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WO2025076306A1 - Éditeurs de base à efficacité d'édition de base améliorée - Google Patents

Éditeurs de base à efficacité d'édition de base améliorée Download PDF

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WO2025076306A1
WO2025076306A1 PCT/US2024/049905 US2024049905W WO2025076306A1 WO 2025076306 A1 WO2025076306 A1 WO 2025076306A1 US 2024049905 W US2024049905 W US 2024049905W WO 2025076306 A1 WO2025076306 A1 WO 2025076306A1
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prime
cell
editing
programmable
pemax
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Scot WOLFE
Karthikeyan PONNIENSELVAN
Pengpeng LIU
Jeremy Luban
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University of Massachusetts Amherst
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University of Massachusetts Amherst
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    • 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
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    • 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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/85—Fusion polypeptide containing an RNA binding domain
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13033—Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory

Definitions

  • the present invention relates to improved prime editing systems, and more particularly to prime editing systems having prime editors with improved solubility and dNTP affinity.
  • Prime editing has the potential for broad therapeutic application for a variety of human diseases due to its versatility in performing targeted base conversion, deletions and insertions 1 .
  • a prime editor has 3 core components: Cas9 nickase, M-MLV reverse transcriptase (MMLV-RT), and prime editing guide RNA (pegRNA) 1, 2 .
  • Prime editing efficiency is enhanced by using a sgRNA to direct a nick to the target strand (PE3 system), and a high level of precision can be maintained by directing the nicking guide to recognize sequence changes encoded by the pegRNA (PE3b system) 1, 3 .
  • a prime editor protein comprising a Cas9 nickase fused to a Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase domain, wherein the M-MLV reverse transcriptase domain comprises a V223M and a L435K mutation.
  • M-MLV Moloney Murine Leukemia Virus
  • a method of modifying a double-stranded target DNA sequence in a cell comprising: sequentially introducing into the cell, in the following order: (1) a programmable prime editing system, the programmable prime editing system comprising (i) a prime editor protein comprising a Cas9 nickase fused to a M-MLV reverse transcriptase domain and (ii) a pegRNA comprising a spacer sequence having a region of complementarity to a target strand of the double-stranded target DNA sequence; and (2) a nicking sgRNA.
  • the M-MLV reverse transcriptase domain comprises a V223M and a L435K mutation.
  • the programmable prime editing system comprises an inhibitor of SAMHDL
  • the inhibitor of SAMHDl may be Vpx, Vpx (S13E), EBV BGLF4, HHV-6/7 U69, HCMV UL97, or KSHV ORF36.
  • the nicking sgRNA is introduced into the cell 2-36 hours after introducing the programmable prime editing system into the cell. In other embodiments, the nicking sgRNA is introduced into the cell 6-24 hours after introducing the programmable prime editing system into the cell.
  • the nicking sgRNA is introduced into the cell by a second method selected from the group consisting of electroporation, transfection, viral delivery, virus-like particle delivery, and lipid nanoparticle delivery.
  • Fig. 1C is a chart showing PEmax, PEmax* (V223M) and PEmax** (V223M & L435K) protein and mRNA mediated prime editing efficiency with synthetic pegRNA/sgRNA delivered by electroporation in iMyoblasts and primary T cells.
  • PE3 editing at HEK4 (+4+5 GG to CC) in iMyoblasts, and at FANCF (+5 G to T) in iMyoblasts and in activated primary T cells, respectively (n 3 independent replicates).
  • PAM sequence is boxed, protospacer sequence is underlined, and edited bases are indicated in lowercase letters.
  • Figure 1C discloses SEQ ID NOS 2-5, respectively, from left to right and top to bottom.
  • Figure ID discloses SEQ ID NOS 6-7, respectively, in order of appearance.
  • Fig. IE shows violin plots of fold-change in PE2 or Twin-PE editing efficiency of prime editor variants compared to PEmax in HEK293T cells under different conditions (standard, co-delivery of VPX or supplementation of dNs). Horizontal white lines indicate quartiles. Medians are indicated by the black horizontal lines.
  • Two-way ANOVA was used to compare the intended edit from multiple prime editor architectures under different conditions, and standard PEmax editing was used as control for multiple comparisons. * indicates P ⁇ 0.05, ** indicates P ⁇ 0.01, *** indicates P ⁇ 0.001, **** indicates P ⁇ 0.0001, ns indicates not significant.
  • Fig. IF is a chart showing co-delivery of VPX mRNA significantly improves prime editing efficiency in primary T cells and SC-derived islet cells.
  • Co-delivery of VPX mRNA with PEmax mRNA increases prime editing at FANCF with PE3b system in SC-Derived islet cells.
  • Two-way ANOVA was used to compare the intended edit from multiple prime editor architectures under different conditions. * indicates P ⁇ 0.05, *** indicates P ⁇ 0.001, **** indicates P ⁇ 0.0001, ns indicates not significant.
  • Figure IF discloses SEQ ID NOS 8-11, respectively, from left to right and top to bottom.
  • FIG. 2A is a schematic overview of simultaneous and sequential delivery of PE3b Nicking sgRNA for PE3b prime editing.
  • PAM sequences are boxed, protospacer regions are underlined, and edited sequences are indicated as lowercase letters.
  • the protospacer for PE3b nicking sgRNAs is indicated on bottom strand.
  • Figure 2B discloses SEQ ID NOS 9 and 11, respectively, in order of appearance.
  • Figure 2C discloses SEQ ID NOS 12-13, respectively, in order of appearance.
  • Figure 2D discloses SEQ ID NOS 14-15, respectively, in order of appearance.
  • Figure 2E discloses SEQ ID NOS 16-17, respectively, in order of appearance.
  • Fig. 2F is a violin plot showing a comparison of precise prime editing between simultaneous and sequential delivery of PE3b nicking sgRNA across multiple endogenous genomic loci and human cell types, with (+VPX) and without (-VPX) co- delivery of Vpx.
  • White lines indicate quartiles. Medians are indicated by black horizontal lines.
  • Fig. 2G is a schematic overview of a combination of methods used to boost prime editing efficiency.
  • Cas9 or “Cas9 nuclease” refers to an RNA-guided nuclease comprising a Cas9 domain, or a fragment thereof (e.g., a protein comprising an active or inactive DNA cleavage domain of Cas9, and/or the gRNA binding domain of Cas9).
  • a “Cas9 domain” as used herein, is a protein fragment comprising an active or inactive cleavage domain of Cas9 and/or the gRNA binding domain of Cas9.
  • a “Cas9 protein” is a full length Cas9 protein.
  • a Cas9 nuclease is also referred to sometimes as a casnl nuclease or a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-associated nuclease.
  • CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements, and conjugative plasmids).
  • CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids.
  • CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA).
  • tracrRNA transencoded small RNA
  • rnc endogenous ribonuclease 3
  • Cas9 domain The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA.
  • Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer.
  • the target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3 '-5' exonucleolytically.
  • DNA-binding and cleavage typically requires protein and both RNAs.
  • single guide RNAs can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816-821(2012), the entire contents of which are hereby incorporated by reference.
  • Cas9 recognizes a short motif absent from the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus non-self Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g., “Complete genome sequence of an Ml 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.
  • Cas9 and Cas9 nickases derived from the aforementioned species, as well as variants thereof, are also contemplated as part of the present disclosure. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on U.S. Patent No.
  • a Cas9 nuclease comprises one or more mutations that partially impair or inactivate the DNA cleavage domain.
  • U.S. Patent No. 11,447,770 is hereby incorporated by reference for its disclosure of Cas9.
  • nickase refers to a Cas9 with one of its two nuclease domains inactivated. This enzyme is capable of cleaving only one strand of a target DNA.
  • U.S. Patent No. 11,447,770 is hereby incorporated by reference for its disclosure of Cas9 nickases.
  • DNA synthesis template As used herein, the terms “DNA synthesis template,” “reverse transcriptase template,” and “RTT” are used interchangeably to refer to the region or portion of the extension arm of a pegRNA that is utilized as a template strand by a polymerase, such as reverse transcriptase, of a prime editor to encode a 3' replacement DNA flap that contains a desired edit and which then, through the mechanism of prime editing, replaces the corresponding endogenous strand of DNA at the target site.
  • a polymerase such as reverse transcriptase
  • 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 5'-to-3' direction.
  • a first element is 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 SNP is upstream of a Cas9-induced nick site if the SNP is on the 5' side of the nick site.
  • 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.
  • a SNP is downstream of a Cas9-induced nick site if the SNP is on the 3' side of the nick site.
  • the nucleic acid molecule can be a DNA (double or single stranded). RNA (double or single stranded), or a hybrid of DNA and RNA.
  • the analysis is the same for single strand nucleic acid molecule and a double strand molecule since the terms upstream and downstream are in reference to only a single strand of a nucleic acid molecule, except that one needs to select which strand of the double stranded molecule is being considered.
  • the strand of a double stranded DNA which can be used to determine the positional relativity of at least two elements is the “sense” or “coding” strand.
  • a “sense” strand is the segment within double-stranded DNA that runs from 5' to 3', and which is complementary to the antisense strand of DNA, or template strand, which runs from 3' to 5'.
  • a SNP nucleobase is “downstream” of a promoter sequence in a genomic DNA (which is double- stranded) if the SNP nucleobase is on the 3' side of the promoter on the sense or coding strand.
  • extension arm refers to a nucleotide sequence component of a pegRNA which provides several functions, including a primer binding site (PBS) and a DNA synthesis template (also referred to as an “reverse transcriptase template” or “RTT”) for reverse transcriptase.
  • PBS primer binding site
  • RTT reverse transcriptase template
  • the extension arm is located at the 3' end of the guide RNA.
  • the extension arm comprises the following components in a 5' to 3' direction: the DNA synthesis template and the primer binding site.
  • the preferred arrangement of the DNA synthesis template and primer binding site is in the 5' to 3' direction such that the reverse transcriptase, once primed by an annealed primer sequence, polymerizes a single strand of DNA using the DNA synthesis template as a complementary template strand.
  • the extension arm may also be described as comprising generally two regions: a primer binding site (PBS) and a DNA synthesis template.
  • the primer binding site binds to a primer sequence that is formed from the endogenous DNA strand of the target site when it becomes nicked by the prime editor complex, thereby exposing a 3' end on the endogenous nicked strand.
  • the binding of the primer sequence to the primer binding site on the extension arm of the pegRNA creates a duplex region with an exposed 3' end (i.e., the 3' of the primer sequence), which then provides a substrate for reverse transcriptase to begin polymerizing a single strand of DNA from the exposed 3' end along the length of the DNA synthesis template.
  • the sequence of the single strand DNA product is the complement of the DNA synthesis template.
  • Polymerization continues towards the 5' of the DNA synthesis template (or extension arm) until polymerization terminates.
  • the DNA synthesis template represents the portion of the extension arm that is encoded into a single strand DNA product (i.e., the 3' single strand DNA flap containing the desired genetic edit information) by the polymerase of the prime editor complex and which ultimately replaces the corresponding endogenous DNA strand of the target site that sits immediate downstream of the PE-induced nick site.
  • polymerization of the DNA synthesis template continues towards the 5' end of the extension arm until a termination event.
  • Polymerization may terminate in a variety of ways, including, but not limited to (a) reaching a 5' terminus of the pegRNA (e.g., in the case of the 5' extension arm wherein the DNA polymerase simply runs out of template), (b) reaching an impassable RNA secondary structure (e.g., hairpin or stem/loop), or (c) reaching a replication termination signal, e.g., a specific nucleotide sequence that blocks or inhibits the polymerase, or a nucleic acid topological signal, such as, supercoiled DNA or RNA.
  • a 5' terminus of the pegRNA e.g., in the case of the 5' extension arm wherein the DNA polymerase simply runs out of template
  • an impassable RNA secondary structure e.g., hairpin or stem/loop
  • a replication termination signal e.g., a specific nucleotide sequence that blocks or inhibits the polymerase, or a nucleic acid topological signal, such as,
  • fusion protein refers to a hybrid polypeptide which comprises protein domains from at least two different proteins.
  • One protein may be located at the amino-terminal (N-terminal) portion of the fusion protein or at the carboxy -terminal (C-terminal) protein thus forming an “amino-terminal fusion protein” or a “carboxy-terminal fusion protein,” respectively.
  • a protein may comprise different domains, for example, a nucleic acid binding domain (e.g., the gRNA binding domain of Cas9 that directs the binding of the protein to a target site) and a nucleic acid cleavage domain or a catalytic domain of a nucleic-acid editing protein.
  • a fusion protein may comprise a Cas9 nickase fused to a reverse transcriptase. Such a fusion protein is referred to herein as a “prime editor protein.”
  • guide RNA is a particular type of guide nucleic acid which is mostly commonly associated with a Cas protein of a CRISPR-Cas9 and which associates with Cas9, directing the Cas9 protein to a specific sequence in a DNA molecule that includes complementarity to a spacer sequence of the guide RNA.
  • this term also embraces the equivalent guide nucleic acid molecules that associate with Cas9 equivalents, homologs, orthologs, or paralogs, whether naturally occurring or non-naturally occurring (e.g., engineered or recombinant), and which otherwise program the Cas9 equivalent to localize to a specific target nucleotide sequence.
