2K-DMG, the structural lipid is cholesterol, and the phospholipid is DLPC. In some embodiments, the PEG lipids is PEG2K-DMG, the structural lipid is cholesterol, and the phospholipid is DOPS. In some embodiments, the PEG lipids is PEG-DMG, the structural lipid is cholesterol, and the phospholipid is a mixture of a phosphatidylcholine lipid and a sphingolipid. In some embodiments, the PEG lipids is PEG- DMG, the structural lipid is cholesterol, and the phospholipid is a mixture of a phosphatidylcholine lipid and phosphatidylserine lipid.
  • the PEG lipids is PEG-DMG, the structural lipid is cholesterol, and the phospholipid is a mixture of a phosphatidylcholine lipid and a phosphoethanolamine lipid.
  • the PEG lipids is PEG-DMG, the structural lipid is cholesterol, and the phospholipid is a mixture of a sphingolipid and phosphatidylserine lipid.
  • the PEG lipids is PEG- DMG, the structural lipid is cholesterol, and the phospholipid is a mixture of a sphingolipid and a phosphoethanolamine lipid.
  • the LNP comprises about 33mol% ionizable lipid, about 20mol% of a sphingolipid, about 20mol% of a non-sphingolipid phospholipid, about 25mol% cholesterol and about 2mol% of a PEGylated lipid. In certain embodiments, the LNP comprises about 33mol% ionizable lipid, about 10mol% of a sphingolipid, about 30mol% of a non-sphingolipid phospholipid, about 25mol% cholesterol and about 2mol% of a PEGylated lipid.
  • the LNP comprises about 33mol% ionizable lipid, about 30mol% of a sphingolipid, about 10mol% of a non-sphingolipid phospholipid, about 25mol% cholesterol and about 2mol% of a PEGylated lipid. In certain embodiments, the LNP comprises about 33mol% ionizable lipid, about 20mol% sphingomyelin, about 20mol% of a DSPC, about 25mol% cholesterol and about 2mol% of a PEGylated lipid.
  • the LNP comprises about 33mol% ionizable lipid, about 10mol% sphingomyelin, about 30mol% of a DSPC, about 25mol% cholesterol and about RNG043-WO1 PCT Application 2mol% of a PEGylated lipid. In certain embodiments, the LNP comprises about 33mol% ionizable lipid, about 30mol% sphingomyelin, about 10mol% of a DSPC, about 25mol% cholesterol and about 2mol% of a PEGylated lipid.
  • the LNP comprises about 33mol% ionizable lipid, about 25mol% cholesterol, about 2mol% of a PEGylated lipid, and about 40% of a mixture of phosphatidylcholine, phosphatidylserine, phosphoethanolamine, and sphingoid lipids.
  • the LNP comprises about 33mol% ionizable lipid, about 25mol% cholesterol, about 2mol% of a PEGylated lipid, and about 40% of a mixture of phosphatidylcholine, phosphatidylserine, phosphoethanolamine, and sphingoid lipids, wherein each of the phosphatidylcholine, phosphatidylserine, phosphoethanolamine, and sphingoid lipids is present in an amount less than 30 mol% of the total lipid component of the LNP.
  • the LNP comprises about 33mol% ionizable lipid, about 25mol% cholesterol, about 2mol% of a PEGylated lipid, and about 40% of a mixture of phosphatidylcholine, phosphatidylserine, phosphoethanolamine, and sphingoid lipids, wherein each of the phosphatidylcholine, phosphatidylserine, phosphoethanolamine, and sphingoid lipids is present in an amount less than 25 mol% of the total lipid component of the LNP.
  • LNP is any one of the aforementioned in this paragraph wherein the PEG lipid is PEG2k-DMG.
  • LNP is any one of the aforementioned in this paragraph wherein the PEG lipid is PEG2k-DSPE.
  • LNP comprises about 33 mol % ionizable lipid, about 40 mol % DSPC, about 25 mol % cholesterol, and about 2 mol % of PEG lipid.
  • LNP comprises about 33 mol % ionizable lipid, about 40 mol % sphingomyelin, about 25 mol % cholesterol, and about 2 mol % of PEG lipid.
  • LNP comprises about 33 mol % ionizable lipid, about 40 mol % DOPE, about 25 mol % cholesterol, and about 2 mol % of PEG lipid. In another particular embodiment, LNP comprises about 33 mol % ionizable lipid, about 40 mol % DOPC, about 25 mol % cholesterol, and about 2 mol % of PEG lipid. In another particular embodiment, LNP comprises about 33 mol % ionizable lipid, about 40 mol % DLPC, about 25 mol % cholesterol, and about 2 mol % of PEG lipid.
  • LNP comprises about 33 mol % ionizable lipid, about 40 mol % DOPS, about 25 mol % cholesterol, and about 2 mol % of PEG lipid. In another particular embodiment, LNP comprises about 33 mol % ionizable lipid, about 40 mol % phospholipid, about 25 mol % cholesterol, and about 2 mol % of PEG lipid. In another particular embodiment, LNP comprises about 33 mol % ionizable lipid, about 20 mol % sphingomyelin, about 20 mol% DSPC, about 25 mol % cholesterol, and about 2 mol % of PEG lipid.
  • LNP is any one of the aforementioned in this RNG043-WO1 PCT Application paragraph wherein the PEG lipid is PEG2k-DMG. In certain embodiments, LNP is any one of the aforementioned in this paragraph wherein the PEG lipid is PEG2k-DSPE. [00469] In certain embodiments, the LNP comprises about 43mol% ionizable lipid, about 15mol% of a sphingolipid, about 15mol% of a non-sphingolipid phospholipid, about 25mol% cholesterol and about 2mol% of a PEGylated lipid.
  • the LNP comprises about 33mol% ionizable lipid, about 25mol% of a sphingolipid, about 15mol% of a non-sphingolipid phospholipid, about 25mol% cholesterol and about 2mol% of a PEGylated lipid. In certain embodiments, the LNP comprises about 33mol% ionizable lipid, about 15mol% of a sphingolipid, about 25mol% of a non-sphingolipid phospholipid, about 25mol% cholesterol and about 2mol% of a PEGylated lipid.
  • the PEG lipid is PEG2K-DSPE, the structural lipid is cholesterol, and the phospholipid is a mixture of DSPC and sphingomyelin.
  • the PEG lipid is PEG2K-DMG, the structural lipid is cholesterol, and the phospholipid is a mixture of DSPC and sphingomyelin.
  • the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 40mol% cholesterol and about 1.5mol% PEG2K-DSPE.
  • the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 40mol% cholesterol and about 1.5mol% PEG2K-DMG. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 39mol% cholesterol and about 2.5mol% PEG2K-DSPE.
  • the lipid component includes about 48.5 mol % ionizable lipid, about 10 mol % phospholipid, about 38.5 mol % structural lipid, and about 3 mol % of PEG lipid.
  • the lipid component includes about 48.5 mol % ionizable lipid, about 10 mol % phospholipid, about 38 mol % structural lipid, and about 3.5 mol % of PEG lipid.
  • the PEG lipid is PEG2K-DPPE, the structural lipid is cholesterol, and the phospholipid is a DSPC or a mixture of DSPC and sphingomyelin.
  • the PEG lipid is PEG2K-DPPE, the structural lipid is cholesterol, and the phospholipid is a mixture of DSPC and sphingomyelin.
  • the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 40mol% cholesterol and about 1.5mol% PEG2K-DPPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 39.5 mol% cholesterol and about 2 mol% PEG2K- DPPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 39mol% cholesterol and about 2.5mol% PEG2K-DPPE.
  • a phospholipid such as DSPC
  • the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 39mol% cholesterol and about 2.5mol% PEG2K-DPPE.
  • the LNP comprises about 48.5mol% ionizable lipid, RNG043-WO1 PCT Application about 10mol% of a phospholipid (such as DSPC), about 38.5 mol% cholesterol and about 3 mol% PEG2K-DPPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 38 mol% cholesterol and about 3.5mol% PEG2K-DPPE. In some embodiments, the PEG lipid is PEG2K-DMG, the structural lipid is cholesterol, and the phospholipid is a DSPC or a mixture of DSPC and sphingomyelin.
  • the PEG lipid is PEG2K-DMG
  • the structural lipid is cholesterol
  • the phospholipid is a mixture of DSPC and sphingomyelin.
  • the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 40mol% cholesterol and about 1.5mol% PEG2K-DMG.
  • the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 39.5 mol% cholesterol and about 2 mol% PEG2K-DMG.
  • the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 39mol% cholesterol and about 2.5mol% PEG2K-DMG. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 38.5 mol% cholesterol and about 3 mol% PEG2K- DMG. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 38 mol% cholesterol and about 3.5mol% PEG2K-DMG.
  • a phospholipid such as DSPC
  • the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 38 mol% cholesterol and about 3.5mol% PEG2K-DMG.
  • the PEG lipid is PEG2K-DSPE, the structural lipid is cholesterol, and the phospholipid is a DSPC or a mixture of DSPC and sphingomyelin.
  • the PEG lipid is PEG2K-DSPE, the structural lipid is cholesterol, and the phospholipid is a mixture of DSPC and sphingomyelin.
  • the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 40mol% cholesterol and about 1.5mol% PEG2K-DSPE.
  • the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 39.5 mol% cholesterol and about 2 mol% PEG2K-DSPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 39mol% cholesterol and about 2.5mol% PEG2K-DSPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 38.5 mol% cholesterol and about 3 mol% PEG2K- DSPE.
  • the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 38 mol% cholesterol and about 3.5mol% PEG2K-DSPE.
  • the LNP further comprises a targeting moiety.
  • the targeting moiety is an antibody or a fragment thereof.
  • RNG043-WO1 PCT Application [00472]
  • a nanoparticle composition of the present disclosure is formulated to provide a specific N:P ratio.
  • the N:P ratio of the composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in an RNA active agent (e.g., a linear or circular mRNA payload). In general, a lower N:P ratio is preferred.
  • the one or more enzymes, lipids, and amounts thereof is selected to provide an N:P ratio from about 2:1 to about 30:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1. In certain embodiments, the N:P ratio is from about 2:1 to about 8:1.
  • the N:P ratio is from about 5:1 to about 8:1.
  • the N:P ratio is about 5.0:1, about 5.5:1, about 5.67:1, about 6.0:1, about 6.5:1, or about 7.0:1.
  • the characteristics of a nanoparticle composition may depend on the components thereof.
  • a nanoparticle composition including cholesterol as a structural lipid may have different characteristics than a nanoparticle composition that includes a different structural lipid.
  • the characteristics of a nanoparticle composition may depend on the absolute or relative amounts of its components. For instance, a nanoparticle composition including a higher molar fraction of a phospholipid may have different characteristics than a nanoparticle composition including a lower molar fraction of a phospholipid.
  • Nanoparticle compositions may be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) may be used to examine the morphology and size distribution of a nanoparticle composition. Dynamic light scattering or potentiometry (e.g., potentiometric titrations) may be used to measure Zeta potentials. Dynamic light scattering may also be utilized to determine particle sizes.
  • microscopy e.g., transmission electron microscopy or scanning electron microscopy
  • Dynamic light scattering or potentiometry e.g., potentiometric titrations
  • Dynamic light scattering may also be utilized to determine particle sizes.
  • the mean size of a nanoparticle composition may be between 10s of nm and 100s of nm, e.g., measured by dynamic light scattering (DLS).
  • DLS dynamic light scattering
  • the mean size may be from about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm.
  • the mean size of a nanoparticle composition may be from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about RNG043-WO1 PCT Application 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, or from about 90 nm to about 100 nm.
  • the mean size of a nanoparticle composition may be from about 70 nm to about 100 nm. In a particular embodiment, the mean size may be about 80 nm. In other embodiments, the mean size may be about 100 nm.
  • a nanoparticle composition may be relatively homogenous.
  • a polydispersity index may be used to indicate the homogeneity of a nanoparticle composition, e.g., the particle size distribution of the nanoparticle compositions. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution.
  • a nanoparticle composition may have a polydispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25.
  • the Zeta potential of a nanoparticle composition may be used to indicate the electrokinetic potential of the composition.
  • the Zeta potential may describe the surface charge of a nanoparticle composition.
  • Nanoparticle compositions with relatively low charges, positive or negative, are generally desirable, as more highly charged species may interact undesirably with cells, tissues, and other elements in the body.
  • the Zeta potential of a nanoparticle composition may be from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about -10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV, to about +20 mV, from about 0 mV to about +15 m
  • the efficiency of encapsulation of a payload describes the amount of payload that is encapsulated or otherwise associated with a nanoparticle composition after preparation, relative to the initial amount provided.
  • the encapsulation efficiency is desirably high (e.g., close to 100%).
  • the encapsulation efficiency may be measured, for example, by comparing the amount of payload in a solution containing the nanoparticle composition before and after breaking up the nanoparticle composition with one or more organic solvents or detergents. Fluorescence may be used to measure the amount of free payload in a solution.
  • the encapsulation efficiency of a therapeutic and/or prophylactic may be at least 50%, for example 50%, 55%, 60%.65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In certain embodiments, the encapsulation efficiency may be at least 90%.
  • lipids and their method of preparation can be those disclosed in U.S. Patent Nos.8,569,256, 5,965,542 and U.S.
  • a nanoparticle composition may include any substance useful in pharmaceutical compositions.
  • the nanoparticle composition may include one or more pharmaceutically acceptable excipients or accessory ingredients such as, but not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulating aids, disintegrants, fillers, glidants, liquid vehicles, binders, surface active agents, isotonic agents, thickening or emulsifying agents, buffering agents, lubricating agents, oils, preservatives, and other species.
  • Excipients such as waxes, butters, coloring agents, coating agents, flavorings, and perfuming agents may also be included.
  • the pharmaceutically acceptable excipients can be those described in Remington’s The Science and Practice of Pharmacy, 21 st Edition, A. R. Gennaro: Lippincott, Williams & Wilkins, Baltimore, Md., 2006).
  • other different lipids or liposomal formulations including nanoparticles and methods of administration can include, but are not limited to, those disclosed in U.S. Patent Publication 20030203865, 20020150626, 20030032615, and 20040048787, which are specifically incorporated by reference to the extent they disclose formulations and other related aspects of administration and delivery of nucleic acids.
  • RNG043-WO1 PCT Application methods used for forming particles can include the methods disclosed in U.S. Pat.
  • the LNP encapsulates the engineered retron, e.g., an engineered nucleic acid construct, ncRNA, vector system, RNA molecule, and/or engineered nucleic acid-enzyme construct as described herein.
  • the engineered retron e.g., an engineered nucleic acid construct, ncRNA, vector system, RNA molecule, and/or engineered nucleic acid-enzyme construct as described herein.
  • the one or more structural lipids are selected from the group consisting of cholesterol, fecosterol, beta sitosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, prednisolone, dexamethasone, prednisone, and hydrocortisone.
  • the one or more PEGylated lipids are selected from the group consisting of PEG-c-DOMG, PEG-DMG, PEG- DLPE, PEG-DMPE, PEG-DPPC, and PEG-DSPE.
  • the one or more phospholipids are selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1.2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocho line (POPC), 1,2-di-O-octadecenyl-
  • DOPE 1,2-dio
  • the lipid nanoparticle comprises about 48.5 mol % ionizable lipid, about 10 mol % phospholipid, about 40 mol % structural lipid, and about 1.5 mol% of PEG lipid.
  • the lipid nanoparticle comprises about 48.5 mol % ionizable lipid, about 10 mol % phospholipid, about 39 mol % structural lipid, and about 2.5 mol% of PEG lipid.
  • the LNP further comprises a targeting moiety RNG043-WO1 PCT Application operably connected to the LNP.
  • the LNP further comprises one or more additional components selected from the group consisting of DDAB, EPC, 14PA, 18BMP, DODAP, DOTAP, and C12-200.
  • the engineered retron can be used for gene transfer, which may be performed under ex vivo or in vivo conditions.
  • Ex vivo gene therapy refers to the isolation of cells from a subject, the delivery of a nucleic acid into cells in vitro, and the return of the modified cells back into the subject. This may involve the collection of a biological sample comprising cells from the subject. For example, blood can be obtained by venipuncture, and solid tissue samples can be obtained by surgical techniques according to methods well known in the art.
  • the subject who receives the cells is also the subject from whom the cells are harvested or obtained, which provides the advantage that the donated cells are autologous.
  • cells can be obtained from another subject (e.g., a donor), a culture of cells from a donor, or from established cell culture lines. Accordingly, in some embodiments the cells are allogeneic to the recipient.
  • Cells may be obtained from the same or a different species than the subject to be treated, but preferably are of the same species, and more preferably of the same immunological profile as the subject.
  • Such cells can be obtained, for example, from a biological sample comprising cells from a close relative or matched donor, then transfected with nucleic acids (e.g., comprising an engineered retron), and administered to a subject in need of genome modification, for example, for treatment of a disease or condition.
  • nucleic acids e.g., comprising an engineered retron
  • the engineered retron can be introduced in vivo (e.g., used in gene therapy) by physically delivering the engineered retron to a subject. Examples of physically introducing the engineered retron includes via injections, electroporation and transfection (e.g., calcium-mediated or liposome tranfection, or the like). C.
  • the retron-based gene editing systems and/or components thereof may be delivered by way of LNPs as described herein.
  • the retron-based gene editing systems may be delivered by LNPs into cells, tissues, organs, or organisms.
  • the retron-based gene editing systems and/or the individual or combined components thereof may be delivered as DNA molecules (e.g., encoded on one or more plasmids), RNA molecules (e.g., guide RNAs for targeting a programmable nuclease or linear or circular mRNAs coding for the retron RTs or programmable nuclease components of the retron-based gene editing systems), proteins (e.g., retron polypeptides, accessory proteins RNG043-WO1 PCT Application having other functions (e.g., recombinases, nucleases, polymerases, ligases, deaminases, or reverse transcriptases), or protein-nucleic acid complexes (e.g., complexes between a guide RNA and a programmable nuclease protein or fusion protein comprising a retron RT).
  • DNA molecules e.g., encoded on one or more plasmids
  • RNA molecules e.g.,
  • DNA, RNA, protein, or nucleoprotein corresponding to and/or encoding the retron-based gene editing systems or components thereof comprise the LNP cargo or payloads.
  • the LNP cargo or payloads may comprise nucleic acid payloads, including coding payloads such as linear and circular mRNA for encoding the various components of the retron-based editing system. 1.
  • Nucleic acid payloads [00489]
  • the LNP compositions described herein can be used to deliver a nucleic acid or polynucleotide payload, e.g., DNA or a coding or noncoding RNA.
  • the retron-based editing compositions described herein can include a nucleic acid or polynucleotide payload, e.g., DNA or a coding or noncoding RNA.
  • the retron gene editing systems may comprise one or more coding mRNA (circular or linear) for encoding retron RT and other accessory proteins (e.g., programmable nuclease) and these RNA components may be delivered by LNPs.
  • a LNP is capable of delivering a polynucleotide to a target cell, tissue, or organ.
  • a polynucleotide in its broadest sense of the term, includes any compound and/or substance that is or can be incorporated into an oligonucleotide chain.
  • Exemplary polynucleotides for use in accordance with the present disclosure include, but are not limited to, one or more of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) including messenger mRNA (mRNA), hybrids thereof, RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that induce triple helix formation, aptamers, vectors, etc.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • mRNA messenger mRNA
  • RNAi-inducing agents RNAi agents
  • siRNAs siRNAs
  • shRNAs miRNAs
  • antisense RNAs antisense RNAs
  • RNAs useful in the compositions and methods described herein can be selected from the group consisting of but are not limited to, shortimers, antagomirs, antisense, ribozymes, short interfering RNA (siRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), Dicer substrate RNA (dsRNA), short hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof.
  • a polynucleotide is mRNA.
  • a polynucleotide is circular RNA.
  • a polynucleotide encodes a protein, e.g., a nucleobase editing enzyme.
  • a polynucleotide may encode any polypeptide of interest, including any naturally or non-naturally occurring or otherwise modified polypeptide.
  • a polypeptide may be of any size and may have any secondary structure or activity.
  • a polypeptide encoded by an mRNA may have a therapeutic effect when expressed in a cell.
  • RNG043-WO1 PCT Application [00492]
  • a polynucleotide is an siRNA.
  • An siRNA may be capable of selectively knocking down or down regulating expression of a gene of interest. For example, an siRNA could be selected to silence a gene associated with a particular disease, disorder, or condition upon administration to a subject in need thereof of a nanoparticle composition including the siRNA.
  • An siRNA may comprise a sequence that is complementary to an mRNA sequence that encodes a gene or protein of interest.
  • the siRNA may be an immunomodulatory siRNA.
  • a polynucleotide is an shRNA or a vector or plasmid encoding the same.
  • An shRNA may be produced inside a target cell upon delivery of an appropriate construct to the nucleus. Constructs and mechanisms relating to shRNA are well known in the relevant arts.
  • a polynucleotide may include a first region of linked nucleosides encoding a polypeptide of interest (e.g., a coding region), a first flanking region located at the 5'-terminus of the first region (e.g., a 5'-UTR), a second flanking region located at the 3'-terminus of the first region (e.g., a 3'-UTR), at least one 5'-cap region, and a 3'-stabilizing region.
  • a polynucleotide further includes a poly-A region or a Kozak sequence (e.g., in the 5'-UTR).
  • polynucleotides may contain one or more intronic nucleotide sequences capable of being excised from the polynucleotide.
  • a polynucleotide e.g., an mRNA
  • a polynucleotide may include a 5' cap structure, a chain terminating nucleotide, a stem loop, a polyA sequence, and/or a polyadenylation signal. Any one of the regions of a nucleic acid may include one or more alternative components (e.g., an alternative nucleoside).
  • the 3'-stabilizing region may contain an alternative nucleoside such as an L- nucleoside, an inverted thymidine, or a 2'-O-methyl nucleoside and/or the coding region, 5'- UTR, 3'-UTR, or cap region may include an alternative nucleoside such as a 5-substituted uridine (e.g., 5-methoxyu ridine), a 1-substituted pseudouridine (e.g., 1-methyl pseudouridine or 1-ethyl-pseudouridine), and/or a 5-substituted cytidine (e.g., 5-methyl-cytidine).
  • a 5-substituted uridine e.g., 5-methoxyu ridine
  • a 1-substituted pseudouridine e.g., 1-methyl pseudouridine or 1-ethyl-pseudouridine
  • cytidine e.g., 5-methyl-cy
  • a polynucleotide contains only naturally occurring nucleosides. [00495] In some cases, a polynucleotide is greater than 30 nucleotides in length. In another embodiment, the poly nucleotide molecule is greater than 35 nucleotides in length. In another embodiment, the length is at least 40 nucleotides. In another embodiment, the length is at least 45 nucleotides. In another embodiment, the length is at least 55 nucleotides. In another embodiment, the length is at least 50 nucleotides. In another embodiment, the length is at least 60 nucleotides. In another embodiment, the length is at least 80 nucleotides. In another embodiment, the length is at least 90 nucleotides.
  • the length is at least RNG043-WO1 PCT Application 100 nucleotides. In another embodiment, the length is at least 120 nucleotides. In another embodiment, the length is at least 140 nucleotides. In another embodiment, the length is at least 160 nucleotides. In another embodiment, the length is at least 180 nucleotides. In another embodiment, the length is at least 200 nucleotides. In another embodiment, the length is at least 250 nucleotides. In another embodiment, the length is at least 300 nucleotides. In another embodiment, the length is at least 350 nucleotides. In another embodiment, the length is at least 400 nucleotides. In another embodiment, the length is at least 450 nucleotides.
  • the length is at least 500 nucleotides. In another embodiment, the length is at least 600 nucleotides. In another embodiment, the length is at least 700 nucleotides. In another embodiment, the length is at least 800 nucleotides. In another embodiment, the length is at least 900 nucleotides. In another embodiment, the length is at least 1000 nucleotides. In another embodiment, the length is at least 1100 nucleotides. In another embodiment, the length is at least 1200 nucleotides. In another embodiment, the length is at least 1300 nucleotides. In another embodiment, the length is at least 1400 nucleotides. In another embodiment, the length is at least 1500 nucleotides.
  • the length is at least 1600 nucleotides. In another embodiment, the length is at least 1800 nucleotides. In another embodiment, the length is at least 2000 nucleotides. In another embodiment, the length is at least 2500 nucleotides. In another embodiment, the length is at least 3000 nucleotides. In another embodiment, the length is at least 4000 nucleotides. In another embodiment, the length is at least 5000 nucleotides, or greater than 5000 nucleotides.
  • a polynucleotide molecule, formula, composition or method associated therewith comprises one or more polynucleotides comprising features as described in WO2002/098443, WO2003/051401, WO2008/052770, WO2009/127230, WO2006/122828, WO2008/083949, WO2010/088927, WO2010/037539, WO2004/004743, WO2005/016376, WO2006/024518, WO2007/095976, WO2008/014979, WO2008/077592, WO2009/030481, WO2009/095226, WO2011/069586, WO2011/026641, WO2011/144358, WO2012/019780, WO2012/013326, WO2012/089338, WO2012/113513, WO2012/116811, WO2012/116810, WO2013/113502, WO2013/113501, WO2013/11350
  • a polynucleotide comprises one or more microRNA binding sites.
  • a microRNA binding site is recognized by a microRNA in a non-target organ.
  • a microRNA binding site is recognized by a microRNA in the liver.
  • a microRNA binding site is recognized by a microRNA in hepatic cells.
  • an RNA of the present disclosure comprises one or more phosphonate modifications selected from a phosphorothioate linkage (PS), phosphorodithioate linkage (PS2), methylphosphonate linkage (MP), methoxypropylphosphonate linkage (MOP), 5’-(E)-vinylphosphonate linkage (5’-(E)-VP), 5’- Methyl Phosphonate linkage (5’-MP), (S)-5’-C-methyl with phosphate linkage, 5’- phosphorothioate linkage (5’-PS), and a peptide nucleic acid linkage (PNA).
  • PS phosphorothioate linkage
  • PS2 phosphorodithioate linkage
  • MOP methoxypropylphosphonate linkage
  • 5’-(E)-vinylphosphonate linkage 5’-(E)-VP
  • 5’- Methyl Phosphonate linkage 5’-MP
  • S -5’-C-methyl
  • an RNA of the present disclosure comprises one or more ribose modifications selected from a 2’-O-methyl (2’-OMe), 2’-O-methoxyethyl (2’-O-MOE), 2’-deoxy-2’-fluoro (2’-F), 2’-arabino-fluoro (2’-Ara-F), 2’-O-benzyl, 2’-O-methyl-4-pyridine (2’-O-CH2Py(4)), Locked nucleic acid (LNA), (S)-cET-BNA, tricyclo-DNA (tcDNA), PMO, Unlocked Nucleic Acid (UNA) and glycol nucleic acid (GNA).
  • the RNA comprises a Locked Nucleic Acid (LNA) comprising a methyl bridge, an ethyl bridge, a propyl bridge, a butyl bridge or an optionally substituted variant of any of the aforementioned.
  • LNA Locked Nucleic Acid
  • an RNA of the present disclosure comprises one or more modified bases selected from a pseudouridine ( ⁇ ), 2’thiouridine (s2U), N6’-methyladenosine (m 6 A), 5’methylcytidine (m 5 C), 5’fluoro2’-deoxyuridine, N-ethylpiperidine 7’-EAA triazole modified adenine, N-ethylpiperidine 6’triazole modified adenine, 6’pheynlpyrrolo-cytosine (PhpC), 2’,4’-difluorotoluyl ribonucleoside (rF), and 5’-nitroindole.
  • the LNPs of the present disclosure may comprise a payload having at least one single stranded DNA.
  • the single stranded DNA is a linear single stranded DNA.
  • the single stranded DNA is a circular single stranded DNA.
  • the payload further comprises a nucleobase editing system, such as an enzyme or polynucleotide encoding an enzyme capable of independently or co-dependently editing, modifying, or altering a target polynucleotide sequence or a target transcript comprising a nucleic acid sequence.
  • the circular single stranded DNA (CiSSD) payload can be those described in PCT Publication WO2020142730A1, which is incorporated by reference herein in its entirety.
  • the CiSSD is a donor template for use as part of RNG043-WO1 PCT Application a nucleobase editing system for targeted genome modification.
  • the CiSSD comprises a DNA insert, a 5’ homology arm, and a 3’ homology arm. In some embodiments, the DNA insert is located between the 5’ homology arm and the 3’ homology arm.
  • Homology arms refer to a series of nucleotides that are complementary to a series of nucleotides in an endogenous DNA sequence in the target region.
  • the homology arms flanking the DNA insert allow for specific insertion of the DNA insert in the target region.
  • a target region is a nucleic acid sequence where a desired insertion or modification occurs.
  • the DNA insert is at least 1 nucleotide. In certain embodiments, the DNA insert is at least about 0.5 kb, 2 kb, 2.5 kb, 5 kb, 10 kb, 20 kb, 40 kb, 80 kb, 100 kb, 150 kb, or 200 kb.
  • the length of the DNA insert is about 0.5 kb to 5 kb, about 1 kb to 5 kb, about 1 kb to 10 kb, about 1.6 kb to 5 kb, about 1.6 kb to 10 kb, about 2 kb to 5 kb, about 2 kb to 20 kb, about 2.5 kb to 5 kb, about 2.5 kb to 10 kb, about 2.5 kb to 20 kb, and about 5kb to 100 kb.
  • the DNA insert size may range from about 1 kb to about 3 kb, about 3 kb to about 6 kb, about 6 kb to about 9 kb, about 9 kb to about 12 kb, about 12 kb to about 15 kb, about 15 kb to about 18 kb, or about 18 kb to about 21 kb.
  • the DNA insert may comprise a nucleotide sequence that encodes a maker or a reporter, e.g., a fluorescent marker, an antibiotic marker, or any suitable marker.
  • the insert may include a nucleotide sequence encoding a reporter (e.g, GFP, RFP, or any suitable reporter) or a recombinase.
  • the reporter is an N-terminal GFP fusion reporter.
  • the DNA insert may comprise a nucleotide sequence that encodes a transcription unit, wherein each transcription unit can produce a cellular product (e.g, protein or RNA).
  • the DNA insert may comprise a nucleotide sequence that encodes a protein, e.g, an immunomodulatory protein (e.g, a cytokine), an antibody, a chimeric antigen receptor (CAR), a growth factor, a T cell receptor, or another protein.
  • the CiSSD comprises a DNA insert that can be inserted at a nucleotide break in a target region of genomic DNA.
  • the break is a double stranded break (DSB).
  • the break is a single stranded DNA break or a nick.
  • Precision gene editing techniques e.g, CRISPR, create a break near a desired sequence change (target sequence).
  • CRISPR can be applied to produce deletions, disruptions, insertions, replacements, and repairs.
  • the components of template donors for these different RNG043-WO1 PCT Application modifications is generally the same, consisting of three basic elements: a 5’ homology arm, a DNA insert, and a 3’ homology arm.
  • CRISPR-based gene editing can generate gene knockouts by disrupting the gene sequence, however, efficiency for inserting exogenous DNA (knock-in) or replacement of genomic sequences is very poor using current methods.
  • CiSSDs may be used with CRISPR by generating a knock-in modification.
  • Double-stranded breaks can be introduced by any suitable mechanism, including, for example, by gene-editing systems using CRISPR, zinc finger nuclease, TALEN nuclease (Transcription Activator-Like Effector Nuclease), or meganuclease as described previously.
  • CRISPR genome editing system generates a targeted DSB using the CRISPR programmable DNA endonuclease that can be targeted to a specific DNA sequence (target sequence) by a small “guide” RNA (crRNA).
  • Guide RNAs for use in CRISPR-based modification z.e., crRNAs and tracrRNAs
  • an LNP of the present disclosure comprises a CiSSD disclosed herein and further comprises a precision gene editing system component such as a CRISPR, zinc finger nuclease, TALEN nuclease (Transcription Activator-Like Effector Nuclease), or meganuclease, or any other nucleobase editing system known in the art.
  • a precision gene editing system component such as a CRISPR, zinc finger nuclease, TALEN nuclease (Transcription Activator-Like Effector Nuclease), or meganuclease, or any other nucleobase editing system known in the art.
  • the single stranded DNA (SSD) payload can be those described in PCT Publication WO2020232286A1, which is incorporated by reference herein in its entirety.
  • the SSD comprises an engineered initiator sequence and an engineered terminator sequence from a filamentous bacteriophage, and a DNA sequence of interest, wherein the DNA sequence of interest is located 3’ to the engineered initiator sequence and 5’ to the engineered terminator sequence.
  • the SSD comprises a selectable marker.
  • the single stranded DNA (SSD) payload can be made by a method described in PCT Publication WO2020232286A1.
  • the SSD is made by a method comprising: (a) culturing a host cell under conditions suitable for producing a ssDNA from the DNA sequence of interest in the engineered nucleic acid and the plurality of bacteriophage proteins from the nucleic acid helper plasmid; (b) allowing the ssDNA and the plurality of bacteriophage proteins to assemble into an engineered phage; and (c) collecting the engineered phage.
  • the method further comprises extracting the SSD from the engineered phage.
  • at least 90% of the SSD is the same length as the DNA sequence of interest.
  • the single stranded DNA (SSD) payload can be those described in PCT Publication WO2022011082A1, which is incorporated by reference herein in its entirety.
  • the SSD comprises a first sequence from a filamentous bacteriophage, the first sequence having both initiator and terminator functions; a second sequence that is identical to the first sequence; and a single-strand DNA sequence of interest that is located between the first sequence and the second sequence.
  • the SSD further comprises a selectable marker.
  • the SSD is circular.
  • the SSD is linear.
  • the single stranded DNA (SSD) payload can be made by a method described in PCT Publication WO2022011082A1.
  • the method comprises culturing a host cell under conditions suitable for producing the single- stranded DNA from the single-strand DNA sequence of interest in the isolated nucleic acid and producing the bacteriophage proteins from the nucleic acid helper plasmid; allowing the single-stranded DNA and bacteriophage proteins to assemble into an engineered phage; and collecting the engineered phage.
  • the host cell comprises an isolated nucleic acid that includes: a first sequence from a filamentous bacteriophage, the first sequence having both initiator and terminator functions; a second sequence that is identical to the first sequence; and a single-strand DNA sequence of interest that is located between the first sequence and the second sequence, and a nucleic acid helper plasmid for expressing bacteriophage proteins capable of assembling a single-strand DNA into a bacteriophage.
  • the method further comprises extracting the SSD from the engineered phage. [00512] In certain embodiments, at least 90% of the SSD is the same length as the DNA sequence of interest.
  • the SSD is between 100 and 20,000 nucleotides in length. In certain embodiments, the SSD is circular.
  • the single stranded DNA (SSD) payload can be those described in PCT Publication WO2021055616A1, which is incorporated by reference herein in its entirety. 3. Linear mRNA payloads RNG043-WO1 PCT Application [00514]
  • the LNP-based pharmaceutical compositions described herein, e.g., LNP-based gene editing systems may include one or more linear mRNA molecules or linear mRNA payloads.
  • the mRNA payloads may encode one or more components of the herein described gene editing systems.
  • an mRNA payload may encode an amino acid sequence-programmable DNA binding domain (e.g., retron RTs or programmable nucleases) or a nucleic acid sequence-programmable DNA binding domain (e.g., CRISPR Cas9, CRISPR Cas12a, CRISPR Cas12f, CRISPR Cas13a, CRISPR Cas13b, or TnpB).
  • an amino acid sequence-programmable DNA binding domain e.g., retron RTs or programmable nucleases
  • a nucleic acid sequence-programmable DNA binding domain e.g., CRISPR Cas9, CRISPR Cas12a, CRISPR Cas12f, CRISPR Cas13a, CRISPR Cas13b, or TnpB.
  • mRNA payloads may also encode, depending upon the nature of the gene editing system, one or more effector domains that provide various functionalities that facilitate changes in nucleotide sequence and/or gene expression, such as, but not limited to, single- strand DNA binding proteins, nucleases, endonucleases, exonucleases, deaminases (e.g., cytidine deaminases or adenosine deaminases), polymerases (e.g., reverse transcriptases), integrases, recombinases, etc., and fusion proteins comprising one or more functional domains linked together.
  • deaminases e.g., cytidine deaminases or adenosine deaminases
  • polymerases e.g., reverse transcriptases
  • integrases e.g., recombinases, etc.
  • fusion proteins comprising one or more functional domains linked together.
  • RNA Ribonucleic acid
  • the nitrogenous bases include adenine (A), guanine (G), uracil (U), and cytosine (C).
  • A adenine
  • G guanine
  • U uracil
  • C cytosine
  • RNA mostly exists in the single-stranded form but can also exists double-stranded in certain circumstances. The length, form and structure of RNA is diverse depending on the purpose of the RNA.
  • the length of an RNA can vary from a short sequence (e.g., siRNA) to a long sequences (e.g., lncRNA), can be linear (e.g., mRNA) or circular (e.g., oRNA), and can either be a coding (e.g., mRNA) or a non-coding (e.g., lncRNA) sequence.
  • a short sequence e.g., siRNA
  • lncRNA long sequences
  • mRNA e.g., mRNA
  • oRNA e.g., oRNA
  • the LNP-based gene editing systems, RNA therapeutics and pharmaceutical compositions thereof described herein can be used to deliver a mRNA payload that is a linear mRNA molecule.
  • the mRNA payload may comprise one or more nucleotide sequences that encode a product of interest, such as, but not limited to a component of a gene editing system (e.g., an endonuclease, a prime editor, etc.) and/or a therapeutic protein.
  • the RNA payload may be a linear mRNA.
  • messenger RNA refers to any polynucleotide which encodes a protein of interest and which is capable of being translated to produce the encoded protein of interest in vitro, in vivo, in situ or ex vivo.
  • a mRNA molecule comprises at least a coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap and a poly-A tail.
  • UTR 5' untranslated region
  • 3' UTR 3' UTR
  • 5' cap 5' cap
  • poly-A tail one or more structural and/or chemical modifications or alterations may be included in the RNA which can reduce the innate immune response of a cell in which the mRNA is introduced.
  • a "structural" feature or modification is one in which two or more linked nucleotides are inserted, deleted, duplicated, inverted or randomized in a nucleic acid without significant chemical modification to the nucleotides themselves.
  • a coding region of interest in an mRNA used herein may encode a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, an octapeptide, a nonapeptide, or a decapeptide.
  • the mRNA may encode a peptide of 2-30 amino acids, e.g.5-30, 10-30, 2-25, 5-25, 10-25, or 10-20 amino acids.
  • the mRNA may encode a peptide of at least 10, 11, 12, 13, 14, 15, 17, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids, or a peptide that is no longer than 10, 11, 12, 13, 14, 15, 17, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids.
  • the length of the region of the mRNA encoding a product of interest is greater than about 30 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 or up to and including 100,000 nucleotides).
  • the mRNA has a total length that spans from about 30 to about 100,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1,000 to 1,500, from 1,000 to 1,500, from 500 to 2,000, from 500 to 3,000
  • the region or regions flanking the region encoding the product of interest may range independently from 15-1,000 nucleotides in length (e.g., greater than 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, and 900 nucleotides or at least 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, and 1,000 nucleotides).
  • 15-1,000 nucleotides in length e.g., greater than 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, and 1,000 nucleotides.
  • the mRNA comprises a tailing sequence which can range from absent to 500 nucleotides in length (e.g., at least 60, 70, 80, 90, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 nucleotides).
  • the tailing region is a polyA tail
  • the length may be determined in units of or as a function of polyA Binding Protein binding.
  • the polyA tail is long enough to bind at least 4 monomers of PolyA Binding Protein.
  • PolyA Binding Protein monomers bind to stretches of approximately 38 nucleotides. As such, it has been observed that polyA tails of about 80 nucleotides and 160 nucleotides are functional.
  • the mRNA comprises a capping sequence which comprises a single cap or a series of nucleotides forming the cap.
  • the capping sequence may be from 1 to 10, e.g.2-9, 3-8, 4-7, 1-5, 5-10, or at least 2, or 10 or fewer nucleotides in length.
  • the caping sequence is absent.
  • the mRNA comprises a region comprising a start codon. The region comprising the start codon may range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length.
  • the mRNA comprises a region comprising a stop codon.
  • the region comprising the stop codon may range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length.
  • the mRNA comprises a region comprising a restriction sequence.
  • the region comprising the restriction sequence may range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length.
  • the mRNA payloads of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions thereof described herein may comprise at least one untranslated region (UTR) which flanks the region encoding the product of interest and/or is incorporated within the mRNA molecule. UTRs are transcribed by not translated.
  • the mRNA payloads can include 5’ UTR sequences and 3’ UTR sequences, as well as internal UTRs.
  • the RNA payloads of the present disclosure may comprise one or more regions or parts which act or function as an untranslated region.
  • the nucleic acid may comprise one or more of these untranslated regions (UTRs). Wild-type untranslated regions of a nucleic acid are transcribed but not translated. In mRNA, the 5′ UTR starts at the transcription start site and continues to the start codon but does not include the start codon; whereas, the 3′ UTR starts immediately following the stop codon and continues until the transcriptional termination signal. There is growing body of evidence about the regulatory roles played by the UTRs in terms of stability of the nucleic acid molecule and translation.
  • RNA payload molecules e.g., linear and circular mRNA molecules
  • the specific features can also be incorporated to ensure controlled down-regulation of the transcript in case they are misdirected to undesired organs sites.
  • a variety of 5′UTR and 3′UTR sequences are known and available in the art.
  • the mRNA payloads of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions thereof described herein may comprise at least one UTR that may be selected from any UTR sequence listed in Tables 19 or 20 of U.S.
  • the mRNA payloads of the LNP-based gene editing systems, RNA therapeutics and pharmaceutical compositions thereof described herein, may comprise at least one 5′ UTR.
  • a 5′ UTR is region of an mRNA that is directly upstream (5′) from the start codon (the first codon of an mRNA transcript translated by a ribosome).
  • a 5′ UTR does not encode a protein (is non-coding).
  • Natural 5′UTRs have features that play roles in translation initiation. They harbor signatures like Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes.
  • Kozak sequences have the consensus CCR(A/G)CCAUGG, where R is a purine (adenine or guanine) RNG043-WO1 PCT
  • R is a purine (adenine or guanine) RNG043-WO1 PCT
  • Three bases upstream of the start codon (AUG) which is followed by another ‘G’.5′UTR also have been known to form secondary structures which are involved in elongation factor binding.5’ UTR sequences are also known to be important for ribosome recruitment to the mRNA and have been reported to play a role in translation, for example, as described in Hinnebusch A, et al., (2016) Science, 352:6292: 1413-6.
  • 5’ UTR sequences may confer increased half-life, increased expression and/or increased activity of a polypeptide encoded by the RNA payload described herein.
  • the RNA payload constructs contemplated herein may include 5’UTRs that are found in nature and those that are not.
  • the 5’UTRs can be synthetic and/or can be altered in sequence with respect to a naturally occurring 5’UTR.
  • Such altered 5’UTRs can include one or more modifications relative to a naturally occurring 5’UTR, such as, for example, an insertion, deletion, or an altered sequence, or the substitution of one or more nucleotide analogs in place of a naturally occurring nucleotide.
  • the 5' UTR starts at the transcription start site and continues to the start codon but does not include the start codon; whereas, the 3 'UTR starts immediately following the stop codon and continues until the transcriptional termination signal. While not wishing to be bound by theory, the UTRs may have a regulatory role in terms of translation and stability of the nucleic acid.
  • Natural 5' UTRs usually include features which have a role in translation initiation as they tend to include Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes.
  • Kozak sequences have the consensus CCR(A/G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another 'G'.5'UTR also have been known to form secondary structures which are involved in elongation factor binding.
  • the 5’ UTR comprises a sequence provided in the abovementioned Table X or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a 5’ UTR sequence provided in the abovementioned Table Y or a variant or a fragment thereof (e.g., a fragment that lacks the first one, two, three, four, five, or six nucleotides of the 5’ UTR sequence provided in the abovementioned Table Y).
  • the 5’ UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of the sequences of the abovementioned Table Y.
  • a 5′ UTR is a synthetic UTR, i.e., does not occur in nature.
  • Synthetic UTRs include UTRs that have been mutated to improve their properties, e.g., which increase gene expression as well as those which are completely synthetic.
  • exemplary 5′ UTRs can RNG043-WO1 PCT Application include Xenopus or human derived alpha-globin or beta-globin, as disclosed in US8,278,063 and US9,012,219, human cytochrome b-245 polypeptide, and hydroxysteroid (17b) dehydrogenase, and Tobacco etch virus.
  • CMV immediate-early 1 (IE1) gene as disclosed in US20140206753 and WO2013/185069, the sequence GGGAUCCUACC (SEQ ID NO: 19451), as disclosed in WO2014144196, may also be used.
  • 5′ UTR of a TOP gene is a 5′ UTR of a TOP gene lacking the 5′ TOP motif (the oligopyrimidine tract), such as the 5’ UTR disclosed in WO/2015101414, WO2015101415, WO/2015/062738, WO2015024667, WO2015024667, a 5′ UTR element derived from ribosomal protein Large 32 (L32) gene as disclosed in WO/2015101414, WO2015101415, WO/2015/062738, 5′ UTR element derived from the 5′UTR of an hydroxysteroid (17- ⁇ ) dehydrogenase 4 gene (HSD17B4) as disclosed in WO2015024667, or a 5′ UTR element derived from the 5′ UTR of ATP5A1 as disclosed in WO2015024667 can be used.
  • L32 ribosomal protein Large 32
  • an internal ribosome entry site is used as a substitute for a 5′ UTR.
  • a 5′ UTR of the present disclosure comprises a sequence selected from SEQ ID NO:19452 (GGGAAAUAAG AGAGAAAAGA AGAGUAAGAA GAAAUAUAAG AGCCACC), and SEQ ID NO:19453 (GGGAAATAAG AGAGAAAAGA AGAGTAAGAA GAAATATAAG AGCCACC).
  • SEQ ID NO:19452 GGGAAAUAAG AGAGAAAAGA AGAGTAAGAA GAAATATAAG AGCCACC.
  • 3' UTR regions the mRNA payloads of the LNP-based base editing systems, RNA therapeutics and pharmaceutical compositions thereof described herein, may comprise at least one 3′ UTR.3′ UTRs may be heterologous or synthetic.
  • a 3′ UTR is region of an mRNA that is directly downstream (3′) from the stop codon (the codon of an mRNA transcript that signals a termination of translation).
  • a 3′ UTR does not encode a protein (is non-coding).
  • Natural or wild type 3′ UTRs are known to have stretches of adenosines and uridines embedded in them. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into three classes, for example, as described in Chen et al, 1995: Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD contain class I AREs.
  • AREs possess two or more overlapping UUAUUUA(U/A)(U/A) nonamers. Molecules containing this type of AREs include GM-CSF and TNF- ⁇ . Class III ARES are less well defined. These U rich regions do not contain an AUUUA motif. c-Jun and Myogenin are two well-studied examples of this class. Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented RNG043-WO1 PCT Application to increase the stability of mRNA. HuR binds to AREs of all the three classes.
  • AU rich elements can be separated into three classes, for example, as described in Chen et al., 1995: Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD contain class I AREs.
  • AREs possess two or more overlapping UUAUUUA(U/A)(U/A) nonamers. Molecules containing this type of AREs include GM-CSF and TNF-a. Class III ARES are less well defined. These U rich regions do not contain an AUUUA motif. c-Jun and Myogenin are two well-studied examples of this class. Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA. HuR binds to AREs of all the three classes.
  • AREs 3' UTR AU rich elements
  • AREs 3' UTR AU rich elements
  • one or more copies of an ARE can be introduced to make mRNA less stable and thereby curtail translation and decrease production of the resultant protein.
  • AREs can be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein.
  • the introduction of features often expressed in genes of target organs the stability and protein production of the mRNA can be enhanced in a specific organ and/or tissue.
  • the feature can be a UTR.
  • the feature can be introns or portions of introns sequences.
  • 5′ UTRs that are heterologous or synthetic may be used with any desired 3′ UTR sequence.
  • a heterologous 5′ UTR may be used with a synthetic 3′ UTR with a heterologous 3′ UTR.
  • Non-UTR sequences may also be used as regions or subregions within an RNA payload construct.
  • introns or portions of introns sequences may be incorporated into regions of nucleic acid of the disclosure. Incorporation of intronic sequences may increase protein production as well as nucleic acid levels.
  • the polypeptide coding region of interest in an mRNA payload may be flanked by a 5′ UTR which may contain a strong Kozak translational initiation signal and/or a 3′ UTR which may include an oligo(dT) sequence for templated addition of a poly-A tail.
  • a 5′ UTR may comprise a first polynucleotide fragment and a second polynucleotide fragment from the same and/or different genes such as the 5′ UTRs described in US Patent Application Publication No.20100293625 and PCT/US2014/069155, herein incorporated by reference in its entirety [00548] It should be understood that any UTR from any gene may be incorporated into the regions of an RNA payload molecule (e.g., a linear mRNA). Furthermore, multiple wild- type UTRs of any known gene may be utilized. It is also within the scope of the present disclosure to provide artificial UTRs which are not variants of wild type regions.
  • UTRs or portions thereof may be placed in the same orientation as in the transcript from which they were selected or may be altered in orientation or location.
  • a 5′ or 3′ UTR may be inverted, shortened, lengthened, made with one or more other 5′ UTRs or 3′ UTRs.
  • the term “altered” as it relates to a UTR sequence means that the UTR has been changed in some way in relation to a reference sequence.
  • a 3′ UTR or 5′ UTR may be altered relative to a wild-type or native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides.
  • any of these changes producing an “altered” UTR (whether 3′ or 5′) comprise a variant UTR.
  • a double, triple or quadruple UTR such as a 5′ UTR or 3′ UTR may be used.
  • a “double” UTR is one in which two copies of the same UTR are encoded either in series or substantially in series.
  • a double beta- globin 3′ UTR may be used as described in US Patent publication 20100129877, the contents of which are incorporated herein by reference in its entirety.
  • flanking regions are selected from a family of transcripts whose proteins share a common function, structure, feature or property.
  • polypeptides of interest may belong to a family of proteins which are expressed in a particular RNG043-WO1 PCT Application cell, tissue or at some time during development.
  • the UTRs from any of these genes may be swapped for any other UTR of the same or different family of proteins to create a new polynucleotide.
  • a “family of proteins” is used in the broadest sense to refer to a group of two or more polypeptides of interest which share at least one function, structure, feature, localization, origin, or expression pattern.
  • the untranslated region may also include translation enhancer elements (TEE).
  • TEE translation enhancer elements
  • the TEE may include those described in US Application No. 20090226470, herein incorporated by reference in its entirety, and those known in the art.
  • the mRNA payloads of the LNP-based base editing systems, RNA therapeutics and pharmaceutical compositions thereof described herein, may comprise a 5’ cap structure.
  • the 5' cap structure of an mRNA is involved in nuclear export, increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP), which is responsible for mRNA stability in the cell and translation competency through the association of CBP with poly(A) binding protein to form the mature cyclic mRNA species.
  • CBP mRNA Cap Binding Protein
  • the cap further assists the removal of 5' proximal introns removal during mRNA splicing.
  • Endogenous mRNA molecules may be 5'-end capped generating a 5'-ppp-5'- triphosphate linkage between a terminal guanosine cap residue and the 5'-terminal transcribed sense nucleotide of the mRNA molecule. This 5'-guanylate cap may then be methylated to generate an N7-methyl-guanylate residue.
  • the ribose sugars of the terminal and/or ante- terminal transcribed nucleotides of the 5' end of the mRNA may optionally also be 2'-0- methylated.5'-decapping through hydrolysis and cleavage of the guanylate cap structure may target a nucleic acid molecule, such as an mRNA molecule, for degradation.
  • Modifications to mRNA may generate a non-hydrolyzable cap structure preventing decapping and thus increasing mRNA half-life. Because cap structure hydrolysis requires cleavage of 5'-ppp-5' phosphorodiester linkages, modified nucleotides may be used during the capping reaction. For example, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, MA) may be used with a-thio-guanosine nucleotides according to the manufacturer's instructions to create a phosphorothioate linkage in the 5'-ppp-5' cap.
  • a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, MA) may be used with a-thio-guanosine nucleotides according to the manufacturer's instructions to create a phosphorothioate linkage in the 5'-ppp-5' cap.
  • Additional modified guanosine nucleotides may be used such as a-methyl- phosphonate and seleno-phosphate nucleotides.
  • Additional modifications include, but are not limited to, 2'-0-methylation of the ribose sugars of 5 '-terminal and/or 5'-anteterminal nucleotides of the mRNA (as mentioned RNG043-WO1 PCT Application above) on the 2'-hydroxyl group of the sugar ring.
  • Multiple distinct 5 '-cap structures can be used to generate the 5 '-cap of a nucleic acid molecule, such as an mRNA molecule.
  • Cap analogs which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e. endogenous, wild-type or physiological) 5'-caps in their chemical structure, while retaining cap function. Cap analogs may be chemically (i.e. non-enzymatically) or enzymatically synthesized and/or linked to a nucleic acid molecule.
  • the Anti-Reverse Cap Analog (ARCA) cap contains two guanines linked by a 5 '-5 '-triphosphate group, wherein one guanine contains an N7 methyl group as well as a 3'-0-methyl group (i.e., N7,3'-0-dimethyl-guanosine-5'-triphosphate-5 '-guanosine (m 7 G-3'mppp-G; which may equivalently be designated 3' O-Me-m7G(5')ppp(5')G).
  • the 3'-0 atom of the other, unmodified, guanine becomes linked to the 5'-terminal nucleotide of the capped nucleic acid molecule (e.g.
  • mRNA an mRNA
  • the N7- and 3'-0-methlyated guanine provides the terminal moiety of the capped nucleic acid molecule (e.g. mRNA).
  • mCAP Another exemplary cap is mCAP, which is similar to ARCA but has a 2'-0- methyl group on guanosine (i.e., N7,2'-0-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m 7 Gm-ppp-G).
  • cap analogs allow for the concomitant capping of a nucleic acid molecule in an in vitro transcription reaction, up to 20% of transcripts can remain uncapped.
  • mRNA may also be capped post-transcriptionally, using enzymes, in order to generate more authentic 5'-cap structures.
  • the phrase "more authentic" refers to a feature that closely mirrors or mimics, either structurally or functionally, an endogenous or wild type feature.
  • a "more authentic" feature is better representative of an endogenous, wild-type, natural or physiological cellular function and/or structure as compared to synthetic features or analogs, etc., of the prior art, or which outperforms the corresponding endogenous, wild-type, natural or physiological feature in one or more respects.
  • Non-limiting examples of more authentic 5 'cap structures are those which, among other things, have enhanced binding of cap binding proteins, increased half-life, reduced susceptibility to 5' endonucleases and/or reduced 5'decapping, as compared to synthetic 5 'cap structures known in the art (or to a wild- type, natural or physiological 5 'cap structure).
  • recombinant Vaccinia Virus Capping Enzyme and recombinant 2'-0-methyltransferase enzyme can create a canonical 5 '-5 RNG043-WO1 PCT Application '-triphosphate linkage between the 5 '-terminal nucleotide of an mRNA and a guanine cap nucleotide wherein the cap guanine contains an N7 methylation and the 5 '-terminal nucleotide of the mRNA contains a 2'-0-methyl.
  • Capl structure Such a structure is termed the Capl structure.
  • Cap structures include, but are not limited to, 7mG(5 * )ppp(5 * )N,pN2p (cap 0), 7mG(5 * )ppp(5 * )NlmpNp (cap 1), and 7mG(5 * )-ppp(5')NlmpN2mp (cap 2).
  • the 5' terminal caps may include endogenous caps or cap analogs.
  • a 5' terminal cap may comprise a guanine analog.
  • Useful guanine analogs include, but are not limited to, inosine, Nl-methyl-guanosine, 2'fluoro- guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2- azido-guanosine.
  • IRES Sequences [00566]
  • the mRNA payloads of the LNP-based gene editing systems, RNA therapeutics and pharmaceutical compositions thereof described herein, may comprise one or more IRES sequences.
  • the mRNA may contain an internal ribosome entry site (IRES).
  • IRES internal ribosome entry site
  • An IRES plays an important role in initiating protein synthesis in absence of the 5' cap structure.
  • An IRES may act as the sole ribosome binding site, or may serve as one of multiple ribosome binding sites of an mRNA.
  • An mRNA that contains more than one functional ribosome binding site may encode several peptides or polypeptides that are translated independently by the ribosomes.
  • IRES sequences that can be used include without limitation, those from picornaviruses (e.g.
  • FMDV pest viruses
  • CFFV pest viruses
  • PV polio viruses
  • ECMV encephalomyocarditis viruses
  • FMDV foot-and-mouth disease viruses
  • HCV hepatitis C viruses
  • CSFV classical swine fever viruses
  • MLV murine leukemia virus
  • SIV simian immune deficiency viruses
  • CrPV cricket paralysis viruses
  • the IRES is from Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, Simian Virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, Human poliovirus 1, Plautia stali intestine virus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulata virus-1, Human Immunodeficiency Virus type 1, Homalodisca coagulata virus-1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, Foot and mouth disease virus, Human enterovirus RNG043-WO1 PCT Application 71, Equine rhinitis virus, Ectropis obliqua picorna-like virus, Encephalomyocarditis virus, Drosophila C Virus, Human coxsackievirus B3, Crucifer tobamovirus, Cricket paralysis
  • the mRNA payloads of the LNP-based gene editing systems, RNA therapeutics and pharmaceutical compositions thereof described herein, may comprise a poly-A tail.
  • a long chain of adenine nucleotides may be added to a polynucleotide such as an mRNA molecules in order to increase stability.
  • the 3' end of the transcript may be cleaved to free a 3' hydroxyl.
  • poly-A polymerase adds a chain of adenine nucleotides to the free 3' hydroxyl end.
  • polyadenylation adds a poly-A tail of a certain length.
  • the length of a poly-A tail is greater than 30 nucleotides in length (SEQ ID NO: 19937).
  • the poly-A tail is greater than 35 RNG043-WO1 PCT Application nucleotides in length (SEQ ID NO: 19938) (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides) and no more than about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, or 3000 nucleotides in length.
  • the mRNA includes a poly- A tail from about 30 to about 3,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 750, from 30 to 1,000, from 30 to 1,500, from 30 to 2,000, from 30 to 2,500, from 50 to 100, from 50 to 250, from 50 to 500, from 50 to 750, from 50 to 1 ,000, from 50 to 1,500, from 50 to 2,000, from 50 to 2,500, from 50 to 3,000, from 100 to 500, from 100 to 750, from 100 to 1,000, from 100 to 1,500, from 100 to 2,000, from 100 to 2,500, from 100 to 3,000, from 500 to 750, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 2,500, from 500 to 3,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 2,500, from 1,000 to 3,000, from 1,500 to 2,000, from 1,500 to 2,500, from 1,500 to 3,000, from 1,500
  • the poly-A tail is designed relative to the length of the overall mRNA. This design may be based on the length of the region coding for a target of interest, the length of a particular feature or region (such as a flanking region), or based on the length of the ultimate product expressed from the mRNA. [00573] In this context the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% greater in length than the mRNA or feature thereof. The poly-A tail may also be designed as a fraction of mRNA to which it belongs.
  • the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct or the total length of the construct minus the poly-A tail.
  • engineered binding sites and conjugation of mRNA for poly-A binding protein may enhance expression.
  • multiple distinct mRNA may be linked together to the PABP (Poly-A binding protein) through the 3'-end using modified nucleotides at the 3 '-terminus of the poly-A tail. Transfection experiments can be conducted in relevant cell lines at and protein production can be assayed by ELISA at 12hr, 24hr, 48hr, 72 hr and day 7 post-transfection.
  • the mRNA are designed to include a polyA-G quartet.
  • the G-quartet is a cyclic hydrogen bonded array of four guanine nucleotides that can be formed by G-rich sequences in both DNA and RNA.
  • the G-quartet is incorporated at the end of the poly-A tail. Stop Codons RNG043-WO1 PCT Application
  • the mRNA payloads of the LNP-based gene editing systems, RNA therapeutics and pharmaceutical compositions thereof described herein may comprise one or more translation stop codons.
  • a stop element as used herein refers to a nucleic acid sequence comprising a stop codon.
  • the stop codon can be selected from TGA, TAA and TAG in the case of DNA, or from UGA, UAA and UAG in the case of RNA.
  • a stop element comprises two consecutive stop codons.
  • a stop element comprises three consecutive stop codons. In an embodiment, a stop element comprises four consecutive stop codons. In an embodiment, a stop element comprises five consecutive stop codons. [00578] In some embodiments, the mRNA may include one stop codon. In some embodiments, the mRNA may include two stop codons. In some embodiments, the mRNA may include three stop codons. In some embodiments, the mRNA may include at least one stop codon. In some embodiments, the mRNA may include at least two stop codons. In some embodiments, the mRNA may include at least three stop codons. As non-limiting examples, the stop codon may be selected from TGA, TAA and TAG.
  • the stop codon may be selected from one or more of the following stop elements of Table Z: Table Z: Additional stop elements of linear mRNA Nucleotide sequence (5’ to 3’) Sequence Identifier RNG043-WO1 PCT Application UAAAGCUCC NA UAGGGUUAA NA additional stop codon.
  • the addition stop codon may be TAA.
  • the mRNA payloads of the LNP-based gene editing systems, RNA therapeutics and pharmaceutical compositions thereof described herein may comprise one or more regulatory elements, including, but not limited to microRNA (miRNA) binding sites, structured mRNA sequences and/or motifs, artificial binding sites to bind to endogenous nucleic acid binding molecules, and combinations thereof.
  • miRNA microRNA
  • nucleotides and nucleosides of the present disclosure comprise standard nucleoside residues such as those present in transcribed RNA (e.g. A, G, C, or U).
  • nucleotides and nucleosides of the present disclosure comprise standard deoxyribonucleosides such as those present in DNA (e.g.
  • the mRNA payloads of the LNP-based gene editing systems, RNA therapeutics and pharmaceutical compositions thereof described herein comprise, in some embodiments, comprises at least one chemical modification.
  • chemical modification and “chemically modified” refer to modification with respect to adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C) ribonucleosides or deoxyribnucleosides in at least one of their position, pattern, percent or population.
  • RNA polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • polynucleotides in some embodiments, comprise various (more than one) different modifications.
  • a particular region of a polynucleotide contains one, two or more (optionally different) nucleoside or nucleotide modifications.
  • a modified RNA polynucleotide e.g., a modified mRNA polynucleotide
  • introduced to a cell or organism exhibits reduced degradation in the cell or organism, respectively, relative to an unmodified polynucleotide.
  • a modified RNA polynucleotide e.g., a modified mRNA polynucleotide
  • introduced into a cell or organism may exhibit reduced immunogenicity in the cell or organism, respectively (e.g., a reduced innate response).
  • Polynucleotides may comprise modifications that are naturally-occurring, non-naturally-occurring or the polynucleotide may comprise a combination of naturally-occurring and non-naturally- occurring modifications.
  • Polynucleotides may include any useful modification, for example, of a sugar, a nucleobase, or an internucleoside linkage (e.g., to a linking phosphate, to a phosphodiester linkage or to the phosphodiester backbone).
  • Polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • RNA polynucleotides such as mRNA polynucleotides
  • polynucleotides in some embodiments, comprise non-natural modified nucleotides that are introduced during synthesis or post-synthesis of the polynucleotides to achieve desired functions or properties.
  • the modifications may be present on an internucleotide linkages, purine or pyrimidine bases, or sugars.
  • the modification may be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain. Any of the regions of a polynucleotide may be chemically modified.
  • nucleosides and nucleotides of a polynucleotide e.g., RNA polynucleotides, such as mRNA polynucleotides.
  • a “nucleoside” refers to a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”).
  • a “nucleotide” refers to a nucleoside, including a phosphate group.
  • Modified nucleotides may by synthesized by any useful method, such as, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides.
  • Polynucleotides may comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages may be RNG043-WO1 PCT Application standard phosphodiester linkages, in which case the polynucleotides would comprise regions of nucleotides.
  • Modified nucleotide base pairing encompasses not only the standard adenosine- thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and/or modified nucleotides comprising non-standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures.
  • non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine or uracil.
  • polynucleotides include a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.
  • modified nucleobases in polynucleotides are selected from the group consisting of pseudouridine ( ⁇ ), N1-methylpseudouridine (m 1 ⁇ ), N1-ethylpseudouridine, 2-thiouridine, 4′- thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl- pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2- thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1- methyl-pseudouridine, 4-thio-pseudouridine, 5-aza
  • polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • RNA polynucleotides include a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.
  • modified nucleobases in polynucleotides are selected from the group consisting of 1- methyl-pseudouridine (m 1 ⁇ ), 5-methoxy-uridine (mo 5 U), 5-methyl-cytidine (m 5 C), pseudouridine ( ⁇ ), ⁇ -thio-guanosine and ⁇ -thio-adenosine.
  • polynucleotides includes a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.
  • polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • polynucleotides comprise pseudouridine ( ⁇ ) and 5-methyl-cytidine (m 5 C).
  • polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • polynucleotides comprise 1-methyl-pseudouridine (m 1 ⁇ ).
  • polynucleotides e.g., RNA RNG043-WO1 PCT Application polynucleotides, such as mRNA polynucleotides
  • polynucleotides comprise 1-methyl-pseudouridine (m 1 ⁇ ) and 5-methyl-cytidine (m 5 C).
  • polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • 2-thiouridine s 2 U
  • polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • 2-thiouridine s 2 U
  • polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • 2- thiouridine 2- thiouridine and 5-methyl-cytidine (m 5 C).
  • polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • RNA polynucleotides comprise methoxy-uridine (mo 5 U).
  • polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • 5-methoxy-uridine mo 5 U
  • 5-methyl-cytidine m 5 C
  • polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • polynucleotides comprise 2′-O-methyl uridine and 5-methyl-cytidine (m 5 C).
  • polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • N6-methyl-adenosine m 6 A
  • polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • N6-methyl-adenosine m 6 A
  • 5- methyl-cytidine methyl-cytidine
  • polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • RNA polynucleotides are uniformly modified (e.g., fully modified, modified throughout the entire sequence) for a particular modification.
  • a polynucleotide can be uniformly modified with 5-methyl-cytidine (m 5 C), meaning that all cytosine residues in the mRNA sequence are replaced with 5-methyl-cytidine (m 5 C).
  • m 5 C 5-methyl-cytidine
  • a polynucleotide can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue such as those set forth above.
  • nucleobases and nucleosides having a modified cytosine include N4- acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5- hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), and 2- thio-5-methyl-cytidine.
  • a modified nucleobase is a modified uridine.
  • a modified nucleobase is a modified cytosine.
  • nucleosides having a modified uridine include 5-cyano uridine, and 4′-thio uridine.
  • the polynucleotides of the present disclosure may be partially or fully modified along the entire length of the molecule.
  • one or more or all or a given type of nucleotide e.g., purine or pyrimidine, or any one or more or all of A, G, U, C
  • nucleotides X in a polynucleotide of the present disclosure are modified nucleotides, wherein X may any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+CorA+G+C.
  • the polynucleotide may contain from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e., any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 20% to 95%, from 20% to 100%, from
  • the polynucleotides may contain at a minimum 1% and at maximum 100% modified nucleotides, or any intervening percentage, such as at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides.
  • the polynucleotides may contain a modified pyrimidine such as a modified uracil or cytosine.
  • At least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the uracil in the polynucleotide is replaced with a modified uracil (e.g., a 5-substituted uracil).
  • the modified uracil can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures).
  • cytosine in the polynucleotide is replaced with a modified cytosine (e.g., a 5-substituted cytosine).
  • the modified cytosine can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures). 4.
  • the LNP-based pharmaceutical compositions described herein may include one or more circular mRNA molecules or “oRNAs.”
  • the circular mRNA payloads may encode one or more components of the herein described gene editing systems or other therapeutic protein of interest.
  • a circular mRNA payload may encode an amino acid sequence-programmable DNA binding domain (e.g., retron RTs, CRISPR nucleases, TALENS and zinc finger-binding domains) or a nucleic acid sequence-programmable DNA binding domain (e.g., CRISPR Cas9, CRISPR Cas12a, CRISPR Cas12f, CRISPR Cas13a, CRISPR Cas13b, or TnpB).
  • amino acid sequence-programmable DNA binding domain e.g., retron RTs, CRISPR nucleases, TALENS and zinc finger-binding domains
  • a nucleic acid sequence-programmable DNA binding domain e.g., CRISPR Cas9, CRISPR Cas12a, CRISPR Cas12f, CRISPR Cas13a, CRISPR Cas13b, or TnpB.
  • the circular mRNA payloads may also encode, depending upon the nature of the gene editing system, one or more effector domains that provide various functionalities that facilitate changes in nucleotide sequence and/or gene expression, such as, but not limited to, single-strand DNA binding proteins, nucleases, endonucleases, exonucleases, deaminases (e.g., cytidine deaminases or adenosine deaminases), polymerases (e.g., reverse transcriptases), integrases, recombinases, etc., and fusion proteins comprising one or more functional domains linked together.
  • deaminases e.g., cytidine deaminases or adenosine deaminases
  • polymerases e.g., reverse transcriptases
  • integrases e.g., recombinases, etc.
  • fusion proteins comprising one or more functional domains linked together.
  • Circular RNA (oRNA) described herein are polyribonucleotides that form a continuous structure through covalent or non-covalent bonds. Due to the circular structure, oRNAs have improved stability, increased half-life, reduced immunogenicity, and/or improved functionality (e.g., of a function described herein) compared to a corresponding linear RNA.
  • an oRNA binds a target. In some embodiments, an oRNA binds a substrate. In some embodiments, an oRNA binds a target and binds a substrate of the target. In some embodiments, an oRNA binds a target and mediates modulation of a substrate of the target.
  • an oRNA brings together a target and its substrate to mediate modification of the substrate, e.g., post-translational modification. In some embodiments, an oRNA brings together a target and its substrate to mediate a cellular process (e.g., alters protein degradation or signal transduction) involving the substrate. In some embodiments, a target is a target protein and a substrate is a substrate protein.
  • an oRNA comprises a conjugation moiety for binding to chemical compound.
  • the conjugation moiety can be a modified polyribonucleotide.
  • the chemical compound can be conjugated to the oRNA by the conjugation moiety.
  • the chemical compound binds to a target and mediates modulation of a substrate of the target.
  • an oRNA binds a substrate of a target and a chemical compound conjugated to the oRNA by the conjugation moiety binds the target to bring RNG043-WO1 PCT Application together the target and its substrate to mediate modification of the substrate, e.g., post- translational modification.
  • an oRNA binds a substrate of a target and a chemical compound conjugated to the oRNA by the conjugation moiety binds the target to bring together the target and its substrate to mediate modification of the substrate to mediate a cellular process (e.g., alters protein degradation or signal transduction) involving the substrate.
  • a target is a target protein and a substrate is a substrate protein.
  • the oRNA may be non-immunogenic in a mammal (e.g., a human, non-human primate, rabbit, rat, and mouse).
  • the oRNA may be capable of replicating or replicates in a cell from an aquaculture animal (e.g., fish, crabs, shrimp, oysters etc.), a mammalian cell, a cell from a pet or zoo animal (e.g., cats, dogs, lizards, birds, lions, tigers and bears etc.), a cell from a farm or working animal (e.g., horses, cows, pigs, chickens etc.), a human cell, cultured cells, primary cells or cell lines, stem cells, progenitor cells, differentiated cells, germ cells, cancer cells (e.g., tumorigenic, metastatic), non-tumorigenic cells (e.g., normal cells), fetal cells, embryonic cells, adult cells, mitotic cells, non-mitotic cells, or any combination thereof.
  • an aquaculture animal e.g., fish, crabs, shrimp, oysters etc.
  • a mammalian cell e.g., a cell from a
  • a pharmaceutical composition comprising: a circular RNA comprising, in the following order, a 3’ group I intron fragment, an Internal Ribosome Entry Site (IRES), an expression sequence encoding a polypeptide (e.g., a nucleobase editing system or component thereof), and a 5’ group I intron fragment, and a transfer vehicle comprising at least one of (i) an ionizable lipid, (ii) a structural lipid, and (iii) a PEG-modified lipid, wherein the transfer vehicle is capable of delivering the circular RNA polynucleotide to a cell (e.g., a human cell, such as an immune cell present in a human subject), such that the polypeptide is translated in the cell.
  • a transfer vehicle comprising at least one of (i) an ionizable lipid, (ii) a structural lipid, and (iii) a PEG-modified lipid, wherein the transfer vehicle is capable of delivering the circular
  • the pharmaceutical composition is formulated for intravenous administration to the human subject in need thereof.
  • the 3’ group I intron fragment and 5’ group I intron fragment are Anabaena group I intron fragments.
  • the 3’ intron fragment and 5’ intron fragment are defined by the L9a-5 permutation site in the intact intron.
  • the 3’ intron fragment and 5’ intron fragment are defined by the L8-2 permutation site in the intact intron.
  • the IRES is from Taura syndrome virus, Tiiatoma virus, Theiler's encephalomyelitis virus, Simian Virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, Human poliovirus 1, Plautia stall intestine virus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulata virus- 1, Human Immunodeficiency Virus type 1, Homalodisca coagulata virus- 1, Himetobi P virus, Hepatitis RNG043-WO1 PCT Application C virus, Hepatitis A virus, Hepatitis GB virus , Foot and mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Ectropis obliqua picoma-like virus, Encephalomyocarditis virus, Drosophila C Virus, Human coxsackievirus B3, Crucifer tobamovirus, Cricket paralysis
  • the IRES comprises a CVB3 IRES or a fragment or variant thereof.
  • the pharmaceutical composition comprises a first internal spacer between the 3’ group I intron fragment and the IRES, and a second internal spacer between the expression sequence and the 5’ group I intron fragment.
  • the first and second internal spacers each have a length of about 10 to about 60 nucleotides.
  • the circular mRNA comprises a nucleotide sequence encoding a polypeptide of interest, such as a nucleobase editing system or therapeutic protein (e.g., a CAR or TCR complex protein).
  • the LNP-based nucleobase editing systems, RNA therapeutics, and pharmaceutical compositions described herein further comprise a targeting moiety.
  • the targeting moiety mediates receptor-mediated endocytosis or direct fusion of the delivery vehicle (LNPs) into selected cells of a selected cell population or tissue in the absence of cell isolation or purification.
  • the targeting moiety is capable of binding to a protein selected from the group CD3, CD4, CD8, CDS, CD7, PD-1, 4-1BB, CD28, Clq, and CD2.
  • the targeting moiety comprises an antibody specific for a macrophage, dendritic cell, NK cell, NKT, or T cell antigen.
  • the targeting moiety comprises a scFv, nanobody, peptide, minibody, polynucleotide aptamer, heavy chain variable region, light chain variable region or fragment thereof.
  • the LNP-based nucleobase editing systems, RNA therapeutics, and pharmaceutical compositions described herein are administered in an amount effective to treat a disease in the human subject (e.g., wherein the disease can be cancer, muscle disorder, or CNS disorder, etc.).
  • the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions have an enhanced safety profile when compared to a pharmaceutical composition comprising T cells or vectors comprising exogenous DNA encoding the same polypeptide.
  • the LNP-based nucleobase editing systems and pharmaceutical compositions thereof are administered in an amount effective to induce a desire precise edit in a genome.
  • the LNP-based nucleobase editing systems and pharmaceutical compositions have an enhanced safety profile when compared to state of the art gene editing delivery compositions.
  • the present disclosure provides a circular RNA comprising, in the following order, a 3’ group I intron fragment, an Internal Ribosome Entry Site (IRES), an expression sequence encoding a polypeptide (e.g., a nucleobase editing system or component thereof), and a 5’ group I intron fragment.
  • the 3’ group I intron fragment and 5’ group I intron fragment are Anabaena group I intron fragments.
  • the 3’ intron fragment and 5’ intron fragment are defined by the L9a-5 permutation site in the intact intron.
  • the 3’ intron fragment and 5’ intron fragment are defined by the L8-2 permutation site in the intact intron.
  • the IRES comprises a CVB3 IRES or a fragment or variant thereof.
  • RNG043-WO1 PCT Application [00618]
  • the circular RNA comprises a first internal spacer between the 3’ group I intron fragment and the IRES, and a second internal spacer between the expression sequence and the 5’ group I intron fragment.
  • the first and second internal spacers each have a length of about 10 to about 60 nucleotides.
  • the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics, and pharmaceutical compositions described herein comprises of natural nucleotides.
  • the circular RNA further comprises a second expression sequence encoding a therapeutic protein.
  • the therapeutic protein comprises a checkpoint inhibitor.
  • the therapeutic protein comprises a cytokine.
  • the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics, and pharmaceutical compositions described herein consists of natural nucleotides.
  • the circular RNA payload LNP-based nucleobase editing systems, RNA therapeutics, and pharmaceutical compositions described herein comprises a nucleotide sequence that is codon optimized, either partially or fully.
  • the circular RNA is optimized to lack at least one microRNA binding site present in an equivalent pre-optimized polynucleotide.
  • the circular RNA is optimized to lack at least one endonuclease susceptible site present in an equivalent pre-optimized polynucleotide.
  • the circular RNA is optimized to lack at least one RNA-editing susceptible site present in an equivalent pre-optimized polynucleotide.
  • the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics, and pharmaceutical compositions described herein has an in vivo functional half- life in humans greater than that of an equivalent linear RNA having the same expression sequence.
  • the circular RNA has a length of about 100 nucleotides to about 10 kilobases.
  • the circular RNA has a functional half-life of at least about 20 hours.
  • the circular RNA has a duration of therapeutic effect in a human cell of at least about 20 hours.
  • the circular RNA has a duration of therapeutic effect in a human cell greater than or equal to that of an equivalent linear RNA comprising the same expression sequence.
  • the circular RNA has a functional half-life in a human cell greater than or equal to that of an equivalent linear RNA comprising the same expression sequence.
  • the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics, and pharmaceutical compositions described herein has a half-life of at least that of a linear counterpart.
  • the oRNA has a half-life that is increased over that of a linear counterpart. In some embodiments, the half-life is increased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or greater.
  • the oRNA has a half-life or persistence in a cell for at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours (1 day), 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or longer or any time therebetween.
  • the oRNA has a half-life or persistence in a cell for no more than about 10 mins to about 7 days, or no more than about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 24 hours (1 day), 36 hours (1.5 days), 48 hours (2 days),60 hours (2.5 days), 72 hours (3 days), 4 days, 5 days, 6 days, or 7 days.
  • the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics, and pharmaceutical compositions described herein has a half-life or persistence in a cell while the cell is dividing.
  • the oRNA has a half-life or persistence in a cell post division.
  • the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics, and pharmaceutical compositions described herein has a half-life or persistence in a dividing cell for greater than about 10 minutes to about 30 days, or at least about 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 24 hours (1 day), 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or longer or any time therebetween.
  • the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics, and pharmaceutical compositions described herein modulates a cellular function, e.g., transiently or long term.
  • the cellular function is stably altered, such as a modulation that persists for at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours (1 day), 2 days, RNG043-WO1 PCT Application 3, days, 4days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or longer.
  • the cellular function is transiently altered, e.g., such as a modulation that persists for no more than about 30 mins to about 7 days, or no more than about 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours (1 day), 36 hours (1.5 days), 48 hours (2 days), 60 hours (2.5 days), 72 hours(3 days), 4 days, 5 days, 6 days, or 7 days.
  • a modulation that persists for no more than about 30 mins to about 7 days, or no more than about 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours
  • the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions described herein is at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 1,000 nucleotides, at least about 2,000 nucleotides, at least about 5,000 nucleotides, at least about 6,000 nucleotides, at least about 7,000 nucleotides, at least about 8,000 nucleotides, at least about 9,000 nucleotides, at least about 10,000 nucleotides, at least about 12,000 nucleotides, at least about 14,000 nucleotides, at least about 15,000 nucleotides, at least about 100
  • the oRNA may be of a sufficient size to accommodate a binding site for a ribosome.
  • the maximum size of the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics, and pharmaceutical compositions described herein may be limited by the ability of packaging and delivering the RNA to a target.
  • the size of the oRNA is a length sufficient to encode polypeptides, and thus, lengths of at least 20,000 nucleotides, at least 15,000 nucleotides, at least 10,000 nucleotides, at least 7,500 nucleotides, or at least 5,000 nucleotides, at least 4,000 nucleotides, at least 3,000 nucleotides, at least 2,000 nucleotides, at least 1,000 nucleotides, at least 500 nucleotides, at least 400 nucleotides, at least 300 nucleotides, at least 200 nucleotides, at least 100 nucleotides may be useful.
  • the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics, and pharmaceutical compositions described herein comprises one or more elements described elsewhere herein.
  • the RNG043-WO1 PCT Application elements may be separated from one another by a spacer sequence or linker.
  • the elements may be separated from one another by 1 nucleotide, 2 nucleotides, about 5 nucleotides, about 10 nucleotides, about 15 nucleotides, about 20 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides, about 80 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 400 nucleotides, about 500 nucleotides, about 600 nucleotides, about 700 nucleotides, about 800 nucleotides, about 900 nucleotides, about 1000 nucleotides, up to about 1 kb, at least about 1000 nucleotides.
  • one or more elements are contiguous with one another, e.g., lacking a spacer element.
  • one or more elements is conformationally flexible. In some embodiments, the conformational flexibility is due to the sequence being substantially free of a secondary structure.
  • the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions described herein comprises a secondary or tertiary structure that accommodates a binding site for a ribosome, translation, or rolling circle translation.
  • the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions described herein comprises particular sequence characteristics.
  • the oRNA may comprise a particular nucleotide composition.
  • the oRNA may include one or more purine rich regions (adenine or guanosine).
  • the oRNA may include one or more purine rich regions (adenine or guanosine).
  • the oRNA may include one or more AU rich regions or elements (AREs).
  • the oRNA may include one or more adenine rich regions.
  • the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics, and pharmaceutical compositions described herein comprises one or more modifications described elsewhere herein.
  • the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics, and pharmaceutical compositions described herein comprises one or more expression sequences and is configured for persistent expression in a cell of a subject in vivo.
  • the oRNA is configured such that expression of the one or more expression sequences in the cell at a later time point is equal to or higher than an earlier time point.
  • the expression of the one or more expression RNG043-WO1 PCT Application sequences can be either maintained at a relatively stable level or can increase over time.
  • the expression of the expression sequences can be relatively stable for an extended period of time. For instance, in some cases, the expression of the one or more expression sequences in the cell over a time period of at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23 or more days does not decrease by 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%.
  • the expression of the one or more expression sequences in the cell is maintained at a level that does not vary by more than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% for at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23 or more days.
  • Regulatory Elements [00637]
  • the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions described herein comprises one or more regulatory elements.
  • a "regulatory element” is a sequence that modifies expression of an expression sequence, e.g., a nucleotide sequence encoding a nucleobase editing system or a therapeutic protein, i.e., a coding region of interest (CROI).
  • the regulatory element may include a sequence that is located adjacent to a coding region of interest encoded on the circular RNA payload.
  • the regulatory element may be operatively linked to a nucleotide sequence of the circular RNA that encodes a coding region of interest (e.g., a nucleobase editing system or therapeutic polypeptide).
  • a regulatory element may increase an amount of expression of a coding region of interest encoded on the circular RNA payload as compared to an amount expressed when no regulatory element exists.
  • a regulatory element may comprise a sequence to selectively initiates or activates translation of a coding sequence of interest encoded on the circular RNA payload.
  • a regulatory element may comprise a sequence to initiate degradation of the oRNA or the payload or cargo. Non-limiting examples of the sequence to initiate degradation includes, but is not limited to, riboswitch aptazyme and miRNA binding sites.
  • a regulatory element can modulate translation of a coding region of interest encoded on the oRNA.
  • the modulation can create an increase (enhancer) or decrease (suppressor) in the expression of the coding region of interest.
  • the regulatory element may be located adjacent to the CROI (e.g., on one side or both sides of the CROI).
  • Translation Initiation Sequence RNG043-WO1 PCT Application [00642]
  • a translation initiation sequence functions as a regulatory element.