  • Primary editing guide RNA (or “pegRNA”) is a guide RNA that has been modified and designed for the prime editing methods and systems disclosed herein.
  • a pegRNA associates with a prime editor protein.
  • U.S. Patent No. 11,447,770 is hereby incorporated by reference for its disclosure of guide RNA.
  • RNA refers to a specialized form of a guide RNA that has been modified to include one or more additional sequences for implementing the prime editing methods and systems described herein.
  • the additional sequences comprise (i) a “DNA synthesis template” which encodes (copied by the polymerase, e.g., reverse transcriptase, of the prime editor) a single-stranded DNA which, in turn, has been designed to be (a) homologous with the endogenous target DNA to be edited, and (b) which comprises at least one desired nucleotide change (e.g., a transition, a transversion, a deletion, or an insertion) to be introduced or integrated into the endogenous target DNA; and (ii) a “primer binding site.”
  • the “primer binding site” comprises a sequence that hybridizes to a single-strand DNA sequence having a 3' end generated from the nicked DNA of the R-loop.
  • U.S. Patent No. 11,447,770 is hereby incorporated by reference for its disclosure of pegRNA.
  • PE2 refers to a PE complex comprising a fusion protein comprising a Cas9 nickase and a reverse transcriptase (RT), and a desired pegRNA.
  • RT reverse transcriptase
  • PE3 refers to PE2 plus a second-strand nicking guide RNA (Nk sgRNA) that complexes with the PE2 and introduces a nick in the non-edited DNA strand in order to induce preferential replacement of the edited strand.
  • Nk sgRNA second-strand nicking guide RNA
  • polymerase refers to an enzyme that synthesizes a nucleotide strand and which may be used in connection with the prime editor systems described herein.
  • Reverse transcriptase is a polymerase.
  • U.S. Patent No. 11,447,770 is hereby incorporated by reference for its disclosure of polymerases.
  • the term “prime editing” refers to an approach for gene editing using a nucleic acid programmable fusion protein comprising a Cas9 nickase and a polymerase (e.g., a reverse transcriptase), and specialized guide RNAs that include a DNA synthesis template for encoding desired new genetic information (or deleting genetic information) that is then incorporated into a target DNA sequence.
  • a polymerase e.g., a reverse transcriptase
  • U.S. Patent No. 11,447,770 is hereby incorporated by reference for its disclosure of prime editing.
  • primary editor protein refers to fusion constructs comprising a nucleic acid programmable Cas9 nickase and a polymerase (e.g., reverse transcriptase) that is capable of carrying out prime editing on a target nucleotide sequence in the presence of a pegRNA.
  • polymerase e.g., reverse transcriptase
  • the term “prime editor” may refer to the fusion protein or to the fusion protein complexed with a pegRNA, or to the fusion protein complexed with a second-strand nicking sgRNA.
  • a nicking sgRNA may have a spacer sequence comprising a region of complementarity to the modified sequence of a non-target strand (PE3b) or to a sequence adjacent to the modified sequence of the non-target strand (PE3).
  • a non-target strand is the strand opposite the target strand, the target strand having a sequence complementary to the spacer sequence of a pegRNA, and to which the prime editorpegRNA RNP complex binds.
  • U.S. Patent No. 11,447,770 is hereby incorporated by reference for its disclosure of prime editors and prime editor proteins.
  • primer binding site refers to the nucleotide sequence located on a pegRNA as component of the extension arm (typically at the 3' end of the extension arm) and serves to bind to the primer sequence that is formed after Cas9 nicking of the target site sequence by the prime editor.
  • U.S. Patent No. 11,447,770 is hereby incorporated by reference for its disclosure of primer binding sites.
  • reverse transcriptase describes a class of polymerases characterized as RNA-dependent DNA polymerases. All known reverse transcriptases require a primer to synthesize a DNA transcript from an RNA template. Avian myoblastosis virus (AMV) reverse transcriptase was the first widely used RNA-dependent DNA polymerase (Verma, Biochim. Biophys. Acta 473:1 (1977)). The enzyme has 5 '-3' RNA- directed DNA polymerase activity, 5 '-3' DNA-directed DNA polymerase activity, and RNase H activity.
  • AMV Avian myoblastosis virus
  • RNase H is a processive 5' and 3' ribonuclease specific for the RNA strand for RNA-DNA hybrids (Perbal, A Practical Guide to Molecular Cloning, New York: Wiley & Sons (1984)).
  • Another reverse transcriptase which is used extensively in molecular biology is reverse transcriptase originating from Moloney murine leukemia virus (M-MLV).
  • M-MLV Moloney murine leukemia virus
  • M-MLV reverse transcriptase substantially lacking in RNase H activity has also been described. See, e.g., U.S. Pat. No. 5,244,797.
  • U.S. Patent No. 11,447,770 is hereby incorporated by reference for its disclosure of reverse transcriptases.
  • M-MLV reverse transcriptase domain means a protein domain of a prime editor protein having reverse transcriptase activity and at least 95% amino acid sequence identity with a corresponding contiguous sequence of wild-type M-MLV reverse transcriptase, as known to those of ordinary skill in the art, e.g., as disclosed in U.S. Publication No. US 2021/0292769, incorporated by reference herein at least for its disclosure of an M-MLV reference amino acid sequence and numbering convention used to refer to amino acid residues within the reference M-MLV sequence.
  • amino acid residue numbering for an M-MLV RT domain of a prime editor refers to the amino acid residue numbering of M-MLV reverse transcriptase itself — not the entire prime editor protein — for example, the M-MLV RT amino acid residue numbering disclosed in the reference M-MLV reference amino acid sequence of U.S. Publication No. US 2021/0292769.
  • An M-MLV RT domain includes, but is not limited to, various M-MLV-RT domain prime editor architectures, as known to those of ordinary skill in the art.
  • an M-MLV reverse transcriptase domain includes wild-type M-MLV reverse transcriptase, RNaseH minus M-MLV reverse transcriptase (a deletion of the C-terminal RNaseH domain of M-MLV RT), and an N-terminal deletion of an M-MLV-RT.
  • target site refers to a sequence within a nucleic acid molecule that is edited by a prime editor (PE) disclosed herein.
  • the target site further refers to the target sequence within a nucleic acid molecule to which a complex of the prime editor (PE) and gRNA binds.
  • variants should be taken to mean the exhibition of qualities that have a pattern that deviates from what occurs in nature, e.g., a variant Cas9 is a Cas9 comprising one or more changes in amino acid residues as compared to a wild type Cas9 amino acid sequence.
  • variants 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.
  • 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.
  • “Engineered pegRNA” or “epegRNA” is a pegRNA comprising a 3’ structured RNA pseudoknot (3’ of the PBS of the pegRNA) that protects the 3' extension arm from degradation by exonucleases.
  • epegRNAs are described in Liu et al., International PCT Application PCT/US2021/052097, filed September 24, 2021, published as WO/2022067130 on March 31, 2022, the contents of which are incorporated herein by reference for its disclosure of epegRNA.
  • the prime editor proteins disclosed herein form a complex with (e.g., bind or associate with) one or more RNA(s) that is not a target for cleavage.
  • an RNA-programmable nuclease such as a prime editor protein, when in a complex with an RNA, may be referred to as a ribonucleoprotein complex, ribonucleoprotein, RNP, PE RNP, or RNP complex.
  • the bound RNA(s) may be, for example, a pegRNA, epegRNA, or sgRNA.
  • Melting temperature is the temperature at which one half of the strands of a population of duplexed nucleic acid will dissociate to become single-stranded.
  • the duplexed nucleic acid is a RNA:DNA duplex.
  • Melting temperature is a function of both the sequence and the length of the duplex.
  • hybridizable and derivatives thereof or “complementary” it is meant that a nucleic acid (e.g. RNA) 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],
  • A adenine
  • U uracil
  • G guanine
  • C cytosine
  • G/U base-pairing is 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.
  • a guanine (G) of a protein-binding segment (dsRNA duplex) of a subject DNA-targeting RNA molecule is considered complementary to a uracil (U), and vice versa.
  • G guanine
  • U uracil
  • An “inhibitor of SAMHD1” means a compound that inhibits or reduces the activity of SAMHD1, directly or indirectly (e.g., by increasing degradation of SAMHD1 or inhibiting its enzymatic activity), thereby causing an increase in intracellular dNTP levels.
  • Examples of an inhibitor of SAMHD1 include, but are not limited to, Vpx and variants thereof (Vpx (S13E) described by Miyakawa et al. Nat Commun.
  • Prime editing efficiency in primary cells may be hampered by inherent properties of MMLV-RT.
  • most prior optimization of prime editing components has been performed in rapidly dividing mammalian cells 1, 9 > n .
  • intracellular dNTP concentration can be 100-fold lower in post-mitotic or quiescent cells than cycling transformed cell lines 16 ' 19 , this scarcity may suppress MMLV-RT processivity due to its low affinity for dNTPs 18, 20 .
  • MMLV-RT -dependent protein aggregation during purification of the PEmax prime editor protein 7 , which suggests that its solubility may limit prime editing activity.
  • a prime editor protein comprising a Cas9 nickase fused to a Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase domain having mutations (1) V223M, which increases dNTP affinity and (2) L435K, which increases prime editor solubility, significantly improves prime editing rates.
  • M-MLV Moloney Murine Leukemia Virus
  • a prime editor protein comprising a Cas9 nickase fused to a Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase domain, wherein the M-MLV reverse transcriptase domain comprises a V223M and a L435K mutation is disclosed.
  • an isolated nucleic acid encoding the prime editor protein having the V223M and L435K mutations is contemplated as part of the present disclosure.
  • the present disclosure also contemplates a programmable prime editing system for modification of a double-stranded target DNA sequence comprising a target strand and a complementary non-target strand, the system comprising a prime editor protein having the V223M and L435K mutations; and a prime editing guide RNA (pegRNA) comprising a spacer sequence having a region of complementarity to the target strand of the double-stranded target DNA sequence.
  • the prime editing system comprises a nucleic acid encoding the prime editor protein and/or a nucleic acid encoding the pegRNA.
  • the programmable prime editing system further comprises an inhibitor of SAMHD1 known to those of skill in the art, now and in the future.
  • the inhibitor of SAMHD1 may, e.g., be Vpx, Vpx (S13E), EBV BGLF4, HHV-6/7 U69, HCMV UL97, or KSHV ORF36.
  • the inhibitor of SAMHD1 comprised by the system is a protein.
  • the system comprises a nucleic acid encoding the inhibitor of SAMHD1.
  • a prime editing system of the present disclosure may be a twin prime editing (Twin PE) system.
  • Twin PE systems utilize a prime editor protein and two pegRNAs targeting opposite strands of locus to be modified, allowing for longer sequence edits.
  • the programmable prime editing system comprises a nicking sgRNA.
  • the prime editing system comprises a nucleic acid encoding the nicking sgRNA.
  • a method of modifying a double-stranded target DNA sequence comprising contacting the double-stranded target DNA sequence with the prime editor protein and the pegRNA.
  • a method of modifying a double-stranded target DNA sequence in a cell comprising: sequentially introducing into the cell, in the following order: (1) a programmable prime editing system, the programmable prime editing system comprising (i) a prime editor protein comprising a Cas9 nickase fused to a M-MLV reverse transcriptase domain and (ii) a pegRNA comprising a spacer sequence having a region of complementarity to a target strand of the double-stranded target DNA sequence; and (2) a nicking sgRNA.
  • the prime editor may be introduced into the cell by introducing a nucleic acid encoding the prime editor into the cell. In some embodiments, the prime editor may be introduced into the cell by introducing the prime editor protein into the cell. In some embodiments, the pegRNA may be introduced into the cell by introducing a nucleic acid encoding the pegRNA into the cell. In some embodiments, the pegRNA may be introduced into the cell by introducing the pegRNA into the cell. In some embodiments, the nicking sgRNA may be introduced into the cell by introducing the nicking sgRNA into the cell. In some embodiments, the nicking sgRNA may be introduced into the cell by introducing a nucleic acid encoding the nicking sgRNA into the cell.
  • the M-MLV reverse transcriptase domain comprises a V223M and a L435K mutation.
  • the programmable prime editing system further comprises an inhibitor of SAMHD1.
  • the inhibitor of SAMHD1 may be Vpx, Vpx (S13E), EBV BGLF4, HHV-6/7 U69, HCMV UL97, or KSHV ORF36.
  • the inhibitor of SAMHD1 is a protein introduced into the cell.
  • the inhibitor of SAMHD1 is introduced into the cell by introducing a nucleic acid encoding the inhibitor of SAMHD1.
  • the nicking sgRNA is introduced into the cell 2-96 hours after introducing the programmable prime editing system into the cell. In some embodiments, the nicking sgRNA is introduced into the cell 2-72 hours after introducing the programmable prime editing system into the cell. In some embodiments, the nicking sgRNA is introduced into the cell 2-48 hours after introducing the programmable prime editing system into the cell. In some embodiments, the nicking sgRNA is introduced into the cell 2- 36 hours after introducing the programmable prime editing system into the cell. In other embodiments, the nicking sgRNA is introduced into the cell 6-24 hours after introducing the programmable prime editing system into the cell.