  • the translation initiation sequence comprises an AUG/ATG codon.
  • a translation initiation sequence comprises any eukaryotic start codon such as, but not limited to, AUG/ATG, CUG/CTG, GUG/GTG, UUG/TTG, ACG, AUC/ATC, AUU, AAG, AUA/ATA, or AGG.
  • a translation initiation sequence comprises a Kozak sequence.
  • translation begins at an alternative translation initiation sequence, e.g., translation initiation sequence other than AUG/ATG codon, under selective conditions, e.g., stress induced conditions.
  • the translation of the circular polyribonucleotide may begin at alternative translation initiation sequence, such as ACG.
  • the circular polyribonucleotide translation may begin at alternative translation initiation sequence, CUG/CTG.
  • the translation may begin at alternative translation initiation sequence, GUG/GTG.
  • the translation may begin at a repeat-associated non-AUG (RAN) sequence such as an alternative translation initiation sequence that includes short stretches of repetitive RNA e.g. CGG, GGGGCC, CAG, or CTG.
  • RAN repeat-associated non-AUG
  • the oRNA encodes a polypeptide or peptide and may comprise a translation initiation sequence.
  • the translation initiation sequence may comprise, but is not limited to a start codon, a non-coding start codon, a Kozak sequence or a Shine- Dalgarno sequence.
  • the translation initiation sequence may be located adjacent to the payload or cargo (e.g., on one side or both sides of the coding region of interest).
  • the translation initiation sequence provides conformational flexibility to the oRNA.
  • the translation initiation sequence is within a substantially single stranded region of the oRNA.
  • the oRNA may include more than 1 start codon such as, but not limited to, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15 or more than 15 start codons. Translation may initiate on the first start codon or may initiate downstream of the first start codon. [00646] In some embodiments, the oRNA may initiate at a codon which is not the first start codon, e.g., AUG.
  • Translation of the circular polyribonucleotide may initiate at an alternative translation initiation sequence, such as, but not limited to, ACG, AGG, AAG, CUG/CTG, GUG/GTG, AUA/ATA, AUU/ATT, UUG/TTG.
  • translation begins at an alternative translation initiation sequence under selective conditions, e.g., stress induced conditions.
  • the translation of the oRNA may RNG043-WO1 PCT Application begin at alternative translation initiation sequence, such as ACG.
  • the oRNA translation may begin at alternative translation initiation sequence, CUG/CTG.
  • the oRNA translation may begin at alternative translation initiation sequence, GTG/GUG.
  • the oRNA may begin translation at a repeat-associated non-AUG (RAN) sequence, such as an alternative translation initiation sequence that includes short stretches of repetitive RNA e.g. CGG, GGGGCC, CAG, CTG.
  • RAN repeat-associated non-AUG
  • IRES Sequences [00647]
  • the oRNA described herein comprises an internal ribosome entry site (IRES) element capable of engaging an eukaryotic ribosome.
  • IRES internal ribosome entry site
  • the IRES element is at least about 5 nucleotides, at least about 8 nucleotides, at least about 9 nucleotides, at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 250 nucleotides, at least about 350 nucleotides, or at least about 500 nucleotides.
  • the IRES element is derived from the DNA of an organism including, but not limited to, a virus, a mammal, and a Drosophila.
  • viral DNA may be derived from, but is not limited to, picornavirus complementary DNA (cDNA), with encephalomyocarditis virus (EMCV) cDNA and poliovirus cDNA.
  • cDNA picornavirus complementary DNA
  • EMCV encephalomyocarditis virus
  • Drosophila DNA from which an IRES element is derived includes, but is not limited to, an Antennapedia gene from Drosophila melanogaster.
  • the IRES element is at least partially derived from a virus, for instance, it can be derived from a viral IRES element, such as ABPV_IGRpred, AEV, ALPV_IGRpred, BQCV_IGRpred, BVDV1_1-385, BVDV1_29-391, CrPV_5NCR, CrPV_IGR, crTMV_IREScp, crTMV_IRESmp75, crTMV_IRESmp228, crTMV_IREScp, crTMV_IREScp, CSFV, CVB3, DCV_IGR, EMCV-R, EoPV_5NTR, ERAV 245-961, ERBV 162-920, EV71_1-748, FeLV-Notch2, FMDV_type_C, GBV-A, GBV-B, GBV-C, gypsy_env, gypsyD5, gypsyD2, HAV_HM175,
  • a viral IRES element such
  • the IRES element is at least partially derived from a cellular IRES, such as AML1/RUNX1, Antp-D, Antp-DE, Antp-CDE, Apaf-1, Apaf-1, AQP4, AT1R_var1, RNG043-WO1 PCT Application AT1R_var2, AT1R_var3, AT1R_var4, BAG1_p36delta236 nt, BAG1_p36, BCL2, BiP_-222_- 3, c-IAP1_285-1399, c-IAP1_1313-1462, c-jun, c-myc, Cat-1224, CCND1, DAPS, eIF4G, eIF4GI-ext, eIF4GII, eIF4GII-long, ELG1, ELH, FGF1A,FMR1, Gtx-133-141, Gtx-1-166, Gtx-1-120, Gtx-1-196, hairless, HAP4, H
  • the IRES is an IRES sequence from Coxsackievirus B3 (CVB3), the protein coding region encodes Guassia luciferase (Gluc), and the spacer sequences are polyA-C.
  • the IRES if present, is at least about 50 nucleotides in length.
  • the vector comprises an IRES that comprises a natural sequence.
  • the vector comprises an IRES that comprises a synthetic sequence.
  • An IRES may act as the sole ribosome binding site, or may serve as one of multiple ribosome binding sites of an mRNA.
  • a polynucleotide containing more than one functional ribosome binding site may encode several peptides or polypeptides that are translated independently by the ribosomes (e.g., multicistronic mRNA).
  • polynucleotides are provided with an IRES, further optionally provided is a second translatable region.
  • IRES sequences that can be used according to the present disclosure include without limitation, those from picornaviruses (e.g., FMDV), pest viruses (CFFV), polio viruses (PV), encephalomyocarditis viruses (ECMV), foot-and mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical Swine fever viruses (CSFV), murine leukemia virus (MLV), simian immune deficiency viruses (SIV) or cricket paralysis viruses (CrPV).
  • picornaviruses e.g., FMDV
  • CFFV pest viruses
  • PV polio viruses
  • ECMV encephalomyocarditis viruses
  • FMDV foot-and mouth disease viruses
  • HCV hepatitis C viruses
  • CSFV classical Swine fever viruses
  • MLV murine leukemia virus
  • SIV simian immune deficiency viruses
  • CrPV cricket paralysis viruses
  • the oRNA includes one or more coding regions of interest (i.e., also referred to as product expression sequences) which encode polypeptides of interest, including but not limited to nucleobase editing system and therapeutic proteins.
  • the product expression sequences may or may not have a termination element.
  • the oRNA includes one or more product expression sequences that lack a termination element, such that the oRNA is continuously translated. RNG043-WO1 PCT Application [00654] Exclusion of a termination element may result in rolling circle translation or continuous expression of the encoded peptides or polypeptides as the ribosome will not stall or fall-off.
  • rolling circle translation expresses continuously through the product expression sequence.
  • one or more product expression sequences in the oRNA comprise a termination element.
  • not all of the product expression sequences in the oRNA comprise a termination element. In such instances, the product expression sequence may fall off the ribosome when the ribosome encounters the termination element and terminates translation.
  • Rolling Circle Translation [00657] In some embodiments, once translation of the oRNA is initiated, the ribosome bound to the oRNA does not disengage from the oRNA before finishing at least one round of translation of the oRNA. In some embodiments, the oRNA as described herein is competent for rolling circle translation.
  • the ribosome bound to the oRNA does not disengage from the oRNA before finishing at least 2 rounds, at least 3 rounds, at least 4 rounds, at least 5 rounds, at least 6 rounds, at least 7 rounds, at least 8 rounds,at least 9 rounds, at least 10 rounds, at least 11 rounds, at least 12 rounds, at least 13 rounds, at least 14 rounds, at least 15 rounds, at least 20 rounds, at least 30 rounds, at least 40 rounds, at least 50 rounds, at least 60 rounds, at least 70 rounds, at least 80 rounds, at least 90 rounds, at least 100 rounds, at least 150 rounds, at least 200 rounds, at least 250 rounds, at least 500 rounds, at least 1000 rounds, at least 1500 rounds, at least 2000 rounds, at least 5000 rounds, at least 10000 rounds, at least 10 5 rounds, or at least 10 6 rounds of translation of the oRNA.
  • the rolling circle translation of the oRNA leads to generation of polypeptide that is translated from more than one round of translation of the oRNA.
  • the oRNA comprises a stagger element, and rolling circle translation of the oRNA leads to generation of polypeptide product that is generated from a single round of translation or less than a single round of translation of the oRNA.
  • Circularization [00659]
  • a linear RNA may be cyclized, or concatemerized.
  • the linear RNA may be cyclized in vitro prior to formulation and/or delivery.
  • the linear RNA may be cyclized within a cell.
  • the mechanism of cyclization or concatemerization may occur through at least 3 different routes: 1) chemical, 2) enzymatic, and 3) ribozyme catalyzed.
  • the newly formed 5'-/3'-linkage may be intramolecular or intermolecular.
  • the 5'-end and the 3 '-end of the nucleic acid contain chemically reactive groups that, when close together, form a new covalent linkage between the 5 '-end and the 3 '-end of the molecule.
  • the 5 '-end may contain an NHS-ester reactive group and the 3 '-end may contain a 3'-amino-terminated nucleotide such that in an organic solvent the 3'-amino-terminated nucleotide on the 3 '-end of a synthetic mRNA molecule will undergo a nucleophilic attack on the 5 '-NHS-ester moiety forming a new 5 '-/3 '-amide bond.
  • T4 RNA ligase may be used to enzymatically link a 5'- phosphorylated nucleic acid molecule to the 3'-hydroxyl group of a nucleic acid forming a new phosphorodiester linkage.
  • ⁇ g of a nucleic acid molecule is incubated at 37°C for 1 hour with 1-10 units of T4 RNA ligase (New England Biolabs, Ipswich, MA) according to the manufacturer's protocol.
  • the ligation reaction may occur in the presence of a split oligonucleotide capable of base-pairing with both the 5'-and 3'-region in juxtaposition to assist the enzymatic ligation reaction.
  • either the 5 '-or 3 '-end of the cDNA template encodes a ligase ribozyme sequence such that during in vitro transcription, the resultant nucleic acid molecule can contain an active ribozyme sequence capable of ligating the 5 '-end of a nucleic acid molecule to the 3 '-end of a nucleic acid molecule.
  • the ligase ribozyme may be derived from the Group I Intron, Group I Intron, Hepatitis Delta Virus, Hairpin ribozyme or may be selected by SELEX (systematic evolution of ligands by exponential enrichment).
  • the ribozyme ligase reaction may take 1 to 24 hours at temperatures between 0 and 37°C.
  • the oRNA is made via circularization of a linear RNA.
  • the following elements are operably connected to each other and, in some embodiments, arranged in the following sequence: a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′ splice site dinucleotide, c.) a protein coding or noncoding region, d.) a 5′ group I intron fragment containing a 5′ splice site dinucleotide, and e.) a 3′ homology arm.
  • the vector allows production of a circular RNA that is translatable and/or biologically active inside eukaryotic cells.
  • the biologically active RNA is, for example, an miRNA sponge, or long noncoding RNA.
  • the following elements are operably connected to each other and arranged in the following sequence: a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′ splice site dinucleotide, c.) optionally, a 5′ spacer sequence, d.) RNG043-WO1 PCT Application optionally, an internal ribosome entry site (IRES), e.) a protein coding or noncoding region, f.) optionally, a 3′ spacer sequence, g.) a 5′ group I intron fragment containing a 5′ splice site dinucleotide, and h.) a 3′ homology arm.
  • IRS internal ribosome entry site
  • the vector allows production of a circular RNA that is translatable and/or biologically active inside eukaryotic cells.
  • the following elements are operably connected to each other and arranged in the following sequence: a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′ splice site dinucleotide, c.) a 5′ spacer sequence, d.) an internal ribosome entry site (IRES), e.) a protein coding or noncoding region, f.) a 5′ group I intron fragment containing a 5′ splice site dinucleotide, and g.) a 3′ homology arm.
  • IRS internal ribosome entry site
  • the vector allows production of a circular RNA that is translatable and/or biologically active inside eukaryotic cells.
  • the following elements are operably connected to each other and arranged in the following sequence: a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′ splice site dinucleotide, c.) a 5′ spacer sequence, d.) a protein coding or noncoding region, e.) a 3′ spacer sequence, f.) a 5′ group I intron fragment containing a 5′ splice site dinucleotide, and g.) a 3′ homology arm.
  • the vector allows production of a circular RNA that is translatable and/or biologically active inside eukaryotic cells.
  • the following elements are operably connected to each other and arranged in the following sequence: a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′ splice site dinucleotide, c.) an internal ribosome entry site (IRES), d.) a protein coding or noncoding region, e.) a 3′ spacer sequence, f) a 5′ group I intron fragment containing a 5′ splice site dinucleotide, and g.) a 3′ homology arm.
  • the vector allows production of a circular RNA that is translatable and/or biologically active inside eukaryotic cells.
  • the following elements are operably connected to each other and arranged in the following sequence: a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′ splice site dinucleotide, c.) a protein coding or noncoding region, d.) a 3′ spacer sequence, e.) a 5′ group I intron fragment containing a 5′ splice site dinucleotide, and f.) a 3′ homology arm.
  • the vector allows production of a circular RNA that is translatable and/or biologically active inside eukaryotic cells.
  • the following elements are operably connected to each other and arranged in the following sequence: a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′ splice site dinucleotide, c.) a 5′ spacer sequence, d.) a protein coding RNG043-WO1 PCT Application or noncoding region, e.) a 5′ group I intron fragment containing a 5′ splice site dinucleotide, and f.) a 3′ homology arm.
  • the vector allows production of a circular RNA that is translatable and/or biologically active inside eukaryotic cells.
  • the following elements are operably connected to each other and arranged in the following sequence: a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′ splice site dinucleotide, c.) an internal ribosome entry site (IRES), d.) a protein coding or noncoding region, e.) a 5′ group I intron fragment containing a 5′ splice site dinucleotide, and f) a 3′ homology arm.
  • the vector allows production of a circular RNA that is translatable and/or biologically active inside eukaryotic cells.
  • the following elements are operably connected to each other and arranged in the following sequence: a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′ splice site dinucleotide, c.) a 5′ spacer sequence, d.) an internal ribosome entry site (IRES), e.) a protein coding or noncoding region, f) a 3′ spacer sequence, g.) a 5′ group I intron fragment containing a 5′ splice site dinucleotide, and h.) a 3′ homology arm.
  • the vector allowing production of a circular RNA that is translatable and/or biologically active inside eukaryotic cells.
  • the 3′ group I intron fragment and/or the 5′ group I intron fragment is from a Cyanobacterium Anabaena sp. pre-tRNA-Leu gene or T4 phage Td gene.
  • the 3′ group I intron fragment and/or the 5′ group I intron fragment is from a Cyanobacterium Anabaena sp. pre-tRNA-Leu gene.
  • the protein coding region encodes a protein of eukaryotic or prokaryotic origin.
  • the protein coding region encodes human protein or non-human protein.
  • the protein coding region encodes one or more antibodies.
  • the protein coding region encodes human antibodies.
  • the protein coding region encodes a protein selected from hFIX, SP-B, VEGF-A, human methylmalonyl-CoA mutase (hMUT), CFTR, cancer self- antigens, and additional gene editing enzymes like Cpf1, zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs).
  • the protein coding region encodes a protein for therapeutic use.
  • the human antibody encoded by the protein coding region is an anti-HIV antibody. In one embodiment, the antibody encoded by the protein coding region is a bispecific antibody. In one embodiment, the bispecific antibody is specific for CD19 and CD22. In another embodiment, the bispecific antibody is specific for CD3 and CLDN6. In one embodiment, the protein coding region encodes a protein for diagnostic use. In one embodiment, the protein coding region RNG043-WO1 PCT Application encodes Gaussia luciferase (Gluc), Firefly luciferase (Fluc), enhanced green fluorescent protein (eGFP), human erythropoietin (hEPO), or Cas9 endonuclease.
  • Gluc Gaussia luciferase
  • Fluc Firefly luciferase
  • eGFP enhanced green fluorescent protein
  • hEPO human erythropoietin
  • the 5′ homology arm is about 5-50 nucleotides in length. In another embodiment, the 5′ homology arm is about 9-19 nucleotides in length. In some embodiments, the 5′ homology arm is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the 5′ homology arm is no more than 50, 45, 40, 35, 30, 25 or 20 nucleotides in length. In some embodiments, the 5′ homology arm is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 nucleotides in length. [00678] In one embodiment, the 3′ homology arm is about 5-50 nucleotides in length.
  • the 3′ homology arm is about 9-19 nucleotides in length. In some embodiments, the 3′ homology arm is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the 3′ homology arm is no more than 50, 45, 40, 35, 30, 25 or 20 nucleotides in length. In some embodiments, the 3′ homology arm is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 nucleotides in length. [00679] In one embodiment, the 5′ spacer sequence is at least 10 nucleotides in length. In another embodiment, the 5′ spacer sequence is at least 15 nucleotides in length.
  • the 5′ spacer sequence is at least 30 nucleotides in length. In some embodiments, the 5′ spacer sequence is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or 30 nucleotides in length. In some embodiments, the 5′ spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30 nucleotides in length. In some embodiments the 5′ spacer sequence is between 20 and 50 nucleotides in length.
  • the 5′ spacer sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length.
  • the 5′ spacer sequence is a polyA sequence.
  • the 5′ spacer sequence is a polyA-C sequence.
  • the 3′ spacer sequence is at least 10 nucleotides in length.
  • the 3′ spacer sequence is at least 15 nucleotides in length.
  • the 3′ spacer sequence is at least 30 nucleotides in length.
  • the 3′ spacer sequence is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or 30 nucleotides in length. In some embodiments, the 3′ spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30 nucleotides in length. In some embodiments the 3′ spacer sequence is between 20 and 50 nucleotides in length. In certain embodiments, the 3′ spacer sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length.
  • the 3′ spacer sequence is a polyA sequence.
  • the 5′ spacer sequence is a polyA-C sequence.
  • Extracellular Circularization [00681]
  • the linear RNA is cyclized, or concatemerized using a chemical method to form an oRNA.
  • the 5'-end and the 3'-end of the nucleic acid e.g., a linear RNA
  • the 5'-end and the 3'-end of the nucleic acid includes chemically reactive groups that, when close together, may form a new covalent linkage between the 5'-end and the 3'-end of the molecule.
  • the 5'- end may contain an NHS-ester reactive group and the 3'-end may contain a 3'-amino- terminated nucleotide such that in an organic solvent the 3'-amino-terminated nucleotide on the 3'-end of a linear RNA will undergo a nucleophilic attack on the 5'-NHS-ester moiety forming a new 5'-/3'-amide bond.
  • a DNA or RNA ligase may be used to enzymatically link a 5'-phosphorylated nucleic acid molecule (e.g., a linear RNA) to the 3'-hydroxyl group of a nucleic acid (e.g., a linear nucleic acid) forming a new phosphorodiester linkage.
  • a linear RNA is incubated at 37°C for 1 hour with 1-10 units of T4 RNA ligase according to the manufacturer's protocol.
  • the ligation reaction may occur in the presence of a linear nucleic acid capable of base-pairing with both the 5'-and 3'-region in juxtaposition to assist the enzymatic ligation reaction.
  • the ligation is splint ligation where a single stranded polynucleotide (splint), like a single stranded RNA, can be designed to hybridize with both termini of a linear RNA, so that the two termini can be juxtaposed upon hybridization with the single-stranded splint.
  • Splint ligase can thus catalyze the ligation of the juxtaposed two termini of the linear RNA, generating an oRNA.
  • a DNA or RNA ligase may be used in the synthesis of the oRNA.
  • the ligase may be a circ ligase or circular ligase.
  • either the 5'-or 3'-end of the linear RNA can encode a ligase ribozyme sequence such that during in vitro transcription, the resultant linear RNA includes an active ribozyme sequence capable of ligating the 5'-end of the linear RNA to the 3'- end of the linear RNA.
  • the ligase ribozyme may be derived from the Group I Intron, Hepatitis Delta Virus, Hairpin ribozyme or may be selected by SELEX (systematic evolution of ligands by exponential enrichment).
  • a linear RNA may be cyclized or concatemerized by using at least one non-nucleic acid moiety.
  • the at least one non-nucleic acid moiety may react with regions or features near the 5' terminus and/or near the 3' terminus of the linear RNA in order to cyclize or concatermerize the linear RNA.
  • the at least one RNG043-WO1 PCT Application non-nucleic acid moiety may be located in or linked to or near the 5' terminus and/or the 3' terminus of the linear RNA.
  • the non-nucleic acid moieties contemplated may be homologous or heterologous.
  • the non-nucleic acid moiety may be a linkage such as a hydrophobic linkage, ionic linkage, a biodegradable linkage and/or a cleavable linkage.
  • the non-nucleic acid moiety is a ligation moiety.
  • the non-nucleic acid moiety may be an oligonucleotide or a peptide moiety, such as an aptamer or a non-nucleic acid linker as described herein.
  • a linear RNA may be cyclized or concatemerized due to a non-nucleic acid moiety that causes an attraction between atoms, molecular surfaces at, near or linked to the 5' and 3' ends of the linear RNA.
  • one or more linear RNA may be cyclized or concatemerized by intermolecular forces or intramolecular forces.
  • intermolecular forces include dipole-dipole forces, dipole-induced dipole forces, induced dipole-induced dipole forces, Van der Waals forces, and London dispersion forces.
  • the linear RNA may comprise a ribozyme RNA sequence near the 5' terminus and near the 3' terminus.
  • the ribozyme RNA sequence may covalently link to a peptide when the sequence is exposed to the remainder of the ribozyme.
  • the peptides covalently linked to the ribozyme RNA sequence near the 5' terminus and the 3' terminus may associate with each other causing a linear RNA to cyclize or concatemerize.
  • the peptides covalently linked to the ribozyme RNA near the 5' terminus and the 3' terminus may cause the linear RNA to cyclize or concatemerize after being subjected to ligated using various methods known in the art such as, but not limited to, protein ligation.
  • the linear RNA may include a 5' triphosphate of the nucleic acid converted into a 5' monophosphate, e.g., by contacting the 5' triphosphate with RNA 5' pyrophosphohydrolase (RppH) or an ATP diphosphohydrolase (apyrase).
  • converting the 5' triphosphate of the linear RNA into a 5' monophosphate may occur by a two- step reaction comprising: (a) contacting the 5' nucleotide of the linear RNA with a phosphatase (e.g., Antarctic Phosphatase, Shrimp Alkaline Phosphatase, or Calf Intestinal Phosphatase) to remove all three phosphates; and (b) contacting the 5' nucleotide after step (a) with a kinase (e.g., Polynucleotide Kinase) that adds a single phosphate.
  • a phosphatase e.g., Antarctic Phosphatase, Shrimp Alkaline Phosphatase, or Calf Intestinal Phosphatase
  • a kinase e.g., Polynucleotide Kinase
  • RNA may be circularized using the methods described in WO2017222911 and WO2016197121, the contents of each of which are herein incorporated by reference in their entirety.
  • RNA may be circularized, for example, by back splicing of a non-mammalian exogenous intron or splint ligation of the 5' and 3 ' ends of a linear RNA.
  • the circular RNA is produced from a recombinant nucleic acid encoding the target RNA to be made circular.
  • the method comprises: a) producing a recombinant nucleic acid encoding the target RNA to be made circular, wherein the recombinant nucleic acid comprises in 5' to 3 ' order: i) a 3 ' portion of an exogenous intron comprising a 3' splice site, ii) a nucleic acid sequence encoding the target RNA, and iii) a 5 ' portion of an exogenous intron comprising a 5 ' splice site; b) performing transcription, whereby RNA is produced from the recombinant nucleic acid; and c) performing splicing of the RNA, whereby the RNA circularizes to produce a oRNA.
  • circular RNAs generated with exogenous introns are recognized by the immune system as "non-self” and trigger an innate immune response.
  • circular RNAs generated with endogenous introns are recognized by the immune system as "self” and generally do not provoke an innate immune response, even if carrying an exon comprising foreign RNA.
  • circular RNAs can be generated with either an endogenous or exogenous intron to control immunological self/non-self discrimination as desired.
  • Numerous intron sequences from a wide variety of organisms and viruses are known and include sequences derived from genes encoding proteins, ribosomal RNA (rRNA), or transfer RNA (tRNA).
  • Circular RNAs can be produced from linear RNAs in a number of ways.
  • circular RNAs are produced from a linear RNA by backsplicing of a downstream 5' splice site (splice donor) to an upstream 3' splice site (splice acceptor).
  • Circular RNAs can be generated in this manner by any nonmammalian splicing method.
  • linear RNAs containing various types of introns including self-splicing group I introns, self- splicing group II introns, spliceosomal introns, and tRNA introns can be circularized.
  • group I and group II introns have the advantage that they can be readily used for production of circular RNAs in vitro as well as in vivo because of their ability to undergo self- splicing due to their autocatalytic ribozyme activity.
  • circular RNAs can be produced in vitro from a linear RNA by chemical or enzymatic ligation of the 5' and 3' ends of the RNA.
  • RNG043-WO1 PCT Application chemical ligation can be performed, for example, using cyanogen bromide (BrCN) or ethyl-3- (3'-dimethylaminopropyl) carbodiimide (EDC) for activation of a nucleotide phosphomonoester group to allow phosphodiester bond formation, such as, for example, as described in Sokolova (1988) FEBS Lett 232: 153-155; Dolinnaya et al. (1991) Nucleic Acids Res., 19:3067-3072; Fedorova (1996) Nucleosides Nucleotides Nucleic Acids 15: 1137-1147; herein incorporated by reference.
  • BrCN cyanogen bromide
  • EDC ethyl-3- (3'-dimethylaminopropyl) carbodiimide
  • enzymatic ligation can be used to circularize RNA.
  • exemplary ligases that can be used include T4 DNA ligase (T4 Dnl), T4 RNA ligase 1 (T4 Rnl 1), and T4 RNA ligase 2 (T4 Rnl 2).
  • T4 Dnl T4 DNA ligase
  • T4 Rnl 1 T4 RNA ligase 1
  • T4 Rnl 2 T4 RNA ligase 2
  • splint ligation using an oligonucleotide splint that hybridizes with the two ends of a linear RNA can be used to bring the ends of the linear RNA together for ligation.
  • Hybridization of the splint which can be either a DNA or a RNA, orientates the 5 '-phosphate and 3' -OH of the RNA ends for ligation.
  • ligation can be performed using either chemical or enzymatic techniques, as described above.
  • Enzymatic ligation can be performed, for example, with T4 DNA ligase (DNA splint required), T4 RNA ligase 1 (RNA splint required) or T4 RNA ligase 2 (DNA or RNA splint).
  • Chemical ligation, such as with BrCN or EDC, in some cases is more efficient than enzymatic ligation if the structure of the hybridized splint-RNA complex interferes with enzymatic activity.
  • the oRNA may further comprise an internal ribosome entry site (IRES) operably linked to an RNA sequence encoding a polypeptide.
  • IRS internal ribosome entry site
  • an IRES permits the translation of one or more open reading frames from a circular RNA.
  • the IRES element attracts a eukaryotic ribosomal translation initiation complex and promotes translation initiation, for example, as described in Kaufman et al., Nuc. Acids Res. (1991) 19:4485-4490; Gurtu et al., Biochem. Biophys. Res. Comm. (1996) 229:295-298; Rees et al., BioTechniques (1996) 20: 102-110; Kobayashi et al., BioTechniques (1996) 21 :399- 402; and Mosser et al., BioTechniques 199722150-161).
  • the circularization efficiency of the circularization methods provided herein is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100%. In some embodiments, the circularization efficiency of the circularization methods provided herein is at least about 40%.
  • Splicing Element [00698] In some embodiments, the oRNA includes at least one splicing element.
  • the splicing element can be a complete splicing element that can mediate splicing of the oRNA or RNG043-WO1 PCT
  • the spicing element can be a residual splicing element from a completed splicing event.
  • a splicing element of a linear RNA can mediate a splicing event that results in circularization of the linear RNA, thereby the resultant oRNA comprises a residual splicing element from such splicing-mediated circularization event.
  • the residual splicing element is not able to mediate any splicing. In other cases, the residual splicing element can still mediate splicing under certain circumstances.
  • the splicing element is adjacent to at least one expression sequence.
  • the oRNA includes a splicing element adjacent each expression sequence.
  • the splicing element is on one or both sides of each expression sequence, leading to separation of the expression products, e.g., peptide(s) and or polypeptide(s).
  • the oRNA includes an internal splicing element that when replicated the spliced ends are joined together.
  • Some examples may include miniature introns ( ⁇ 100 nt) with splice site sequences and short inverted repeats (30-40 nt) such as AluSq2, AluJr, and AluSz, inverted sequences in flanking introns, Alu elements in flanking introns, and motifs found in (suptable4 enriched motifs) cis-sequence elements proximal to backsplice events such as sequences in the 200 bp preceding (upstream of) or following (downstream from) a backsplice site with flanking exons.
  • the oRNA includes at least one repetitive nucleotide sequence described elsewhere herein as an internal splicing element.
  • the repetitive nucleotide sequence may include repeated sequences from the Alu family of introns, such as those described in US Patent No. 11,058,706.
  • the oRNA may include canonical splice sites that flank head-to-tail junctions of the oRNA.
  • the oRNA may include a bulge-helix-bulge motif, comprising a 4-base pair stem flanked by two 3-nucleotide bulges. Cleavage occurs at a site in the bulge region, generating characteristic fragments with terminal 5'-hydroxyl group and 2', 3'-cyclic phosphate.
  • the oRNA may include a sequence that mediates self- ligation.
  • sequences that can mediate self-ligation include a self- circularizing intron, e.g., a 5' and 3' slice junction, or a self-circularizing catalytic intron such as a Group I, Group II or Group III Introns.
  • Non-limiting examples of group I intron self- splicing sequences may include self-splicing permuted intron-exon sequences derived from T4 bacteriophage gene td, and the intervening sequence (IVS) rRNA of Tetrahymena. RNG043-WO1 PCT Application Other Circularization Methods [00703]
  • linear RNA may include complementary sequences, including either repetitive or nonrepetitive nucleic acid sequences within individual introns or across flanking introns.
  • the oRNA includes a repetitive nucleic acid sequence.
  • the repetitive nucleotide sequence includes poly CA or poly UG sequences.
  • the oRNA includes at least one repetitive nucleic acid sequence that hybridizes to a complementary repetitive nucleic acid sequence in another segment of the oRNA, with the hybridized segment forming an internal double strand.
  • the complementary sequences are found at the 5' and 3' ends of the linear RNA.
  • the complementary sequences include about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more paired nucleotides.
  • chemical methods of circularization may be used to generate the oRNA. Such methods may include, but are not limited to click chemistry (e.g., alkyne- and azide-based methods, or clickable bases), olefin metathesis, phosphoramidate ligation, hemiaminal-imine crosslinking, base modification, and any combination thereof.
  • enzymatic methods of circularization may be used to generate the oRNA.
  • a ligation enzyme e.g., DNA or RNA ligase
  • DNA or RNA ligase may be used to generate a template of the oRNA or complement, a complementary strand of the oRNA, or the oRNA.
  • any of the circular polynucleotides as taught in for example U.S. Provisional Application No.61/873,010 filed Sep.3, 2013 or U.S. Patent No.10,709,779, may be used herein. The contents of these references are incorporated herein by reference in their entirety.
  • any of the circular RNAs, methods for making circular RNAs, circular RNA compositions that are described in the following publications are contemplated herein and are incorporated by reference in their entireties are part of the instant specification: US Patents US 11,352,640, US 11,352,641, US 11,203,767, US 10,683,498, US 5,773,244, and US 5,766,903; US Application Publications US 2022/0177540, US 2021/0371494, US 2022/0090137, US 2019/0345503, and US 2015/0299702; and PCT Application Publications WO 2021/226597, WO 2019/236673, WO 2017/222911, WO2016/187583, WO2014/082644 and WO 1997/007825.
  • kits comprising engineered retrons (e.g., engineered nucleic acid constructs, or engineered nucleic acid-enzyme constructs) as described herein.
  • the kit provides an engineered retron construct or a vector system comprising such a retron construct.
  • the engineered retron construct included in the kit, comprises a heterologous sequence capable of providing a cell with a nucleic acid encoding a protein or regulatory RNA of interest, a cellular barcode, a donor polynucleotide suitable for use in gene editing, e.g., by homology directed repair (HDR) or recombination-mediated genetic engineering (recombineering), or a CRISPR protospacer DNA sequence for use in molecular recording.
  • HDR homology directed repair
  • recombination-mediated genetic engineering recombineering
  • CRISPR protospacer DNA sequence for use in molecular recording.
  • Other agents may also be included in the kit such as transfection agents, host cells, suitable media for culturing cells, buffers, and the like.
  • agents can be provided in liquid or solid form in any convenient packaging (e.g., stick pack, dose pack, etc.).
  • the agents of a kit can be present in the same or separate containers.
  • the kits may contain any one or more of the components described herein in one or more containers.
  • the components may be prepared sterilely, packaged in a syringe and shipped refrigerated. Alternatively it may be housed in a vial or other container for storage. A second container may have other components prepared sterilely.
  • the kits may include the active agents premixed and shipped in a vial, tube, or other container.
  • kits may have a variety of forms, such as a blister pouch, a shrink wrapped pouch, a vacuum sealable pouch, a sealable thermoformed tray, or a similar pouch or tray form, with the accessories loosely packed within the pouch, one or more tubes, containers, a box or a bag.
  • the kits may be sterilized after the accessories are added, thereby allowing the individual accessories in the container to be otherwise unwrapped.
  • the kits can be sterilized using any appropriate sterilization techniques, such as radiation sterilization, heat sterilization, or other sterilization methods known in the art.
  • kits may also include other components, depending on the specific application, for example, containers, cell media, salts, buffers, reagents, syringes, needles, a fabric, such as gauze, for applying or removing a disinfecting agent, disposable gloves, a support for the agents prior to administration, etc.
  • kits comprising a nucleic acid construct comprising a nucleotide sequence encoding the various components of the retron-based editing system described herein.
  • the subject kits may further include (in some embodiments) instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit.
  • RNG043-WO1 PCT Application One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, and the like. Yet another form of these instructions is a computer readable medium, e.g., diskette, compact disk (CD), flash drive, and the like, on which the information has been recorded. Yet another form of these instructions that may be present is a website address which may be used via the internet to access the information at a remote site. In some embodiments, information provided on the website is periodically updated to provide, for example, the most up-to-date information.
  • a suitable medium or substrate e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, and the like.
  • a computer readable medium e.g., diskette, compact disk (CD), flash drive, and the like
  • a website address which may be used
  • the written instructions may be in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which can also reflect approval by the agency of manufacture, use or sale for animal administration.
  • “promoted” includes all methods of doing business including methods of education, hospital and other clinical instruction, scientific inquiry, drug discovery or development, academic research, pharmaceutical industry activity including pharmaceutical sales, and any advertising or other promotional activity including written, oral and electronic communication of any form, associated with the disclosure.
  • kits comprising one or more nucleic acid constructs (e.g., one or more mRNA or circular RNA molecules encoding the components of the retron-based genome editing system)
  • nucleic acid constructs can be based on RNA molecules, i.e., and “all-RNA system.”
  • each of the components of the editing system could be expressed from a mRNA molecule, which would be delivered to a target cell by one or more delivery methods (e.g., LNP delivery).
  • One aspect of the disclosure provides an isolated host cell that includes one or more of the compositions described herein, including, but not limited to, engineered retrons and/or retron components, engineered ncRNAs, engineered msDNA, engineered RT, nucleic acid molecules encoding the engineered retrons and/or retron components, and vector or vector systems encoding the engineered retrons and/or retron components, and any combinations thereof.
  • the host cell is a prokaryotic cell, an archaeal cell, or a eukaryotic host cell.
  • the eukaryotic host cell is a mammalian cell, such as a human cell, a non-human cell, or a non-human mammalian cell.
  • the host cell is an artificial cell or genetically modified cell.
  • the host cell is in vitro, such as a tissue culture cell.
  • the host cell is within a living host organism.
  • RNG043-WO1 PCT Application [00713] Cells that may contain any of the compositions described herein. The methods described herein are used to deliver recombinant retrons or components thereof into a eukaryotic cell (e.g., a mammalian cell, such as a human cell).
  • the cell is in vitro (e.g., cultured cell.
  • the cell is in vivo (e.g., in a subject such as a human subject).
  • the cell is ex vivo (e.g., isolated from a subject and may be administered back to the same or a different subject).
  • the present disclosure contemplates the use of any suitable host cell.
  • the cell host can be a mammalian cell. Mammalian cells of the present disclosure include human cells, primate cells (e.g., vero cells), rat cells (e.g., GH3 cells, OC23 cells) or mouse cells (e.g., MC3T3 cells).
  • human cell lines including, without limitation, human embryonic kidney (HEK) cells, HeLa cells, cancer cells from the National Cancer Institute's 60 cancer cell lines (NCI60), DU145 (prostate cancer) cells, Lncap (prostate cancer) cells, MCF-7 (breast cancer) cells, MDA-MB-438 (breast cancer) cells, PC3 (prostate cancer) cells, T47D (breast cancer) cells, THP-1 (acute myeloid leukemia) cells, U87 (glioblastoma) cells, SHSY5Y human neuroblastoma cells (cloned from a myeloma) and Saos- 2 (bone cancer) cells.
  • HEK human embryonic kidney
  • HeLa cells cancer cells from the National Cancer Institute's 60 cancer cell lines (NCI60)
  • DU145 (prostate cancer) cells Lncap (prostate cancer) cells
  • MCF-7 breast cancer
  • MDA-MB-438 breast cancer
  • PC3 prostate cancer
  • the cells can be human embryonic kidney (HEK) cells (e.g., HEK 293 or HEK 293T cells).
  • the cells can be stem cells (e.g., human stem cells) such as, for example, pluripotent stem cells (e.g., human pluripotent stem cells including human induced pluripotent stem cells (hiPSCs)).
  • stem cell refers to a cell with the ability to divide for indefinite periods in culture and to give rise to specialized cells.
  • a pluripotent stem cell refers to a type of stem cell that is capable of differentiating into all tissues of an organism, but not alone capable of sustaining full organismal development.
  • a human induced pluripotent stem cell refers to a somatic (e.g., mature or adult) cell that has been reprogrammed to an embryonic stem cell-like state by being forced to express genes and factors important for maintaining the defining properties of embryonic stem cells, for example, as described in Takahashi and Yamanaka, Cell 126 (4): 663–76, 2006, incorporated by reference herein).
  • Human induced pluripotent stem cells express stem cell markers and are capable of generating cells characteristic of all three germ layers (ectoderm, endoderm, mesoderm).
  • a host cell is transiently or non-transiently transfected with one or more delivery systems described herein, including virus-based systems, virus-like particle systems, and non-virus-base delivery, including LNPs and liposomes.
  • a cell is transfected as it naturally occurs in a subject.
  • a cell that is transfected is taken from a subject, i.e., ex vivo transfection.
  • the cell is derived from cells taken from a subject, such as a cell line.
  • a wide variety of cell lines for tissue culture are known in the art.
  • cell lines include, but are not limited to, C8161, CCRF-CEM, MOLT, mIMCD- 3, NHDF, HeLa-S3, Huh1, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, TF1, CTLL-2, C1R, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH- 77, Calu1, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelial, BA
  • Cell lines are available from a variety of sources known to those with skill in the art, for example, as described in the American Type Culture Collection (ATCC) (Manassus, Va.).
  • ATCC American Type Culture Collection
  • a cell transfected with one or more retron delivery systems described herein is used to establish a new cell line comprising one or more nucleic acid molecules encoding the recombinant retron-based gene editing systems described herein, or encoding at last a component of said systems (e.g., a recombinant ncRNA or a recombinant retron RT).
  • ATCC American Type Culture Collection
  • a cell transfected with one or more retron delivery systems described herein is used to establish a new cell line comprising one or more nucleic acid molecules encoding the recombinant retron-based gene editing systems described herein, or encoding at last a component of said systems (e.g., a recombinant ncRNA or a recombin
  • compositions RNG043-WO1 PCT Application [00717]
  • the engineered genome editing systems described herein, or one or more components thereof may be provided as pharmaceutical compositions.
  • one or more LNPs or other non-virus-based delivery system comprising one or more circular or linear RNA molecules encoding each of the components of the retron-based genome editing system may be formulated as a pharmaceutical composition for administering to a subject in need (e.g., a human in need of gene editing).
  • Formulations can include, without limitation, saline, liposomes, lipid nanoparticles, polymers, peptides, proteins, cells transfected with viral vectors (e.g., for transfer or transplantation into a subject) and combinations thereof.
  • Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology.
  • pharmaceutical composition refers to compositions comprising at least one active ingredient and optionally one or more pharmaceutically acceptable excipients.
  • such preparatory methods include the step of associating the active ingredient with an excipient and/or one or more other accessory ingredients.
  • the phrase “active ingredient” generally refers an engineered retron as described herein.
  • a pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
  • a “unit dose” refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • compositions comprising any of the various components of the recombinant retron-based genome editing systems described herein, including, but not limited to, engineered retrons and/or retron components, engineered ncRNAs, engineered msDNA, engineered RT, nucleic acid molecules encoding the engineered retrons and/or retron components, programmable nucleases (e.g., RNA-guided nucleases), guide RNAs, and vector or vector systems encoding the engineered retrons and/or retron components, and any combinations thereof.
  • programmable nucleases e.g., RNA-guided nucleases
  • guide RNAs e.g., RNA-guided nucleases
  • vector or vector systems encoding the engineered retrons and/or retron components, and any combinations thereof.
  • pharmaceutical composition refers to a composition formulated for pharmaceutical use.
  • the pharmaceutical composition further comprises a pharmaceutically RNG043-WO1 PCT Application acceptable carrier.
  • the pharmaceutical composition comprises additional agents (e.g. for specific delivery, increasing half-life, or other therapeutic compounds).
  • pharmaceutically-acceptable carrier means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the compound from one site (e.g., the delivery site) of the body, to another site (e.g., organ, tissue or portion of the body).
  • manufacturing aid e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid
  • solvent encapsulating material involved in carrying or transporting the compound from one site (e.g., the delivery site) of the body, to another site (e.g., organ, tissue or portion
  • a pharmaceutically acceptable carrier is “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the tissue of the subject (e.g., physiologically compatible, sterile, physiologic pH, etc.).
  • materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil;
  • the pharmaceutical composition is formulated for delivery to a subject, e.g., for gene editing.
  • Suitable routes of administrating the pharmaceutical composition described herein include, without limitation: topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, RNG043-WO1 PCT Application intravesical, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseus, periocular, intratumoral, intracerebral, and intracerebroventricular administration.
  • the pharmaceutical composition described herein is administered locally to a diseased site (e.g., tumor site).
  • the pharmaceutical composition described herein is administered to a subject by injection, by means of a catheter, by means of a suppository, or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including a membrane, such as a sialastic membrane, or a fiber.
  • the pharmaceutical composition described herein is delivered in a controlled release system.
  • a pump may be used, such as the one described in Langer, 1990, Science 249:1527-1533; Sefton, 1989, CRC Crit. Ref. Biomed.
  • polymeric materials can be used, such as the ones described in Medical Applications of Controlled Release (Langer and Wise eds., CRC Press, Boca Raton, Fla., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., Wiley, New York, 1984); Ranger and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem.23:61; Levy et al., 1985, Science 228:190; During et al., 1989, Ann.
  • the pharmaceutical composition is formulated in accordance with routine procedures as a composition adapted for intravenous or subcutaneous administration to a subject, e.g., a human.
  • pharmaceutical composition for administration by injection are solutions in sterile isotonic aqueous buffer.
  • the pharmaceutical can also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • the pharmaceutical is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.
  • a pharmaceutical composition for systemic administration may be a liquid, e.g., sterile saline, lactated Ringer’s or Hank’s solution.
  • the pharmaceutical composition can be in solid forms and re-dissolved or suspended immediately prior to use. Lyophilized forms are also contemplated.
  • the pharmaceutical composition can be contained within a lipid particle or vesicle, such as a liposome or microcrystal or LNP, which is also suitable for parenteral administration.
  • the particles can be of any suitable structure, such as unilamellar or plurilamellar, so long as compositions are contained therein.
  • compounds can be entrapped in “stabilized plasmid- lipid particles” (SPLP) containing the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), low levels (5-10 mol%) of cationic lipid, and stabilized by a polyethyleneglycol (PEG) coating, such as described in Zhang Y. P. et al., Gene Ther.1999, 6:1438-47.
  • SPLP stabilized plasmid- lipid particles
  • DOPE fusogenic lipid dioleoylphosphatidylethanolamine
  • PEG polyethyleneglycol
  • Positively charged lipids such as N-[1-(2,3-dioleoyloxi)propyl]-N,N,N- trimethyl-amoniummethylsulfate, or “DOTAP,” are particularly preferred for such particles and vesicles.
  • methods of preparing such lipid particles can include the methods disclosed in U.S. Patent Nos.4,880,635; 4,906,477; 4,911,928; 4,917,951; 4,920,016; and 4,921,757; each of which is incorporated herein by reference.
  • the pharmaceutical composition can be provided as a pharmaceutical kit comprising (a) a container containing a recombinant retron-based genome editing system or one or more components thereof in lyophilized form and (b) a second container containing a pharmaceutically acceptable diluent (e.g., sterile water) for injection.
  • a pharmaceutically acceptable diluent e.g., sterile water
  • the pharmaceutically acceptable diluent can be used for reconstitution or dilution of the lyophilized system of the present disclosure.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • an article of manufacture containing materials useful for the treatment of the diseases described above is included.
  • the article of manufacture comprises a container and a label.
  • Suitable containers include, for example, bottles, vials, syringes, and test tubes.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition that is effective for treating a disease described herein and may have a sterile access port.
  • the container may be an intravenous solution bag or a vial having a stopper pierce- able by a hypodermic injection needle.
  • the active agent in the composition is a compound of the present disclosure.
  • the label on or associated with the container RNG043-WO1 PCT Application indicates that the composition is used for treating the disease of choice.
  • the article of manufacture may further comprise a second container comprising a pharmaceutically- acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution.
  • the engineered retron can be used in any suitable organism.
  • the organism is a eukaryote.
  • the organism is an animal.
  • the animal is a fish, an amphibian, a reptile, a mammal, or a bird.
  • the animal is a farm animal or agriculture animal.
  • Non-limiting examples of farm and agriculture animals include horses, goats, sheep, swine, cattle, llamas, alpacas, and birds, e.g., chickens, turkeys, ducks, and geese.
  • the animal is a non-human primate, e.g., baboons, capuchin monkeys, chimpanzees, lemurs, macaques, marmosets, tamarins, spider monkeys, squirrel monkeys, and vervet monkeys.
  • the animal is a pet.
  • Non-limiting examples of pets include dogs, cats horses, wolfs, rabbits, ferrets, gerbils, hamsters, chinchillas, fancy rats, guinea pigs, canaries, parakeets, and parrots.
  • the organism is a plant. Plants that may be transfected with an engineered retron include monocots and dicots.
  • Particular examples include, but are not limited to, corn (maize), sorghum, wheat, sunflower, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape, Brassica sp., alfalfa, rye, millet, safflower, peanuts, sweet potato, cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, oats, vegetables, ornamentals, and conifers.
  • Vegetables include, but are not limited to, crucifers, peppers, tomatoes, lettuce, green beans, lima beans, peas, and members of the genus Cucumis such as cucumber, cantaloupe, and musk melon.
  • Ornamentals include, but are not limited to, azalea, hydrangea, hibiscus, roses, tulips, daffodils, petunias, carnation, poinsettia, and chrysanthemum.
  • heterologous nucleic acid sequences can be added to the subject engineered retron to provide a cell with a heterologous nucleic acid encoding a protein or regulatory RNA of interest, a cellular barcode, a donor polynucleotide suitable for use in gene editing, e.g., by homology directed repair (HDR) or recombination-mediated genetic engineering (recombineering), or a CRISPR protospacer DNA sequence for use in molecular RNG043-WO1 PCT Application recording, as discussed further below.
  • HDR homology directed repair
  • recombination-mediated genetic engineering recombineering
  • CRISPR protospacer DNA sequence for use in molecular RNG043-WO1 PCT Application recording, as discussed further below.
  • the engineered retrons described herein may be used for research tools, such as kits, functional genomics assays, and generating engineered cell lines and animal models for research and drug screening.
  • the kit may comprise one or more reagents in addition to the engineered retron, such as a buffer, a control reagent, a control vector, a control RNA polynucleotide, a reagent for in vitro production of the polypeptide from DNA, and adaptors for sequencing.
  • a buffer can be, for example, a stabilization buffer, a reconstituting buffer, a diluting buffer, a wash buffer, or a buffer for introducing a polypeptide and/or polynucleotide of the kit into a cell.
  • a kit can comprise one or more additional reagents specific for plants.
  • One or more additional reagents for plants can include, for example, soil, nutrients, plants, seeds, spores, Agrobacterium, a T-DNA vector, and a pBINAR vector.
  • a retron is engineered with a heterologous nucleic acid sequence encoding a donor polynucleotide suitable for use with nuclease genome editing system.
  • the nuclease is designed to specifically target a location proximal to the desired edit (the nuclease should be designed such that it will not cut the target once the edit is properly installed).
  • the nuclease e.g., CAS or non-CAS
  • a heterologous nucleic acid sequence is inserted into the retron msd.
  • the heterologous nucleic acid sequence has 10-100 or more bp of homologous nucleic acid sequence to the genome on both sides of the desired edit.
  • the desired edit (insertion, deletion, or mutation) is in between the homologous sequence.
  • donor polynucleotides comprise a sequence comprising an intended genome edit flanked by a pair of homology arms responsible for targeting the donor polynucleotide to the target locus to be edited in a cell.
  • the donor polynucleotide typically comprises a 5 ⁇ homology arm that hybridizes to a 5 ⁇ genomic target sequence and a 3 ⁇ homology arm that hybridizes to a 3 ⁇ genomic target sequence.
  • the homology arms are referred to herein as 5 ⁇ and 3 ⁇ (i.e., upstream and downstream) homology arms, which relate to the relative position of the homology arms to the nucleotide sequence comprising the intended RNG043-WO1 PCT Application edit within the donor polynucleotide.
  • the 5 ⁇ and 3 ⁇ homology arms hybridize to regions within the target locus in the genomic DNA to be modified, which are referred to herein as the “5 ⁇ target sequence” and “3 ⁇ target sequence,” respectively.
  • the homology arm must be sufficiently complementary for hybridization to the target sequence to mediate homologous recombination between the donor polynucleotide and genomic DNA at the target locus.
  • a homology arm may comprise a nucleotide sequence having at least about 80-100% sequence identity to the corresponding genomic target sequence, including any percent identity within this range, such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto, wherein the nucleotide sequence comprising the intended edit can be integrated into the genomic DNA by HDR at the genomic target locus recognized (i.e., having sufficient complementary for hybridization) by the 5 ⁇ and 3 ⁇ homology arms.
  • the corresponding homologous nucleotide sequences in the genomic target sequence flank a specific site for cleavage and/or a specific site for introducing the intended edit.
  • the distance between the specific cleavage site and the homologous nucleotide sequences can be several hundred nucleotides. In some embodiments, the distance between a homology arm and the cleavage site is 200 nucleotides or less (e.g., 0, 10, 20, 30, 50, 75, 100, 125, 150, 175, and 200 nucleotides).
  • the donor polynucleotide is substantially identical to the target genomic sequence, across its entire length except for the sequence changes to be introduced to a portion of the genome that encompasses both the specific cleavage site and the portions of the genomic target sequence to be altered.
  • a homology arm can be of any length, e.g.10 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 300 nucleotides or more, 350 nucleotides or more, 400 nucleotides or more, 450 nucleotides or more, 500 nucleotides or more, 1000 nucleotides (1 kb) or more, 5000 nucleotides (5 kb) or more, 10000 nucleotides (10 kb) or more, etc.
  • the 5 ⁇ and 3 ⁇ homology arms are substantially equal in length to one another.
  • the 5 ⁇ and 3 ⁇ homology arms are not necessarily equal in length to one another.
  • one homology arm may be 30% shorter or less than the other homology arm, 20% shorter or less than the other homology arm, 10% shorter or less than the other homology arm, 5% shorter or less than the other homology arm, 2% shorter or less than the other homology RNG043-WO1 PCT Application arm, or only a few nucleotides less than the other homology arm.
  • the 5 ⁇ and 3 ⁇ homology arms are substantially different in length from one another, e.g.
  • the donor polynucleotide may be used in combination with an RNA-guided nuclease, which is targeted to a particular genomic sequence (i.e., genomic target sequence to be modified) by a guide RNA.
  • a target-specific guide RNA comprises a nucleotide sequence that is complementary to a genomic target sequence, and thereby mediates binding of the nuclease-gRNA complex by hybridization at the target site.
  • the gRNA can be designed with a sequence complementary to the sequence of a minor allele to target the nuclease-gRNA complex to the site of a mutation.
  • the mutation may comprise an insertion, a deletion, or a substitution.
  • the mutation may include a single nucleotide variation, gene fusion, translocation, inversion, duplication, frameshift, missense, nonsense, or other mutation associated with a phenotype or disease of interest.
  • the targeted minor allele may be a common genetic variant or a rare genetic variant.
  • the gRNA is designed to selectively bind to a minor allele with single base-pair discrimination, for example, to allow binding of the nuclease-gRNA complex to a single nucleotide polymorphism (SNP).
  • the gRNA may be designed to target disease-relevant mutations of interest for the purpose of genome editing to remove the mutation from a gene.
  • the gRNA can be designed with a sequence complementary to the sequence of a major or wild-type allele to target the nuclease-gRNA complex to the allele for the purpose of genome editing to introduces a mutation into a gene in the genomic DNA of the cell, such as an insertion, deletion, or substitution.
  • RNA-guided nuclease used for genome modification is a clustered regularly interspersed short palindromic repeats (CRISPR) system Cas nuclease.
  • CRISPR clustered regularly interspersed short palindromic repeats
  • RNA-guided Cas nuclease capable of catalyzing site- directed cleavage of DNA to allow integration of donor polynucleotides by the HDR mechanism can be used in genome editing, including CRISPR system Class 1, Type I, II, or III Cas nucleases; Class 2, Type II nuclease (such as Cas9); a Class 2, Type V nuclease (such as Cpfl), or a Class 2, Type VI nuclease (such as C2c2).
  • Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9 (Csnl or Csxl2), CaslO, CaslOd, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (CasA), Cse2 (CasB), Cse3 RNG043-WO1 PCT Application (CasE), Cse4 (CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csx
  • a Class 1, type II CRISPR system Cas9 endonuclease is used.
  • Cas9 nucleases from any species, or biologically active fragments, variants, analogs, or derivatives thereof that retain Cas9 endonuclease activity i.e., catalyze site-directed cleavage of DNA to generate double-strand breaks
  • the Cas9 need not be physically derived from an organism but may be synthetically or recombinantly produced.
  • Cas9 sequences from a number of bacterial species are well known in the art and listed in the National Center for Biotechnology Information (NCBI) database.
  • the genomic target site will typically comprise a nucleotide sequence that is complementary to the gRNA and may further comprise a protospacer adjacent motif (PAM).
  • PAM protospacer adjacent motif
  • the target site comprises 20-30 base pairs in addition to a 3 or more base pair PAM.
  • the first nucleotide of a PAM can be any nucleotide, while the two or more other nucleotides will depend on the specific Cas9 protein that is chosen.
  • Exemplary PAM sequences are known to those of skill in the art and include, without limitation, NNG, NGN, NAG, and NGG, wherein N represents any nucleotide.
  • the allele targeted by a gRNA comprises a mutation that creates a PAM within the allele, wherein the PAM promotes binding of the Cas9-gRNA complex to the allele.
  • the gRNA is 5-50 nucleotides, 10-30 nucleotides, 15- 25 nucleotides, 18-22 nucleotides, or 19-21 nucleotides in length, or any length between the stated ranges, including, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in length.
  • the guide RNA may be a single guide RNA comprising crRNA and tracrRNA sequences in a single RNA molecule, or the guide RNA may comprise two RNA molecules with crRNA and tracrRNA sequences residing in separate RNA molecules.
  • C2c1 (Cas12b) is another class II CRISPR/Cas system RNA-guided nuclease that may be used.
  • C2cl similarly to Cas9, depends on both a crRNA and tracrRNA for guidance to target sites.
  • RNA-guided Fokl nucleases can comprise fusions of inactive Cas9 (dCas9) and the Fokl endonuclease (FokI-dCas9), wherein the dCas9 portion confers guide RNA-dependent targeting on Fokl, such as, for example, the ones described in Havlicek et al. (2017) Mol. Ther.25(2):342-355, Pan et al. (2016) Sci Rep.6:35794, Tsai et al. (2014) Nat Biotechnol.32(6):569-576; herein incorporated by reference.
  • dCas9 inactive Cas9
  • the RNA-guided nuclease is provided in the form of a protein, optionally where the nuclease is complexed with a gRNA to form a ribonucleoprotein (RNP) complex.
  • the RNA-guided nuclease is provided by a nucleic acid encoding the RNA-guided nuclease, such as an RNA (e.g., messenger RNA) or DNA (expression vector).
  • the RNA-guided nuclease and the gRNA are both provided by vectors, such as the vectors and the vector system described in other parts of the application (all incorporated herein by reference). Both can be expressed by a single vector or separately on different vectors.
  • the vectors encoding the RNA-guided nuclease and gRNA may be included in the vector system comprising the engineered retron msr gene, msd gene and ret gene sequences.
  • the RNA-guided nuclease is fused to the RT and/or the msDNA.
  • the RNP complex may be administered to a subject or delivered into a cell by methods known in the art, such as those described in U.S. Pat. No. 11,390,884, which is incorporated by reference herein in its entirety.
  • the endonuclease/gRNA ribonucleoprotein (RNP) complexes are delivered to cells by electroporation. Direct delivery of the RNP complex to a subject or cell eliminates the need for expression from nucleic acids (e.g., transfection of plasmids encoding Cas9 and gRNA).
  • Codon usage may be optimized to further improve production of an RNA- guided nuclease and/or reverse transcriptase (RT) in a particular cell or organism.
  • a nucleic acid encoding an RNA-guided nuclease or reverse transcriptase can be modified to substitute codons having a higher frequency of usage in a yeast cell, a bacterial cell, a human cell, a non-human cell, a mammalian cell, a rodent cell, a mouse cell, a rat cell, or any other host cell of interest, as compared to the naturally occurring polynucleotide sequence.
  • the protein can be transiently, conditionally, or constitutively expressed in the cell.
  • the engineered retron used for genome editing with nuclease genome editing systems can further include accessory or enhancer proteins for recombination.
  • recombination enhancers can include nonhomologous end joining (NHEJ) inhibitors (e.g., inhibitor of DNA ligase IV, a KU inhibitor (e.g., KU70 or KU80), a DNA-PKc inhibitor, or an artemis inhibitor) and homologous directed repair (HDR) promoters, or both, that can enhance or improve more precise genome editing and/or the efficiency of RNG043-WO1 PCT Application homologous recombination.
  • NHEJ nonhomologous end joining
  • HDR homologous directed repair
  • the recombination accessory or enhancers can comprise C-terminal binding protein interacting protein (CtIP), cyclinB2, Rad family members (e.g. Rad50, Rad51, Rad52, etc).
  • CtIP C-terminal binding protein interacting protein
  • cyclinB2 Rad family members
  • Rad50, Rad51, Rad52, etc C-terminal binding protein interacting protein
  • HDR may be enhanced by using Cas9 nuclease associated (e.g.
  • an N-terminal fragment of CtIP called HE for HDR enhancer, may be sufficient for HDR stimulation and requires the CtIP multimerization domain and CDK phosphorylation sites to be active.
  • HDR stimulation by the Cas9-HE fusion depends on the guide RNA used, and therefore the guide RNA will be designed accordingly.
  • any target gene or sequence in a host cell can be edited or modified for a desired trait, including but not limited to: Myostatin (e.g., GDF8) to increase muscle growth; Pc POLLED to induce hairlessness; KISS1R to induce bore taint; Dead end protein (dnd) to induce sterility; Nano2 and DDX to induce sterility; CD163 to induce PRRSV resistance; RELA to induce ASFV resilience; CD18 to induce Mannheimia (Pasteurella) haemolytica resilience; NRAMPl to induce tuberculosis resilience; Negative regulators of muscle mass (e.g., Myostatin) to increase muscle mass.
  • Myostatin e.g., GDF8
  • Pc POLLED to induce hairlessness
  • KISS1R to induce bore taint
  • Dead end protein (dnd) to induce sterility
  • Nano2 and DDX to induce sterility
  • CD163 to induce PRRSV resistance
  • RELA
  • target genome or epigenetic modifications include cells with monogenic diseases or disorders.
  • Various monogenic diseases include but are not limited to: Adenosine Deaminase (ADA) Deficiency; Alpha-1 Antitrypsin Deficiency; Cystic Fibrosis; Duchenne Muscular Dystrophy; Galactosemia; Hemochromatosis; Huntington’s Disease; Maple Syrup Urine Disease; Marfan Syndrome; Neurofibromatosis Type 1; Pachyonychia Congenita; Phenylkeotnuria; Severe Combined Immunodeficiency; Sickle Cell Disease; Smith-Lemli-Opitz Syndrome; Tay-Sachs Disease; hereditary tyrosinemia I; Influenza; SARS- CoV-2; Alzheimer’s disease; Parkinson’s disease.
  • ADA Adenosine Deaminase
  • Alpha-1 Antitrypsin Deficiency Cystic Fibrosis
  • Duchenne Muscular Dystrophy Galactosemia
  • Hemochromatosis Huntington
  • Target sequences related to certain diseases and disorders are known in some cases.
  • Target sequences or target editing sites include disease-associated or causative RNG043-WO1 PCT Application mutations for one or more of 10,000 monogenic disorders.
  • a list of target sequences can be generated based on the monogenic disorders.
  • ADA Adenosine Deaminase
  • Alpha-1 Antitrypsin Deficiency Cystic Fibrosis
  • Duchenne Muscular Dystrophy Galactosemia; Hemochromatosis; Huntington’s Disease; Maple Syrup Urine Disease; Marfan Syndrome; Neurofibromatosis Type 1; Pachyonychia Congenita; Phenylkeotnuria; Severe Combined Immunodeficiency; Sickle Cell Disease; Smith-Lemli-Opitz Syndrome; and Tay-Sachs Disease.
  • the disease- associated gene can be associated with a polygenic disorder selected from the group consisting of: heart disease; high blood pressure; Alzheimer’s disease; arthritis; diabetes; cancer; and obesity.
  • the gene editing systems disclosed herein may also be used to treat the following genetic disorders by editing a defect in the disease-associated gene, as follows: Genetic disease Disease gene Arenoleukodystrophy (ALD) ABCD1 Agammaglobulinemia non-Bruton type IGHM Alport syndrome COL4A5 Amyloid neuropathy – Andrade disease TTR Angioneurotic oedema C1NH Alpha1-antitrypsin deficiency SERPINEA 1 Bartter syndrome type 4 BSND Blepharophimosis - ptosis - epicanthus inversus FOXL2 syndrome (BEPS) Brugada Sindrome - Long QT syndrome-3 SCN5A Bruton agammaglobulinemia tyrosine kinase BTK Ceroid lipof
  • the guide RNA directs a programmable nuclease of a retron-based editing system to the target site or the targeted polynucleotide sequence; and optionally forms a ribonucleoprotein complex with the polypeptide and the guide RNA.
  • Additional therapeutic applications for the genome editing systems disclosed herein include base editing, prime editing, gene insertions and/or deletions.
  • Diagnostic applications for the genome editing system include probes, diagnostics, theranostics.
  • the editing system comprising the heterologous nucleic acid sequence can be used in a variety of applications, several non-limiting examples of which are described herein. In general, the editing system can be used in any suitable organism.
  • the organism is a eukaryote.
  • the organism is an animal.
  • the animal is a fish, an amphibian, a reptile, a mammal, or a bird.
  • the animal is a farm animal or agriculture animal.
  • farm and agriculture animals include horses, goats, sheep, swine, cattle, llamas, alpacas, and birds, e.g., chickens, turkeys, ducks, and geese.
  • the animal is a non-human primate, e.g., RNG043-WO1 PCT Application baboons, capuchin monkeys, chimpanzees, lemurs, macaques, marmosets, tamarins, spider monkeys, squirrel monkeys, and vervet monkeys.
  • the animal is a pet.
  • Non-limiting examples of pets include dogs, cats, horses, rabbits, ferrets, gerbils, hamsters, chinchillas, fancy rats, guinea pigs, canaries, parakeets, and parrots.
  • the organism is a plant.
  • Plants that may be transfected with an Cas12a editing system include monocots and dicots. Particular examples include, but are not limited to, corn (maize), sorghum, wheat, sunflower, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape, Brassica sp., alfalfa, rye, millet, safflower, peanuts, sweet potato, cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, oats, vegetables, ornamentals, and conifers.
  • Vegetables include, but are not limited to, crucifers, peppers, tomatoes, lettuce, green beans, lima beans, peas, and members of the genus Cucumis such as cucumber, cantaloupe, and musk melon.
  • Ornamentals include, but are not limited to, azalea, hydrangea, hibiscus, roses, tulips, daffodils, petunias, carnation, poinsettia, and chrysanthemum.
  • heterologous nucleic acid sequences can be added to the subject editing system to provide a cell with a heterologous nucleic acid encoding a protein or regulatory RNA of interest, a cellular barcode, a donor polynucleotide suitable for use in gene editing, e.g., by homology directed repair (HDR) or recombination-mediated genetic engineering (recombineering), or a CRISPR protospacer DNA sequence for use in molecular recording, as discussed further below.
  • HDR homology directed repair
  • recombination-mediated genetic engineering recombineering
  • CRISPR protospacer DNA sequence for use in molecular recording, as discussed further below.
  • heterologous sequences may be inserted, for example, into the msr locus or the msd locus such that the heterologous sequence is transcribed by the retron reverse transcriptase as part of the msDNA product.
  • the editing systems described herein may be used for research tools, such as kits, functional genomics assays, and generating engineered cell lines and animal models for research and drug screening.
  • the kit may comprise one or more reagents in addition to the editing system, such as a buffer, a control reagent, a control vector, a control RNA polynucleotide, a reagent for in vitro production of the polypeptide from DNA, and adaptors for sequencing.
  • a buffer can be, for example, a stabilization buffer, a reconstituting buffer, a diluting buffer, a wash buffer, or a buffer for introducing a polypeptide and/or polynucleotide of the kit into a cell.
  • a kit can comprise one or more additional reagents specific for plants.
  • One or more additional reagents for plants can include, RNG043-WO1 PCT Application for example, soil, nutrients, plants, seeds, spores, Agrobacterium, a T-DNA vector, and a pBINAR vector.
  • Therapeutic Applications [00772] Also provided herein are methods of diagnosing, prognosing, treating, and/or preventing a disease, state, or condition in or of a subject, using the engineered gene editing systems of the present disclosure.
  • the methods of diagnosing, prognosing, treating, and/or preventing a disease, state, or condition in or of a subject can include modifying a polynucleotide in a subject or cell thereof using a composition, system, or component thereof of the engineered retron as described herein, and/or include detecting a diseased or healthy polynucleotide in a subject or cell thereof using a composition, system, or component thereof of the engineered retron as described herein.
  • the method of treatment or prevention can include using a composition, system, or component of the engineered retron to modify a polynucleotide of an infectious organism (e.g.
  • the method of treatment or prevention can include using a composition, system, or component of the engineered retron to modify a polynucleotide of an infectious organism or symbiotic organism within a subject.
  • the composition, system, and components of the engineered retron can be used to develop models of diseases, states, or conditions.
  • the composition, system, and components of the engineered retron can be used to detect a disease state or correction thereof, such as by a method of treatment or prevention described herein.
  • the composition, system, and components of the engineered retron can be used to screen and select cells that can be used, for example, as treatments or preventions described herein.
  • the composition, system, and components thereof can be used to develop biologically active agents that can be used to modify one or more biologic functions or activities in a subject or a cell thereof.
  • the method can include delivering a composition, system, and/or component of the engineered retron to a subject or cell thereof, or to an infectious or symbiotic organism by a suitable delivery technique and/or composition. Once administered, the components can operate as described elsewhere herein to elicit a nucleic acid modification event.
  • the nucleic acid modification event can occur at the genomic, RNG043-WO1 PCT Application epigenomic, and/or transcriptomic level. DNA and/or RNA cleavage, gene activation, and/or gene deactivation can occur.
  • composition, system, and components of the engineered retron as described elsewhere herein can be used to treat and/or prevent a disease, such as a genetic and/or epigenetic disease, in a subject; to treat and/or prevent genetic infectious diseases in a subject, such as bacterial infections, viral infections, fungal infections, parasite infections, and combinations thereof; to modify the composition or profile of a microbiome in a subject, which can in turn modify the health status of the subject; to modify cells ex vivo, which can then be administered to the subject whereby the modified cells can treat or prevent a disease or symptom thereof; or to treat mitochondrial diseases, where the mitochondrial disease etiology involves a mutation in the mitochondrial DNA.
  • a disease such as a genetic and/or epigenetic disease
  • genetic infectious diseases in a subject such as bacterial infections, viral infections, fungal infections, parasite infections, and combinations thereof
  • modify the composition or profile of a microbiome in a subject which can in turn modify the health status of the subject
  • a method of treating a subject comprising inducing gene editing by transforming the subject with the Cas effector(s), and encoding and expressing in vivo the remaining portions of the composition, system, (e.g., RNA, guides), complex or component of the engineered retron.
  • a suitable repair template may also be provided by the engineered retron as described herein elsewhere.
  • a method of treating a subject e.g., a subject in need thereof, comprising inducing transcriptional activation or repression by transforming the subject with the systems or compositions herein.
  • a method of inducing one or more polynucleotide modifications in a eukaryotic or prokaryotic cell or component thereof (e.g. a mitochondria) of a subject, infectious organism, and/or organism of the microbiome of the subject can include the introduction, deletion, or substitution of one or more nucleotides RNG043-WO1 PCT Application at a target sequence of a polynucleotide of one or more cell(s).
  • the modification can occur in vitro, ex vivo, in situ, or in vivo.
  • the method of treating or inhibiting a condition or a disease caused by one or more mutations in a genomic locus in a eukaryotic organism or a non- human organism can include manipulation of a target sequence within a coding, non-coding or regulatory element of said genomic locus in a target sequence in a subject or a non-human subject in need thereof comprising modifying the subject or a non -human subject by manipulation of the target sequence and wherein the condition or disease is susceptible to treatment or inhibition by manipulation of the target sequence including providing treatment comprising delivering a composition comprising the particle delivery system or the delivery system or the virus particle of any one of the above embodiment or the cell of any one of the above embodiment.
  • particle delivery system or the delivery system or the virus vector (in viral particle) of any one of the above embodiment or the cell of any one of the above embodiment in ex vivo or in vivo gene or genome editing; or for use in in vitro, ex vivo or in vivo gene therapy.
  • target polynucleotide modification using the subject engineered retron and the associated composition, vectors, system and methods comprises addition, deletion, or substitution of 1-about 10k nucleotides at each target sequence of said polynucleotide of said cell(s).
  • the modification can include the addition, deletion, or substitution of at least 1, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, 100, 200, 250, 300, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 5000, 6000, 7000, 8000, 9000, 10,000 or more nucleotides at each target sequence.
  • formation of system or complex results in cleavage, nicking, and/or another modification of one or both strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence.
  • a method of modifying a target polynucleotide in a cell to treat or prevent a disease can include allowing a composition, system, or component of the subject engineered retron to bind to the target polynucleotide, e.g., to effect cleavage, nicking, or other modification as the composition, system, is capable of said target polynucleotide, thereby modifying the target polynucleotide, wherein the composition, system, or component thereof, complex with a guide sequence, and hybridize said guide sequence to a target sequence within the target polynucleotide, wherein said guide sequence is optionally linked to a tracr mate sequence, which in turn can hybridize to a tracr sequence.
  • modification can include cleaving or nicking one or two strands at the location of the target sequence by one or more components of the composition, system, or component thereof.
  • the engineered retron and the associated compositions, systems, vectors, uses, and methods of use can be used to treat diseases of the circulatory system.
  • the treatment can be carried out by using an AAV or a lentiviral vector to deliver the engineered retron, composition, system, and/or vector described herein to modify hematopoietic stem cells (HSCs) or iPSCs in vivo or ex vivo.
  • HSCs hematopoietic stem cells
  • the treatment can be carried out by correcting HSCs or iPSCs as to the disease using a composition, system, herein or a component thereof, wherein the composition, system, optionally includes a suitable HDR repair template (e.g., a template in the msDNA of the engineered retron).
  • the treatment or prevention for treating a circulatory system or blood disease can include modifying a human cord blood cell.
  • the treatment or prevention for treating a circulatory system or blood disease can include modifying a granulocyte colony-stimulating factor-mobilized peripheral blood cell (mPB) with any modification described herein.
  • the human cord blood cell or mPB can be CD34 + .

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Abstract

Disclosed herein relates to a chimeric gene editing system comprising a modified retron editing system for precise and efficient gene editing. Additionally, a chimeric gene editing system that comprises one or more components of a prime editing system (or a prime editor-like system) with one or more components of a retron editing system, is disclosed, providing an entirely new gene editing system with distinct properties, capabilities and advantages.