  • the nicking sgRNA is introduced into the cell 1, 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,
  • the programmable prime editing system is introduced into the cell by a first method selected from the group consisting of electroporation, transfection, viral delivery, virus-like particle delivery, and lipid nanoparticle delivery.
  • the nicking sgRNA is introduced into the cell by a second method selected from the group consisting of electroporation, transfection, viral delivery, virus-like particle delivery, and lipid nanoparticle delivery.
  • Example 1 PEmax** (V223M and L435K MMLV-RT mutations)
  • MMLV-RT sequence variants were screened for their impact on PEmax prime editing activity in transformed cell lines (HEK293T and U2OS cells) and primary fibroblasts.
  • MMLV-RT processivity under limiting dNTP levels has been enhanced by a Q221R mutation 22 and a variety of mutations at position 223 22-24 .
  • V223M changes the MMLV-RT active site from an onco-retroviral RT to a more efficient lentiviral- like RT, reducing its K m for dNTPs by 2 to 4-fold 22-24 .
  • V223A and V223Y mutations have been previously introduced in MMLV-RT to improve prime editing rates 11, 23 .
  • V223A Q221R and three different V223 mutations (V223A, V223M, and V223Y) individually on prime editing rates at multiple target sites under subsaturating editor levels to discern differences in activity.
  • V223M PEmax*
  • V223Y modestly enhanced prime editing rates, with V223M displaying the most substantial improvement in activity (Fig. 1A).
  • MMLV-RT solubility has been improved by a 24 amino acid N-terminal truncation or a L435K mutation without substantial impact on its catalytic performance 26 .
  • the N-terminal MMLV-RT truncation reduced prime editing efficiency
  • the L435K mutation significantly enhanced prime editing efficiency (Fig. 1A)
  • purified PEmax protein formed aggregates when concentrated above 30 pM.
  • purified PEmax** protein could be concentrated to 140 pM without visible aggregates.
  • the improved solubility of the PEmax** prime editor protein resulted in a 7-fold increase in protein yield.
  • PEmax** displayed significantly higher enzymatic activity at subsaturating dNTP levels (0.05 to 0.15 pM dNTPs), consistent with the improved dNTP affinity of the V223M mutation 22 .
  • Example 4 VPX Co-delivery and Deoxynucleoside Supplementation Increase Prime Editing Efficiency
  • SAMHD1 is a deoxynucleotide triphosphohydrolase that maintains low dNTP levels in cells that are not undergoing DNA replication, which restricts infection by retroviruses 30, 31 .
  • Many viruses subvert SAMHD1 activity to promote infectivity 32 .
  • HIV-2 packages an accessory protein - VPX - within the virion that targets SAMHD1 for degradation via the cullin 4-based (CRL4) E3 ubiquitin ligase pathway 33 ' 34 .
  • SAMHD1 inactivation increases intracellular dNTP levels, which promotes reverse transcription and successful viral infection in quiescent cells 31, 35 .
  • An alternate approach for increasing intracellular dNTP levels in vitro is supplementation of deoxynucleosides (dNs) in growth media 36 ’ 38 .
  • dNs deoxynucleosides
  • VPX protein or mRNA increased precise editing rates for PEmax, PEmax* or PEmax** at the FANCF locus in resting T cells (data not shown).
  • VPX mRNA co-delivery significantly increased prime editing rates at the CCR5 locus in activated T cells, where a TWIN-PE approach 44 with Cas9-NG 45 PEmax** efficiently introduced the CCR5 deIta32 deletion (-40%) that is associated with HIV-1 R5-trophic virus resistance 46 ' 48 (Fig. IF).
  • co-delivery of VPX mRNA significantly improved prime editing efficiency with PEmax** in stem cell-derived islet (SC-islet) cells 49, 50 (Fig. IF).
  • Co-delivery of VPX increased the ratio of precise to imprecise prime editing outcomes, while also modestly increasing the rate of pegRNA scaffold insertions 11 (data not shown).
  • VPX co-delivery with PEmax** may be used with LNPs or eVLPs 46 to improve editing outcomes in various organ systems in vivo.
  • DNA polymerase-based prime editing systems may provide an alternate method to overcome low dNTP levels that restrict prime editing in quiescent and post-mitotic cells due to their superior processivity and dNTP affinity relative to RTs 41,42 .
  • Example 5 Sequential Delivery of Nicking sgRNA Increases Prime Editing Rates Compared to Simultaneous Delivery of Nicking sgRNA in PE3b Prime Editing System
  • the PE3b prime editing system provides an efficient method to increase prime editing rates while reducing unwanted editing byproducts through delivery of a nicking guide RNA that recognizes the modified sequence introduced by prime editing 1, 3 . Because the protospacer recognized by the nicking sgRNA contains prime editing directed mutations, nicking of the unmodified genomic strand should occur after MMLV-RT polymerization of the pegRNA-encoded template sequence into the genome 1, 3 . Since the nicking sgRNA has higher affinity for the prime editor than the pegRNA, prime editing rates can be reduced by competition between the sgRNA and pegRNA for the prime editor 7 .
  • Sequential PE3b editing with PEmax** and VPX mRNA co-delivery increases precise editing rates by ⁇ 5-fold over standard PE3b editing with PEmax in primary fibroblasts and immortalized cell lines (Fig. 2F-G).
  • Sequential PE3b editing involves the delivery of Cas9 H840A nickase and the nicking sgRNA, where the additional nickase may also help to stimulate precise editing outcomes. Because the nicking sgRNA in PE3b systems is programmed to recognize a precisely edited sequence 1 ’ 3 , the co-delivery of nicking sgRNA with the pegRNA can only initially produce unproductive competition by complexing with the prime editor 7 . Sequential delivery prevents guide RNA competition under conditions of limiting prime editor protein.
  • Expression plasmids used for pegRNAs have been previously described 27 .
  • Mammalian expression plasmids for different PEmax variants (N terminal 24aa deletion, Q221R, V223A, V223M, V223Y, L435K, L435K/Q221R, L435K/V223A, L435K/V223M, L435K/V223Y) were generated by site-directed mutagenesis on pCMV-PEmax (pCMV- PEmax and pCMV-PE6d are gifts from David Liu, Addgene plasmid #174820 and #207854).
  • NG-PEmax mammalian expression vector the
  • LI 111R/D1135V/G1218R/E1219F/A1322R/R1335V/T1337R (VRVRFRR) fragment 45 was synthesized by IDT and Gibson-cloned into the PEmax mammalian expression and IVT plasmids (Addgene plasmid #174820 and #204472) digested with Pmll/Rsr2 enzymes. All plasmids used for transient transfection experiments were purified with an endotoxin removal step (ZymoPURE Plasmid Miniprep Kit from Zymo Research).
  • PCR products were Gibson-cloned into the PEmax mRNA plasmid (Addgene plasmid #204472) digested with EcoRI enzyme.
  • VPX coding sequence was amplified from pscALPS gag-gfp/deltavpx vector (Addgene #115807) and inserted into PEmax mRNA plasmid (Addgene plasmid #204472) digested by Sall and EcoRI to replace the PEmax coding sequence.
  • the VPX Q76A vector was generated from the VPX mRNA vector by Gibson cloning.
  • SAMHDl coding sequence was amplified from U2OS cell cDNA library, then Gibson-cloned into the pCMV-PEmax or PEmax IVT mRNA plasmid to replace the PEmax coding sequence.
  • a PBS sequence length was chosen that has a Tm close to 37°C.
  • the PBS length for all pegRNAs used in this study except the PCSK9 pegRNA were designed using the MELTING 5 program.
  • pegLIT peglit.liugroup.us
  • RTT reverse transcriptase template
  • PEmax, PEmax*, PEmax**, PE6d, SAMHDl, VPX and VPXQ76A IVT mRNA plasmid were linearized using Pmel to cleave after the polyA tail.
  • mRNA was transcribed from 500 ng purified linearized template using the HiScribe T7 High-Yield RNA Synthesis Kit (New England BioLabs) with co-transcriptional capping by CleanCap AG (TriLink Biotechnologies) and full replacement of UTP with Nl-Methylpseudouridine-5’- triphosphate (TriLink Biotechnologies). After 1 hour of in vitro transcription, the DNA template was digested by 1 uL DNasel (Thermo Fisher Scientific) for 15 min.
  • RNA Clean & Concentrator-25 kit from Zymo Research, then purified mRNA was dissolved in nuclease-free water. The resulting mRNA was quantified with a NanoDrop One UV-Vis spectrophotometer (Thermo Fisher Scientific) and stored at - 80°C.
  • PEmax protein purification followed our previously described protocol 7 . Briefly, PEmax and PEmax** protein expression constructs were introduced into E. coli Rosetta2(DE3)pLysS cells (EMD Millipore). Bacteria were grown at 37°C in baffle flasks to an OD600 of ⁇ 0.6, then pre-chilled in an ice bath for 10 minutes and shifted to growth at 18°C. At an OD600 of -0.8 the cells were induced for 16 hours with IPTG (0.7 mM final concentration).
  • cells were pelleted by centrifugation (3500 g, 20 min) and then resuspended with Nickel-NTA buffer (20 mM TRIS + 1 M NaCl + 20 mM imidazole + 1 mM TCEP, pH 7.5) supplemented with HALT Protease Inhibitor Cocktail, EDTA-Free (100X) [ThermoFisher] and lysed with LM-20 Microfluidizer (Microfluidics) following the manufacturer’s instructions. The lysate was transferred to a centrifuge tube and spun at 20,000 g for 20 minutes.
  • the clarified lysate was then purified with Ni-NTA resin (Qiagen) in batch mode, washed with wash buffer (20 mM TRIS + 1 M NaCl + 20 mM imidazole + 1 mM TCEP, pH 7.5) and eluted with an elution buffer (20 mM TRIS, 500 mM NaCl, 250 mM Imidazole, 10% w/v glycerol, pH 7.5).
  • the eluted proteins were dialyzed overnight at 4°C in 20 mM HEPES, 500 mM NaCl, 1 mM EDTA, 10% w/v (8% v/v) glycerol, pH 7.5.
  • the proteins were step-dialyzed from 500 mMNaCl to 250 mM NaCl to 200 mM NaCl (not exceeding one hour incubation per step; final dialysis buffer: 20 mM HEPES, 200 mM NaCl, 1 mM EDTA, 10% w/v glycerol, pH 7.5).
  • the anion exchange column was removed prior to the elution of the prime editor protein from the cation exchange column.
  • the primary prime editor protein peak fractions were dialyzed into 20 mM HEPES pH 7.5, 300 mM NaCl and then concentrated to ⁇ 30pM for PEmax and 140pM for PEmax** using a 100 kDa amicon ultra centrifugal filter (UFC910008, Millipore).
  • HEK293T cells and U2OS cells were purchased from ATCC.
  • Patient-derived fibroblasts containing the T158M mutation were a gift from the Rett Syndrome Research Trust.
  • These cell lines were maintained in Dulbecco’s Modified Eagle’s Medium supplemented with 10% FBS at 37°C and 5% CO2.
  • CFF-16HBEge CFTR A508 (16HBE) cells were a gift of the Cystic Fibrosis Foundation.
  • 16HBE cells were grown at 37 °C/5% CO2 in MEM (Gibco) supplemented with 10% FBS (Gibco) and 1% Penicillin/Streptomycin (Gibco). Plates/flasks for 16HBE cell growth were prepared by incubating a thin layer of coating solution [LHC-8 basal medium (Gibco), 1.34 pl/ml Bovine serum albumin 7.5% (Gibco), 10 pl/ml Bovine collagen solution (Gibco), Type 1 (Advanced BioMatrix), 10 pl/ml Fibronectin from human plasma (Thermo Fisher Scientific)] at 37 °C/5% CO2 for 2-3 h followed by thorough removal of coating solution and storage at 4 °C until use.
  • coating solution [LHC-8 basal medium (Gibco), 1.34 pl/ml Bovine serum albumin 7.5% (Gibco), 10 pl/ml Bovine collagen solution (Gibco), Type 1 (Advanced BioMatrix), 10
  • HEXA I278+TATC EbvB Cells were purchased from Coriell (GM11852). HEXA EbvB cells were cultured in RPMI 1640 medium (Gibco) with 20% of Fetal Bovine Serum (Gibco), 4 mM L-glutamine (twice the normal culture concentration, Gibco) and 1% Penicillin/Streptomycin at 37 °C/5% CO2.
  • Deoxynucleosides (Sigma-Aldrich) were resuspended in nuclease-free water at 25 mM each, filter-sterilized, aliquoted and stored at -20°C freezer.
  • dN deoxynucleoside
  • cells were cultured in the presence of a specific amount of dNs 12 hours before transfection or electroporation.
  • 50 pM of each dN (final concentration) was added to the media for HEK293T cells and U2OS cells, and 30 pM of each dN was added to the media for Fibroblast cells.
  • HEK293T and U2OS cells were plated 40,000 cells per well in a 48-well plate. 24 hours later, the cells were co-transfected with 1000 ng prime editor plasmid and 330 ng pegRNA plasmid using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. 250 ng SAMHD1 or VPX expression plasmid was included for overexpression of these factors.