Description

RNG043-WO1 PCT Application NICKASE-RETRON TEMPLATE-BASED PRECISION EDITING SYSTEM AND METHODS OF USE SEQUENCE LISTING [0001] This application contains a sequencing listing filed in electronic form in eXentsible Markup Language (XML) format entitled 60676WO_CRF_sequencelisting, created on September 30, 2024, and having a size of 36,013,000 bytes. The content of the sequence listing is incorporate herein in its entirety. This application incorporates by reference the Sequence Listings filed in U.S. Application No.18/087,673, International Application No. PCT/US2023/061038, filed January 20, 2023, and International Application No. PCT/US2023/072872, filed August 24, 2023, including without limitation each of the disclosed retron reverse transcriptases amino acid and nucleotide sequence described therein and each of the disclosed retron ncRNA nucleotide sequences. The contents of said Sequence Listings are incorporated herein in their entireties. TECHNICAL FIELD [0002] The present disclosure generally relates to systems, methods and pharmaceutical compositions used for precise genome editing, including nucleic acid edits, insertions, replacements, and deletions at targeted and precise genome sites, wherein said systems, methods, and compositions are based on a chimeric editing system comprising one or more components of a prime editor and one or more components of a retron editor. BACKGROUND OF THE INVENTION [0003] Gene editing tools encompass a diverse set of technologies that can make many types of genetic alterations in various contexts. These technologies have evolved over the last couple of decades to provide a range of user-programmable editing tools that include ZFN (zinc finger) nuclease editing systems, meganuclease editing systems, and TALENS (transcription activator-like effector nucleases). The past decade has seen an explosive growth in a new generation of gene editing systems based on components from bacterial immune pathways, including CRISPR (clustered regularly interspaced short palindromic repeats) and the associated CRISPR-associated proteins (e.g., CRISPR-Cas9), for example, as described in Jinek et al., “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,” Science, Vol.337 (6096), pp.816-821, meganuclease editors, for example, as described, Boissel et al., “megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering,” Nucleic Acids Research 42: pp.2591-2601, and bacterial retron systems, RNG043-WO1 PCT Application for example, as described, in Schubert et al., “High-throughput functional variant screens via in vivo production of single-stranded DNA,” PNAS, April 27, 2021, Vol.118(18), pp.1-10. [0004] CRISPR-Cas9 has been derivatized in numerous ways to expand upon its guide RNA- based programmable double-strand cutting activity to form systems ranging from finding alternative CRISPR Cas nuclease enzymes having different PAM requirements and cutting properties (e.g., Cas12a, Cas12f, Cas13a, and Cas13b) forto base editing, for example, as described in Komor et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage,” Nature, May 19, 2016, 533 (7603); pp.420-424 [cytosine base editors or CBEs] and Gaudelli et al., “Programmable base editing of A-T to G-C in genomic DNA without DNA cleavage,” Nature, Vol.551, pp.464-471 [adenine base editors or ABEs]) to prime editing, for example, as described in Anzalone et al., “Search-and-replace genome editing without double-strand breaks or donor DNA,” Nature, Dec 2019, 576 (7789): pp.149-157, to twin prime editing, for example, as described in Anzalone et al., “Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing,” Nature Biotechnology, Dec 9, 2021, vol.40, pp.731-740, to epigenetic editing, for example, as described in Kungulovski and Jeltsch, “Epigenome Editing: State of the Art, Concepts, and Perspective,” Trends in Genetics, Vol.32, 206, pp.101-113, and to CRISPR-directed integrase editing, for example, as described in Yarnell et al., “Drag-and-drop genome insertion of large sequences without double-stranded DNA cleavage using CRISPR- directed integrases,” Nature Biotechnology, Nov 24, 2022, doi.org/10.1038/s41587-022- 01527-4. [0005] Despite vast progress in the gene editing field over the last ten years through the development of a wide variety of gene editing tools based on CRISPR enzymes (e.g., base editors and prime editors) and other editing tools (e.g., retrons), new and improved editing systems that go beyond the capabilities of currently known editing systems are insatiably desired in the art. SUMMARY OF THE INVENTION [0006] In one aspect, the present disclosure describes a chimeric gene editing system that comprises one or more components of site-specific gene editing system (e.g., a prime editing system or a prime editor-like system) with one or more components of a retron editing system, producing an entirely new gene editing system with distinct properties, capabilities and advantages. Fruther provided include modified retrons and retron-editing systems for improving the gene editing specificity and efficiency. RNG043-WO1 PCT Application [0007] In certain embodiments, the chimeric gene editing system comprises (a) a nickase component, (b) a guide RNA that complexes with the nickase component and directs it to a target DNA sequence, (c) a polymerase component, (d) and a polymerase template sequence, wherein the polymerase template sequence is provided by a modified retron ncRNA comprising the polymerase template sequence (herein referred to as a “templated ncRNA” or “tncRNA”). In various embodiments, the tncRNA comprises an altered structural configuration relative to a wildtype retron ncRNA wherein the alterations include (i) a linker joining the 5’ end of the ncRNA a1 region to the 3’ end of the ncRNA a2 region, (ii) a deletion of the wildtype msd region or portion thereof, or (iii) installation of a single-strand RNA sequence comprising the polymerase template in place of the deleted msd sequence. The chimeric gene editing system may also optionally comprise a retron reverse transcriptase (RT) to convert the templated ncRNA to a cognate msDNA, which is referred to herein as the “templated msDNA” or “tmsDNA”. [0008] In certain other embodiments, the chimeric gene editing system comprises (a) a nickase component or a nucleic acid sequence encoding same (e.g., a mRNA), (b) a guide RNA that complexes with the nickase component and directs it to a target DNA sequence, (c) a polymerase component or a nucleic acid sequence encoding same (e.g., a mRNA), (d) and a polymerase template sequence, wherein the polymerase template sequence is provided by a modified retron ncRNA comprising the polymerase template sequence (herein referred to as a “templated ncRNA” or “tncRNA”). In various embodiments, the tncRNA comprises an altered structural configuration relative to a wildtype retron ncRNA wherein sthe alterations include (i) a linker joining the 5’ end of the ncRNA a1 region to the 3’ end of the ncRNA a2 region, (ii) a deletion of the wildtype msd region or portion thereof, or (iii) installation of a single-strand RNA sequence comprising the polymerase template in place of the deleted msd sequence. The chimeric gene editing system may also optionally comprise a retron RT (or a nucleic acid sequence encoding a retron RT) to convert the templated ncRNA to a cognate msDNA, which is referred to herein as the “templated msDNA” or “tmsDNA”. [0009] Referring to the figures, FIG.1 provides a schematic of a prime editor which includes (a) a reverse transcriptase component fused to a (b) CRISPR Cas9 nickase component, and which is complexed with (c) a prime editor guide RNA (pegRNA) (i.e., the PE:pegRNA complex). The pegRNA comprises a (i) primer binding site (PBS) and (ii) a template sequence. The mechanism of prime editing is described in the art. The PE:pegRNA complex binds to a target DNA at a site specified by the spacer sequence of the pegRNA. The Cas9 nickase then nicks one strand, generating a 3’-ended flap of endogenous DNA (“the 3’ flap”). RNG043-WO1 PCT Application The PBS, located on the pegRNA, binds to the 3’ flap and the edited RNA sequence is reverse transcribed from the 3’ flap against the template portion of the pegRNA by the reverse transcriptase of the PE:pegRNA complex. The reverse transcription product effectively extends from the endogenous 3’ flap to form a newly synthesized single strand of DNA (i.e., the RT product or edited strand). The edited strand displaces the endogenous strand sitting immediately downstream of the nick on the nicked strand, forming a 5’ flap of displaced endogenous DNA, which is removed by cell enzymes. The edited DNA is permanently installed onto both strands as a result of endogenous DNA repair enzymes and a round of DNA replication. This process can be enhanced by using an additional normal-length guide RNA that is programmed to install (by again complexing with the PE) a second nick into the unedited strand. This particular DNA lesion is recognized by the cells, compelling the cell to repair the unedited strand to match as the complement of the edited strand. This process leaves both strands in the edited configuration, thereby permanently installing the edit. [0010] Referring to FIG.2, a retron editing system is depicted. Retrons encode and transcribe as a single RNA, which comprises a non-coding RNA (ncRNA) portion and a portion encoding a specialized reverse transcriptase (RT) (see Panel 1). The retron ncRNA (msr and msd) is the precursor of the hybrid molecule that eventually forms, and it initially folds into a typical RNA secondary structure that is recognized by the accompanying RT. As shown in Panel 2, the translated RT typically recognizes certain secondary structures in the ncRNA, and binds the RNA template downstream from the msd region. The RT initiates reverse transcription of the RNA towards its 5ʹ end, starting from the 2’-end of a conserved guanosine (G) residue found immediately after a double-stranded RNA structure (the a1/a2 region) within the ncRNA. A portion of the ncRNA serves as a template for reverse transcription, and reverse transcription terminates before reaching the msr locus. During reverse transcription, cellular RNase H degrades the segment of the ncRNA that serves as template, but not other parts of the ncRNA. As shown in Panel 3, the result of the reverse transcription, the msDNA, remains covalently attached to the RNA template via the 2’-5ʹ phosphodiester bond, and base-pairs with the RNA template using the 3ʹ end of the msDNA. [0011] As described at a high level in FIG.3, the present disclosure describes a chimeric gene editing system comprising one or more components of a prime editor system and one or more components of a retron editor. The resulting chimeric editing system is referred to herein as a “nickase/retron template precision editing system.” RNG043-WO1 PCT Application [0012] The present disclosure contemplates a variety of configurations and interchangeable components that may constitute different embodiments envisioned for the nickase/retron template precision editing system described herein. [0013] In various embodiments, the nickase/retron template precision editing system described herein may comprise: (1) a nickase or a nucleic acid sequence encoding a nickase (e.g., a mRNA encoding a nickase); (2) a guide RNA that complexes with the nickase and directs the nickase to a target DNA sequence; (3) a polymerase or a nucleic acid sequence encoding a polymerase (e.g., a mRNA encoding a polymerase); and (4) a polymerase template sequence for synthesis of an edit strand to be incorporated into a target site, wherein the polymerase template sequence is provided by a templated ncRNA. [0014] In various embodiments, the tncRNA comprises an altered structural configuration relative to a wildtype retron ncRNA wherein the alterations include, but are not limited to, (i) a linker joining the 5’ end of the ncRNA a1 region to the 3’ end of the ncRNA a2 region, (ii) a deletion of the wildtype msd region or a portion thereof, or/and (iii) installation of a single- strand RNA sequence comprising the polymerase template in place of the deleted msd sequence. The polymerase template may also comprise a primer binding site which anneals to an endogenous 3’ flap produced as a result of introducing a nick at the target site by the nickase. The annealing of the primer binding site to the endogenous 3’ flap creates a polymerization initiation site for synthesis of a new strand of single-stranded DNA (“the edited strand” or “the edited flap”) from the 3’ end of the endogenous flap and using the polymerase template as instructions. The edited strand displaces the endogenous strand sitting immediately downstream of the nick on the nicked strand, forming a 5’ flap of displaced endogenous DNA, which is removed by cell enzymes. The edited flap is permanently installed onto both strands as a result of endogenous DNA repair enzymes and DNA replication. This process can be enhanced by using an additional normal-length guide RNA that is programmed to install (by again complexing with the nickase) a second nick into the unedited strand. This particular DNA lesion is recognized by the cells, compelling the cell to repair the unedited strand to match as the complement of the edited strand. This process leaves both strands in the edited configuration, thereby permanently installing the edit. RNG043-WO1 PCT Application [0015] The chimeric gene editing system shown in FIG.3 may also optionally comprise a retron RT (or a nucleic acid sequence encoding a retron RT) to convert the templated ncRNA to a cognate msDNA, which is referred to herein as the “templated msDNA” or “tmsDNA”. [0016] In various embodiments of the nickase/retron template precision editing system described herein, the polymerase template may be provided directly by a templated ncRNA (e.g., as shown in FIG.6). In other embodiments, the polymerase template of the nickase/retron template precision editing system described herein may be provided by a templated ncRNA and a retron RT which converts the ncRNA to a templated msDNA, which then effectivley provides the templating function for the polymerase to synthesize and edited strand for incorporation. This embodiment is depicted in FIG.5. [0017] In various embodiments, the nickase/retron template precision editing system described herein comprises a templated ncRNA and/or a templated msDNA, as illustrated in FIG.4. Referring to panel A, a non-folded wildtype ncRNA is depicted. The molecule comprises in the 5’ to 3’ direction an a1 region, an msr region, an msd region, and an a2 region. The ncRNA folds as represented by panel B, whereby the a1/a2 regions comprises regions of reverse complementarity thereby forming a duplex. In addition, the msr region forms 1 or more stem loops, as well as in the msd region. Refer to FIG.2 for a detailed schematic on how the ncRNA is conventionally converted to an msRNA by a retron RT. By contrast, panels C and D are components of the nickase/retron template precision editing system described herein. Panel C illustrates an embodiment of a templated ncRNA. As depicted, the tncRNA comprises an altered structural configuration relative to a wildtype retron ncRNA (e.g., as shown in Panel B) wherein said alterations include, but are not limited to, (i) a linker joining the 5’ end of the ncRNA a1 region to the 3’ end of the ncRNA a2 region, (ii) a deletion of the wildtype msd region or a portion thereof, and (iii) installation of a single-strand RNA sequence comprising the polymerase template ((i) or (ii)) in place of the deleted msd sequence. The polymerase template may also comprise a primer binding site ((i) or (ii)) which anneals to an endogenous 3’ flap produced as a result of introducing a nick at the target site by the nickase. Panel D depicts a structure of the templated msDNA, which is generated after the the single-stranded RNA sequence comprising the polymerase template is reversed transcribed. [0018] FIG.5 depicts one embodiment of the nickase/retron template precision editing system described herein. In this embodiment, a templated ncRNA is converted to a templated msDNA by a retron RT. In parallel, a complex comprising a fusion protein comprising a nickase and a polymerase and a guide RNA binds to and cuts an unedited target DNA to introduce a nick in the DNA (“nicked DNA”). Next, the templated msDNA (comprising a polymerase template RNG043-WO1 PCT Application with a PBS) associates with the nicked DNA by annealing the PBS to the endogenous 3’ flap immediately upstream of the nick on the nicked strand. The annealed PBS provides an initiation point for the polymerase (e.g., an DNA-dependent DNA polymerase) of the fusion protein to synthesize a new strand of DNA from the available 3’ end of the endogenous 3’ flap templated against the polymerase template. This results in an edited 3’ flap which displaces the 5’ endogenous flap formed downstream of the nick site and becomes incorporated into the DNA following cellular DNA repair and replication processes. In some embodiments, a second site nicking guide RNA may be provided to introduce a nick in the non-edited strand downstream of the original nick site. The second nick site induces the cell to replace the unedited strand to form a reverse complement against the 3’ edited flap, thereby introducing the edit into both strands. Replication ensures permanent incorporation of the edit into both strands. [0019] FIG.6 depicts another embodiment of the nickase/retron template precision editing system described herein. In this embodiment, a templated ncRNA directly provides the polymerase template function (without the need to convert it to msDNA by a retron RT). In parallel, a complex comprising a fusion protein comprising a nickase and a polymerase and a guide RNA binds to and cuts an unedited target DNA to introduce a nick in the DNA (“nicked DNA”). Next, the templated ncRNA (comprising a polymerase template with a PBS) associates with the nicked DNA by annealing the PBS to the endogenous 3’ flap immediately upstream of the nick on the nicked strand. The annealed PBS provides an initiation point for the polymerase (e.g., a RNA-dependent DNA polymerase) of the fusion protein to synthesize a new strand of DNA from the available 3’ end of the endogenous 3’ flap templated against the polymerase template. This results in an edited 3’ flap which displaces the 5’ endogenous flap formed downstream of the nick site and becomes incorporated into the DNA following cellular DNA repair and replication processes. In some embodiments, a second site nicking guide RNA may be provided to introduce a nick in the non-edited strand downstream of the original nick site. The second nick site induces the cell to replace the unedited strand to form a reverse complement against the 3’ edited flap, thereby introducing the edit into both strands. Replication ensures permanent incorporation of the edit into both strands. [0020] FIG.7A through FIG.7D provides a more detailed look at the mechanism of a nickase/retron template precision editing system described herein, which relies on use of an msDNA (i.e., corresponding to the general configuration of FIG.5). FIG.7A depicts the association of a complex comprising a fusion protein comprising a nickase and a polymerase complexed with a guide RNA. The black triangle represents a single nuclease catalytic RNG043-WO1 PCT Application activity. FIG.7B shows the binding of the guide RNA to a target site by annealing to a specific complementary sequence therein, which then results in the action of the nuclease thereby introducing a singular nick in the top strand (which will become the “edited strand”). In FIG.7C, the region immediately upstream of the nick forms a 3’ flap region, which anneals to an inbound templated msDNA in the PBS of the msDNA. In this embodiment, the PBS is at the 3’ end of the msDNA, and the template region is upstream of the PBS. Also, as this embodiment involves an msDNA, the polymerase template (comprising both the PBS and the template) is a single strand of DNA which is joined to the msDNA by a 2’ to 5’ connection with a guanosine. Given the template is DNA, the polymerase can be a DNA-dependent DNA polymerase. Next, as shown in FIG.7D in panel (1), the polymerase synthesizes a new strand of DNA from the 3’ end of the endogenous flap over and instructed by the template of the msDNA. Once the complex and msDNA dissasemble, the newly synthesized DNA strand displaces the endogenous DNA, forming a 5’ endogenous flap which is removed by host enzymes (panel (2)). Lastly, the edit is incorporated into both strands as shown in panel (3) as a result of celluar repair and replication processes that preferably edit the endogenous strand to match as the reverse complement of the first-edited strand. [0021] In various embodiments, the nickase/retron template precision editing system described herein includes both a guide RNA (to guide the nickase to the target site) and a retron templated ncRNA. In some embodiments, the guide RNA and the ncRNA may be fused together, as shown for example in FIG.8A (PBS = primer binding site and RTT = reverse transcriptase template). In other embodiments, the guide RNA and the ncRNA can be delivered separately and do not need to be joined, as shown in FIG.8B. In still other embodiments, as illustrated in FIG.8C, the structure of the ncRNA may be further minimized by removing all or a portion of the a1/a2 stem/loop. In that embodiment, the guide RNA may be fused to the ncRNA or unfused. Other configurations on delivering ncRNAs and guide RNAs are depicted in FIG.8E. [0022] In various other embodiments, the ncRNA used herein may be comprise one or more stabilizing modifications, including stable nucleotide analogs, structures such as a 3’ hairpin, or circularized. Such embodiments are shown in FIG.8D. [0023] In another embodiment, shown in FIG.9, the nickase/retron template precision editing system may be configured as a dual editing system to create a pair of complementary edited strands (as shown in panel (2), which are then recombined into the target DNA, as shown in panel (3). In this embodiment, as shown in panel (1), the system involves a first template ncRNA and a first nickase-polymerase-guide RNA complex (dotted gray circle) that nicks a RNG043-WO1 PCT Application first site and creates a first single-stranded DNA edited flap. In this case, since the ncRNA is used directly (i.e., without the msDNA), a retron RT is provided to convert the ncRNA to msDNA. In addition, since the ncRNA is all-RNA, the polymerase is an RNA-dependent DNA polymerase so that it may synthesize the edited DNA strands against an RNA template. [0024] FIG.10A and FIG.10B depict yet another embodiment which is differs from the system of FIG.9 in that the ncRNAs each have a first and second PBS. As shown in panel (1), a first ncRNA comprising a polymerase template with flanking PBS sites (PBS1 and PBS2). Panel (2) depicts that as a first step the RT (e.g., retron RT) of a first fusion protein (the first fusion protein comprises a nickase (dotted black circle) and RT) synthesizes DNA against the first RTT from the 3’ of the endogenous 3’ flap, extending over the entire RTT and the PBS2 downstream of the RTT. The PBS2 is encoded into the resulting first edited DNA flap a counterpart PBS2’ DNA sequence, which anneals to a second 3’ endogenous flap formed at a second nick site as shown in Panel 3. DNA is again synthesized by a second editing fusion protein (e.g. by a DNA-dependent DNA polymerase, which in theory could be an engineered retron RT or another DNA-dependent DNA polymerase). The second synthesis uses the first synthesis product as a template. Similarly, in panel (4), the double stranded DNA duplex is then incorporated into the DNA thereby installing the edited strands. [0025] In various embodiments, depicted in FIG.11, each of the components of the nickase/retron template precision editing system may comprises various modifications resulting from optimization, engineering, mutagenesis, substitution of orthologs, and other engineering processes to change, alter, or improve editing functions, stability, and processivity, and/or other characteristics. [0026] In still other aspects, as depicted in FIG.12, the disclosure provides pharmaceutical compositions, such as lipid nanoparticles (LNPs) comprising an appropriate set of cargo elements that constitute the components of the nickase/retron template precision editing system. Any delivery system may be used to delivery the nickase/retron template precision editing systems as proteins, nucleic acids, or protein-nucleic acid complexes, etc. For example, the nickase/retron template precision editing system may be delivered in an all-RNA configuration comprising one or more mRNAs encoding a nickase, a polymerase, and an optionally a retron RT (i.e., where the polymerase template is an msDNA such that it is converted from a ncRNA), as well as function RNA components such as targeting guide RNAs, second nicking guide RNAs, and ncRNAs. [0027] In another aspect, the present disclosure further provides nucleic acid molecules encoding the gene editing system components (e.g., a recombinant ncRNA and/or a RNG043-WO1 PCT Application recombinant retron RT). In still another aspect, the present disclosure provides genome editing systems comprising recombinant retron components (e.g., recombinant ncRNA and/or recombinant RT), programmable nickases, and guide RNAs. In a further aspect, the disclosure provides nucleic acid molecules encoding the described genome editing systems and said components thereof, as well as polypeptides making up the components of said genome editing systems. In yet another aspect, the disclosure provides vectors for transferring and/or expressing said genome editing systems, e.g., under in vitro, ex vivo, and in vivo conditions. In still another aspect, the disclosure provides cell-delivery compositions and methods, including compositions for passive and/or active transport to cells (e.g., plasmids), delivery by virus- based recombinant vectors (e.g., AAV and/or lentivirus vectors), delivery by non-virus-based systems (e.g., liposomes and LNPs), and delivery by virus-like particles. Depending on the delivery system employed, the genome editing systems described herein may be delivered in the form of DNA (e.g., plasmids or DNA-based virus vectors), RNA (e.g., ncRNA and mRNA delivered by LNPs), a mixture of DNA and RNA, protein (e.g., virus-like particles), and ribonucleoprotein (RNP) complexes. Any suitable combinations of approaches for delivering the components of the herein disclosed retron-based genome editing systems may be employed. In one embodiment, each of the components of the retron-based genome editing system is delivered by an all-RNA system, e.g., the delivery of one or more RNA molecules (e.g., mRNA and/or ncRNA) by one or more LNPs, wherein the one or more RNA molecules form the ncRNA and guide RNA (as needed) and/or are translated into the polypeptide components (e.g., the RT and a programmable nuclease). In yet another aspect, the disclosure provides methods for genome editing by introducing a retron-based genome editing system described herein into a cell (e.g., under in vitro, in vivo, or ex vivo conditions) comprising a target edit site, thereby resulting in an edit at the target edit. In other aspects, the disclosure provides formulations comprising any of the aforementioned components for delivery to cells and/or tissues, including in vitro, in vivo, and ex vivo delivery, recombinant cells and/or tissues modified by the recombinant retron-based genome modification systems and methods described herein, and methods of modifying cells by conducting genome editing and related DNA donor-dependent methods, such as recombineering, or cell recording, using the herein disclosed retron-based genome modification systems. The disclosure also provides methods of making the recombinant retrons, retron-based genome modification systems, vectors, compositions, and formulations described herein, as well as to pharmaceutical compositions and kits for modifying cells under in vitro, in vivo, and ex vivo conditions that comprise the herein disclosed genome editing and/or modification systems. RNG043-WO1 PCT Application [0028] In an embodiment, this disclosure or the inventions herein provide a gene editing system comprising one or more delivery vehicles, wherein: the delivery vehicle(s) comprise RNA cargo; the RNA cargo comprises (a) at least one mRNA molecule encoding (i) a nucleic acid programmable nuclease (e.g., a nickase) and (ii) a polymerase (e.g., a DNA-dependent DNA polymerase, an RNA-dependent DNA polymerase, e.g., a retron reverse transcriptase), (b) an engineered templated retron ncRNA, and (c) guide RNA for the programmable nuclease; and each delivery vehicle contains (a)(i) and/or (a)(ii) and/or (b) and/or (c); whereby one delivery vehicle or more than one delivery vehicle delivers (a)(i), (a)(ii), (b), and (c). [0029] In an embodiment, in the gene editing system, (a)(i) and (a)(ii) comprise a single mRNA molecule encoding the nucleic acid programmable nuclease and the polymerase (e.g, retron reverse transcriptase). [0030] In an embodiment, in the gene editing system, (a)(i) and (a)(ii) are encoded and expressed as a fusion protein. [0031] In an embodiment, in the gene editing system (a)(i) and (a)(ii) are encoded and expressed as a fusion protein and the fusion protein comprises the C-terminal end of the nucleic acid programmable nuclease fused to the N-terminal end of the polymerase (e.g. retron reverse transcriptase (nuclease:polymerase)); or the fusion protein comprises the N-terminal end of the nucleic acid programmable nuclease fused to the C-terminal end of the retron reverse transcriptase (polymerase:nuclease fusion). [0032] In an embodiment, in the gene editing system, (a)(i) and (a)(ii) comprise a first mRNA molecule encoding the nucleic acid programmable nuclease and a second mRNA molecule encoding the polymerase (e.g., retron reverse transcriptase). [0033] In an embodiment, in the gene editing system, (c) is separate from (a)(i), (a)(ii) and (b) or is provided in trans. [0034] In an embodiment, in the gene editing system, (b) the engineered retron ncRNA, and (c) the guide RNA are fused or are provided in cis. [0035] In an embodiment, in the gene editing system, (b) the engineered retron ncRNA, and (c) the guide RNA are fused or are provided in cis and the guide RNA is fused to the 5’ end of the retron ncRNA. [0036] In an embodiment, in the gene editing system, (b) the engineered retron ncRNA, and (c) the guide RNA are fused or are provided in cis and the guide RNA is fused to the 3’ end of the retron ncRNA. [0037] In an embodiment, in the gene editing system, (b) the engineered retron ncRNA, and (c) the guide RNA are fused or are provided in cis and the engineered ncRNA comprises a first RNG043-WO1 PCT Application guide RNA fused to the 5’ end of the retron ncRNA, and a second guide RNA fused to the 3’ end of the retron ncRNA, and the first and second guide RNAs target different sequences. Thus, on a broader scale, in an embodiment, in the gene editing system, (c) guide RNA for the programmable nuclease, can comprise one or more guides that target the same or different target sequences. Such guide RNA(s) in an embodiment, can be single guide RNA(s) or sgRNA(s); for instance, when the nucleic acid programmable nuclease comprises a Cas9. [0038] In an embodiment, in the gene editing system, the one or more delivery vehicles comprise a liposome or a lipid nanoparticle (LNP). [0039] In an embodiment, in the gene editing system, (a) the at least one mRNA molecule encoding (i) the nucleic acid programmable nuclease and (ii) the polymerase (e.g., retron reverse transcriptase), and (b) the engineered retron ncRNA, are in the same delivery vehicle. [0040] In an embodiment, in the gene editing system, (a) the at least one mRNA molecule encoding (i) the nucleic acid programmable nuclease and (ii) the polymerase (e.g., retron reverse transcriptase), and (b) the engineered retron ncRNA, are in separate delivery vehicles. [0041] In an embodiment, in the gene editing system, the nucleic acid programmable nuclease and the polymerase (e.g., retron reverse transcriptase) are encoded on separate mRNA molecules and those separate mRNA molecules of (a)(i) and (a)(ii) are contained in the same delivery vehicle. [0042] In an embodiment, in the gene editing system, the nucleic acid programmable nuclease and the polymerase (e.g., retron reverse transcriptase) are encoded on separate mRNA molecules and those separate mRNA molecules of (a)(i) and (a)(ii) are contained in different delivery vehicles. [0043] In an embodiment, in the gene editing system, the engineered retron ncRNA includes a polymerase template comprising a PBS and a template sequence comprising an intended edit to be integrated at a target sequence in a cell, and wherein the polymerase template comprises one or more regions of homology to the endogenous sequence downstream of a nick site to facilitate integration of the edited strand. The editing systems can be used in an animal cell, or a mammalian cell (e.g., a primate, a non-human primate, or a domesticated mammal such as a cat or dog or horse) or a human cell; for instance to correct, address, treat, mitigate a genetic condition in the animal, mammal, domesticated mammal, cat, dog, horse or human. Such can be done in plant cells to introduce mutations that give rise to favorable phenotypic characteristics such as disease resistance or other favorable plant trait(s). RNG043-WO1 PCT Application [0044] In an embodiment, in the gene editing system, the nucleic acid programmable nuclease comprises a Cas9 nuclease, a TnpB nuclease, or a Cas12a nuclease. Preferably, the nuclease is a nickase such that only one of the two strands in the target region is cut. [0045] In various embodiments, the templated ncRNA may be derived from a baseline or wildtype ncRNA, such as any of those ncRNAs disclosed in Table B or a nucleotide sequence of Table B of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872 (each are incorporated herein by reference in their entireties, including their Sequence Listings), or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with a sequence from Table B of any of the aforementioned applications. The polymerase template can be heterologous to a cell. Alternatively, the polymerase template can be endogenous to the cell. For instance, the cell can contain a sequence that is typical for those in a population having a disease state and the donor polynucleotide can be a sequence that is typical for those in the population not having a non-disease state (e.g., the donor can be for a genetic correction or repair of a cell to modify the cell from having a mutation or modification that gives rise to a disease state to having a sequence typical of not having the disease state). [0046] In an embodiment of the gene editing system, the gene editing system can comprise any combination(s) of the foregoing embodiments of the gene editing system. [0047] In an embodiment, this disclosure or the inventions herein provide a cell, such as an isolated cell comprising the gene editing system disclosed herein, such as in any of the foregoing paragraphs. In an embodiment, the cell, e.g., isolated cell, can be a eukaryotic cell. In an embodiment, the eukaryotic cell can be a plant cell or an animal cell or a mammalian cell, e.g., an isolated plant cell or an isolated animal cell or an isolated mammalian cell. In an embodiment, the mammalian cell, e.g., an isolated mammalian cell, can be a human cell. In an embodiment, the cell can be a prokaryotic cell, e.g., a bacterial cell. In such an embodiment where the cell is a bacterial cell, the donor polynucleotide can code for antibiotic susceptibility; and thus, the present disclosure and any inventions disclosed herein can involve a means for addressing antibiotic resistant bacteria by rendering such bacteria susceptible to antibiotics (and a subject to whom the gene editing system is administered can also then receive antibiotics to which the bacteria are rendered susceptible by the gene editing system). [0048] In an embodiment, this disclosure or the inventions herein provide a composition comprising: a) the gene editing system disclosed herein, such as in any of the foregoing paragraphs; and b)a pharmaceutically or veterinarily acceptable carrier. In an embodiment, in the composition the delivery vehicle can comprise a lipid nanoparticle comprising: a) one or RNG043-WO1 PCT Application more ionizable lipids; b) one or more structural lipids; c) one or more PEGylated lipids; and d) one or more phospholipids. In an embodiment, in the composition the one or more ionizable lipids comprises an ionizable lipid set forth in Table 2 of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038. [0049] In an embodiment, this disclosure or the inventions herein provide uses of the gene editing system embodiments and/or the compositions disclosed herein, such as in any of the foregoing paragraphs; for instance, use in modifying a cell or genetically modifying a cell, e.g., a eukaryotic or a prokaryotic cell and/or an animal cell and/or a mammalian and/or a human cell and/or a bacterial cell and/or a plant cell, in vivo, in vitro or ex vivo (e.g., any cell discussed herein wherein the cell comprises an isolated cell). In an embodiment, this disclosure or the inventions herein provide uses of the gene editing system embodiments and/or the compositions disclosed herein, such as in any of the foregoing paragraphs; for instance, use in treating or addressing a genetic condition of a subject, [0050] In an embodiment, this disclosure or the inventions herein provide methods of genetically modifying a cell comprising: contacting a gene editing system as herein discussed, such as in any of the foregoing paragraphs, or a composition as herein discussed, such as in any of the foregoing paragraphs (which comprises a gene editing system as herein discussed, such as in any of the foregoing paragraphs), advantageously a gene editing system that includes a sequence of interest encoding a donor polynucleotide comprising an intended edit to be integrated at a target sequence in a cell, said method comprising contacting the composition or the gene editing system with the cell, thereby delivering the RNA cargo to the cell, wherein: the nucleic acid programmable nuclease forms a complex with the guide RNA, wherein said guide RNA directs the complex to the target sequence; the nucleic acid programmable nuclease creates a double-stranded break in in the target sequence; the retron reverse transcriptase and engineered retron ncRNA create msDNA that comprises the donor polynucleotide; and the donor polynucleotide becomes integrated at the target sequence; whereby editing the cell is genetically modified. In an embodiment, the cell can be a eukaryotic or a prokaryotic cell or an animal cell or a mammalian cell or a human cell or a bacterial cell or a plant cell. [0051] In another aspect, the present disclosure provides a non-coding RNA (ncRNA) variant comprising a reference retron ncRNA with one or more modifications, wherein the reference retron ncRNA comprises from 5’ to 3’ direction: a1 region, first branching guanosine, msr, msd, and a2 region, and the one or more modifications comprises (i) linkage of the a1 region and the a2 region, (ii) deletion of at least a portion of the msr; (iii) deletion of at least a portion RNG043-WO1 PCT Application of the msd, (iv) addition of a single-stranded RNA comprising a polymerase template, or (v) addition of an RNA motif. [0052] In an embodiment, the one or more modifications comprise the linkage of the a1 region to the a2 region. [0053] In an embodiment of any one of the preceding embodiments, the one or more modifications comprise the linkage of the a1 region and the a2 region, wherein the linkage of the a1 region and the a2 region is formed via a linker joining the 5’ end of the a1 region to the 3’ end of the a2 region. In an embodiment of any one of the preceding embodiments, the one or more modifications comprise the linkage of the a1 region and the a2 region, wherein the linkage of the a1 region and the a2 region is formed by directly joining the 5’ end of the a1 region to the 3’ end of the a2 region without a linker. [0054] In an embodiment of any one of the preceding embodiments, the a1 region and the a2 region are partially or completely complementary to each other and the a1 region and the a2 region form a stem-loop structure after the linkage. [0055] In an embodiment of any one of the preceding embodiments, the ncRNA variant further comprises a second branching guanosine. [0056] In an embodiment of any one of the preceding embodiments, formation of the linkage of the a1 region and the a2 region circularizes the ncRNA variant. [0057] In an embodiment of any one of the preceding embodiments, the one or more modifications further comprises a breakage between the msd and the a2 region, thereby generating the ncRNA variant comprising from 5’ to 3’ direction: the a2 region, the linkage between the a1 and the a2 region, the a1 region, the first branching guanosine, the msr, and the msd. [0058] In an embodiment of any one of the preceding embodiments, the one or more modifications comprises the deletion of at least a portion of the msr. In an embodiment, the portion deleted from the msr comprises a spacer between two stem-loops in the msr or a portion of the spacer. In an embodiment, the portion deleted from the msr comprises a portion of the spacer, whereby the deletion preserves a remaining 1-15 base pairs between the two stem-loops in the msr, as compared to the reference retron ncRNA. In an embodiment, the deletion preserves a remaining 1 base pair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs, 6 base pairs, 7 base pairs, 8 base pairs, 9 base pairs, 10 base pairs, 11 base pairs, 12 base pairs, 13 base pairs, 14 base pairs, or 15 base pairs between the two stem-loops in the msr, as compared to the reference retron ncRNA. In an embodiment, the portion deleted from the msr comprises the entirety of the spacer between two stem-loops in the msr, as compared to the RNG043-WO1 PCT Application reference retron ncRNA. In an embodiment, the portion deleted from the msr comprises a stem-loop in the msr or a portion thereof. [0059] In an embodiment of any one of the preceding embodiments, the one or more modifications comprises the deletion of at least a portion of the msd. In an embodiment, the deleted portion of the msd comprises a spacer located between a stem-loop in the msd and the a2 region or a portion of the spacer. In an embodiment, the deleted portion in the msd comprises a portion of the spacer located between the stem-loop in the msd and the a2 region, whereby the deletion preserves a remaining 1-15 base pairs between the stem-loop in the msd and the a2 region, as compared to the reference retron ncRNA. In an embodiment, the deletion preserves a remaining 1 base pair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs, 6 base pairs, 7 base pairs, 8 base pairs, 9 base pairs, 10 base pairs, 11 base pairs, 12 base pairs, 13 base pairs, 14 base pairs, or 15 base pairs between the stem-loop in the msd and the a2 region, as compared to the reference retron ncRNA. In an embodiment, the deleted portion in the msd comprises the entire spacer between the stem-loop in the msd and the a2 region. In an embodiment, the deleted portion in the msd further comprises the stem-loop in the msd, thereby the deletion comprises deletion of the entire spacer between the stem-loop in the msd and the a2 region and the stem-loop in the msd. [0060] In an embodiment of any one of the preceding embodiments, the one or more modifications comprises the addition of the single-stranded RNA comprising the polymerase template. In an embodiment, the one or more modifications comprise the addition of the single-stranded RNA comprising the polymerase template and the deletion of at least a portion of msd, and the single-stranded RNA comprising the polymerase template is added to the deleted portion of the msd. In an embodiment, the single-stranded RNA comprising the polymerase template comprises a pair of homology arms specific to a target locus. In an embodiment, each of the homology arms comprises 5-200 nucleotides. In an embodiment, each of the homology arms comprises between 5 and 100 nucleotides, between 5 and 50 nucleotides, between 10 and 50 nucleotides, between 25 and 50 nucleotides, between 5 and 25 nucleotides, between 10 and 25 nucleotides, between 10 and 20 nucleotides, between 20 and 30 nucleotides, between 30 and 40 nucleotides, or between 40 to 50 nucleotides. In an embodiment, the single-stranded RNA comprising the polymerase template further comprises a donor sequence for integration into a target locus. In an embodiment, the donor sequence comprises 2-100 nucleotides, 2-50 nucleotides, 2-25 nucleotides, 5-25 nucleotides, 5-15 nucleotides, 5-10 nucleotides, 10-30 nucleotides, 10-20 nucleotides or 1-10 nucleotides. RNG043-WO1 PCT Application [0061] In an embodiment of any one of the preceding embodiments, the one or more modifications comprises the addition of the RNA motif. In an embodiment, the RNA motif is a MS2 stem-loop or a 3’ tail of U7 small nuclear RNA (snRNA). [0062] In an embodiment of any one of the preceding embodiments, the one or more modifications comprise: (a) a combination of (i) and (ii): the linkage of the a1 region and the a2 region and the deletion of at least a portion of the msr, (b) a combination of (i) and (iii): the linkage of the a1 region and the a2 region and the deletion of at least a portion of the msd, (c) a combination of (i) and (iv): the linkage of the a1 region and the a2 region and the addition of the single-stranded RNA comprising the polymerase template, (d) a combination of (i) and (v): the linkage of the a1 region and the a2 region and the addition of the RNA motif, (e) a combination of (ii) and (iii): the deletion of at least a portion of the msr and the deletion of at least a portion of the msd, (f) a combination of (ii) and (iv): the deletion of at least a portion of the msr and the addition of the single-stranded RNA comprising the polymerase template, (g) a combination of (ii) and (v): the deletion of at least a portion of the msr and the addition of the RNA motif at the 5’ end or 3’ end, or (h) a combination of (iv) and (v): the addition of the single-stranded RNA sequence comprising the polymerase template and the addition of the RNA motif at the 5’ end or 3’ end. [0063] In an embodiment of any one of the preceding embodiments, the one or more modifications comprise: (a) a combination of (i), (ii), and (iii): the linkage of the a1 region and the a2 region, the deletion of at least a portion of the msr, and the deletion of at least a portion of the msd, (b) a combination of (i), (ii), and (iv): the linkage of the a1 region and the a2 region, the deletion of at least a portion of the msr, and the addition of the single-stranded RNA comprising the polymerase template, (c) a combination of (i), (iii), and (iv): the linkage of the a1 region and the a2 region, the deletion of at least a portion of the msd, and the addition of the single-stranded RNA sequence comprising the polymerase template, or (d) a combination of (ii), (iii), and (iv): the deletion of at least a portion of the msr, the deletion of at least a portion of the msd, and the addition of the RNA motif at the 5’ end or 3’ end. [0064] In an embodiment of any one of the preceding embodiments, the one or more modifications comprise: (i) the linkage of the a1 region and the a2 region, (ii) the deletion of at least a portion of the msr, (iii) the deletion of at least a portion of the msd, (iv) the addition of the single-stranded RNA sequence comprising the polymerase template, and (v) the addition of the RNA motif at the 5’ end or 3’ end region. [0065] In an embodiment of any one of the preceding embodiments, the one or more modifications further comprises substitution, insertion, or deletion of one or more nucleotides, RNG043-WO1 PCT Application or a combination thereof. In an embodiment, the substitution, insertion, or deletion is capable of modulating activity of a first polymerase or a second polymerase, production of a single stranded DNA from the ncRNA variant, or immunogenicity of the ncRNA variant. [0066] In an embodiment of any one of the preceding embodiments, the reference retron ncRNA comprises a sequence of a naturally occurring retron or a portion thereof. [0067] In an embodiment of any one of the preceding embodiments, the reference retron ncRNA comprises a sequence selected from SEQ ID NO: 3980-4178, 11231-11429, 4671- 4825, 11922-12075, 4980-5143, 12229-12392, 367-368, 427-441, 494-521, 526-527, 536, 626, 649, 660-668, 675, 679, 687-692, 695, 697, 703, 716, 721-722, 751-763, 767, 770-1411, 1456- 1462, 7624-7625, 7684-7698, 7751-7778, 7783-7784, 7793, 7883, 7906, 7917-7925, 7932, 7936, 7944-7949, 7952, 7954, 7960, 7973, 7978-7979, 8008-8020, 8024, 8027-8667, 8712- 8718, 1529-1569, 8784-8823, 6697-6701, 13943-13947, 4179-4670, 11430-11921, 4884-4909, 12134-12159, 6919-6972, 14163-14215, 2786-2866, 2887-2938, 10039-10119, 10140-10191, 4826-4863, 12076-12113, 4864-4875, 12114-12125, 6974-7002, 14217-14244, 2598-2600, 2759-2785, 9851-9853, 10012-10038, 2445-2582, 9699-9836, 1983-2158, 9237-9412, 1612- 1982, 8866-9236, 2601-2678, 9854-9931, 2679-2758, 9932-10011, 3442-3603, 10694-10855, 3604-3708, 10856-10959, 2939-3441, 3709-3979, 5177-5192, 10192-10693, 10960-111230, 12426-12441, 7003-7033, 14245-14275, 7054-7133, 14296-14374, 7034-7049, 14276-14291, 6835-6918, 14079-14162, 6823-6834, 14068-14078, 298-366, 369-373, 442-493, 522-525, 528-535, 537, 551-554, 557, 560-625, 672-674, 680-681, 684-686, 696, 698, 702, 723-742, 764-766, 1412-1453, 1463-1466, 1571-1577, 7555-7623, 2626-7630, 7699-7750, 7785-7792, 7794, 7808-7811, 7814, 7817-7882, 7929-7931, 7937-7938, 7941-7943, 7953, 7955, 7959, 7980-7999, 8021-8023, 8668-8706, 8708-8709, 8719-8722, 8825-8831, 374-426, 539-550, 555-556, 558-559, 671, 682-683, 743, 745-750, 7631-7683, 7796-7807, 7812-7813, 7815- 7816, 7928, 7939-7940, 800, 8002-8007, 5942-6665, 13189-13911, 1-297, 715, 1580-1603, 7258-7554, 7972, 8834-8857, 705-714, 7962-7971, 6681-6694, 13927-13940, 6788-6803, 14033-14048, 1469-1526, 5147-5151, 8725-8781, 12396-12400, 2159-2428, 9413-9682, 646- 648, 7903-7905, 2592-2595, 9846-9849, 676-678, 717-720, 7933-7935, 7974-7977, 538, 669, 704, 7795, 7926, 7961, 8710, 670, 699-701, 7927, 7956-7958, 4917-4979, 12167-12228, 4910- 4916, 12160-12166, 5195-5941, 12444-13188, 627-645, 650-659, 693-694, 744, 768-769, 1451, 1455, 1467-1468, 1527-1528, 1570, 1578, 1579, 1604-1611, 2429-2444, 2583-2591, 2596-2597, 2867-2886, 4876-4883, 5144-5146, 5152-5176, 5193-5194, 6666-6680, 6695- 6696, 6702-6787, 6804-6822, 6973, 7050-7053, 7134-7257, 7884-7902, 7907-7916, 7950- 7951, 8001, 8025-8026, 8707, 8711, 8723-8724, 8782-8783, 8824, 8832-8833, 8858-8865, RNG043-WO1 PCT Application 9683-9698, 9837-9845, 9850, 10120-10139, 12126-12133, 12393-12395, 12401-12425, 12442-12443, 13912-13926, 13941-13942, 13948-14032, 14049-14067, 14216, 14292-14295, 14375-14498, 16886-17078, 17478-17622, 17677-17756, 14831-14833, 14838, 14847, 14850- 15460, 17079-17477, 17660-17676, 19031-19080, 16414-16516, 17623-17659, 19081-19108, 16397-16413, 16195-16320, 15779-15925, 15476-15778, 16321-16366, 16367-16396, 16705- 16814, 16815-16885, 16517-16704, 18949-19030, 14657-14716, 14778-14824, 14834, 14835- 14836, 14839, 15461-15475, 14717-14777, 14841-14846, 18413-18936, 14499-14656, 18939, 15926-16178, 14837, 17757-18412, 14825-14830, 14840, 14848-14849, 16179-16194, 18937- 18938, and 18940-18948 (Table A or Table B of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872 or Table 31A of PCT Publication WO2024044723A1; SEQ ID NOs: 19543-19733 of the PCT). [0068] In an embodiment of any one of the preceding embodiments, the ncRNA variant comprises a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to any one of Table A or Table B of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872 or Table 31A of PCT Publication WO2024044723A1 (SEQ ID NOs: 19543-19733 of the PCT). [0069] In an embodiment of any one of the preceding embodiments, the ncRNA variant comprises a sequence selected from RTX3_6342_msr_stem_var1 (SEQ ID NO: 19644), RTX3_6342_msr_stem_var20 (SEQ ID NO: 19663), RTX3_6342_msr_stem_var5 (SEQ ID NO: 19648), RTX3_6342_a1a2_var6 (SEQ ID NO: 19548), RTX3_6342_a1a2_var10 (SEQ ID NO: 19552), RTX3_6342_a1a2_var15 (SEQ ID NO: 19557), RTX3_6342_a1a2_var16 (SEQ ID NO: 19558), RTX3_6342_a1a2_var19 (SEQ ID NO: 19561), RTX3_6342_a1a2_var20 (SEQ ID NO: 19562), RTX3_6342_a1a2_var21 (SEQ ID NO: 19563), RTX3_6342_a1a2_var26 (SEQ ID NO: 19568), RTX3_6342_a1a2_var27 (SEQ ID NO: 19569), and RTX3_6342_a1a2_var32 (SEQ ID NO: 19574). In an embodiment of any one of the preceding embodiments, the ncRNA variant comprises one or more modifications in RTX3_6342_msr_stem_var1 (SEQ ID NO: 19644), RTX3_6342_msr_stem_var20 (SEQ ID NO: 19663), RTX3_6342_msr_stem_var5 (SEQ ID NO: 19648), RTX3_6342_a1a2_var6 (SEQ ID NO: 19548), RTX3_6342_a1a2_var10 (SEQ ID NO: 19552), RNG043-WO1 PCT Application RTX3_6342_a1a2_var15 (SEQ ID NO: 19557), RTX3_6342_a1a2_var16 (SEQ ID NO: 19558), RTX3_6342_a1a2_var19 (SEQ ID NO: 19561), RTX3_6342_a1a2_var20 (SEQ ID NO: 19562), RTX3_6342_a1a2_var21 (SEQ ID NO: 19563), RTX3_6342_a1a2_var26 (SEQ ID NO: 19568), RTX3_6342_a1a2_var27 (SEQ ID NO: 19569), or RTX3_6342_a1a2_var32 (SEQ ID NO: 19574) compared to RTX3_6342_WT (SEQ ID NO: 19734). [0070] In an embodiment of any one of the preceding embodiments, the ncRNA variant comprises a sequence selected from msR Spacer Del-1 (SEQ ID NO: 19939), msD Spacer Del- 1 (SEQ ID NO: 19940), msD Spacer Del-2 (SEQ ID NO: 19941), and msR Spacer Del-1/msD Spacer Del-2 (SEQ ID NO: 19942). In an embodiment of any one of the preceding embodiments, the ncRNA variant comprises one or more modifications in msR Spacer Del-1 (SEQ ID NO: 19939), msD Spacer Del-1 (SEQ ID NO: 19940), msD Spacer Del-2 (SEQ ID NO: 19941), or msR Spacer Del-1/msD Spacer Del-2 (SEQ ID NO: 19942) compared to WT R6342 ncRNA (SEQ ID NO: 19734). [0071] In an embodiment of any one of the preceding embodiments, the ncRNA variant comprises a sequence selected from Alt1 msR Spacer Del- (SEQ ID NO: 19947)1, Alt1 msD Spacer Del-1 (SEQ ID NO: 19948), Alt1 msD Spacer Del-2 (SEQ ID NO: 19949), Alt1 msR Spacer Del-1/msD Spacer Del-2 (SEQ ID NO: 19950), Alt1 msR Spacer Del-2/msD Del-3 (SEQ ID NO: 19951), Alt1 msR Spacer Del-2/msD Del-3 MS2 (SEQ ID NO: 19952), and Alt1 msR Spacer Del-2/msD Del-3 U7 (SEQ ID NO: 19953). In an embodiment of any one of the preceding embodiments, the ncRNA variant comprises one or more modifications in Alt1 msR Spacer Del- (SEQ ID NO: 19947)1, Alt1 msD Spacer Del-1 (SEQ ID NO: 19948), Alt1 msD Spacer Del-2 (SEQ ID NO: 19949), Alt1 msR Spacer Del-1/msD Spacer Del-2 (SEQ ID NO: 19950), Alt1 msR Spacer Del-2/msD Del-3 (SEQ ID NO: 19951), Alt1 msR Spacer Del- 2/msD Del-3 MS2 (SEQ ID NO: 19952), or Alt1 msR Spacer Del-2/msD Del-3 U7 (SEQ ID NO: 19953) compared to WT R6342 ncRNA (SEQ ID NO: 19734). [0072] In an embodiment of any one of the preceding embodiments, the ncRNA variant comprises a sequence selected from msR spacer del_30HA (SEQ ID NO: 19955), msR spacer del_45HA (SEQ ID NO: 19956), WT PAM mt, (SEQ ID NO: 19957) Alt del1+del2 PAM mt (SEQ ID NO: 19958), or Alt del1+del2+U7 PAM mt (SEQ ID NO: 19959), and Alt del1+del2 25bp ins (SEQ ID NO: 19960). In an embodiment of any one of the preceding embodiments, the ncRNA variant comprises one or more modifications in msR spacer del_30HA (SEQ ID NO: 19955), msR spacer del_45HA (SEQ ID NO: 19956), WT PAM mt, (SEQ ID NO: 19957) Alt del1+del2 PAM mt (SEQ ID NO: 19958), or Alt del1+del2+U7 PAM mt (SEQ ID NO: RNG043-WO1 PCT Application 19959), or Alt del1+del225bp ins (SEQ ID NO: 19960)compared to WT R6342 ncRNA (SEQ ID NO: 19734). [0073] In an embodiment of any one of the preceding embodiments, the one or more modifications further comprises addition of 2’O methyl or phosphorothioate bond at the 5’ or 3’ end of the ncRNA variant. In an embodiment, the one or more modifications further comprises the addition of the 2’O methyl or the phosphorothioate bond at both the 5’ and 3’ ends of the ncRNA variant. [0074] In another aspect, the present disclosure provides a retron variant comprising the ncRNA variant of any one of the preceding embodiments and an RNA encoding a first polymerase, optionally downstream of the msd of the ncRNA variant. [0075] In an embodiment, the first polymerase is a reverse transcriptase, optionally wherein the reverse transcriptase is originated from a naturally occurring retron or retron-like sequence. [0076] In an embodiment of any one of the preceding embodiments, the first polymerase is a reverse transcriptase selected from: EcoI-RT, Efe1-RT, Mva1-RT, Cex1-RT, Eco8-RT, Vap1- RT, and Vro1-RT. In an embodiment, the reverse transcriptase is originated from a same naturally occurring retron as the reference retron ncRNA. [0077] In another aspect, the present disclosure provides a chimeric gene editing composition comprising: (a) the ncRNA variant of any one of the preceding embodiments, or the retron variant of any one of the preceding embodiments, (b) a nuclease or a first mRNA encoding the nuclease, (c) a guide RNA (gRNA) associated with the nuclease, and (d) optionally, a second polymerase or a second mRNA encoding the second polymerase. [0078] In an embodiment, the chimeric gene editing composition comprises the second polymerase or the second mRNA encoding the second polymerase, wherein the second polymerase is a reverse transcriptase. In an embodiment, the chimeric gene editing composition comprises the second polymerase or the second mRNA encoding the second polymerase, wherein the second polymerase is a DNA polymerase. [0079] In an embodiment of any one of the preceding embodiments, the nuclease is a nickase. In an embodiment, the nickase comprises a Cas9 nuclease, a Cas9(D10A) nuclease, a TnpB nuclease, or a Cas12a nuclease. [0080] In an embodiment of any one of the preceding embodiments, the ncRNA variant or the retron variant is linked to the gRNA directly or indirectly. In an embodiment, the ncRNA variant or the retron variant, the gRNA, and the first mRNA encoding the nuclease are linked directly or indirectly. RNG043-WO1 PCT Application [0081] In an embodiment of any one of the preceding embodiments, the chimeric gene editing composition further comprises delivery vehicles. In an embodiment, the delivery vehicles encapsulate one or more components selected from: (a) the ncRNA variant or the retron variant, (b) the nuclease or the first mRNA encoding the nuclease, (c) the gRNA associated with the nuclease, and (d) optionally, the second polymerase or the second mRNA encoding the second polymerase. In an embodiment, the delivery vehicle is a lipid nanoparticle. [0082] In another aspect, the present disclosure provides one or more polynucleotides comprising a coding sequence of the ncRNA variant of any one of the preceding embodiments or the retron variant of any one of the preceding embodiments. [0083] In an embodiment, the one or more polynucleotides further comprise a coding sequence of a nuclease or a coding sequence of a gRNA. [0084] In an embodiment, the one or more polynucleotides further comprise a coding sequence of a nuclease and a coding sequence of a gRNA. [0085] In an embodiment of any one of the preceding embodiments, the one or more polynucleotides further comprise a coding sequence of the first polymerase. [0086] In an embodiment of any one of the preceding embodiments, the one or more polynucleotides further comprise a coding sequence of a second polymerase. [0087] In an embodiment of any one of the preceding embodiments, the one or more polynucleotides further comprise one or more promoters, wherein each of the one or more promoters is operably linked to (i) the coding sequence of the ncRNA variant or the retron variant, (ii) a coding sequence of a nuclease, (iii) a coding sequence of a gRNA, (iv) a coding sequence of the first polymerase, or (v) a coding sequence of a second polymerase. [0088] In another aspect, the present disclosure provides a vector comprising the one or more polynucleotides of any one of the preceding embodiments. [0089] In an embodiment, the vector is a plasmid or a viral vector, optionally wherein the viral vector is an AAV or lentiviral vector. [0090] In another aspect, the present disclosure provides a method of editing a target DNA, the method comprising interacting the target DNA with the chimeric gene editing composition of any one of the preceding embodiments, the one or more polynucleotides of any one of the preceding embodiments, or the vector of any one of the preceding embodiments. [0091] In an embodiment, the ncRNA variant in the chimeric gene editing composition, the one or more polynucleotides, or the vector comprises a pair of homology arms specific to a target locus and specific to the target DNA. RNG043-WO1 PCT Application [0092] In an embodiment of any one of the preceding embodiments, the interacting step is performed in vivo or in vitro. [0093] In another aspect, the present disclosure provides a chimeric gene editing composition comprising: (a) a non-coding RNA (ncRNA) comprising a single-stranded RNA comprising a polymerase template, (b) a nickase or a first mRNA encoding the nickase, (c) a guide RNA (gRNA) associated with the nickase, and (d) optionally, a second polymerase or a second mRNA encoding the second polymerase. [0094] In an embodiment, the chimeric gene editing composition comprises the second polymerase or the second mRNA encoding the second polymerase, wherein the second polymerase is a reverse transcriptase. [0095] In an embodiment, the chimeric gene editing composition comprises the second polymerase or the second mRNA encoding the second polymerase, wherein the second polymerase is a DNA polymerase. [0096] In an embodiment of any one of the preceding embodiments, the nickase comprises a Cas9 nuclease, a Cas9(D10A) nuclease, a TnpB nuclease, or a Cas12a nuclease. [0097] In ame embodiments of any one of the preceding embodiments, the ncRNA is linked to the gRNA directly or indirectly. In an embodiment, the ncRNA, the gRNA, and the first mRNA encoding the nickase are linked directly or indirectly. BRIEF DESCRIPTION OF THE DRAWINGS [0098] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [0099] FIG.1 depicts a prime editing system. [00100] FIG.2 depicts a retron editing system. [00101] FIG.3 depicts the chimeric editing system disclosed herein, i.e., the nickase retron template precision editing system which comprises one or more prime editing components and one or more components of a retron editor. [00102] FIG.4A-4D compares the structures of a wildtype ncRNA (A and B) with a templated ncRNA (C) and cognate templated msDNA (D) which are used in the gene editing system described herein. RNG043-WO1 PCT Application [00103] FIG.5 illustrates the process of editing with a gene editing system described herein that utilizes a ncRNA and a retron RT to generate the msDNA to use as the polymerase template to edit the target DNA. [00104] FIG.6 illustrates the process of editing with a gene editing system described herein that utilizes a ncRNA and a retron RT to use the ncRNA as the polymerase template to edit the target DNA. [00105] FIG.7A-7D illustrates the mechanism of editing with the gene editing system described herein. [00106] FIG.8A-8C illustrates various configurations envisioned for the guide RNA and ncRNA components of the gene editing systems described herein. [00107] FIG.8D illustrates various modifications that may be introduced in the templated ncRNAs described herein. [00108] FIG.8E illustrates various configurations envisioned for the guide RNA and ncRNA components of the gene editing systems described herein. [00109] FIG.9 illustrates an dual-editor embodiment of the gene editing system described herein. [00110] FIG.10A-10B illustrates an dual-editor embodiment of the gene editing system described herein. [00111] FIG.11 is a schematic illustrating various modifications of the gene editing system described herein. [00112] FIG.12 is a schematic of an LNP formulation comprising RNA cargo for delivery the gene editing system of the disclosure to a cell, tissue, or patient under in vitro, in vivo, or ex vivo conditions. [00113] FIG.13 is a scatter plot from a pooled screen assay, where a1:a2 variants were identified that showed higher performance than WT retron R6342. [00114] FIG.14 is a scatter plot showing various a1a2 variants identified in the pooled screen assay from FIG.13, graphed by ssDNA production versus melt temperature. [00115] FIG.15 is a graph summarizing edit% as determined by Exact Inserts, Inserts w/indels, and inserts w/ SNPs for WT R6342, a variant lacking an msr spacer and stem (Del1), a variant lacking a msd spacer (Del2) and a variant lacking both (Del1+Del2). [00116] FIG.16 is a set of graphs and a graphic showing the effects of deletion of the msr stem on editing efficiency. Overall, deletion of the msR stem resulted in near complete loss of precise gene editing. RNG043-WO1 PCT Application [00117] FIG.17 is a set of graphs and a graphic showing the effects of altering the a1a2 stem on editing efficiency. These results suggest that the a1:a2 stem structure, instead of sequence, is the important feature for the editing output for ncRNA. [00118] FIG.18 is a set of graphs and a graphic showing the effects of deleting the msD and msR spacer on editing efficiency. Deleting msR spacer alone did not affect the gene editing outcome while msD deletion variants exhibited lower editing efficiency, partially due to the decrease in indel rate in these cell lines. When combined, deletion of both msR spacer and msD spacer resulted in robust editing outcome comparable to WT, suggesting that the ncRNA can be shortened at msD and msR spacer region without a compromise in its function. [00119] FIG.19 is a set of graphs and a graphic showing the effects of a fused a1a2 ncRNA configuration on editing efficiency. A fused a1a2 design (ALT) with both long and short R6342 retron ncRNA (R6342L and R6342S, respectively) was generated. The alternative design with the fused a1a2 loop did not impact editing efficiency for R6342L ncRNA and slightly reduced the editing efficiency of R6342S ncRNA. [00120] FIG.20 is a set of graphs and a graphic showing the effects of deletion of the msR and msD spacers with a fused a1a2 ncRNA configuration on editing efficiency. Deletion of the spacer sequences generally reduced editing efficiencyas compared to WT, but overall the modified constructs still demonstrated appreciable activity. [00121] FIG.21 is a set of graphs and a graphic showing the effects of deletion of the msD stem loop in combination with the msD and msR spacers. ncRNA with an msD stem- loop deletion with a further shortening of the msD spacer and a complete removal of the msR spacer, with the fused a1a2 stem (Alt1 msR Spacer Del-2/msD Del-3) only suffered a small negative effect on gene editing. Incorporation of a MS2 stem-loop at 3’ end of the ncRNA significantly reduced the editing efficiency of such ncRNA (Alt1 msR Spacer Del-2/msD Del- 3 MS2), while the 3’ tail from a snRNA, U7, maintained similar editing efficiency (Alt1 msR Spacer Del-2/msD Del-3 MS2). Together, this demonstrates high level of editing when deleting msD spacer and stem-loop elements, in addition to msR spacer elements. [00122] FIG.22 is a set of graphs and a graphic showing that the U7 snRNA sequence strongly boosted ssDNA production in the short ncRNA. ssDNA production with short ncRNA with deletions of msD, and msR stem-loop was measured. It was found that such ncRNA variants result in a 6-fold reduction in ssDNA production. Incorporation of an MS2 stem-loop (Alt1 msR Spacer Del-2/msD Del-3 MS2) boosted the ssDNA production while incorporation U7 sequence (Alt1 msR Spacer Del-2/msD Del-3 U7) shows a higher enhancement of the ssDNA. RNG043-WO1 PCT Application [00123] FIG.23 is a set of graphs showing that a minimization variant ncRNA with msr spacer deletion allowed up to 20% editing in human hematopoietic stem cells (HSCs) for M (ATG)>T (ACC) mutation installation in the UBA1 gene at 2.5 fold higher efficiency than WT version. [00124] FIG.24 is a set of graphs showing ncRNA with msr/msd spacer deletion and alternative configuration (as presented in FIG.19) allowed up to 40% editing in human hematopoietic stem cells (HSC) for PAM mutation installation or 25 bp insertion in AAVS1 gene, at 2.5 fold higher efficiency than WT version. DEFINITIONS [00125] All technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this the present disclosure belongs. The following references provide one of skill in the art with a general definition of many of the terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed.1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Margham, The Harper Collins Dictionary of Biology (1991). [00126] In some examples, general methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference. [00127] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. RNG043-WO1 PCT Application [00128] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a ncRNA” includes a plurality of ncRNAs and reference to “the reverse transcriptase” includes reference to one or more RTs and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation. For example, claims may be drafted to exclude certain RT sequences. [00129] It is appreciated that certain features of the present disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the present disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the present disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub- combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub combination was individually and explicitly disclosed herein. Biologically active [00130] As used herein, the term “biologically active” refers to a characteristic of an agent (e.g., DNA, RNA, or protein) that has activity in a biological system (including in vitro and in vivo biological system), and particularly in a living organism, such as in a mammal, including human and non-human mammals. For instance, an agent when administered to an organism has a biological effect on that organism, is considered to be biologically active. Bulge [00131] As used herein, the term “bulge” refers to a small region of unpaired base(s) that interrupts a “stem” of base-paired nucleotides. The bulge may comprise one or two single-stranded or unbase-paired nucleotides joined at both ends by base-paired nucleotides of the stem. The bulge can be symmetrical (viz., the two unbase-paired single-stranded regions have the same number of nucleotides), or asymmetrical (viz., the unbase-paired single stranded region(s) have different or unequal numbers of nucleotides), or there is only one unbase-paired nucleotide on one strand. A bulge can be described as A/B (such as a “2/2 bulge,” or a “1/0 bulge”) wherein A represents the number of unpaired nucleotides on the upstream strand of the RNG043-WO1 PCT Application stem, and B represents the number of unpaired nucleotides on the downstream strand of the stem. An upstream strand of a bulge is more 5ʹ to a downstream strand of the bulge in the primary nucleotide sequence. cDNA [00132] As used hereing, the term “cDNA” refers to a strand of DNA copied from an RNA template, e.g., by a reverse transcriptase. Cognate [00133] The term “cognate” refers to two biomolecules that normally interact or co-exist in nature. Complementary [00134] As used herein, the terms “complementary” or “substantially complementary" are meant to refer to a nucleic acid (e.g., RNA, DNA) that comprises a sequence of nucleotides that enables it to non-covalently bind, i.e., form Watson-Crick base pairs and/or G/U base pairs, “anneal”, or “hybridize,” to another nucleic acid in a sequence-specific, antiparallel, manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and/or in vivo conditions of temperature and solution ionic strength. Standard Watson-Crick base-pairing includes: adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C) [DNA, RNA]. In addition, for hybridization between two RNA molecules (e.g., dsRNA), and for hybridization of a DNA molecule with an RNA molecule (e.g., when a DNA target nucleic acid base pairs with a guide RNA, etc.): guanine (G) can also base pair with uracil (U). For example, G/U base-pairing is at least partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anti-codon base-pairing with codons in mRNA. Thus, in the context of this disclosure, a guanine (G) is considered complementary to both a uracil (U) and to an adenine (A). For example, when a G/U base-pair can be made at a given nucleotide position of a dsRNA duplex of a guide RNA molecule, the position is not considered to be non-complementary, but is instead considered to be complementary. [00135] It is understood that the sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable or hybridizable. Moreover, a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a bulge, a loop structure or hairpin structure, etc.). A polynucleotide can comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence complementarity to a target region within the RNG043-WO1 PCT Application target nucleic acid sequence to which it will hybridize. For example, an antisense nucleic acid in which 18 of 20 nucleotides of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary nucleotides. Percent complementarity between particular stretches of nucleic acid sequences within nucleic acids can be determined using any convenient method. Example methods include BLAST programs (basic local alignment search tools) and PowerBLAST programs, such as those described in Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656), the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), e.g., using default settings, which uses the algorithm of Smith and Waterman as described in Adv. Appl. Math., 1981, 2, 482-489, and the like. DNA-guided nuclease [00136] As used herein, an “DNA-guided nuclease” is a type of “programmable nuclease,” and a specific type of “nucleic acid-guided nuclease.” An example of a DNA- guided nuclease is reported in Varshney et al., DNA-guided genome editing using structure- guided endonucleases, Genome Biology, 2016, 17(1), 187, which may be used in the context of the present disclosure and is incorporated herein by reference. As used herein, the term “DNA-guided nuclease” or “DNA-guided endonuclease” refers to a nuclease that associates covalently or non-covalently with a guide RNA thereby forming a complex between the guide RNA and the DNA-guided nuclease. The guide RNA comprises a spacer sequence which comprises a nucleotide sequence having complementarity with a strand of a target DNA sequence. Thus, the DNA-guided nuclease is indirectly guided or programmed to localize to a specific site in a DNA molecule through its association with the guide RNA, which directly binds or anneals to a strand of the target DNA through its complementarity region via Watson- Crick base-pairing. DNA regulatory sequences [00137] As used herein, the terms “DNA regulatory sequences,” “control elements,” and “regulatory elements,” can be used interchangeably herein to refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and/or regulate transcription of a non-coding sequence (e.g., guide RNA) or a coding sequence and/or regulate translation of a mRNA into an encoded polypeptide. RNG043-WO1 PCT Application Donor nucleic acid [00138] By a “donor nucleic acid” or “donor polynucleotide” or “donor DNA” or “HDR donor DNA” it is meant a single-stranded DNA to be inserted at a site cleaved by a programmable nuclease (e.g., a CRISPR/Cas effector protein; a TALEN; a ZFN; a meganuclease) (e.g., after dsDNA cleavage, after nicking a target DNA, after dual nicking a target DNA, and the like). The donor polynucleotide can contain sufficient homology to a genomic sequence at the target site, e.g.70%, 80%, 85%, 90%, 95%, or 100% homology with the nucleotide sequences flanking the target site, e.g., within about 200 bases or less of the target site, e.g., within about 190 bases or less of the target site, e.g., within about 180 bases or less of the target site, e.g., within about 170 bases or less of the target site, e.g., within about 160 bases or less of the target site, e.g., within about 150 bases or less of the target site, e.g., within about 140 bases or less of the target site, e.g., within about 130 bases or less of the target site, e.g., within about 120 bases or less of the target site, e.g., within about 110 bases or less of the target site, e.g., within about 100 bases or less of the target site, e.g., within about 90 bases or less of the target site, e.g., within about 80 bases or less of the target site, e.g., within about 70 bases or less of the target site, e.g., within about 60 bases or less of the target site, e.g., 50 bases or less of the target site, e.g., within about 30 bases, within about 15 bases, within about 10 bases, within about 5 bases, or immediately flanking the target site, to support homology-directed repair between it and the genomic sequence to which it bears homology. Encodes [00139] As used herein, a DNA sequence that “encodes” a particular RNA is a DNA nucleotide sequence that is transcribed into RNA. A DNA polynucleotide may encode an RNA (mRNA) that is translated into protein (and therefore the DNA and the mRNA both encode the protein), or a DNA polynucleotide may encode an RNA that is not translated into protein (e.g. tRNA, rRNA, microRNA (miRNA), a “non-coding” RNA (ncRNA), a guide RNA, etc.). In the case of retrons, the retron DNA may encode the ncRNA loci (which includes the msr and msd regions) as well as a retron RT. Engineered retron [00140] As used herein, the term “engineered retron” or equivalently, “recombinant retron” or “retron variant” refers to a retron that does not occur in nature. In one embodiment, engineered retrons can include wildtype or naturally-occurring retrons that are modified to contain at least one modification, including a single nucleotide substitution, insertion, or deletion, or a substitution, insertion, or deletion of more than one nucleotide, i.e., up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, RNG043-WO1 PCT Application 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or up to 100, or up to 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or up to 2000 nucleotides substituted, inserted, or deleted from a starting point retron (e.g., a wildtype retron). Where more than one nucleotide of a starting point retron (e.g., a wildtype retron) is substituted, deleted, or inserted, the nucleotides may be contiguous or non- contiguous. While an engineered retron as a whole is not naturally-occurring, it may include components such as nucleotide sequences that do occur in nature. For example, an engineered retron can have nucleotide sequences from different organisms (e.g., from different bacteria species), or from completely synthetic / artificial / recombinant nucleic acid sequences. Thus, an engineered retron can have a bacterial nucleotide sequence, a human nucleotide sequence, a viral nucleotide sequence, and/or a synthetic / artificial / recombinant nucleotide sequence, and/or combinations of such sequences. An example of modifications of the recombinant retrons disclosed herein include the insertion of a heterologous nucleic acid sequence in a retron, for example, inserted into the ncRNA locus, such as in the msr or the msd loci. Linking guide RNA molecules to the 5ʹ and/or 3ʹ ends (i.e., linking one at the 5ʹ end of a ncRNA and/or one at the 3ʹ end of a ncRNA) also represent another modification envisioned by the recombinant retrons disclosed herein. In such embodiments, the guide RNA molecules may also be categorized or referred to more generally as types of heterologous nucleic acid sequences used to modify starting point retrons. Exosomes [00141] As used herein, the term “exosomes” refer to small membrane bound vesicles with an endocytic origin. Without wishing to be bound by theory, exosomes are generally released into an extracellular environment from host/progenitor cells post fusion of multivesicular bodies the cellular plasma membrane. As such, exosomes can include components of the progenitor membrane in addition to designed components (e.g. engineered retron). Exosome membranes are generally lamellar, composed of a bilayer of lipids, with an aqueous inter-nanoparticle space. Expression vector [00142] As used herein, the term “expression vector” or “expression construct” refers to a vector that includes one or more expression control sequences, and an “expression control sequence” is a DNA sequence that controls and regulates the transcription and/or translation of another DNA sequence. Suitable expression vectors include, without limitation, plasmids and RNG043-WO1 PCT Application viral vectors derived from, for example, bacteriophage, baculoviruses, tobacco mosaic virus, herpes viruses, cytomegalovirus, retroviruses, vaccinia viruses, adenoviruses, and adeno- associated viruses. Numerous vectors and expression systems are commercially available, such as from Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA), and Invitrogen/Life Technologies (Carlsbad, CA). The present disclosure comprehends recombinant vectors that may include viral vectors, bacterial vectors, protozoan vectors, DNA vectors, or recombinants thereof. Guide RNA [00143] The RNA molecule that binds to a programmable nuclease of a retron-based gene editing systems and which targets the nuclease to a specific location within the targeted polynucleotide sequence is referred to herein as the “guide RNA” or “guide RNA polynucleotide” (also referred to herein as a “guide RNA” or “gRNA” or “crRNA”). In certain embodiments (depending on the particular nuclease to which it interacts with), a guide RNA comprises two segments, a “DNA-targeting segment” and a “protein-binding segment.” By “segment” it is meant a segment/section/region of a molecule, e.g., a contiguous stretch of nucleotides in an RNA. As an illustrative, non-limiting example, a protein-binding segment of a guide RNA can comprise base pairs 5-20 of the RNA molecule that is 40 base pairs in length; and the DNA-targeting segment can comprise base pairs 21-40 of the RNA molecule that is 40 base pairs in length. The definition of “segment,” unless otherwise specifically defined in a particular context, is not limited to a specific number of total base pairs, is not limited to any particular number of base pairs from a given RNA molecule, is not limited to a particular number of separate molecules within a complex, and may include regions of RNA molecules that are of any total length and may or may not include regions with complementarity to other molecules. [00144] The DNA-targeting segment (or “DNA-targeting sequence”) comprises a nucleotide sequence that is complementary to a specific sequence within a targeted polynucleotide sequence (the complementary strand of the targeted polynucleotide sequence) designated the “protospacer-like” sequence herein. The protein-binding segment (or “protein- binding sequence”) interacts with a site-directed modifying polypeptide. When the site- directed modifying polypeptide is a CRISPR nuclease, site-specific cleavage of the targeted polynucleotide sequence may occur at locations determined by both (i) base-pairing complementarity between the guide RNA and the targeted polynucleotide sequence; and (ii) a short motif (referred to as the protospacer adjacent motif (PAM)) in the targeted polynucleotide sequence. RNG043-WO1 PCT Application Heterologous nucleic acid sequence [00145] As used herein, the term “heterologous nucleic acid” refers to a genotypically distinct entity from that of the rest of the entity to which it is compared or into which it is introduced or incorporated. For example, a polynucleotide introduced by genetic engineering techniques into a different cell type is a heterologous polynucleotide (e.g., DNA or RNA) and, if expressed, can encode a heterologous polypeptide. Similarly, a cellular sequence (e.g., a gene or portion thereof) that is incorporated into a viral vector is a heterologous nucleotide sequence with respect to the vector. In some embodiments, the heterologous sequence inserted into the wild-type retron regions does not naturally insert into such regions (e.g., the engineered retron with the inserted heterologous sequence is not naturally existing). For example, the heterologous sequence can be from the same species of bacteria in which the wild-type retron is normally found, so long as the heterologous sequence is not naturally inserted in the wild-type retron at the location in which the heterologous sequence is inserted. In certain embodiments, the heterologous sequence is a mammalian sequence (e.g., a human sequence), or a reverse complement thereof. Heterologous nucleic acid sequences introduced into retrons can including without limitation guide RNA sequences, HDR donor templates, protein-encoding genes, or non-coding functional RNA elements (e.g., stem-loops, hairpins, and bulges). Homology-directed repair [00146] As used herein, “homology-directed repair (HDR)” refers to the specialized form DNA repair that takes place, for example, during repair of double-strand breaks in cells. This process requires nucleotide sequence homology, uses a “donor” molecule to template repair of a “target” molecule (i.e., the one that experienced the double-strand break), and leads to the transfer of genetic information from the donor to the target. Homology-directed repair may result in an alteration of the sequence of the target molecule (e.g., insertion, deletion, mutation), if the donor polynucleotide differs from the target molecule and part or all of the sequence of the donor polynucleotide is incorporated into the targeted polynucleotide sequence. Identical [00147] As used herein, the term “identical” refers to two or more sequences or subsequences which are the same. In addition, the term “substantially identical,” as used herein, refers to two or more sequences which have a percentage of sequential units which are the same when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a comparison algorithm or by manual RNG043-WO1 PCT Application alignment and visual inspection. By way of example only, two or more sequences may be “substantially identical” if the sequential units are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a specified region. Such percentages to describe the “percent identity” of two or more sequences. The identity of a sequence can exist over a region that is at least about 75-100 sequential units in length, over a region that is about 50 sequential units in length, or, where not specified, across the entire sequence. This definition also refers to the complement of a test sequence. [00148] Alternatively, substantially identical or similarity exists when a nucleic acid or fragment thereof hybridizes to another nucleic acid, to a strand of another nucleic acid, or to the complementary strand thereof, under stringent hybridization conditions. “Stringent hybridization conditions” and “stringent wash conditions” in the context of nucleic acid hybridization experiments depend upon a number of different physical parameters. Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, solvents, the base composition of the hybridizing species, length of the complementary regions, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. One having ordinary skill in the art knows how to vary these parameters to achieve a particular stringency of hybridization. Lipid nanoparticle (LNP) [00149] As used herein, the term “lipid nanoparticle” or LNP refers to a type of lipid particle delivery system formed of small solid or semi-solid particles possessing an exterior lipid layer with a hydrophilic exterior surface that is exposed to the non-LNP environment, an interior space which may aqueous (vesicle like) or non-aqueous (micelle like), and at least one hydrophobic inter-membrane space. LNP membranes may be lamellar or non-lamellar and may be comprised of 1, 2, 3, 4, 5 or more layers. In some embodiments, LNPs may comprise a nucleic acid (e.g. engineered retron) into their interior space, into the inter membrane space, onto their exterior surface, or any combination thereof. In some embodiments, an LNP of the present disclosure comprises an ionizable lipid, a structural lipid, a PEGylated lipid (aka PEG lipid), and a phospholipid. In alternative embodiments, an LNP comprises an ionizable lipid, a structural lipid, a PEGylated lipid (aka PEG lipid), and a zwitterionic amino acid lipid. [00150] Further discuss of liposomes can be found, for example, in Tenchov et al., “Lipid Nanoparticles – From Liposomes to mRNA Vaccine Delivery, a Landscape of Diversity and Advancement,” ACS Nano, 2021, 15, pp.16982-17015 (the contents of which are incorporated by reference). RNG043-WO1 PCT Application Linker [00151] As used herein, the term “linker” refers to a molecule linking or joining two other molecules or moieties. The linker can be an amino acid sequence in the case of a linker joining two fusion proteins. For example, an RNA-guided nuclease (e.g., Cas12a) can be fused to a retron reverse transcriptase by an amino acid linker sequence. The linker can also be a nucleotide sequence in the case of joining two nucleotide sequences together. For example, in the instant case, a ncRNA at its 5ʹ and/or 3ʹ ends may be linked by a nucleotide sequence linker to one or more guide RNAs. In other embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5-100 amino acids in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40- 45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150- 200 amino acids in length. Longer or shorter linkers are also contemplated. Liposomes [00152] As used herein, the term “liposomes” refer to a type of lipid particle delivery system comprising small vesicles that contain at least one lipid membrane surrounding an aqueous inner-nanoparticle space that is generally not derived from a progenitor/host cell. Liposomes are a versatile carrier platform in that they are capable of transporting hydrophobic or hydrophilic molecules, including small molecules, proteins, and nucleic acids into cells. They were the earliest developed generation of nanoscale medicine delivery platform. Numerous liposomal drug formulations have been approved for human medicines, e.g., Doxil, a lipid nanoparticle formulation of the antitumor agent doxorubicin. In some examples, the liposomes can include those described in Tenchov et al., “Lipid Nanoparticles – From Liposomes to mRNA Vaccine Delivery, a Landscape of Diversity and Advancement,” ACS Nano, 2021, 15, pp.16982-17015 (the contents of which are incorporated by reference). Loop [00153] As used herein, the term “loop” in a polynucleotide refers to a single stranded stretch of one or more nucleotides, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, wherein the most 5ʹ nucleotide and the most 3ʹ nucleotide of the loop are each linked to a base-paired nucleotide in a stem. Micelles [00154] As used herein, the term “micelles” refer to small particles which do not have an aqueous intra-particle space. Nanoparticle RNG043-WO1 PCT Application [00155] As used herein, the term “nanoparticle” refers to any nanoscale particle typically ranging in size from about 1 nm to 1000 nm. Nuclear localization sequence (NLS) [00156] As used herein, the term“nuclear localization sequence” or“NLS” refers to an amino acid sequence that promotes import of a protein (e.g., a RNA-guided nuclease) into the cell nucleus, for example, by nuclear transport. For example, NLS sequences are described in Plank et al., international PCT application, PCT/EP2000/011690, filed November 23, 2000, published as WO/2001/038547 on May 31, 2001, the contents of which are incorporated herein by reference for its disclosure of exemplary nuclear localization sequences. Nucleic acid [00157] As used herein, the term “nucleic acid” or “nucleic acid molecule” or “nucleic acid sequence” or “polynucleotide” generally refer to deoxyribonucleic or ribonucleic oligonucleotides in either single- or double-stranded form. The term may also encompass oligonucleotides containing known analogues of natural nucleotides. The term also may also encompass nucleic acid-like structures with synthetic backbones, such as, for example, in Eckstein, 1991; Baserga et al., 1992; Milligan, 1993; WO 97/03211; WO 96/39154; Mata, 1997; Strauss-Soukup, 1997; and Samstag, 1996. The term encompasses both ribonucleic acid (RNA) and DNA, including cDNA (including RT DNA), genomic DNA, synthetic, synthesized (e.g., chemically synthesized) DNA, and/or DNA (or RNA) containing nucleic acid analogs. The nucleotides Adenine (A), Thymine (T), Guanine (G) and Cytosine (C) also may (or may not) encompass nucleotide modifications, e.g., methylated and/or hydroxylated nucleotides, e.g., Cytosine (C) encompasses 5-methylcytosine and 5- hydroxymethylcytosine. Nucleic acid-guided nuclease [00158] As used herein, the term “nucleic acid-guided nuclease” or “nucleic acid-guided endonuclease” refers to a nuclease that associates covalently or non-covalently with a guide nucleic acid (e.g., a guide RNA or a guide DNA) thereby forming a complex between the guide nucleic acid and the nucleic acid-guided nuclease. The guide nucleic acid comprises a spacer sequence which comprises a nucleotide sequence having complementarity with a strand of a target DNA sequence. Thus, the nucleic acid-guided nuclease is indirectly guided or programmed to localize to a specific site in a DNA molecule through its association with the guide nucleic acid, which directly binds or anneals to a strand of the target DNA through its complementarity region via Watson-Crick base-pairing. In some embodiments, the nucleic acid-guided nuclease will include a DNA-binding activity (e.g., as in the case for CRISPR Cas9). Most commonly, the nucleic acid-guided nuclease is programmed by associating with a RNG043-WO1 PCT Application guide RNA molecule and in such cases the nuclease may be called “RNA-guided nuclease.” When programmed by a guide DNA, the nuclease may be called a “DNA-guided nuclease.” Nucleic acid-guided, RNA-guided, or DNA-guided nucleases may also be referred to as “programmable nucleases,” which also include other classes of programmable nucleases which associate with specific DNA sequences through amino acid / nucleotide sequence recognition (e.g., zinc fingers nucleases (ZFN) and transcription activator like effector nucleases (TALEN)) rather than through guide RNAs. In addition, any nuclease contemplated herein may also be engineered to remove, inactivate, or otherwise eliminate one or more nuclease activities (e.g., by introducing a nuclease-inactiving mutation in the active site(s) of a nuclease). A nuclease that has been modified to remove, inactivate, or otherwise eliminate all nuclease activity may be referred to as a “dead” nuclease. A dead nuclease is not able to cut either strand of a double-stranded DNA molecule. A nuclease that has been modified to remove, inactivate, or otherwise eliminate at least one nuclease activity but which still retains at least one nuclease activity may be referred to as a “nickase” nuclease. A nickase nuclease cuts one strand of a double-stranded DNA molecule, but not both strands. For example, a CRISPR Cas9 naturally comprises two distinct nuclease activity domains, namely, the HNH domain and the RuvC domain. The HNH domain cuts the strand of DNA bound to the guide RNA and the RuvC domain cuts the protospacer strand. One can obtain a nickase Cas9 by inactivating either the HNH domain or the RuvC domain. One can obtain a dead Cas9 by inactivating both the HNH domain and the RuvC domain. Other RNA-guided nuclease may be similarly converted to nickases and/or dead nucleases by inactivating one or more of the existing nuclease domains. Operably linked [00159] As used herein, the term “operably linked” or “under transcriptional control,” when used in conjunction with the description of a promoter, refers to the correct location and orientation in relation to a polynucleotide (e.g., a coding sequence) to control the initiation of transcription by RNA polymerase and expression of the coding sequence, such as one for the msr gene, msd gene, and/or the ret gene. Other transcriptional control regulatory elements (e.g., enhancer sequences, transcription factor binding sites) may also be operably linked to a gene if their location relative to a gene controls or regulates the expression of the gene. Programmable nuclease [00160] As used herein, the term “programmable nuclease” is meant to refer to a polypeptide that has the property of selective localization to a specific desired nucleotide sequence target in a nucleic acid molecule (e.g., to a specific gene target) due to one or more RNG043-WO1 PCT Application targeting functions. Such targeting functions can include one or more DNA-binding domains, such as zinc finger domains characteristic of many different types of DNA binding proteins or TALE domains characteristic of TALEN proteins. Such targeting function may also include the ability to associate and/or form a complex with a guide RNA, which then localizes to a specific site on the DNA which bears a sequence that is complementary to a portion of the guide RNA (i.e., the spacer of the guide RNA). In some embodiments, the programmable nuclease may be a single protein which comprises both a domain that binds directly (e.g., a ZF protein) or indirectly (e.g., an RNA-guided protein) to a target DNA site, as well as a nuclease domain. In other embodiments, the programmable nuclease may be a composite of two or more separate proteins or domains (from different proteins) which together provide the necessary functions of selective DNA binding and nuclease activity. For example, the programmable nuclease may comprise a (a) nuclease-inactive RNA-guided nuclease (which still is capable of binding a guide RNA, localizing to a target DNA, and binding to the target DNA, but not capable of cutting or nicking the strands) fused to a (b) nuclease protein or domain, such as a FokI nuclease. Promoter [00161] As used herein, the term“promoter” is art-recognized and refers to a nucleic acid molecule with a sequence recognized by the cellular transcription machinery and which is able to initiate transcription of a downstream gene. A promoter can be constitutively active, meaning that the promoter is always active in a given cellular context, or conditionally active, meaning that the promoter is only active in the presence of a specific condition. For example, a conditional promoter may only be active in the presence of a specific protein that connects a protein associated with a regulatory element in the promoter to the basic transcriptional machinery, or only in the absence of an inhibitory molecule. Within the promoter sequence will be found a transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain “TATA” boxes and “CAT” boxes. Various promoters, including inducible promoters, may be used to drive expression by the various vectors of the present disclosure. Recombinant nucleic acid [00162] A “recombinant nucleic acid” or “recombinant nucleotide” refers to a molecule that is constructed by joining nucleic acid molecules, which optionally may self-replicate in a live cell. Retron RNG043-WO1 PCT Application [00163] As used herein, the term “retron” refers to a specific type of naturally-occuring and distinct DNA sequence found in the genome of many bacteria which typically encodes three distinct components, namely, (a) a non-coding RNA (“ncRNA”) (comprising continguous inverted sequences (msr and msd), (b) a reverse transcriptase (RT)-coding gene (ret), and (c) in many cases, a retron-associated gene of unknown function. Retrons are particularly defined by their unique ability to produce a satellite DNA known as msDNA (multicopy single-stranded DNA). The ncRNA (comprising the msr and msd elements) and the ret gene are transcribed as a single polycitronic RNA transcript which processed into the ncRNA transcript and a transcript encoding the ret gene. The ncRNA then becomes folded into a specific secondary structure. Once translated, the RT then binds the folded ncRNA and reverse transcribes the msd region to form a single strand of cDNA (the msDNA) that remains covalently attached to the RNA template via a 2’-5ʹ phophodiester bond and base-pairing between the 3ʹ ends of the msDNA and the RNA template. See FIG.2 which provides a schematic of the production of an msDNA from a naturally-occurring retron. Retron component [00164] As used herein, the term “retron component” refers to a distinct element or feature of a retron, namely (a) a non-coding RNA (“ncRNA”) (comprising continguous inverted sequences (msr and msd), (b) a reverse transcriptase (RT)-coding gene (ret), and (c) in many cases, a retron-associated gene of unknown function. RNA-guided nuclease [00165] As used herein, an “RNA-guided nuclease” is a type of “programmable nuclease,” and a specific type of “nucleic acid-guided nuclease.” As used herein, the term “RNA-guided nuclease” or “RNA-guided endonuclease” refers to a nuclease that associates covalently or non-covalently with a guide RNA thereby forming a complex between the guide RNA and the RNA-guided nuclease. The guide RNA comprises a spacer sequence which comprises a nucleotide sequence having complementarity with a strand of a target DNA sequence. Thus, the RNA-guided nuclease is indirectly guided or programmed to localize to a specific site in a DNA molecule through its association with the guide RNA, which directly binds or anneals to a strand of the target DNA through its complementarity region via Watson- Crick base-pairing. Sequence identity [00166] As used herein, the term “sequence identity” refers to the overall relatedness between polymeric molecules, e.g., between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of the percent RNG043-WO1 PCT Application identity of two polynucleotide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). For example, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; each of which is incorporated herein by reference. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller as described in CABIOS, 1989, 4:11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna CMP matrix. Methods commonly employed to determine percent identity between sequences can include, but are not limited to those disclosed in Carillo, H. and Lipman, D., SIAM J Applied Math., 48:1073 (1988); incorporated herein by reference. Techniques for determining identity are codified in publicly available computer programs. Exemplary computer software to determine homology between two sequences can include, but are not limited to, GCG program package as described in Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984), BLASTP, BLASTN, and FASTA as described in Altschul, S. F. et al., J. Molec. Biol., 215, 403 (1990). RNG043-WO1 PCT Application [00167] It is noted that when this disclosure speaks to a polypeptide (including anywhere in this specification, including in Table A of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872, and the Examples) having a percent identity with respect to another amino acid sequence (a reference amino acid sequence), such as a polypeptide at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to another amino acid sequence (a reference amino acid sequence), it is advantageous that in the polypeptide having a percent identity to the reference amino acid sequence conserved regions of the reference amino acid sequence (e.g., conserved when compared with other retron RTs, such as those identified herein) be preserved and/or that the polypeptide has at least one activity selected from reverse transcriptase; endonuclease activity; endoribonuclease activity, or RNA-guided DNase activity and/or that the polypeptide of which comprises: a. one or more α-helical recognition lobe (REC) and a nuclease lobe (NUC); b. a Wedge (WED), α-helical recognition lobe (REC), PAM-interacting (PI), RuvC nuclease, Bridge Helix (BH) and NUC domains; or c. one or more domains selected from RuvC, REC, WED, BH, PI and NUC domains and/or that the polypeptide recognizes or binds a guide RNA or ncRNA as the case may be. Likewise, when this disclosure speaks to a nucleic acid sequence or molecule having a percent identity with respect to a nucleic acid sequence having a percent identity with respect to another nucleic acid sequence or molecule (a reference nucleic acid sequence), such as a nucleic acid sequence at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to another nucleic acid sequence, it is advantageous that in the nucleic acid sequence that has a percent identity to the reference nucleic acid sequence that conserved regions of the reference nucleic acid sequence (e.g., conserved when compared with other retron ncRNAs, such as those identified herein) be preserved and/or that in the polypeptide that is expressed from the nucleic acid sequence that has a percent identity to the reference nucleic acid sequence that the polypeptide contain conserved region(s) (e.g., conserved when compared with other retron sequences, such as those identified herein) and/or that the polypeptide has at least one activity selected from reverse transcriptase; endonuclease RNG043-WO1 PCT Application activity; endoribonuclease activity, or RNA-guided DNase activity and/or that the polypeptide of which comprises: a. one or more α-helical recognition lobe (REC) and a nuclease lobe (NUC); b. a Wedge (WED), α-helical recognition lobe (REC), PAM-interacting (PI), RuvC nuclease, Bridge Helix (BH) and NUC domains; or c. one or more domains selected from RuvC, REC, WED, BH, PI and NUC domains and/or that the polypeptide recognizes or binds a guide RNA. Subject [00168] As used herein, the term“subject” refers to an individual organism, for example, an individual mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, a goat, a cattle, a cat, or a dog. In some embodiments, the subject is a vertebrate, an amphibian, a reptile, a fish, an insect, a fly, or a nematode. In some embodiments, the subject is a research animal. In some embodiments, the subject is genetically engineered, e.g., a genetically engineered non-human subject. The subject may be of either sex and at any stage of development. The terms “individual,” “subject,” “host,” and “patient,” used interchangeably herein. Stem [00169] As used herein, the term “stem” refers to two or more base pairs, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more base pairs, formed by inverted repeat sequences connected at a “tip,” where the more 5ʹ or “upstream” strand of the stem bends to allows the more 3ʹ or “downstream” strand to base-pair with the upstream strand. The number of base pairs in a stem is the “length” of the stem. The tip of the stem is typically at least 3 nucleotides, but can be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more nucleotides. Larger tips with more than 5 nucleotides are also referred to as a “loop.” An otherwise continuous stem may be interrupted by one or more bulges as defined herein. The number of unpaired nucleotides in the bulge(s) are not included in the length of the stem. The position of a bulge closest to the tip can be described by the number of base pairs between the bulge and the tip (e.g., the bulge is 4 bps from the tip). The position of the other bulges (if any) further away from the tip can be described by the number of base pairs in the stem between the bulge in question and the tip, excluding any unpaired bases of other bulges in between. Synthetic or artificial nucleic acid [00170] A “synthetic or artificial nucleic acid” refers nucleic acids that are non-naturally occurring sequences. Such sequences do not originate from, or are not known to be present in RNG043-WO1 PCT Application any living organism (e.g., based on sequence search in existing sequence databases). Recombinant nucleic acids and synthetic nucleic acids also include those molecules that result from the replication of either of the foregoing. Engineered nucleic acid constructs of the present disclosure, such as the engineer ed retron described herein, may be encoded by a single molecule (e.g., encoded by or present on the same plasmid or other suitable vector) or by multiple different molecules (e.g., multiple independently-replicating vectors). Target site [00171] As used herein, a “target site” as used herein is a polynucleotide (e.g., DNA such as genomic DNA) that includes a site or specific locus (“target site” or “target sequence”) targeted by a recombinant retron genome modification system disclosed herein. In the context of retron genome modification systems disclosed herein that comprise an RNA-guided nuclease, a target sequence is the sequence to which the guide sequence of a guide nucleic acid (e.g., guide RNA) will hybridize. For example, the target site (or target sequence) 5ʹ- GTCAATGGACC-3ʹ (SEQ ID NO:19933) within a target nucleic acid is targeted by (or is bound by, or hybridizes with, or is complementary to) the sequence 5ʹ-GGTCCATTGAC-3ʹ (SEQ ID NO:19934). Suitable hybridization conditions include physiological conditions normally present in a cell. For a double stranded target nucleic acid, the strand of the target nucleic acid that is complementary to and hybridizes with the guide RNA is referred to as the “complementary strand” or “target strand”; while the strand of the target nucleic acid that is complementary to the “target strand” (and is therefore not complementary to the guide RNA) is referred to as the “non-target strand” or “non-complementary strand.” Treatment [00172] As used herein, the terms “treatment,” “treat,” and “treating,” refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed and/or after a disease has been diagnosed. In other embodiments, treatment may be administered in the absence of symptoms, e.g., to prevent or delay onset of a symptom or inhibit onset or progression of a disease. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their recurrence. Upstream and downstream RNG043-WO1 PCT Application [00173] As used herein, the terms “upstream” and “downstream” are terms of relativity that define the linear position of at least two elements located in a nucleic acid molecule (whether single or double-stranded) that is orientated in a 5ʹ-to-3ʹ direction. A first element is said to be upstream of a second element in a nucleic acid molecule where the first element is positioned somewhere that is 5ʹ to the second element. Conversely, a first element is downstream of a second element in a nucleic acid molecule where the first element is positioned somewhere that is 3ʹ to the second element. Variant [00174] As used herein the term “variant” should be taken to mean the exhibition of qualities that have a pattern that deviates from what occurs in nature, e.g., a variant retron RT is retron RT comprising one or more changes in amino acid residues as compared to a wild type retron RT amino acid sequence. The term“variant” encompasses homologous proteins having at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% percent identity with a reference sequence and having the same or substantially the same functional activity or activities as the reference sequence. The term also encompasses mutants, truncations, or domains of a reference sequence, and which display the same or substantially the same functional activity or activities as the reference sequence. Vector [00175] As used herein, the term “vector” permits or facilitates the transfer of a polynucleotide from one environment to another. It is a replicon such as a plasmid, phage, or cosmid into which another DNA segment may be inserted so as to bring about the replication of the inserted segment (e.g., the subject engineered retron). Generally, a vector is capable of replication when associated with the proper control elements. The term “vector” may include cloning and expression vectors, as well as viral vectors and integrating vectors. Wild type [00176] As used herein the term “wild type” is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene, protein, or characteristic as it occurs in nature as distinguished from mutant or variant forms. DETAILED DESCRIPTION [00177] The present disclosure provides systems, methods and compositions used for precise genome editing, including installing nucleic acid insertions, replacements, and deletions at targeted and precise genome sites, wherein said systems, methods, and compositions are based on novel and/or modified retrons or components thereof, such as modified versions of the retron RTs of Table X of U.S. Application No.18/087,673, or RNG043-WO1 PCT Application International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872, modified versions of the ncRNAs of Table A of U.S. Application No. 18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872, and modified versions of the RTs of Table B of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872. [00178] In one aspect, the present disclosure provides a non-coding RNA variant (ncRNA variant) comprising a reference retron non-coding RNA (ncRNA) with one or more modifications. The reference retron ncRNA can comprise from 5’ to 3’ direction: a1 region, first branching guanosine, msr, msd, and a2 region, In some embodiments, the reference retron ncRNA further comprises second branching guanisine. In some embodiments, the one or more modifications comprises (i) linkage of the a1 region and the a2 region, (ii) deletion of at least a portion of the msr; (iii) deletion of at least a portion of the msd, (iv) addition of a single- stranded RNA comprising a polymerase template, or (v) addition of an RNA motif. The ncRNA variant can be incorporated into a recombinant or engineered retron and provide improved functionality and/or properties compared to the reference retron. [00179] Accordingly, in one aspect, the present disclosure provides recombinant retrons comprising one or more genetic modifications which improves the functionality and/or properties of a retron. Such genetic modifications can include a mutation, insertion, deletion, inversion, replacement, substitution, or translocation of one or more contiguous or non- contiguous nucleobases in a nucleic acid molecule encoding a retron or a component of a retron, such as an ncRNA or a reverse transcriptase. In various aspects, the retron that becomes modifed with the one or more genetic modifications (i.e., the “pre-modified” or “unmodified” retron or retron component) is a naturally occurring retron or retron component (e.g., naturally occuring ncRNA of Table A of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872 or RT) with the ability to facilitate homology-dependent recombination (or HDR) in a cell, thereby resulting in a relative increase in the concentrations or amounts of msDNA comprising a DNA donor template. In particular embodiments, the recombinant retrons are based on and/or derived from a naturally-occurring retron, such as any retron- related sequence provided by Table X of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872 (the introduction of the one or more genetic modifications into a set of 7257 previously unknown retrons discovered through computational methods described herein (e.g., see RNG043-WO1 PCT Application Examples). In other examples, the recombinant retrons can be based on introducing the one or more genetic modifications into previously available retron sequences, as described in “Mestre et al., Systematic Prediction of Genes Functionally Associated with Bacterial Retrons and Classification of The Encoded Tripartite Systems, Nucleic Acids Research, Volume 48, Issue 22, 16 December 2020, Pages 12632-12647” (incorporated herein by reference)) to achieve recombinant retrons with the enhanced ability to produce increased concentrations or amounts of msDNA comprising a DNA donor template. [00180] In another aspect, the present disclosure further provides nucleic acid molecules encoding the recombinant retrons and/or recombinant retron components (e.g., a recombinant ncRNA and/or a recombinant retron RT). In still another aspect, the present disclosure provides genome editing systems comprising recombinant retron components (e.g., recombinant ncRNA and/or recombinant RT), programmable nucleases (e.g., RNA-guided nucleases, such as CRISPR-Cas proteins, ZFPs, and TALENS), and guide RNAs (in the case where RNA-guide nucleases are used in said genome editing systems). In a further aspect, the disclosure provides nucleic acid molecules encoding the described genome editing systems and said components thereof, as well as polypeptides making up the components of said genome editing systems. In yet another aspect, the disclosure provides vectors for transferring and/or expressing the genome editing systems, e.g., under in vitro, ex vivo, and in vivo conditions. In still another aspect, the disclosure provides cell-delivery compositions and methods, including compositions for passive and/or active transport to cells (e.g., plasmids), delivery by virus- based recombinant vectors (e.g., AAV and/or lentivirus vectors), delivery by non-virus-based systems (e.g., liposomes and LNPs), and delivery by virus-like particles. Depending on the delivery system employed, the retron-based genome editing systems described herein may be delivered in the form of DNA (e.g., plasmids or DNA-based virus vectors), RNA (e.g., ncRNA and mRNA delivered by LNPs), a mixture of DNA and RNA, protein (e.g., virus-like particles), and ribonucleoprotein (RNP) complexes. Any suitable combinations of approaches for delivering the components of the herein disclosed retron-based genome editing systems may be employed. In one embodiment, each of the components of the retron-based genome editing system is delivered by an all-RNA system, e.g., the delivery of one or more RNA molecules (e.g., mRNA and/or ncRNA) by one or more LNPs, wherein the one or more RNA molecules form the ncRNA and guide RNA (as needed) and/or are translated into the polypeptide components (e.g., the RT and a programmable nuclease). In yet another aspect, the disclosure provides methods for genome editing by introducing a retron-based genome editing system described herein into a cell (e.g., under in vitro, in vivo, or ex vivo conditions) RNG043-WO1 PCT Application comprising a target edit site, thereby resulting in an edit at the target edit. In other aspects, the disclosure provides formulations comprising any of the aforementioned components for delivery to cells and/or tissues, including in vitro, in vivo, and ex vivo delivery, recombinant cells and/or tissues modified by the recombinant retron-based genome modification systems and methods described herein, and methods of modifying cells by conducting genome editing and related DNA donor-dependent methods, such as recombineering, or cell recording, using the herein disclosed retron-based genome modification systems. The disclosure also provides methods of making the recombinant retrons, retron-based genome modification systems, vectors, compositions, and formulations described herein, as well as to pharmaceutical compositions and kits for modifying cells under in vitro, in vivo, and ex vivo conditions that comprise the herein disclosed genome editing and/or modification systems. [00181] Described herein are engineered retrons comprising one or more heterologous nucleic acids. The one or more heterologous nucleic acids may be inserted, for example, at or within a location selected from: the msd locus, upstream of the msr locus, upstream of the msd locus, and downstream of the msd locus. In some embodiments, the engineered retrons have structural improvements over their naturally existing counterparts or wild-type retrons at least with respect to the encoded ncRNA and/or the reverse transcriptase (RT), such that the engineered retron or the encoded ncRNA thereof, when delivered to a host cell, such as a mammalian host cell, exhibits various functional improvements over its naturally existing/wild-type retron elements. [00182] Exemplary, but non-limiting, functional improvements may include any one or more of the features described herein. For example, in some embodiments, the engineered retron may comprise a sequence modification (e.g., insertion, deletion, and/or substitution of one or more nucleotide(s)) in the msr locus and/or the msd locus that: i) modulates (e.g., enhances) reverse transcription, processivity, accuracy/fidelity, and/or production of the msDNA (e.g., in the mammalian cell); ii) modulates (e.g., reduces) immunogenicity of the ncRNA encoded by the engineered retron (e.g., encoded by the msr locus and/or the msd locus) in a host (e.g., a host comprising the mammalian cell); iii) comprises a nucleotide sequence that modulates (e.g., inhibits or antagonizes) a function of the msDNA; and/or iv) modulates (e.g., improves) efficiency of targeted genomic engineering. [00183] Thus, in general, the engineered retron is an engineered nucleic acid construct comprising: a) a first polynucleotide encoding a non-coding RNA (ncRNA), the first polynucleotide comprising: i) an msr locus encoding the msr RNA portion of a multi-copy RNG043-WO1 PCT Application single-stranded DNA (msDNA); and ii) an msd locus encoding the msd RNA portion of the msDNA; and b) one or more heterologous nucleic acids inserted at or within a location selected from: the msd locus, upstream of the msr locus, upstream of the msd locus, and downstream of the msd locus. [00184] The engineered nucleic acid construct (e.g., the engineered retron or retron variant) may further comprise a second polynucleotide encoding a reverse transcriptase (RT), or a portion thereof, wherein the encoded RT is capable of synthesizing a DNA copy of at least a portion of the msd locus encoding the msDNA. [00185] In certain embodiments, the engineered retron of the present disclosure encodes a reverse transcriptase (RT) or a functional domain thereof, comprising: i) a polypeptide listed in Table A of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polypeptide listed in Table A of U.S. Application No. 18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872; and/or ii) a polypeptide listed in any one of Table C of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872. In some embodiments, the RT does not comprise a polypeptide listed in Table X of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872. [00186] In certain embodiments, the engineered retron of the present disclosure encodes a reverse transcriptase (RT) or a functional domain thereof, comprising: i) a polynucleotide listed in Table A of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872, or a polynucleotide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polynucleotide listed in Table A of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or RNG043-WO1 PCT Application International Application No. PCT/US2023/072872; and/or ii) a consensus polynucleotide sequence listed in Table C of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872. In some embodiments, the polynucleotide encoding the RT does not comprise a polynucleotide of Table X of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872. [00187] In certain embodiments, the engineered retron of the present disclosure encodes an ncRNA comprising: (I) an ncRNA listed in Table B of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872, or an ncRNA having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to an ncRNA in Table B of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872. [00188] In certain embodiments, the engineered retron of the present disclosure encodes an ncRNA and a reverse transcriptase (RT) or a functional domain thereof, wherein the ncRNA and the RT or functional domain thereof are as described above. [00189] Specifically, in such embodiment, the ncRNA may comprise: (I) an ncRNA listed in Table B of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872, or an ncRNA having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to an ncRNA listed in Table B of U.S. Application No. 18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872. [00190] Also in such embodiment, the reverse transcriptase (RT) or functional domain thereof comprises: (A) i) a polypeptide listed in Table A of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least RNG043-WO1 PCT Application 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polypeptide listed in Table A of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872; and/or ii) a polypeptide listed in Table C of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872; optionally, the RT does not comprise a polypeptide listed in Table X of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872; OR (B) i) a polynucleotide listed in Table A of U.S. Application No. 18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872, or a polynucleotide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polynucleotide in Table A of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872; and/or optionally, the polynucleotide encoding the RT does not comprise a polynucleotide in Table X of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872. [00191] In certain embodiments, the engineered nucleic acid construct comprises: 1) an msr locus (that encodes the msr RNA portion of an msDNA); 2) an msd locus encoding the msd RNA portion of the msDNA; 3) a sequence encoding a retron reverse transcriptase (RT), wherein the msd RNA is capable of being reverse transcribed to form the msDNA by the retron reverse transcriptase (RT); and, 4) a heterologous nucleic acid inserted at or within the msd locus, upstream of the msr locus, upstream or downstream of the msd locus; wherein the engineered nucleic acid construct is engineered based on and/or to resemble a secondary structure of a wild-type or consensus retron encoding a wild-type or consensus retron ncRNA encompassed by: a) any one of the sequences and/or structures as depicted in any one of SEQ ID NOs: of Table B and/or FIGs.2-27 of of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872; or b) a variant of a), having: i) up to 1, 2, or 3 (e.g., up to 1) nucleotide changes per 10 red lettered-nucleotides; ii) up to 4, 5, or 6 (e.g., up to 1 or 2) nucleotide changes per 10 black RNG043-WO1 PCT Application lettered-nucleotides; and/or iii) up to 7, 8, or 9 (e.g., up to 3 or 4) nucleotide changes per 10 grey lettered-nucleotides; and/or optionally further comprising: i) 7, 8, 9, or 10 (e.g., 9 or 10) nucleotides present per 10 red-circled nucleotides; ii) 6, 7, 8, 9, or 10 (e.g., 8, 9 or 10) nucleotides present per 10 black-circled nucleotides; iii) 4, 5, 6, 7, 8, 9, or 10 (e.g., 6, 7, 8, 9 or 10) nucleotides present per 10 grey-circled nucleotides; and/or iv) 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., 4, 5, 6, 7, 8, 9, or 10) nucleotides present per 10 white-circled nucleotides; wherein the ncRNA does not comprise an ncRNA associated with the sequences of Table X of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872. [00192] The engineered nucleic acid construct (e.g., the engineered retron) may comprise one or more sequence modifications (e.g., an insertion, deletion, and/or substitution of one or more nucleotide(s)) in the msr locus and/or the msd locus that: a) modulates (e.g., enhances) reverse transcription, processivity, accuracy/fidelity, and/or production of the msDNA (e.g., in the mammalian cell); b) modulates (e.g., reduces) immunogenicity of ncRNA encoded by the engineered retron (e.g., the msr locus and/or the msd locus) in a host (e.g., a host comprising the mammalian cell); c) modulates (e.g., inhibits, either permanently or transiently) a function of the msDNA; and/or d) modulates (e.g., improves) efficiency of targeted genome editing / engineering. [00193] In some embodiments, the engineered nucleic acid construct (e.g., the engineered retron) is engineered based on and/or to resemble a secondary structure of a wild- type or consensus retron encoding a wild-type or consensus retron ncRNA encompassed by: a) the sequence of any one of Table B ncRNA sequences and/or the structure depicted in any one of FIGs.2-27 of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872; or b) a variant of a), having: i) up to 1, 2, or 3 (e.g., up to 1) nucleotide changes per 10 red lettered- nucleotides; ii) up to 4, 5, or 6 (e.g., up to 1 or 2) nucleotide changes per 10 black lettered- nucleotides; and/or iii) up to 7, 8, or 9 (e.g., up to 3 or 4) nucleotide changes per 10 grey lettered-nucleotides; and/or optionally further comprising: i) 7, 8, 9, or 10 (e.g., 9 or 10) nucleotides present per 10 red-circled nucleotides; ii) 6, 7, 8, 9, or 10 (e.g., 8, 9 or 10) nucleotides present per 10 black-circled nucleotides; iii) 4, 5, 6, 7, 8, 9, or 10 (e.g., 6, 7, 8, 9 or 10) nucleotides present per 10 grey-circled nucleotides; and/or iv) 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., 4, 5, 6, 7, 8, 9, or 10) nucleotides present per 10 white-circled nucleotides. [00194] Another aspect of the disclosure provides a vector system comprising a vector comprising the engineered retron described herein. RNG043-WO1 PCT Application [00195] Another aspect of the disclosure provides an isolated host cell comprising the engineered retron described herein, or the vector system described herein. [00196] Another aspect of the disclosure provides a pharmaceutical composition comprising the engineered retron described herein, or the vector system described herein. [00197] Another aspect of the disclosure provides a delivery vehicle comprising the engineered retron described herein or the ncRNA encoded by the engineered retron described herein, the vector or vector system described herein, the host cell described herein, or the pharmaceutical composition described herein. [00198] Another aspect of the disclosure provides a kit comprising the engineered retron described herein or the ncRNA encoded by the engineered retron described herein, and optionally instructions for genetically modifying a cell using the engineered retron described herein or the ncRNA encoded by the engineered retron described herein. [00199] Another aspect of the disclosure provides a method of modifying a target DNA sequence in a host cell (e.g., a mammalian cell), the method comprising introducing into the host cell (e.g., the mammalian cell) the engineered retron of the present disclosure, the ncRNA encoded by the engineered retron of the present disclosure, or the vector / vector system described herein, to allow the production of the msDNA in the host cell (e.g., mammalian cell), wherein at least a part of the heterologous nucleic acid in the msDNA is integrated into the genome of the host (e.g., mammalian) cell at the target DNA sequence. Optionally, the target sequence is recognized by a suitable nuclease, such as a CRISPR/Cas effector enzyme, a ZFN, a TALEN, a meganuclease, TnpB, IscB, or a restriction endonuclease (RE), and a double- stranded break (DSB) is created by the nuclease to facilitate / promote the insertion of the part of the heterologous nucleic acid into the target sequence. Further optionally, the target sequence modified / inserted by the part of the heterologous nucleic acid can no longer be recognized by the nuclease to re-create a DSB. [00200] Another aspect of the disclosure provides a use of the engineered retron in the various methods described herein. [00201] Another aspect of the disclosure provides a genome editing system comprising: a) nuclease capable of acting at a target site on a genome (e.g. human genome), such as a CRISPR/Cas effector enzyme, a ZFN, a TALEN, a meganuclease, TnpB, IscB, or a restriction endonuclease (RE); and b) an engineered retron described herein, or an ncRNA encoded thereby, or a vector or a vector system comprising or encoding the same. Optionally, the nuclease may be linked to one or more element(s) of the engineered retron or the encoded ncRNA. For example, in one embodiment, the nuclease may be linked (e.g., fused or RNG043-WO1 PCT Application conjugated) to the reverse transcriptase of the engineered retron described herein. In another embodiment, the nuclease may engage/bind to form a complex with a nucleic acid guide sequence (such as a single-guided RNA of a Cas enzyme), wherein the guide sequence is linked to the ncRNA and/or msDNA of the engineered retron described herein. [00202] Another aspect of the disclosure provides an enhanced genome editing system, comprising the genome editing system of the disclosure connected to a biomolecule that modulates host DNA repair, in order to, for example, modulate (e.g., enhance) the incorporation of the heterologous nucleic acid sequence into a genome (e.g., human genome). [00203] With the general aspect of the disclosure described herein, specific aspects and embodiments of the disclosure are further described in the sections below. It should be understood that any one embodiment of the disclosure, including those described only in the examples or the claims, or only in one section herein below, can be combined with any one or more additional embodiments of the present disclosure, unless such combination is expressly disclaimed or are improper. A. Chimeric gene editing composition [00204] The present disclosure describes a chimeric gene editing system that combines one or more site-specific gene editing components (e.g., prime editing or CRISPR/Cas9) with one or more components of a retron editing system, including a retron RT and a modified retron ncRNA. The chimeric gene editing composition comprises (a) an ncRNA, optionally comprising a polymerase template sequence and a first polymerase, (b) a nuclease or a first mRNA encoding the nuclease, (c) a guide RNA (gRNA) associated with the nuclease, and (d) optionally, a second polymerase or a second mRNA encoding the second polymerase. [00205] In some embodiments, the chimeric gene editing composition comprises (a) a nickase component, (b) a guide RNA that complexes with the nickase component and directs it to a target DNA sequence, (c) a polymerase component, and (d) a polymerase template sequence, wherein the polymerase template sequence is provided by a modified retron ncRNA comprising the polymerase template sequence (herein referred to as a “templated ncRNA” or “tncRNA”). In various embodiments, the chimeric gene editing composition comprises an ncRNA variant having an altered structural configuration relative to a reference retron ncRNA (e.g., wildtype retron ncRNA) wherein the alterations include (i) a linker joining the 5’ end of the ncRNA a1 region to the 3’ end of the ncRNA a2 region, (ii) a deletion of the wildtype msd region or portion thereof, (iii) a deletion of the wildtype msr region or portion thereof,iii) installation of a single-strand RNA sequence comprising the polymerase template e.g., in place of the deleted msd sequence, and/or iv) addition of an RNA motif. The chimeric gene editing RNG043-WO1 PCT Application system may also optionally comprise a retron RT to convert the templated ncRNA to a cognate msDNA, which is referred to herein as the “templated msDNA” or “tmsDNA”. [00206] The gene editing systems described herein may also include any templated ncRNA that is derived from any ncRNA known in the art, including any of those described in Table B of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872 (each are incorporated herein by reference in their entireties, including their Sequence Listings), or a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with a sequence from Table B of any of the aforementioned applications. [00207] Retrons were originally discovered in 1984 in Myxococcus xanthus bacterium when a short, multi-copy single-stranded DNA (msDNA) that is abundantly present in the bacterial cell was identified. Since then, a number of naturally existing retrons have been found in many prokaryotes such as bacteria. [00208] Retrons encode and transcribe as a single RNA, which comprises a non-coding RNA (ncRNA) portion and a portion encoding a specialized reverse transcriptase (RT). The retron ncRNA (msr and msd) is the precursor of the hybrid molecule that eventually forms, and it initially folds into a typical RNA secondary structure that is recognized by the accompanying RT. The translated RT typically recognizes certain secondary structures in the ncRNA, and binds the RNA template downstream from the msd region. The RT initiates reverse transcription of the RNA towards its 5ʹ end, starting from the 2’-end of a conserved guanosine (G) residue found immediately after a double-stranded RNA structure (the a1/a2 region) within the ncRNA. A portion of the ncRNA serves as a template for reverse transcription, and reverse transcription terminates before reaching the msr locus. During reverse transcription, cellular RNase H degrades the segment of the ncRNA that serves as template, but not other parts of the ncRNA. The result of the reverse transcription, the msDNA, remains covalently attached to the RNA template via the 2’-5ʹ phosphodiester bond, and base-pairs with the RNA template using the 3ʹ end of the msDNA. See FIG.2 for a general or typical organization of the retron coding sequence, including the RT encoding sequence and the msr and msd loci, as well as the synthesis of the msDNA by reverse transcription of the initial ncRNA transcript. [00209] The templated retron ncRNAs may be constructed by modifying a starting point retron DNA sequence encoding a ncRNA (the msr/msd region) (such as any of those of aforementioned Table B). A starting point retron DNA sequence encoding an ncRNA (a reference retron ncRNA) may be modified in any number of ways and can including one modification or more than one modification. RNG043-WO1 PCT Application [00210] Retron ncRNA sequences may be further modified to contain at least one nucleotide modification, including a single nucleotide substitution, insertion, or deletion, or a substitution, insertion, or deletion of more than one nucleotide, i.e., up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or up to 100, or up to 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or up to 2000 nucleotides substituted, inserted, or deleted from a starting point retron (e.g., a wildtype retron). Where more than one nucleotide of a starting point retron (e.g., a wildtype retron) is substituted, deleted, or inserted, the nucleotides may be contiguous or non-contiguous. While an engineered retron as a whole is not naturally-occurring, it may include components such as nucleotide sequences that do occur in nature. For example, an engineered retron can have nucleotide sequences from different organisms (e.g., from different bacteria species), or from completely synthetic / artificial / recombinant nucleic acid sequences. Thus, an engineered retron can have a bacterial nucleotide sequence, a human nucleotide sequence, a viral nucleotide sequence, and/or a synthetic / artificial / recombinant nucleotide sequence, and/or combinations of such sequences. An example of modifications of the recombinant retrons disclosed herein include the insertion of a heterologous nucleic acid sequence in a retron, for example, inserted into the ncRNA locus, such as in the msr or the msd loci. Linking guide RNA molecules to the 5ʹ and/or 3ʹ ends (i.e., linking one at the 5ʹ end of a ncRNA and/or one at the 3ʹ end of a ncRNA) also represents another modification contemplated by the recombinant retrons disclosed herein. In such embodiments, the guide RNA molecules may also be categorized or referred to more generally as types of heterologous nucleic acid sequences used to modify starting point retrons. Examples of such modifications are depicted in FIG.8A-8E. [00211] In addition to the DNA encoding the ncRNA, the DNA encoding the RT may also be modified to obtain a recombinant RT. For example, the RT-encoding DNA may modified to contain at least one nucleotide modification, including a single nucleotide substitution, insertion, or deletion, or a substitution, insertion, or deletion of more than one nucleotide, i.e., up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, RNG043-WO1 PCT Application 99, or up to 100, or up to 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or up to 2000 nucleotides substituted, inserted, or deleted from a starting point retron (e.g., a wildtype retron) within the RT gene. [00212] Such modifications to the DNA encoding ncRNA and/or RT may modulate the function of the ncRNA and/or RT in various ways, including i) modulating (e.g., enhancing) reverse transcription, processivity, accuracy/fidelity, and/or production of the msDNA (e.g., in the mammalian cell); ii) modulating (e.g., reducing) immunogenicity of ncRNA (msr locus and msd locus) encoded by the engineered retron in a host (e.g., a host comprising the mammalian cell); iii) modulating (e.g., inhibits, either permanently or transiently) a function of the msDNA; and/or iv) modulating (e.g., improving) efficiency of targeted genome editing / engineering. [00213] In one embodiment, the present disclosure provides recombinant retrons having the general structure of: a) an msr locus; b) an msd locus encoding the msd RNA portion of the msDNA; c) a sequence encoding a retron reverse transcriptase (RT) (optionally in trans to the ncRNA), wherein the msd RNA is capable of being reverse transcribed (e.g., in a host cell such as a mammalian cell) to form an msDNA by the retron reverse transcriptase (RT); and/or d) a polymerase template nucleic acid capable of being transcribed. In some embodiments, the retrons have a general structure comprising from 5’ to 3’ direction: a1 region, first branching guanosine, msr, msd, and a2 region. [00214] The engineered retrons of the present disclosure are optionally structurally further modified to include one or more heterologous nucleic acids. The engineered retron may be further modified to provide various functional improvements, such as (without limitation), to enhance the production of msDNA in a cell (e.g., a mammalian cell, including a human cell). [00215] In certain embodiments, the engineered retron of the present disclosure encodes a reverse transcriptase (RT) or a functional domain thereof, comprising: i) a polypeptide listed in aforementioned Table A, or ii) a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polypeptide listed in aforementioned Table A. [00216] In certain embodiments, the engineered retron of the present disclosure encodes a reverse transcriptase (RT) or a functional domain thereof, comprising: i) a polynucleotide RNG043-WO1 PCT Application listed in aforementioned Table A, or a polynucleotide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polynucleotide of Table A of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872 and/or ii) a consensus polynucleotide sequence listed in aforementioned Table A. [00217] In certain embodiments, the templated ncRNA is derived from a ncRNA comprising: (I) an ncRNA listed in aforementioned Table B, or (II) an ncRNA having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to an ncRNA of aforementioned Table B. [00218] Engineered ncRNAs of the present disclosure can diverge in size from ncRNAs on which they are based and the proportion of the the ncRNA retained in the engineered ncRNA can vary. In certain embodiments the retained amount of the ncRNA is about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 88%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or from 50% to 80%, or from 60% to 85%, of from 70% to 90%, or from 80% to 95%, or from 85% to 98%, or from 90% to 99%, or all of the ncRNA. [00219] In certain embodiments, the engineered retron of the present disclosure encodes an ncRNA and a reverse transcriptase (RT) or a functional domain thereof, wherein the ncRNA and the RT or functional domain thereof are as described above. [00220] In some examples, the engineered retrons are engineered based on clades defined on retron/retron RTs, in which the retrons are associated with a tripartite system composed of the ncRNA, the RT and an additional protein or RT-fused domain with diverse enzymatic functions, such as, for example, as described in “Mestre et al., Systematic Prediction of Genes Functionally Associated with Bacterial Retrons and Classification of The Encoded Tripartite Systems, Nucleic Acids Research, Volume 48, Issue 22, 16 December 2020, Pages 12632-12647” (incorporated herein by reference). While the clades are based primarily upon naturally occurring ncRNA and retron/retron RT, and an additional protein or RT-fused domain, the clades, for the purpose of serving as the templates for the subject engineered RNG043-WO1 PCT Application retrons, are not limited to naturally occurring sequences. Rather, the clades can also encompass non-naturally occurring ncRNA and RT, including, without limitation, recombinant, modified or altered, chimeric, hybrid, synthetic, artificial, etc. [00221] Thus, according to the instant disclosure, retrons may be considered phylogenetically related based on a Neighbor-Joining algorithm of at least 75% (of at least 1000 replicates) and a Poisson correction distance measurement of no more than 0.05, based on alignment of the retron RT. Alternatively or in addition, retrons may be considered phylogenetically related when / if the same RT, or closely related RT, can recognize the secondary structures of the ncRNA of the retrons and reserve transcribe the retrons to produce msDNA. [00222] In certain embodiments, sequence alignments between different retron sequences (e.g., ncRNA and/or RT (protein and/or nucleic acid) sequences) or secondary structure generations are based on software known to one of ordinary skill in the art. [00223] The retron ncRNA sequences including msr and msd sequences within the same clade may be highly conserved at certain positions, while being less conserved at other positions. [00224] Unless specifically indicated otherwise, the a1 and a2 regions are both single- stranded and substantially reverse complementary to each other, forming a stem with optional interruption by a symmetric or asymmetric bulge, with optional one or more 5ʹ and/or 3ʹ overhang/unpaired nucleotide(s), wherein the a1 region generally ends before (e.g., ends immediately 5ʹ to) the conserved branching guanosine (G) providing the 2’-OH for reverse transcription priming. [00225] In some embodiments, the sequence change comprises a mutated, reduced, or eliminated bulge in the a1/a2 stem region, including sequence change(s) in one (i.e., a1 or a2) strand, or both a1 and a2 strands. [00226] For example, in some embodiments, the sequence change comprises deleting nucleotides from a1, a2, or both a1 and a2, such that the size of the bulge is reduced, or a symmetrical bulge becomes asymmetrical or vice versa, or a bulge is eliminated. [00227] In some embodiments, the sequence change comprises replacing/substituting nucleotides in a1, a2, or both a1 and a2, such that previously unpaired bases in the bulge become base-paired. [00228] In some embodiments, the sequence change comprises replacing an unpaired purine base with one or more unpaired pyrimidine base(s). RNG043-WO1 PCT Application [00229] In some embodiments, the sequence change comprises replacing an unpaired pyrimidine base with one or more unpaired purine base(s). [00230] In some embodiments, the sequence change comprises replacing one unpaired purine base (e.g., A or G) with another unpaired purine base (e.g., G or A, respectively). [00231] In some embodiments, the sequence change comprises replacing one unpaired pyrimidine base (e.g., T/U or C) with another unpaired pyrimidine base (e.g., C or T/U, respectively). [00232] In some embodiments, the sequence change comprises an extension or shortening of a1, a2, or both a1 and a2. [00233] For example, the length of a1 can be shortened by deleting 5ʹ overhang, deleting any upstream bulge nucleotides, deleting bases involved in base-pairing. Likewise, the length of a1 can be extended by adding 5ʹ overhang, adding any upstream bulge nucleotides, adding bases involved in base-pairing. [00234] In some embodiments, the length of a2 can be shortened by deleting 5ʹ overhang, deleting any downstream bulge nucleotides, deleting bases involved in base-pairing. Likewise, the length of a2 can be extended by adding 5ʹ overhang, adding any downstream bulge nucleotides, adding bases involved in base-pairing. Retron and ncRNA variants [00235] The ncRNA variants provided herein comprise one or more modifications compared to a reference retron ncRNA. The one or more modifications can comprise (i) linkage of the a1 region and the a2 region, (ii) deletion of at least a portion of the msr; (iii) deletion of at least a portion of the msd, (iv) addition of a single-stranded RNA comprising a polymerase template, and/or (v) addition of an RNA motif. [00236] In some embodiments, the ncRNAs disclosed herein may be modified by introducing additional RNA motifs into the ncRNAs, e.g., at the 5ʹ and 3ʹ termini of the ncRNAs, or even at positions therein between (e.g., in the msr or msd regions) to improve transcriptional production and/or stability and/or function (e.g., RT-DNA production). Such structures may include, but are not limited to RNA hairpins, RNA step-loops, RNA quadruplexes, cap structures, and poly(A) tails, or ribozyme functions and the like. Also, ncRNAs could also be modified to include one or more nuclear localization sequences. [00237] Additional RNA motifs could also improve RT processivity of the ncRNA or enhance ncRNA activity by enhancing RT binding. Addition of dimerization motifs - such as kissing loops or a GNRA tetraloop/tetraloop receptor pair - at the 5ʹ and 3ʹ termini of the ncRNA could also result in effective circularization of the ncRNA, improving stability. RNG043-WO1 PCT Application Additionally, it is envisioned that addition of these motifs could enable the physical separation of ncRNA components, e.g., separation of the msr and msd regions. Short 5ʹ extensions or 3’ extensions to the ncRNA that form a small toehold hairpin at either or both ends of the ncRNA could also compete favorably against the annealing of intracomplementary regions along the length of the ncRNA. Finally, kissing loops could also be used to recruit other RNAs or proteins to the genomic site and enable swapping of RT activity from one RNA to the other. [00238] ncRNAs could be further improved via directed evolution, in an analogous fashion to how protein function can be improved. Directed evolution could enhance ncRNA recognition by RT and/or reduce off-site targeting and/or indels and/or improve precise editing efficiency. [00239] The present disclosure contemplates any such ways to further improve the stability and/or functionality of the ncRNAs disclosed here. [00240] In some embodiments, the RNAs (including the guide RNAs and the ncRNAs) used in the compositions of the disclosure have undergone a chemical or biological modification to render them more stable. Exemplary modifications to an RNA include the depletion of a base (e.g., by deletion or by the substitution of one nucleotide for another) or modification of a base, for example, the chemical modification of a base. The phrase "chemical modifications" as used herein, includes modifications which introduce chemistries which differ from those seen in naturally occurring RNA, for example, covalent modifications such as the introduction of modified nucleotides, (e.g., nucleotide analogs, or the inclusion of pendant groups which are not naturally found in such mRNA molecules). [00241] Other suitable polynucleotide modifications that may be incorporated into the RNAs used in the compositions of the disclosure include, but are not limited to, 4'- thio- modified bases: 4'-thio-adenosine, 4'-thio-guanosine, 4'-thio-cytidine, 4'-thio-uridine, 4'- thio- 5-methyl-cytidine, 4'-thio-pseudouridine, and 4'-thio-2-thiouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2- thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1- carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5- taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1- taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl- pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1- methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy- pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl- RNG043-WO1 PCT Application cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2- thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio- 1- methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza- zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy- cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl- pseudoisocytidine, 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza- adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7- deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6- isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis- hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6- threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6- dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio- guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6- thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2- methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1- methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio- guanosine, and combinations thereof. The term modification also includes, for example, the incorporation of non-nucleotide linkages or modified nucleotides into the mRNA sequences of the present disclosure (e.g., modifications to one or both of the 3' and 5' ends of an mRNA molecule encoding a functional protein or enzyme). Such modifications include the addition of bases to an mRNA sequence (e.g., the inclusion of a poly A tail or a longer poly A tail), the alteration of the 3' UTR or the 5' UTR, complexing the mRNA with an agent (e.g., a protein or a complementary nucleic acid molecule), and inclusion of elements which change the structure of an RNA molecule (e.g., which form secondary structures). [00242] In some embodiments, RNAs (e.g., ncRNAs) include a 5' cap structure. A 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5'5'5 triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp (5'(A,G(5')ppp(5')A and G(5')ppp(5')G. Naturally occurring cap structures comprise a 7- methyl guanosine that is linked via a triphosphate bridge to the 5'-end of the first transcribed RNG043-WO1 PCT Application nucleotide, resulting in a dinucleotide cap of m7G(5')ppp(5')N, where N is any nucleoside. In vivo, the cap is added enzymatically; the cap is added in the nucleus and is catalyzed by the enzyme guanylyl transferase. The addition of the cap to the 5' terminal end of RNA occurs immediately after initiation of transcription. The terminal nucleoside is typically a guanosine, and is in the reverse orientation to all the other nucleotides, i.e., G(5')ppp(5')GpNpNp. [00243] Additional cap analogs include, but are not limited to, a chemical structures selected from the group consisting of m7GpppG, m7GpppA, m7GpppC; unmethylated cap analogs (e.g., GpppG); dimethylated cap analog (e.g., m2,7GpppG), trimethylated cap analog (e.g., m2,2,7GpppG), dimethylated symmetrical cap analogs (e.g., m7Gpppm7G), or anti reverse cap analogs (e.g., ARCA; m7,2'OmeGpppG, m72'dGpppG, m7,3'OmeGpppG, m7,3'dGpppG and their tetraphosphate derivatives), for example, as described in Jemielity, J. et al., "Novel 'anti-reverse' cap analogs with superior translational properties", RNA, 9: 1108- 1122 (2003). [00244] Typically, the presence of a "tail" serves to protect the RNA (e.g., ncRNA) from exonuclease degradation. A poly A or poly U tail is thought to stabilize natural messengers and synthetic sense RNA. Therefore, in certain embodiments a long poly A or poly U tail can be added to an RNA molecule thus rendering the RNA more stable. Poly A or poly U tails can be added using a variety of art-recognized techniques. For example, long poly A tails can be added to synthetic or in vitro transcribed RNA using poly A polymerase as described in Yokoe, et al. Nature Biotechnology.1996; 14: 1252-1256. A transcription vector can also encode long poly A tails. In addition, poly A tails can be added by transcription directly from PCR products. In some examples, poly A may also be ligated to the 3' end of a sense RNA with RNA ligase, such as, for example, as described in Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1991 edition)). [00245] Typically, the length of a poly A or poly U tail can be at least about 10, 50, 100, 200, 300, 400 at least 500 nucleotides. In some embodiments, a poly-A tail on the 3' terminus of mRNA typically includes about 10 to 300 adenosine nucleotides (e.g., about 10 to 200 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 100 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60 adenosine nucleotides). In some embodiments, mRNAs include a 3' poly(C) tail structure. A suitable poly-C tail on the 3' terminus of mRNA typically include about 10 to 200 cytosine nucleotides (e.g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides). The RNG043-WO1 PCT Application poly-C tail may be added to the poly-A or poly U tail or may substitute the poly-A or poly U tail. [00246] RNAs according to the present disclosure (e.g., ncRNAs) may be synthesized according to any of a variety of known methods. For example, RNAs according to the present disclosure may be synthesized via in vitro transcription (IVT). Briefly, IVT is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and/or RNAse inhibitor. The exact conditions will vary according to the specific application. An improved method of IVT of a ncRNA is disclosed in Example 5 herein. [00247] In a particular embodiment (as exemplified in Example 6 herein), the ncRNAs can comprise an MS2 modification, a specific RNA hairpin structure recognized in nature by a certain MS2-binding protein. This domain can help to stabilize the ncRNA and improve the editing efficiency. The disclosure contemplates other similar modifications. In some examples, other such MS2-like domains can include, for example, the ones descirbed in Johansson et al.,“RNA recognition by the MS2 phage coat protein,” Sem Virol., 1997, Vol.8(3): 176-185; Delebecque et al.,“Organization of intracellular reactions with rationally designed RNA assemblies,” Science, 2011, Vol.333: 470-474; Mali et al.,“Cas9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering,” Nat. Biotechnol., 2013, Vol.31: 833- 838; and Zalatan et al.,“Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds,” Cell, 2015, Vol.160: 339-350, each of which are incorporated herein by reference in their entireties. In some examples, other systems include the PP7 hairpin, which specifically recruits the PCP protein, and the “com” hairpin, which specifically recruits the Com protein, for example, as described in Zalatan et al. The nucleotide sequence of the MS2 hairpin (or equivalently referred to as the“MS2 aptamer”) is: GCCAACATGAGGATCACCCATGTCTGCAGGGCC (SEQ ID NO:19935). Polymerase [00248] In various embodiments, the chimeric gene editing composition includes one or more polymerases, which can be an retron RT. The retron RT in such embodiments would handle converting a template ncRNA to a cognate templated msDNA, as well as being the RNA-dependent DNA polymerase functioning on the target DNA at the nick site (see Figures for details mechanism of action). RNG043-WO1 PCT Application [00249] The retron RTs that may be used with the gene editing system described herein may be any retron RT described in the art, including any of those described in Table A of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872 (each are incorporated herein by reference in their entireties, including their Sequence Listings), or a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with a sequence from Table A of any of the aforementioned applications. [00250] Reverse transcriptases (RTs, also known as RNA-directed DNA polymerases) are enzymes present in all three domains of life, which are DNA polymerase using RNA as a template. Reverse transcriptases of the present disclosure are used to reverse transcribe template msd RNA into single-stranded msDNA. [00251] The reverse transcriptase or a functional domain thereof that may be used in the instant disclosure includes prokaryotic and eukaryotic RT, provided that the RT functions within the host to generate a donor polynucleotide sequence from the RNA template (e.g., an RNA template from the retron transcript ncRNA). [00252] In certain embodiments, suitable RT sequences (including amino acid sequences and the encoding polynucleotide sequences) are provided in Table A of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872. [00253] In certain embodiments, the nucleotide sequence of a native or wild-type RT is modified, for example, using known codon optimization techniques, so that expression within the desired host is optimized. [00254] In certain embodiments, the RT domain of a reverse transcriptase is used in the present disclosure, so long as it is compatible with the engineered retron of the present disclosure. The domain may include only the RNA-dependent DNA polymerase activity. In certain embodiments, the RT domain is non-mutagenic, i.e., does not cause mutation in the donor polynucleotide (e.g., during the reverse transcriptase process). In certain embodiments, the RT domain may be non-retron RT in origin, e.g., a viral RT or a human endogenous RTs. In certain embodiments, the RT domain is retron RT or diversity-generating retroelements (DGRs) RT. In certain embodiments, the RT may be less mutagenic than a counterpart wildtype RT. In certain embodiments, the RT is not mutagenic. [00255] In some embodiments, a reverse transcriptase is encoded by a retron ret gene, which may accompany the cognate msr and msd loci and specifically recognize the secondary structure of the cognate ncRNA transcript. RNG043-WO1 PCT Application [00256] In some embodiments, the RT may be obtained from prokaryotic or eukaryotic cells. Most reverse transcriptases (80%) can be phylogenetically clustered into three major lineages: group II introns, diversity-generating retroelements (DGRs), and retrons. Other clades of RTs include abortive infection (Abi) RTs, CRISPR-Cas-associated RTs, Group II- like (G2L), the unknown groups (UG), and rvt elements. [00257] In some embodiments, the RT gene is a cognate RT, a retron RT from a species within the same species or clade of the cognate RT, or a retron RT not within the same clade of the cognate RT such as an unrelated RT or an engineered RT. In some examples, the non- retron related RT are RTs from group II introns, diversity-generating retroelements (DGRs), abortive infection (Abi) RTs, CRISPR-Cas-associated RTs, Group II-like (G2L), the unknown groups (UG), and rvt elements, for example, as described in Mestre et al., Nucleic Acids Research, Volume 48, Issue 22, 16 December 2020, Pages 12632-12647; and Mestere et al., UG/Abi: “A Highly Diverse Family of Prokaryotic Reverse Transcriptases Associated With Defense Functions,” doi.org/10.1101/2021.12.02.470933 (incorporated herein by reference). [00258] In some embodiments, the RT are from clades related to retron/retron-like sequences. In some embodiments, the RT are selected from RTs provided in Table A of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872. In some embodiments, the RT is not an RT associated with the sequences identified in Table X of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872. [00259] In prokaryotic retron systems, the RT gene is typically located downstream from the ncRNA (msr and msd) locus. In the engineered retron, the RT position can differ from the natural or wild type retrons. In some embodiments, the RT gene can be provided in cis, such as either upstream or downstream of the msr locus or the msd locus. In certain embodiments, the RT gene is provided in trans, such as provided separately in a vector of the vector system described herein, wherein the ncRNA coding msr and msd sequences are provided in a different vector of the vector system described herein. [00260] In some embodiments, the RT is modified (e.g., insertion, deletion, and/or substitution of one or more nucleotide(s)) or codon optimized to enhance activity or processivity. [00261] In certain embodiments, a cryptic stop signal is removed from the RT thereby allowing generation of longer ssDNAs. RNG043-WO1 PCT Application [00262] In certain embodiments, the RT is from a retron which encodes msDNA, such as, for example, RTs described in US 6,017,737; US5,849,563; US5,780,269; US5,436,141; US5,405,775; US5,320,958; CA2,075,515; all of which are incorporated by reference herein in their entireties. [00263] In some embodiments, the engineered retron further comprises a polynucleotide (e.g., a DNA molecule) encoding a reverse transcriptase (RT) or a portion thereof. In some embodiments, the encoded RT or portion thereof is capable of synthesizing a DNA copy of at least a portion of the msd locus encoding the msDNA. [00264] In some embodiments, the polynucleotide (e.g., a DNA molecule) encoding the RT comprises a polynucleotide listed in Table A of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872, or a polynucleotide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polynucleotide listed in Table A of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872. [00265] In some embodiments, the polynucleotide encoding the RT encodes a polypeptide listed in Table A of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polypeptide listed in Table A of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872; and/or a polypeptide of Table C of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872. [00266] In some embodiments, the polynucleotide encoding the RT does not comprise a polynucleotide listed in Table X of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872. RNG043-WO1 PCT Application [00267] Once translated, the RT binds the ncRNA template downstream from the msd locus, forming an RT-RNA complex, and initiating reverse transcription of the RNA towards its 5ʹ end. Accordingly, in certain aspects the disclosure relates to an engineered nucleic acid- enzyme construct comprising: a. a non-coding RNA (ncRNA) comprising: i) an msr locus encoding the msr RNA portion of a multi-copy single-stranded DNA (msDNA); and ii) an msd locus encoding the msd RNA portion of the msDNA; b. a heterologous nucleic acid inserted at or within a location selected from: the msd locus, upstream of the msr locus, upstream of the msd locus, and downstream of the msd locus; and c. a reverse transcriptase (RT), or a domain thereof comprising: i) a polypeptide listed in the abovementioned Table A, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polypeptide listed in the abovementioned Table A; and/or ii) a polypeptide listed in the abovementioned Table C. In some embodiments, the RT does not comprise a polypeptide listed in the abovementioned Table X. [00268] In certain aspects, the disclosure relates to an engineered nucleic acid-enzyme construct comprising: a) a non-coding RNA (ncRNA) comprising: i) an msr locus encoding the msr RNA portion of a multi-copy single-stranded DNA (msDNA); and ii) an msd locus encoding the msd RNA portion of the msDNA, b) a heterologous nucleic acid inserted at or within a location selected from: the msd locus; upstream of the msr locus; upstream of the msd locus; and downstream of the msd locus; and c) a reverse transcriptase (RT), or a portion thereof, wherein the RT is capable of synthesizing a DNA copy of at least a portion of the msd locus encoding the msDNA, and wherein the ncRNA and/or the RT is any one of the present disclosure described herein. [00269] In certain aspects, the disclosure relates to an engineered nucleic acid-enzyme construct comprising: a) a non-coding RNA (ncRNA) comprising: i) an msr locus encoding the msr RNA portion of a multi-copy single-stranded DNA (msDNA); and ii) an msd locus encoding the msd RNA portion of the msDNA; b) a heterologous nucleic acid inserted at or within a location selected from: the msd locus, upstream of the msr locus, upstream of the msd locus, and downstream of the msd locus; and c) a reverse transcriptase (RT) or a domain thereof: wherein the RT comprises: i) an RT listed in the abovementioned Table A, or an RT having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least RNG043-WO1 PCT Application 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to an RT listed in the abovementioned Table A; and/or ii) a consensus sequence listed in the abovementioned Table C; and wherein, the RT does not comprise a sequence from the abovementioned Table X. [00270] In some embodiments of the nucleic-acid enzyme constructs described herein, the ncRNA comprises: i) an ncRNA listed in the abovementioned Table B, or ii) an ncRNA having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to an ncRNA listed in the abovementioned Table B; and/or optionally wherein the ncRNA is not an ncRNA from the retons of the abovementioned Table X. [00271] In some embodiments, the RT is linked to components such as RNA-guided and non-RNA guided nucleases. The linked maybe via a peptide bond or a short linker peptide in a fusion protein. Suitable linker peptides include flexible linkers such as those comprising G or S repeats, such as G4S (SEQ ID NO: 19511) repeat units or GS repeat units, with 1-20 repeats (SEQ ID NO: 19936), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 repeats. [00272] In certain embodiments, the RT is chemically linked or conjugated to the RNA- guided and non-RNA guided nucleases via non-peptide bonds. Such protein conjugates may be delivered directly to a host cell, either together with the nucleic acid component of the engineered retron described herein, or separately. [00273] In some embodiments, the RT is linked to a DNA-repair modulating biomolecule (e.g., NHEJ peptide inhibitors). Nuclease [00274] The chimeric gene editing composition disclosed herein comprises a nuclease. In some embodiments, the nuclease is a double-stranded endonuclease or a nickase. [00275] In certain embodiments, the chimeric gene editing system comprises (a) a nickase component, (b) a guide RNA that complexes with the nickase component and directs it to a target DNA sequence, (c) a polymerase component, and/or (d) and a polymerase template sequence, wherein the polymerase template sequence is provided by a modified retron ncRNA comprising the polymerase template sequence (herein referred to as a “templated ncRNA” or RNG043-WO1 PCT Application “tncRNA”). In various embodiments, the tncRNA comprises an altered structural configuration relative to a wildtype retron ncRNA wherein the alterations include (i) a linker joining the 5’ end of the ncRNA a1 region to the 3’ end of the ncRNA a2 region, (ii) a deletion of the wildtype msd region or portion thereof, and/or (iii) installation of a single-strand RNA sequence comprising the polymerase template in place of the deleted msd sequence. The chimeric gene editing system may also optionally comprise a retron RT to convert the templated ncRNA to a cognate msDNA, which is referred to herein as the “templated msDNA” or “tmsDNA”. [00276] The nickase component of the chimeric editing system described herein may be any programmable CRISPR nickase, including nickases based on CRISPR system Class 1, Type I, II, or III Cas nucleases; Class 2, Type II nuclease (such as Cas9); a Class 2, Type V nuclease (such as Cpfl), or a Class 2, Type VI nuclease (such as C2c2). Examples of Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9 (Csnl or Csxl2), CaslO, CaslOd, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966, and homologs or modified versions thereof. [00277] In some embodiments, a Class 1, type II CRISPR system Cas9 endonuclease is used. Cas9 nucleases from any species, or biologically active fragments, variants, analogs, or derivatives thereof that retain Cas9 endonuclease activity (i.e., catalyze site-directed cleavage of DNA to generate double-strand breaks) may be used to perform genome modification as described herein. The Cas9 need not be physically derived from an organism but may be synthetically or recombinantly produced. In some examples, Cas9 sequences from a number of bacterial species are well known in the art and listed in the National Center for Biotechnology Information (NCBI) database, for example, NCBI entries for Cas9 from: Streptococcus pyogenes (WP 002989955, WP_038434062, WP_011528583); Campylobacter jejuni (WP_022552435, YP 002344900), Campylobacter coli (WP 060786116); Campylobacter fetus (WP 059434633); Corynebacterium ulcerans (NC_015683, NC_017317); Corynebacterium diphtheria (NC_016782, NC_016786); Enterococcus faecalis (WP 033919308); Spiroplasma syrphidicola (NC 021284); Prevotella intermedia (NC 017861); Spiroplasma taiwanense (NC 021846); Streptococcus iniae (NC 021314); Belliella baltica (NC 018010); Psychroflexus torquisl (NC O 18721); Streptococcus thermophilus (YP 820832), Streptococcus mutans (WP 061046374, WP 024786433); Listeria innocua (NP 472073); Listeria monocytogenes (WP RNG043-WO1 PCT Application 061665472); Legionella pneumophila (WP 062726656); Staphylococcus aureus (WP_001573634); Francisella tularensis (WP_032729892, WP_014548420), Enterococcus faecalis (WP 033919308); Lactobacillus rhamnosus (WP 048482595, WP_032965177); and Neisseria meningitidis (WP_061704949, YP_002342100); all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference in their entireties. Any of these sequences or a variant thereof comprising a sequence having at least about 70- 100% sequence identity thereto, including any percent identity within this range, such as 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used for genome editing, as described herein and in some examples, as described in Fonfara et al. (2014) Nucleic Acids Res. 42(4):2577-90; Kapitonov et al. (2015) J. Bacterid.198(5): 797-807, Shmakov et al. (2015) Mol. Cell.60(3):385- 397, and Chylinski et al. (2014) Nucleic Acids Res.42(10):6091-6105). [00278] In another embodiment, the CRISPR nuclease from Prevotella and Francisella 1 (Cpfl, or Cas12a) is used. Cpfl is another class II CRISPR/Cas system RNA-guided nuclease with similarities to Cas9 and may be used analogously. Unlike Cas9, Cpfl does not require a tracrRNA and only depends on a crRNA in its guide RNA, which provides the advantage that shorter guide RNAs can be used with Cpfl for targeting than Cas9. Cpfl is capable of cleaving either DNA or RNA. The PAM sites recognized by Cpfl have the sequences 5ʹ-YTN-3ʹ (where “Y” is a pyrimidine and “N” is any nucleobase) or 5ʹ-TTN-3ʹ, in contrast to the G-rich PAM site recognized by Cas9. Cpfl cleavage of DNA produces double-stranded breaks with a sticky-ends having a 4 or 5 nucleotide overhang. In some examples, additional information on examples of Cpfl is disclosed in Ledford et al. (2015) Nature.526 (7571):17-17, Zetsche et al. (2015) Cell.163 (3):759-771, Murovec et al. (2017) Plant Biotechnol. J.15(8):917-926, Zhang et al. (2017) Front. Plant Sci.8:177, Fernandes et al. (2016) Postepy Biochem.62(3):315-326; herein incorporated by reference. [00279] C2c1 (Cas12b) is another class II CRISPR/Cas system RNA-guided nuclease that may be used. C2cl, similarly to Cas9, depends on both a crRNA and tracrRNA for guidance to target sites, such as, for example, those disclosed in Shmakov et al. (2015) Mol Cell.60(3):385-397, Zhang et al. (2017) Front Plant Sci.8:177; herein incorporated by reference. [00280] In one aspect, a nucleic acid sequence-programmable DNA binding domain can be associated with or complexed with at least one guide nucleic acid (e.g., guide RNA or a pegRNA), which localizes the DNA binding domain to a DNA sequence that comprises a DNA strand (i.e., a target strand) that is complementary to the guide nucleic acid, or a portion RNG043-WO1 PCT Application thereof (e.g., the spacer of a guide RNA which anneals to the protospacer of the DNA target). In other words, the guide nucleic-acid “programs” the DNA binding domain (e.g., Cas9 or equivalent) to localize and bind to complementary sequence of the protospacer in the DNA. [00281] Any suitable nucleic acid sequence-programmable DNA binding domain may be used in the prime editors described herein. In various embodiments, the nucleic acid sequence-programmable DNA binding domain may be any Class 2 CRISPR-Cas system, including any type II, type V, or type VI CRISPR-Cas enzyme. Given the rapid development of CRISPR-Cas as a tool for genome editing, there have been constant developments in the nomenclature used to describe and/or identify CRISPR-Cas enzymes, such as Cas9 and Cas9 orthologs. In some examples, CRISPR-Cas nomenclature is discussed in Makarova et al., “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?,” The CRISPR Journal, Vol.1. No.5, 2018, the entire contents of which are incorporated herein by reference. [00282] Without being bound by theory, the mechanism of action of certain CRISPR Cas enzymes contemplated herein includes the step of forming an R-loop whereby the Cas protein induces the unwinding of a double-strand DNA target, thereby separating the strands in the region bound by the Cas protein. The guide RNA spacer then hybridizes to the “target strand” at a region that is complementary to the protospacer sequence of the DNA. In some embodiments, the Cas protein may include one or more nuclease activities, which then cut the DNA leaving various types of lesions. For example, the Cas protein may comprises a nuclease activity that cuts the non-target strand at a first location, and/ or cuts the target strand at a second location. Depending on the nuclease activity, the target DNA can be cut to form a “double-stranded break” whereby both strands are cut. In other embodiments, the target DNA can be cut at only a single site, i.e., the DNA is “nicked” on one strand. Exemplary Cas proteins with different nuclease activities include “Cas9 nickase” (“nCas9”) and a deactivated Cas9 having no nuclease activities (“dead Cas9” or “dCas9”). [00283] The below description of various Cas proteins which can be used in connection with the presently disclosed LNP-delivered gene editing systems is not meant to be limiting in any way. The gene editing systems may comprise the canonical SpCas9, or any ortholog Cas9 protein, or any variant Cas9 protein—including any naturally occurring variant, mutant, or otherwise engineered version of Cas9—that is known or which can be made or evolved through a directed evolutionary or otherwise mutagenic process. In various embodiments, the Cas9 or Cas9 variants have a nickase activity, i.e., only cleave one strand of the target DNA sequence. In other embodiments, the Cas9 or Cas9 variants have inactive nucleases, i.e., are “dead” Cas9 proteins. Other variant Cas9 proteins that may be used are those having a smaller RNG043-WO1 PCT Application molecular weight than the canonical SpCas9 (e.g., for easier delivery) or having modified or rearranged primary amino acid structure. [00284] The gene editing systems described herein may also comprise Cas9 equivalents, including Cas12a (Cpf1) and Cas12b1 proteins. The Cas proteins usable herein (e.g., SpCas9, Cas9 variant, or Cas9 equivalents) may also contain various modifications that alter/enhance their PAM specificities. The present disclosure contemplates any Cas9, Cas9 variant, or Cas9 equivalent which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% sequence identity to a reference Cas9 sequence, such as a reference SpCas9 canonical sequence of Streptococcus pyogenes M1 (Accession No. Q99ZW2) (SEQ ID NO: 2027): MDKKYSIGLD IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR HSIKKNLIGA LLFDSGETAE ATRLKRTARR RYTRRKNRIC YLQEIFSNEM AKVDDSFFHR LEESFLVEED KKHERHPIFG NIVDEVAYHE KYPTIYHLRK KLVDSTDKAD LRLIYLALAH MIKFRGHFLI EGDLNPDNSD VDKLFIQLVQ TYNQLFEENP INASGVDAKA ILSARLSKSR RLENLIAQLP GEKKNGLFGN LIALSLGLTP NFKSNFDLAE DAKLQLSKDT YDDDLDNLLA QIGDQYADLF LAAKNLSDAI LLSDILRVNT EITKAPLSAS MIKRYDEHHQ DLTLLKALVR QQLPEKYKEI FFDQSKNGYA GYIDGGASQE EFYKFIKPIL EKMDGTEELL VKLNREDLLR KQRTFDNGSI PHQIHLGELH AILRRQEDFY PFLKDNREKI EKILTFRIPY YVGPLARGNS RFAWMTRKSE ETITPWNFEE VVDKGASAQS FIERMTNFDK NLPNEKVLPK HSLLYEYFTV YNELTKVKYV TEGMRKPAFL SGEQKKAIVD LLFKTNRKVT VKQLKEDYFK KIECFDSVEI SGVEDRFNAS LGTYHDLLKI IKDKDFLDNE ENEDILEDIV LTLTLFEDRE MIEERLKTYA HLFDDKVMKQ LKRRRYTGWG RLSRKLINGI RDKQSGKTIL DFLKSDGFAN RNFMQLIHDD SLTFKEDIQK AQVSGQGDSL HEHIANLAGS PAIKKGILQT VKVVDELVKV MGRHKPENIV IEMARENQTT QKGQKNSRER MKRIEEGIKE LGSQILKEHP VENTQLQNEK LYLYYLQNGR DMYVDQELDI NRLSDYDVDH IVPQSFLKDD SIDNKVLTRS DKNRGKSDNV PSEEVVKKMK NYWRQLLNAK LITQRKFDNL TKAERGGLSE LDKAGFIKRQ LVETRQITKH VAQILDSRMN TKYDENDKLI REVKVITLKS KLVSDFRKDF QFYKVREINN YHHAHDAYLN AVVGTALIKK YPKLESEFVY GDYKVYDVRK MIAKSEQEIG KATAKYFFYS NIMNFFKTEI TLANGEIRKR PLIETNGETG EIVWDKGRDF ATVRKVLSMP QVNIVKKTEV RNG043-WO1 PCT Application QTGGFSKESI LPKRNSDKLI ARKKDWDPKK YGGFDSPTVA YSVLVVAKVE KGKSKKLKSV KELLGITIME RSSFEKNPID FLEAKGYKEV KKDLIIKLPK YSLFELENGR KRMLASAGEL QKGNELALPS KYVNFLYLAS HYEKLKGSPE DNEQKQLFVE QHKHYLDEII EQISEFSKRV ILADANLDKV LSAYNKHRDK PIREQAENII HLFTLTNLGA PAAFKYFDTT IDRKRYTSTK EVLDATLIHQ SITGLYETRI DLSQLGGD [00285] The Cas proteins contemplated herein embrace CRISPR Cas 9 proteins, as well as Cas9 equivalents, variants (e.g., Cas9 nickase (nCas9) or nuclease inactive Cas9 (dCas9)) homologs, orthologs, or paralogs, whether naturally occurring or non-naturally occurring (e.g., engineered or recombinant), and may include a Cas9 equivalent from any Class 2 CRISPR system (e.g., type II, V, VI), including Cas12a (Cpf1), Cas12e (CasX), Cas12b1 (C2c1), Cas12b2, Cas12c (C2c3), C2c4, C2c8, C2c5, C2c10, C2c9 Cas13a (C2c2), Cas13d, Cas13c (C2c7), Cas13b (C2c6), and Cas13b. In some examples, further Cas-equivalents are described in Makarova et al., “C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector,” Science 2016; 353(6299) and Makarova et al., “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?,” The CRISPR Journal, Vol.1. No.5, 2018, the contents of which are incorporated herein by reference. [00286] The terms “Cas9” or “Cas9 nuclease” or “Cas9 moiety” or “Cas9 domain” embrace any naturally occurring Cas9 from any organism, any naturally-occurring Cas9 equivalent or functional fragment thereof, any Cas9 homolog, ortholog, or paralog from any organism, and any mutant or variant of a Cas9, naturally-occurring or engineered. The term Cas9 is not meant to be particularly limiting and may be referred to as a “Cas9 or equivalent.” Exemplary Cas9 proteins are further described in the art and are incorporated herein by reference. As noted herein, Cas9 nuclease sequences and structures are well known to those of skill in the art. In some examples, the Cas9 nuclease sequences and structures can be those described in “Complete genome sequence of an M1 strain of Streptococcus pyogenes.” Ferretti et al., J.J., McShan W.M., Ajdic D.J., Savic D.J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A.N., Kenton S., Lai H.S., Lin S.P., Qian Y., Jia H.G., Najar F.Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S.W., Roe B.A., McLaughlin R.E., Proc. Natl. Acad. Sci. U.S.A.98:4658-4663(2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski K., Sharma C.M., Gonzales K., Chao Y., Pirzada Z.A., Eckert M.R., Vogel J., Charpentier E., Nature 471:602-607(2011); and “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K., RNG043-WO1 PCT Application Fonfara I., Hauer M., Doudna J.A., Charpentier E.Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference). [00287] In certain embodiments, a polynucleotide programmable nucleotide binding domain of a nucleobase editor itself comprises one or more domains. In one embodiment, a polynucleotide programmable nucleotide binding domain comprises one or more nuclease domains. In some embodiments, the nuclease domain of a polynucleotide programmable nucleotide binding domain comprises an endonuclease or an exonuclease. In some embodiments, the endonuclease cleaves a single strand of a double-stranded nucleobase. In some embodiments, the endonuclease cleaves both strands of a double-stranded nucleobase molecule. In some embodiments, the polynucleotide programmable nucleotide binding domain is a deoxyribonuclease. In some embodiments, the polynucleotide programmable nucleotide binding domain is a ribonuclease. [00288] In some embodiments, the nuclease domain of a polynucleotide programmable nucleotide binding domain can cut zero, one, or two strands of a target polynucleotide. In some embodiments, the polynucleotide programmable nucleotide binding domain comprises a nickase domain. Herein the term “nickase” refers to a polynucleotide programmable nucleotide binding domain comprising a nuclease domain that is capable of cleaving only one strand of the two strands in a duplexed nucleobase molecule (e.g., DNA). In some embodiments, the nickase is derived from a fully catalytically active (e.g., natural) form of a polynucleotide programmable nucleotide binding domain by introducing one or more mutations into the active polynucleotide programmable nucleotide binding domain. In certain embodiments, where a polynucleotide programmable nucleotide binding domain comprises a nickase domain derived from Cas9. [00289] In some embodiments, the Cas9-derived nickase has one or more mutations in the RuvC-1 domain. In one embodiment, the Cas9-derived nickase has a D10A mutation in the RuvC-1 domain. In some embodiments, the Cas9-derived nickase has one or more mutations in the REC Lobe domain. In one embodiment, the Cas9-derived nickase has a N497A, R661A, and/or Q695A mutation in the REC Lobe domain. In some embodiment, the Cas9-derived nickase has one or more mutations in the HNH domain. In one embodiment, the Cas9-derived nickase has H840A, N863A, and/or D839A in the HNH domain. [00290] In certain embodiments, in the SpCas9-derived nickase, the residue H840 retains catalytic activity and can thereby cleave a single strand of the nucleobase duplex. In certain embodiments, a Cas9-derived nickase domain can comprise an H840A mutation, while the amino acid residue at position 10 remains a D. In certain embodiments, a Cas9-derived RNG043-WO1 PCT Application nickase domain can comprise an N863A mutation, while the amino acid residue at position 10 remains a D. In some embodiments, the nickase is derived from a fully catalytically active (e.g., natural) form of a polynucleotide programmable nucleotide binding domain by removing all or a portion of a nuclease domain that is not required for the nickase activity. In certain embodiments, where a polynucleotide programmable nucleotide binding domain comprises a nickase domain derived from Cas9, the Cas9-derived nickase domain comprises a deletion of all or a portion of the RuvC domain or the HNH domain. [00291] In certain embodiments, the nucleobase editing system is or comprises a CRISPR-Cas editor or Cas9 disclosed and described in one or more of US Application Publications US2015/0045546A1, US2019/0264232A1, US2018/0258417A1, and PCT Publications WO2013141680A1 and WO2021173359A1, each of which is incorporated by reference herein in their entirety. [00292] Any of the above CRISPR-Cas editor embodiments or any variants, modifications, or derivatives thereof are contemplated herein to be delivered by the LNP systems disclosed in this specification for gene editing in cells, tissues, and/or organs under in vitro, ex vivo, or in vivo conditions. The various components described herein may be configured and delivered in any suitable manner. Any of the descriptions presented in this section are not intended to be strictly limiting. Prime editors [00293] The chimeric gene editing systems described herein may include a prime editor or one or more components thereof, in combination with retron editing components. [00294] Prime editing technology is a gene editing technology that can make targeted insertions, deletions, and all transversion and transition point mutations in a target genome. Without wishing to be bound by any particular theory, the prime editing process may search and replace endogenous sequences in a target polynucleotide. The spacer sequence of a prime editing guide RNA (“PEgRNA” or “pegRNA”) recognizes and anneals with a search target sequence in a target strand of a double stranded target polynucleotide, e.g., a double stranded target DNA. A prime editing complex may generate a nick in the target DNA on the edit strand which is the complementary strand of the target strand. The prime editing complex may then use a free 3’ end formed at the nick site of the edit strand to initiate DNA synthesis, where a “primer binding site sequence” (PBS) of the PEgRNA complexes with the free 3’ end, and a single stranded DNA is synthesized (by reverse transcriptase) using an editing template of the PEgRNA as a template. As used herein, a “primer binding site” is a single-stranded portion of the PEgRNA that comprises a region of complementarity to the RNG043-WO1 PCT Application PAM strand (i.e., the non-target strand or the edit strand). The PBS is complementary or substantially complementary to a sequence on the PAM strand of the double stranded target DNA that is immediately upstream of the nick site. [00295] The term “prime editor (PE)” refers to the polypeptide or polypeptide components involved in prime editing, or any polynucleotide(s) encoding the polypeptide or polypeptide components. In various embodiments, a prime editor includes a polypeptide domain having DNA binding activity and a polypeptide domain having DNA polymerase activity. In some embodiments, the prime editor further comprises a polypeptide domain having nuclease activity. In some embodiments, the polypeptide domain having DNA binding activity comprises a nuclease domain or nuclease activity. In some embodiments, the polypeptide domain having nuclease activity comprises a nickase, or a fully active nuclease. As used herein, the term “nickase” refers to a nuclease capable of cleaving only one strand of a double-stranded DNA target. In some embodiments, the prime editor comprises a polypeptide domain that is an inactive nuclease. In some embodiments, the polypeptide domain having programmable DNA binding activity comprises a nucleic acid guided DNA binding domain, for example, a CRISPR-Cas protein, for example, a Cas9 nickase, a Cpf1 nickase, or another CRISPR-Cas nuclease. In some embodiments, the polypeptide domain having DNA polymerase activity comprises a template-dependent DNA polymerase, for example, an RNA- dependent DNA polymerase. In some embodiments, the DNA polymerase is a reverse transcriptase. In some embodiments, the prime editor comprises additional polypeptides involved in prime editing, for example, a polypeptide domain having 5’ endonuclease activity, e.g., a 5' endogenous DNA flap endonucleases (e.g., FEN1), for helping to drive the prime editing process towards the edited product formation. In some embodiments, the prime editor further comprises an RNA-protein recruitment polypeptide, for example, a MS2 coat protein. [00296] A prime editor may be engineered. In some embodiments, the polypeptide components of a prime editor do not naturally occur in the same organism or cellular environment. In some embodiments, the polypeptide components of a prime editor may be of different origins or from different organisms. In some embodiments, a prime editor comprises a DNA binding domain and a DNA polymerase domain that are derived from different species. In some embodiments, a prime editor comprises a Cas polypeptide (DNA binding domain) and a reverse transcriptase polypeptide (DNA polymerase) that are derived from different species. For example, a prime editor may comprise a S. pyogenes Cas9 polypeptide and a Moloney murine leukemia virus (M-MLV) reverse transcriptase polypeptide. In some embodiments, a RNG043-WO1 PCT Application prime editor comprises an engineered pentamutant M-MLV RT. In some embodiments, a primer editor engineered to reduce size and improve efficiency is used. [00297] In some embodiments, polypeptide domains of a prime editor may be fused or linked by a peptide linker to form a fusion protein. In other embodiments, a prime editor comprises one or more polypeptide domains provided in trans as separate proteins, which are capable of being associated to each other through non-peptide linkages or through aptamers or recruitment sequences. For example, a prime editor may comprise a DNA binding domain and a reverse transcriptase domain associated with each other by an RNA-protein recruitment aptamer, e.g., a MS2 aptamer, which may be linked to a PEgRNA. Prime editor polypeptide components may be encoded by one or more polynucleotides in whole or in part. In some embodiments, a single polynucleotide, construct, or vector encodes the prime editor fusion protein. In some embodiments, multiple polynucleotides, constructs, or vectors each encode a polypeptide domain or portion of a domain of a prime editor, or a portion of a prime editor fusion protein. For example, a prime editor fusion protein may comprise an N-terminal portion fused to an intein-N and a C-terminal portion fused to an intein-C, each of which is individually encoded by an AAV vector. [00298] The editing template may comprise one or more intended nucleotide edits compared to the endogenous double stranded target DNA sequence. Accordingly, the newly synthesized single stranded DNA also comprises the nucleotide edit(s) encoded by the editing template. Through removal of the editing target sequence on the edit strand of the double stranded target DNA and DNA repair mechanism, the newly synthesized single stranded DNA replaces the editing target sequence, and the desired nucleotide edit(s) are incorporated into the double stranded target DNA. [00299] In some embodiments, prime editing was first described in Anzalone et al., “Search-and-replace genome editing without double-strand breaks or donor DNA,” Nature, Dec 2019, 576 (7789): pp.149-157, which is incorporated herein in its entirety. In some examples, prime editing has subsequently been described and detailed in numerous follow-on publications, including but not limited to, for example, (i) Liu et al., “Prime editing: a search and replace tool with versatile base changes,” Yi Chuan, Nov.20, 2022, 44(11): 993-1008; (ii) Lu C et al., “Prime Editing: An All-Rounder for Genome Editing. Int J Mol Sci.2022 Aug 30;23(17):9862; (iii) Velimirovic M, Zanetti LC, Shen MW, Fife JD, Lin L, Cha M, Akinci E, Barnum D, Yu T, Sherwood RI. Peptide fusion improves prime editing efficiency. Nat Commun.2022 Jun 18;13(1):3512. doi: 10.1038/s41467-022-31270-y. PMID: 35717416; PMCID: PMC9206660; (iv) Velimirovic M, Zanetti LC, Shen MW, Fife JD, Lin L, Cha M, RNG043-WO1 PCT Application Akinci E, Barnum D, Yu T, Sherwood RI. Peptide fusion improves prime editing efficiency. Nat Commun.2022 Jun 18;13(1):3512. doi: 10.1038/s41467-022-31270-y. PMID: 35717416; PMCID: PMC9206660; (v) Habib O, Habib G, Hwang GH, Bae S. Comprehensive analysis of prime editing outcomes in human embryonic stem cells. Nucleic Acids Res.2022 Jan 25;50(2):1187-1197. doi: 10.1093/nar/gkab1295. PMID: 35018468; PMCID: PMC8789035; (vi) Marzec M, Brąszewska-Zalewska A, Hensel G. Prime Editing: A New Way for Genome Editing. Trends Cell Biol.2020 Apr;30(4):257-259. doi: 10.1016/j.tcb.2020.01.004. Epub 2020 Jan 27. PMID: 32001098; (vii) Tao R, Wang Y, Jiao Y, Hu Y, Li L, Jiang L, Zhou L, Qu J, Chen Q, Yao S. Bi-PE: bi-directional priming improves CRISPR/Cas9 prime editing in mammalian cells. Nucleic Acids Res.2022 Jun 24;50(11):6423-6434. doi: 10.1093/nar/gkac506. PMID: 35687127; PMCID: PMC9226529; (viii) Nelson JW, Randolph PB, Shen SP, Everette KA, Chen PJ, Anzalone AV, An M, Newby GA, Chen JC, Hsu A, Liu DR. Engineered pegRNAs improve prime editing efficiency. Nat Biotechnol.2022 Mar;40(3):402-410. doi: 10.1038/s41587-021-01039-7. Epub 2021 Oct 4. Erratum in: Nat Biotechnol.2021 Dec 8; PMID: 34608327; PMCID: PMC8930418; (ix) Doman JL, Sousa AA, Randolph PB, Chen PJ, Liu DR. Designing and executing prime editing experiments in mammalian cells. Nat Protoc.2022 Nov;17(11):2431-2468. doi: 10.1038/s41596-022-00724-4. Epub 2022 Aug 8. PMID: 35941224; PMCID: PMC9799714; (x) Jiao Y, Zhou L, Tao R, Wang Y, Hu Y, Jiang L, Li L, Yao S. Random-PE: an efficient integration of random sequences into mammalian genome by prime editing. Mol Biomed.2021 Nov 18;2(1):36. doi: 10.1186/s43556-021-00057-w. PMID: 35006470; PMCID: PMC8607425; (xi) Awan MJA, Ali Z, Amin I, Mansoor S. Twin prime editor: seamless repair without damage. Trends Biotechnol. 2022 Apr;40(4):374-376. doi: 10.1016/j.tibtech.2022.01.013. Epub 2022 Feb 10. PMID: 35153078; and (xii) Doman JL, Pandey S, Neugebauer ME, An M, Davis JR, Randolph PB, McElroy A, Gao XD, Raguram A, Richter MF, Everette KA, Banskota S, Tian K, Tao YA, Tolar J, Osborn MJ, Liu DR. Phage-assisted evolution and protein engineering yield compact, efficient prime editors. Cell.2023 Aug 31;186(18):3983-4002.e26. doi: 10.1016/j.cell.2023.07.039. PMID: 37657419; PMCID: PMC10482982, all of which are incorporated herein by reference. [00300] In some examples, prime editing has been described and disclosed in numerous published patent applications, each of which their entire contents, amino acid sequences, nucleotide sequences, and all disclosures therein are incorporated herein by reference in their entireties: International Application No. PCT/US2022/074628, filed August 5, 2022; International Application No. PCT/US2022/074088, filed July 23, 2022; International RNG043-WO1 PCT Application Application No. PCT/US2022/073819, filed July 16, 2022; International Application No. PCT/US2022/035613, filed June 29, 2022; International Application No. PCT/US2022/036230, filed July 6, 2022; International Application No. PCT/US2022/032267, filed June 3, 2022, European Application No. EP21707651.2, filed February 19, 2021; International Application No. PCT/EP2022/062223, filed May 5, 2022; U.S. Application No. 17/219,635, filed March 31, 2021; International Application No. PCT/CN2022/080595, filed March 14, 2022; International Application No. PCT/US2022/023175, filed April 1, 2022; International Application No. PCT/US2022/021879, filed March 25, 2022; International Application No. PCT/US2022/020392, filed March 15, 2022; U.S. Application No. 17/219,672, filed March 31, 2021, now U.S. Patent No.11,447,770, issued September 20, 2022; International Application No. PCT/CN2022/077097, filed February 21, 2022; International Application No. PCT/US2022/015260, filed February 4, 2022; International Application No. PCT/KR2022/001611, filed January 28, 2022; International Application No. PCT/IN2022/050017, filed January 7, 2022; International Application No. PCT/US2022/012054, filed January 11, 2022; U.S. Application No.17/427,040, filed July 29, 2021, now U.S. Patent No.11,384,353, issued July 12, 2022; International Application No. PCT/US2021/052097, filed September 24, 2021; International Application No. PCT/KR2021/017534, filed November 25, 2021; International Application No. PCT/CN2021/130059, filed November 11, 2021; International Application No. PCT/US2021/057908, filed November 3, 2021; International Application No. PCT/US2021/058079, filed November 4, 2021; International Application No. PCT/KR2021/013326, filed September 29, 2021; International Application No. PCT/US2021/052097, filed September 24, 2021; International Application No. PCT/KR2021/010740, filed August 12, 2021; U.S. Application No.17/427,040, filed July 29, 2021; International Application No. PCT/US2021/044924, filed August 6, 2021; International Application No. PCT/KR2021/009794, filed July 28, 2021; International Application No. PCT/US2021/031439, filed May 7, 2021; International Application No. PCT/US2021/034996, filed May 28, 2021; International Application No. PCT/KR2021/005244, filed April 26, 2021; International Application No. PCT/KR2021/005031, filed April 21, 2021; International Application No. PCT/US2020/023730, filed March 19, 2020; International Application No. PCT/US2020/023713, filed March 19, 2020; International Application No. PCT/EP2021/054228, filed February 19, 2021; International Application No. PCT/US2020/067535, filed December 30, 2020; International Application No. PCT/US2020/059149, filed November 5, 2020; International Application No. RNG043-WO1 PCT Application PCT/US2020/055959, filed October 16, 2020; International Application No. PCT/US2020/055156, filed October 9, 2020; International Application No. PCT/US2020/023553, filed March 19, 2020; International Application No. PCT/US2020/023583, filed March 19, 2020; International Application No. PCT/US2020/023730, filed March 19, 2020; International Application No. PCT/US2020/023721, filed March 19, 2020; International Application No. PCT/US2020/023728, filed March 19, 2020; International Application No. PCT/US2020/023732, filed March 19, 2020; International Application No. PCT/US2020/023712, filed March 19, 2020; International Application No. PCT/US2020/023725, filed March 19, 2020; International Application No. PCT/US2020/023713, filed March 19, 2020; International Application No. PCT/US2020/023727, filed March 19, 2020; International Application No. PCT/US2020/023724, filed March 19, 2020; International Application No. PCT/US2020/023583, filed March 19, 2020; International Application No. PCT/US2020/023723, filed March 19, 2020; International Application No. PCT/CN2020/074218, filed February 3, 2020; U.S. Application No.15/616,756, filed June 7, 2017, now U.S. Patent No.10,189,831, issued January 29, 2019; International Application No. PCT/US2018/042040, filed July 13, 2018; U.S. Application No.15/164,208, filed May 25, 2016, now U.S. Patent No.10,150,955, issued December 11, 2018; International Application No. PCT/US2017/050690, September 8, 2017; U.S. Application No.11/502,819, filed August 10, 2006, now U.S. Patent No.9,783,791, issued October 10, 2017; and U.S. Application No. 13/277,763, filed October 20, 2011, now U.S. Patent No.9,458,484, issued October 4, 2016. [00301] In some embodiments, the chimeric gene editing composition comprises a prime editing system or a polynucleotide encoding a prime editing system. In some embodiments, the cargo comprises a component of a prime editing system or a polynucleotide encoding a component of a prime editing system. [00302] Prime editing is a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas fused to an engineered reverse transcriptase, also referred to as a prime editor, which is programmable using a prime editing guide RNA (“pegRNA”) that both specifies the target site and encodes the desired edit, such as, for example, as described in Anzalone et al., Nature 2019. Prime editing bypasses the need for DNA donor templates by using a prime editor having nickase or catalytically impaired enzymatic activity. RNG043-WO1 PCT Application [00303] A prime editing system comprises a prime editor. The prime editor (“PE”) comprises a catalytically impaired Cas protein fused to an engineered reverse transcriptase which can precisely and permanently edit one or more target nucleobases in a target polynucleotide. [00304] In some embodiments, the prime editor comprises an engineered Moloney murine leukemia virus (“M-MLV”) reverse transcriptase (“RT”) fused to a Cas-H840A nickase (called “PE2”). In some embodiments, the prime editor comprises an engineered M-MLV RT fused to a Cas9-H840A nickase. In some embodiments, the prime editor comprises an engineered M-MLV RT fused to a Streptococcus pyogenes Cas9 (spCas9)-H840A nickase. PE modifications include increased PAM flexibility to increase the utility of PE2 editing, expanding the coverage of targetable pathogenic variants in the ClinVar database that can now be prime edited to 94.4%. [00305] In some embodiments, the prime editing system further comprises a prime editing guide RNA (“pegRNA”). In some embodiments, the cargo comprises a pegRNA or a polynucleotide encoding a pegRNA. [00306] In some embodiments, the prime editing system further comprises a second guide RNA targeting the complementary strand, allowing the Cas9 nickase to also nick the non-edited strand (called “PE3”), which biases mismatch DNA repair in favor of the edited sequence. In some embodiments, the second guide RNA is designed to recognize the complementary strand of DNA only after the PE3 edit has occurred (called “PE3b”), which reduces indel formation. [00307] In some embodiments, the prime editing system comprises an uracil glycosylase inhibitor. In some embodiments, the prime editing system comprises a Cas9 protein fused to an uracil glycosylase inhibitor. In some embodiments, the cargo comprises an uracil glycosylase inhibitor or a polynucleotide encoding an uracil glycosylase inhibitor. In some embodiments, the cargo comprises a Cas9 protein fused to an uracil glycosylase inhibitor or a polynucleotide encoding a Cas9 protein fused to an uracil glycosylase inhibitor. [00308] Any of the above prime editor embodiments or variants, modifications, or derivatives thereof are contemplated herein to be delivered by the LNP systems disclosed in this specification for gene editing in cells, tissues, and/or organs under in vitro, ex vivo, or in vivo conditions. The various components described herein may be configured and delivered in any suitable manner. Any of the descriptions presented in this section are not intended to be strictly limiting. guide RNAs RNG043-WO1 PCT Application [00309] The present disclosure further provides guide RNAs for use in accordance with the disclosed nucleic acid programmable DNA binding proteins (e.g., Cas9) for use in methods of editing. The disclosure provides guide RNAs that are designed to recognize target sequences. Such gRNAs may be designed to have guide sequences (or “spacers”) having complementarity to a target sequence. Such gRNAs may be designed to have not only a guide sequences having complementarity to a target sequence to be edited, but also to have a backbone sequence that interacts specifically with the nucleic acid programmable DNA binding protein. [00310] In some embodiments, the guide RNA may be 15-100 nucleotides in length and comprise a sequence of at least 10, at least 15, or at least 20 contiguous nucleotides that is complementary to a target nucleotide sequence. The guide RNA may comprise a spacer sequence of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides that is complementary to a target nucleotide sequence. In some cases, the guide sequence has a length in a range of from 17-30 nucleotides (nt) (e.g., from 17-25, 17-22, 17-20, 19-30, 19-25, 19-22, 19-20, 20-30, 20-25, or 20-22 nt). In some cases, the guide sequence has a length in a range of from 17-25 nucleotides (nt) (e.g., from 17- 22, 17-20, 19-25, 19-22, 19-20, 20-25, or 20-22 nt). In some cases, the guide sequence has a length of 17 or more nt (e.g., 18 or more, 19 or more, 20 or more, 21 or more, or 22 or more nt; 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, etc.). In some cases, the guide sequence has a length of 19 or more nt (e.g., 20 or more, 21 or more, or 22 or more nt; 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, etc.). In some cases, the guide sequence has a length of 17 nt. In some cases, the guide sequence has a length of 18 nt. In some cases, the guide sequence has a length of 19 nt. In some cases, the guide sequence has a length of 20 nt. In some cases, the guide sequence has a length of 21 nt. In some cases, the guide sequence has a length of 22 nt. In some cases, the guide sequence has a length of 23 nt. [00311] In some cases, the spacer sequence has a length of from 15 to 50 nucleotides (e.g., from 15 nucleotides (nt) to 20 nt, from 20 nt to 25 nt, from 25 nt to 30 nt, from 30 nt to 35 nt, from 35 nt to 40 nt, from 40 nt to 45 nt, or from 45 nt to 50 nt). [00312] A subject guide RNA can interact with a target nucleic acid (e.g., double stranded DNA (dsDNA), single stranded DNA (ssDNA), single stranded RNA (ssRNA), or double stranded RNA (dsRNA)) in a sequence-specific manner via hybridization (i.e., base pairing). [00313] The guide RNA can be modified to hybridize to any desired target sequence (e.g., while taking the PAM into account, e.g., when targeting a dsDNA target) within a target RNG043-WO1 PCT Application nucleic acid (e.g., a eukaryotic target nucleic acid such as genomic DNA). In some cases, the percent complementarity between the spacer sequence of the guide and the target site of the target nucleic acid is 60% or more (e.g., 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the spacer and the target site of the target nucleic acid is 80% or more (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the spacer and the target site of the target nucleic acid is 90% or more (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the spacer and the target site of the target nucleic acid is 100%. [00314] In some cases, the percent complementarity between the spacer sequence and the target site of the target nucleic acid is 100% over an at least 5-nucleotide contiguous region of the spacer. In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid over an at least 6-nucleotide contiguous region of the spacer is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid over an at least 7-nucleotide contiguous region of the spacer is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid over an at least 8-nucleotide contiguous region of the spacer is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid over an at least 9-nucleotide contiguous region of the spacer is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid over an at least 10-nucleotide contiguous region of the spacer is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid over an at least 11-nucleotide contiguous region of the spacer is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In RNG043-WO1 PCT Application some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid over an at least 12-nucleotide contiguous region of the spacer is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid over an at least 13-nucleotide contiguous region of the spacer is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid over an at least 14-nucleotide contiguous region of the spacer is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid over an at least 15-nucleotide contiguous region of the spacer is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid over an at least 16-nucleotide contiguous region of the spacer is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid over an at least 17-nucleotide contiguous region of the spacer is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid over an at least 18-nucleotide contiguous region of the spacer is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid over an at least 19-nucleotide contiguous region of the spacer is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid over an at least 20-nucleotide contiguous region of the spacer is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the RNG043-WO1 PCT Application target nucleic acid over an at least 21-nucleotide contiguous region of the spacer is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid over an at least 22-nucleotide contiguous region of the spacer is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). [00315] In some cases, the percent complementarity between the spacer sequence and the target site of the target nucleic acid is 100% over an at least 5-10 nucleotide contiguous region of the spacer. In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid over an at least 6-11 nucleotide contiguous region of the spacer is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid over an at least 7-12 nucleotide contiguous region of the spacer is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid over an at least 8-13 nucleotide contiguous region of the spacer is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid over an at least 9-14 nucleotide contiguous region of the spacer is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid over an at least 10-15 nucleotide contiguous region of the spacer is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid over an at least 11-16 nucleotide contiguous region of the spacer is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid over an at least 12-17 nucleotide contiguous region of the spacer is 60% or more (e.g., 70% or more, 75% or more, 80% or RNG043-WO1 PCT Application more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid over an at least 13-18 nucleotide contiguous region of the spacer is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid over an at least 14-19 nucleotide contiguous region of the spacer is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid over an at least 15-20 nucleotide contiguous region of the spacer is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid over an at least 16-21 nucleotide contiguous region of the spacer is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid over an at least 17-22 nucleotide contiguous region of the spacer is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid over an at least 18-23 nucleotide contiguous region of the spacer is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid over an at least 19-24 nucleotide contiguous region of the spacer is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid over an at least 20-25 nucleotide contiguous region of the spacer is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid over an at least 21-26 nucleotide contiguous region of the spacer is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or RNG043-WO1 PCT Application 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid over an at least 22-27 nucleotide contiguous region of the spacer is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). [00316] In various embodiments, the guide RNAs may have a scaffold or core region that complexes with a cognate nucleic acid programmable DNA binding protein (e.g., CRISPR Cas9 or Cas12a). In some cases, a guide scaffold can have two stretches of nucleotides that are complementary to one another and hybridize to form a double stranded RNA duplex (dsRNA duplex). Thus, in some cases, the protein binding segment of a guide RNA includes a dsRNA duplex. In some embodiments, the dsRNA duplex region includes a range of from 5-25 base pairs (bp) (e.g., from 5-22, 5-20, 5-18, 5-15, 5-12, 5-10, 5-8, 8-25, 8-22, 8-18, 8-15, 8-12, 12- 25, 12-22, 12-18, 12- 15, 13-25, 13-22, 13-18, 13-15, 14-25, 14-22, 14-18, 14-15, 15-25, 15- 22, 15-18, 17-25, 17-22, or 17-18 bp, e.g., 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, etc.). In some cases, the dsRNA duplex region includes a range of from 6-15 base pairs (bp) (e.g., from 6-12, 6-10, or 6-8 bp, e.g., 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, etc.). In some cases, the duplex region includes 5 or more bp (e.g., 6 or more, 7 or more, or 8 or more bp). In some cases, the duplex region includes 6 or more bp (e.g., 7 or more, or 8 or more bp). In some cases, not all nucleotides of the duplex region are paired, and therefore the duplex forming region can include a bulge. The term “bulge” herein is used to mean a stretch of nucleotides (which can be one nucleotide) that do not contribute to a double stranded duplex, but which are surrounded 5’ and 3’ by nucleotides that do contribute, and as such a bulge is considered part of the duplex region. In some cases, the dsRNA includes 1 or more bulges (e.g., 2 or more, 3 or more, 4 or more bulges). In some cases, the dsRNA duplex includes 2 or more bulges (e.g., 3 or more, 4 or more bulges). In some cases, the dsRNA duplex includes 1-5 bulges (e.g., 1-4, 1-3, 2-5, 2-4, or 2-3 bulges). [00317] Thus, in some cases, the stretches of nucleotides that hybridize to one another to form the dsRNA duplex in a guide scaffold region have 70%-100% complementarity (e.g., 75%-100%, 80%-10%, 85%-100%, 90%- 100%, 95%-100% complementarity) with one another. In some cases, the stretches of nucleotides that hybridize to one another to form the dsRNA duplex have 70%-100% complementarity (e.g., 75%-100%, 80%-10%, 85%-100%, 90%-100%, 95%-100% complementarity) with one another. In some cases, the stretches of nucleotides that hybridize to one another to form the dsRNA duplex have 85%-100% complementarity (e.g., 90%-100%, 95%-100% complementarity) with one another. In some cases, the stretches of nucleotides that hybridize to one another to form the dsRNA duplex RNG043-WO1 PCT Application have 70%-95% complementarity (e.g., 75%-95%, 80%-95%, 85%-95%, 90%-95% complementarity) with one another. In other words, in some cases, the dsRNA duplex includes two stretches of nucleotides that have 70%-100% complementarity (e.g., 75%-100%, 80%- 10%, 85%-100%, 90%-100%, 95%-100% complementarity) with one another. In some cases, the dsRNA duplex includes two stretches of nucleotides that have 85%-100% complementarity (e.g., 90%-100%, 95%-100% complementarity) with one another. In some cases, the dsRNA duplex includes two stretches of nucleotides that have 70%-95% complementarity (e.g., 75%- 95%, 80%-95%, 85%-95%, 90%-95% complementarity) with one another. [00318] In various embodiments, the scaffold region of a guide RNA can also include one or more (1, 2, 3, 4, 5, etc.) mutations relative to a naturally occurring scaffold region. For example, in some cases a base pair can be maintained while the nucleotides contributing to the base pair from each segment can be different. In some cases, the duplex region of a subject guide RNA includes more paired bases, less paired bases, a smaller bulge, a larger bulge, fewer bulges, more bulges, or any convenient combination thereof, as compared to a naturally occurring duplex region (of a naturally occurring guide RNA). [00319] Examples of various guide RNAs can be found in the art, and in some cases variations similar to those introduced into Cas9 guide RNAs can also be introduced into guide RNAs of the present disclosure (e.g., mutations to the dsRNA duplex region, extension of the 5’ or 3’ end for added stability for to provide for interaction with another protein, and the like). In some examples, the guide RNAs can be the guide RNAs described in Jinek et al., Science. 2012 Aug 17;337(6096):816-21; Chylinski et al., RNA Biol.2013 May;10(5):726- 37; Ma et al., Biomed Res Int.2013;2013:270805; Hou et al., Proc Natl Acad Sci U S A.2013 Sep 24;110(39):15644-9; Jinek et al., Elife.2013;2:e00471; Pattanayak et al., Nat Biotechnol.2013 Sep;31(9):839-43; Qi et al, Cell.2013 Feb 28 ; 152(5): 1173-83 ; Wang et al., Cell.2013 May 9;153(4):910-8; Auer et al., Genome Res.2013 Oct 31; Chen et al., Nucleic Acids Res.2013 Nov 1 ;41(20):el9; Cheng et al., Cell Res.2013 Oct;23(10):1163-71; Cho et al., Genetics.2013 Nov;195(3):1177-80; DiCarlo et al., Nucleic Acids Res.2013 Apr;41(7):4336-43; Dickinson et al., Nat Methods.2013 Oct;10(10):1028-34; Ebina et al., Sci Rep.2013;3:2510; Fujii et. al, Nucleic Acids Res.2013 Nov l;41(20):el87; Hu et al., Cell Res.2013 Nov;23(ll):1322-5; Jiang et al., Nucleic Acids Res.2013 Nov l;41(20):el88; Larson et al., Nat Protoc.2013 Nov;8(l l):2180-96; Mali et. at., Nat Methods.2013 Oct;10(10):957-63; Nakayama et al., Genesis.2013 Dec;51(12):835-43; Ran et al., Nat Protoc.2013 Nov;8(l l):2281-308; Ran et al., Cell.2013 Sep 12;154(6):1380-9; Upadhyay et al., G3 (Bethesda).2013 Dec 9;3(12):2233-8; Walsh et al., Proc Natl Acad Sci U S A.2013 Sep 24;110(39):15514-5; Xie et al., Mol Plant.2013 Oct 9; RNG043-WO1 PCT Application Yang et al., Cell.2013 Sep 12;154(6):1370-9; Briner et al., Mol Cell.2014 Oct 23;56(2):333- 9; and U.S. patents and patent applications: 8,906,616; 8,895,308; 8,889,418; 8,889,356; 8,871,445; 8,865,406; 8,795,965; 8,771,945; 8,697,359; 20140068797; 20140170753; 20140179006; 20140179770; 20140186843; 20140186919; 20140186958; 20140189896; 20140227787; 20140234972; 20140242664; 20140242699; 20140242700; 20140242702; 20140248702; 20140256046; 20140273037; 20140273226; 20140273230; 20140273231; 20140273232; 20140273233; 20140273234; 20140273235; 20140287938; 20140295556; 20140295557; 20140298547; 20140304853; 20140309487; 20140310828; 20140310830; 20140315985; 20140335063; 20140335620; 20140342456; 20140342457; 20140342458; 20140349400; 20140349405; 20140356867; 20140356956; 20140356958; 20140356959; 20140357523; 20140357530; 20140364333; and 20140377868; all of which are hereby incorporated by reference in their entirety. [00320] In one embodiment, the guide RNAs (including pegRNAs) contemplated herein comprise non-naturally occurring nucleic acids and/or non-naturally occurring nucleotides and/or nucleotide analogs, and/or chemical modifications. Non-naturally occurring nucleic acids can include, for example, mixtures of naturally and non-naturally occurring nucleotides. Non-naturally occurring nucleotides and/or nucleotide analogs may be modified at the ribose, phosphate, and/or base moiety. In an embodiment of the present disclosure, a guide RNA (including pegRNA) component nucleic acid comprises ribonucleotides and non- ribonucleotides. In one such embodiment, a guide RNA (including pegRNA) component comprises one or more ribonucleotides and one or more deoxyribonucleotides. In an embodiment of the present disclosure, the guide RNA (including pegRNA) component comprises one or more non-naturally occurring nucleotide or nucleotide analog such as a nucleotide with phosphorothioate linkage, a locked nucleic acid (LNA) nucleotides comprising a methylene bridge between the 2' and 4' carbons of the ribose ring, or bridged nucleic acids (BNA). [00321] Other examples of modified nucleotides include 2'-O-methyl analogs, 2'-deoxy analogs, or 2'-fluoro analogs. Further examples of modified bases include, but are not limited to, 2-aminopurine, 5-bromo-uridine, pseudouridine, inosine, 7-methylguanosine. Examples of coRNA chemical modifications include, without limitation, incorporation of 2'-O-methyl (M), 2'-O-methyl 3 'phosphorothioate (MS), S-constrained ethyl(cEt), or 2'-O-methyl 3 'thioPACE (MSP) at one or more terminal nucleotides. Such chemically modified oRNA components can comprise increased stability and increased activity as compared to unmodified oRNA components, though on-target vs. off-target specificity is not predictable. In some RNG043-WO1 PCT Application embodiments, the modified nucleotides can be those described in Hendel, 2015, Nat Biotechnol.33(9):985-9, doi: 10.1038/nbt.3290, published online 29 June 2015 Ragdarm et al., 0215, PNAS, E7110-E7111; Allerson et al., J. Med. Chem.2005, 48:901-904; Bramsen et al., Front. Genet., 2012, 3:154; Deng et al., PNAS, 2015, 112: 11870-11875; Sharma et al., MedChemComm., 2014, 5: 1454-1471; Li et al., Nature Biomedical Engineering, 2017, 1, 0066 D01: 10.1038/s41551-017-0066). In one embodiment, the 5’ and/or 3’ end of a guide RNA (including pegRNA) component is modified by a variety of functional moieties including fluorescent dyes, polyethylene glycol, cholesterol, proteins, or detection tags, such as, for example, as described in Kelly et al., 2016, J. Biotech.233:74-83). In one embodiment, a guide RNA (including pegRNA) component comprises ribonucleotides in a region that binds to a target sequence and one or more deoxyribonucletides and/or nucleotide analogs in a region that binds to a nucleic acid programmable DNA binding protein (e.g., Cas9 nickase). [00322] In an embodiment, deoxyribonucleotides and/or nucleotide analogs are incorporated in engineered guide RNA (including pegRNA) component structures. In one embodiment, 3-5 nucleotides at either the 3’ or the 5’ end of a guide RNA (including pegRNA) component are chemically modified. In one embodiment, only minor modifications are introduced in the seed region, such as 2’-F modifications. In one embodiment, 2’-F modification is introduced at the 3’ end of a guide RNA (including pegRNA) component. In one embodiment, three to five nucleotides at the 5’ and/or the 3’ end of the reRNA component are chemically modified with 2’ -O-methyl (M), 2’-O-methyl 3’ phosphorothioate (MS), S- constrained ethyl(cEt), or 2’ -O-methyl 3’ thioPACE (MSP). Such modification can enhance genome editing efficiency, such as, for example, those described in Hendel et al., Nat. Biotechnol. (2015) 33(9): 985-989). In one embodiment, all of the phosphodiester bonds of a guide RNA (including pegRNA) component are substituted with phosphorothioates (PS) for enhancing levels of gene disruption. In one embodiment, more than five nucleotides at the 5’ and/or the 3’ end of the guide RNA (including pegRNA) component are chemically modified with 2’-0-Me, 2’-F or S-constrained ethyl(cEt). Such chemically modified guide RNA (including pegRNA) component can mediate enhanced levels of gene disruption, such as, for example, those described in Ragdarm et al., 2015, PNAS, E7110-E7111. In an embodiment of the present disclosure, a guide RNA (including pegRNA) component is modified to comprise a chemical moiety at its 3’ and/or 5’ end. Such moieties include, but are not limited to amine, azide, alkyne, thio, dibenzocyclooctyne (DBCO), or Rhodamine. In certain embodiment, the chemical moiety is conjugated to the guide RNA (including pegRNA) component by a linker, such as an alkyl chain. In one embodiment, the chemical moiety of the modified nucleic acid RNG043-WO1 PCT Application component can be used to attach the guide RNA (including pegRNA) component to another molecule, such as DNA, RNA, protein, or nanoparticles. Such chemically modified guide RNA (including pegRNA) component can be used to identify or enrich cells generically edited by a gene editing system described herein. [00323] In some examples, other guide RNA (including pegRNA) modifications are described in Kim, D.Y., Lee, J.M., Moon, S.B. et al. Efficient CRISPR editing with a hypercompact Cas12f1 and engineered guide RNAs delivered by adeno-associated virus. Nat Biotechnol 40, 94–102 (2022). [00324] Accordingly, in various aspects of the present disclosure, the guide RNA (including pegRNA) are modified in one or more locations within the molecule: MS1, an internal penta(uridinylate) (UUUUU) sequence in the tracrRNA; MS2, the 3′ terminus of the crRNA; MS3, the ‘stem 1’ region of the tracrRNA; MS4, the tracrRNA–crRNA complementary region; and MS5, the ‘stem 2’ region of the tracrRNA. [00325] Various aspects of the present disclosure provide methods and compositions for improved guide RNA (including pegRNA) stability via chemical modifications, such as, for examples, the methods and compositions described in Braasch, D. A., Jensen, S., Liu, Y., Kaur, K., Arar, K., White, M. A., et al. (2003). RNA interference in mammalian cells by chemically-modified RNA. Biochemistry 42, 7967–7975. doi: 10.1021/bi0343774. Chiu, Y. L., and Rana, T. M. (2003). siRNA function in RNAi: a chemical modification analysis. RNA 9, 1034–1048. doi: 10.1261/rna.5103703. Behlke, M. A. (2008). Chemical modification of siRNAs for in vivo use. Oligonucleotides18, 305–319. doi: 10.1089/oli.2008.0164. Bennett, C. F., and Swayze, E. E. (2010). RNA targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform. Annu. Rev. Pharmacol. Toxicol.50, 259–293. doi: 10.1146/annurev.pharmtox.010909.105654. Deleavey, G. F., and Damha, M. J. (2012). Designing chemically modified oligonucleotides for targeted gene silencing. Chem. Biol.19, 937–954. doi: 10.1016/j.chembiol.2012.07.011. Lennox, K. A., and Behlke, M. A. (2020). Chemical modifications in RNA interference and CRISPR/Cas genome editing reagents. Methods Mol. Biol.2115, 23–55. doi: 10.1007/978-1-0716-0290-4_2. [00326] In some examples, Hendel et al. improved guide RNA stability by chemically modifying gRNA ends to reduce degradation by exonucleases and RNA nuclease. See Hendel, A., Bak, R. O., Clark, J. T., Kennedy, A. B., Ryan, D. E., Roy, S., et al. (2015a). Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat. Biotechnol.33, 985–989. doi: 10.1038/nbt.3290. Chemical modifications of gRNAs may enable more efficient and safer gene-editing in primary cells suitable for clinical applications. RNG043-WO1 PCT Application [00327] In some examples, a review of types of chemical modifications is provided in Allen, Daniel et al. “Using Synthetically Engineered Guide RNAs to Enhance CRISPR Genome Editing Systems in Mammalian Cells.” Frontiers in genome editing vol.2617910.28 Jan.2021, doi:10.3389/fgeed.2020.617910. [00328] Accordingly, in various embodiments of the present disclosure, the genome editing system comprising a guide RNA (including pegRNA) and further comprises one or more chemical modifications selected from, but not limited to the modifications described in Allen et al. [00329] In exemplary embodiments, chemical modifications to the guide RNA (including pegRNA) include modifications on the ribose rings and phosphate backbone of guide RNA (including pegRNA) and modifications at the 2′OH include 2′-O-Me, 2′-F, and 2′F- ANA. More extensive ribose modifications include 2′F-4′-Cα-OMe and 2′,4′-di-Cα-OMe combine modification at both the 2′ and 4′ carbons. Phosphodiester modifications include sulfide-based Phosphorothioate (PS) or acetate-based phosphonoacetate alterations. Combinations of the ribose and phosphodiester modifications have given way to formulations such as 2′-O-methyl 3′phosphorothioate (MS), or 2′-O-methyl-3′-thioPACE (MSP), and 2′-O- methyl-3′-phosphonoacetate (MP) RNAs. Locked and unlocked nucleotides such as locked nucleic acid (LNA), bridged nucleic acids (BNA), S-constrained ethyl (cEt), and unlocked nucleic acid (UNA) are examples of sterically hindered nucleotide modifications. Modifications to make a phosphodiester bond between the 2′ and 5′ carbons (2′,5′-RNA) of adjacent RNAs as well as a butane 4-carbon chain link between adjacent RNAs have been described. [00330] In certain embodiments, the ncRNA and the guide RNA can be delivered as a single molecule, i.e., with the guide RNA fused to the 5’ and/or 3’ end of the ncRNA. The ncRNA may have guide RNAs located at both ends in some embodiments. [00331] In other embodiments, the guide RNA and ncRNA may be provided and/or delivered as separate components. Separation of the guide RNA from the ncRNA can result in increased editing efficiency. [00332] In still other embodiments, a ncRNA-gRNA fusion may be co-delivered with a separate guide RNA. B. Delivery Systems and Methods of Delivery Overview [00333] In yet another aspect, the disclosure provides compositions for transferring and/or expressing the retron-based gene editing systems, e.g., under in vitro, ex vivo, and in RNG043-WO1 PCT Application vivo conditions. In still another aspect, the disclosure provides cell-delivery compositions and methods, including compositions for passive and/or active transport to cells (e.g., plasmids), delivery by virus-based recombinant vectors (e.g., AAV and/or lentivirus vectors), delivery by non-virus-based systems (e.g., liposomes and LNPs), and delivery by virus-like particles of the retron-based gene editing systems described herein. Depending on the delivery system employed, the retron-based gene editing systems described herein may be delivered in the form of DNA (e.g., plasmids or DNA-based virus vectors), RNA (e.g., guide RNA and mRNA delivered by LNPs), a mixture of DNA and RNA, protein (e.g., virus-like particles), a mixture of DNA or RNA and protein, and ribonucleoprotein (RNP) complexes. Any suitable combinations of approaches for delivering the components of the herein disclosed retron-based gene editing systems may be employed. [00334] The retron-based gene editing systems and/or components thereof can be delivered by any known delivery system such as those described above, including (a) without vectors (e.g., electroporation), (b) viral delivery systems and (c) non-viral delivery systems. Viral delivery systems include expression vectors, adeno-associated virus (AAV) vectors, retroviral vectors, lentiviral vectors, and the like. An expression construct can be replicated in a living cell, or it can be made synthetically. Non-viral delivery systems include without limitation lipid particles (e.g. Lipid nanoparticles (LNPs)), non-lipid nanoparticles, exosomes, liposomes, micelles, viral particles, stable nucleic-acid-lipid particles (SNALPs), lipoplexes/polyplexes, DNA nanoclews, Gold nanoparticles, iTOP, Streptolysin O (SLO), multifunctional envelope-type nanodevice (MEND), lipid-coated mesoporous silica particles, inorganic nanoparticles, and polymeric delivery technology (e.g., polymer-based particles). [00335] In some examples, delivery of nucleic acid modalities, including RNA therapeutics, can be described further in Paunovska K, Loughrey D, Dahlman JE. Drug delivery systems for RNA therapeutics. Nat Rev Genet.2022 May;23(5):265-280. doi: 10.1038/s41576-021-00439-4. Epub 2022 Jan 4. PMID: 34983972; PMCID: PMC8724758; Hong CA, Nam YS. Functional nanostructures for effective delivery of small interfering RNA therapeutics. Theranostics.2014 Sep 19;4(12):1211-32. doi: 10.7150/thno.8491. PMID: 25285170; PMCID: PMC4183999; Liu F, Wang C, Gao Y, Li X, Tian F, Zhang Y, Fu M, Li P, Wang Y, Wang F. Current Transport Systems and Clinical Applications for Small Interfering RNA (siRNA) Drugs. Mol Diagn Ther.2018 Oct;22(5):551-569. doi: 10.1007/s40291-018- 0338-8. PMID: 29926308; Zhang Y, Almazi JG, Ong HX, Johansen MD, Ledger S, Traini D, Hansbro PM, Kelleher AD, Ahlenstiel CL. Nanoparticle Delivery Platforms for RNAi Therapeutics Targeting COVID-19 Disease in the Respiratory Tract. Int J Mol Sci.2022 Feb RNG043-WO1 PCT Application 22;23(5):2408. doi: 10.3390/ijms23052408. PMID: 35269550; PMCID: PMC8909959; Zhang M, Hu S, Liu L, Dang P, Liu Y, Sun Z, Qiao B, Wang C. Engineered exosomes from different sources for cancer-targeted therapy. Signal Transduct Target Ther.2023 Mar 15;8(1):124. doi: 10.1038/s41392-023-01382-y. PMID: 36922504; PMCID: PMC10017761; Hastings ML, Krainer AR. RNA therapeutics. RNA.2023 Apr;29(4):393-395. doi: 10.1261/rna.079626.123. PMID: 36928165; PMCID: PMC10019368; Miele E, Spinelli GP, Miele E, Di Fabrizio E, Ferretti E, Tomao S, Gulino A. Nanoparticle-based delivery of small interfering RNA: challenges for cancer therapy. Int J Nanomedicine.2012;7:3637-57. doi: 10.2147/IJN.S23696. Epub 2012 Jul 20. PMID: 22915840; PMCID: PMC3418108, each of which are incorporated by reference in their entireties. [00336] The engineered retron-based gene editing systems (or vectors containing them) may be introduced into any type of cell, including any cell from a prokaryotic, eukaryotic, or archaeon organism, including bacteria, archaea, fungi, protists, plants (e.g., monocotyledonous and dicotyledonous plants); and animals (e.g., vertebrates and invertebrates). Examples of animals that may be transfected with an engineered retron-based gene editing systems include, without limitation, vertebrates such as fish, birds, mammals (e.g., human and non-human primates, farm animals, pets, and laboratory animals), reptiles, and amphibians. [00337] The engineered retron-based gene editing systems (or components thereof) can be introduced into a single cell or a population of cells. Cells from tissues, organs, and biopsies, as well as recombinant cells, genetically modified cells, cells from cell lines cultured in vitro, and artificial cells (e.g., nanoparticles, liposomes, polymersomes, or microcapsules encapsulating nucleic acids) may all be transfected with the engineered retron-based gene editing systems. [00338] The engineered retron-based gene editing systems (or components thereof) can be introduced into cellular fragments, cell components, or organelles (e.g., mitochondria in animal and plant cells, plastids (e.g., chloroplasts) in plant cells and algae). [00339] Cells may be cultured or expanded after transfection with the engineered retron- based gene editing systems. [00340] Methods of introducing nucleic acids into a host cell are well known in the art. Commonly used methods include chemically induced transformation, typically using divalent cations (e.g., CaCl2), dextran-mediated transfection, polybrene mediated transfection, lipofectamine and LT-1 mediated transfection, electroporation, protoplast fusion, encapsulation of nucleic acids in liposomes, and direct microinjection of the nucleic acids comprising Cas12a editing systems into nuclei, such as, for example, the methods described in Sambrook et al. RNG043-WO1 PCT Application (2001) Molecular Cloning, a laboratory manual, 3rd edition, Cold Spring Harbor Laboratories, New York, Davis et al. (1995) Basic Methods in Molecular Biology, 2nd edition, McGraw- Hill, and Chu et al. (1981) Gene 13:197; herein incorporated by reference in their entireties. [00341] Plant cells may also be targeted by the retron-based gene editing systems (or components thereof) disclosed herein. In some examples, methods for genetic transformation of plant cells can be those described in in US2022/0145296, and U.S. Pat. Nos.8,575,425; 7,692,068; 8,802,934; 7,541,517; Rakoczy-Trojanowska, M. (2002) Cell Mol Biol Lett.7:849- 858; Jones et al. (2005) Plant Methods 1:5; Rivera et al. (2012) Physics of Life Reviews 9:308- 345; Bartlett et al. (2008) Plant Methods 4:1-12; Bates, G. W. (1999) Methods in Molecular Biology 111:359-366; Binns and Thomashow (1988) Annual Reviews in Microbiology 42:575-606; Christou, P. (1992) The Plant Journal 2:275-281; Christou, P. (1995) Euphytica 85:13-27; Tzfira et al. (2004) TRENDS in Genetics 20:375-383; Yao et al. (2006) Journal of Experimental Botany 57:3737-3746; Zupan and Zambryski (1995) Plant Physiology 107:1041- 1047; and Jones et al. (2005) Plant Methods 1:5, each of which is herein incorporated by reference in its entirety. [00342] The plant cells that have been transformed may be grown into a transgenic organism, such as a plant, in accordance with conventional methods for example, such as those described in McCormick et al. (1986) Plant Cell Reports 5:81-84. [00343] Plant material that may be transformed with the retron-based gene editing systems (or components thereof) described herein includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like. Progeny, variants, and mutants of the regenerated plants are also included within the scope of the disclosure, provided that these parts comprise the genetic modification introduced by the retron-based gene editing systems. Further provided is a processed plant product or byproduct that retains the genetic modification introduced by the retron-based gene editing systems. [00344] The retron-based gene editing systems described herein may be used to produce transgenic plants with desired phenotypes, including but not limited to, increased disease resistance (e.g., increased viral, bacterial of fungal resistance), increased insect resistance, increased drought resistance, increased yield, and altered fruit ripening characteristics, sugar and oil composition, and color. [00345] In some embodiments involving retron-based gene editing systems, the retron msr gene, msd gene, and/or ret gene can be expressed in vitro from a vector, such as in an in RNG043-WO1 PCT Application vitro transcription system. The resulting ncRNA or msDNA can be isolated before being packaged and/or formulated for direct delivery into a host cell. For example, the isolated ncRNA or msDNA can be packaged/formulated in a delivery vehicle such as lipid nanoparticles as described in other sections. [00346] In some embodiments involving retron-based gene editing systems, the retron msr gene, msd gene, and/or ret gene are expressed in vivo from a vector within a cell. The retron msr gene, msd gene, and/or ret gene can be introduced into a cell with a single vector or in multiple separate vectors to produce msDNA in a host subject. [00347] In other embodiments, the retron msr gene, msd gene, and/or ret gene, and any other components of the retron-based genome editing systems described herein (e.g., guide RNA in trans, programmable nuclease (e.g., in trans)) may be expressed in vivo from RNA delivered to the cell. The retron msr gene, msd gene, and/or ret gene can be introduced into a cell with a single vector or in multiple separate vectors to produce msDNA in a host subject. [00348] Vectors and/or nucleic acid molecules encoding the recombinant retron-based genome editing system or components thereof can include control elements operably linked to the retron sequences, which allow for the production of msDNA either in vitro, or in vivo in the subject species. For example, in embodiments relating to retron-based gene editing systems, the retron msr gene, msd gene, and/or ret gene can be operably linked to a promoter to allow expression of the retron reverse transcriptase and/or the msDNA product. In some embodiments, heterologous sequences encoding desired products of interest (e.g., polynucleotide encoding polypeptide or regulatory RNA, donor polynucleotide for gene editing, or protospacer DNA for molecular recording) may be inserted in the msr gene and/or msd gene. [00349] In some embodiments, the retron-based gene editing systems are produced by a vector system comprising one or more vectors. [00350] Numerous vectors are available for use in the vector or vector system, including but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. All-RNA format [00351] In various embodiments, the retron-based gene editing systems (or components thereof) disclosed herein may be delivered in an “all-RNA” format. As used herein, the term “all-RNA” format refers to the fact that each of the components of a retron editing system (e.g., the retron RT, the programmable nuclease, the sgRNA, and the ncRNA) are delivered and/or administered as RNA (e.g., coding RNA or non-coding RNA). In some embodiments, RNG043-WO1 PCT Application the RNA components may be delivered to cells and/or tissues by direct means, such as electroporation or transfection. In other embodiments, the RNA components may be delivered to cells and/or tissues by way of a delivery vehicle, such as an LNP or liposome. [00352] In various embodiments, the retron editing systems described herein may comprise a coding RNA (e.g., linear or circular mRNA) that encodes a retron reverse transcriptase (e.g., any RT from the abovementioned Table X or the abovementioned Table A), a coding RNA (e.g., linear or circular mRNA) that encodes a programmable nuclease (e.g., a Cas9, Cas12a, or TnpB nuclease), a retron ncRNA (e.g., a ncRNA from the abovementioned Table B), and a guide RNA for targeting the programmable nuclease to a particular desired target sequence. [00353] In some embodiments, RT and nuclease components may be encoded on the same coding RNA molecule. The proteins may also be expressed from separate coding RNA molecules. In still other embodiments, the RT and the nuclease components can be fused together as a singular fusion polypeptide having an RT domain and a nuclease domain optionally joined by a linker. [00354] In addition, in some embodiments, the ncRNA and the guide RNA may be fused together as a single RNA molecule. For example, the guide RNA may be located at the 5’ end of the ncRNA. In other embodiments, the guide RNA may be located at the 3’ end of the ncRNA. In some embodiments, the ncRNA may comprise a guide RNA at both the 3’ and the 5’ ends of the ncRNA. [00355] In still other embodiments, the ncRNA and the guide RNA may be separate molecules, i.e., delivered separately. [00356] In still other embodiments, the retron editing system may include both a ncRNA-guide RNA fusion and an additional guide RNA provided as a separate molecule. [00357] In various embodiments, the different RNA components of the all-RNA retron editing system can be combined and administered (e.g., directly or within a delivery vehicle) in different ratios. In some embodiments, the ratios of such RNA components or species can be expressed as molar ratios. [00358] For example, the molar ratio of RT coding RNA to nuclease coding RNA can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20. Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 RNG043-WO1 PCT Application to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40. [00359] In another example, the molar ratio of nuclease coding RNA to RT coding RNA can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20. Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40. [00360] In still another example, the molar ratio of ncRNA or ncRNA-guide RNA fusion to separate guide RNA can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20. Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40. [00361] In still another example, the molar ratio of separate guide RNA to ncRNA or ncRNA-guide RNA fusion can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20. Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40. [00362] In still another example, the molar ratio of ncRNA to separate guide RNA can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20. Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40. [00363] In still another example, the molar ratio of separate guide RNA to ncRNA can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20. Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, RNG043-WO1 PCT Application from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40. [00364] In still another example, the molar ratio of a coding RNA (e.g., encoding RT and/or nuclease) to ncRNA or ncRNA-guide RNA fusion, as the case may be, can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20. Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40. [00365] In still another example, the molar ratio of a coding RNA encoding a retron RT to ncRNA or ncRNA-guide RNA fusion, as the case may be, can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20. Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40. [00366] In still another example, the molar ratio of a coding RNA encoding a programmable nuclease to ncRNA or ncRNA-guide RNA fusion, as the case may be, can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20. Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40. [00367] In still another example, the molar ratio of a coding RNA encoding a retron RT or a nuclease to a separate guide RNA can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20. Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40. [00368] In still another example, the molar ratio of a separate guide RNA to a coding RNA encoding a retron RT or a nuclease can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, RNG043-WO1 PCT Application about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20. Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40. [00369] In certain embodiments, the amount of ncRNA-sgRNA relative to RT mRNA is augmented. In certain embodiments the RT mRNA: ncRNA-sgRNA ratio is about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20. Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40. In certain embodiments, an RT-Cas9 (or Cas9-RT) fusion is encoded by an mRNA. In certain embodiments, the RT-Cas9 mRNA: ncRNA-sgRNA ratio is about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20. Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40. In certain embodiments, multiple genetic loci are targeted hence the ncRNA-sgRNA includes a mixture of ncRNA-sgRNA species and the same ratios and ranges are applicable. RNA-protein or DNA-protein format [00370] In various embodiments, the retron-based gene editing systems (or components thereof) disclosed herein may be delivered in a composition comprising protein, DNA and/or RNAs. For example, some components of a retron editing system (e.g., the sgRNA, and the ncRNA) are delivered as RNA and other components (e.g., the retron RT, the programmable nuclease) are delivered as proteins. [00371] In some embodiments, the RNA or DNA components and the protein components are delivered to cells or tissues by the same means, such as electroporation or transfection, LNP, liposome, exosomes, etc. In other embodiments, the RNA or DNA components and the protein components are be delivered to cells or tissues by different types of delivery vehicles. RNG043-WO1 PCT Application [00372] In some embodiments, the RNA or DNA components and the protein components are delivered concurrently. In some embodiments, the RNA or DNA components and the protein components are delivered separately, e.g., sequentially. Viral vector delivery [00373] In various embodiments, the retron-based gene editing systems described herein may be delivered in viral vectors. [00374] Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus (AAV) vectors, retroviral vectors, lentiviral vectors, and the like. An expression construct can be replicated in a living cell, or it can be made synthetically. [00375] In some embodiments, the nucleic acid comprising an retron-based gene editing system (or component thereof) is under transcriptional control of a promoter. In some embodiments, the promoter is competent for initiating transcription of an operably linked coding sequence by a RNA polymerase I, II, or III. [00376] Exemplary promoters for mammalian cell expression include the SV40 early promoter, a CMV promoter such as the CMV immediate early promoter, such as, for example, as described in U.S. Patent Nos.5,168,062 and 5,385,839, incorporated herein by reference in their entireties), the mouse mammary tumor virus LTR promoter, the adenovirus major late promoter (Ad MLP), and the herpes simplex virus promoter, among others. Other nonviral promoters, such as a promoter derived from the murine metallothionein gene, will also find use for mammalian expression. [00377] In some examples, exemplary promoters for plant cell expression include the CaMV 35S promoter for example as described in Odell et al., 1985, Nature 313:810-812; the rice actin promoter for example as described in McElroy et al., 1990, Plant Cell 2:163-171; the ubiquitin promoter for example as described in Christensen et al., 1989, Plant Mol. Biol. 12:619-632; and Christensen et al., 1992, Plant Mol. Biol.18:675-689; the pEMU promoter for example as described in Last et al., 1991, Theor. Appl. Genet.81:581-588; and the MAS promoter for example as described in Velten et al., 1984, EMBO J.3:2723-2730. [00378] In additional embodiments, the retron-based vectors may also comprise tissue- specific promoters to start expression only after it is delivered into a specific tissue. Non- limiting exemplary tissue-specific promoters include B29 promoter, CD14 promoter, CD43 promoter, CD45 promoter, CD68 promoter, desmin promoter, elastase- 1 promoter, endoglin promoter, fibronectin promoter, Flt-1 promoter, GFAP promoter, GPIIb promoter, ICAM- 2 RNG043-WO1 PCT Application promoter, INF-b promoter, Mb promoter, Nphsl promoter, OG-2 promoter, SP-B promoter, SYN1 promoter, and WASP promoter. [00379] In some examples, these and other promoters can be obtained from or incorporated into commercially available plasmids, using techniques as described in Sambrook et al., supra. [00380] In some embodiments, one or more enhancer elements is/are used in association with the promoter to increase expression levels of the constructs. Examples include the SV40 early gene enhancer, as described in Dijkema et al., EMBOJ (1985) 4:761, the enhancer/promoter derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus, as described in Gorman et al., Proc. Natl. Acad. Sci. USA (1982b) 79:6777, and elements derived from human CMV, as described in Boshart et al., Cell (1985) 41:521, such as elements included in the CMV intron A sequence. All such sequences are incorporated herein by reference. [00381] In one embodiment, an expression vector for expressing a retron-based gene editing system (or component thereof) comprises a promoter operably linked to a polynucleotide encoding the components. The components of the retron-based gene editing system may be configured as individual gene transcripts or as fused constructs. For example, the nuclease component may be fused with the reverse transcriptase component. In another example, the ncRNA component may be fused with the guide RNA component. In another example, the nuclease component may be fused with the reverse transcriptase component, but the guide RNA and the ncRNA are separate. In other embodiments, the guide RNA and ncRNA components may be fused, but the reverse transcripase and nuclease components are separately provided. Any functional combinations of fused components and non-fused components are contemplated. [00382] In some embodiments, the vector or vector system also comprises a transcription terminator/polyadenylation signal. Examples of such sequences include, but are not limited to, those derived from SV40, as described in Sambrook et al., supra, as well as a bovine growth hormone terminator sequence, as described in U.S. Patent No.5,122,458. [00383] Additionally, 5ʹ- UTR sequences can be placed adjacent to the coding sequence to further enhance the expression. Such sequences may include UTRs comprising an internal ribosome entry site (IRES). Inclusion of an IRES permits the translation of one or more open reading frames from a vector. In some examples, the IRES element attracts a eukaryotic ribosomal translation initiation complex and promotes translation initiation, for example, as described in Kaufman et al., Nuc. Acids Res. (1991) 19:4485-4490; Gurtu et al., Biochem. RNG043-WO1 PCT Application Biophys. Res. Comm. (1996) 229:295-298: Rees et al., BioTechniques (1996) 20:102-110; Kobayashi et al., BioTechniques (1996) 21:399-402; and Mosser et al., BioTechniques (199722 ISO- 161)c . In some examples, a multitude of IRES sequences are known and include sequences derived from a wide variety of viruses, such as, for example, from leader sequences of picomaviruses such as the encephalomyocarditis virus (EMCV) UTR, for example, as described in Jang et al. Virol. (1989) 63:1651-1660, the polio leader sequence, the hepatitis A virus leader, the hepatitis C virus IRES, human rhinovirus type 2 IRES, for example, as described in Dobrikova et al., Proc. Natl. Acad. Sci. (2003) 100(251:15125-151301), an IRES element from the foot and mouth disease virus, for example, as described in Ramesh et al., Nucl. Acid Res. (1996) 24:2697-2700, a giardiavirus IRES, for example, as described in Garlapati et al., J Biol. Chem. (2004) 279(51):3389-33971, and the like. In some examples, a variety of nonviral IRES sequences will also find use herein, including, but not limited to IRES sequences from yeast, as well as the human angiotensin II type 1 receptor IRES for example as described in Martin et al., Mol. Cell Endocrinol. (2003) 212:51-61, fibroblast growth factor IRESs (FGF-1 IRES and FGF-2 IRES, for example as described in Martineau et al. (2004) Mol. Cell. Biol.24(17): 7622-7635), vascular endothelial growth factor IRES for example as described in Baranick et al. (2008) Proc. Natl. Acad Sci. U.S.A.105(12):4733-4738, Stein et al. (1998) Mol. Cell. Biol.18(6):3112-3119, Bert et al. (2006) RNA 12(6): 1074-1083, and insulin-like growth factor 2 IRES for example as described in Pedersen et al. (2002) Biochem. J.363(Pt l):37-44. [00384] These elements are commercially available in plasmids sold, e.g., by Clontech (Mountain View, CA), Invivogen (San Diego, CA), Addgene (Cambridge, MA) and GeneCopoeia (Rockville, MD). See also IRESite: The database of experimentally verified IRES structures (iresite.org). An IRES sequence may be included in a vector, for example, to express multiple bacteriophage recombination proteins for recombineering or an RNA-guided nuclease (e.g., Cas9) for HDR in combination with a retron reverse transcriptase from an expression cassette. [00385] In some embodiments, a polynucleotide encoding a viral self-cleaving 2A peptide, such as a T2A peptide, can be used to allow production of multiple protein products (e.g., Cas9, bacteriophage recombination proteins, retron reverse transcriptase) from a single vector or a single transcription unit under one promoter. One or more 2A linker peptides can be inserted between the coding sequences in the multicistronic construct. The 2A peptide, which is self-cleaving, allows co-expressed proteins from the multicistronic construct to be produced at equimolar levels. In some examples, 2A peptides from various viruses may be used, RNG043-WO1 PCT Application including, but not limited to 2A peptides derived from the foot-and-mouth disease virus, equine rhinitis A virus, Jhosea asigna virus and porcine teschovirus, such as those described in Kim et al. (2011) PLoS One 6(4): el8556, Trichas et al. (2008) BMC Biol.6:40, Provost et al. (2007) Genesis 45(10): 625-629, Furler et al. (2001) Gene Ther.8(11):864-873; herein incorporated by reference in their entireties. [00386] In some embodiments, the expression construct comprises a plasmid suitable for transforming a bacterial host. Numerous bacterial expression vectors are known to those of skill in the art, and the selection of an appropriate vector is a matter of choice. Bacterial expression vectors include, but are not limited to, pACYC177, pASK75, pBAD, pBADM, pBAT, pCal, pET, pETM, pGAT, pGEX, pHAT, pKK223, pMal, pProEx, pQE, and pZA31. In some examples, bacterial plasmids may contain antibiotic selection markers (e.g., ampicillin, kanamycin, erythromycin, carbenicillin, streptomycin, or tetracycline resistance), a lacZ gene (b-galactosidase produces blue pigment from x-gal substrate), fluorescent markers (e.g., GFP. mCherry), or other markers for selection of transformed bacteria, such as, for example, those described in Sambrook et al., supra. [00387] In other embodiments, the expression construct comprises a plasmid suitable for transforming a yeast cell. Yeast expression plasmids typically contain a yeast-specific origin of replication (ORI) and nutritional selection markers (e.g., HIS3, URA3, LYS2, LEU2, TRP1, METIS, ura4+, leul+, ade6+), antibiotic selection markers (e.g., kanamycin resistance), fluorescent markers (e.g., mCherry), or other markers for selection of transformed yeast cells. The yeast plasmid may further contain components to allow shuttling between a bacterial host (e.g., E coli) and yeast cells. A number of different types of yeast plasmids are available including yeast integrating plasmids (Yip), which lack an ORI and are integrated into host chromosomes by homologous recombination; yeast replicating plasmids (YRp), which contain an autonomously replicating sequence (ARS) and can replicate independently; yeast centromere plasmids (YCp), which are low copy vectors containing a part of an ARS and part of a centromere sequence (CEN); and yeast episomal plasmids (YEp), which are high copy number plasmids comprising a fragment from a 2 micron circle (a natural yeast plasmid) that allows for 50 or more copies to be stably propagated per cell. [00388] In other embodiments, the expression construct does not comprise a plasmid suitable for transforming a yeast cell. [00389] In other embodiments, the expression construct comprises a virus or engineered construct derived from a viral genome. A number of viral based systems have been developed for gene transfer into mammalian cells. In some examples, the viral based systems include RNG043-WO1 PCT Application adenoviruses, retroviruses (g-retroviruses and lentiviruses), poxviruses, adeno-associated viruses, baculoviruses, and herpes simplex viruses, such as, for example, those described in Wamock et al. (2011) Methods Mol. Biol.737:1-25; Walther et al. (2000) Drugs 60(2):249- 271; and Lundstrom (2003) Trends Biotechnol.21(3): 117-122; herein incorporated by reference in their entireties). The ability of certain viruses to enter cells via receptor-mediated endocytosis, to integrate into host cell genomes and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells. [00390] For example, retroviruses provide a convenient platform for gene delivery systems. Selected sequences can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. In some examples, the retroviral system can be the one described in U.S. Pat. No.5,219,740; Miller and Rosman (1989) BioTechniques 7:980- 990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Bums et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop.3:102-109; and Ferry et al. (2011) Curr. Pharm. Des.17(24): 2516-2527). Lentiviruses are a class of retroviruses that are particularly useful for delivering polynucleotides to mammalian cells because they are able to infect both dividing and nondividing cells, such as, for example, as described in Lois et al. (2002) Science 295:868- 872; Durand et al. (2011) Viruses 3(2): 132-159; herein incorporated by reference). [00391] A number of adenoviral vectors have also been described. Unlike retroviruses which integrate into the host genome, adenoviruses persist extrachromosomally thus minimizing the risks associated with insertional mutagenesis. [00392] Additionally, various adeno-associated vims (AAV) vector systems have been developed for gene delivery. In some examples, AAV vectors can be readily constructed using techniques as described in U.S. Pat. Nos.5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 (published 23 January 1992) and WO 93/03769 (published 4 March 1993); Lebkowski et al., Molec. Cell. Biol. (1988) 8:3988-3996; Vincent et al., Vaccines 90 (1990) (Cold Spring Harbor LaboratoryPress); Carter, B. J. Current Opinion in Biotechnology (1992) 3:533-539; Muzyczka, N. Current Topics in Microbiol and Immunol. (1992) 158:97-129; Kotin, R. M. Human Gene Therapy (1994) 5:793-801; Shelling and Smith, Gene Therapy (1994) 1:165-169; and Zhou et al., J. Exp. Med. (1994) 179:1867-1875. [00393] In some examples, another vector system useful for delivering nucleic acids encoding the Cas12a editing system components is the enterically administered recombinant RNG043-WO1 PCT Application poxvirus vaccines as described by Small, Jr., P. A., et al. (U.S. Pat. No.5,676,950, issued Oct. 14, 1997, herein incorporated by reference). [00394] Other viral vectors include those derived from the pox family of viruses, including vaccinia virus and avian poxvirus. By way of example, vaccinia virus recombinants expressing a nucleic acid molecule of interest (e.g., Cas12a editing system) can be constructed as follows. The DNA encoding the particular nucleic acid sequence is first inserted into an appropriate vector so that it is adjacent to a vaccinia promoter and flanking vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK). This vector is then used to transfect cells which are simultaneously infected with vaccinia. Homologous recombination serves to insert the vaccinia promoter plus the gene encoding the sequences of interest into the viral genome. The resulting TK-recombinant can be selected by culturing the cells in the presence of 5- bromodeoxyuridine and picking viral plaques resistant thereto. [00395] In some embodiments, avipoxviruses, such as the fowlpox and canarypox viruses, can also be used to deliver the nucleic acid molecules of interest. The use of an avipox vector is particularly desirable in human and other mammalian species since members of the avipox genus can only productively replicate in susceptible avian species and therefore are not infective in mammalian cells. Methods for producing recombinant avipoxviruses are known in the art and employ genetic recombination, as described above with respect to the production of vaccinia viruse, such as, for example, the methods described in WO 91/12882; WO 89/03429; and WO 92/03545. [00396] In some examples, molecular conjugate vectors, such as the adenovirus chimeric vectors described in Michael et al., J. Biol. Chem. (1993) 268:6866-6869 and Wagner et al., Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can also be used for gene delivery. [00397] Members of the alphavirus genus, such as, but not limited to, vectors derived from the Sindbis virus (SIN), Semliki Forest virus (SFV), and Venezuelan Equine Encephalitis virus (VEE), will also find use as viral vectors for delivering the polynucleotides of the present disclosure. In some examples, the Sindbis-virus derived vectors can be those described in Dubensky et al. (1996) J. Virol.70:508-519; and International Publication Nos. WO 95/07995, WO 96/17072; as well as, Dubensky, Jr., T. W., et al., U.S. Pat. No.5,843,723, issued Dec.1, 1998, and Dubensky, Jr., T. W., U.S. Patent No.5,789,245, issued Aug.4, 1998, which are each herein incorporated by reference. In some examples, particularly preferred are chimeric alphavirus vectors comprised of sequences derived from Sindbis virus and Venezuelan equine encephalitis virus, such as, for example, those described in Perri et al. (2003) J. Virol.77: RNG043-WO1 PCT Application 10394-10403 and International Publication Nos. WO 02/099035, WO 02/080982, WO 01/81609, and WO 00/61772; each herein incorporated by reference in their entireties. [00398] A vaccinia-based infection/transfection system can be conveniently used to provide for inducible, transient expression of the nucleic acids of interest (e.g., Cas12a editing system) in a host cell. In this system, cells are first infected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase. This polymerase displays exquisite specificity in that it only transcribes templates bearing T7 promoters. Following infection, cells are transfected with the nucleic acid of interest, driven by a T7 promoter. The polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA. The method provides for high level, transient, cytoplasmic production of large quantities of RNA, such as, for example, as described in Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990) 87:6743- 6747; Fuerst et al., Proc. Natl. Acad. Sci. USA (1986) 83:8122-8126. [00399] In other approaches to infection with vaccinia or avipox virus recombinants, or to the delivery of nucleic acids using other viral vectors, an amplification system can be used that will lead to high level expression following introduction into host cells. Specifically, a T7 RNA polymerase promoter preceding the coding region for T7 RNA polymerase can be engineered. Translation of RNA derived from this template will generate T7 RNA polymerase which in turn will transcribe more templates. Concomitantly, there will be a cDNA whose expression is under the control of the T7 promoter. Thus, some of the T7 RNA polymerase generated from translation of the amplification template RNA will lead to transcription of the desired gene. Because some T7 RNA polymerase is required to initiate the amplification, T7 RNA polymerase can be introduced into cells along with the template(s) to prime the transcription reaction. The polymerase can be introduced as a protein or on a plasmid encoding the RNA polymerase. In some examples, the T7 system can be those described in International Publication No. WO 94/26911; Studier and Moffatt, J. Mol. Biol. (1986) 189:113-130; Deng and Wolff, Gene (1994) 143:245-249; Gao et al., Biochem. Biophys. Res. Commun. (1994) 200:1201-1206; Gao and Huang, Nuc. Acids Res. (1993) 21:2867-2872; Chen et al., Nuc. Acids Res. (1994) 22:2114-2120; and U.S. Pat. No.5,135,855. [00400] Insect cell expression systems, such as baculovirus systems, can also be used, such as, for example those described in Baculovirus and Insect Cell Expression Protocols (Methods in Molecular Biology, D.W. Murhammer ed., Humana Press, 2nd edition, 2007) and L. King The Baculovirus Expression System: A laboratory guide (Springer, 1992). Materials and methods for baculovirus/insect cell expression systems are commercially available in kit RNG043-WO1 PCT Application form from, inter alia, Thermo Fisher Scientific (Waltham, MA) and Clontech (Mountain View, CA). [00401] Plant expression systems can also be used for transforming plant cells. Generally, such systems use virus-based vectors to transfect plant cells with heterologous genes. In some examples, such systems can include those described in Porta et al., Mol. Biotech. (1996) 5:209-221; and Hackland et al., Arch. Virol. (1994) 139:1-22. [00402] To obtain expression of the retron-based editing systems (or components thereof), the expression constructs and/or RNA components must be delivered into a cell. This delivery may be accomplished in vitro, as in laboratory procedures for transforming cells lines, or in vivo or ex vivo, as in the treatment of certain disease states. One mechanism for delivery is via viral infection where the expression construct is encapsulated in an infectious viral particle. Non-viral delivery methods [00403] Several non-viral methods for the transfer of expression constructs are available for delivering the retron-based editing systems or components thereof into cells also are contemplated. These include the use of calcium phosphate precipitation, DEAE-dextran, electroporation, direct microinjection, DNA-loaded liposomes, lipofectamine-DNA complexes, cell sonication, gene bombardment using high velocity microprojectiles, and receptor-mediated transfection. For example, the non-viral methods can include those described in Graham and Van Der Eb (1973) Virology 52:456-467; Chen and Okayama (1987) Mol. Cell Biol.7:2745- 2752; Rippe et al. (1990) Mol. Cell Biol.10:689-695; Gopal (1985) Mol. Cell Biol.5:1188- 1190; Tur-Kaspa et al. (1986) Mol. Cell. Biol.6:716-718; Potter et al. (1984) Proc. Natl. Acad. Sci. USA 81:7161-7165); Harland and Weintraub (1985) J. Cell Biol.101:1094-1099); Nicolau & Sene (1982) Biochim. Biophys. Acta 721:185-190; Fraley et al. (1979) Proc. Natl. Acad. Sci. USA 76:3348-3352; Fechheimer et al. (1987) Proc Natl. Acad. Sci. USA 84:8463- 8467; Yang et al. (1990) Proc. Natl. Acad. Sci. USA 87:9568-9572; Wu and Wu (1987) J. Biol. Chem.262:4429-4432; Wu and Wu (1988) Biochemistry 27:887-892; herein incorporated by reference). Some of these techniques may be successfully adapted for in vivo or ex vivo use. [00404] In some embodiments, nucleic acid molecules encoding the retron-based editing systems or components thereof may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation). In yet further embodiments, the nucleic acid may be stably maintained in the RNG043-WO1 PCT Application cell as a separate, episomal segment of DNA. Such nucleic acid segments or episomes encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed. [00405] In some embodiments, expression constructs encoding the retron-based editing systems or components thereof may simply consist of naked recombinant DNA or plasmids comprising nucleotide sequences encoding said retron-based editing systems or components thereof. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well. For example, Dubensky et al. (Proc. Natl. Acad. Sci. USA (1984) 81:7529-7533) successfully injected polyomavirus DNA in the form of calcium phosphate precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection. In another example, Benvenisty & Neshif (Proc. Natl. Acad. Sci. USA (1986) 83:9551-9555) also demonstrated that direct intraperitoneal injection of calcium phosphate- precipitated plasmids results in expression of the transfected genes. It is envisioned that DNA encoding a retron-based editing system or components thereof of interest may also be transferred in a similar manner in vivo and express retron products. [00406] In still another embodiment, DNA expression constructs encoding the retron- based editing systems or components thereof may be transferred into cells by particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them, such as, for example, as described in Klein et al. (1987) Nature 327:70-73. Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force, for example, as described in Yang et al. (1990) Proc. Natl. Acad. Sci. USA 87:9568-9572. The microprojectiles may consist of biologically inert substances, such as tungsten or gold beads. Liposomes [00407] In a further embodiment, constructs encoding the retron-based editing systems or components thereof may be delivered using liposomes. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid RNG043-WO1 PCT Application components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers, for example, as described in Ghosh & Bachhawat (1991) Liver Diseases, Targeted Diagnosis and Therapy Using Specific Receptors and Ligands, Wu et al. (Eds.), Marcel Dekker, NY, 87-104. Also contemplated is the use of lipofectamine-DNA complexes. [00408] In some embodiments, the liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA, for example, as described in Kaneda et al. (1989) Science 243:375-378. In other embodiments, the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-I), for example, described in Kato et al. (1991) J. Biol. Chem.266(6):3361-3364. [00409] In yet further embodiments, the liposome may be complexed or employed in conjunction with both HVJ and HMG-I. Where a bacterial promoter is employed in the DNA construct, it also will be desirable to include within the liposome an appropriate bacterial polymerase. [00410] Liposomes can be made from several different types of lipids, e.g., phospholipids. A liposome may comprise natural phospholipids and lipids such as l,2- distearoryl-sn-glycero-3 -phosphatidyl choline (DSPC), sphingomyelin, egg phosphatidylcholines, monosialoganglioside, or any combination thereof. [00411] Several other additives may be added to liposomes in order to modify their structure and properties. For instance, liposomes may further comprise cholesterol, sphingomyelin, and/or 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), e.g., to increase stability and/or to prevent the leakage of the liposomal inner cargo. [00412] In one embodiment, the liposome comprises a transport polymer, which may optionally be branched, comprising at least 10 amino acids and a ratio of histidine to non- histidine amino acids greater than 1.5 and less than 10. The branched transport polymer can comprise one or more backbones, one or more terminal branches, and optionally, one or more non-terminal branches, such as, for example, as described in U.S. Patent No.7,070,807, incorporated herein by reference in its entirety. In one embodiment, the transport polymer is a Histidine-Lysine co-polymer (HKP) used to package and deliver mRNA and other cargo, for example, as described in U.S. Patent Nos., 7,163,695, and 7,772,201, incorporated herein by reference in their entireties, [00413] In one embodiment, the lipid particles may be stable nucleic acid lipid particles (SNALPs). SNALPs may comprise an ionizable lipid (DLinDMA) (e.g., cationic at low pH), a RNG043-WO1 PCT Application neutral helper lipid, cholesterol, a diffusible polyethylene glycol (PEG)-lipid, or any combination thereof. In some examples, SNALPs may comprise synthetic cholesterol, dipalmitoylphosphatidylcholine, 3-N-[(w-methoxy polyethylene glycol)2000)carbamoyl]-l,2- dimyrestyloxypropylamine, and cationic l,2-dilinoleyloxy-3-N,Ndimethylaminopropane. In some examples, SNALPs may comprise synthetic cholesterol, l,2-distearoyl-sn-glycero-3- phosphocholine, PEG- eDMA, and 1,2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane (DLinDMA). Polymer based vehicles [00414] In one embodiment, the retron-based editing systems or components thereof may be encapsulated by delivery vehicles that comprise polymer-based particles (e.g., nanoparticles). In one embodiment, the polymer-based particles may mimic a viral mechanism of membrane fusion. The polymer-based particles may be a synthetic copy of Influenza virus machinery and form transfection complexes with various types of nucleic acids ((siRNA, miRNA, plasmid DNA or snucleic acid component, mRNA) that cells take up via the endocytosis pathway, a process that involves the formation of an acidic compartment. The low pH in late endosomes acts as a chemical switch that renders the particle surface hydrophobic and facilitates membrane crossing. Once in the cytosol, the particle releases its payload for cellular action. This Active Endosome Escape technology is safe and maximizes transfection efficiency as it is using a natural uptake pathway. In one embodiment, the polymer-based particles may comprise alkylated and carboxyalkylated branched polyethylenimine. In some examples, the polymer-based particles are VIROMER, e.g., VIROMER RNAi, VIROMER RED, VIROMER mRNA. In some examples, methods of delivering the systems and compositions herein include those described in Bawage SS et al., Synthetic mRNA expressed Cast 3a mitigates RNA virus infections, biorxiv.org/content/10.1101/370460vl. full doi: doi.org/10.1101/370460, Viromer® RED, a powerful tool for transfection of keratinocytes. doi: 10.13140/RG.2.2.16993.61281, Viromer® Transfection - Factbook 2018: technology, product overview, users' data., doi: 10.13140/RG.2.2.23912.16642. Exosomes [00415] The delivery vehicles may comprise exosomes. Exosomes include membrane bound extracellular vesicles, which can be used to contain and delivery various types of biomolecules, such as proteins, carbohydrates, lipids, and nucleic acids, and complexes thereof (e.g., RNPs). In some examples, the exosomes can include those described in Schroeder A, et al., J Intern Med.2010 Jan;267(l):9-21; El-Andaloussi S, et al., Nat Protoc.2012 Dec;7(12):2112-26; Uno Y, et al., Hum Gene Ther.2011 Jun;22(6):711-9; Zou W, et al., Hum RNG043-WO1 PCT Application Gene Then 2011 Apr;22(4):465-75. Exemplary exosomes can be generated from 293F cells, with mRNA-loaded exosomes driving higher mRNA expression than mRNA loaded LNPs in some instances, for example, as described in J. Biol. Chem. (2021) 297(5) 101266. [00416] In some examples, the exosome may form a complex (e.g., by binding directly or indirectly) to one or more components of the cargo. In certain examples, a molecule of an exosome may be fused with first adapter protein and a component of the cargo may be fused with a second adapter protein. The first and the second adapter protein may specifically bind each other, thus associating the cargo with the exosome. Examples of such exosomes can include those described in Ye Y, et al., Biomater Sci.2020 Apr 28. doi: 10.1039/d0bm00427h. Receptor-mediated delivery [00417] Other expression constructs encoding the retron-based editing systems or components thereof are receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific, such as, for example, as described in Wu and Wu (1993) Adv. Drug Delivery Rev.12:159- 167). Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA-binding agent. Several ligands have been used for receptor-mediated gene transfer. In some examples, the ligands can include asialoorosomucoid (ASOR) and transferrin, as described in Wu and Wu (1987), supra; Wagner et al. (1990) Proc. Natl. Acad. Sci. USA 87(9):3410- 3414). In some examples, the ligands can include synthetic neoglycoprotein, which recognizes the same receptor as ASOR, which can be used as a gene delivery vehicle, as described in Ferkol et al. (1993) FASEB J.7:1081-1091; Perales et al. (1994) Proc. Natl. Acad. Sci. USA 91(9):4086-4090. In some examples, the ligands can include epidermal growth factor (EGF) has also been used to deliver genes to squamous carcinoma cells, for example, as described in Myers, EPO 0273085. [00418] In other embodiments, delivery vehicle comprising one or more expression constructs encoding the retron-based editing systems or components thereof may comprise a ligand and a liposome, such as, for example, those described in Nicolau et al. (Methods Enzymol. (1987) 149:157-176), employed lactosy 1-ceramide, a galactose-terminal asialoganglioside, incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes. Thus, it is feasible that a nucleic acid encoding a particular gene also may be specifically delivered into a cell by any number of receptor-ligand systems with or without liposomes. Also, antibodies to surface antigens on cells can similarly be used as targeting moieties. RNG043-WO1 PCT Application [00419] In some embodiments, the promoters that may be used in the retron-based editing systems or components thereof described herein may be constitutive, inducible, or tissue-specific. In some embodiments, the promoters may be a constitutive promoters. Non- limiting exemplary constitutive promoters include cytomegalovirus immediate early promoter (CMV), simian virus (SV40) promoter, adenovirus major late (MLP) promoter, Rous sarcoma virus (RSV) promoter, mouse mammary tumor virus (MMTV) promoter, phosphoglycerate kinase (PGK) promoter, elongation factor-alpha (EFla) promoter, ubiquitin promoters, actin promoters, tubulin promoters, immunoglobulin promoters, a functional fragment thereof, or a combination of any of the foregoing. In some embodiments, the promoter may be a CMV promoter. In some embodiments, the promoter may be a truncated CMV promoter. In other embodiments, the promoter may be an EFla promoter. In some embodiments, the promoter may be an inducible promoter. Non-limiting exemplary inducible promoters include those inducible by heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohol. In some embodiments, the inducible promoter may be one that has a low basal (non-induced) expression level, such as, e.g., the Tet-On® promoter (Clontech). In some embodiments, the promoter may be a tissue-specific promoter. In some embodiments, the tissue-specific promoter is exclusively or predominantly expressed in liver tissue. Non-limiting exemplary tissue-specific promoters include B29 promoter, CD14 promoter, CD43 promoter, CD45 promoter, CD68 promoter, desmin promoter, elastase- 1 promoter, endoglin promoter, fibronectin promoter, Flt-1 promoter, GFAP promoter, GPIIb promoter, ICAM- 2 promoter, INF-b promoter, Mb promoter, Nphsl promoter, OG-2 promoter, SP-B promoter, SYN1 promoter, and WASP promoter. Lipid Nanoparticle Compositions [00420] In one aspect, the present disclosure further provides delivery systems for delivery of a therapeutic payload (e.g., the RNA payloads described herein which may encode a polypeptide of interest, e.g., a nucleobase editing system or a therapeutic protein) disclosed herein. In some embodiments, a delivery system suitable for delivery of the therapeutic payload disclosed herein comprises a lipid nanoparticle (LNP) formulation. [00421] In some embodiments, an LNP of the present disclosure comprises an ionizable lipid, a structural lipid, a PEGylated lipid (aka PEG lipid), and a phospholipid. In alternative embodiments, an LNP comprises an ionizable lipid, a structural lipid, a PEGylated lipid (aka PEG lipid), and a zwitterionic amino acid lipid. In some embodiments, an LNP further comprises a 5th lipid, besides any of the aforementioned lipid components. In some embodiments, the LNP encapsulates one or more elements of the active agent of the present RNG043-WO1 PCT Application disclosure. In some embodiments, an LNP further comprises a targeting moiety covalently or non-covalently bound to the outer surface of the LNP. In some embodiments, the targeting moiety is a targeting moiety that binds to, or otherwise facilitates uptake by, cells of a particular organ system. [00422] In some embodiments, an LNP has a diameter of at least about 20nm, 30 nm, 40nm, 50nm, 60nm, 70nm, 80nm, or 90nm. In some embodiments, an LNP has a diameter of less than about 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, or 160nm. In some embodiments, an LNP has a diameter of less than about 100nm. In some embodiments, an LNP has a diameter of less than about 90nm. In some embodiments, an LNP has a diameter of less than about 80nm. In some embodiments, an LNP has a diameter of about 60-100nm. In some embodiments, an LNP has a diameter of about 75-80nm. [00423] In some embodiments, the lipid nanoparticle compositions of the present disclosure are described according to the respective molar ratios of the component lipids in the formulation. As a non-limiting example, the mol-% of the ionizable lipid may be from about 10 mol-% to about 80 mol-%. As a non-limiting example, the mol-% of the ionizable lipid may be from about 20 mol-% to about 70 mol-%. As a non-limiting example, the mol-% of the ionizable lipid may be from about 30 mol-% to about 60 mol-%. As a non-limiting example, the mol-% of the ionizable lipid may be from about 35 mol-% to about 55 mol-%. As a non- limiting example, the mol-% of the ionizable lipid may be from about 40 mol-% to about 50 mol-%. [00424] In some embodiments, the mol-% of the phospholipid may be from about 1 mol-% to about 50 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 2 mol-% to about 45 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 3 mol-% to about 40 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 4 mol-% to about 35 mol-%. In some embodiments, the mol- % of the phospholipid may be from about 5 mol-% to about 30 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 10 mol-% to about 20 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 5 mol-% to about 20 mol-%. [00425] In some embodiments, the mol-% of the structural lipid may be from about 10 mol-% to about 80 mol-%. In some embodiments, the mol-% of the structural lipid may be from about 20 mol-% to about 70 mol-%. In some embodiments, the mol-% of the structural lipid may be from about 30 mol-% to about 60 mol-%. In some embodiments, the mol-% of the structural lipid may be from about 35 mol-% to about 55 mol-%. In some embodiments, the mol-% of the structural lipid may be from about 40 mol-% to about 50 mol-%. RNG043-WO1 PCT Application [00426] In some embodiments, the mol-% of the PEG lipid may be from about 0.1 mol- % to about 10 mol-%. In some embodiments, the mol-% of the PEG lipid may be from about 0.2 mol-% to about 5 mol-%. In some embodiments, the mol-% of the PEG lipid may be from about 0.5 mol-% to about 3 mol-%. In some embodiments, the mol-% of the PEG lipid may be from about 1 mol-% to about 2 mol-%. In some embodiments, the mol-% of the PEG lipid may be about 1.5 mol-%. In some embodiments, the mol-% of the PEG lipid may be about 2.5 mol- %. i. Ionizable lipids [00427] In some embodiments, an LNP disclosed herein comprises an ionizable lipid. In some embodiments, an LNP comprises two or more ionizable lipids. [00428] Described below are a number of exemplary ionizable lipids of the present disclosure. [00429] In some examples, an LNP of the present disclosure comprises an ionizable lipid, such as those disclosed in US 2023/0053437; US 2019/0240354; US 2010/0130588; US 2021/0087135; WO 2021/204179; US 2021/0128488; US 2020/0121809; US 2017/0119904; US 2013/0108685; US 2013/0195920; US 2015/0005363; US 2014/0308304; US 2013/0053572; WO 2019/232095A1; WO 2021/077067; WO 2019/152557; US 2017/0210697; or WO 2019/089828A1, each of which is incorporated by reference herein in their entirety. [00430] In some examples, an LNP described herein comprises an ionizable lipid, such as those disclosed in PCT Publications WO2023044343A1, WO2023044333A1, WO2023122752A1, WO2024044728A1 and WO2023196931A1 and PCT Application PCT/US2024/019990, each of which is incorporated by reference herein, in its entirety. [00431] In some examples, an LNP described herein comprises a lipid, e.g., an ionizable lipid, such as those disclosed in US Application publication US2017/0119904, which is incorporated by reference herein, in its entirety. [00432] In some examples, an LNP described herein comprises a lipid, e.g., an ionizable lipid, such as those disclosed in PCT Application publication WO2021/204179, which is incorporated by reference herein, in its entirety. [00433] In some examples, an LNP described herein comprises a lipid, e.g., an ionizable lipid, such as those disclosed in PCT Application WO2022/251665A1, which is incorporated by reference herein, in its entirety. RNG043-WO1 PCT Application [00434] In some examples, an LNP of the present disclosure comprises an ionizable lipid, such as those disclosed in PCT Application Publication WO2023044343A1, which is incorporated by reference herein, in its entirety. [00435] In some examples, an LNP of the present disclosure comprises an ionizable lipid, such as those disclosed in PCT Application Publication WO2023044333A1, which is incorporated by reference herein, in its entirety. [00436] In some examples, an LNP of the present disclosure comprises an ionizable lipid, such as those disclosed in PCT Publication WO2023122752A1, which is incorporated by reference herein, in its entirety. [00437] In some examples, an LNP of the present disclosure comprises an ionizable lipid, such as those disclosed in PCT Application PCT/US2023/065477, which is incorporated by reference herein, in its entirety. ii. Structural lipids [00438] In some embodiments, an LNP comprises a structural lipid. Structural lipids can be selected from the group consisting of, but are not limited to, cholesterol, fecosterol, fucosterol, beta sitosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, cholic acid, sitostanol, litocholic acid, tomatine, ursolic acid, alpha-tocopherol, Vitamin D3, Vitamin D2, Calcipotriol, botulin, lupeol, oleanolic acid, beta-sitosterol-acetate and mixtures thereof. In some embodiments, the structural lipid is cholesterol. In some examples, the structural lipid is a cholesterol analogue, such as the one disclosed by Patel, et al., Nat Commun., 11, 983 (2020), which is incorporated herein by reference in its entirety. In some embodiments, the structural lipid includes cholesterol and a corticosteroid (such as prednisolone, dexamethasone, prednisone, and hydrocortisone), or any combinations thereof. In some examples, a structural lipid can be a structural lipid as described in international patent application WO2019152557A1, which is incorporated herein by reference in its entirety. [00439] In some embodiments, a structural lipid is a cholesterol analog. Using a cholesterol analog may enhance endosomal escape, for example, as described in Patel et al., Naturally-occurring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA, Nature Communications (2020), which is incorporated herein by reference. [00440] In some embodiments, a structural lipid is a phytosterol. Using a phytosterol may enhance endosomal escape, for example, as described in Herrera et al., Illuminating endosomal escape of polymorphic lipid nanoparticles that boost mRNA delivery, Biomaterials Science (2020), which is incorporated herein by reference. RNG043-WO1 PCT Application [00441] In some embodiments, a structural lipid contains plant sterol mimetics for enhanced endosomal release. iii. PEGylated lipids [00442] A PEGylated lipid is a lipid modified with polyethylene glycol. As used herein, PEGylated lipid and PEG lipid are used interchangeably to refer to the same concept. [00443] In some embodiments, an LNP comprises one, two or more PEGylated lipid or PEG-modified lipid. A PEGylated lipid may be selected from the non-limiting group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG- modified dialkylglycerols, and mixtures thereof. For example, a PEG lipid may be PEG-c- DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid. [00444] In some embodiments, the PEGylated lipid is selected from (R)-2,3- bis(octadecyloxy)propyl-1-(methoxypoly(ethyleneglycol)2000)propylcarbamate, PEG-S-DSG, PEG-S-DMG, PEG-PE, PEG-PAA, PEG-OH DSPE C18, PEG-DSPE, PEG-DSG, PEG-DPG, PEG-DOMG, PEG-DMPE Na, PEG-DMPE, PEG-DMG2000, PEG-DMG C14, PEG-DMG 2000, PEG-DMG, PEG-DMA, PEG-Ceramide C16, PEG-C-DOMG, PEG-c-DMOG, PEG-c- DMA, PEG-cDMA, PEGA, PEG750-C-DMA, PEG400, PEG2k-DMG, PEG2k-C11, PEG2000-PE, PEG2000P, PEG2000-DSPE, PEG2000-DOMG, PEG2000-DMG, PEG2000-C- DMA, PEG2000, PEG200, PEG(2k)-DMG, PEG DSPE C18, PEG DMPE C14, PEG DLPE C12, PEG Click DMG C14, PEG Click C12, PEG Click C10, N(Carbonyl- methoxypolyethylenglycol-2000)-l,2-distearoyl-sn-glycero3-phosphoethanolamine, Myrj52, mPEG-PLA, MPEG-DSPE, mPEG3000-DMPE, MPEG-2000-DSPE, MPEG2000-DSPE, mPEG2000-DPPE, mPEG2000-DMPE, mPEG2000-DMG, mDPPE-PEG2000, l,2-distearoyl- sn-glycero-3-phosphoethanolamine-PEG2000, HPEG-2K-LIPD, Folate PEG-DSPE, DSPE- PEGMA 500, DSPE-PEGMA, DSPE-PEG6000, DSPE-PEG5000, DSPE-PEG2K-NAG, DSPE-PEG2k, DSPE-PEG2000maleimide, DSPE-PEG2000, DSPE-PEG, DSG-PEGMA, DSG-PEG5000, DPPE-PEG-2K, DPPE-PEG, DPPE-mPEG2000, DPPE-mPEG, DPG- PEGMA, DOPE-PEG2000, DMPE-PEGMA, DMPE-PEG2000, DMPE-Peg, DMPE- mPEG2000, DMG-PEGMA, DMG-PEG2000, DMG-PEG, distearoyl-glycerol- polyethyleneglycol, Cl8PEG750, CI8PEG5000, CI8PEG3000, CI8PEG2000, CI6PEG2000, CI4PEG2000, C18-PEG5000, C18PEG, C16PEG, C16 mPEG (polyethylene glycol) 2000 Ceramide, C14-PEG-DSPE200, C14-PEG2000, C14PEG2000, C14-PEG 2000, C14-PEG, C14PEG, 14:0-PEG2KPE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG2000, (R)- RNG043-WO1 PCT Application 2,3-bis(octadecyloxy)propyl-1-(methoxypoly(ethyleneglycol)2000)propylcarbamate, (PEG)-C- DOMG, PEG-C-DMA, and DSPE-PEG-X. [00445] In some examples, the LNP comprises a PEGylated lipid, such as, for example, a PEGylated lipid as disclosed in one of US 2019/0240354; US 2010/0130588; US 2021/0087135; WO 2021/204179; US 2021/0128488; US 2020/0121809; US 2017/0119904; US 2013/0108685; US 2013/0195920; US 2015/0005363; US 2014/0308304; US 2013/0053572; WO 2019/232095A1; WO 2021/077067; WO 2019/152557; US 2015/0203446; US 2017/0210697; US 2014/0200257; or WO 2019/089828A1, each of which is incorporated by reference herein in their entirety. [00446] In some embodiments, the LNP comprises a PEGylated lipid substitute in place of the PEGylated lipid. All embodiments disclosed herein that contemplate a PEGylated lipid should be understood to also apply to PEGylated lipid substitutes. In some examples, the LNP comprises a polysarcosine-lipid conjugate, such as those disclosed in US 2022/0001025 A1, which is incorporated by reference herein in its entirety. [00447] In some examples, the LNP comprises a PEGylated lipid, such as those disclosed and described in PCT Application PCT/US2023/072878, which is incorporated by reference herein, in its entirety. [00448] In some examples an LNP described herein comprises a PEG lipid, such as theose disclosed in PCT Publications WO2023044343A1, WO2023044333A1, WO2023122752A1, WO2024044728A1 and WO2023196931A1 and PCT Application PCT/US2024/019990, each of which is incorporated by reference herein, in its entirety. iv. Phospholipids [00449] In some embodiments, an LNP of the present disclosure comprises a phospholipid. Phospholipids useful in the compositions and methods may be selected from the non-limiting group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3- phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1.2- dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphocho line (POPC), 1,2-di-O-octadecenyl-sn- glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuc cinoyl-sn- glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero- 3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2- RNG043-WO1 PCT Application diphytanoylsn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero- 3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3- phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2- dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sodium (S)-2- ammonio-3-((((R)-2-(oleoyloxy)-3- (stearoyloxy)propoxy)oxidophosphoryl)oxy)propanoate (L-α-phosphatidylserine; Brain PS), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphoethanolamine (DMPE), dimyristoylphosphatidylglycerol (DMPG), dioleoyl-phosphatidylethanolamine4- (N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dioleoylphosphatidylglycerol (DOPG), 1,2-dioleoyl-sn-glycero-3-(phospho-L-serine) (DOPS), acell-fusogenicphospholipid (DPhPE), dipalmitoylphosphatidylethanolamine (DPPE), 1,2-Dielaidoyl-sn-phosphatidylethanolamine (DEPE), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylcholine (DSPC), distearoyl-phosphatidyl-ethanolamine (DSPE), distearoyl phosphoethanolamineimidazole (DSPEI), 1,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), egg phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3- phosphate (18:1 PA; DOPA), ammonium bis((S)-2-hydroxy-3-(oleoyloxy)propyl) phosphate (18:1 DMP; LBPA), 1,2-dioleoyl-sn-glycero-3-phospho-(1’-myo-inositol) (DOPI; 18:1 PI), 1,2-distearoyl-sn-glycero-3-phospho-L-serine (18:0 PS), 1,2-dilinoleoyl- sn-glycero-3-phospho-L-serine (18:2 PS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L- serine (16:0-18:1 PS; POPS), 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (18:0- 18:1 PS), 1-stearoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine (18:0-18:2 PS), 1-oleoyl- 2-hydroxy-sn-glycero-3-phospho-L-serine (18:1 Lyso PS), 1-stearoyl-2-hydroxy-sn- glycero-3-phospho-L-serine (18:0 Lyso PS), and sphingomyelin. In some embodiments, an LNP includes DSPC. In certain embodiments, an LNP includes DOPE. In some embodiments, an LNP includes both DSPC and DOPE. [00450] In some embodiments, an LNP comprises a phospholipid selected from 1- pentadecanoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-myristoyl-2-palmitoyl-sn- glycero-3-phosphocholine, 1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine, 1- palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-stearoyl-sn-glycero-3- phosphocholine, 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine, 1-palmitoyl-2-linoleoyl- sn-glycero-3-phosphocholine, 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine, 1-stearoyl-2-myristoyl-sn- RNG043-WO1 PCT Application glycero-3-phosphocholine, 1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine, 1- stearoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-stearoyl-2-linoleoyl-sn-glycero-3- phosphocholine, 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, 1-stearoyl-2- docosahexaenoyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-myristoyl-sn-glycero-3- phosphocholine, 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-stearoyl- sn-glycero-3-phosphocholine, 1-palmitoyl-2-acetyl-sn-glycero-3-phosphocholine, 1,2- dioleoyl-sn-glycero-3-phospho-(1’-myo-inositol-3’,4’-bisphosphate), 1,2-dioleoyl-sn- glycero-3-phospho-(1’-myo-inositol-3’,5’-bisphosphate), 1,2-dioleoyl-sn-glycero-3- phospho-(1’-myo-inositol-4’,5’-bisphosphate), 1,2-dioleoyl-sn-glycero-3-phospho-(1'- myo-inositol-3',4',5'-trisphosphate), 1,2-dioleoyl-sn-glycero-3-phospho-(1’-myo-inositol- 3’-phosphate), 1,2-dioleoyl-sn-glycero-3-phospho-(1’-myo-inositol-4’-phosphate), 1,2- dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol-5'-phosphate), 1,2-dioleoyl-sn-glycero-3- phospho-(1’-myo-inositol), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine, and 1-(8Z- octadecenoyl)-2-palmitoyl-sn-glycero-3-phosphocholine. [00451] In some examples, a phospholipid tail may be modified in order to promote endosomal escape as described in U.S. Application Publication 2021/0121411, which is incorporated herein by reference. [00452] In some examples, the LNP can a phospholipid, such as those disclosed in US 2019/0240354; US 2010/0130588; US 2021/0087135; WO 2021/204179; US 2021/0128488; US 2020/0121809; US 2017/0119904; US 2013/0108685; US 2013/0195920; US 2015/0005363; US 2014/0308304; US 2013/0053572; WO 2019/232095A1; WO 2021/077067; WO 2019/152557; US 2017/0210697; or WO 2019/089828A1, each of which is incorporated by reference herein in their entirety. vi. Targeting moieties [00453] In some embodiments, the lipid nanoparticle further comprises a targeting moiety. The targeting moiety may be an antibody or a fragment thereof. The targeting moiety may be capable of binding to a target antigen. [00454] In some embodiments, the pharmaceutical composition comprises a targeting moiety that is operably connected to a lipid nanoparticle. In some embodiments, the targeting moiety is capable of binding to a target antigen. In some embodiments, the target antigen is expressed in a target organ. In some embodiments, the target antigen is expressed more in the target organ than it is in the liver. [00455] In some examples, the targeting moiety is an antibody, such as those described in WO2016189532A1, which is incorporated herein by reference. For example, in some RNG043-WO1 PCT Application embodiments, the targeted particles are conjugated to a specific anti-CD38 monoclonal antibody (mAb), which allows specific delivery of the siRNAs encapsulated within the particles at a greater percentage to B-cell lymphocytes malignancies (such as MCL) than to other subtypes of leukocytes. [00456] In some embodiments, the lipid nanoparticles may be targeted when conjugated/attached/associated with a targeting moiety such as an antibody. vii. Zwitterionic amino lipids [00457] In some embodiments, an LNP comprises a zwitterionic lipid. In some embodiments, an LNP comprising a zwitterionic lipid does not comprise a phospholipid. [00458] Zwitterionic amino lipids have been shown to be able to self-assemble into LNPs without phospholipids to load, stabilize, and release mRNAs intracellularly, for example, as described in U.S. Patent Publication 20210121411, which is incorporated herein by reference in its entirety. In some examples, zwitterionic, ionizable cationic and permanently cationic helper lipids can enable tissue-selective mRNA delivery and CRISPR-Cas9 gene editing in spleen, liver and lungs as described in Liu et al., Membrane-destablizing ionizable phospholipids for organ-selective mRNA delivery and CRISPR-Cas gene editing, Nat Mater. (2021), which is incorporated herein by reference in its entirety. [00459] The zwitterionic lipids may have head groups containing a cationic amine and an anionic carboxylate, such as the ones described in Walsh et al., Synthesis, Characterization and Evaluation of Ionizable Lysine-Based Lipids for siRNA Delivery, Bioconjug Chem. (2013), which is incorporated herein by reference in its entirety. Ionizable lysine-based lipids containing a lysine head group linked to a long-chain dialkylamine through an amide linkage at the lysine α-amine may reduce immunogenicity, for example, as described in Walsh et al., Synthesis, Characterization and Evaluation of Ionizable Lysine-Based Lipids for siRNA Delivery, Bioconjug Chem. (2013). viii. Additional lipid components [00460] In some embodiments, the LNP compositions of the present disclosure further comprise one or more additional lipid components capable of influencing the tropism of the LNP. In some examples, the LNP further comprises at least one lipid selected from DDAB, EPC, 14PA, 18BMP, DODAP, DOTAP, and C12-200, such as described in Cheng, et al. Nat Nanotechnol.2020 April; 15(4): 313–320.; Dillard, et al. PNAS 2021 Vol.118 No.52. [00461] In some embodiments, the LNP compositions of the present disclosure comprise, or further comprise one or more lipids selected from 1,2-di-O-octadecenyl-sn- glycero-3-phosphocholine (18:0 Diether PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine RNG043-WO1 PCT Application (18:3 PC), Acylcarnosine (AC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), N- oleoyl-sphingomyelin (SPM) (C18:l), N-lignoceryl SPM (C24:0), N-nervonoylshphingomyelin (C24:l), Cardiolipin (CL), l,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC8- 9PC), dicetyl phosphate (DCP), dihexadecyl phosphate (DCP1), 1,2-Dipalmitoylglycerol-3- hemisuccinate (DGSucc), short-chain bis-n-heptadecanoyl phosphatidylcholine (DHPC), dihexadecoyl-phosphoethanolamine (DHPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), l,2-dilauroyl-sn-glycero-3-PE (DLPE), dimyristoyl glycerol hemisuccinate (DMGS), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphoethanolamine (DMPE), dimyristoylphosphatidylglycerol (DMPG), dioleyloxybenzylalcohol (DOBA), 1,2- dioleoylglyceryl-3-hemisuccinate (DOGHEMS), N-[2-(2-{2-[2-(2,3-Bis-octadec-9-enyloxy- propoxy)-ethoxy]-ethoxy}-ethoxy)-ethyl]-3-(3,4,5-1rihydroxy-6-hydroxymethyl-1etrahydro- pyran-2-ylsulfanyl)-propionamide (DOGP4αMan), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dioleoyl-phosphatidylethanolamine4-(N- maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dioleoylphosphatidylglycerol (DOPG), 1,2-dioleoyl-sn-glycero-3-(phospho-L-serine) (DOPS), acell-fusogenicphospholipid (DPhPE), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylcholine (DSPC), distearoyl-phosphatidyl-ethanolamine (DSPE), distearoyl phosphoethanolamineimidazole (DSPEI), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), egg phosphatidylcholine (EPC), histaminedistearoylglycerol (HDSG), 1,2-Dipalmitoylglycerol-hemisuccinate-Nα- Histidinyl-Hemisuccinate (HistSuccDG), N-(5'-hydroxy-3'-oxypentyl)-10-12- pentacosadiynamide (h-Pegi-PCDA), 2-[l-hexyloxyethyl]-2-devinylpyropheophorbide-a (HPPH), hydrogenatedsoybeanphosphatidylcholine (HSPC), 1,2-Dipalmitoylglycerol-O-α- histidinyl-Nα-hemisuccinate (IsohistsuccDG), mannosialized dipalmitoylphosphatidylethanolamine (ManDOG), l,2-Dioleoyl-sn-Glycero-3- Phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide] (MCC-PE), 1,2- diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16:0 PE), 1-myristoyl-2-hydroxy-sn- glycero-phosphocholine (MHPC), a thiol-reactive maleimide headgroup lipid e.g.1,2-dioleoyl- sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)but-yramid (MPB-PE), Nervonic Acid (NA), sodium cholate (NaChol), l,2-dioleoyl-sn-glycero-3- [phosphoethanolamine-N-dodecanoyl (NC12-DOPE), 1-oleoyl-2-cholesteryl hemisuccinoyl- sn-glycero-3-phosphocholine (OChemsPC), phosphatidylethanolamine lipid (PE), PE lipid conjugated with polyethylene glycol(PEG) (e.g., polyethylene glycol- distearoylphosphatidylethanolamine lipid (PEG-PE)), phosphatidylglycerol (PG), partially RNG043-WO1 PCT Application hydrogenated soy phosphatidylchloline (PHSPC), phosphatidylinositol lipid (PI), phosphotidylinositol-4-phosphate (PIP), palmitoyloleoylphosphatidylcholine (POPC), phosphatidylethanolamine (POPE), palmitoyloleyolphosphatidylglycerol (POPG), phosphatidylserine (PS), lissamine rhodamine B-phosphatidylethanolamine lipid (Rh-PE), purified soy-derived mixture of phospholipids (SIOO), phosphatidylcholine (SM), 18-1-trans- PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), soybean phosphatidylcholine (SPC), sphingomyelins (SPM), alpha,alpha-trehalose-6,6'-dibehenate (TDB), l,2-dielaidoyl-sn- glycero-3-phophoethanolamine (transDOPE), ((23S,5R)-3-(bis(hexadecyloxy)methoxy)-5-(5- methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2- yl)methylmethylphosphate, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2- diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl- sn-glycero-3-phosphocholine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleyl-sn-glycero-3- phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 16-O-monomethyl PE, 16-O-dimethyl PE, and dioleylphosphatidylethanolamine. ix. LNP pharmaceutical compositions [00462] In some embodiments, a nanoparticle includes an ionizable lipid, a phospholipid, a PEG lipid, and a structural lipid. In certain embodiments, the lipid component of the nanoparticle composition includes about 30 mol % to about 60 mol % ionizable lipid, about 0 mol % to about 30 mol % phospholipid, about 18.5 mol % to about 48.5 mol % structural lipid, and about 0 mol% to about 10 mol% of PEG lipid, provided that the total mol % does not exceed 100%. In some embodiments, the lipid component of the nanoparticle composition includes about 35 mol % to about 55 mol % ionizable lipid, about 5 mol % to about 25 mol % phospholipid, about 30 mol % to about 40 mol % structural lipid, and about 0 mol % to about 10 mol % of PEG lipid. In a particular embodiment, the lipid component includes about 50 mol % ionizable lipid, about 10 mol % phospholipid, about 38.5 mol % structural lipid, and about 1.5 mol% of PEG lipid. In another particular embodiment, the lipid component includes about 40 mol % ionizable lipid, about 20 mol % phospholipid, about 38.5 mol % structural lipid, and about 1.5 mol % of PEG lipid. In another particular embodiment, the lipid component includes about 48.5 mol % ionizable lipid, about 10 mol % phospholipid, about 40 mol % structural lipid, and about 1.5 mol % of PEG lipid. In another particular embodiment, the lipid component includes about 48.5 mol % ionizable lipid, about 10 mol % phospholipid, about 39 mol % structural lipid, and about 2.5 mol % of PEG lipid. In some RNG043-WO1 PCT Application embodiments, the phospholipid may be DOPE or DSPC. In other embodiments, the PEG lipid may be PEG-DMG and/or the structural lipid may be cholesterol. The amount of active agent in a nanoparticle composition may depend on the size, composition, desired target and/or application, or other properties of the nanoparticle composition as well as on the properties of the active agent. For example, the amount of active agent useful in a nanoparticle composition may depend on the size, sequence, and other characteristics of the active agent. The relative amounts of active agent and other elements (e.g., lipids) in a nanoparticle composition may also vary. In some embodiments, the wt/wt ratio of the lipid component to an enzyme in a nanoparticle composition may be from about 5:1 to about 60:1, such as 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, and 60:1. The amount of a enzyme in a nanoparticle composition may, for example, be measured using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy). [00463] In some embodiments, the lipid component of the nanoparticle comprises: (a) about 1 mol% to about 2 mol% of PEG lipid; (b) about 25 mol% to about 40 mol% structural lipid; (c) about 20 mol% to about 45 mol% phospholipid, non-ionizable lipid or zwitterionic lipid; and (d) about 30 mol% to about 60 mol% of an ionizable lipid. [00464] In some embodiments, the lipid component of the nanoparticle comprises: (a) about 2 mol% of PEG lipid; (b) about 25 mol% structural lipid; (c) about 40 mol% phospholipid, non-ionizable lipid or zwitterionic lipid; and (d) about 33 mol% of an ionizable lipid. [00465] In some embodiments, the lipid component of the nanoparticle comprises: (a) about 2.5 mol% of PEG lipid; (b) about 39 mol% structural lipid; (c) about 10 mol% phospholipid, non-ionizable lipid or zwitterionic lipid; and (d) about 48.5 mol% of an ionizable lipid. [00466] In some embodiments, the lipid component of the nanoparticle comprises: (a) about 1.5 mol% of PEG lipid; (b) about 40 mol% structural lipid; (c) about 10 mol% phospholipid, non-ionizable lipid or zwitterionic lipid; and (d) about 48.5 mol% of an ionizable lipid. [00467] In certain embodiments, the lipid component of the nanoparticle composition comprises about 30 mol % to about 60 mol % ionizable lipid, about 0 mol % to about 30 mol % phospholipid, about 18.5 mol % to about 48.5 mol % structural lipid, and about 0 mol% to about 10 mol% of PEG lipid, provided that the total mol % does not exceed 100%. In certain embodiments, the lipid component of the nanoparticle composition comprises about 20 mol % to about 45 mol % ionizable lipid, about 30 mol % to about 60 mol % phospholipid, about 10 RNG043-WO1 PCT Application mol % to about 30 mol % structural lipid, and about 0 mol% to about 10 mol% of PEG lipid, provided that the total mol % does not exceed 100%. In some embodiments, the lipid component of the nanoparticle composition comprises about 35 mol % to about 55 mol % ionizable lipid, about 5 mol % to about 25 mol % phospholipid, about 30 mol % to about 40 mol % structural lipid, and about 0 mol % to about 10 mol % of PEG lipid, provided that the total mol % does not exceed 100%. In some embodiments, the lipid component of the nanoparticle composition comprises about 30 mol % to about 40 mol % ionizable lipid, about 35 mol % to about 45 mol % phospholipid, about 20 mol % to about 30 mol % structural lipid, and about 0.5 mol % to about 5 mol % of PEG lipid, provided that the total mol % does not exceed 100%. In certain embodiments, the lipid component of the nanoparticle composition comprises about 25 mol % to about 45 mol % ionizable lipid, about 35 mol % to about 50 mol % phospholipid, about 10 mol % to about 25 mol % structural lipid, and about 1 mol% to about 5 mol% of PEG lipid, provided that the total mol % does not exceed 100%. In a particular embodiment, the lipid component comprises about 50 mol % ionizable lipid, about 10 mol % phospholipid, about 38.5 mol % structural lipid, and about 1.5 mol% of PEG lipid. In another particular embodiment, the lipid component comprises about 40 mol % ionizable lipid, about 20 mol % phospholipid, about 38.5 mol % structural lipid, and about 1.5 mol % of PEG lipid. In another particular embodiment, the lipid component comprises about 48.5 mol % ionizable lipid, about 10 mol % phospholipid, about 40 mol % structural lipid, and about 1.5 mol % of PEG lipid. In another particular embodiment, the lipid component comprises about 48.5 mol % ionizable lipid, about 10 mol % phospholipid, about 39 mol % structural lipid, and about 2.5 mol % of PEG lipid. In another particular embodiment, the lipid component comprises about 33 mol % ionizable lipid, about 40 mol % phospholipid, about 25 mol % structural lipid, and about 2 mol % of PEG lipid. In some embodiments, the phospholipid is DOPE or DSPC. In some embodiments, the phospholipid is DSPC. In some embodiments, the phospholipid is a sphingolipid. In some embodiments, the phospholipid is a sphingomyelin. In other embodiments, the PEG lipid is PEG-DMG (eg. PEG2K-DMG). In other embodiments, the PEG lipid is PEG-DSPE (eg. PEG2K-DSPE). In other embodiments, the PEG lipid is PEG- DMPE (eg. PEG2K-DMPE). In other embodiments, the structural lipid is cholesterol. In other embodiments, the PEG lipid is PEG-DMG and/or the structural lipid is cholesterol. In some embodiments, the PEG lipids is PEG2K-DMG, the structural lipid is cholesterol, and the phospholipid is DSPC. In some embodiments, the PEG lipids is PEG2K-DMG, the structural lipid is cholesterol, and the phospholipid is sphingomyelin. In some embodiments, the PEG lipids is PEG-DMG, the structural lipid is cholesterol, and the phospholipid is a mixture of RNG043-WO1 PCT Application DSPC and sphingomyelin. In certain embodiments, the LNP comprises about 33mol% ionizable lipid (eg. at least one ionizable lipid of a Formula described herein), about 40mol% of a sphingolipid, about 25mol% cholesterol and about 2mol% PEG2K-DMG. In some embodiments, the PEG lipids is PEG2K-DSPE, the structural lipid is cholesterol, and the phospholipid is DSPC. In some embodiments, the PEG lipids is PEG2K-DSPE, the structural lipid is cholesterol, and the phospholipid is sphingomyelin. In some embodiments, the PEG lipids is PEG-DSPE, the structural lipid is cholesterol, and the phospholipid is a mixture of DSPC and sphingomyelin. In some embodiments, the PEG lipids is PEG2K-DMG, the structural lipid is cholesterol, and the phospholipid is DOPE. In some embodiments, the PEG lipids is PEG2K-DMG, the structural lipid is cholesterol, and the phospholipid is DOPC. In some embodiments, the PEG lipids is PEG2K-DMG, the structural lipid is cholesterol, and the phospholipid is DLPC. In some embodiments, the PEG lipids is PEG2K-DMG, the structural lipid is cholesterol, and the phospholipid is DOPS. In some embodiments, the PEG lipids is PEG-DMG, the structural lipid is cholesterol, and the phospholipid is a mixture of a phosphatidylcholine lipid and a sphingolipid. In some embodiments, the PEG lipids is PEG- DMG, the structural lipid is cholesterol, and the phospholipid is a mixture of a phosphatidylcholine lipid and phosphatidylserine lipid. In some embodiments, the PEG lipids is PEG-DMG, the structural lipid is cholesterol, and the phospholipid is a mixture of a phosphatidylcholine lipid and a phosphoethanolamine lipid. In some embodiments, the PEG lipids is PEG-DMG, the structural lipid is cholesterol, and the phospholipid is a mixture of a sphingolipid and phosphatidylserine lipid. In some embodiments, the PEG lipids is PEG- DMG, the structural lipid is cholesterol, and the phospholipid is a mixture of a sphingolipid and a phosphoethanolamine lipid. In certain embodiments, the LNP comprises about 33mol% ionizable lipid, about 20mol% of a sphingolipid, about 20mol% of a non-sphingolipid phospholipid, about 25mol% cholesterol and about 2mol% of a PEGylated lipid. In certain embodiments, the LNP comprises about 33mol% ionizable lipid, about 10mol% of a sphingolipid, about 30mol% of a non-sphingolipid phospholipid, about 25mol% cholesterol and about 2mol% of a PEGylated lipid. In certain embodiments, the LNP comprises about 33mol% ionizable lipid, about 30mol% of a sphingolipid, about 10mol% of a non-sphingolipid phospholipid, about 25mol% cholesterol and about 2mol% of a PEGylated lipid. In certain embodiments, the LNP comprises about 33mol% ionizable lipid, about 20mol% sphingomyelin, about 20mol% of a DSPC, about 25mol% cholesterol and about 2mol% of a PEGylated lipid. In certain embodiments, the LNP comprises about 33mol% ionizable lipid, about 10mol% sphingomyelin, about 30mol% of a DSPC, about 25mol% cholesterol and about RNG043-WO1 PCT Application 2mol% of a PEGylated lipid. In certain embodiments, the LNP comprises about 33mol% ionizable lipid, about 30mol% sphingomyelin, about 10mol% of a DSPC, about 25mol% cholesterol and about 2mol% of a PEGylated lipid. In certain embodiments, the LNP comprises about 33mol% ionizable lipid, about 25mol% cholesterol, about 2mol% of a PEGylated lipid, and about 40% of a mixture of phosphatidylcholine, phosphatidylserine, phosphoethanolamine, and sphingoid lipids. In certain embodiments, the LNP comprises about 33mol% ionizable lipid, about 25mol% cholesterol, about 2mol% of a PEGylated lipid, and about 40% of a mixture of phosphatidylcholine, phosphatidylserine, phosphoethanolamine, and sphingoid lipids, wherein each of the phosphatidylcholine, phosphatidylserine, phosphoethanolamine, and sphingoid lipids is present in an amount less than 30 mol% of the total lipid component of the LNP. In certain embodiments, the LNP comprises about 33mol% ionizable lipid, about 25mol% cholesterol, about 2mol% of a PEGylated lipid, and about 40% of a mixture of phosphatidylcholine, phosphatidylserine, phosphoethanolamine, and sphingoid lipids, wherein each of the phosphatidylcholine, phosphatidylserine, phosphoethanolamine, and sphingoid lipids is present in an amount less than 25 mol% of the total lipid component of the LNP. In certain embodiments, LNP is any one of the aforementioned in this paragraph wherein the PEG lipid is PEG2k-DMG. In certain embodiments, LNP is any one of the aforementioned in this paragraph wherein the PEG lipid is PEG2k-DSPE. [00468] In another particular embodiment, LNP comprises about 33 mol % ionizable lipid, about 40 mol % DSPC, about 25 mol % cholesterol, and about 2 mol % of PEG lipid. In another particular embodiment, LNP comprises about 33 mol % ionizable lipid, about 40 mol % sphingomyelin, about 25 mol % cholesterol, and about 2 mol % of PEG lipid. In another particular embodiment, LNP comprises about 33 mol % ionizable lipid, about 40 mol % DOPE, about 25 mol % cholesterol, and about 2 mol % of PEG lipid. In another particular embodiment, LNP comprises about 33 mol % ionizable lipid, about 40 mol % DOPC, about 25 mol % cholesterol, and about 2 mol % of PEG lipid. In another particular embodiment, LNP comprises about 33 mol % ionizable lipid, about 40 mol % DLPC, about 25 mol % cholesterol, and about 2 mol % of PEG lipid. In another particular embodiment, LNP comprises about 33 mol % ionizable lipid, about 40 mol % DOPS, about 25 mol % cholesterol, and about 2 mol % of PEG lipid. In another particular embodiment, LNP comprises about 33 mol % ionizable lipid, about 40 mol % phospholipid, about 25 mol % cholesterol, and about 2 mol % of PEG lipid. In another particular embodiment, LNP comprises about 33 mol % ionizable lipid, about 20 mol % sphingomyelin, about 20 mol% DSPC, about 25 mol % cholesterol, and about 2 mol % of PEG lipid. In certain embodiments, LNP is any one of the aforementioned in this RNG043-WO1 PCT Application paragraph wherein the PEG lipid is PEG2k-DMG. In certain embodiments, LNP is any one of the aforementioned in this paragraph wherein the PEG lipid is PEG2k-DSPE. [00469] In certain embodiments, the LNP comprises about 43mol% ionizable lipid, about 15mol% of a sphingolipid, about 15mol% of a non-sphingolipid phospholipid, about 25mol% cholesterol and about 2mol% of a PEGylated lipid. In certain embodiments, the LNP comprises about 33mol% ionizable lipid, about 25mol% of a sphingolipid, about 15mol% of a non-sphingolipid phospholipid, about 25mol% cholesterol and about 2mol% of a PEGylated lipid. In certain embodiments, the LNP comprises about 33mol% ionizable lipid, about 15mol% of a sphingolipid, about 25mol% of a non-sphingolipid phospholipid, about 25mol% cholesterol and about 2mol% of a PEGylated lipid. In some embodiments, the PEG lipid is PEG2K-DSPE, the structural lipid is cholesterol, and the phospholipid is a mixture of DSPC and sphingomyelin. In some embodiments, the PEG lipid is PEG2K-DMG, the structural lipid is cholesterol, and the phospholipid is a mixture of DSPC and sphingomyelin. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 40mol% cholesterol and about 1.5mol% PEG2K-DSPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 40mol% cholesterol and about 1.5mol% PEG2K-DMG. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 39mol% cholesterol and about 2.5mol% PEG2K-DSPE. [00470] In another particular embodiment, the lipid component includes about 48.5 mol % ionizable lipid, about 10 mol % phospholipid, about 38.5 mol % structural lipid, and about 3 mol % of PEG lipid. In another particular embodiment, the lipid component includes about 48.5 mol % ionizable lipid, about 10 mol % phospholipid, about 38 mol % structural lipid, and about 3.5 mol % of PEG lipid. In some embodiments, the PEG lipid is PEG2K-DPPE, the structural lipid is cholesterol, and the phospholipid is a DSPC or a mixture of DSPC and sphingomyelin. In some embodiments, the PEG lipid is PEG2K-DPPE, the structural lipid is cholesterol, and the phospholipid is a mixture of DSPC and sphingomyelin. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 40mol% cholesterol and about 1.5mol% PEG2K-DPPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 39.5 mol% cholesterol and about 2 mol% PEG2K- DPPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 39mol% cholesterol and about 2.5mol% PEG2K-DPPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, RNG043-WO1 PCT Application about 10mol% of a phospholipid (such as DSPC), about 38.5 mol% cholesterol and about 3 mol% PEG2K-DPPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 38 mol% cholesterol and about 3.5mol% PEG2K-DPPE. In some embodiments, the PEG lipid is PEG2K-DMG, the structural lipid is cholesterol, and the phospholipid is a DSPC or a mixture of DSPC and sphingomyelin. In some embodiments, the PEG lipid is PEG2K-DMG, the structural lipid is cholesterol, and the phospholipid is a mixture of DSPC and sphingomyelin. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 40mol% cholesterol and about 1.5mol% PEG2K-DMG. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 39.5 mol% cholesterol and about 2 mol% PEG2K-DMG. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 39mol% cholesterol and about 2.5mol% PEG2K-DMG. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 38.5 mol% cholesterol and about 3 mol% PEG2K- DMG. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 38 mol% cholesterol and about 3.5mol% PEG2K-DMG. In some embodiments, the PEG lipid is PEG2K-DSPE, the structural lipid is cholesterol, and the phospholipid is a DSPC or a mixture of DSPC and sphingomyelin. In some embodiments, the PEG lipid is PEG2K-DSPE, the structural lipid is cholesterol, and the phospholipid is a mixture of DSPC and sphingomyelin. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 40mol% cholesterol and about 1.5mol% PEG2K-DSPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 39.5 mol% cholesterol and about 2 mol% PEG2K-DSPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 39mol% cholesterol and about 2.5mol% PEG2K-DSPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 38.5 mol% cholesterol and about 3 mol% PEG2K- DSPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 38 mol% cholesterol and about 3.5mol% PEG2K-DSPE. [00471] In some embodiments, the LNP further comprises a targeting moiety. In some embodiments, the targeting moiety is an antibody or a fragment thereof. RNG043-WO1 PCT Application [00472] In some embodiments, a nanoparticle composition of the present disclosure is formulated to provide a specific N:P ratio. The N:P ratio of the composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in an RNA active agent (e.g., a linear or circular mRNA payload). In general, a lower N:P ratio is preferred. The one or more enzymes, lipids, and amounts thereof is selected to provide an N:P ratio from about 2:1 to about 30:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1. In certain embodiments, the N:P ratio is from about 2:1 to about 8:1. In other embodiments, the N:P ratio is from about 5:1 to about 8:1. For example, the N:P ratio is about 5.0:1, about 5.5:1, about 5.67:1, about 6.0:1, about 6.5:1, or about 7.0:1. [00473] The characteristics of a nanoparticle composition may depend on the components thereof. For example, a nanoparticle composition including cholesterol as a structural lipid may have different characteristics than a nanoparticle composition that includes a different structural lipid. Similarly, the characteristics of a nanoparticle composition may depend on the absolute or relative amounts of its components. For instance, a nanoparticle composition including a higher molar fraction of a phospholipid may have different characteristics than a nanoparticle composition including a lower molar fraction of a phospholipid. Characteristics may also vary depending on the method and conditions of preparation of the nanoparticle composition. Nanoparticle compositions may be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) may be used to examine the morphology and size distribution of a nanoparticle composition. Dynamic light scattering or potentiometry (e.g., potentiometric titrations) may be used to measure Zeta potentials. Dynamic light scattering may also be utilized to determine particle sizes. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) may also be used to measure multiple characteristics of a nanoparticle composition, Such as particle size, polydispersity index, and Zeta potential. [00474] The mean size of a nanoparticle composition may be between 10s of nm and 100s of nm, e.g., measured by dynamic light scattering (DLS). For example, the mean size may be from about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the mean size of a nanoparticle composition may be from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about RNG043-WO1 PCT Application 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, or from about 90 nm to about 100 nm. In certain embodiments, the mean size of a nanoparticle composition may be from about 70 nm to about 100 nm. In a particular embodiment, the mean size may be about 80 nm. In other embodiments, the mean size may be about 100 nm. [00475] A nanoparticle composition may be relatively homogenous. A polydispersity index may be used to indicate the homogeneity of a nanoparticle composition, e.g., the particle size distribution of the nanoparticle compositions. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. A nanoparticle composition may have a polydispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. [00476] The Zeta potential of a nanoparticle composition may be used to indicate the electrokinetic potential of the composition. For example, the Zeta potential may describe the surface charge of a nanoparticle composition. Nanoparticle compositions with relatively low charges, positive or negative, are generally desirable, as more highly charged species may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the Zeta potential of a nanoparticle composition may be from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about -10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV, to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV, to about +15 mV, or from about +5 mV to about +10 mV. [00477] The efficiency of encapsulation of a payload describes the amount of payload that is encapsulated or otherwise associated with a nanoparticle composition after preparation, relative to the initial amount provided. The encapsulation efficiency is desirably high (e.g., close to 100%). The encapsulation efficiency may be measured, for example, by comparing the amount of payload in a solution containing the nanoparticle composition before and after breaking up the nanoparticle composition with one or more organic solvents or detergents. Fluorescence may be used to measure the amount of free payload in a solution. For the RNG043-WO1 PCT Application nanoparticle compositions described herein, the encapsulation efficiency of a therapeutic and/or prophylactic may be at least 50%, for example 50%, 55%, 60%.65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In certain embodiments, the encapsulation efficiency may be at least 90%. [00478] In some examples, lipids and their method of preparation can be those disclosed in U.S. Patent Nos.8,569,256, 5,965,542 and U.S. Patent Publication Nos.2016/0199485, 2016/0009637, 2015/0273068, 2015/0265708, 2015/0203446, 2015/0005363, 2014/0308304, 2014/0200257, 2013/086373, 2013/0338210, 2013/0323269, 2013/0245107, 2013/0195920, 2013/0123338, 2013/0022649, 2013/0017223, 2012/0295832, 2012/0183581, 2012/0172411, 2012/0027803, 2012/0058188, 2011/0311583, 2011/0311582, 2011/0262527, 2011/0216622, 2011/0117125, 2011/0091525, 2011/0076335, 2011/0060032, 2010/0130588, 2007/0042031, 2006/0240093, 2006/0083780, 2006/0008910, 2005/0175682, 2005/017054, 2005/0118253, 2005/0064595, 2004/0142025, 2007/0042031, 1999/009076 and PCT Pub. Nos. WO 99/39741, WO 2017/117528, WO 2017/004143, WO 2017/075531, WO 2015/199952, WO 2014/008334, WO 2013/086373, WO 2013/086322, WO 2013/016058, WO 2013/086373, WO2011/141705, and WO 2001/07548 and Semple et. al, Nature Biotechnology, 2010, 28, 172-176, the full disclosures of which are herein incorporated by reference in their entirety for all purposes. [00479] A nanoparticle composition may include any substance useful in pharmaceutical compositions. For example, the nanoparticle composition may include one or more pharmaceutically acceptable excipients or accessory ingredients such as, but not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulating aids, disintegrants, fillers, glidants, liquid vehicles, binders, surface active agents, isotonic agents, thickening or emulsifying agents, buffering agents, lubricating agents, oils, preservatives, and other species. Excipients such as waxes, butters, coloring agents, coating agents, flavorings, and perfuming agents may also be included. In some examples, the pharmaceutically acceptable excipients can be those described in Remington’s The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro: Lippincott, Williams & Wilkins, Baltimore, Md., 2006). In some examples, other different lipids or liposomal formulations including nanoparticles and methods of administration can include, but are not limited to, those disclosed in U.S. Patent Publication 20030203865, 20020150626, 20030032615, and 20040048787, which are specifically incorporated by reference to the extent they disclose formulations and other related aspects of administration and delivery of nucleic acids. In some exmaples, RNG043-WO1 PCT Application methods used for forming particles can include the methods disclosed in U.S. Pat. Nos. 5,844,107, 5,877,302, 6,008,336, 6,077,835, 5,972,901, 6,200,801, and 5,972,900, which are incorporated by reference for those aspects. [00480] In some embodiments, the LNP encapsulates the engineered retron, e.g., an engineered nucleic acid construct, ncRNA, vector system, RNA molecule, and/or engineered nucleic acid-enzyme construct as described herein. [00481] In some embodiments, the one or more structural lipids are selected from the group consisting of cholesterol, fecosterol, beta sitosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, prednisolone, dexamethasone, prednisone, and hydrocortisone. In some embodiments, the one or more PEGylated lipids are selected from the group consisting of PEG-c-DOMG, PEG-DMG, PEG- DLPE, PEG-DMPE, PEG-DPPC, and PEG-DSPE. [00482] In some embodiments, the one or more phospholipids are selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1.2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocho line (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuc cinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1- hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3- phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn- glycero-3-phosphocholine, 1,2-diphytanoylsn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2- diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3- phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin. [00483] In some embodiments, the lipid nanoparticle comprises about 48.5 mol % ionizable lipid, about 10 mol % phospholipid, about 40 mol % structural lipid, and about 1.5 mol% of PEG lipid. [00484] In some embodiments, the lipid nanoparticle comprises about 48.5 mol % ionizable lipid, about 10 mol % phospholipid, about 39 mol % structural lipid, and about 2.5 mol% of PEG lipid. In some embodiments, the LNP further comprises a targeting moiety RNG043-WO1 PCT Application operably connected to the LNP. In some embodiments, the LNP further comprises one or more additional components selected from the group consisting of DDAB, EPC, 14PA, 18BMP, DODAP, DOTAP, and C12-200. [00485] In some embodiments, the engineered retron can be used for gene transfer, which may be performed under ex vivo or in vivo conditions. Ex vivo gene therapy refers to the isolation of cells from a subject, the delivery of a nucleic acid into cells in vitro, and the return of the modified cells back into the subject. This may involve the collection of a biological sample comprising cells from the subject. For example, blood can be obtained by venipuncture, and solid tissue samples can be obtained by surgical techniques according to methods well known in the art. [00486] Usually, but not always, the subject who receives the cells (e.g., the recipient) is also the subject from whom the cells are harvested or obtained, which provides the advantage that the donated cells are autologous. However, cells can be obtained from another subject (e.g., a donor), a culture of cells from a donor, or from established cell culture lines. Accordingly, in some embodiments the cells are allogeneic to the recipient. Cells may be obtained from the same or a different species than the subject to be treated, but preferably are of the same species, and more preferably of the same immunological profile as the subject. Such cells can be obtained, for example, from a biological sample comprising cells from a close relative or matched donor, then transfected with nucleic acids (e.g., comprising an engineered retron), and administered to a subject in need of genome modification, for example, for treatment of a disease or condition. [00487] In other embodiments, the engineered retron can be introduced in vivo (e.g., used in gene therapy) by physically delivering the engineered retron to a subject. Examples of physically introducing the engineered retron includes via injections, electroporation and transfection (e.g., calcium-mediated or liposome tranfection, or the like). C. Payloads [00488] The retron-based gene editing systems and/or components thereof may be delivered by way of LNPs as described herein. In various embodiments, the retron-based gene editing systems may be delivered by LNPs into cells, tissues, organs, or organisms. Depending on the chosen format, the retron-based gene editing systems and/or the individual or combined components thereof may be delivered as DNA molecules (e.g., encoded on one or more plasmids), RNA molecules (e.g., guide RNAs for targeting a programmable nuclease or linear or circular mRNAs coding for the retron RTs or programmable nuclease components of the retron-based gene editing systems), proteins (e.g., retron polypeptides, accessory proteins RNG043-WO1 PCT Application having other functions (e.g., recombinases, nucleases, polymerases, ligases, deaminases, or reverse transcriptases), or protein-nucleic acid complexes (e.g., complexes between a guide RNA and a programmable nuclease protein or fusion protein comprising a retron RT). These DNA, RNA, protein, or nucleoprotein corresponding to and/or encoding the retron-based gene editing systems or components thereof comprise the LNP cargo or payloads. In various embodiments, the LNP cargo or payloads may comprise nucleic acid payloads, including coding payloads such as linear and circular mRNA for encoding the various components of the retron-based editing system. 1. Nucleic acid payloads [00489] In various embodiments, the LNP compositions described herein can be used to deliver a nucleic acid or polynucleotide payload, e.g., DNA or a coding or noncoding RNA. [00490] In various embodiments, the retron-based editing compositions described herein can include a nucleic acid or polynucleotide payload, e.g., DNA or a coding or noncoding RNA. For example, the retron gene editing systems may comprise one or more coding mRNA (circular or linear) for encoding retron RT and other accessory proteins (e.g., programmable nuclease) and these RNA components may be delivered by LNPs. [00491] In some embodiments, a LNP is capable of delivering a polynucleotide to a target cell, tissue, or organ. A polynucleotide, in its broadest sense of the term, includes any compound and/or substance that is or can be incorporated into an oligonucleotide chain. Exemplary polynucleotides for use in accordance with the present disclosure include, but are not limited to, one or more of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) including messenger mRNA (mRNA), hybrids thereof, RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that induce triple helix formation, aptamers, vectors, etc. RNAs useful in the compositions and methods described herein can be selected from the group consisting of but are not limited to, shortimers, antagomirs, antisense, ribozymes, short interfering RNA (siRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), Dicer substrate RNA (dsRNA), short hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof. In some embodiments, a polynucleotide is mRNA. In some embodiments, a polynucleotide is circular RNA. In some embodiments, a polynucleotide encodes a protein, e.g., a nucleobase editing enzyme. A polynucleotide may encode any polypeptide of interest, including any naturally or non-naturally occurring or otherwise modified polypeptide. A polypeptide may be of any size and may have any secondary structure or activity. In some embodiments, a polypeptide encoded by an mRNA may have a therapeutic effect when expressed in a cell. RNG043-WO1 PCT Application [00492] In other embodiments, a polynucleotide is an siRNA. An siRNA may be capable of selectively knocking down or down regulating expression of a gene of interest. For example, an siRNA could be selected to silence a gene associated with a particular disease, disorder, or condition upon administration to a subject in need thereof of a nanoparticle composition including the siRNA. An siRNA may comprise a sequence that is complementary to an mRNA sequence that encodes a gene or protein of interest. In some embodiments, the siRNA may be an immunomodulatory siRNA. [00493] In some embodiments, a polynucleotide is an shRNA or a vector or plasmid encoding the same. An shRNA may be produced inside a target cell upon delivery of an appropriate construct to the nucleus. Constructs and mechanisms relating to shRNA are well known in the relevant arts. [00494] A polynucleotide may include a first region of linked nucleosides encoding a polypeptide of interest (e.g., a coding region), a first flanking region located at the 5'-terminus of the first region (e.g., a 5'-UTR), a second flanking region located at the 3'-terminus of the first region (e.g., a 3'-UTR), at least one 5'-cap region, and a 3'-stabilizing region. In some embodiments, a polynucleotide further includes a poly-A region or a Kozak sequence (e.g., in the 5'-UTR). In some cases, polynucleotides may contain one or more intronic nucleotide sequences capable of being excised from the polynucleotide. In some embodiments, a polynucleotide (e.g., an mRNA) may include a 5' cap structure, a chain terminating nucleotide, a stem loop, a polyA sequence, and/or a polyadenylation signal. Any one of the regions of a nucleic acid may include one or more alternative components (e.g., an alternative nucleoside). For example, the 3'-stabilizing region may contain an alternative nucleoside such as an L- nucleoside, an inverted thymidine, or a 2'-O-methyl nucleoside and/or the coding region, 5'- UTR, 3'-UTR, or cap region may include an alternative nucleoside such as a 5-substituted uridine (e.g., 5-methoxyu ridine), a 1-substituted pseudouridine (e.g., 1-methyl pseudouridine or 1-ethyl-pseudouridine), and/or a 5-substituted cytidine (e.g., 5-methyl-cytidine). In some embodiments, a polynucleotide contains only naturally occurring nucleosides. [00495] In some cases, a polynucleotide is greater than 30 nucleotides in length. In another embodiment, the poly nucleotide molecule is greater than 35 nucleotides in length. In another embodiment, the length is at least 40 nucleotides. In another embodiment, the length is at least 45 nucleotides. In another embodiment, the length is at least 55 nucleotides. In another embodiment, the length is at least 50 nucleotides. In another embodiment, the length is at least 60 nucleotides. In another embodiment, the length is at least 80 nucleotides. In another embodiment, the length is at least 90 nucleotides. In another embodiment, the length is at least RNG043-WO1 PCT Application 100 nucleotides. In another embodiment, the length is at least 120 nucleotides. In another embodiment, the length is at least 140 nucleotides. In another embodiment, the length is at least 160 nucleotides. In another embodiment, the length is at least 180 nucleotides. In another embodiment, the length is at least 200 nucleotides. In another embodiment, the length is at least 250 nucleotides. In another embodiment, the length is at least 300 nucleotides. In another embodiment, the length is at least 350 nucleotides. In another embodiment, the length is at least 400 nucleotides. In another embodiment, the length is at least 450 nucleotides. In another embodiment, the length is at least 500 nucleotides. In another embodiment, the length is at least 600 nucleotides. In another embodiment, the length is at least 700 nucleotides. In another embodiment, the length is at least 800 nucleotides. In another embodiment, the length is at least 900 nucleotides. In another embodiment, the length is at least 1000 nucleotides. In another embodiment, the length is at least 1100 nucleotides. In another embodiment, the length is at least 1200 nucleotides. In another embodiment, the length is at least 1300 nucleotides. In another embodiment, the length is at least 1400 nucleotides. In another embodiment, the length is at least 1500 nucleotides. In another embodiment, the length is at least 1600 nucleotides. In another embodiment, the length is at least 1800 nucleotides. In another embodiment, the length is at least 2000 nucleotides. In another embodiment, the length is at least 2500 nucleotides. In another embodiment, the length is at least 3000 nucleotides. In another embodiment, the length is at least 4000 nucleotides. In another embodiment, the length is at least 5000 nucleotides, or greater than 5000 nucleotides. [00496] In some examples, a polynucleotide molecule, formula, composition or method associated therewith comprises one or more polynucleotides comprising features as described in WO2002/098443, WO2003/051401, WO2008/052770, WO2009/127230, WO2006/122828, WO2008/083949, WO2010/088927, WO2010/037539, WO2004/004743, WO2005/016376, WO2006/024518, WO2007/095976, WO2008/014979, WO2008/077592, WO2009/030481, WO2009/095226, WO2011/069586, WO2011/026641, WO2011/144358, WO2012/019780, WO2012/013326, WO2012/089338, WO2012/113513, WO2012/116811, WO2012/116810, WO2013/113502, WO2013/113501, WO2013/113736, WO2013/143698, WO2013/143699, WO2013/143700, WO2013/120626, WO2013/120627, WO2013/120628, WO2013/120629, WO2013/174409, WO2014/127917, WO2015/024669, WO2015/024668, WO2015/024667, WO2015/024665, WO2015/024666, WO2015/024664, WO2015/101415, WO2015/101414, WO2015/024667, WO2015/062738, WO2015/101416, all of which are incorporated by reference herein. RNG043-WO1 PCT Application [00497] In some embodiments, a polynucleotide comprises one or more microRNA binding sites. In some embodiments, a microRNA binding site is recognized by a microRNA in a non-target organ. In some embodiments, a microRNA binding site is recognized by a microRNA in the liver. In some embodiments, a microRNA binding site is recognized by a microRNA in hepatic cells. [00498] In certain embodiments, an RNA of the present disclosure comprises one or more phosphonate modifications selected from a phosphorothioate linkage (PS), phosphorodithioate linkage (PS2), methylphosphonate linkage (MP), methoxypropylphosphonate linkage (MOP), 5’-(E)-vinylphosphonate linkage (5’-(E)-VP), 5’- Methyl Phosphonate linkage (5’-MP), (S)-5’-C-methyl with phosphate linkage, 5’- phosphorothioate linkage (5’-PS), and a peptide nucleic acid linkage (PNA). In certain embodiments, an RNA of the present disclosure comprises one or more ribose modifications selected from a 2’-O-methyl (2’-OMe), 2’-O-methoxyethyl (2’-O-MOE), 2’-deoxy-2’-fluoro (2’-F), 2’-arabino-fluoro (2’-Ara-F), 2’-O-benzyl, 2’-O-methyl-4-pyridine (2’-O-CH2Py(4)), Locked nucleic acid (LNA), (S)-cET-BNA, tricyclo-DNA (tcDNA), PMO, Unlocked Nucleic Acid (UNA) and glycol nucleic acid (GNA). In certain embodiments, the RNA comprises a Locked Nucleic Acid (LNA) comprising a methyl bridge, an ethyl bridge, a propyl bridge, a butyl bridge or an optionally substituted variant of any of the aforementioned. In certain embodiments, an RNA of the present disclosure comprises one or more modified bases selected from a pseudouridine (ψ), 2’thiouridine (s2U), N6’-methyladenosine (m6A), 5’methylcytidine (m5C), 5’fluoro2’-deoxyuridine, N-ethylpiperidine 7’-EAA triazole modified adenine, N-ethylpiperidine 6’triazole modified adenine, 6’pheynlpyrrolo-cytosine (PhpC), 2’,4’-difluorotoluyl ribonucleoside (rF), and 5’-nitroindole. 2. Single-stranded DNA payloads [00499] In various embodiments, the LNPs of the present disclosure may comprise a payload having at least one single stranded DNA. In certain embodiments, the single stranded DNA is a linear single stranded DNA. In certain embodiments, the single stranded DNA is a circular single stranded DNA. In certain embodiments, the payload further comprises a nucleobase editing system, such as an enzyme or polynucleotide encoding an enzyme capable of independently or co-dependently editing, modifying, or altering a target polynucleotide sequence or a target transcript comprising a nucleic acid sequence. [00500] In certain examples, the circular single stranded DNA (CiSSD) payload can be those described in PCT Publication WO2020142730A1, which is incorporated by reference herein in its entirety. In certain embodiments, the CiSSD is a donor template for use as part of RNG043-WO1 PCT Application a nucleobase editing system for targeted genome modification. In certain embodiments, the CiSSD comprises a DNA insert, a 5’ homology arm, and a 3’ homology arm. In some embodiments, the DNA insert is located between the 5’ homology arm and the 3’ homology arm. Homology arms as used herein refer to a series of nucleotides that are complementary to a series of nucleotides in an endogenous DNA sequence in the target region. The homology arms flanking the DNA insert allow for specific insertion of the DNA insert in the target region. A target region is a nucleic acid sequence where a desired insertion or modification occurs. [00501] In certain embodiments, the DNA insert is at least 1 nucleotide. In certain embodiments, the DNA insert is at least about 0.5 kb, 2 kb, 2.5 kb, 5 kb, 10 kb, 20 kb, 40 kb, 80 kb, 100 kb, 150 kb, or 200 kb. In certain embodiments, the length of the DNA insert is about 0.5 kb to 5 kb, about 1 kb to 5 kb, about 1 kb to 10 kb, about 1.6 kb to 5 kb, about 1.6 kb to 10 kb, about 2 kb to 5 kb, about 2 kb to 20 kb, about 2.5 kb to 5 kb, about 2.5 kb to 10 kb, about 2.5 kb to 20 kb, and about 5kb to 100 kb. In some embodiments, the DNA insert size may range from about 1 kb to about 3 kb, about 3 kb to about 6 kb, about 6 kb to about 9 kb, about 9 kb to about 12 kb, about 12 kb to about 15 kb, about 15 kb to about 18 kb, or about 18 kb to about 21 kb. [00502] In some embodiments, the DNA insert may comprise a nucleotide sequence that encodes a maker or a reporter, e.g., a fluorescent marker, an antibiotic marker, or any suitable marker. A “marker” or “reporter” as used herein means a feature that allows for identification and selection of a desired cell, e.g., by fluorescence or antibiotic resistance. For example, the insert may include a nucleotide sequence encoding a reporter (e.g, GFP, RFP, or any suitable reporter) or a recombinase. For example, the reporter is an N-terminal GFP fusion reporter. [00503] In some embodiments, the DNA insert may comprise a nucleotide sequence that encodes a transcription unit, wherein each transcription unit can produce a cellular product (e.g, protein or RNA). In some embodiments, the DNA insert may comprise a nucleotide sequence that encodes a protein, e.g, an immunomodulatory protein (e.g, a cytokine), an antibody, a chimeric antigen receptor (CAR), a growth factor, a T cell receptor, or another protein. [00504] In certain embodiments, the CiSSD comprises a DNA insert that can be inserted at a nucleotide break in a target region of genomic DNA. In some embodiments, the break is a double stranded break (DSB). In certain embodiments, the break is a single stranded DNA break or a nick. Precision gene editing techniques, e.g, CRISPR, create a break near a desired sequence change (target sequence). CRISPR can be applied to produce deletions, disruptions, insertions, replacements, and repairs. The components of template donors for these different RNG043-WO1 PCT Application modifications is generally the same, consisting of three basic elements: a 5’ homology arm, a DNA insert, and a 3’ homology arm. CRISPR-based gene editing can generate gene knockouts by disrupting the gene sequence, however, efficiency for inserting exogenous DNA (knock-in) or replacement of genomic sequences is very poor using current methods. In certain embodiments, CiSSDs may be used with CRISPR by generating a knock-in modification. Double-stranded breaks can be introduced by any suitable mechanism, including, for example, by gene-editing systems using CRISPR, zinc finger nuclease, TALEN nuclease (Transcription Activator-Like Effector Nuclease), or meganuclease as described previously. Briefly, the CRISPR genome editing system generates a targeted DSB using the CRISPR programmable DNA endonuclease that can be targeted to a specific DNA sequence (target sequence) by a small “guide” RNA (crRNA). Guide RNAs for use in CRISPR-based modification (z.e., crRNAs and tracrRNAs) may be generated by any suitable method. In certain embodiments, crRNAs and tracrRNAs may be chemically synthesized. In other embodiments, a single guide RNA (sgRNA) may be constructed and synthesized by in vitro transcription. [00505] In certain embodiments, an LNP of the present disclosure comprises a CiSSD disclosed herein and further comprises a precision gene editing system component such as a CRISPR, zinc finger nuclease, TALEN nuclease (Transcription Activator-Like Effector Nuclease), or meganuclease, or any other nucleobase editing system known in the art. [00506] In certain eexamples, the single stranded DNA (SSD) payload can be those described in PCT Publication WO2020232286A1, which is incorporated by reference herein in its entirety. [00507] In certain embodiments, the SSD comprises an engineered initiator sequence and an engineered terminator sequence from a filamentous bacteriophage, and a DNA sequence of interest, wherein the DNA sequence of interest is located 3’ to the engineered initiator sequence and 5’ to the engineered terminator sequence. In certain embodiments, the SSD comprises a selectable marker. [00508] In certain examples, the single stranded DNA (SSD) payload can be made by a method described in PCT Publication WO2020232286A1. In certain embodiments, the SSD is made by a method comprising: (a) culturing a host cell under conditions suitable for producing a ssDNA from the DNA sequence of interest in the engineered nucleic acid and the plurality of bacteriophage proteins from the nucleic acid helper plasmid; (b) allowing the ssDNA and the plurality of bacteriophage proteins to assemble into an engineered phage; and (c) collecting the engineered phage. In certain embodiments, the method further comprises extracting the SSD from the engineered phage. RNG043-WO1 PCT Application [00509] In certain embodiments, at least 90% of the SSD is the same length as the DNA sequence of interest. In certain embodiments, at least 95% of the ssDNA is the same length as the DNA sequence of interest. In certain embodiments, the SSD is between 100 and 20,000 nucleotides in length. In certain embodiments, the ssDNA is circular. [00510] In certain examples, the single stranded DNA (SSD) payload can be those described in PCT Publication WO2022011082A1, which is incorporated by reference herein in its entirety. In certain embodiments, the SSD comprises a first sequence from a filamentous bacteriophage, the first sequence having both initiator and terminator functions; a second sequence that is identical to the first sequence; and a single-strand DNA sequence of interest that is located between the first sequence and the second sequence. In certain embodiments, the SSD further comprises a selectable marker. In certain embodiments, the SSD is circular. In certain embodiments, the SSD is linear. [00511] In certain examples, the single stranded DNA (SSD) payload can be made by a method described in PCT Publication WO2022011082A1. In certain embodiments, the method comprises culturing a host cell under conditions suitable for producing the single- stranded DNA from the single-strand DNA sequence of interest in the isolated nucleic acid and producing the bacteriophage proteins from the nucleic acid helper plasmid; allowing the single-stranded DNA and bacteriophage proteins to assemble into an engineered phage; and collecting the engineered phage. In certain embodiments, the host cell comprises an isolated nucleic acid that includes: a first sequence from a filamentous bacteriophage, the first sequence having both initiator and terminator functions; a second sequence that is identical to the first sequence; and a single-strand DNA sequence of interest that is located between the first sequence and the second sequence, and a nucleic acid helper plasmid for expressing bacteriophage proteins capable of assembling a single-strand DNA into a bacteriophage. In certain embodiments, the method further comprises extracting the SSD from the engineered phage. [00512] In certain embodiments, at least 90% of the SSD is the same length as the DNA sequence of interest. In certain embodiments, at least 95% of the ssDNA is the same length as the DNA sequence of interest. In certain embodiments, the SSD is between 100 and 20,000 nucleotides in length. In certain embodiments, the SSD is circular. [00513] In certain examples, the single stranded DNA (SSD) payload can be those described in PCT Publication WO2021055616A1, which is incorporated by reference herein in its entirety. 3. Linear mRNA payloads RNG043-WO1 PCT Application [00514] In various embodiments, the LNP-based pharmaceutical compositions described herein, e.g., LNP-based gene editing systems, may include one or more linear mRNA molecules or linear mRNA payloads. In various embodiments, the mRNA payloads may encode one or more components of the herein described gene editing systems. For example, an mRNA payload may encode an amino acid sequence-programmable DNA binding domain (e.g., retron RTs or programmable nucleases) or a nucleic acid sequence-programmable DNA binding domain (e.g., CRISPR Cas9, CRISPR Cas12a, CRISPR Cas12f, CRISPR Cas13a, CRISPR Cas13b, or TnpB). [00515] mRNA payloads may also encode, depending upon the nature of the gene editing system, one or more effector domains that provide various functionalities that facilitate changes in nucleotide sequence and/or gene expression, such as, but not limited to, single- strand DNA binding proteins, nucleases, endonucleases, exonucleases, deaminases (e.g., cytidine deaminases or adenosine deaminases), polymerases (e.g., reverse transcriptases), integrases, recombinases, etc., and fusion proteins comprising one or more functional domains linked together. [00516] Ribonucleic acid (RNA) is a molecule that is made up of nucleotides, which are ribose sugars attached to nitrogenous bases and phosphate groups. The nitrogenous bases include adenine (A), guanine (G), uracil (U), and cytosine (C). Generally, RNA mostly exists in the single-stranded form but can also exists double-stranded in certain circumstances. The length, form and structure of RNA is diverse depending on the purpose of the RNA. For example, the length of an RNA can vary from a short sequence (e.g., siRNA) to a long sequences (e.g., lncRNA), can be linear (e.g., mRNA) or circular (e.g., oRNA), and can either be a coding (e.g., mRNA) or a non-coding (e.g., lncRNA) sequence. [00517] In various embodiments, the LNP-based gene editing systems, RNA therapeutics and pharmaceutical compositions thereof described herein can be used to deliver a mRNA payload that is a linear mRNA molecule. In embodiments, the mRNA payload may comprise one or more nucleotide sequences that encode a product of interest, such as, but not limited to a component of a gene editing system (e.g., an endonuclease, a prime editor, etc.) and/or a therapeutic protein. [00518] In some embodiments, the RNA payload may be a linear mRNA. As used herein, the term "messenger RNA" (mRNA) refers to any polynucleotide which encodes a protein of interest and which is capable of being translated to produce the encoded protein of interest in vitro, in vivo, in situ or ex vivo. RNG043-WO1 PCT Application [00519] Generally, a mRNA molecule comprises at least a coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap and a poly-A tail. In some aspects, one or more structural and/or chemical modifications or alterations may be included in the RNA which can reduce the innate immune response of a cell in which the mRNA is introduced. As used herein, a "structural" feature or modification is one in which two or more linked nucleotides are inserted, deleted, duplicated, inverted or randomized in a nucleic acid without significant chemical modification to the nucleotides themselves. Because chemical bonds will necessarily be broken and reformed to affect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural modifications will result in a different sequence of nucleotides. For example, the polynucleotide "ATCG" may be chemically modified to "AT-5meC-G". [00520] Generally, a coding region of interest in an mRNA used herein may encode a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, an octapeptide, a nonapeptide, or a decapeptide. In another embodiment, the mRNA may encode a peptide of 2-30 amino acids, e.g.5-30, 10-30, 2-25, 5-25, 10-25, or 10-20 amino acids. The mRNA may encode a peptide of at least 10, 11, 12, 13, 14, 15, 17, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids, or a peptide that is no longer than 10, 11, 12, 13, 14, 15, 17, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids. [00521] Generally, the length of the region of the mRNA encoding a product of interest is greater than about 30 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 or up to and including 100,000 nucleotides). [00522] In some embodiments, the mRNA has a total length that spans from about 30 to about 100,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1,000 to 1,500, from 1,000 to RNG043-WO1 PCT Application 2,000, from 1,000 to 3,000, from 1,000 to 5,000, from 1,000 to 7,000, from 1,000 to 10,000, from 1 ,000 to 25,000, from 1,000 to 50,000, from 1,000 to 70,000, from 1,000 to 100,000, from 1,500 to 3,000, from 1,500 to 5,000, from 1,500 to 7,000, from 1,500 to 10,000, from 1 ,500 to 25,000, from 1,500 to 50,000, from 1,500 to 70,000, from 1,500 to 100,000, from 2,000 to 3,000, from 2,000 to 5,000, from 2,000 to 7,000, from 2,000 to 10,000, from 2,000 to 25,000, from 2,000 to 50,000, from 2,000 to 70,000, and from 2,000 to 100,000 nucleotides). [00523] In some embodiments, the region or regions flanking the region encoding the product of interest may range independently from 15-1,000 nucleotides in length (e.g., greater than 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, and 900 nucleotides or at least 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, and 1,000 nucleotides). [00524] In some embodiments, the mRNA comprises a tailing sequence which can range from absent to 500 nucleotides in length (e.g., at least 60, 70, 80, 90, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 nucleotides). Where the tailing region is a polyA tail, the length may be determined in units of or as a function of polyA Binding Protein binding. In this embodiment, the polyA tail is long enough to bind at least 4 monomers of PolyA Binding Protein. PolyA Binding Protein monomers bind to stretches of approximately 38 nucleotides. As such, it has been observed that polyA tails of about 80 nucleotides and 160 nucleotides are functional. [00525] In some embodiments, the mRNA comprises a capping sequence which comprises a single cap or a series of nucleotides forming the cap. The capping sequence may be from 1 to 10, e.g.2-9, 3-8, 4-7, 1-5, 5-10, or at least 2, or 10 or fewer nucleotides in length. In some embodiments, the caping sequence is absent. [00526] In some embodiments, the mRNA comprises a region comprising a start codon. The region comprising the start codon may range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length. [00527] In some embodiments, the mRNA comprises a region comprising a stop codon. The region comprising the stop codon may range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length. [00528] In some embodiments, the mRNA comprises a region comprising a restriction sequence. The region comprising the restriction sequence may range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length. Untranslated Regions (UTRs) RNG043-WO1 PCT Application [00529] In various embodiments, the mRNA payloads of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions thereof described herein, may comprise at least one untranslated region (UTR) which flanks the region encoding the product of interest and/or is incorporated within the mRNA molecule. UTRs are transcribed by not translated. The mRNA payloads can include 5’ UTR sequences and 3’ UTR sequences, as well as internal UTRs. [00530] The RNA payloads of the present disclosure may comprise one or more regions or parts which act or function as an untranslated region. Where nucleic acids are designed to encode at least one polypeptide of interest, the nucleic acid may comprise one or more of these untranslated regions (UTRs). Wild-type untranslated regions of a nucleic acid are transcribed but not translated. In mRNA, the 5′ UTR starts at the transcription start site and continues to the start codon but does not include the start codon; whereas, the 3′ UTR starts immediately following the stop codon and continues until the transcriptional termination signal. There is growing body of evidence about the regulatory roles played by the UTRs in terms of stability of the nucleic acid molecule and translation. The regulatory features of a UTR can be incorporated into the RNA payload molecules (e.g., linear and circular mRNA molecules) of the present disclosure to, among other things, enhance the stability of the molecule. The specific features can also be incorporated to ensure controlled down-regulation of the transcript in case they are misdirected to undesired organs sites. A variety of 5′UTR and 3′UTR sequences are known and available in the art. [00531] In various examples, the mRNA payloads of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions thereof described herein, may comprise at least one UTR that may be selected from any UTR sequence listed in Tables 19 or 20 of U.S. Patent No.10,709,779, which is incorporated herein by reference. 5' UTR regions [00532] In various embodiments, the mRNA payloads of the LNP-based gene editing systems, RNA therapeutics and pharmaceutical compositions thereof described herein, may comprise at least one 5′ UTR. [00533] A 5′ UTR is region of an mRNA that is directly upstream (5′) from the start codon (the first codon of an mRNA transcript translated by a ribosome). A 5′ UTR does not encode a protein (is non-coding). Natural 5′UTRs have features that play roles in translation initiation. They harbor signatures like Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCR(A/G)CCAUGG, where R is a purine (adenine or guanine) RNG043-WO1 PCT Application three bases upstream of the start codon (AUG), which is followed by another ‘G’.5′UTR also have been known to form secondary structures which are involved in elongation factor binding.5’ UTR sequences are also known to be important for ribosome recruitment to the mRNA and have been reported to play a role in translation, for example, as described in Hinnebusch A, et al., (2016) Science, 352:6292: 1413-6. In addition, 5’ UTR sequences may confer increased half-life, increased expression and/or increased activity of a polypeptide encoded by the RNA payload described herein. [00534] In various embodiments, the RNA payload constructs contemplated herein may include 5’UTRs that are found in nature and those that are not. For example, the 5’UTRs can be synthetic and/or can be altered in sequence with respect to a naturally occurring 5’UTR. Such altered 5’UTRs can include one or more modifications relative to a naturally occurring 5’UTR, such as, for example, an insertion, deletion, or an altered sequence, or the substitution of one or more nucleotide analogs in place of a naturally occurring nucleotide. [00535] The 5' UTR starts at the transcription start site and continues to the start codon but does not include the start codon; whereas, the 3 'UTR starts immediately following the stop codon and continues until the transcriptional termination signal. While not wishing to be bound by theory, the UTRs may have a regulatory role in terms of translation and stability of the nucleic acid. [00536] Natural 5' UTRs usually include features which have a role in translation initiation as they tend to include Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCR(A/G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another 'G'.5'UTR also have been known to form secondary structures which are involved in elongation factor binding. [00537] In an embodiment, the 5’ UTR comprises a sequence provided in the abovementioned Table X or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a 5’ UTR sequence provided in the abovementioned Table Y or a variant or a fragment thereof (e.g., a fragment that lacks the first one, two, three, four, five, or six nucleotides of the 5’ UTR sequence provided in the abovementioned Table Y). In an embodiment, the 5’ UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of the sequences of the abovementioned Table Y. Table Y – Exemplary nucleotide sequences of 5’ UTRs 5’ UTR Nucleotide Sequence Sequence Identifier
Figure imgf000148_0001
RNG043-WO1 PCT Application ggaaaucgca aaauuugcuc uucgcguuag auuucuuuua guuuucucgc SEQ ID NO: 19422 aacuagcaag cuuuuuguuc ucgccgccgc c
Figure imgf000149_0001
RNG043-WO1 PCT Application ggaaacccgc ccaagcgacc ccaacauauc agcaguugcc caaucccaac SEQ ID NO: 19438 ucccaacaca auccccaagc aacgccgcc
Figure imgf000150_0001
[00538] In some embodiments of the disclosure, a 5′ UTR is a heterologous UTR, i.e., is a UTR found in nature associated with a different mRNA. In another embodiment, a 5′ UTR is a synthetic UTR, i.e., does not occur in nature. Synthetic UTRs include UTRs that have been mutated to improve their properties, e.g., which increase gene expression as well as those which are completely synthetic. In some examples, exemplary 5′ UTRs can RNG043-WO1 PCT Application include Xenopus or human derived alpha-globin or beta-globin, as disclosed in US8,278,063 and US9,012,219, human cytochrome b-245 polypeptide, and hydroxysteroid (17b) dehydrogenase, and Tobacco etch virus. In some examples, CMV immediate-early 1 (IE1) gene, as disclosed in US20140206753 and WO2013/185069, the sequence GGGAUCCUACC (SEQ ID NO: 19451), as disclosed in WO2014144196, may also be used. In another examples, 5′ UTR of a TOP gene is a 5′ UTR of a TOP gene lacking the 5′ TOP motif (the oligopyrimidine tract), such as the 5’ UTR disclosed in WO/2015101414, WO2015101415, WO/2015/062738, WO2015024667, WO2015024667, a 5′ UTR element derived from ribosomal protein Large 32 (L32) gene as disclosed in WO/2015101414, WO2015101415, WO/2015/062738, 5′ UTR element derived from the 5′UTR of an hydroxysteroid (17-β) dehydrogenase 4 gene (HSD17B4) as disclosed in WO2015024667, or a 5′ UTR element derived from the 5′ UTR of ATP5A1 as disclosed in WO2015024667 can be used. In one embodiment, an internal ribosome entry site (IRES) is used as a substitute for a 5′ UTR. [00539] In some embodiments, a 5′ UTR of the present disclosure comprises a sequence selected from SEQ ID NO:19452 (GGGAAAUAAG AGAGAAAAGA AGAGUAAGAA GAAAUAUAAG AGCCACC), and SEQ ID NO:19453 (GGGAAATAAG AGAGAAAAGA AGAGTAAGAA GAAATATAAG AGCCACC). 3' UTR regions [00540] In various embodiments, the mRNA payloads of the LNP-based base editing systems, RNA therapeutics and pharmaceutical compositions thereof described herein, may comprise at least one 3′ UTR.3′ UTRs may be heterologous or synthetic. [00541] A 3′ UTR is region of an mRNA that is directly downstream (3′) from the stop codon (the codon of an mRNA transcript that signals a termination of translation). A 3′ UTR does not encode a protein (is non-coding). Natural or wild type 3′ UTRs are known to have stretches of adenosines and uridines embedded in them. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into three classes, for example, as described in Chen et al, 1995: Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U/A)(U/A) nonamers. Molecules containing this type of AREs include GM-CSF and TNF-α. Class III ARES are less well defined. These U rich regions do not contain an AUUUA motif. c-Jun and Myogenin are two well-studied examples of this class. Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented RNG043-WO1 PCT Application to increase the stability of mRNA. HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3′ UTR of nucleic acid molecules will lead to HuR binding and thus, stabilization of the message in vivo. [00542] 3' UTRs are known to have stretches of adenosines and uridines embedded in them. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into three classes, for example, as described in Chen et al., 1995: Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U/A)(U/A) nonamers. Molecules containing this type of AREs include GM-CSF and TNF-a. Class III ARES are less well defined. These U rich regions do not contain an AUUUA motif. c-Jun and Myogenin are two well-studied examples of this class. Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA. HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3' UTR of nucleic acid molecules will lead to HuR binding and thus, stabilization of the message in vivo. [00543] Introduction, removal or modification of 3' UTR AU rich elements (AREs) can be used to modulate the stability of the mRNA payloads described herein. For example, one or more copies of an ARE can be introduced to make mRNA less stable and thereby curtail translation and decrease production of the resultant protein. Alternatively, AREs can be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein. [00544] In some embodiments, the introduction of features often expressed in genes of target organs the stability and protein production of the mRNA can be enhanced in a specific organ and/or tissue. As a non-limiting example, the feature can be a UTR. As another example, the feature can be introns or portions of introns sequences. [00545] Those of ordinary skill in the art will understand that 5′ UTRs that are heterologous or synthetic may be used with any desired 3′ UTR sequence. For example, a heterologous 5′ UTR may be used with a synthetic 3′ UTR with a heterologous 3′ UTR. [00546] Non-UTR sequences may also be used as regions or subregions within an RNA payload construct. For example, introns or portions of introns sequences may be incorporated into regions of nucleic acid of the disclosure. Incorporation of intronic sequences may increase protein production as well as nucleic acid levels. RNG043-WO1 PCT Application [00547] Combinations of features may be included in flanking regions and may be contained within other features. For example, the polypeptide coding region of interest in an mRNA payload may be flanked by a 5′ UTR which may contain a strong Kozak translational initiation signal and/or a 3′ UTR which may include an oligo(dT) sequence for templated addition of a poly-A tail. In some examples, a 5′ UTR may comprise a first polynucleotide fragment and a second polynucleotide fragment from the same and/or different genes such as the 5′ UTRs described in US Patent Application Publication No.20100293625 and PCT/US2014/069155, herein incorporated by reference in its entirety [00548] It should be understood that any UTR from any gene may be incorporated into the regions of an RNA payload molecule (e.g., a linear mRNA). Furthermore, multiple wild- type UTRs of any known gene may be utilized. It is also within the scope of the present disclosure to provide artificial UTRs which are not variants of wild type regions. These UTRs or portions thereof may be placed in the same orientation as in the transcript from which they were selected or may be altered in orientation or location. Hence a 5′ or 3′ UTR may be inverted, shortened, lengthened, made with one or more other 5′ UTRs or 3′ UTRs. As used herein, the term “altered” as it relates to a UTR sequence, means that the UTR has been changed in some way in relation to a reference sequence. For example, a 3′ UTR or 5′ UTR may be altered relative to a wild-type or native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides. Any of these changes producing an “altered” UTR (whether 3′ or 5′) comprise a variant UTR. [00549] In some embodiments, a double, triple or quadruple UTR such as a 5′ UTR or 3′ UTR may be used. As used herein, a “double” UTR is one in which two copies of the same UTR are encoded either in series or substantially in series. In some examples, a double beta- globin 3′ UTR may be used as described in US Patent publication 20100129877, the contents of which are incorporated herein by reference in its entirety. [00550] It is also within the scope of the present disclosure to have patterned UTRs. As used herein “patterned UTRs” are those UTRs which reflect a repeating or alternating pattern, such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than 3 times. In these patterns, each letter, A, B, or C represent a different UTR at the nucleotide level. [00551] In some embodiments, flanking regions are selected from a family of transcripts whose proteins share a common function, structure, feature or property. For example, polypeptides of interest may belong to a family of proteins which are expressed in a particular RNG043-WO1 PCT Application cell, tissue or at some time during development. The UTRs from any of these genes may be swapped for any other UTR of the same or different family of proteins to create a new polynucleotide. As used herein, a “family of proteins” is used in the broadest sense to refer to a group of two or more polypeptides of interest which share at least one function, structure, feature, localization, origin, or expression pattern. [00552] The untranslated region may also include translation enhancer elements (TEE). As a non-limiting example, the TEE may include those described in US Application No. 20090226470, herein incorporated by reference in its entirety, and those known in the art. 5' Capping [00553] In various embodiments, the mRNA payloads of the LNP-based base editing systems, RNA therapeutics and pharmaceutical compositions thereof described herein, may comprise a 5’ cap structure. [00554] The 5' cap structure of an mRNA is involved in nuclear export, increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP), which is responsible for mRNA stability in the cell and translation competency through the association of CBP with poly(A) binding protein to form the mature cyclic mRNA species. The cap further assists the removal of 5' proximal introns removal during mRNA splicing. [00555] Endogenous mRNA molecules may be 5'-end capped generating a 5'-ppp-5'- triphosphate linkage between a terminal guanosine cap residue and the 5'-terminal transcribed sense nucleotide of the mRNA molecule. This 5'-guanylate cap may then be methylated to generate an N7-methyl-guanylate residue. The ribose sugars of the terminal and/or ante- terminal transcribed nucleotides of the 5' end of the mRNA may optionally also be 2'-0- methylated.5'-decapping through hydrolysis and cleavage of the guanylate cap structure may target a nucleic acid molecule, such as an mRNA molecule, for degradation. [00556] Modifications to mRNA may generate a non-hydrolyzable cap structure preventing decapping and thus increasing mRNA half-life. Because cap structure hydrolysis requires cleavage of 5'-ppp-5' phosphorodiester linkages, modified nucleotides may be used during the capping reaction. For example, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, MA) may be used with a-thio-guanosine nucleotides according to the manufacturer's instructions to create a phosphorothioate linkage in the 5'-ppp-5' cap. [00557] Additional modified guanosine nucleotides may be used such as a-methyl- phosphonate and seleno-phosphate nucleotides. [00558] Additional modifications include, but are not limited to, 2'-0-methylation of the ribose sugars of 5 '-terminal and/or 5'-anteterminal nucleotides of the mRNA (as mentioned RNG043-WO1 PCT Application above) on the 2'-hydroxyl group of the sugar ring. Multiple distinct 5 '-cap structures can be used to generate the 5 '-cap of a nucleic acid molecule, such as an mRNA molecule. [00559] Cap analogs, which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e. endogenous, wild-type or physiological) 5'-caps in their chemical structure, while retaining cap function. Cap analogs may be chemically (i.e. non-enzymatically) or enzymatically synthesized and/or linked to a nucleic acid molecule. [00560] For example, the Anti-Reverse Cap Analog (ARCA) cap contains two guanines linked by a 5 '-5 '-triphosphate group, wherein one guanine contains an N7 methyl group as well as a 3'-0-methyl group (i.e., N7,3'-0-dimethyl-guanosine-5'-triphosphate-5 '-guanosine (m7G-3'mppp-G; which may equivalently be designated 3' O-Me-m7G(5')ppp(5')G). The 3'-0 atom of the other, unmodified, guanine becomes linked to the 5'-terminal nucleotide of the capped nucleic acid molecule (e.g. an mRNA). The N7- and 3'-0-methlyated guanine provides the terminal moiety of the capped nucleic acid molecule (e.g. mRNA). [00561] Another exemplary cap is mCAP, which is similar to ARCA but has a 2'-0- methyl group on guanosine (i.e., N7,2'-0-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m7Gm-ppp-G). [00562] While cap analogs allow for the concomitant capping of a nucleic acid molecule in an in vitro transcription reaction, up to 20% of transcripts can remain uncapped. This, as well as the structural differences of a cap analog from an endogenous 5 '-cap structures of nucleic acids produced by the endogenous, cellular transcription machinery, may lead to reduced translational competency and reduced cellular stability. [00563] mRNA may also be capped post-transcriptionally, using enzymes, in order to generate more authentic 5'-cap structures. As used herein, the phrase "more authentic" refers to a feature that closely mirrors or mimics, either structurally or functionally, an endogenous or wild type feature. That is, a "more authentic" feature is better representative of an endogenous, wild-type, natural or physiological cellular function and/or structure as compared to synthetic features or analogs, etc., of the prior art, or which outperforms the corresponding endogenous, wild-type, natural or physiological feature in one or more respects. Non-limiting examples of more authentic 5 'cap structures are those which, among other things, have enhanced binding of cap binding proteins, increased half-life, reduced susceptibility to 5' endonucleases and/or reduced 5'decapping, as compared to synthetic 5 'cap structures known in the art (or to a wild- type, natural or physiological 5 'cap structure). For example, recombinant Vaccinia Virus Capping Enzyme and recombinant 2'-0-methyltransferase enzyme can create a canonical 5 '-5 RNG043-WO1 PCT Application '-triphosphate linkage between the 5 '-terminal nucleotide of an mRNA and a guanine cap nucleotide wherein the cap guanine contains an N7 methylation and the 5 '-terminal nucleotide of the mRNA contains a 2'-0-methyl. Such a structure is termed the Capl structure. This cap results in a higher translational-competency and cellular stability and a reduced activation of cellular pro-inflammatory cytokines, as compared, e.g., to other 5 'cap analog structures known in the art. Cap structures include, but are not limited to, 7mG(5*)ppp(5*)N,pN2p (cap 0), 7mG(5*)ppp(5*)NlmpNp (cap 1), and 7mG(5*)-ppp(5')NlmpN2mp (cap 2). [00564] In some embodiments, the 5' terminal caps may include endogenous caps or cap analogs. [00565] In some embodiments, a 5' terminal cap may comprise a guanine analog. Useful guanine analogs include, but are not limited to, inosine, Nl-methyl-guanosine, 2'fluoro- guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2- azido-guanosine. IRES Sequences [00566] In various embodiments, the mRNA payloads of the LNP-based gene editing systems, RNA therapeutics and pharmaceutical compositions thereof described herein, may comprise one or more IRES sequences. [00567] In some embodiments, the mRNA may contain an internal ribosome entry site (IRES). First identified as a feature Picorna virus RNA, IRES plays an important role in initiating protein synthesis in absence of the 5' cap structure. An IRES may act as the sole ribosome binding site, or may serve as one of multiple ribosome binding sites of an mRNA. An mRNA that contains more than one functional ribosome binding site may encode several peptides or polypeptides that are translated independently by the ribosomes. Non-limiting examples of IRES sequences that can be used include without limitation, those from picornaviruses (e.g. FMDV), pest viruses (CFFV), polio viruses (PV), encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV), murine leukemia virus (MLV), simian immune deficiency viruses (SIV) or cricket paralysis viruses (CrPV). [00568] In some embodiments, the IRES is from Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, Simian Virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, Human poliovirus 1, Plautia stali intestine virus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulata virus-1, Human Immunodeficiency Virus type 1, Homalodisca coagulata virus-1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, Foot and mouth disease virus, Human enterovirus RNG043-WO1 PCT Application 71, Equine rhinitis virus, Ectropis obliqua picorna-like virus, Encephalomyocarditis virus, Drosophila C Virus, Human coxsackievirus B3, Crucifer tobamovirus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black Queen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA1, Human AML1/RUNX1, Drosophila antennapedia, Human AQP4, Human AT1R, Human BAG-1, Human BCL2, Human BiP, Human c-IAP1, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIF1 alpha, Human n.myc, Mouse Gtx, Human p27kip1, Human PDGF2/c-sis, Human p53, Human Pim-1, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae YAP1, tobacco etch virus, turnip crinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, Picobirnavirus, HCV QC64, Human Cosavirus E/D, Human Cosavirus F, Human Cosavirus JMY, Rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, Salivirus A SH1, Salivirus FHB, Salivirus NG-J1, Human Parechovirus 1, Crohivirus B, Yc-3, Rosavirus M-7, Shanbavirus A, Pasivirus A, Pasivirus A 2, Echovirus E14, Human Parechovirus 5, Aichi Virus, Hepatitis A Virus HA16, Phopivirus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J, Human Pegivirus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, Pegivirus A 1220, Pasivirus A 3, Sapelovirus, Rosavirus B, Bakunsa Virus, Tremovirus A, Swine Pasivirus 1, PLV-CHN, Pasivirus A, Sicinivirus, Hepacivirus K, Hepacivirus A, BVDV1, Border Disease Virus, BVDV2, CSFV-PK15C, SF573 Dicistrovirus, Hubei Picorna-like Virus, CRPV, Salivirus A BNS, Salivirus A BN2, Salivirus A 02394, Salivirus A GUT, Salivirus A CH, Salivirus A SZ1, Salivirus FHB, CVB3, CVB1, Echovirus 7, CVBS, EVA71, CVA3, CVA12, EV24 or an aptamer to eIF4G. Poly-A tails and 3’ stabilizing regions [00569] In various embodiments, the mRNA payloads of the LNP-based gene editing systems, RNA therapeutics and pharmaceutical compositions thereof described herein, may comprise a poly-A tail. [00570] During RNA processing, a long chain of adenine nucleotides (poly-A tail) may be added to a polynucleotide such as an mRNA molecules in order to increase stability. Immediately after transcription, the 3' end of the transcript may be cleaved to free a 3' hydroxyl. Then poly-A polymerase adds a chain of adenine nucleotides to the free 3' hydroxyl end. The process, called polyadenylation, adds a poly-A tail of a certain length. [00571] In some embodiments, the length of a poly-A tail is greater than 30 nucleotides in length (SEQ ID NO: 19937). In another embodiment, the poly-A tail is greater than 35 RNG043-WO1 PCT Application nucleotides in length (SEQ ID NO: 19938) (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides) and no more than about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, or 3000 nucleotides in length. In some embodiments, the mRNA includes a poly- A tail from about 30 to about 3,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 750, from 30 to 1,000, from 30 to 1,500, from 30 to 2,000, from 30 to 2,500, from 50 to 100, from 50 to 250, from 50 to 500, from 50 to 750, from 50 to 1 ,000, from 50 to 1,500, from 50 to 2,000, from 50 to 2,500, from 50 to 3,000, from 100 to 500, from 100 to 750, from 100 to 1,000, from 100 to 1,500, from 100 to 2,000, from 100 to 2,500, from 100 to 3,000, from 500 to 750, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 2,500, from 500 to 3,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 2,500, from 1,000 to 3,000, from 1,500 to 2,000, from 1,500 to 2,500, from 1,500 to 3,000, from 2,000 to 3,000, from 2,000 to 2,500, and from 2,500 to 3,000). [00572] In some embodiments, the poly-A tail is designed relative to the length of the overall mRNA. This design may be based on the length of the region coding for a target of interest, the length of a particular feature or region (such as a flanking region), or based on the length of the ultimate product expressed from the mRNA. [00573] In this context the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% greater in length than the mRNA or feature thereof. The poly-A tail may also be designed as a fraction of mRNA to which it belongs. In this context, the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct or the total length of the construct minus the poly-A tail. Further, engineered binding sites and conjugation of mRNA for poly-A binding protein may enhance expression. [00574] Additionally, multiple distinct mRNA may be linked together to the PABP (Poly-A binding protein) through the 3'-end using modified nucleotides at the 3 '-terminus of the poly-A tail. Transfection experiments can be conducted in relevant cell lines at and protein production can be assayed by ELISA at 12hr, 24hr, 48hr, 72 hr and day 7 post-transfection. [00575] In some embodiments, the mRNA are designed to include a polyA-G Quartet. The G-quartet is a cyclic hydrogen bonded array of four guanine nucleotides that can be formed by G-rich sequences in both DNA and RNA. In this embodiment, the G-quartet is incorporated at the end of the poly-A tail. Stop Codons RNG043-WO1 PCT Application [00576] In various embodiments, the mRNA payloads of the LNP-based gene editing systems, RNA therapeutics and pharmaceutical compositions thereof described herein, may comprise one or more translation stop codons. Translational stop codons, UAA, UAG, and UGA, are an important component of the genetic code and signal the termination of translation of an mRNA. During protein synthesis, stop codons interact with protein release factors and this interaction can modulate ribosomal activity thus having an impact translation, for example, as described in Tate WP, et al., (2018) Biochem Soc Trans, 46(6):1615-162. [00577] A stop element as used herein, refers to a nucleic acid sequence comprising a stop codon. The stop codon can be selected from TGA, TAA and TAG in the case of DNA, or from UGA, UAA and UAG in the case of RNA. In an embodiment, a stop element comprises two consecutive stop codons. In an embodiment, a stop element comprises three consecutive stop codons. In an embodiment, a stop element comprises four consecutive stop codons. In an embodiment, a stop element comprises five consecutive stop codons. [00578] In some embodiments, the mRNA may include one stop codon. In some embodiments, the mRNA may include two stop codons. In some embodiments, the mRNA may include three stop codons. In some embodiments, the mRNA may include at least one stop codon. In some embodiments, the mRNA may include at least two stop codons. In some embodiments, the mRNA may include at least three stop codons. As non-limiting examples, the stop codon may be selected from TGA, TAA and TAG. [00579] In other embodiments, the stop codon may be selected from one or more of the following stop elements of Table Z: Table Z: Additional stop elements of linear mRNA Nucleotide sequence (5’ to 3’) Sequence Identifier