  • To determine editing rates at endogenous genomic loci cells were cultured 3 days following transfection, after which the media was removed, the cells were harvested, and genomic DNA was isolated using QIAamp DNA mini kit (QIAGEN) according to the manufacturer’s instructions. The editing rates were then determined by targeted amplicon deep sequencing.
  • PEmax mRNA mixed with synthetic pegRNA/sgRNA mixtures or RNPs programmed with synthetic pegRNA/sgRNA were delivered by electroporation using the NEON Nucleofection System 10 pL kit (Thermo Fisher Scientific). Simultaneous or sequential delivery are used for some mRNA-based editing experiments in Fibroblast cells, HBE cells and EbvB cells.
  • nicking guide was not included in the initial electroporation. 12 to 16 hours after electroporation, 500 ng SpCas9 H840A nickase or SpCas9-NG H840A nickase mRNA and 50 pmol synthetic sgRNA (IDT, for sequential PE3b approaches; Supplementary Table) were transfected into the cells using Lipofectamine MessengerMAX Reagent.
  • IDT for sequential PE3b approaches; Supplementary Table
  • iMy oblast cells were resuspended into 10 pl of NEON Buffer R, mixed with Ipg PEmax variant mRNA, 100 pmol synthetic pegRNA (IDT) and 50pmol sgRNA (IDT), and then electroporated using NEON Nucleofection System 10 pL kit (Thermo Fisher Scientific, pulse voltage 1,500 V, pulse width 20 ms, 1 pulse). After electroporation, the iMy oblast cells were plated into a 6-well plate with HMP growth medium.
  • PEmax or PEmax** protein was incubated with 200 pmol of synthetic pegRNA (IDT) and 15 pmol of sgRNA (IDT) in R buffer to a total volume of 10 pL for 15 min at room temperature for complex formation.
  • 200k cells were electroporated with 10 pL of PEmax RNP complex using the same electroporation and culture conditions described above for mRNA delivery.
  • gDNA was isolated 3 days after electroporation from each group and stored at -80°C for Illumina library preparation for targeted amplicon deep sequencing.
  • PBMCs peripheral blood mononuclear cells
  • CD4+ T cell enrichment was confirmed by determining the percentage of CD3+/CD4+ cells via flow cytometry.
  • Isolated CD4+ T cells were cultured in complete RPMI-IL2 media (RPMI-1640 media (cat# 11875093, Thermofisher Scientific) supplemented with 10% heat-inactivated Cosmic Calf Serum (cat#SH30087.03, GE Life Sciences), 25 mM HEPES pH 7.2 (cat#25- 060-CI, Corning), 20 mM GlutaMAX (cat#3505-061, Gibco), 1 mM Sodium pyruvate (cat#25-000-CI, Coming), IX MEM non-essential amino acids (cat#25-025-CI, Corning), 1% penicillin-streptomycin (cat#l 5140-122, Gibco), and 1 :2000 human interleukin-2 (made in-house from IL-2 expressing cell line).
  • primary CD4+ T cells were activated with anti-CD3/CD28 antibodies (cat#10971, Stemcell Technologies).
  • activated primary CD4+ T cells 5xl0 5 cells per sample were pelleted by centrifugation for 5 min at 300 g and resuspended in 11 mL NEON Buffer T (Thermo Fisher Scientific). The cell solution was added to a mix of 1.5 mg PEmax (or the variants) mRNA, 120 pmol synthetic pegRNA (Integrated DNA Technologies), 30 pmol synthetic sgRNA (Synthego), and 0 to 1 mg VPX (or VPX Q76A ) mRNA.
  • Synthetic pegRNAs and sgRNAs were dissolved in TE buffer (10 mM Tris-HCl, pH 8.0; 0.1 mM EDTA). Mock control electroporations were performed with 3 mL NEON Buffer T without any RNA added. Electroporation on the NEON Transfection System (Thermo Fisher Scientific) was carried out using 10 mL NEON tips with the following parameters: 1,400 V, 10 ms, three pulses. Cells were plated in 600 mL fresh T cell media in a 24-well plate. 2 days after electroporation, ImL fresh T cell media was added to cells.
  • CD4+ T cells were allowed to recover in RPMI-IL2 media for 72 hours post nucleofection before genomic extraction using the Qiagen QiAamp DNA Blood Mini Kit (cat#51104, Qiagen).
  • Qiagen QiAamp DNA Blood Mini Kit catalog#51104, Qiagen.
  • VPX co-delivery experiments 0.5 - 4 pg of VPX protein (ab267924, Abeam) or 500 ng of VPX/VPX Q76A mRNA was added to the electroporation mixture described above.
  • HuES8-derived SC-islet cells were maintained and differentiated as described 60 in protocol version 8, except the medium used for Stage 6 was the Stage 7 medium described by Balboa and colleagues 61 , modified to include non-essential amino acids and to omit ZM447439, T3, and NAC.
  • differentiating cultures were treated throughout Stage 5 and for Days 1-7 of Stage 6 with 1 pM aphi dicolin (A-0781, Sigma).
  • GSIS glucose-stimulated insulin secretion
  • SC-islet clusters were washed twice MCDB131 medium (CM134-050, GenDepot) supplemented with glutagro (25- 015-CI, Corning) and 5.6 mM glucose and preincubated at 37° C for 1 h containing MCDB13 1 medium containing glutagro and 2.5 mM glucose (basal).
  • Clusters were then challenged with MCDB131 + glutagro containing basal glucose (2.5 mM), high glucose (20 mM), followed by depolarization with MCDB131 + glutagro containing 2.5 mM glucose + 20 mM KC1. Each treatment lasted 1 h, after which supernatant was collected and human insulin quantified using a human insulin TRF -PINCER® Assay (#IHTR1010, Mediomics).
  • SC-islet cells were dissociated and stained with the following: BV786 mouse anti-Ki-67 (cat. #563756, BD Biosciences), Alexa Fluor 790 mouse anti-glucagon (cat. #sc514592 AF790, Santa Cruz Biotechnology), Alexa Fluor 647 mouse anti-C-peptide (cat. #565831, BD Biosciences), and PE mouse anti-Sox2 (cat. #560291, BD Biosciences).
  • Flow cytometry data were acquired using a FACSymphonyTM Al cytometer (BD Biosciences) and analyzed using FlowJo software (BD Biosciences).
  • Islet reaggregation and DNA extraction Aggrewell 800 24-well plates (cat. #34811, STEMCELL Technologies) were prepared by adding 500 mL of anti-adherence rinsing solution (cat. #07010, STEMCELL Technologies) to each well. The plate was spun at 1300 x g for 5 minutes and wells were rinsed with 2 mL of warm complete CMRL 1066 media. This media was aspirated and replaced with 1 mL of warm complete CMRL 1066 media. The electroporated samples were then pipetted evenly into each well of the plate and the plate was centrifuged at 100 x g for 3 min.