Figure imgf000159_0001
RNG043-WO1 PCT Application UAAAGCUCC NA UAGGGUUAA NA
Figure imgf000160_0001
additional stop codon. In a further embodiment the addition stop codon may be TAA. MicroRNA binding sites and other regulatory elements [00581] In various embodiments, the mRNA payloads of the LNP-based gene editing systems, RNA therapeutics and pharmaceutical compositions thereof described herein, may comprise one or more regulatory elements, including, but not limited to microRNA (miRNA) binding sites, structured mRNA sequences and/or motifs, artificial binding sites to bind to endogenous nucleic acid binding molecules, and combinations thereof. Chemically unmodified nucleotides [00582] In some embodiments, the mRNA payloads of the LNP-based gene editing systems, RNA therapeutics and pharmaceutical compositions thereof described herein are not chemically modified and comprises the standard ribonucleotides consisting of adenosine, guanosine, cytosine and uridine. In some embodiments, nucleotides and nucleosides of the present disclosure comprise standard nucleoside residues such as those present in transcribed RNA (e.g. A, G, C, or U). In some embodiments, nucleotides and nucleosides of the present disclosure comprise standard deoxyribonucleosides such as those present in DNA (e.g. dA, dG, dC, or dT). Chemically modified nucleotides [00583] In some embodiments, the mRNA payloads of the LNP-based gene editing systems, RNA therapeutics and pharmaceutical compositions thereof described herein comprise, in some embodiments, comprises at least one chemical modification. [00584] The terms “chemical modification” and “chemically modified” refer to modification with respect to adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C) ribonucleosides or deoxyribnucleosides in at least one of their position, pattern, percent or population. Generally, these terms do not refer to the ribonucleotide modifications in naturally occurring 5′-terminal mRNA cap moieties. With respect to a polypeptide, the term “modification” refers to a modification relative to the canonical set 20 amino acids. RNG043-WO1 PCT Application Polypeptides, as provided herein, are also considered “modified” if they contain amino acid substitutions, insertions or a combination of substitutions and insertions. [00585] Polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides), in some embodiments, comprise various (more than one) different modifications. In some embodiments, a particular region of a polynucleotide contains one, two or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, a modified RNA polynucleotide (e.g., a modified mRNA polynucleotide), introduced to a cell or organism, exhibits reduced degradation in the cell or organism, respectively, relative to an unmodified polynucleotide. In some embodiments, a modified RNA polynucleotide (e.g., a modified mRNA polynucleotide), introduced into a cell or organism, may exhibit reduced immunogenicity in the cell or organism, respectively (e.g., a reduced innate response). [00586] Modifications of polynucleotides include, without limitation, those described herein. Polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) may comprise modifications that are naturally-occurring, non-naturally-occurring or the polynucleotide may comprise a combination of naturally-occurring and non-naturally- occurring modifications. Polynucleotides may include any useful modification, for example, of a sugar, a nucleobase, or an internucleoside linkage (e.g., to a linking phosphate, to a phosphodiester linkage or to the phosphodiester backbone). [00587] Polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides), in some embodiments, comprise non-natural modified nucleotides that are introduced during synthesis or post-synthesis of the polynucleotides to achieve desired functions or properties. The modifications may be present on an internucleotide linkages, purine or pyrimidine bases, or sugars. The modification may be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain. Any of the regions of a polynucleotide may be chemically modified. [00588] The present disclosure provides for modified nucleosides and nucleotides of a polynucleotide (e.g., RNA polynucleotides, such as mRNA polynucleotides). A “nucleoside” refers to a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”). A “nucleotide” refers to a nucleoside, including a phosphate group. Modified nucleotides may by synthesized by any useful method, such as, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides. Polynucleotides may comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages may be RNG043-WO1 PCT Application standard phosphodiester linkages, in which case the polynucleotides would comprise regions of nucleotides. [00589] Modified nucleotide base pairing encompasses not only the standard adenosine- thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and/or modified nucleotides comprising non-standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures. One example of such non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine or uracil. Any combination of base/sugar or linker may be incorporated into polynucleotides of the present disclosure. [00590] In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) include a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases. [00591] In some embodiments, modified nucleobases in polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) are selected from the group consisting of pseudouridine (ψ), N1-methylpseudouridine (m1ψ), N1-ethylpseudouridine, 2-thiouridine, 4′- thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl- pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2- thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1- methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5- methoxyuridine and 2′-O-methyl uridine. In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) include a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases. [00592] In some embodiments, modified nucleobases in polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) are selected from the group consisting of 1- methyl-pseudouridine (m1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine (ψ), α-thio-guanosine and α-thio-adenosine. In some embodiments, polynucleotides includes a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases. [00593] In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise pseudouridine (ψ) and 5-methyl-cytidine (m5C). In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise 1-methyl-pseudouridine (m1ψ). In some embodiments, polynucleotides (e.g., RNA RNG043-WO1 PCT Application polynucleotides, such as mRNA polynucleotides) comprise 1-methyl-pseudouridine (m1ψ) and 5-methyl-cytidine (m5C). In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise 2-thiouridine (s2U). In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise 2- thiouridine and 5-methyl-cytidine (m5C). In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise methoxy-uridine (mo5U). In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise 5-methoxy-uridine (mo5U) and 5-methyl-cytidine (m5C). In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise 2′-O- methyl uridine. In some embodiments polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise 2′-O-methyl uridine and 5-methyl-cytidine (m5C). In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise N6-methyl-adenosine (m6A). In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise N6-methyl-adenosine (m6A) and 5- methyl-cytidine (mC). [00594] In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) are uniformly modified (e.g., fully modified, modified throughout the entire sequence) for a particular modification. For example, a polynucleotide can be uniformly modified with 5-methyl-cytidine (m5C), meaning that all cytosine residues in the mRNA sequence are replaced with 5-methyl-cytidine (m5C). Similarly, a polynucleotide can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue such as those set forth above. [00595] Exemplary nucleobases and nucleosides having a modified cytosine include N4- acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5- hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), and 2- thio-5-methyl-cytidine. [00596] In some embodiments, a modified nucleobase is a modified uridine. Exemplary nucleobases and In some embodiments, a modified nucleobase is a modified cytosine. nucleosides having a modified uridine include 5-cyano uridine, and 4′-thio uridine. [00597] The polynucleotides of the present disclosure may be partially or fully modified along the entire length of the molecule. For example, one or more or all or a given type of nucleotide (e.g., purine or pyrimidine, or any one or more or all of A, G, U, C) may be uniformly modified in a polynucleotide of the present disclosure, or in a given predetermined sequence region thereof (e.g., in the mRNA including or excluding the polyA tail). In some RNG043-WO1 PCT Application embodiments, all nucleotides X in a polynucleotide of the present disclosure (or in a given sequence region thereof) are modified nucleotides, wherein X may any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+CorA+G+C. [00598] The polynucleotide may contain from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e., any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%). It will be understood that any remaining percentage is accounted for by the presence of unmodified A, G, U, or C. [00599] The polynucleotides may contain at a minimum 1% and at maximum 100% modified nucleotides, or any intervening percentage, such as at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides. For example, the polynucleotides may contain a modified pyrimidine such as a modified uracil or cytosine. In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the uracil in the polynucleotide is replaced with a modified uracil (e.g., a 5-substituted uracil). The modified uracil can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures). In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the cytosine in the polynucleotide is replaced with a modified cytosine (e.g., a 5-substituted cytosine). The modified cytosine can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures). 4. Circular mRNA payloads RNG043-WO1 PCT Application [00600] In various embodiments, the LNP-based pharmaceutical compositions described herein, e.g., LNP-based gene editing systems, may include one or more circular mRNA molecules or “oRNAs.” In various embodiments, the circular mRNA payloads may encode one or more components of the herein described gene editing systems or other therapeutic protein of interest. For example, a circular mRNA payload may encode an amino acid sequence-programmable DNA binding domain (e.g., retron RTs, CRISPR nucleases, TALENS and zinc finger-binding domains) or a nucleic acid sequence-programmable DNA binding domain (e.g., CRISPR Cas9, CRISPR Cas12a, CRISPR Cas12f, CRISPR Cas13a, CRISPR Cas13b, or TnpB). [00601] The circular mRNA payloads may also encode, depending upon the nature of the gene editing system, one or more effector domains that provide various functionalities that facilitate changes in nucleotide sequence and/or gene expression, such as, but not limited to, single-strand DNA binding proteins, nucleases, endonucleases, exonucleases, deaminases (e.g., cytidine deaminases or adenosine deaminases), polymerases (e.g., reverse transcriptases), integrases, recombinases, etc., and fusion proteins comprising one or more functional domains linked together. [00602] Circular RNA (oRNA) described herein are polyribonucleotides that form a continuous structure through covalent or non-covalent bonds. Due to the circular structure, oRNAs have improved stability, increased half-life, reduced immunogenicity, and/or improved functionality (e.g., of a function described herein) compared to a corresponding linear RNA. [00603] In some embodiments, an oRNA binds a target. In some embodiments, an oRNA binds a substrate. In some embodiments, an oRNA binds a target and binds a substrate of the target. In some embodiments, an oRNA binds a target and mediates modulation of a substrate of the target. In some embodiments, an oRNA brings together a target and its substrate to mediate modification of the substrate, e.g., post-translational modification. In some embodiments, an oRNA brings together a target and its substrate to mediate a cellular process (e.g., alters protein degradation or signal transduction) involving the substrate. In some embodiments, a target is a target protein and a substrate is a substrate protein. [00604] In some embodiments, an oRNA comprises a conjugation moiety for binding to chemical compound. The conjugation moiety can be a modified polyribonucleotide. The chemical compound can be conjugated to the oRNA by the conjugation moiety. In some embodiments, the chemical compound binds to a target and mediates modulation of a substrate of the target. In some embodiments, an oRNA binds a substrate of a target and a chemical compound conjugated to the oRNA by the conjugation moiety binds the target to bring RNG043-WO1 PCT Application together the target and its substrate to mediate modification of the substrate, e.g., post- translational modification. In some embodiments, an oRNA binds a substrate of a target and a chemical compound conjugated to the oRNA by the conjugation moiety binds the target to bring together the target and its substrate to mediate modification of the substrate to mediate a cellular process (e.g., alters protein degradation or signal transduction) involving the substrate. In some embodiments, a target is a target protein and a substrate is a substrate protein. [00605] In some embodiments, the oRNA may be non-immunogenic in a mammal (e.g., a human, non-human primate, rabbit, rat, and mouse). [00606] In some embodiments, the oRNA may be capable of replicating or replicates in a cell from an aquaculture animal (e.g., fish, crabs, shrimp, oysters etc.), a mammalian cell, a cell from a pet or zoo animal (e.g., cats, dogs, lizards, birds, lions, tigers and bears etc.), a cell from a farm or working animal (e.g., horses, cows, pigs, chickens etc.), a human cell, cultured cells, primary cells or cell lines, stem cells, progenitor cells, differentiated cells, germ cells, cancer cells (e.g., tumorigenic, metastatic), non-tumorigenic cells (e.g., normal cells), fetal cells, embryonic cells, adult cells, mitotic cells, non-mitotic cells, or any combination thereof. [00607] In one aspect, provided herein is a pharmaceutical composition comprising: a circular RNA comprising, in the following order, a 3’ group I intron fragment, an Internal Ribosome Entry Site (IRES), an expression sequence encoding a polypeptide (e.g., a nucleobase editing system or component thereof), and a 5’ group I intron fragment, and a transfer vehicle comprising at least one of (i) an ionizable lipid, (ii) a structural lipid, and (iii) a PEG-modified lipid, wherein the transfer vehicle is capable of delivering the circular RNA polynucleotide to a cell (e.g., a human cell, such as an immune cell present in a human subject), such that the polypeptide is translated in the cell. [00608] In some embodiments, the pharmaceutical composition is formulated for intravenous administration to the human subject in need thereof. In some embodiments, the 3’ group I intron fragment and 5’ group I intron fragment are Anabaena group I intron fragments. [00609] In certain embodiments, the 3’ intron fragment and 5’ intron fragment are defined by the L9a-5 permutation site in the intact intron. In certain embodiments, the 3’ intron fragment and 5’ intron fragment are defined by the L8-2 permutation site in the intact intron. [00610] In some embodiments, the IRES is from Taura syndrome virus, Tiiatoma virus, Theiler's encephalomyelitis virus, Simian Virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, Human poliovirus 1, Plautia stall intestine virus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulata virus- 1, Human Immunodeficiency Virus type 1, Homalodisca coagulata virus- 1, Himetobi P virus, Hepatitis RNG043-WO1 PCT Application C virus, Hepatitis A virus, Hepatitis GB virus , Foot and mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Ectropis obliqua picoma-like virus, Encephalomyocarditis virus, Drosophila C Virus, Human coxsackievirus B3, Crucifer tobamovirus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black Queen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA1, Human AML1/RUNX1, Drosophila antennapedia, Human AQP4, Human AT1R, Human BAG-1, Human BCL2, Human BiP, Human c-IAPl, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIFl alpha, Human n.myc, Mouse Gtx, Human p27kipl, Human PDGF2/c-sis, Human p53, Human Pim-1, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae YAP1, tobacco etch virus, turnip crinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, Picobirnavirus, HCV QC64, Human Cosavirus E/D, Human Cosavirus F, Human Cosavirus JMY, Rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, Salivirus A SHI, Salivirus FHB, Salivirus NG-J1, Human Parechovirus 1, Crohivirus B, Yc-3, Rosavirus M-7, Shanbavirus A, Pasivirus A, Pasivirus A 2, Echovirus E14, Human Parechovirus 5, Aichi Virus, Hepatitis A Virus HA 16, Phopivirus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J, Human Pegivirus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, Pegivirus A 1220, Pasivirus A 3, Sapelovirus, Rosavirus B, Bakunsa Virus, Tremovirus A, Swine Pasivirus 1, PLV-CHN, Pasivirus A, Sicinivirus, Hepacivirus K, Hepacivirus A, BVDV1, Border Disease Virus, BVDV2, CSFV- PK15C, SF573 Dicistravirus, Hubei Picoma-like Virus, CRPV, Salivirus A BN5, Salivirus A BN2, Salivirus A 02394, Salivirus A GUT, Salivirus A CH, Salivirus A SZ1, Salivirus FHB, CVB3, CVB1, Echovirus 7, CVB5, EVA71, CVA3, CVA12, EV24 or an aptamer to eIF4G. [00611] In some embodiments, the IRES comprises a CVB3 IRES or a fragment or variant thereof. In some embodiments, the pharmaceutical composition comprises a first internal spacer between the 3’ group I intron fragment and the IRES, and a second internal spacer between the expression sequence and the 5’ group I intron fragment. In certain embodiments, the first and second internal spacers each have a length of about 10 to about 60 nucleotides. [00612] In some embodiments, the circular mRNA comprises a nucleotide sequence encoding a polypeptide of interest, such as a nucleobase editing system or therapeutic protein (e.g., a CAR or TCR complex protein). RNG043-WO1 PCT Application [00613] In some embodiments, the LNP-based nucleobase editing systems, RNA therapeutics, and pharmaceutical compositions described herein further comprise a targeting moiety. In certain embodiments, the targeting moiety mediates receptor-mediated endocytosis or direct fusion of the delivery vehicle (LNPs) into selected cells of a selected cell population or tissue in the absence of cell isolation or purification. In certain embodiments, the targeting moiety is capable of binding to a protein selected from the group CD3, CD4, CD8, CDS, CD7, PD-1, 4-1BB, CD28, Clq, and CD2. In certain embodiments, the targeting moiety comprises an antibody specific for a macrophage, dendritic cell, NK cell, NKT, or T cell antigen. In certain embodiments, the targeting moiety comprises a scFv, nanobody, peptide, minibody, polynucleotide aptamer, heavy chain variable region, light chain variable region or fragment thereof. [00614] In some embodiments, the LNP-based nucleobase editing systems, RNA therapeutics, and pharmaceutical compositions described herein are administered in an amount effective to treat a disease in the human subject (e.g., wherein the disease can be cancer, muscle disorder, or CNS disorder, etc.). In some embodiments, the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions have an enhanced safety profile when compared to a pharmaceutical composition comprising T cells or vectors comprising exogenous DNA encoding the same polypeptide. [00615] In some embodiments, the LNP-based nucleobase editing systems and pharmaceutical compositions thereof are administered in an amount effective to induce a desire precise edit in a genome. In some embodiments, the LNP-based nucleobase editing systems and pharmaceutical compositions have an enhanced safety profile when compared to state of the art gene editing delivery compositions. [00616] In another aspect, the present disclosure provides a circular RNA comprising, in the following order, a 3’ group I intron fragment, an Internal Ribosome Entry Site (IRES), an expression sequence encoding a polypeptide (e.g., a nucleobase editing system or component thereof), and a 5’ group I intron fragment. [00617] In some embodiments, the 3’ group I intron fragment and 5’ group I intron fragment are Anabaena group I intron fragments. In certain embodiments, the 3’ intron fragment and 5’ intron fragment are defined by the L9a-5 permutation site in the intact intron. In certain embodiments, the 3’ intron fragment and 5’ intron fragment are defined by the L8-2 permutation site in the intact intron. In certain embodiments, the IRES comprises a CVB3 IRES or a fragment or variant thereof. RNG043-WO1 PCT Application [00618] In some embodiments, the circular RNA comprises a first internal spacer between the 3’ group I intron fragment and the IRES, and a second internal spacer between the expression sequence and the 5’ group I intron fragment. [00619] In certain embodiments, the first and second internal spacers each have a length of about 10 to about 60 nucleotides. [00620] In some embodiments, the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics, and pharmaceutical compositions described herein comprises of natural nucleotides. In some embodiments, the circular RNA further comprises a second expression sequence encoding a therapeutic protein. In some embodiments, the therapeutic protein comprises a checkpoint inhibitor. In certain embodiments, the therapeutic protein comprises a cytokine. [00621] In some embodiments, the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics, and pharmaceutical compositions described herein consists of natural nucleotides. [00622] In some embodiments, the circular RNA payload LNP-based nucleobase editing systems, RNA therapeutics, and pharmaceutical compositions described herein comprises a nucleotide sequence that is codon optimized, either partially or fully. In some embodiments, the circular RNA is optimized to lack at least one microRNA binding site present in an equivalent pre-optimized polynucleotide. In some embodiments, the circular RNA is optimized to lack at least one endonuclease susceptible site present in an equivalent pre-optimized polynucleotide. In some embodiments, the circular RNA is optimized to lack at least one RNA-editing susceptible site present in an equivalent pre-optimized polynucleotide. [00623] In some embodiments, the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics, and pharmaceutical compositions described herein has an in vivo functional half- life in humans greater than that of an equivalent linear RNA having the same expression sequence. In some embodiments, the circular RNA has a length of about 100 nucleotides to about 10 kilobases. In some embodiments, the circular RNA has a functional half-life of at least about 20 hours. In some embodiments, the circular RNA has a duration of therapeutic effect in a human cell of at least about 20 hours. In some embodiments, the circular RNA has a duration of therapeutic effect in a human cell greater than or equal to that of an equivalent linear RNA comprising the same expression sequence. In some embodiments, the circular RNA has a functional half-life in a human cell greater than or equal to that of an equivalent linear RNA comprising the same expression sequence. RNG043-WO1 PCT Application [00624] In some embodiments, the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics, and pharmaceutical compositions described herein has a half-life of at least that of a linear counterpart. In some embodiments, the oRNA has a half-life that is increased over that of a linear counterpart. In some embodiments, the half-life is increased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or greater. In some embodiments, the oRNA has a half-life or persistence in a cell for at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours (1 day), 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or longer or any time therebetween. In some embodiments, the oRNA has a half-life or persistence in a cell for no more than about 10 mins to about 7 days, or no more than about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 24 hours (1 day), 36 hours (1.5 days), 48 hours (2 days),60 hours (2.5 days), 72 hours (3 days), 4 days, 5 days, 6 days, or 7 days. [00625] In some embodiments, the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics, and pharmaceutical compositions described herein has a half-life or persistence in a cell while the cell is dividing. In some embodiments, the oRNA has a half-life or persistence in a cell post division. [00626] In certain embodiments, the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics, and pharmaceutical compositions described herein has a half-life or persistence in a dividing cell for greater than about 10 minutes to about 30 days, or at least about 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 24 hours (1 day), 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or longer or any time therebetween. [00627] In some embodiments, the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics, and pharmaceutical compositions described herein modulates a cellular function, e.g., transiently or long term. In certain embodiments, the cellular function is stably altered, such as a modulation that persists for at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours (1 day), 2 days, RNG043-WO1 PCT Application 3, days, 4days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or longer. In certain embodiments, the cellular function is transiently altered, e.g., such as a modulation that persists for no more than about 30 mins to about 7 days, or no more than about 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours (1 day), 36 hours (1.5 days), 48 hours (2 days), 60 hours (2.5 days), 72 hours(3 days), 4 days, 5 days, 6 days, or 7 days. [00628] In some embodiments, the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions described herein is at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 1,000 nucleotides, at least about 2,000 nucleotides, at least about 5,000 nucleotides, at least about 6,000 nucleotides, at least about 7,000 nucleotides, at least about 8,000 nucleotides, at least about 9,000 nucleotides, at least about 10,000 nucleotides, at least about 12,000 nucleotides, at least about 14,000 nucleotides, at least about 15,000 nucleotides, at least about 16,000 nucleotides, at least about 17,000 nucleotides, at least about 18,000 nucleotides, at least about 19,000 nucleotides, or at least about 20,000 nucleotides. In some embodiments, the oRNA may be of a sufficient size to accommodate a binding site for a ribosome. [00629] In some embodiments, the maximum size of the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics, and pharmaceutical compositions described herein may be limited by the ability of packaging and delivering the RNA to a target. In some embodiments, the size of the oRNA is a length sufficient to encode polypeptides, and thus, lengths of at least 20,000 nucleotides, at least 15,000 nucleotides, at least 10,000 nucleotides, at least 7,500 nucleotides, or at least 5,000 nucleotides, at least 4,000 nucleotides, at least 3,000 nucleotides, at least 2,000 nucleotides, at least 1,000 nucleotides, at least 500 nucleotides, at least 400 nucleotides, at least 300 nucleotides, at least 200 nucleotides, at least 100 nucleotides may be useful. [00630] In some embodiments, the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics, and pharmaceutical compositions described herein comprises one or more elements described elsewhere herein. In some embodiments, the RNG043-WO1 PCT Application elements may be separated from one another by a spacer sequence or linker. In some embodiments, the elements may be separated from one another by 1 nucleotide, 2 nucleotides, about 5 nucleotides, about 10 nucleotides, about 15 nucleotides, about 20 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides, about 80 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 400 nucleotides, about 500 nucleotides, about 600 nucleotides, about 700 nucleotides, about 800 nucleotides, about 900 nucleotides, about 1000 nucleotides, up to about 1 kb, at least about 1000 nucleotides. [00631] In some embodiments, one or more elements are contiguous with one another, e.g., lacking a spacer element. [00632] In some embodiments, one or more elements is conformationally flexible. In some embodiments, the conformational flexibility is due to the sequence being substantially free of a secondary structure. [00633] In some embodiments, the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions described herein comprises a secondary or tertiary structure that accommodates a binding site for a ribosome, translation, or rolling circle translation. [00634] In some embodiments, the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions described herein comprises particular sequence characteristics. For example, the oRNA may comprise a particular nucleotide composition. In some such embodiments, the oRNA may include one or more purine rich regions (adenine or guanosine). In some such embodiments, the oRNA may include one or more purine rich regions (adenine or guanosine).In some embodiments, the oRNA may include one or more AU rich regions or elements (AREs). In some embodiments, the oRNA may include one or more adenine rich regions. [00635] In some embodiments, the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics, and pharmaceutical compositions described herein comprises one or more modifications described elsewhere herein. [00636] In some embodiments, the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics, and pharmaceutical compositions described herein comprises one or more expression sequences and is configured for persistent expression in a cell of a subject in vivo. In some embodiments, the oRNA is configured such that expression of the one or more expression sequences in the cell at a later time point is equal to or higher than an earlier time point. In such embodiments, the expression of the one or more expression RNG043-WO1 PCT Application sequences can be either maintained at a relatively stable level or can increase over time. The expression of the expression sequences can be relatively stable for an extended period of time. For instance, in some cases, the expression of the one or more expression sequences in the cell over a time period of at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23 or more days does not decrease by 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%. In some cases, in some cases, the expression of the one or more expression sequences in the cell is maintained at a level that does not vary by more than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% for at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23 or more days. Regulatory Elements [00637] In some embodiments, the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions described herein comprises one or more regulatory elements. As used herein, a "regulatory element" is a sequence that modifies expression of an expression sequence, e.g., a nucleotide sequence encoding a nucleobase editing system or a therapeutic protein, i.e., a coding region of interest (CROI). The regulatory element may include a sequence that is located adjacent to a coding region of interest encoded on the circular RNA payload. The regulatory element may be operatively linked to a nucleotide sequence of the circular RNA that encodes a coding region of interest (e.g., a nucleobase editing system or therapeutic polypeptide). [00638] In some embodiments, a regulatory element may increase an amount of expression of a coding region of interest encoded on the circular RNA payload as compared to an amount expressed when no regulatory element exists. [00639] In some embodiments, a regulatory element may comprise a sequence to selectively initiates or activates translation of a coding sequence of interest encoded on the circular RNA payload. [00640] In some embodiments, a regulatory element may comprise a sequence to initiate degradation of the oRNA or the payload or cargo. Non-limiting examples of the sequence to initiate degradation includes, but is not limited to, riboswitch aptazyme and miRNA binding sites. [00641] In some embodiments, a regulatory element can modulate translation of a coding region of interest encoded on the oRNA. The modulation can create an increase (enhancer) or decrease (suppressor) in the expression of the coding region of interest. The regulatory element may be located adjacent to the CROI (e.g., on one side or both sides of the CROI). Translation Initiation Sequence RNG043-WO1 PCT Application [00642] In some embodiments, a translation initiation sequence functions as a regulatory element. In some embodiments, the translation initiation sequence comprises an AUG/ATG codon. In some embodiments, a translation initiation sequence comprises any eukaryotic start codon such as, but not limited to, AUG/ATG, CUG/CTG, GUG/GTG, UUG/TTG, ACG, AUC/ATC, AUU, AAG, AUA/ATA, or AGG. In some embodiments, a translation initiation sequence comprises a Kozak sequence. In some embodiments, translation begins at an alternative translation initiation sequence, e.g., translation initiation sequence other than AUG/ATG codon, under selective conditions, e.g., stress induced conditions. As a non-limiting example, the translation of the circular polyribonucleotide may begin at alternative translation initiation sequence, such as ACG. As another non-limiting example, the circular polyribonucleotide translation may begin at alternative translation initiation sequence, CUG/CTG. As another non-limiting example, the translation may begin at alternative translation initiation sequence, GUG/GTG. As yet another non-limiting example, the translation may begin at a repeat-associated non-AUG (RAN) sequence such as an alternative translation initiation sequence that includes short stretches of repetitive RNA e.g. CGG, GGGGCC, CAG, or CTG. [00643] In some embodiments, the oRNA encodes a polypeptide or peptide and may comprise a translation initiation sequence. The translation initiation sequence may comprise, but is not limited to a start codon, a non-coding start codon, a Kozak sequence or a Shine- Dalgarno sequence. The translation initiation sequence may be located adjacent to the payload or cargo (e.g., on one side or both sides of the coding region of interest). [00644] In some embodiments, the translation initiation sequence provides conformational flexibility to the oRNA. In some embodiments, the translation initiation sequence is within a substantially single stranded region of the oRNA. [00645] The oRNA may include more than 1 start codon such as, but not limited to, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15 or more than 15 start codons. Translation may initiate on the first start codon or may initiate downstream of the first start codon. [00646] In some embodiments, the oRNA may initiate at a codon which is not the first start codon, e.g., AUG. Translation of the circular polyribonucleotide may initiate at an alternative translation initiation sequence, such as, but not limited to, ACG, AGG, AAG, CUG/CTG, GUG/GTG, AUA/ATA, AUU/ATT, UUG/TTG. In some embodiments, translation begins at an alternative translation initiation sequence under selective conditions, e.g., stress induced conditions. As a non-limiting example, the translation of the oRNA may RNG043-WO1 PCT Application begin at alternative translation initiation sequence, such as ACG. As another non-limiting example, the oRNA translation may begin at alternative translation initiation sequence, CUG/CTG. As yet another non-limiting example, the oRNA translation may begin at alternative translation initiation sequence, GTG/GUG. As yet another non-limiting example, the oRNA may begin translation at a repeat-associated non-AUG (RAN) sequence, such as an alternative translation initiation sequence that includes short stretches of repetitive RNA e.g. CGG, GGGGCC, CAG, CTG. IRES Sequences [00647] In some embodiments, the oRNA described herein comprises an internal ribosome entry site (IRES) element capable of engaging an eukaryotic ribosome. In some embodiments, the IRES element is at least about 5 nucleotides, at least about 8 nucleotides, at least about 9 nucleotides, at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 250 nucleotides, at least about 350 nucleotides, or at least about 500 nucleotides. In one embodiment, the IRES element is derived from the DNA of an organism including, but not limited to, a virus, a mammal, and a Drosophila. Such viral DNA may be derived from, but is not limited to, picornavirus complementary DNA (cDNA), with encephalomyocarditis virus (EMCV) cDNA and poliovirus cDNA. In one embodiment, Drosophila DNA from which an IRES element is derived includes, but is not limited to, an Antennapedia gene from Drosophila melanogaster. [00648] In some embodiments, the IRES element is at least partially derived from a virus, for instance, it can be derived from a viral IRES element, such as ABPV_IGRpred, AEV, ALPV_IGRpred, BQCV_IGRpred, BVDV1_1-385, BVDV1_29-391, CrPV_5NCR, CrPV_IGR, crTMV_IREScp, crTMV_IRESmp75, crTMV_IRESmp228, crTMV_IREScp, crTMV_IREScp, CSFV, CVB3, DCV_IGR, EMCV-R, EoPV_5NTR, ERAV 245-961, ERBV 162-920, EV71_1-748, FeLV-Notch2, FMDV_type_C, GBV-A, GBV-B, GBV-C, gypsy_env, gypsyD5, gypsyD2, HAV_HM175, HCV_type_1a, HiPV_IGRpred, HIV-1, HoCV1_IGRpred, HRV-2, IAPV_IGRpred, idefix, KBV_IGRpred, LINE-1_ORF1_-101_to_-1, LINE-1_ORF1- 302_to_-202, LINE-1_ORF2-138_to_-86, LINE-1_ORF1_-44to_-1, PSIV_IGR, PV_type1_Mahoney,PV_type3_Leon, REV-A, RhPV_5NCR, RhPV_IGR, SINV1_IGRpred, SV40_661-830, TMEV, TMV_UI_IRESmp228, TRV_5NTR, TrV_IGR, or TSV_IGR. In some embodiments, the IRES element is at least partially derived from a cellular IRES, such as AML1/RUNX1, Antp-D, Antp-DE, Antp-CDE, Apaf-1, Apaf-1, AQP4, AT1R_var1, RNG043-WO1 PCT Application AT1R_var2, AT1R_var3, AT1R_var4, BAG1_p36delta236 nt, BAG1_p36, BCL2, BiP_-222_- 3, c-IAP1_285-1399, c-IAP1_1313-1462, c-jun, c-myc, Cat-1224, CCND1, DAPS, eIF4G, eIF4GI-ext, eIF4GII, eIF4GII-long, ELG1, ELH, FGF1A,FMR1, Gtx-133-141, Gtx-1-166, Gtx-1-120, Gtx-1-196, hairless, HAP4, HIF1a, hSNM1, Hsp101, hsp70, hsp70, Hsp90, IGF2_leader2, Kv1.4_1.2, L-myc, LamB1_-335_-1, LEF1, MNT_75-267, MNT_36-160, MTG8a, MYB, MYT2_997-1152, n-MYC, NDST1, NDST2, NDST3, NDST4L, NDST4S, NRF_-653_-17, NtHSF1, ODC1, p27kip1, 03_128-269, PDGF2/c-sis, Pim-1, PITSLRE_p58, Rbm3, reaper, Scamper, TFIID, TIF4631, Ubx_1-966, Ubx_373-961, UNR, Ure2, UtrA, VEGF-A-133-1, XIAP_5-464, XIAP_305-466, or YAP1. [00649] In another embodiment, the IRES is an IRES sequence from Coxsackievirus B3 (CVB3), the protein coding region encodes Guassia luciferase (Gluc), and the spacer sequences are polyA-C. [00650] In some embodiments, the IRES, if present, is at least about 50 nucleotides in length. In one embodiment, the vector comprises an IRES that comprises a natural sequence. In one embodiment, the vector comprises an IRES that comprises a synthetic sequence. [00651] An IRES may act as the sole ribosome binding site, or may serve as one of multiple ribosome binding sites of an mRNA. A polynucleotide containing more than one functional ribosome binding site may encode several peptides or polypeptides that are translated independently by the ribosomes (e.g., multicistronic mRNA). When polynucleotides are provided with an IRES, further optionally provided is a second translatable region. Examples of IRES sequences that can be used according to the present disclosure include without limitation, those from picornaviruses (e.g., FMDV), pest viruses (CFFV), polio viruses (PV), encephalomyocarditis viruses (ECMV), foot-and mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical Swine fever viruses (CSFV), murine leukemia virus (MLV), simian immune deficiency viruses (SIV) or cricket paralysis viruses (CrPV). Termination Element [00652] In some embodiments, the oRNA includes one or more coding regions of interest (i.e., also referred to as product expression sequences) which encode polypeptides of interest, including but not limited to nucleobase editing system and therapeutic proteins. In various embodiments, the product expression sequences may or may not have a termination element. [00653] In some embodiments, the oRNA includes one or more product expression sequences that lack a termination element, such that the oRNA is continuously translated. RNG043-WO1 PCT Application [00654] Exclusion of a termination element may result in rolling circle translation or continuous expression of the encoded peptides or polypeptides as the ribosome will not stall or fall-off. In such an embodiment, rolling circle translation expresses continuously through the product expression sequence. [00655] In some embodiments, one or more product expression sequences in the oRNA comprise a termination element. [00656] In some embodiments, not all of the product expression sequences in the oRNA comprise a termination element. In such instances, the product expression sequence may fall off the ribosome when the ribosome encounters the termination element and terminates translation. Rolling Circle Translation [00657] In some embodiments, once translation of the oRNA is initiated, the ribosome bound to the oRNA does not disengage from the oRNA before finishing at least one round of translation of the oRNA. In some embodiments, the oRNA as described herein is competent for rolling circle translation. In some embodiments, during rolling circle translation, once translation of the oRNA is initiated, the ribosome bound to the oRNA does not disengage from the oRNA before finishing at least 2 rounds, at least 3 rounds, at least 4 rounds, at least 5 rounds, at least 6 rounds, at least 7 rounds, at least 8 rounds,at least 9 rounds, at least 10 rounds, at least 11 rounds, at least 12 rounds, at least 13 rounds, at least 14 rounds, at least 15 rounds, at least 20 rounds, at least 30 rounds, at least 40 rounds, at least 50 rounds, at least 60 rounds, at least 70 rounds, at least 80 rounds, at least 90 rounds, at least 100 rounds, at least 150 rounds, at least 200 rounds, at least 250 rounds, at least 500 rounds, at least 1000 rounds, at least 1500 rounds, at least 2000 rounds, at least 5000 rounds, at least 10000 rounds, at least 105 rounds, or at least 106 rounds of translation of the oRNA. [00658] In some embodiments, the rolling circle translation of the oRNA leads to generation of polypeptide that is translated from more than one round of translation of the oRNA. In some embodiments, the oRNA comprises a stagger element, and rolling circle translation of the oRNA leads to generation of polypeptide product that is generated from a single round of translation or less than a single round of translation of the oRNA. Circularization [00659] In one embodiment, a linear RNA may be cyclized, or concatemerized. In some embodiments, the linear RNA may be cyclized in vitro prior to formulation and/or delivery. In some embodiments, the linear RNA may be cyclized within a cell. RNG043-WO1 PCT Application [00660] In some embodiments, the mechanism of cyclization or concatemerization may occur through at least 3 different routes: 1) chemical, 2) enzymatic, and 3) ribozyme catalyzed. The newly formed 5'-/3'-linkage may be intramolecular or intermolecular. [00661] In the first route, the 5'-end and the 3 '-end of the nucleic acid contain chemically reactive groups that, when close together, form a new covalent linkage between the 5 '-end and the 3 '-end of the molecule. The 5 '-end may contain an NHS-ester reactive group and the 3 '-end may contain a 3'-amino-terminated nucleotide such that in an organic solvent the 3'-amino-terminated nucleotide on the 3 '-end of a synthetic mRNA molecule will undergo a nucleophilic attack on the 5 '-NHS-ester moiety forming a new 5 '-/3 '-amide bond. [00662] In the second route, T4 RNA ligase may be used to enzymatically link a 5'- phosphorylated nucleic acid molecule to the 3'-hydroxyl group of a nucleic acid forming a new phosphorodiester linkage. In an example reaction, ^g of a nucleic acid molecule is incubated at 37°C for 1 hour with 1-10 units of T4 RNA ligase (New England Biolabs, Ipswich, MA) according to the manufacturer's protocol. The ligation reaction may occur in the presence of a split oligonucleotide capable of base-pairing with both the 5'-and 3'-region in juxtaposition to assist the enzymatic ligation reaction. [00663] In the third route, either the 5 '-or 3 '-end of the cDNA template encodes a ligase ribozyme sequence such that during in vitro transcription, the resultant nucleic acid molecule can contain an active ribozyme sequence capable of ligating the 5 '-end of a nucleic acid molecule to the 3 '-end of a nucleic acid molecule. The ligase ribozyme may be derived from the Group I Intron, Group I Intron, Hepatitis Delta Virus, Hairpin ribozyme or may be selected by SELEX (systematic evolution of ligands by exponential enrichment). The ribozyme ligase reaction may take 1 to 24 hours at temperatures between 0 and 37°C. [00664] In some embodiments, the oRNA is made via circularization of a linear RNA. [00665] In some embodiments, the following elements are operably connected to each other and, in some embodiments, arranged in the following sequence: a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′ splice site dinucleotide, c.) a protein coding or noncoding region, d.) a 5′ group I intron fragment containing a 5′ splice site dinucleotide, and e.) a 3′ homology arm. In certain embodiments, the vector allows production of a circular RNA that is translatable and/or biologically active inside eukaryotic cells. In some embodiments, the biologically active RNA is, for example, an miRNA sponge, or long noncoding RNA. [00666] In some embodiments, the following elements are operably connected to each other and arranged in the following sequence: a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′ splice site dinucleotide, c.) optionally, a 5′ spacer sequence, d.) RNG043-WO1 PCT Application optionally, an internal ribosome entry site (IRES), e.) a protein coding or noncoding region, f.) optionally, a 3′ spacer sequence, g.) a 5′ group I intron fragment containing a 5′ splice site dinucleotide, and h.) a 3′ homology arm. In certain embodiments, the vector allows production of a circular RNA that is translatable and/or biologically active inside eukaryotic cells. [00667] In some embodiments, the following elements are operably connected to each other and arranged in the following sequence: a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′ splice site dinucleotide, c.) a 5′ spacer sequence, d.) an internal ribosome entry site (IRES), e.) a protein coding or noncoding region, f.) a 5′ group I intron fragment containing a 5′ splice site dinucleotide, and g.) a 3′ homology arm. In some embodiments, the vector allows production of a circular RNA that is translatable and/or biologically active inside eukaryotic cells. [00668] In some embodiments, the following elements are operably connected to each other and arranged in the following sequence: a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′ splice site dinucleotide, c.) a 5′ spacer sequence, d.) a protein coding or noncoding region, e.) a 3′ spacer sequence, f.) a 5′ group I intron fragment containing a 5′ splice site dinucleotide, and g.) a 3′ homology arm. In some embodiments, the vector allows production of a circular RNA that is translatable and/or biologically active inside eukaryotic cells. [00669] In some embodiments, the following elements are operably connected to each other and arranged in the following sequence: a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′ splice site dinucleotide, c.) an internal ribosome entry site (IRES), d.) a protein coding or noncoding region, e.) a 3′ spacer sequence, f) a 5′ group I intron fragment containing a 5′ splice site dinucleotide, and g.) a 3′ homology arm. In some embodiments, the vector allows production of a circular RNA that is translatable and/or biologically active inside eukaryotic cells. [00670] In some embodiments, the following elements are operably connected to each other and arranged in the following sequence: a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′ splice site dinucleotide, c.) a protein coding or noncoding region, d.) a 3′ spacer sequence, e.) a 5′ group I intron fragment containing a 5′ splice site dinucleotide, and f.) a 3′ homology arm. In some embodiments, the vector allows production of a circular RNA that is translatable and/or biologically active inside eukaryotic cells. [00671] In some embodiments, the following elements are operably connected to each other and arranged in the following sequence: a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′ splice site dinucleotide, c.) a 5′ spacer sequence, d.) a protein coding RNG043-WO1 PCT Application or noncoding region, e.) a 5′ group I intron fragment containing a 5′ splice site dinucleotide, and f.) a 3′ homology arm. In some embodiments, the vector allows production of a circular RNA that is translatable and/or biologically active inside eukaryotic cells. [00672] In some embodiments, the following elements are operably connected to each other and arranged in the following sequence: a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′ splice site dinucleotide, c.) an internal ribosome entry site (IRES), d.) a protein coding or noncoding region, e.) a 5′ group I intron fragment containing a 5′ splice site dinucleotide, and f) a 3′ homology arm. In some embodiments, the vector allows production of a circular RNA that is translatable and/or biologically active inside eukaryotic cells. [00673] In some embodiments, the following elements are operably connected to each other and arranged in the following sequence: a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′ splice site dinucleotide, c.) a 5′ spacer sequence, d.) an internal ribosome entry site (IRES), e.) a protein coding or noncoding region, f) a 3′ spacer sequence, g.) a 5′ group I intron fragment containing a 5′ splice site dinucleotide, and h.) a 3′ homology arm. In some embodiments, the vector allowing production of a circular RNA that is translatable and/or biologically active inside eukaryotic cells. [00674] In one embodiment, the 3′ group I intron fragment and/or the 5′ group I intron fragment is from a Cyanobacterium Anabaena sp. pre-tRNA-Leu gene or T4 phage Td gene. [00675] In one embodiment, the 3′ group I intron fragment and/or the 5′ group I intron fragment is from a Cyanobacterium Anabaena sp. pre-tRNA-Leu gene. [00676] In one embodiment, the protein coding region encodes a protein of eukaryotic or prokaryotic origin. In another embodiment, the protein coding region encodes human protein or non-human protein. In some embodiments, the protein coding region encodes one or more antibodies. For example, in some embodiments, the protein coding region encodes human antibodies. In one embodiment, the protein coding region encodes a protein selected from hFIX, SP-B, VEGF-A, human methylmalonyl-CoA mutase (hMUT), CFTR, cancer self- antigens, and additional gene editing enzymes like Cpf1, zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). In another embodiment, the protein coding region encodes a protein for therapeutic use. In one embodiment, the human antibody encoded by the protein coding region is an anti-HIV antibody. In one embodiment, the antibody encoded by the protein coding region is a bispecific antibody. In one embodiment, the bispecific antibody is specific for CD19 and CD22. In another embodiment, the bispecific antibody is specific for CD3 and CLDN6. In one embodiment, the protein coding region encodes a protein for diagnostic use. In one embodiment, the protein coding region RNG043-WO1 PCT Application encodes Gaussia luciferase (Gluc), Firefly luciferase (Fluc), enhanced green fluorescent protein (eGFP), human erythropoietin (hEPO), or Cas9 endonuclease. [00677] In one embodiment, the 5′ homology arm is about 5-50 nucleotides in length. In another embodiment, the 5′ homology arm is about 9-19 nucleotides in length. In some embodiments, the 5′ homology arm is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the 5′ homology arm is no more than 50, 45, 40, 35, 30, 25 or 20 nucleotides in length. In some embodiments, the 5′ homology arm is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 nucleotides in length. [00678] In one embodiment, the 3′ homology arm is about 5-50 nucleotides in length. In another embodiment, the 3′ homology arm is about 9-19 nucleotides in length. In some embodiments, the 3′ homology arm is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the 3′ homology arm is no more than 50, 45, 40, 35, 30, 25 or 20 nucleotides in length. In some embodiments, the 3′ homology arm is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 nucleotides in length. [00679] In one embodiment, the 5′ spacer sequence is at least 10 nucleotides in length. In another embodiment, the 5′ spacer sequence is at least 15 nucleotides in length. In a further embodiment, the 5′ spacer sequence is at least 30 nucleotides in length. In some embodiments, the 5′ spacer sequence is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or 30 nucleotides in length. In some embodiments, the 5′ spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30 nucleotides in length. In some embodiments the 5′ spacer sequence is between 20 and 50 nucleotides in length. In certain embodiments, the 5′ spacer sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length. In one embodiment, the 5′ spacer sequence is a polyA sequence. In another embodiment, the 5′ spacer sequence is a polyA-C sequence. [00680] In one embodiment, the 3′ spacer sequence is at least 10 nucleotides in length. In another embodiment, the 3′ spacer sequence is at least 15 nucleotides in length. In a further embodiment, the 3′ spacer sequence is at least 30 nucleotides in length. In some embodiments, the 3′ spacer sequence is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or 30 nucleotides in length. In some embodiments, the 3′ spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30 nucleotides in length. In some embodiments the 3′ spacer sequence is between 20 and 50 nucleotides in length. In certain embodiments, the 3′ spacer sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length. RNG043-WO1 PCT Application In one embodiment, the 3′ spacer sequence is a polyA sequence. In another embodiment, the 5′ spacer sequence is a polyA-C sequence. Extracellular Circularization [00681] In some embodiments, the linear RNA is cyclized, or concatemerized using a chemical method to form an oRNA. In some chemical methods, the 5'-end and the 3'-end of the nucleic acid (e.g., a linear RNA) includes chemically reactive groups that, when close together, may form a new covalent linkage between the 5'-end and the 3'-end of the molecule. The 5'- end may contain an NHS-ester reactive group and the 3'-end may contain a 3'-amino- terminated nucleotide such that in an organic solvent the 3'-amino-terminated nucleotide on the 3'-end of a linear RNA will undergo a nucleophilic attack on the 5'-NHS-ester moiety forming a new 5'-/3'-amide bond. [00682] In one embodiment, a DNA or RNA ligase may be used to enzymatically link a 5'-phosphorylated nucleic acid molecule (e.g., a linear RNA) to the 3'-hydroxyl group of a nucleic acid (e.g., a linear nucleic acid) forming a new phosphorodiester linkage. In an example reaction, a linear RNA is incubated at 37°C for 1 hour with 1-10 units of T4 RNA ligase according to the manufacturer's protocol. The ligation reaction may occur in the presence of a linear nucleic acid capable of base-pairing with both the 5'-and 3'-region in juxtaposition to assist the enzymatic ligation reaction. In one embodiment, the ligation is splint ligation where a single stranded polynucleotide (splint), like a single stranded RNA, can be designed to hybridize with both termini of a linear RNA, so that the two termini can be juxtaposed upon hybridization with the single-stranded splint. Splint ligase can thus catalyze the ligation of the juxtaposed two termini of the linear RNA, generating an oRNA. [00683] In one embodiment, a DNA or RNA ligase may be used in the synthesis of the oRNA. As a non-limiting example, the ligase may be a circ ligase or circular ligase. [00684] In one embodiment, either the 5'-or 3'-end of the linear RNA can encode a ligase ribozyme sequence such that during in vitro transcription, the resultant linear RNA includes an active ribozyme sequence capable of ligating the 5'-end of the linear RNA to the 3'- end of the linear RNA. The ligase ribozyme may be derived from the Group I Intron, Hepatitis Delta Virus, Hairpin ribozyme or may be selected by SELEX (systematic evolution of ligands by exponential enrichment). [00685] In one embodiment, a linear RNA may be cyclized or concatemerized by using at least one non-nucleic acid moiety. In one aspect, the at least one non-nucleic acid moiety may react with regions or features near the 5' terminus and/or near the 3' terminus of the linear RNA in order to cyclize or concatermerize the linear RNA. In another aspect, the at least one RNG043-WO1 PCT Application non-nucleic acid moiety may be located in or linked to or near the 5' terminus and/or the 3' terminus of the linear RNA. The non-nucleic acid moieties contemplated may be homologous or heterologous. As a non-limiting example, the non-nucleic acid moiety may be a linkage such as a hydrophobic linkage, ionic linkage, a biodegradable linkage and/or a cleavable linkage. As another non-limiting example, the non-nucleic acid moiety is a ligation moiety. As yet another non-limiting example, the non-nucleic acid moiety may be an oligonucleotide or a peptide moiety, such as an aptamer or a non-nucleic acid linker as described herein. [00686] In one embodiment, a linear RNA may be cyclized or concatemerized due to a non-nucleic acid moiety that causes an attraction between atoms, molecular surfaces at, near or linked to the 5' and 3' ends of the linear RNA. As a non-limiting example, one or more linear RNA may be cyclized or concatemerized by intermolecular forces or intramolecular forces. Non-limiting examples of intermolecular forces include dipole-dipole forces, dipole-induced dipole forces, induced dipole-induced dipole forces, Van der Waals forces, and London dispersion forces. Non-limiting examples of intramolecular forces include covalent bonds, metallic bonds, ionic bonds, resonant bonds, agnostic bonds, dipolar bonds, conjugation, hyperconjugation and antibonding. [00687] In one embodiment, the linear RNA may comprise a ribozyme RNA sequence near the 5' terminus and near the 3' terminus. The ribozyme RNA sequence may covalently link to a peptide when the sequence is exposed to the remainder of the ribozyme. In one aspect, the peptides covalently linked to the ribozyme RNA sequence near the 5' terminus and the 3' terminus may associate with each other causing a linear RNA to cyclize or concatemerize. In another aspect, the peptides covalently linked to the ribozyme RNA near the 5' terminus and the 3' terminus may cause the linear RNA to cyclize or concatemerize after being subjected to ligated using various methods known in the art such as, but not limited to, protein ligation. [00688] In some embodiments, the linear RNA may include a 5' triphosphate of the nucleic acid converted into a 5' monophosphate, e.g., by contacting the 5' triphosphate with RNA 5' pyrophosphohydrolase (RppH) or an ATP diphosphohydrolase (apyrase). Alternately, converting the 5' triphosphate of the linear RNA into a 5' monophosphate may occur by a two- step reaction comprising: (a) contacting the 5' nucleotide of the linear RNA with a phosphatase (e.g., Antarctic Phosphatase, Shrimp Alkaline Phosphatase, or Calf Intestinal Phosphatase) to remove all three phosphates; and (b) contacting the 5' nucleotide after step (a) witha kinase (e.g., Polynucleotide Kinase) that adds a single phosphate. RNG043-WO1 PCT Application [00689] In some examples, RNA may be circularized using the methods described in WO2017222911 and WO2016197121, the contents of each of which are herein incorporated by reference in their entirety. [00690] In some embodiments, RNA may be circularized, for example, by back splicing of a non-mammalian exogenous intron or splint ligation of the 5' and 3 ' ends of a linear RNA. In one embodiment, the circular RNA is produced from a recombinant nucleic acid encoding the target RNA to be made circular. As a non-limiting example, the method comprises: a) producing a recombinant nucleic acid encoding the target RNA to be made circular, wherein the recombinant nucleic acid comprises in 5' to 3 ' order: i) a 3 ' portion of an exogenous intron comprising a 3' splice site, ii) a nucleic acid sequence encoding the target RNA, and iii) a 5 ' portion of an exogenous intron comprising a 5 ' splice site; b) performing transcription, whereby RNA is produced from the recombinant nucleic acid; and c) performing splicing of the RNA, whereby the RNA circularizes to produce a oRNA. [00691] While not wishing to be bound by theory, circular RNAs generated with exogenous introns are recognized by the immune system as "non-self" and trigger an innate immune response. On the other hand, circular RNAs generated with endogenous introns are recognized by the immune system as "self" and generally do not provoke an innate immune response, even if carrying an exon comprising foreign RNA. [00692] Accordingly, circular RNAs can be generated with either an endogenous or exogenous intron to control immunological self/non-self discrimination as desired. Numerous intron sequences from a wide variety of organisms and viruses are known and include sequences derived from genes encoding proteins, ribosomal RNA (rRNA), or transfer RNA (tRNA). [00693] Circular RNAs can be produced from linear RNAs in a number of ways. In some embodiments, circular RNAs are produced from a linear RNA by backsplicing of a downstream 5' splice site (splice donor) to an upstream 3' splice site (splice acceptor). Circular RNAs can be generated in this manner by any nonmammalian splicing method. For example, linear RNAs containing various types of introns, including self-splicing group I introns, self- splicing group II introns, spliceosomal introns, and tRNA introns can be circularized. In particular, group I and group II introns have the advantage that they can be readily used for production of circular RNAs in vitro as well as in vivo because of their ability to undergo self- splicing due to their autocatalytic ribozyme activity. [00694] In some embodiments, circular RNAs can be produced in vitro from a linear RNA by chemical or enzymatic ligation of the 5' and 3' ends of the RNA. In some examples, RNG043-WO1 PCT Application chemical ligation can be performed, for example, using cyanogen bromide (BrCN) or ethyl-3- (3'-dimethylaminopropyl) carbodiimide (EDC) for activation of a nucleotide phosphomonoester group to allow phosphodiester bond formation, such as, for example, as described in Sokolova (1988) FEBS Lett 232: 153-155; Dolinnaya et al. (1991) Nucleic Acids Res., 19:3067-3072; Fedorova (1996) Nucleosides Nucleotides Nucleic Acids 15: 1137-1147; herein incorporated by reference. Alternatively, enzymatic ligation can be used to circularize RNA. Exemplary ligases that can be used include T4 DNA ligase (T4 Dnl), T4 RNA ligase 1 (T4 Rnl 1), and T4 RNA ligase 2 (T4 Rnl 2). [00695] In some embodiments, splint ligation using an oligonucleotide splint that hybridizes with the two ends of a linear RNA can be used to bring the ends of the linear RNA together for ligation. Hybridization of the splint, which can be either a DNA or a RNA, orientates the 5 '-phosphate and 3' -OH of the RNA ends for ligation. Subsequent ligation can be performed using either chemical or enzymatic techniques, as described above. Enzymatic ligation can be performed, for example, with T4 DNA ligase (DNA splint required), T4 RNA ligase 1 (RNA splint required) or T4 RNA ligase 2 (DNA or RNA splint). Chemical ligation, such as with BrCN or EDC, in some cases is more efficient than enzymatic ligation if the structure of the hybridized splint-RNA complex interferes with enzymatic activity. [00696] In some embodiments, the oRNA may further comprise an internal ribosome entry site (IRES) operably linked to an RNA sequence encoding a polypeptide. Inclusion of an IRES permits the translation of one or more open reading frames from a circular RNA. In some examples, the IRES element attracts a eukaryotic ribosomal translation initiation complex and promotes translation initiation, for example, as described in Kaufman et al., Nuc. Acids Res. (1991) 19:4485-4490; Gurtu et al., Biochem. Biophys. Res. Comm. (1996) 229:295-298; Rees et al., BioTechniques (1996) 20: 102-110; Kobayashi et al., BioTechniques (1996) 21 :399- 402; and Mosser et al., BioTechniques 199722150-161). [00697] In some embodiments, the circularization efficiency of the circularization methods provided herein is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100%. In some embodiments, the circularization efficiency of the circularization methods provided herein is at least about 40%. Splicing Element [00698] In some embodiments, the oRNA includes at least one splicing element. The splicing element can be a complete splicing element that can mediate splicing of the oRNA or RNG043-WO1 PCT Application the spicing element can be a residual splicing element from a completed splicing event. For instance, in some cases, a splicing element of a linear RNA can mediate a splicing event that results in circularization of the linear RNA, thereby the resultant oRNA comprises a residual splicing element from such splicing-mediated circularization event. In some cases, the residual splicing element is not able to mediate any splicing. In other cases, the residual splicing element can still mediate splicing under certain circumstances. In some embodiments, the splicing element is adjacent to at least one expression sequence. In some embodiments, the oRNA includes a splicing element adjacent each expression sequence. In some embodiments, the splicing element is on one or both sides of each expression sequence, leading to separation of the expression products, e.g., peptide(s) and or polypeptide(s). [00699] In some embodiments, the oRNA includes an internal splicing element that when replicated the spliced ends are joined together. Some examples may include miniature introns (<100 nt) with splice site sequences and short inverted repeats (30-40 nt) such as AluSq2, AluJr, and AluSz, inverted sequences in flanking introns, Alu elements in flanking introns, and motifs found in (suptable4 enriched motifs) cis-sequence elements proximal to backsplice events such as sequences in the 200 bp preceding (upstream of) or following (downstream from) a backsplice site with flanking exons. In some embodiments, the oRNA includes at least one repetitive nucleotide sequence described elsewhere herein as an internal splicing element. In some examples, the repetitive nucleotide sequence may include repeated sequences from the Alu family of introns, such as those described in US Patent No. 11,058,706. [00700] In some embodiments, the oRNA may include canonical splice sites that flank head-to-tail junctions of the oRNA. [00701] In some embodiments, the oRNA may include a bulge-helix-bulge motif, comprising a 4-base pair stem flanked by two 3-nucleotide bulges. Cleavage occurs at a site in the bulge region, generating characteristic fragments with terminal 5'-hydroxyl group and 2', 3'-cyclic phosphate. Circularization proceeds by nucleophilic attack of the 5'-OH group onto the 2', 3'-cyclic phosphate of the same molecule forming a 3', 5'-phosphodiester bridge. [00702] In some embodiments, the oRNA may include a sequence that mediates self- ligation. Non-limiting examples of sequences that can mediate self-ligation include a self- circularizing intron, e.g., a 5' and 3' slice junction, or a self-circularizing catalytic intron such as a Group I, Group II or Group III Introns. Non-limiting examples of group I intron self- splicing sequences may include self-splicing permuted intron-exon sequences derived from T4 bacteriophage gene td, and the intervening sequence (IVS) rRNA of Tetrahymena. RNG043-WO1 PCT Application Other Circularization Methods [00703] In some embodiments, linear RNA may include complementary sequences, including either repetitive or nonrepetitive nucleic acid sequences within individual introns or across flanking introns. In some embodiments, the oRNA includes a repetitive nucleic acid sequence. In some embodiments, the repetitive nucleotide sequence includes poly CA or poly UG sequences. In some embodiments, the oRNA includes at least one repetitive nucleic acid sequence that hybridizes to a complementary repetitive nucleic acid sequence in another segment of the oRNA, with the hybridized segment forming an internal double strand. In some embodiments, repetitive nucleic acid sequences and complementary repetitive nucleic acid sequences from two separate oRNA that hybridize to generate a single oRNA, with the hybridized segments forming internal double strands. In some embodiments, the complementary sequences are found at the 5' and 3' ends of the linear RNA. In some embodiments, the complementary sequences include about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more paired nucleotides. [00704] In some embodiments, chemical methods of circularization may be used to generate the oRNA. Such methods may include, but are not limited to click chemistry (e.g., alkyne- and azide-based methods, or clickable bases), olefin metathesis, phosphoramidate ligation, hemiaminal-imine crosslinking, base modification, and any combination thereof. In some embodiments, enzymatic methods of circularization may be used to generate the oRNA. In some embodiments, a ligation enzyme, e.g., DNA or RNA ligase, may be used to generate a template of the oRNA or complement, a complementary strand of the oRNA, or the oRNA. [00705] In some examples, any of the circular polynucleotides as taught in for example U.S. Provisional Application No.61/873,010 filed Sep.3, 2013 or U.S. Patent No.10,709,779, may be used herein. The contents of these references are incorporated herein by reference in their entirety. In some examples, any of the circular RNAs, methods for making circular RNAs, circular RNA compositions that are described in the following publications are contemplated herein and are incorporated by reference in their entireties are part of the instant specification: US Patents US 11,352,640, US 11,352,641, US 11,203,767, US 10,683,498, US 5,773,244, and US 5,766,903; US Application Publications US 2022/0177540, US 2021/0371494, US 2022/0090137, US 2019/0345503, and US 2015/0299702; and PCT Application Publications WO 2021/226597, WO 2019/236673, WO 2017/222911, WO2016/187583, WO2014/082644 and WO 1997/007825. D. Kits RNG043-WO1 PCT Application [00706] Also provided are kits comprising engineered retrons (e.g., engineered nucleic acid constructs, or engineered nucleic acid-enzyme constructs) as described herein. [00707] In some embodiments, the kit provides an engineered retron construct or a vector system comprising such a retron construct. In some embodiments, the engineered retron construct, included in the kit, comprises a heterologous sequence capable of providing a cell with a nucleic acid encoding a protein or regulatory RNA of interest, a cellular barcode, a donor polynucleotide suitable for use in gene editing, e.g., by homology directed repair (HDR) or recombination-mediated genetic engineering (recombineering), or a CRISPR protospacer DNA sequence for use in molecular recording. Other agents may also be included in the kit such as transfection agents, host cells, suitable media for culturing cells, buffers, and the like. [00708] In the context of a kit, agents can be provided in liquid or solid form in any convenient packaging (e.g., stick pack, dose pack, etc.). The agents of a kit can be present in the same or separate containers. The kits may contain any one or more of the components described herein in one or more containers. The components may be prepared sterilely, packaged in a syringe and shipped refrigerated. Alternatively it may be housed in a vial or other container for storage. A second container may have other components prepared sterilely. Alternatively the kits may include the active agents premixed and shipped in a vial, tube, or other container. [00709] The kits may have a variety of forms, such as a blister pouch, a shrink wrapped pouch, a vacuum sealable pouch, a sealable thermoformed tray, or a similar pouch or tray form, with the accessories loosely packed within the pouch, one or more tubes, containers, a box or a bag. The kits may be sterilized after the accessories are added, thereby allowing the individual accessories in the container to be otherwise unwrapped. The kits can be sterilized using any appropriate sterilization techniques, such as radiation sterilization, heat sterilization, or other sterilization methods known in the art. The kits may also include other components, depending on the specific application, for example, containers, cell media, salts, buffers, reagents, syringes, needles, a fabric, such as gauze, for applying or removing a disinfecting agent, disposable gloves, a support for the agents prior to administration, etc. Some aspects of this disclosure provide kits comprising a nucleic acid construct comprising a nucleotide sequence encoding the various components of the retron-based editing system described herein. [00710] In addition to the above components, the subject kits may further include (in some embodiments) instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. RNG043-WO1 PCT Application One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, and the like. Yet another form of these instructions is a computer readable medium, e.g., diskette, compact disk (CD), flash drive, and the like, on which the information has been recorded. Yet another form of these instructions that may be present is a website address which may be used via the internet to access the information at a remote site. In some embodiments, information provided on the website is periodically updated to provide, for example, the most up-to-date information. The written instructions may be in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which can also reflect approval by the agency of manufacture, use or sale for animal administration. As used herein,“promoted” includes all methods of doing business including methods of education, hospital and other clinical instruction, scientific inquiry, drug discovery or development, academic research, pharmaceutical industry activity including pharmaceutical sales, and any advertising or other promotional activity including written, oral and electronic communication of any form, associated with the disclosure. [00711] Other aspects of this disclosure provide kits comprising one or more nucleic acid constructs (e.g., one or more mRNA or circular RNA molecules encoding the components of the retron-based genome editing system) In various embodiments, all nucleic acid constructs can be based on RNA molecules, i.e., and “all-RNA system.” For example, each of the components of the editing system could be expressed from a mRNA molecule, which would be delivered to a target cell by one or more delivery methods (e.g., LNP delivery). E. Cells [00712] One aspect of the disclosure provides an isolated host cell that includes one or more of the compositions described herein, including, but not limited to, engineered retrons and/or retron components, engineered ncRNAs, engineered msDNA, engineered RT, nucleic acid molecules encoding the engineered retrons and/or retron components, and vector or vector systems encoding the engineered retrons and/or retron components, and any combinations thereof. In some embodiments, the host cell is a prokaryotic cell, an archaeal cell, or a eukaryotic host cell. In some embodiments, the eukaryotic host cell is a mammalian cell, such as a human cell, a non-human cell, or a non-human mammalian cell. In some embodiments, the host cell is an artificial cell or genetically modified cell. In some embodiments, the host cell is in vitro, such as a tissue culture cell. In some embodiments, the host cell is within a living host organism. RNG043-WO1 PCT Application [00713] Cells that may contain any of the compositions described herein. The methods described herein are used to deliver recombinant retrons or components thereof into a eukaryotic cell (e.g., a mammalian cell, such as a human cell). In some embodiments, the cell is in vitro (e.g., cultured cell. In some embodiments, the cell is in vivo (e.g., in a subject such as a human subject). In some embodiments, the cell is ex vivo (e.g., isolated from a subject and may be administered back to the same or a different subject). [00714] The present disclosure contemplates the use of any suitable host cell. For example, the cell host can be a mammalian cell. Mammalian cells of the present disclosure include human cells, primate cells (e.g., vero cells), rat cells (e.g., GH3 cells, OC23 cells) or mouse cells (e.g., MC3T3 cells). There are a variety of human cell lines, including, without limitation, human embryonic kidney (HEK) cells, HeLa cells, cancer cells from the National Cancer Institute's 60 cancer cell lines (NCI60), DU145 (prostate cancer) cells, Lncap (prostate cancer) cells, MCF-7 (breast cancer) cells, MDA-MB-438 (breast cancer) cells, PC3 (prostate cancer) cells, T47D (breast cancer) cells, THP-1 (acute myeloid leukemia) cells, U87 (glioblastoma) cells, SHSY5Y human neuroblastoma cells (cloned from a myeloma) and Saos- 2 (bone cancer) cells. In some embodiments, the cells can be human embryonic kidney (HEK) cells (e.g., HEK 293 or HEK 293T cells). In some embodiments, the cells can be stem cells (e.g., human stem cells) such as, for example, pluripotent stem cells (e.g., human pluripotent stem cells including human induced pluripotent stem cells (hiPSCs)). A stem cell refers to a cell with the ability to divide for indefinite periods in culture and to give rise to specialized cells. A pluripotent stem cell refers to a type of stem cell that is capable of differentiating into all tissues of an organism, but not alone capable of sustaining full organismal development. In some examples, a human induced pluripotent stem cell refers to a somatic (e.g., mature or adult) cell that has been reprogrammed to an embryonic stem cell-like state by being forced to express genes and factors important for maintaining the defining properties of embryonic stem cells, for example, as described in Takahashi and Yamanaka, Cell 126 (4): 663–76, 2006, incorporated by reference herein). Human induced pluripotent stem cells express stem cell markers and are capable of generating cells characteristic of all three germ layers (ectoderm, endoderm, mesoderm). [00715] Some aspects of this disclosure provide cells comprising any of the compositions disclosed herein, including, but not limited to, engineered retrons and/or retron components, engineered ncRNAs, engineered msDNA, engineered RT, nucleic acid molecules encoding the engineered retrons and/or retron components, and vector or vector systems encoding the engineered retrons and/or retron components, and any combinations thereof. In RNG043-WO1 PCT Application some embodiments, a host cell is transiently or non-transiently transfected with one or more delivery systems described herein, including virus-based systems, virus-like particle systems, and non-virus-base delivery, including LNPs and liposomes. In some embodiments, a cell is transfected as it naturally occurs in a subject. In some embodiments, a cell that is transfected is taken from a subject, i.e., ex vivo transfection. In some embodiments, the cell is derived from cells taken from a subject, such as a cell line. A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, C8161, CCRF-CEM, MOLT, mIMCD- 3, NHDF, HeLa-S3, Huh1, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, TF1, CTLL-2, C1R, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH- 77, Calu1, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelial, BALB/3T3 mouse embryo fibroblast, 3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A 172, A20, A253, A431, A-549, ALC, B16, B35, BCP-1 cells, BEAS-2B, bEnd.3, BHK-21, BR 293. BxPC3. C3H-10T1/2, C6/36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO- T, CHO Dhfr -/-, COR-L23, COR-L23/CPR, COR-L23/5010, COR-L23/R23, COS-7, COV- 434, CML T1, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6/AR1, EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepa1c1c7, HL- 60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812, KCL22, KG1, KYO1, LNCap, Ma- Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCK II, MDCK 11, MOR/0.2R, MONO-MAC 6, MTD-1A, MyEnd, NCI- H69/CPR, NCI- H69/LX10, NCI-H69/LX20, NCI-H69/LX4, NIH-3T3, NALM-1, NW-145, OPCN/OPCT cell lines, Peer, PNT-1A/PNT 2, RenCa, RIN-5F, RMA/RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T- 47D, T84, THP1 cell line, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, and transgenic varieties thereof. [00716] Cell lines are available from a variety of sources known to those with skill in the art, for example, as described in the American Type Culture Collection (ATCC) (Manassus, Va.). In some embodiments, a cell transfected with one or more retron delivery systems described herein is used to establish a new cell line comprising one or more nucleic acid molecules encoding the recombinant retron-based gene editing systems described herein, or encoding at last a component of said systems (e.g., a recombinant ncRNA or a recombinant retron RT). F. Pharmaceutical Compositions RNG043-WO1 PCT Application [00717] The engineered genome editing systems described herein, or one or more components thereof (e.g., engineered ncRNAs, engineered msDNA, engineered RT, nucleic acid molecules encoding the engineered retrons and/or retron components, guide RNAs, programmable nucleases) may be provided as pharmaceutical compositions. For example, one or more LNPs or other non-virus-based delivery system comprising one or more circular or linear RNA molecules encoding each of the components of the retron-based genome editing system may be formulated as a pharmaceutical composition for administering to a subject in need (e.g., a human in need of gene editing). [00718] Formulations can include, without limitation, saline, liposomes, lipid nanoparticles, polymers, peptides, proteins, cells transfected with viral vectors (e.g., for transfer or transplantation into a subject) and combinations thereof. [00719] Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. As used herein the term “pharmaceutical composition” refers to compositions comprising at least one active ingredient and optionally one or more pharmaceutically acceptable excipients. [00720] In general, such preparatory methods include the step of associating the active ingredient with an excipient and/or one or more other accessory ingredients. As used herein, the phrase “active ingredient” generally refers an engineered retron as described herein. [00721] A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a “unit dose” refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage. [00722] Other aspects of the present disclosure relate to pharmaceutical compositions comprising any of the various components of the recombinant retron-based genome editing systems described herein, including, but not limited to, engineered retrons and/or retron components, engineered ncRNAs, engineered msDNA, engineered RT, nucleic acid molecules encoding the engineered retrons and/or retron components, programmable nucleases (e.g., RNA-guided nucleases), guide RNAs, and vector or vector systems encoding the engineered retrons and/or retron components, and any combinations thereof. The term“pharmaceutical composition”, as used herein, refers to a composition formulated for pharmaceutical use. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically RNG043-WO1 PCT Application acceptable carrier. In some embodiments, the pharmaceutical composition comprises additional agents (e.g. for specific delivery, increasing half-life, or other therapeutic compounds). [00723] As used here, the term “pharmaceutically-acceptable carrier” means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the compound from one site (e.g., the delivery site) of the body, to another site (e.g., organ, tissue or portion of the body). A pharmaceutically acceptable carrier is “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the tissue of the subject (e.g., physiologically compatible, sterile, physiologic pH, etc.). [00724] Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as“excipient”,“carrier”,“pharmaceutically acceptable carrier” or the like are used interchangeably herein. [00725] In some embodiments, the pharmaceutical composition is formulated for delivery to a subject, e.g., for gene editing. Suitable routes of administrating the pharmaceutical composition described herein include, without limitation: topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, RNG043-WO1 PCT Application intravesical, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseus, periocular, intratumoral, intracerebral, and intracerebroventricular administration. [00726] In some embodiments, the pharmaceutical composition described herein is administered locally to a diseased site (e.g., tumor site). In some embodiments, the pharmaceutical composition described herein is administered to a subject by injection, by means of a catheter, by means of a suppository, or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including a membrane, such as a sialastic membrane, or a fiber. [00727] In other embodiments, the pharmaceutical composition described herein is delivered in a controlled release system. In some examples, a pump may be used, such as the one described in Langer, 1990, Science 249:1527-1533; Sefton, 1989, CRC Crit. Ref. Biomed. Eng.14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med.321:574). In some examples, polymeric materials can be used, such as the ones described in Medical Applications of Controlled Release (Langer and Wise eds., CRC Press, Boca Raton, Fla., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., Wiley, New York, 1984); Ranger and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem.23:61; Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol.25:351; Howard et al., 1989, J. Neurosurg.71:105). In some examples, other controlled release systems are discussed, for example, in Langer, supra. [00728] In some embodiments, the pharmaceutical composition is formulated in accordance with routine procedures as a composition adapted for intravenous or subcutaneous administration to a subject, e.g., a human. In some embodiments, pharmaceutical composition for administration by injection are solutions in sterile isotonic aqueous buffer. Where necessary, the pharmaceutical can also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the pharmaceutical is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the pharmaceutical composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration. RNG043-WO1 PCT Application [00729] A pharmaceutical composition for systemic administration may be a liquid, e.g., sterile saline, lactated Ringer’s or Hank’s solution. In addition, the pharmaceutical composition can be in solid forms and re-dissolved or suspended immediately prior to use. Lyophilized forms are also contemplated. [00730] The pharmaceutical composition can be contained within a lipid particle or vesicle, such as a liposome or microcrystal or LNP, which is also suitable for parenteral administration. The particles can be of any suitable structure, such as unilamellar or plurilamellar, so long as compositions are contained therein. In some examples, compounds can be entrapped in “stabilized plasmid- lipid particles” (SPLP) containing the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), low levels (5-10 mol%) of cationic lipid, and stabilized by a polyethyleneglycol (PEG) coating, such as described in Zhang Y. P. et al., Gene Ther.1999, 6:1438-47. Positively charged lipids such as N-[1-(2,3-dioleoyloxi)propyl]-N,N,N- trimethyl-amoniummethylsulfate, or “DOTAP,” are particularly preferred for such particles and vesicles. In some examples, methods of preparing such lipid particles can include the methods disclosed in U.S. Patent Nos.4,880,635; 4,906,477; 4,911,928; 4,917,951; 4,920,016; and 4,921,757; each of which is incorporated herein by reference. [00731] Further, the pharmaceutical composition can be provided as a pharmaceutical kit comprising (a) a container containing a recombinant retron-based genome editing system or one or more components thereof in lyophilized form and (b) a second container containing a pharmaceutically acceptable diluent (e.g., sterile water) for injection. The pharmaceutically acceptable diluent can be used for reconstitution or dilution of the lyophilized system of the present disclosure. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. [00732] In another aspect, an article of manufacture containing materials useful for the treatment of the diseases described above is included. In some embodiments, the article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. In some embodiments, the container holds a composition that is effective for treating a disease described herein and may have a sterile access port. For example, the container may be an intravenous solution bag or a vial having a stopper pierce- able by a hypodermic injection needle. The active agent in the composition is a compound of the present disclosure. In some embodiments, the label on or associated with the container RNG043-WO1 PCT Application indicates that the composition is used for treating the disease of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically- acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. G. Use and Methods of Use [00733] The gene editing systems can be used in a variety of applications, several non- limiting examples of which are described herein. In general, the engineered retron can be used in any suitable organism. In some embodiments, the organism is a eukaryote. [00734] In some embodiments, the organism is an animal. In some embodiments, the animal is a fish, an amphibian, a reptile, a mammal, or a bird. In some embodiments, the animal is a farm animal or agriculture animal. Non-limiting examples of farm and agriculture animals include horses, goats, sheep, swine, cattle, llamas, alpacas, and birds, e.g., chickens, turkeys, ducks, and geese. In some embodiments, the animal is a non-human primate, e.g., baboons, capuchin monkeys, chimpanzees, lemurs, macaques, marmosets, tamarins, spider monkeys, squirrel monkeys, and vervet monkeys. In some embodiments, the animal is a pet. Non-limiting examples of pets include dogs, cats horses, wolfs, rabbits, ferrets, gerbils, hamsters, chinchillas, fancy rats, guinea pigs, canaries, parakeets, and parrots. [00735] In some embodiments, the organism is a plant. Plants that may be transfected with an engineered retron include monocots and dicots. Particular examples include, but are not limited to, corn (maize), sorghum, wheat, sunflower, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape, Brassica sp., alfalfa, rye, millet, safflower, peanuts, sweet potato, cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, oats, vegetables, ornamentals, and conifers. Vegetables include, but are not limited to, crucifers, peppers, tomatoes, lettuce, green beans, lima beans, peas, and members of the genus Cucumis such as cucumber, cantaloupe, and musk melon. Ornamentals include, but are not limited to, azalea, hydrangea, hibiscus, roses, tulips, daffodils, petunias, carnation, poinsettia, and chrysanthemum. [00736] In some embodiments, heterologous nucleic acid sequences can be added to the subject engineered retron to provide a cell with a heterologous nucleic acid encoding a protein or regulatory RNA of interest, a cellular barcode, a donor polynucleotide suitable for use in gene editing, e.g., by homology directed repair (HDR) or recombination-mediated genetic engineering (recombineering), or a CRISPR protospacer DNA sequence for use in molecular RNG043-WO1 PCT Application recording, as discussed further below. Such heterologous sequences may be inserted, for example, into the msr locus or the msd locus such that the heterologous sequence is transcribed by the retron reverse transcriptase as part of the msDNA product. [00737] In some embodiments, the engineered retrons described herein may be used for research tools, such as kits, functional genomics assays, and generating engineered cell lines and animal models for research and drug screening. The kit may comprise one or more reagents in addition to the engineered retron, such as a buffer, a control reagent, a control vector, a control RNA polynucleotide, a reagent for in vitro production of the polypeptide from DNA, and adaptors for sequencing. A buffer can be, for example, a stabilization buffer, a reconstituting buffer, a diluting buffer, a wash buffer, or a buffer for introducing a polypeptide and/or polynucleotide of the kit into a cell. In some instances, a kit can comprise one or more additional reagents specific for plants. One or more additional reagents for plants can include, for example, soil, nutrients, plants, seeds, spores, Agrobacterium, a T-DNA vector, and a pBINAR vector. Gene Editing [00738] In some embodiments, the gene editing systems described herein are used for genome editing a desired site. A retron is engineered with a heterologous nucleic acid sequence encoding a donor polynucleotide suitable for use with nuclease genome editing system. The nuclease is designed to specifically target a location proximal to the desired edit (the nuclease should be designed such that it will not cut the target once the edit is properly installed). The nuclease (e.g., CAS or non-CAS) can be linked to the retron, either by direct fusion to the RT or by fusion of the msDNA to the gRNA (only applicable for RNA-guided nucleases). A heterologous nucleic acid sequence is inserted into the retron msd. See for example FIG.7C, which shows a marker representing the edit. [00739] In some embodiments, the heterologous nucleic acid sequence has 10-100 or more bp of homologous nucleic acid sequence to the genome on both sides of the desired edit. The desired edit (insertion, deletion, or mutation) is in between the homologous sequence. [00740] In some embodiments, donor polynucleotides comprise a sequence comprising an intended genome edit flanked by a pair of homology arms responsible for targeting the donor polynucleotide to the target locus to be edited in a cell. The donor polynucleotide typically comprises a 5ʹ homology arm that hybridizes to a 5ʹ genomic target sequence and a 3ʹ homology arm that hybridizes to a 3ʹ genomic target sequence. The homology arms are referred to herein as 5ʹ and 3ʹ (i.e., upstream and downstream) homology arms, which relate to the relative position of the homology arms to the nucleotide sequence comprising the intended RNG043-WO1 PCT Application edit within the donor polynucleotide. The 5ʹ and 3ʹ homology arms hybridize to regions within the target locus in the genomic DNA to be modified, which are referred to herein as the “5ʹ target sequence” and “3ʹ target sequence,” respectively. [00741] The homology arm must be sufficiently complementary for hybridization to the target sequence to mediate homologous recombination between the donor polynucleotide and genomic DNA at the target locus. For example, a homology arm may comprise a nucleotide sequence having at least about 80-100% sequence identity to the corresponding genomic target sequence, including any percent identity within this range, such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto, wherein the nucleotide sequence comprising the intended edit can be integrated into the genomic DNA by HDR at the genomic target locus recognized (i.e., having sufficient complementary for hybridization) by the 5ʹ and 3ʹ homology arms. [00742] In some embodiments, the corresponding homologous nucleotide sequences in the genomic target sequence (i.e., the “5ʹ target sequence” and “3ʹ target sequence”) flank a specific site for cleavage and/or a specific site for introducing the intended edit. The distance between the specific cleavage site and the homologous nucleotide sequences (e.g., each homology arm) can be several hundred nucleotides. In some embodiments, the distance between a homology arm and the cleavage site is 200 nucleotides or less (e.g., 0, 10, 20, 30, 50, 75, 100, 125, 150, 175, and 200 nucleotides). In most cases, a smaller distance may give rise to a higher gene targeting rate. In some embodiments, the donor polynucleotide is substantially identical to the target genomic sequence, across its entire length except for the sequence changes to be introduced to a portion of the genome that encompasses both the specific cleavage site and the portions of the genomic target sequence to be altered. [00743] A homology arm can be of any length, e.g.10 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 300 nucleotides or more, 350 nucleotides or more, 400 nucleotides or more, 450 nucleotides or more, 500 nucleotides or more, 1000 nucleotides (1 kb) or more, 5000 nucleotides (5 kb) or more, 10000 nucleotides (10 kb) or more, etc. In some instances, the 5ʹ and 3ʹ homology arms are substantially equal in length to one another. However, in some instances the 5ʹ and 3ʹ homology arms are not necessarily equal in length to one another. For example, one homology arm may be 30% shorter or less than the other homology arm, 20% shorter or less than the other homology arm, 10% shorter or less than the other homology arm, 5% shorter or less than the other homology arm, 2% shorter or less than the other homology RNG043-WO1 PCT Application arm, or only a few nucleotides less than the other homology arm. In other instances, the 5ʹ and 3ʹ homology arms are substantially different in length from one another, e.g. one may be 40% shorter or more, 50% shorter or more, sometimes 60% shorter or more, 70% shorter or more, 80% shorter or more, 90% shorter or more, or 95% shorter or more than the other homology arm. [00744] The donor polynucleotide may be used in combination with an RNA-guided nuclease, which is targeted to a particular genomic sequence (i.e., genomic target sequence to be modified) by a guide RNA. A target-specific guide RNA comprises a nucleotide sequence that is complementary to a genomic target sequence, and thereby mediates binding of the nuclease-gRNA complex by hybridization at the target site. For example, the gRNA can be designed with a sequence complementary to the sequence of a minor allele to target the nuclease-gRNA complex to the site of a mutation. The mutation may comprise an insertion, a deletion, or a substitution. For example, the mutation may include a single nucleotide variation, gene fusion, translocation, inversion, duplication, frameshift, missense, nonsense, or other mutation associated with a phenotype or disease of interest. The targeted minor allele may be a common genetic variant or a rare genetic variant. In some embodiments, the gRNA is designed to selectively bind to a minor allele with single base-pair discrimination, for example, to allow binding of the nuclease-gRNA complex to a single nucleotide polymorphism (SNP). In particular, the gRNA may be designed to target disease-relevant mutations of interest for the purpose of genome editing to remove the mutation from a gene. Alternatively, the gRNA can be designed with a sequence complementary to the sequence of a major or wild-type allele to target the nuclease-gRNA complex to the allele for the purpose of genome editing to introduces a mutation into a gene in the genomic DNA of the cell, such as an insertion, deletion, or substitution. Such genetically modified cells can be used, for example, to alter phenotype, confer new properties, or produce disease models for drug screening. [00745] In some embodiments, the RNA-guided nuclease used for genome modification is a clustered regularly interspersed short palindromic repeats (CRISPR) system Cas nuclease. Any RNA-guided Cas nuclease capable of catalyzing site- directed cleavage of DNA to allow integration of donor polynucleotides by the HDR mechanism can be used in genome editing, including CRISPR system Class 1, Type I, II, or III Cas nucleases; Class 2, Type II nuclease (such as Cas9); a Class 2, Type V nuclease (such as Cpfl), or a Class 2, Type VI nuclease (such as C2c2). Examples of Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9 (Csnl or Csxl2), CaslO, CaslOd, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (CasA), Cse2 (CasB), Cse3 RNG043-WO1 PCT Application (CasE), Cse4 (CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966, and homologs or modified versions thereof. [00746] In some embodiments, a Class 1, type II CRISPR system Cas9 endonuclease is used. Cas9 nucleases from any species, or biologically active fragments, variants, analogs, or derivatives thereof that retain Cas9 endonuclease activity (i.e., catalyze site-directed cleavage of DNA to generate double-strand breaks) may be used to perform genome modification as described herein. The Cas9 need not be physically derived from an organism but may be synthetically or recombinantly produced. Cas9 sequences from a number of bacterial species are well known in the art and listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries for Cas9 from: Streptococcus pyogenes (WP 002989955, WP_038434062, WP_011528583); Campylobacter jejuni (WP_022552435, YP 002344900), Campylobacter coli (WP 060786116); Campylobacter fetus (WP 059434633); Corynebacterium ulcerans (NC_015683, NC_017317); Corynebacterium diphtheria (NC_016782, NC_016786); Enterococcus faecalis (WP 033919308); Spiroplasma syrphidicola (NC 021284); Prevotella intermedia (NC 017861); Spiroplasma taiwanense (NC 021846); Streptococcus iniae (NC 021314); Belliella baltica (NC 018010); Psychroflexus torquisl (NC O 18721); Streptococcus thermophilus (YP 820832), Streptococcus mutans (WP 061046374, WP 024786433); Listeria innocua (NP 472073); Listeria monocytogenes (WP 061665472); Legionella pneumophila (WP 062726656); Staphylococcus aureus (WP_001573634); Francisella tularensis (WP_032729892, WP_014548420), Enterococcus faecalis (WP 033919308); Lactobacillus rhamnosus (WP 048482595, WP_032965177); and Neisseria meningitidis (WP_061704949, YP_002342100); all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference in their entireties. In some examples, any of these sequences or a variant thereof comprising a sequence having at least about 70-100% sequence identity thereto, including any percent identity within this range, such as 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used for genome editing, as described herein or as described in Fonfara et al. (2014) Nucleic Acids Res.42(4):2577-90; Kapitonov et al. (2015) J. Bacterid.198(5): 797-807, Shmakov et al. (2015) Mol. Cell. 60(3):385- 397, and Chylinski et al. (2014) Nucleic Acids Res.42(10):6091-6105); for sequence comparisons and a discussion of genetic diversity and phylogenetic analysis of Cas9. [00747] The genomic target site will typically comprise a nucleotide sequence that is complementary to the gRNA and may further comprise a protospacer adjacent motif (PAM). In RNG043-WO1 PCT Application some embodiments, the target site comprises 20-30 base pairs in addition to a 3 or more base pair PAM. Typically, the first nucleotide of a PAM can be any nucleotide, while the two or more other nucleotides will depend on the specific Cas9 protein that is chosen. Exemplary PAM sequences are known to those of skill in the art and include, without limitation, NNG, NGN, NAG, and NGG, wherein N represents any nucleotide. In some embodiments, the allele targeted by a gRNA comprises a mutation that creates a PAM within the allele, wherein the PAM promotes binding of the Cas9-gRNA complex to the allele. [00748] In some embodiments, the gRNA is 5-50 nucleotides, 10-30 nucleotides, 15- 25 nucleotides, 18-22 nucleotides, or 19-21 nucleotides in length, or any length between the stated ranges, including, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in length. The guide RNA may be a single guide RNA comprising crRNA and tracrRNA sequences in a single RNA molecule, or the guide RNA may comprise two RNA molecules with crRNA and tracrRNA sequences residing in separate RNA molecules. [00749] In another embodiment, the CRISPR nuclease from Prevotella and Francisella 1 (Cpfl, or Cas12a) is used. Cpfl is another class II CRISPR/Cas system RNA-guided nuclease with similarities to Cas9 and may be used analogously. Unlike Cas9, Cpfl does not require a tracrRNA and only depends on a crRNA in its guide RNA, which provides the advantage that shorter guide RNAs can be used with Cpfl for targeting than Cas9. Cpfl is capable of cleaving either DNA or RNA. The PAM sites recognized by Cpfl have the sequences 5ʹ-YTN-3ʹ (where “Y” is a pyrimidine and “N” is any nucleobase) or 5ʹ-TTN-3ʹ, in contrast to the G-rich PAM site recognized by Cas9. Cpfl cleavage of DNA produces double-stranded breaks with a sticky-ends having a 4 or 5 nucleotide overhang. [00750] C2c1 (Cas12b) is another class II CRISPR/Cas system RNA-guided nuclease that may be used. C2cl, similarly to Cas9, depends on both a crRNA and tracrRNA for guidance to target sites. [00751] In yet another embodiment, an engineered RNA-guided Fokl nuclease may be used. In some examples, RNA-guided Fokl nucleases can comprise fusions of inactive Cas9 (dCas9) and the Fokl endonuclease (FokI-dCas9), wherein the dCas9 portion confers guide RNA-dependent targeting on Fokl, such as, for example, the ones described in Havlicek et al. (2017) Mol. Ther.25(2):342-355, Pan et al. (2016) Sci Rep.6:35794, Tsai et al. (2014) Nat Biotechnol.32(6):569-576; herein incorporated by reference. [00752] In other embodiments, any other Cas enzymes and variants described in other sections of the application (all incorporated herein) can be used similarly. RNG043-WO1 PCT Application [00753] In some embodiments, the RNA-guided nuclease is provided in the form of a protein, optionally where the nuclease is complexed with a gRNA to form a ribonucleoprotein (RNP) complex. In some embodiments, the RNA-guided nuclease is provided by a nucleic acid encoding the RNA-guided nuclease, such as an RNA (e.g., messenger RNA) or DNA (expression vector). In some embodiments, the RNA-guided nuclease and the gRNA are both provided by vectors, such as the vectors and the vector system described in other parts of the application (all incorporated herein by reference). Both can be expressed by a single vector or separately on different vectors. The vectors encoding the RNA-guided nuclease and gRNA may be included in the vector system comprising the engineered retron msr gene, msd gene and ret gene sequences. In some embodiments, the RNA-guided nuclease is fused to the RT and/or the msDNA. [00754] In some examples, the RNP complex may be administered to a subject or delivered into a cell by methods known in the art, such as those described in U.S. Pat. No. 11,390,884, which is incorporated by reference herein in its entirety. In some embodiments, the endonuclease/gRNA ribonucleoprotein (RNP) complexes are delivered to cells by electroporation. Direct delivery of the RNP complex to a subject or cell eliminates the need for expression from nucleic acids (e.g., transfection of plasmids encoding Cas9 and gRNA). It also eliminates unwanted integration of DNA segments derived from nucleic acid delivery (e.g., transfection of plasmids encoding Cas9 and gRNA). An endonuclease/gRNA ribonucleoprotein (RNP) complex usually is formed prior to administration. [00755] Codon usage may be optimized to further improve production of an RNA- guided nuclease and/or reverse transcriptase (RT) in a particular cell or organism. For example, a nucleic acid encoding an RNA-guided nuclease or reverse transcriptase can be modified to substitute codons having a higher frequency of usage in a yeast cell, a bacterial cell, a human cell, a non-human cell, a mammalian cell, a rodent cell, a mouse cell, a rat cell, or any other host cell of interest, as compared to the naturally occurring polynucleotide sequence. When a nucleic acid encoding the RNA-guided nuclease or reverse transcriptase is introduced into cells, the protein can be transiently, conditionally, or constitutively expressed in the cell. [00756] In some embodiments, the engineered retron used for genome editing with nuclease genome editing systems can further include accessory or enhancer proteins for recombination. Examples of recombination enhancers can include nonhomologous end joining (NHEJ) inhibitors (e.g., inhibitor of DNA ligase IV, a KU inhibitor (e.g., KU70 or KU80), a DNA-PKc inhibitor, or an artemis inhibitor) and homologous directed repair (HDR) promoters, or both, that can enhance or improve more precise genome editing and/or the efficiency of RNG043-WO1 PCT Application homologous recombination. In some embodiments, the recombination accessory or enhancers can comprise C-terminal binding protein interacting protein (CtIP), cyclinB2, Rad family members (e.g. Rad50, Rad51, Rad52, etc). [00757] CtIP is a transcription factor containing C2H2 zinc fingers that are involved in early steps of homologous recombination. Mammalian CtIP and its orthologs in other eukaryotes promote the resection of DNA double-strand breaks and are essential for meiotic recombination. HDR may be enhanced by using Cas9 nuclease associated (e.g. fused) to an N- terminal domain of CtIP, an approach that forces CtIP to the cleavage site and increases transgene integration by HDR. In some embodiments, an N-terminal fragment of CtIP, called HE for HDR enhancer, may be sufficient for HDR stimulation and requires the CtIP multimerization domain and CDK phosphorylation sites to be active. HDR stimulation by the Cas9-HE fusion depends on the guide RNA used, and therefore the guide RNA will be designed accordingly. [00758] Using the gene editing system described herein, any target gene or sequence in a host cell can be edited or modified for a desired trait, including but not limited to: Myostatin (e.g., GDF8) to increase muscle growth; Pc POLLED to induce hairlessness; KISS1R to induce bore taint; Dead end protein (dnd) to induce sterility; Nano2 and DDX to induce sterility; CD163 to induce PRRSV resistance; RELA to induce ASFV resilience; CD18 to induce Mannheimia (Pasteurella) haemolytica resilience; NRAMPl to induce tuberculosis resilience; Negative regulators of muscle mass (e.g., Myostatin) to increase muscle mass. Diseases and Disorders [00759] Provided herein are methods of treating a disease or disorder, the methods comprising administering to a subject in need thereof a pharmaceutical composition of the present disclosure which includes a gene editing system and/or components thereof. In various embodiments of the present disclosure, target genome or epigenetic modifications include cells with monogenic diseases or disorders. Various monogenic diseases include but are not limited to: Adenosine Deaminase (ADA) Deficiency; Alpha-1 Antitrypsin Deficiency; Cystic Fibrosis; Duchenne Muscular Dystrophy; Galactosemia; Hemochromatosis; Huntington’s Disease; Maple Syrup Urine Disease; Marfan Syndrome; Neurofibromatosis Type 1; Pachyonychia Congenita; Phenylkeotnuria; Severe Combined Immunodeficiency; Sickle Cell Disease; Smith-Lemli-Opitz Syndrome; Tay-Sachs Disease; hereditary tyrosinemia I; Influenza; SARS- CoV-2; Alzheimer’s disease; Parkinson’s disease. [00760] Target sequences related to certain diseases and disorders are known in some cases. Target sequences or target editing sites include disease-associated or causative RNG043-WO1 PCT Application mutations for one or more of 10,000 monogenic disorders. A list of target sequences can be generated based on the monogenic disorders. Common genetic disorders that may be correctable by the gene editing systems described here including but are not limited to: Adenosine Deaminase (ADA) Deficiency; Alpha-1 Antitrypsin Deficiency; Cystic Fibrosis; Duchenne Muscular Dystrophy; Galactosemia; Hemochromatosis; Huntington’s Disease; Maple Syrup Urine Disease; Marfan Syndrome; Neurofibromatosis Type 1; Pachyonychia Congenita; Phenylkeotnuria; Severe Combined Immunodeficiency; Sickle Cell Disease; Smith-Lemli-Opitz Syndrome; and Tay-Sachs Disease. In other embodiments, the disease- associated gene can be associated with a polygenic disorder selected from the group consisting of: heart disease; high blood pressure; Alzheimer’s disease; arthritis; diabetes; cancer; and obesity. [00761] The gene editing systems disclosed herein may also be used to treat the following genetic disorders by editing a defect in the disease-associated gene, as follows: Genetic disease Disease gene Arenoleukodystrophy (ALD) ABCD1 Agammaglobulinemia non-Bruton type IGHM Alport syndrome COL4A5 Amyloid neuropathy – Andrade disease TTR Angioneurotic oedema C1NH Alpha1-antitrypsin deficiency SERPINEA 1 Bartter syndrome type 4 BSND Blepharophimosis - ptosis - epicanthus inversus FOXL2 syndrome (BEPS) Brugada sindrome - Long QT syndrome-3 SCN5A Bruton agammaglobulinemia tyrosine kinase BTK Ceroid lipofuscinosis neuronal type 2 CLN2 Charcot Marie Tooth type 1A (CMT1A) PMP22 Charcot Marie Tooth type X (CMTX) CMTX RNG043-WO1 PCT Application Genetic disease Disease gene Chronic granulomatous disease (CGD) CYBB Cystic Fibrosis (CF) CFTR Congenital adrenal hyperplasia (CAH) CYP21A2 Congenital disorder of glycosylation type Ia (CDG Ia) PMM2 Congenital fibrosis of extraocular muscles 1 (CFEOM1) KIF21A Crigler-Najjar syndrome UGT1A1 Deafness, autosomal recessive CX26 Diamond-Blackfan anemia (DBA) RPS19 Duchenne-Becker muscular dystrophy (DMD/DMB) DMD Duncan disease - X-linked lymphoproliferative SH2D1A syndrome (XLPD) Ectrodactyly ectodermal dysplasia and cleft lip/palate p63 syndrome (EEC) Epidermolysis bullosa dystrophica/pruriginosa COL7A1 Exostoses multiple type I (EXT1) EXT1 Exostoses multiple type II (EXT2) EXT2 Facioscapulohumeral muscular dystrophy FRG1 Factor VII deficiency F7 Familial Mediterranean Fever (FMF) MEFV Fanconi anemia A FANCA Fanconi anemia G FANCG Fragile-X FRAXA Gangliosidosis (GM1) GLB1 Gaucher disease (GD) GBA RNG043-WO1 PCT Application Genetic disease Disease gene Glanzmann thrombasthenia ITGA2B Glucose-6-phosphate dehydrogenase deficiency G6PD Glutaric acidemia I GCDH Haemophilia A F8 Haemophilia B F9 Hand-foot-uterus syndrome HOXD13 Hemophagocytic lymphohistiocytosis familial, type 2 PRF1 (FHL2) Hypomagnesaemia primary CLDN16 HYPOPHOSPHATASIA ALPL Holt-Oram Sindrome (HOS) TBX5 Homocystinuria MTHFR Incontinentia pigmenti NEMO Lesch-Nyhan syndrome HPRT Limb-girdle muscular dystrophy type 2C (LGMD2C) SGCG Long QT syndrome-1 KCNQ1 Mannosidosis Alpha MAN2B1 Marfan syndrome FBN1 Methacrylic Aciduria, deficiency of beta- HIBCH hydroxyisobutyryl-CoA deacylase Mevalonic aciduria MVK Myotonic dystrophy (DM) DMPK Myotonic dystrophy type 2 (DM2) ZNF9 Mucopolysaccharidosis Type I - Hurler syndrome IDUA RNG043-WO1 PCT Application Genetic disease Disease gene Mucopolysaccharidosis Type IIIA - Sanfilippo SGSH sindrome A (MPS3A) Mucopolysaccharidosis Type IIIB - Sanfilippo NAGLU sindrome B (MPS3B) Mucopolysaccharidosis Type VI (MPS VI) - ARSB Maroteaux-Lamy Syndrome Neuronal ceroid lipofuscinosis 1 - Batten's disease PPT1 (CLN1) Niemann-Pick disease SMPD1 Noonan sindrome PTPN11 Pancreatitis, hereditary (PCTT) PRSS1 Paramyotonia congenita (PMC) SCN4A Phenylketonuria PAH Polycystic kidney disease type 1 (PKD1) PKD1 Polycystic kidney disease type 2 (PKD2) PKD2 Polycystic kidney and hepatic disease-1 (ARPKD) PKHD1 Schwartz-Jampel/Stuve-Wiedemann syndrome LIFR Sickle cell anemia HBB Synpolydactyly (SPD1) HOXA13 Smith-Lemli-Opitz syndrome DHCR7 Spastic paraplegia type 3 SPG3A Spinal Muscular Atrophy (SMA) SMN Spinocerebellar ataxia 3 (SCA3) ATXN3 Spinocerebellar ataxia 7 (SCA7) ATXN7 Stargardt disease ABCA4 RNG043-WO1 PCT Application Genetic disease Disease gene Tay Sachs (TSD) HEXA Thalassemia-α mental retardation syndrome ATRX Thalassemia-β HBB Torsion dystonia, early onset (EOTD) DYT1 Tyrosinaemia type 1 FAH Tuberosclerosis 1 TSC1 Tuberosclerosis 2 TSC2 Wiskott-Aldrich Sindrome (WAS) WAS [00762] In addition, the gene editing systems disclosed herein may also be used to treat the following genetic disorders by editing a defect in the disease-associated gene, or in more than one gene associated with a particular disorders, as follows: A B C D E F f t
Figure imgf000208_0001
RNG043-WO1 PCT Application A B C D E F Disease- Most Encoded Type of t n n ia s. of
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RNG043-WO1 PCT Application A B C D E F Disease- Most Encoded Type of t ed
Figure imgf000210_0001
RNG043-WO1 PCT Application A B C D E F Disease- Most Encoded Type of t n e a e l i l
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RNG043-WO1 PCT Application A B C D E F Disease- Most Encoded Type of t or y or d l r n- ei r
Figure imgf000212_0001
RNG043-WO1 PCT Application A B C D E F Disease- Most Encoded Type of t r A in
Figure imgf000213_0001
RNG043-WO1 PCT Application A B C D E F Disease- Most Encoded Type of t r r o e -
Figure imgf000214_0001
RNG043-WO1 PCT Application A B C D E F Disease- Most Encoded Type of t n e or n
Figure imgf000215_0001
RNG043-WO1 PCT Application A B C D E F Disease- Most Encoded Type of t l ul
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RNG043-WO1 PCT Application A B C D E F Disease- Most Encoded Type of t l r
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RNG043-WO1 PCT Application A B C D E F Disease- Most Encoded Type of t l n e n
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RNG043-WO1 PCT Application A B C D E F Disease- Most Encoded Type of t or n e n n e l
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RNG043-WO1 PCT Application A B C D E F Disease- Most Encoded Type of t l or o 1 or e io
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RNG043-WO1 PCT Application A B C D E F Disease- Most Encoded Type of t n e
Figure imgf000221_0001
, , s of the present disclosure, one or more targeted polynucleotide sequence related to certain diseases and disorders, e.g., a genetic mutation, is contacted by a retron-based gene editing system disclosed herein; and a guide RNA, wherein the guide RNA comprises a complementary sequence to that of a targeted polynucleotide sequence. [00764] In some embodiments, the guide RNA directs a programmable nuclease of a retron-based editing system to the target site or the targeted polynucleotide sequence; and optionally forms a ribonucleoprotein complex with the polypeptide and the guide RNA. [00765] Additional therapeutic applications for the genome editing systems disclosed herein include base editing, prime editing, gene insertions and/or deletions. [00766] Diagnostic applications for the genome editing system include probes, diagnostics, theranostics. [00767] The editing system comprising the heterologous nucleic acid sequence can be used in a variety of applications, several non-limiting examples of which are described herein. In general, the editing system can be used in any suitable organism. In some embodiments, the organism is a eukaryote. [00768] In some embodiments, the organism is an animal. In some embodiments, the animal is a fish, an amphibian, a reptile, a mammal, or a bird. In some embodiments, the animal is a farm animal or agriculture animal. Non-limiting examples of farm and agriculture animals include horses, goats, sheep, swine, cattle, llamas, alpacas, and birds, e.g., chickens, turkeys, ducks, and geese. In some embodiments, the animal is a non-human primate, e.g., RNG043-WO1 PCT Application baboons, capuchin monkeys, chimpanzees, lemurs, macaques, marmosets, tamarins, spider monkeys, squirrel monkeys, and vervet monkeys. In some embodiments, the animal is a pet. Non-limiting examples of pets include dogs, cats, horses, rabbits, ferrets, gerbils, hamsters, chinchillas, fancy rats, guinea pigs, canaries, parakeets, and parrots. [00769] In some embodiments, the organism is a plant. Plants that may be transfected with an Cas12a editing system include monocots and dicots. Particular examples include, but are not limited to, corn (maize), sorghum, wheat, sunflower, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape, Brassica sp., alfalfa, rye, millet, safflower, peanuts, sweet potato, cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, oats, vegetables, ornamentals, and conifers. Vegetables include, but are not limited to, crucifers, peppers, tomatoes, lettuce, green beans, lima beans, peas, and members of the genus Cucumis such as cucumber, cantaloupe, and musk melon. Ornamentals include, but are not limited to, azalea, hydrangea, hibiscus, roses, tulips, daffodils, petunias, carnation, poinsettia, and chrysanthemum. [00770] In some embodiments, heterologous nucleic acid sequences can be added to the subject editing system to provide a cell with a heterologous nucleic acid encoding a protein or regulatory RNA of interest, a cellular barcode, a donor polynucleotide suitable for use in gene editing, e.g., by homology directed repair (HDR) or recombination-mediated genetic engineering (recombineering), or a CRISPR protospacer DNA sequence for use in molecular recording, as discussed further below. In embodiments relating to retron-based gene editing systems, such heterologous sequences may be inserted, for example, into the msr locus or the msd locus such that the heterologous sequence is transcribed by the retron reverse transcriptase as part of the msDNA product. [00771] In some embodiments, the editing systems described herein may be used for research tools, such as kits, functional genomics assays, and generating engineered cell lines and animal models for research and drug screening. The kit may comprise one or more reagents in addition to the editing system, such as a buffer, a control reagent, a control vector, a control RNA polynucleotide, a reagent for in vitro production of the polypeptide from DNA, and adaptors for sequencing. A buffer can be, for example, a stabilization buffer, a reconstituting buffer, a diluting buffer, a wash buffer, or a buffer for introducing a polypeptide and/or polynucleotide of the kit into a cell. In some instances, a kit can comprise one or more additional reagents specific for plants. One or more additional reagents for plants can include, RNG043-WO1 PCT Application for example, soil, nutrients, plants, seeds, spores, Agrobacterium, a T-DNA vector, and a pBINAR vector. Therapeutic Applications [00772] Also provided herein are methods of diagnosing, prognosing, treating, and/or preventing a disease, state, or condition in or of a subject, using the engineered gene editing systems of the present disclosure. [00773] Generally, the methods of diagnosing, prognosing, treating, and/or preventing a disease, state, or condition in or of a subject can include modifying a polynucleotide in a subject or cell thereof using a composition, system, or component thereof of the engineered retron as described herein, and/or include detecting a diseased or healthy polynucleotide in a subject or cell thereof using a composition, system, or component thereof of the engineered retron as described herein. [00774] In some embodiments, the method of treatment or prevention can include using a composition, system, or component of the engineered retron to modify a polynucleotide of an infectious organism (e.g. bacterial or virus) within a subject or cell thereof. [00775] In some embodiments, the method of treatment or prevention can include using a composition, system, or component of the engineered retron to modify a polynucleotide of an infectious organism or symbiotic organism within a subject. [00776] In some embodiments, the composition, system, and components of the engineered retron can be used to develop models of diseases, states, or conditions. [00777] In some embodiments, the composition, system, and components of the engineered retron can be used to detect a disease state or correction thereof, such as by a method of treatment or prevention described herein. [00778] In some embodiments, the composition, system, and components of the engineered retron can be used to screen and select cells that can be used, for example, as treatments or preventions described herein. [00779] In some embodiments, the composition, system, and components thereof can be used to develop biologically active agents that can be used to modify one or more biologic functions or activities in a subject or a cell thereof. [00780] In general, the method can include delivering a composition, system, and/or component of the engineered retron to a subject or cell thereof, or to an infectious or symbiotic organism by a suitable delivery technique and/or composition. Once administered, the components can operate as described elsewhere herein to elicit a nucleic acid modification event. In some embodiments, the nucleic acid modification event can occur at the genomic, RNG043-WO1 PCT Application epigenomic, and/or transcriptomic level. DNA and/or RNA cleavage, gene activation, and/or gene deactivation can occur. [00781] The composition, system, and components of the engineered retron as described elsewhere herein can be used to treat and/or prevent a disease, such as a genetic and/or epigenetic disease, in a subject; to treat and/or prevent genetic infectious diseases in a subject, such as bacterial infections, viral infections, fungal infections, parasite infections, and combinations thereof; to modify the composition or profile of a microbiome in a subject, which can in turn modify the health status of the subject; to modify cells ex vivo, which can then be administered to the subject whereby the modified cells can treat or prevent a disease or symptom thereof; or to treat mitochondrial diseases, where the mitochondrial disease etiology involves a mutation in the mitochondrial DNA. [00782] Also provided is a method of treating a subject, e.g., a subject in need thereof, comprising inducing gene editing by transforming the subject with the polynucleotide encoding one or more components of the composition, system, or complex or any of polynucleotides or vectors described herein of the engineered retron, and administering them to the subject. [00783] Also provided is a method of treating a subject, e.g., a subject in need thereof, comprising inducing transcriptional activation or repression of multiple target gene loci by transforming the subject with the polynucleotides or vectors described herein, wherein said polynucleotide or vector encodes or comprises one or more components of composition, system, complex or component of the engineered retron, and comprising multiple Cas effectors. [00784] Also provided is a method of treating a subject, e.g., a subject in need thereof, comprising inducing gene editing by transforming the subject with the Cas effector(s), and encoding and expressing in vivo the remaining portions of the composition, system, (e.g., RNA, guides), complex or component of the engineered retron. A suitable repair template may also be provided by the engineered retron as described herein elsewhere. [00785] Also provided is a method of treating a subject, e.g., a subject in need thereof, comprising inducing transcriptional activation or repression by transforming the subject with the systems or compositions herein. [00786] Also provided is a method of inducing one or more polynucleotide modifications in a eukaryotic or prokaryotic cell or component thereof (e.g. a mitochondria) of a subject, infectious organism, and/or organism of the microbiome of the subject. The modification can include the introduction, deletion, or substitution of one or more nucleotides RNG043-WO1 PCT Application at a target sequence of a polynucleotide of one or more cell(s). The modification can occur in vitro, ex vivo, in situ, or in vivo. [00787] In some embodiments, the method of treating or inhibiting a condition or a disease caused by one or more mutations in a genomic locus in a eukaryotic organism or a non- human organism can include manipulation of a target sequence within a coding, non-coding or regulatory element of said genomic locus in a target sequence in a subject or a non-human subject in need thereof comprising modifying the subject or a non -human subject by manipulation of the target sequence and wherein the condition or disease is susceptible to treatment or inhibition by manipulation of the target sequence including providing treatment comprising delivering a composition comprising the particle delivery system or the delivery system or the virus particle of any one of the above embodiment or the cell of any one of the above embodiment. [00788] Also provided herein is the use of the particle delivery system or the delivery system or the virus vector (in viral particle) of any one of the above embodiment or the cell of any one of the above embodiment in ex vivo or in vivo gene or genome editing; or for use in in vitro, ex vivo or in vivo gene therapy. [00789] Also provided herein are particle delivery systems, non-viral delivery systems, and/or the virus particle of any one of the above embodiments or the cell of any one of the above embodiments used in the manufacture of a medicament for in vitro, ex vivo or in vivo gene or genome editing or for use in in vitro, ex vivo or in vivo gene therapy or for use in a method of modifying an organism or a non-human organism by manipulation of a target sequence in a genomic locus associated with a disease or in a method of treating or inhibiting a condition or disease caused by one or more mutations in a genomic locus in a eukaryotic organism or a non- human organism. [00790] In some embodiments, target polynucleotide modification using the subject engineered retron and the associated composition, vectors, system and methods comprises addition, deletion, or substitution of 1-about 10k nucleotides at each target sequence of said polynucleotide of said cell(s). The modification can include the addition, deletion, or substitution of at least 1, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, 100, 200, 250, 300, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 5000, 6000, 7000, 8000, 9000, 10,000 or more nucleotides at each target sequence. [00791] In some embodiments, formation of system or complex results in cleavage, nicking, and/or another modification of one or both strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence. RNG043-WO1 PCT Application [00792] In some embodiments, a method of modifying a target polynucleotide in a cell to treat or prevent a disease can include allowing a composition, system, or component of the subject engineered retron to bind to the target polynucleotide, e.g., to effect cleavage, nicking, or other modification as the composition, system, is capable of said target polynucleotide, thereby modifying the target polynucleotide, wherein the composition, system, or component thereof, complex with a guide sequence, and hybridize said guide sequence to a target sequence within the target polynucleotide, wherein said guide sequence is optionally linked to a tracr mate sequence, which in turn can hybridize to a tracr sequence. In some embodiments, modification can include cleaving or nicking one or two strands at the location of the target sequence by one or more components of the composition, system, or component thereof. [00793] In some embodiments, the engineered retron and the associated compositions, systems, vectors, uses, and methods of use, can be used to treat diseases of the circulatory system. In some embodiments, the treatment can be carried out by using an AAV or a lentiviral vector to deliver the engineered retron, composition, system, and/or vector described herein to modify hematopoietic stem cells (HSCs) or iPSCs in vivo or ex vivo. In some embodiments, the treatment can be carried out by correcting HSCs or iPSCs as to the disease using a composition, system, herein or a component thereof, wherein the composition, system, optionally includes a suitable HDR repair template (e.g., a template in the msDNA of the engineered retron). [00794] In some embodiments, the treatment or prevention for treating a circulatory system or blood disease can include modifying a human cord blood cell. In some embodiments, the treatment or prevention for treating a circulatory system or blood disease can include modifying a granulocyte colony-stimulating factor-mobilized peripheral blood cell (mPB) with any modification described herein. In some embodiments, the human cord blood cell or mPB can be CD34+. In some embodiments, the cord blood cells or mPB cells modified are autologous. In some embodiments, the cord blood cells or mPB cells are allogenic. In addition to the modification of the disease genes, allogenic cells can be further modified using the composition, system, described herein to reduce the immunogenicity of the cells when delivered to the recipient. The modified cord blood cells or mPB cells can be optionally expanded in vitro. The modified cord blood cell(s) or mPB cells can be derived to a subject in need thereof using any suitable delivery technique. [00795] The composition and system may be engineered to target genetic locus or loci in HSCs. In some embodiments, the components of the systems can be codon-optimized for a eukaryotic cell and especially a mammalian cell, e.g., a human cell, for instance, HSC, or iPSC RNG043-WO1 PCT Application and sgRNA targeting a locus or loci in HSC, such as circulatory disease, can be prepared. These may be delivered via particles, such as the lipid nanoparticle delivery system described herein. The particles may be formed by the components of the systems herein being admixed. [00796] In some embodiments, after ex vivo modification the HSCs or iPCS can be expanded prior to administration to the subject. In some examples, expansion of HSCs can be via any suitable method such as that described by, Lee, “Improved ex vivo expansion of adult hematopoietic stem cells by overcoming CUL4-mediated degradation of HOXB4.” Blood. 2013 May 16;121(20):4082-9. doi: 10.1182/blood-2012-09-455204. Epub 2013 Mar 21. [00797] In some embodiments, the HSCs or iPSCs modified are autologous. In some embodiments, the HSCs or iPSCs are allogenic. In addition to the modification of the disease genes, allogenic cells can be further modified using the composition, system, described herein to reduce the immunogenicity of the cells when delivered to the recipient. [00798] In some embodiments, the engineered retron and the associated compositions, systems, vectors, uses, and methods of use, can be used to treat neurological diseases. In some embodiments, the neurological diseases comprise diseases of the brain and CNS. [00799] Delivery options for the diseases in the brain include encapsulation of the systems in the form of either DNA or RNA into liposomes and conjugating to molecular Trojan horses for trans-blood brain barrier (BBB) delivery. Molecular Trojan horses have been shown to be effective for delivery of B-gal expression vectors into the brain of non-human primates. The same approach can be used to delivery vectors or vector systems of the present disclosure. In other embodiments, an artificial virus can be generated for CNS and/or brain delivery. [00800] In some embodiments, the engineered retron and the associated compositions, systems, vectors, uses, and methods of use, can be used to treat hearing diseases or hearing loss in one or both ears. Deafness is often caused by lost or damaged ear cells that cannot relay signals to auditory neurons. In some examples, the composition, system, or modified cells can be delivered to one or both ears for treating or preventing hearing disease or loss by any suitable method or technique, such as described in US20120328580 (e.g., auricular administration), by intratympanic injection (e.g., into the middle ear), and/or injections into the outer, middle, and/or inner ear; administration in situ, via a catheter or pump as described in U.S.2006/0030837), Jacobsen (U.S. Pat. No.7,206,639), and US20120328580. Cells resulting from such methods can then be transplanted or implanted into a patient in need of such treatment. RNG043-WO1 PCT Application [00801] In some embodiments, the engineered retron and the associated compositions, systems, vectors, uses, and methods of use, can be used to treat diseases in non-dividing cells. Exemplary non-dividing cells include muscle cells or neurons. In some examples, in such cells, homologous recombination (HR) is generally suppressed in the G1 cell-cycle phase, but can be turned back on using art-recognized methods, such as those described in Orthwein et al. (Nature.2015 Dec 17; 528(7582): 422–426). [00802] In some embodiments, the engineered retron and the associated compositions, systems, vectors, uses, and methods of use, can be used to treat diseases of the eye. [00803] In some embodiments, the engineered retron and the associated compositions, systems, vectors, uses, and methods of use, can be used to treat muscle diseases and cardiovascular diseases. [00804] In some embodiments, the engineered retron and the associated compositions, systems, vectors, uses, and methods of use, can be used to treat diseases of the liver and kidney. [00805] In some embodiments, the engineered retron and the associated compositions, systems, vectors, uses, and methods of use, can be used to treat epithelial and lung diseases. [00806] In some embodiments, the engineered retron and the associated compositions, systems, vectors, uses, and methods of use, can be used to treat diseases of the skin. [00807] In some embodiments, the engineered retron and the associated compositions, systems, vectors, uses, and methods of use, can be used to treat cancer. [00808] In some embodiments, the engineered retron and the associated compositions, systems, vectors, uses, and methods of use, can be used in adoptive cell therapy. [00809] In some embodiments, the engineered retron and the associated compositions, systems, vectors, uses, and methods of use, can be used to treat infectious diseases. [00810] In some embodiments, the engineered retron and the associated compositions, systems, vectors, uses, and methods of use, can be used to treat mitochondrial diseases. [00811] All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference. INCORPORATION BY REFERENCE [00812] In one aspect, this instant disclosure relates to a chimeric gene editing system that combine elements of prime editors and elements of retrons. Accordingly, this application references and incorporates by reference in their entireties the following applications relating RNG043-WO1 PCT Application to retrons: U.S. Application No.63/301,936, filed January 21, 2022; U.S. Application No. 63/370,880, filed August 9, 2022; U.S. Application No.63/373,545, filed August 25, 2022; U.S. Application No.18/087,673, filed December 22, 2022 (and published as U.S. Published Application No. US 2023/0235365 A1 on July 27, 2023); U.S. Application No.63/476,900, filed December 22, 2022; International Application No. PCT/US2023/061038, filed January 20, 2023 (which published as WO 2023/141602 on July 27, 2023), U.S. Application No. 63/488,317, filed March 3, 2023, U.S. Application No.63/491,603, filed March 22, 2023; U.S. Application No.63/515,783, filed July 26, 2023; and International Application No. PCT/US2023/072872, filed August 24, 2023, each of which are incorporated herein by reference in their entireties. [00813] In addition, this application references and incorporates by reference in their entireties the following applications and patents relating to prime editors, components thereof, or uses thereof: International Application No. PCT/US2022/074628, filed August 5, 2022; International Application No. PCT/US2022/074088, filed July 23, 2022; International Application No. PCT/US2022/073819, filed July 16, 2022; International Application No. PCT/US2022/035613, filed June 29, 2022; International Application No. PCT/US2022/036230, filed July 6, 2022; International Application No. PCT/US2022/032267, filed June 3, 2022, European Application No. EP21707651.2, filed February 19, 2021; International Application No. PCT/EP2022/062223, filed May 5, 2022; U.S. Application No. 17/219,635, filed March 31, 2021; International Application No. PCT/CN2022/080595, filed March 14, 2022; International Application No. PCT/US2022/023175, filed April 1, 2022; International Application No. PCT/US2022/021879, filed March 25, 2022; International Application No. PCT/US2022/020392, filed March 15, 2022; U.S. Application No. 17/219,672, filed March 31, 2021, now U.S. Patent No.11,447,770, issued September 20, 2022; International Application No. PCT/CN2022/077097, filed February 21, 2022; International Application No. PCT/US2022/015260, filed February 4, 2022; International Application No. PCT/KR2022/001611, filed January 28, 2022; International Application No. PCT/IN2022/050017, filed January 7, 2022; International Application No. PCT/US2022/012054, filed January 11, 2022; U.S. Application No.17/427,040, filed July 29, 2021, now U.S. Patent No.11,384,353, issued July 12, 2022; International Application No. PCT/US2021/052097, filed September 24, 2021; International Application No. PCT/KR2021/017534, filed November 25, 2021; International Application No. PCT/CN2021/130059, filed November 11, 2021; International Application No. PCT/US2021/057908, filed November 3, 2021; International Application No. RNG043-WO1 PCT Application PCT/US2021/058079, filed November 4, 2021; International Application No. PCT/KR2021/013326, filed September 29, 2021; International Application No. PCT/US2021/052097, filed September 24, 2021; International Application No. PCT/KR2021/010740, filed August 12, 2021; U.S. Application No.17/427,040, filed July 29, 2021; International Application No. PCT/US2021/044924, filed August 6, 2021; International Application No. PCT/KR2021/009794, filed July 28, 2021; International Application No. PCT/US2021/031439, filed May 7, 2021; International Application No. PCT/US2021/034996, filed May 28, 2021; International Application No. PCT/KR2021/005244, filed April 26, 2021; International Application No. PCT/KR2021/005031, filed April 21, 2021; International Application No. PCT/US2020/023730, filed March 19, 2020; International Application No. PCT/US2020/023713, filed March 19, 2020; International Application No. PCT/EP2021/054228, filed February 19, 2021; International Application No. PCT/US2020/067535, filed December 30, 2020; International Application No. PCT/US2020/059149, filed November 5, 2020; International Application No. PCT/US2020/055959, filed October 16, 2020; International Application No. PCT/US2020/055156, filed October 9, 2020; International Application No. PCT/US2020/023553, filed March 19, 2020; International Application No. PCT/US2020/023583, filed March 19, 2020; International Application No. PCT/US2020/023730, filed March 19, 2020; International Application No. PCT/US2020/023721, filed March 19, 2020; International Application No. PCT/US2020/023728, filed March 19, 2020; International Application No. PCT/US2020/023732, filed March 19, 2020; International Application No. PCT/US2020/023712, filed March 19, 2020; International Application No. PCT/US2020/023725, filed March 19, 2020; International Application No. PCT/US2020/023713, filed March 19, 2020; International Application No. PCT/US2020/023727, filed March 19, 2020; International Application No. PCT/US2020/023724, filed March 19, 2020; International Application No. PCT/US2020/023583, filed March 19, 2020; International Application No. PCT/US2020/023723, filed March 19, 2020; International Application No. PCT/CN2020/074218, filed February 3, 2020; U.S. Application No.15/616,756, filed June 7, 2017, now U.S. Patent No.10,189,831, issued January 29, 2019; International Application No. PCT/US2018/042040, filed July 13, 2018; U.S. Application No.15/164,208, filed May 25, 2016, now U.S. Patent No.10,150,955, issued December 11, 2018; International Application No. PCT/US2017/050690, September 8, 2017; U.S. Application No.11/502,819, filed August RNG043-WO1 PCT Application 10, 2006, now U.S. Patent No.9,783,791, issued October 10, 2017; and U.S. Application No. 13/277,763, filed October 20, 2011, now U.S. Patent No.9,458,484, issued October 4, 2016.The foregoing applications, and all documents cited therein or during their prosecution (“appln cited documents”) and all documents cited or referenced in the appln cited documents, and all documents cited or referenced herein (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the present disclosure. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. EXAMPLES Example 1: Production of nanoparticle compositions [00814] A nanoparticle composition comprising a stabilized RNA molecule described herein is produced as described in US patent application US20170210697A1, or PCT Publications WO2023044343A1, WO2023044333A1, and WO2023122752A1, each of which is incorporated herein by reference in its entirety. In order to investigate safe and efficacious nanoparticle compositions for use in the delivery of various payloads, including but not limited to the herein disclosed nickase-retron editing systems, a range of formulations are prepared and tested. Specifically, the particular elements and ratios thereof in the lipid component of nanoparticle compositions are optimized. Nanoparticles are made with mixing processes such as microfluidics and T-junction mixing of two fluid streams, one of which contains the genome editing system and the other has the lipid components. Lipid compositions are prepared by combining an ionizable lipid, a phospholipid (such as DOPE or DSPC, obtainable from Avanti Polar Lipids, Alabaster, Ala.), a PEG lipid (such as PEG-DMG, obtainable from Avanti Polar Lipids, Alabaster, Ala.), and a structural lipid (such as cholesterol, obtainable from Sigma-Aldrich, Taufkirchen, Germany, or a cholesterol analog) in ethanol. Lipids are combined to yield desired molar ratios and diluted with water and ethanol. Nanoparticle compositions can be prepared by combining a lipid solution with a solution including the genome editing system. The lipid solution is rapidly injected using, for example, a NanoAssemblr® microfluidic based system, into the genome editing system solution. Solutions of the genome editing system in deionized water can be diluted in citrate buffer or acetate buffer to form a stock solution. Nanoparticle compositions are processed by dialysis to remove RNG043-WO1 PCT Application ethanol and achieve buffer exchange. Formulations are dialyzed against a buffer such as phosphate buffered saline (PBS), Tris-HCl, or sodium citrate, using, for example, Slide-A- Lyzer cassettes (Thermo Fisher Scientific Inc., Rockford, Ill.). The resulting nanoparticle suspension is filtered through sterile filters (Sarstedt, Nümbrecht, Germany) into glass vials and sealed with crimp closures. Alternatively, a Tangential Flow Filtration (TFF) system, such as a Spectrum KrosFlo system, can be used. The method described above induces nano-precipitation and particle formation. Alternative processes including, but not limited to, T-junction and direct injection, can be used to achieve the same nano- precipitation. Example 1a: Exemplary Nanoparticle Formulation Procedure [00815] Ionizable lipids, phospholipids, structural lipids (eg. Cholesterol or other sterols), PEG lipids, and optionally any additional lipid components, including but not limited to targeting ligands and lipids comprising the same, are dissolved in ethanol. The ionizable lipids mole percent (mol %) is from 30-70%, phospholipids mol % can be 5- 20%, sterols mol % can be 20-60%, and PEG lipid mol % can be 0.1-10%. The lipid solution is mixed with an acidic buffer containing genome editing system on a mixing device, such as a NanoAssemblr® microfluidic systems, to form LNPs. To adjust LNP particle size, the volume ratio of lipid solution to genome editing system solution is varied from 1:1 to 20:1, genome editing system concentration in aqueous buffer is from 0.01 mg/mL to 10 mg/mL, N/P ratio is from 1 to 50, and different identities of PEG lipids or other polymers can be used. Flow rate of the mixing streams is also adjusted to alter particle size. After the LNP is formed from the mixing device, aqueous buffer is added to reduce the ethanol concentration. The volume of aqueous buffer is from 0.1 to 100 x volume of LNP solution volume coming out of the mixing device. The LNPs are further dialyzed against aqueous solutions and concentrated to a desired concentration. The particle size of LNPs is measured by dynamic light scattering (DLS), for example, by using a Zetasizer Ultra (Malvern Panalytical). Payload encapsulation efficiency is determined, for example, by Quant-itTM RiboGreen assay. Example 2. Retron ncRNA sequence optimization via high-throughput screening of ncRNA variant library RNG043-WO1 PCT Application [00816] Retrons are native to prokaryotes and hence, the ncRNA sequences can be further engineered to accommodate the application of the retrons in human cells and maximize the efficiency of the retrons in human cells. [00817] To understand and optimize the editing efficiency by retron ncRNA in human cells, a pooled ncRNA library with 191 variants (sequences provided in Table 31A of PCT Publication WO2024044723A1; SEQ ID NOs: 19543-19733 of said PCT, which is incorporated by reference herein in its entirety) plus 2 controls (a positive and negative control) of different lengths and sequences in several key elements (such as, but not limited to, modifications to the lengths of the a1:a2 stem and the msR stem-loop) was developed. Each of these variants was associated with unique barcodes designed in the donor region, and the variant library was synthesized as a pooled oligo library. The synthesized oligo library was cloned into a pooled DNA library, from which pooled ncRNAs were produced by in vitro transcription. RNA library was then transfected to the 293T cell line and cells were harvested at different timepoints post-transfection. Next-generation sequencing was then applied to detect the unique barcodes to measure the abundance of each ncRNA variant, production of the ssDNA donor, and the precise insertion at the edited locus. Through comparing these data sets, the stability and efficacy of each ncRNA variant was scored. From the scoring insight, enhanced ncRNA was optimized/engineered by integrating the most effective modified features. Methods: [00818] 191 ncRNA variants were designed with variations introduced in one or more of the following ncRNA elements: (1) a1a2; (2) msD stem; (3) msR stem; (4) msR loop; (5) termination sequence (within msR spacer region); (6) msD, msR spacer deletions (minimal variants). In addition, one positive control (WT R6342S ncRNA) and a negative control with disrupted reverse transcription priming sites was included. Each of these variants was associated with several unique barcodes (e.g., “ACCTATCATTCANNNNNNNN” (SEQID NO: 19735)) designed in the donor region which targeted the human EMX1 gene. The variant library was synthesized as a pooled oligo library. The synthesized oligo library was assembled into a pooled DNA library, from which pooled ncRNAs were produced by in vitro transcription. Genomic insertion activity vs ssDNA production is shown in FIG.13. Melting temperature (Tm) of different a1a2 variants in the pooled library were measured and plotted against ssDNA production, as shown in FIG.14. Validation of ncRNA pooled screening RNG043-WO1 PCT Application [00819] From the pooled screen assay, a few a1:a2 variants were identified that showed higher genomic insertion than WT R6342. In addition, it was shown that the msR stem sequence is intolerant to changes, and that there were certain non-essential elements in retron ncRNA that can be deleted, such as msR spacer, msD spacer, while the full or near full activity of the retron was retained. The performance of these variants is shown in FIG.13, which displays single strand DNA production on the x-axis and genomic integration at the target site on the y-axis, compared with WT (represented by the dotted lines). [00820] Several msR stem variants were selected from the pooled screen and an EMX1 25bp insert was used as the donor template sequence. ncRNA were produced by in vitro transcription. Purified ncRNAs were co-transfected into the HEK 293T cells using Lipofectamine MessengerMAX along with R6342 reverse transcriptase mRNA, Cas9 mRNA, and sgRNA targeting EMX1 locus. Three days post transfection, cell lysates were collected, and amplicon based NGS was performed to evaluate editing efficiency. Indels and editing were analyzed by CRISPResso2. As shown in FIG.16, three msR variants with modified stem sequences (the stem sequences remained complementary), were completely deficient of editing. They were even lower than a priming guanosine mutant ncRNA (brance guanosine mutant) that was used as a negative control. Such lack of the ncRNA activity was consistent with the pooled screen study and suggested that the msR stem sequence was highly important for an active retron ncRNA to function. [00821] Unlike msR stem, variations in the sequence but not the complementarity in a1a2 stem were well tolerated in the pooled screen study. To validate this result, a few a1a2 variants that exhibited different levels of editing efficiency from the pooled study were selected, with individual ncRNA with EMX125bp inserted as the template. These a1a2 variants exhibited different levels of editing activities when compared to WT R6342 ncRNA (FIG.17). Most of the a1a2 variants performed on a similar level as the WT, validating that the a1a2 stem structure, not the sequence, was required for the editing output of the ncRNA. a1a2_var15, in particular, a ncRNA that had exhibited higher editing efficiency in the pooled screen, had an appreciable increase in editing efficiency. [00822] Additionally, it was tested whether certain elements such as msR spacer and msD spacers were non-essential for the ncRNA activity and can be removed. Deletions were introduced that removed most of the msD/msR spacer sequence. Deleting the msR spacer (Del1) resulted in a greater overall edit percentage, as well as a greater percentage of exact insert of the donor template sequence into the target gene compared to either SNP inserts or indels inserts (FIG.15). Deleting the msR and msD spacer (Del1+Del2) resulted in a RNG043-WO1 PCT Application comparable overall edit rate as compared to the WT construct, while also yielded a slightly higher percentage of exact inserts of the donor template sequence as compared to either SNP inserts or indel inserts, while also had the advantage of being a much smaller and simpler overall construct. Deleting the msR spacer down to 4 bp alone did not affect the gene editing outcome at all (msR Spacer Del-1). msD deletion variants exhibited lower editing efficiency, partially due to the decrease in indel rate in the cell line (FIG.18). When combined, deletions in both the msR and msD spacer still demonstrated a robust editing outcome, suggesting it was possible to shorten ncRNA at both the msD and msR spacer region without a compromise in function. Without intending to be limited to any particular theory, a much shorter ncRNA, demonstrating similar function would have advantages when it comes to packaging such a construct into a pharmaceutical delivery vehicle for therapeutic use. In summary, the main conclusions from ncRNA pooled screen were validated by individual studies of the ncRNA. Variant DNA sequence (changed region vs WT labelled with lower case) Barcode C N ) C N ) C N )
Figure imgf000235_0001
RNG043-WO1 PCT Application CAGGCCAATGGGGAGGACATCGATGTCACACAACCAAAT ATAAGAATTGTTAGCAAGAAATCTATG (SEQ ID NO: 19663) C N ) C N ) C N ) C N )
Figure imgf000236_0001
RNG043-WO1 PCT Application RTX3_6 AacgcgaaacgctggGCCTTTATGCTGTGGTGTTGCGCCACGGTG ACCTATC 342 a1a GAGATTTGTCAAATACACATCATTAGGTTGCGACAAACGG ATTCANN ) C N ) C N ) C N ) C N )
Figure imgf000237_0001
RNG043-WO1 PCT Application GGCCAATGGGGAGGACATCGATGTCACACAACCAAATAT AAGAATTGTTAGttcagataattcttattagtataac (SEQ ID NO: 19568) C N ) C N )
Figure imgf000238_0001
Variant RNA sequence A A U A A A A C
Figure imgf000238_0002
RNG043-WO1 PCT Application AAUGGGGAGGACAUCGAUGUCACACAACCAAUGUUAGCAA GAAAUCUAUGU (SEQ ID NO: 19940) A A A C A A A U A
Figure imgf000239_0001
p . p [00823] In view of the finding that the structure of the a1a2 stem was important for the ncRNA function, without being limited to any particular theory, it was reasoned that by placing the a1 and a2 stem strands in proximity, they were more likely to be folded into the desired conformation. A fused a1a2 design (ALT, as shown in FIG.19), with both long and short R6342 retron ncRNAs (R6342L and R6342S, respectively), was designed. Such designs yielded additional benefits such that the HDR templates produced from reverse transcription were cleaner due to the absence of the 3’ msD stem sequences. As shown in the editing efficiency (FIG.19, left graph), the alternative design with fused a1a2 loop did not impact editing efficiency for R6342L ncRNA and slightly reduced the editing efficiency of R6342S ncRNA. Overall, the fused a1a2 design was as efficient at editing as WT configuration. [00824] Similar spacer deletions of msR and msD (as described in Example 2, FIG.18) were incorporated into the fused a1a2 retron ncRNA design to test if such deletions were well tolerated by the alternative used a1a2 ncRNA configuration. While such deletions led to reduced editing efficiency as compared to WT R6342 ncRNA, especially in the case when both msD spacer and msR spacer are deleted down to 4 bp, the editing activity of deletion variants RNG043-WO1 PCT Application was higher than the editing activity observed with Branching Guanosine mutant control (FIG. 20). [00825] Alternative ncRNA designs that separate the msD stem loop from the reverse transcription template that encodes HDR ssDNA offered a unique opportunity to test if the msD stem loop can be further deleted. msD stem-loop deletion was combined with a further shortening of the msD spacer (to 1 bp) and a complete removal of the msR spacer sequence in the fused a1a2 ncRNA design. It was observed that this ncRNA (Alt1 msR Spacer Del-2/msD Del-3) only suffered a small negative effect on gene editing (FIG.21). Incorporation of a MS2 stem-loop at 3’ end of the ncRNA significantly reduced the editing efficiency of such ncRNA (Alt1 msR Spacer Del-2/msD Del-3 MS2), while the addition of a 3’ tail from a snRNA, U7, maintained similar editing efficiency (Alt1 msR Spacer Del-2/msD Del-3 U7). In combination, similar editing efficiency to that of the WT was maintained in the ncRNA variants when the msD spacer and stem-loop elements were deleted, in addition to msR spacer elements. [00826] It was then determined how much ssDNA was produced in these short ncRNAs with deletions of the msD, and msR stem-loop. A ddPCR assay was performed measuring the genomic DNA copy from the MALAT1 locus, along with the HDR template produced from reverse transcription activity. Normalizing ssDNA concentration to genomic DNA served as a loose estimate of ssDNA copy number per copy of genome. It was found that such ncRNA variants resulted in a reduction in ssDNA production when compared to the WT R6342 ncRNA (FIG.22). Incorporation of a MS2 stem-loop (Alt1 msR Spacer Del-2/msD Del-3 MS2) boosted the ssDNA production while incorporation of the U7 snRNA 3’ end sequence (Alt1 msR Spacer Del-2/msD Del-3 U7) showed an even higher enhancement of the ssDNA production. It was worth noting that the ssDNA level did not strictly correlate with the editing efficiency, suggesting HDR template generation was an important initiating step but its abundance might not be the rate limiting step in Retron based gene editing. Variant RNA sequence A
Figure imgf000240_0001
RNG043-WO1 PCT Application GGCCAAUGGGGAGGACAUCGAUGUCAAUGCCACAGUAU UGGUGCUACGCUGAAGUGUCACAACCAAAUAUAAGAAU U C U G U G U C U C U C
Figure imgf000241_0001
RNG043-WO1 PCT Application CAAAUAUAAGAAUUGUUAGCAAGAAAUCUAUGAAACAU AGAUUUCUUGGCCUUUAUGCUGUGGUGUUGCGCCACGG U C G Q U C A U C A U C
Figure imgf000242_0001
RNG043-WO1 PCT Application Alt1 msR Spacer Del- GGACAAACGGCAGAAGCUGGAGGAGGAAGGGCCUGAGU 2/msD Del-3 MS2 CCGAGCAGAAGAAAACUCGAGCUCUGAAUGACUCCUAU C U C A
Figure imgf000243_0001
a p e . g ee e e o c s s g ca y p o e e g e c e cy primary hematopoietic stem cells [00827] In view of the findings of Examples 2 and 3 (identifying essential/dispensable structural elements in the retron ncRNA), shortened ncRNA variants were chemically synthesized and both the 5’ and 3’ ends were modified with 2’O methyl groups and phosphorothioate bonds to increase the stability of ncRNA in cells. These variants were applied to primary human hematopoietic stem cell (HSC) editing. Bone marrow derived CD34+ hematopoietic progenitor cells were cultured for three days in the presence of SCF, FLT-3L, TPO, and IL-6 to prevent stem cell differentiation. Then, ncRNA variants were introduced to the HSCs together with R6342 RT mRNA, Cas9 mRNA, and UBA1 targeting sgRNA by electroporation. At 72-hour post electroporation, genomic DNA was harvested and analyzed for edited content at UBA1 target site. [00828] FIG.23 shows the result of M (ATG)>T (ACC) mutation installation in the UBA1 gene with a ncRNA variant with msR spacer deletion in human hematopoietic stem cells (HSC). The donor sequence to install M41>T mutation at exon 3 of UBA1 gene was flanked at both sides by either 30 bp or 45 bp homology arms inserted into wild type (WT) ncRNA or msR spacer deletion ncRNA variant. RNG043-WO1 PCT Application [00829] To inhibit the microhomology mediated end repair (MMEJ) pathway and enhance homology dependent repair (HDR), POLQ siRNA cocktail was included in the cargo for some samples. Upon electroporation, those that received MMEJ inhibitor were also treated with non homologous end joining (NHEJ) inhibitor (AZD7648, 0.5uM) for 24 hours then changed with fresh media. The exact insertion of M>T mutation occurred at 5% editing efficiency with wild type ncRNA with the 30 bp homology arms, and editing efficiency went up to 7.5% with NHEJ/MMEJ inhibition. The msR spacer deletion variant slightly improved editing without the added inhihitors, but the editing was improved significantly (2-fold higher than WT) in the presence of NHEJ/MMEJ inhibition. The editing occurred at even higher efficiency with longer homology arm, exceeding 20% (left graph, FIG.23). In the right graph of FIG.23, the rate of indels is shown at the same conditions as shown in the left graph. [00830] Even shorter ncRNA variants were tested with msR/msD spacer deletions in the alternative configuration (presented in FIG.24) for editing the AAVS1 locus in hematopoietic stem cells. The donor sequence to edit AAVS1 gene was flanked at both sides by 30 bp homology arms and was inserted into wild type (WT) ncRNA or ncRNA variant. FIG.24 shows that minimized ncRNA msR/msD spacer deletions variants in the alternative, fused a1/a2 stem configuration achieved significantly higher editing than WT for both a PAM mutation installation(PAM mt) or 25 bp insertion in the AAVS1 gene. With NHEJ/MMEJ inhibition, the efficiency increased up to 40% editing with the variants possessing the a1/a2 fusion, and both the msR/msD spacer deletions, over 2 fold higher than WT ncRNA version. Variant RNA sequence C A A C U C
Figure imgf000244_0001
RNG043-WO1 PCT Application msR spacer del_45HA CAUAGAUUUCUUGGCCUUUAUGCUGUGGUGUUGCGCCAC GGUGGUAGGUUGCGCCUGUGUGUCUCCCUAAACUUGUUC G A C C A G A C G A C A U
Figure imgf000245_0001
phosphorothioate linkages and 2’-OMe modifications.

Claims

RNG043-WO1 PCT Application WHAT IS CLAIMED: 1. A non-coding RNA (ncRNA) variant comprising a reference retron ncRNA with one or more modifications, wherein the reference retron ncRNA comprises from 5’ to 3’ direction: a1 region, first branching guanosine, msr, msd, and a2 region; and the one or more modifications comprises (i) linkage of the a1 region and the a2 region, (ii) deletion of at least a portion of the msr; (iii) deletion of at least a portion of the msd, (iv) addition of a single-stranded RNA comprising a polymerase template, or (v) addition of an RNA motif. 2. The ncRNA variant of claim 1, wherein the one or more modifications comprise the linkage of the a1 region to the a2 region. 3. The ncRNA variant of claim 1 or 2, wherein the one or more modifications comprises the linkage of the a1 region and the a2 region, wherein the linkage of the a1 region and the a2 region is formed via a linker joining the 5’ end of the a1 region to the 3’ end of the a2 region. 4. The ncRNA variant of claim 1 or 2, wherein the one or more modifications comprises the linkage of the a1 region and the a2 region, wherein the linkage of the a1 region and the a2 region is formed by directly joining the 5’ end of the a1 region to the 3’ end of the a2 region without a linker. 5. The ncRNA variant of any one of claims 1-4, wherein the a1 region and the a2 region are partially or completely complementary to each other and the a1 region and the a2 region form a stem-loop structure after the linkage. 6. The ncRNA variant of any one of claims 1-5, wherein the ncRNA variant further comprises a second branching guanosine. 7. The ncRNA variant of any one of claims 2-6, wherein formation of the linkage of the a1 region and the a2 region circularizes the ncRNA variant. 8. The ncRNA variant of any one of claims 2-7, wherein the one or more modifications RNG043-WO1 PCT Application further comprises a breakage between the msd and the a2 region, thereby generating the ncRNA variant comprising from 5’ to 3’ direction: the a2 region, the linkage between the a1 and the a2 region, the a1 region, the first branching guanosine, the msr, and the msd. 9. The ncRNA variant of any one of claims 1-8, wherein the one or more modifications comprises the deletion of at least a portion of the msr. 10. The ncRNA variant of claim 9, wherein the portion deleted from the msr comprises a spacer between two stem-loops in the msr or a portion of the spacer. 11. The ncRNA variant of claim 10, wherein the portion deleted from the msr comprises a portion of the spacer, whereby the deletion preserves a remaining 1-15 base pairs between the two stem-loops in the msr, as compared to the reference retron ncRNA. 12. The ncRNA variant of claim 11, wherein the deletion preserves a remaining 1 base pair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs, 6 base pairs, 7 base pairs, 8 base pairs, 9 base pairs, 10 base pairs, 11 base pairs, 12 base pairs, 13 base pairs, 14 base pairs, or 15 base pairs between the two stem-loops in the msr, as compared to the reference retron ncRNA. 13. The ncRNA variant of claim 10, wherein the portion deleted from the msr comprises the entirety of the spacer between the two stem-loops in the msr, as compared to the reference retron ncRNA. 14. The ncRNA variant of any one of claims 9-13, wherein the portion deleted from the msr comprises a stem-loop in the msr or a portion thereof. 15. The ncRNA variant of any one of claims 1-14, wherein the one or more modifications comprises the deletion of at least a portion of the msd. 16. The ncRNA variant of claim 15, wherein the deleted portion of the msd comprises a spacer located between a stem-loop in the msd and the a2 region or a portion of the spacer. 17. The ncRNA variant of claim 16, wherein the deleted portion in the msd comprises a RNG043-WO1 PCT Application portion of the spacer located between the stem-loop in the msd and the a2 region, whereby the deletion preserves a remaining 1-15 base pairs between the stem-loop in the msd and the a2 region, as compared to the reference retron ncRNA. 18. The ncRNA variant of claim 17, wherein the deletion preserves a remaining 1 base pair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs, 6 base pairs, 7 base pairs, 8 base pairs, 9 base pairs, 10 base pairs, 11 base pairs, 12 base pairs, 13 base pairs, 14 base pairs, or 15 base pairs between the stem-loop in the msd and the a2 region, as compared to the reference retron ncRNA. 19. The ncRNA variant of claim 16, wherein the deleted portion in the msd comprises the entire spacer between the stem-loop in the msd and the a2 region. 20. The ncRNA variant of any one of claims 16-19, wherein the deleted portion in the msd further comprises the stem-loop in the msd, thereby the deletion comprises deletion of the entire spacer between the stem-loop in the msd and the a2 region and the stem-loop in the msd. 21. The ncRNA variant of any one of claims 1-20, wherein the one or more modifications comprises the addition of the single-stranded RNA comprising the polymerase template. 22. The ncRNA variant of claim 21, wherein the one or more modifications comprise the addition of the single-stranded RNA comprising the polymerase template and the deletion of at least a portion of msd, and the single-stranded RNA comprising the polymerase template is added to the deleted portion of the msd. 23. The ncRNA variant of claim 21 or 22, wherein the single-stranded RNA comprising the polymerase template comprises a pair of homology arms specific to a target locus. 24. The method of claim 23, wherein each of the homology arms comprises 5-200 nucleotides. 25. The method of claim 24, wherein each of the homology arms comprises between 5 and 100 nucleotides, between 5 and 50 nucleotides, between 10 and 50 nucleotides, between 25 and 50 nucleotides, between 5 and 25 nucleotides, between 10 and 25 RNG043-WO1 PCT Application nucleotides, between 10 and 20 nucleotides, between 20 and 30 nucleotides, between 30 and 40 nucleotides, or between 40 to 50 nucleotides. 26. The ncRNA variant of any one of claims 21-25, wherein the single-stranded RNA comprising the polymerase template further comprises a donor sequence for integration into a target locus. 27. The ncRNA of claim 26, wherein the donor sequence comprises 2-100 nucleotides, 2- 50 nucleotides, 2-25 nucleotides, 5-25 nucleotides, 5-15 nucleotides, 5-10 nucleotides, 10-30 nucleotides, 10-20 nucleotides or 1-10 nucleotides. 28. The ncRNA variant of any one of claims 1-27, wherein the one or more modifications comprises the addition of the RNA motif. 29. The ncRNA variant of claim 28, wherein the RNA motif is a MS2 stem-loop or a 3’ tail of U7 small nuclear RNA (snRNA). 30. The ncRNA variant of any one of claims 1-29, wherein the one or more modifications comprise: a. a combination of (i) and (ii): the linkage of the a1 region and the a2 region and the deletion of at least a portion of the msr; b. a combination of (i) and (iii): the linkage of the a1 region and the a2 region and the deletion of at least a portion of the msd; c. a combination of (i) and (iv): the linkage of the a1 region and the a2 region and the addition of the single-stranded RNA comprising the polymerase template; d. a combination of (i) and (v): the linkage of the a1 region and the a2 region and the addition of the RNA motif; e. a combination of (ii) and (iii): the deletion of at least a portion of the msr and the deletion of at least a portion of the msd; f. a combination of (ii) and (iv): the deletion of at least a portion of the msr and the addition of the single-stranded RNA comprising the polymerase template; g. a combination of (ii) and (v): the deletion of at least a portion of the msr and the addition of the RNA motif at the 5’ end or 3’ end; or RNG043-WO1 PCT Application h. a combination of (iv) and (v): the addition of the single-stranded RNA sequence comprising the polymerase template and the addition of the RNA motif at the 5’ end or 3’ end. 31. The ncRNA variant of any one of claims 1-30, wherein the one or more modifications comprise: a. a combination of (i), (ii), and (iii): the linkage of the a1 region and the a2 region, the deletion of at least a portion of the msr, and the deletion of at least a portion of the msd; b. a combination of (i), (ii), and (iv): the linkage of the a1 region and the a2 region, the deletion of at least a portion of the msr, and the addition of the single-stranded RNA comprising the polymerase template; c. a combination of (i), (iii), and (iv): the linkage of the a1 region and the a2 region, the deletion of at least a portion of the msd, and the addition of the single-stranded RNA sequence comprising the polymerase template; or d. a combination of (ii), (iii), and (iv): the deletion of at least a portion of the msr, the deletion of at least a portion of the msd, and the addition of the RNA motif at the 5’ end or 3’ end. 32. The ncRNA variant of any one of claims 1-31, wherein the one or more modifications comprise: (i) the linkage of the a1 region and the a2 region, (ii) the deletion of at least a portion of the msr, (iii) the deletion of at least a portion of the msd, (iv) the addition of the single-stranded RNA sequence comprising the polymerase template, and (v) the addition of the RNA motif at the 5’ end or 3’ end region. 33. The ncRNA variant of any one of claims 1-32, wherein the one or more modifications further comprises substitution, insertion, or deletion of one or more nucleotides, or a combination thereof. 34. The ncRNA variant of claim 33, wherein the substitution, insertion, or deletion is capable of modulating activity of a first polymerase or a second polymerase, production of a single stranded DNA from the ncRNA variant, or immunogenicity of the ncRNA variant. RNG043-WO1 PCT Application 35. The ncRNA variant of any one of claims 1-34, wherein the reference retron ncRNA comprises a sequence of a naturally occurring retron or a portion thereof. 36. The ncRNA variant of any one of claims 1-35, wherein the reference retron ncRNA comprises a sequence selected from SEQ ID NO: 3980-4178, 11231-11429, 4671- 4825, 11922-12075, 4980-5143, 12229-12392, 367-368, 427-441, 494-521, 526-527, 536, 626, 649, 660-668, 675, 679, 687-692, 695, 697, 703, 716, 721-722, 751-763, 767, 770-1411, 1456-1462, 7624-7625, 7684-7698, 7751-7778, 7783-7784, 7793, 7883, 7906, 7917-7925, 7932, 7936, 7944-7949, 7952, 7954, 7960, 7973, 7978-7979, 8008- 8020, 8024, 8027-8667, 8712-8718, 1529-1569, 8784-8823, 6697-6701, 13943-13947, 4179-4670, 11430-11921, 4884-4909, 12134-12159, 6919-6972, 14163-14215, 2786- 2866, 2887-2938, 10039-10119, 10140-10191, 4826-4863, 12076-12113, 4864-4875, 12114-12125, 6974-7002, 14217-14244, 2598-2600, 2759-2785, 9851-9853, 10012- 10038, 2445-2582, 9699-9836, 1983-2158, 9237-9412, 1612-1982, 8866-9236, 2601- 2678, 9854-9931, 2679-2758, 9932-10011, 3442-3603, 10694-10855, 3604-3708, 10856-10959, 2939-3441, 3709-3979, 5177-5192, 10192-10693, 10960-111230, 12426-12441, 7003-7033, 14245-14275, 7054-7133, 14296-14374, 7034-7049, 14276- 14291, 6835-6918, 14079-14162, 6823-6834, 14068-14078, 298-366, 369-373, 442- 493, 522-525, 528-535, 537, 551-554, 557, 560-625, 672-674, 680-681, 684-686, 696, 698, 702, 723-742, 764-766, 1412-1453, 1463-1466, 1571-1577, 7555-7623, 2626- 7630, 7699-7750, 7785-7792, 7794, 7808-7811, 7814, 7817-7882, 7929-7931, 7937- 7938, 7941-7943, 7953, 7955, 7959, 7980-7999, 8021-8023, 8668-8706, 8708-8709, 8719-8722, 8825-8831, 374-426, 539-550, 555-556, 558-559, 671, 682-683, 743, 745- 750, 7631-7683, 7796-7807, 7812-7813, 7815-7816, 7928, 7939-7940, 800, 8002- 8007, 5942-6665, 13189-13911, 1-297, 715, 1580-1603, 7258-7554, 7972, 8834-8857, 705-714, 7962-7971, 6681-6694, 13927-13940, 6788-6803, 14033-14048, 1469-1526, 5147-5151, 8725-8781, 12396-12400, 2159-2428, 9413-9682, 646-648, 7903-7905, 2592-2595, 9846-9849, 676-678, 717-720, 7933-7935, 7974-7977, 538, 669, 704, 7795, 7926, 7961, 8710, 670, 699-701, 7927, 7956-7958, 4917-4979, 12167-12228, 4910-4916, 12160-12166, 5195-5941, 12444-13188, 627-645, 650-659, 693-694, 744, 768-769, 1451, 1455, 1467-1468, 1527-1528, 1570, 1578, 1579, 1604-1611, 2429- 2444, 2583-2591, 2596-2597, 2867-2886, 4876-4883, 5144-5146, 5152-5176, 5193- 5194, 6666-6680, 6695-6696, 6702-6787, 6804-6822, 6973, 7050-7053, 7134-7257, 7884-7902, 7907-7916, 7950-7951, 8001, 8025-8026, 8707, 8711, 8723-8724, 8782- RNG043-WO1 PCT Application 8783, 8824, 8832-8833, 8858-8865, 9683-9698, 9837-9845, 9850, 10120-10139, 12126-12133, 12393-12395, 12401-12425, 12442-12443, 13912-13926, 13941-13942, 13948-14032, 14049-14067, 14216, 14292-14295, 14375-14498, 16886-17078, 17478- 17622, 17677-17756, 14831-14833, 14838, 14847, 14850-15460, 17079-17477, 17660- 17676, 19031-19080, 16414-16516, 17623-17659, 19081-19108, 16397-16413, 16195- 16320, 15779-15925, 15476-15778, 16321-16366, 16367-16396, 16705-16814, 16815- 16885, 16517-16704, 18949-19030, 14657-14716, 14778-14824, 14834, 14835-14836, 14839, 15461-15475, 14717-14777, 14841-14846, 18413-18936, 14499-14656, 18939, 15926-16178, 14837, 17757-18412, 14825-14830, 14840, 14848-14849, 16179-16194, 18937-18938, and 18940-18948 (Table A or Table B of U.S. Application No. 18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872) or Table 31A of PCT Publication WO2024044723A1 (SEQ ID NOs: 19543-19733 of said PCT). 37. The ncRNA variant of any one of claims 1-36, comprising a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to any one of the sequences in Table A or Table B of U.S. Application No.18/087,673, or International Application No. PCT/US2023/061038, or International Application No. PCT/US2023/072872 or Table 31A of PCT Publication WO2024044723A1 (SEQ ID NOs: 19543-19733 of said PCT). 38. The ncRNA variant of any one of claims 1-37, comprising a sequence selected from RTX3_6342_msr_stem_var1 (SEQ ID NO: 19644), RTX3_6342_msr_stem_var20 (SEQ ID NO: 19663), RTX3_6342_msr_stem_var5 (SEQ ID NO: 19648), RTX3_6342_a1a2_var6 (SEQ ID NO: 19548), RTX3_6342_a1a2_var10 (SEQ ID NO: 19552), RTX3_6342_a1a2_var15 (SEQ ID NO: 19557), RTX3_6342_a1a2_var16 (SEQ ID NO: 19558), RTX3_6342_a1a2_var19 (SEQ ID NO: 19561), RTX3_6342_a1a2_var20 (SEQ ID NO: 19562), RTX3_6342_a1a2_var21 (SEQ ID NO: 19563), RTX3_6342_a1a2_var26 (SEQ ID NO: 19568), RTX3_6342_a1a2_var27 (SEQ ID NO: 19569), and RTX3_6342_a1a2_var32 (SEQ ID NO: 19574). RNG043-WO1 PCT Application 39. The ncRNA variant of any one of claims 1-38, comprising one or more modifications in RTX3_6342_msr_stem_var1 (SEQ ID NO: 19644), RTX3_6342_msr_stem_var20 (SEQ ID NO: 19663), RTX3_6342_msr_stem_var5 (SEQ ID NO: 19648), RTX3_6342_a1a2_var6 (SEQ ID NO: 19548), RTX3_6342_a1a2_var10 (SEQ ID NO: 19552), RTX3_6342_a1a2_var15 (SEQ ID NO: 19557), RTX3_6342_a1a2_var16 (SEQ ID NO: 19558), RTX3_6342_a1a2_var19 (SEQ ID NO: 19561), RTX3_6342_a1a2_var20 (SEQ ID NO: 19562), RTX3_6342_a1a2_var21 (SEQ ID NO: 19563), RTX3_6342_a1a2_var26 (SEQ ID NO: 19568), RTX3_6342_a1a2_var27 (SEQ ID NO: 19569), or RTX3_6342_a1a2_var32 (SEQ ID NO: 19574) compared to RTX3_6342_WT (SEQ ID NO: 19734). 40. The ncRNA variant of any one of claims 1-38, comprising a sequence selected from msR Spacer Del-1 (SEQ ID NO: 19939), msD Spacer Del-1 (SEQ ID NO: 19940), msD Spacer Del-2 (SEQ ID NO: 19941), and msR Spacer Del-1/msD Spacer Del-2 (SEQ ID NO: 19942). 41. The ncRNA variant of any one of claims 1-38, comprising one or more modifications in msR Spacer Del-1 (SEQ ID NO: 19939), msD Spacer Del-1 (SEQ ID NO: 19940), msD Spacer Del-2 (SEQ ID NO: 19941), or msR Spacer Del-1/msD Spacer Del-2 (SEQ ID NO: 19942) compared to WT R6342 ncRNA (SEQ ID NO: 19734). 42. The ncRNA variant of any one of claims 1-38, comprising a sequence selected from Alt1 msR Spacer Del- (SEQ ID NO: 19947)1, Alt1 msD Spacer Del-1 (SEQ ID NO: 19948), Alt1 msD Spacer Del-2 (SEQ ID NO: 19949), Alt1 msR Spacer Del-1/msD Spacer Del-2 (SEQ ID NO: 19950), Alt1 msR Spacer Del-2/msD Del-3 (SEQ ID NO: 19951), Alt1 msR Spacer Del-2/msD Del-3 MS2 (SEQ ID NO: 19952), and Alt1 msR Spacer Del-2/msD Del-3 U7 (SEQ ID NO: 19953). 43. The ncRNA variant of any one of claims 1-38, comprising one or more modifications in Alt1 msR Spacer Del- (SEQ ID NO: 19947)1, Alt1 msD Spacer Del-1 (SEQ ID NO: 19948), Alt1 msD Spacer Del-2 (SEQ ID NO: 19949), Alt1 msR Spacer Del-1/msD Spacer Del-2 (SEQ ID NO: 19950), Alt1 msR Spacer Del-2/msD Del-3 (SEQ ID NO: 19951), Alt1 msR Spacer Del-2/msD Del-3 MS2 (SEQ ID NO: 19952), or Alt1 msR Spacer Del-2/msD Del-3 U7 (SEQ ID NO: 19953) compared to WT R6342 ncRNA (SEQ ID NO: 19734). RNG043-WO1 PCT Application 44. The ncRNA variant of any one of claims 1-38, comprising a sequence selected from msR spacer del_30HA (SEQ ID NO: 19955), msR spacer del_45HA (SEQ ID NO: 19956), WT PAM mt, (SEQ ID NO: 19957) Alt del1+del2 PAM mt (SEQ ID NO: 19958), or Alt del1+del2+U7 PAM mt (SEQ ID NO: 19959), and Alt del1+del225bp ins (SEQ ID NO: 19960). 45. The ncRNA variant of any one of claims 1-38, comprising one or more modifications in msR spacer del_30HA (SEQ ID NO: 19955), msR spacer del_45HA (SEQ ID NO: 19956), WT PAM mt, (SEQ ID NO: 19957) Alt del1+del2 PAM mt (SEQ ID NO: 19958), or Alt del1+del2+U7 PAM mt (SEQ ID NO: 19959), or Alt del1+del225bp ins (SEQ ID NO: 19960)compared to WT R6342 ncRNA (SEQ ID NO: 19734). 46. The ncRNA variant of any one of claims 1-45, wherein the one or more modifications further comprises addition of 2’O methyl or phosphorothioate bond at the 5’ or 3’ end of the ncRNA variant. 47. The ncRNA variant of claim 46, wherein the one or more modifications further comprises the addition of the 2’O methyl or the phosphorothioate bond at both the 5’ and 3’ ends of the ncRNA variant. 48. A retron variant comprising the ncRNA variant of any one of claims 1-47 and an RNA encoding a first polymerase, optionally downstream of the msd of the ncRNA variant. 49. The retron variant of claim 48, wherein the first polymerase is a reverse transcriptase, optionally wherein the reverse transcriptase is originated from a naturally occurring retron or retron-like sequence. 50. The retron variant of claim 48 or 49, wherein the first polymerase is a reverse transcriptase selected from: EcoI-RT, Efe1-RT, Mva1-RT, Cex1-RT, Eco8-RT, Vap1- RT, and Vro1-RT. 51. The retron variant of claim 50, wherein the reverse transcriptase is originated from a same naturally occurring retron as the reference retron ncRNA. RNG043-WO1 PCT Application 52. A chimeric gene editing composition comprising: a. the ncRNA variant of any one of claims 1-47, or the retron variant of any one of claims 48-51, b. a nuclease or a first mRNA encoding the nuclease, c. a guide RNA (gRNA) associated with the nuclease, and d. optionally, a second polymerase or a second mRNA encoding the second polymerase. 53. The chimeric gene editing composition of claim 52, comprising the second polymerase or the second mRNA encoding the second polymerase, wherein the second polymerase is a reverse transcriptase. 54. The chimeric gene editing composition of claim 52, comprising the second polymerase or the second mRNA encoding the second polymerase, wherein the second polymerase is a DNA polymerase. 55. The chimeric gene editing composition of any one of claims 52-54, wherein the nuclease is a nickase. 56. The chimeric gene editing composition of claim 55, wherein the nickase comprises a Cas9 nuclease, a Cas9(D10A) nuclease, a TnpB nuclease, or a Cas12a nuclease. 57. The chimeric gene editing composition of any one of claims 52-56, wherein the ncRNA variant or the retron variant is linked to the gRNA directly or indirectly. 58. The chimeric gene editing composition of claim 57, wherein the ncRNA variant or the retron variant, the gRNA, and the first mRNA encoding the nuclease are linked directly or indirectly. 59. The chimeric gene editing composition of any one of claims 52-58, further comprising delivery vehicles. 60. The chimeric gene editing composition of claim 59, wherein the delivery vehicles encapsulate one or more components selected from: RNG043-WO1 PCT Application a. the ncRNA variant or the retron variant, b. the nuclease or the first mRNA encoding the nuclease, c. the gRNA associated with the nuclease, and d. optionally, the second polymerase or the second mRNA encoding the second polymerase. 61. The chimeric gene editing composition of claim 59 or 60, wherein the delivery vehicle is a lipid nanoparticle. 62. One or more polynucleotides comprising a coding sequence of the ncRNA variant of any one of claims 1-47 or the retron variant of any one of claims 48-51. 63. The one or more polynucleotides of claim 62, further comprising a coding sequence of a nuclease or a coding sequence of a gRNA. 64. The one or more polynucleotides of claim 62, further comprising a coding sequence of a nuclease and a coding sequence of a gRNA. 65. The one or more polynucleotides of any one of claims 62-64, further comprising a coding sequence of the first polymerase. 66. The one or more polynucleotides of any one of claims 62-65, further comprising a coding sequence of a second polymerase. 67. The one or more polynucleotides of any one of claims 62-66, further comprising one or more promoters, wherein each of the one or more promoters is operably linked to (i) the coding sequence of the ncRNA variant or the retron variant, (ii) a coding sequence of a nuclease, (iii) a coding sequence of a gRNA, (iv) a coding sequence of the first polymerase, or (v) a coding sequence of a second polymerase. 68. A vector comprising the one or more polynucleotides of any one of claims 62-67. 69. The vector of claim 68, wherein the vector is a plasmid or a viral vector, optionally wherein the viral vector is an AAV or lentiviral vector. RNG043-WO1 PCT Application 70. A method of editing a target DNA, comprising interacting the target DNA with the chimeric gene editing composition of any one of claims 52-61, the one or more polynucleotides of any one of claims 62-67, or the vector of claim 68 or 69. 71. The method of claim 70, wherein the ncRNA variant in the chimeric gene editing composition, the one or more polynucleotides, or the vector comprises a pair of homology arms specific to a target locus and specific to the target DNA. 72. The method of claim 70 or 71, wherein the interacting step is performed in vivo or in vitro. 73. A chimeric gene editing composition comprising: a. a non-coding RNA (ncRNA) comprising a single-stranded RNA comprising a polymerase template, b. a nickase or a first mRNA encoding the nickase, c. a guide RNA (gRNA) associated with the nickase, and d. optionally, a second polymerase or a second mRNA encoding the second polymerase. 74. The chimeric gene editing composition of claim 73, comprising the second polymerase or the second mRNA encoding the second polymerase, wherein the second polymerase is a reverse transcriptase. 75. The chimeric gene editing composition of claim 73, comprising the second polymerase or the second mRNA encoding the second polymerase, wherein the second polymerase is a DNA polymerase. 76. The chimeric gene editing composition of any one of claims 73-75, wherein the nickase comprises a Cas9 nuclease, a Cas9(D10A) nuclease, a TnpB nuclease, or a Cas12a nuclease. 77. The chimeric gene editing composition of any one of claims 73-76, wherein the ncRNA RNG043-WO1 PCT Application is linked to the gRNA directly or indirectly. 78. The chimeric gene editing composition of claim 77, wherein the ncRNA, the gRNA, and the first mRNA encoding the nickase are linked directly or indirectly.
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