  • PE-tag based quantitative PCR was described previously 27 . Briefly, 100 pmol synthetic pegRNA (IDT) targeting HEK4 was incubated with 50 pmol prime editor protein in 15 ml PBS for 20 min at room temperature to form RNP. Following complex formation, the PE RNP was mixed with the reaction buffer: dNTPs at a desired concentration (0 ⁇ l mM), 5% glycerol, 100 mM KC1, 10 mM HEPES, pH 7.5, 0.2 mM EDTA, 3 mM MgCh, and 5 mM DTT) and 2 pg purified gDNA in a total volume of 30 pl for 2 h at 37 °C.
  • the reaction was treated with 10 pl RNase A (50 pg/ml) to digest the pegRNA, and the gDNA was purified using a DNeasy Blood & Tissue Kit (Qiagen).
  • the tag incorporation rate was quantified by qPCR with a tag-specific primer and a locus-specific primer. A pair of primers located approximately 2,000 bp upstream of the target site served as a qPCR internal control for gDNA normalization.
  • 500,000 cells were electroplated with different amount of VPX (2 or 4 pg) or VPX Q76A (2 pg) mRNA using the NEON Nucleofection System 10 pL kit (Thermo Fisher Scientific), then transferred into 6-well plate. Cells were harvested 3 days later. To generate the desried number of cells for analysis multiple electroporations were performed in parallel. 2 to 3 million cells were harvested in PBS and lysed for 30 min on ice in RIPA buffer (10 mM Tris-Cl [pH 8.0], 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, and 140 mM NaCl) supplemented with protease inhibitors.
  • RIPA buffer 10 mM Tris-Cl [pH 8.0], 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, and 140 mM NaCl
  • Lysates were clarified by centrifugation (13,000 rpm for 10 min at 4 °C) and the supernatants were collected. Protein samples were quantified with the Bradford assay and resolved by SDS-PAGE, transferred onto polyvinylidene fluoride (PVDF), and probed using the indicated primary antibodies.
  • the membrane was stained with Rabbit anti- Mouse IgG (H+L) HRP-conjugated Secondary Antibody (Thermo), detected with SuperSignalTM West Pico PLUS Chemiluminescent Substrate (Thermo) and visualized with X-ray film. The following primary antibodies were used for staining: GAPDH (Santa Cruz, sc-47724) and SAMHD1 (abeam, ab 128107).
  • Viability testing was carried out for HEK293T cells or fibroblasts supplemented with different concentrations of dNs in their growth media. For each dN concentration, 50K cells were plated in 24-well plate. The cell viability was measured every 24 hours after supplementing with dNs with a different well harvested for each time point. Briefly, prior to trypsinization to release adherent cells, the growth medium containing floating cells from each well was placed into 1.5 ml Eppendorf tube. Adherent cells were released by adding 50ul Trypsin-EDTA (0.25%) solution and incubating at 37 °C for 3 min. The growth medium harvested from the well was used to resuspend the cells by pipetting 2 to 3 times.
  • a single cell suspension was confirmed under the microscope. 20 pL of the cell suspension was transferred to a centrifuge tube and 20 pL of 0.4% trypan blue solution was added. The mixture was incubated at room temperature for 2 minutes. Cell viability within each population was determined using a TC20 cell counter (Bio-Rad).
  • Fibroblasts or primary T cell pellets were washed twice with lx Dulbecco’s phosphate-buffered saline and resuspended in 100 pl of ice-cold 60% methanol. Samples were vortexed vigorously to lyse the cells and then heated at 95 °C for 3 min prior to centrifugation at 12,000 g for 30 s. Supernatants were removed by pipet and passed through 3-kDa cutoff centrifugal filters (Amicon Ultra-0.5 ml, Merck) into collection tubes to remove the majority of remaining macromolecules.
  • the cellular extracts were subjected to a Q5 DNA polymerase and EvaGreen-based assay for dNTPs using dNTP-specific 197-nt templates with conserved primer binding sites as previously described 43 .
  • the baseline and the end-point fluorescence were read at a temperature above the primer annealing temperature (the temperature for baseline and end-point fluorescence to 75 °C for dATP and dCTP, 78 °C for dTTP, and 73.5 °C for dGTP).
  • the polymerization reaction time at 66°C was limited to 55 min for dATP, 40 min for dTTP and dCTP, and 20 min for dGTP detection.
  • Zebrafish were maintained and bred according to standard protocols. Zebrafish embryos obtained from EK (WT) wild-type in-crosses were used for one cell-stage microinjections of PE RNPs. Prior to injections the tek target sequence was verified by Sanger sequencing. For IxRNP, 12 pM pegRNA (synthesized by IDT) and 6 pM PE protein were combined in nuclease-free water. For 2x and 4X RNP, the amount of pegRNA and protein used were scaled up 2-fold and 4-fold respectively. Complexes were incubated at room temperature for 5 minutes and then 2 nl was injected into single-cell embryos. Injected embryos were incubated at 28.5 °C overnight. Twenty-four hours post injection embryos were assessed for toxicity and genomic DNA was extracted from 20 normally developing embryos using the Qiagen DNeasy Blood and Tissue kit (Qiagen). Injections were performed in three independent replicates.
  • Qiagen DNeasy Blood and Tissue kit
  • PEmax and PEmax** mRNA were in vitro transcribed and purified as described above. For in vivo mouse experiments, an additional round of purification using cellulose was performed to remove dsRNA contaminants 62 .
  • the purified PEmax/PEmax** mRNA (40pg) was mixed with Pcsk9 synthetic pegRNA (15 g, IDT) and nicking sgRNA (5 g, IDT) in lOmM citrate buffer.
  • RNA mix constituted the aqueous component for forming RNA-loaded LNPs by microfluidic mixing using the NanoAssemblr Ignite (Precision Nanosystems) with a 3 : 1 aqueous: ethanol flow rate ratio and a total flow rate of 12mL/min.
  • the ethanol component consisted of a lipid mix of D-Lin-MC3-DMA (MedChem Express: HY-112251), DSPC (Sigma-Aldrich: Pl 138), cholesterol (Sigma-Aldrich: C8667), and DMG-PEG at a 50:10:38.5: 1.5 molar ratio 63 .
  • a lipid to nucleic acid weight ratio of 20 was used to determine mixing volume.
  • LNPs were dialyzed (Sigma- Aldrich: PURX35O5O) overnight at 4°C in IxDPBS (Thermo: 14190250), sterile filtered (Thermo: 723-2520), and concentrated (Millipore: UFC8100) to 150j.il per dose. Quality control was performed by dynamic light scattering (Malvern ZetaSizer) to assess particle diameter & RiboGreen (Invitrogen: R11490) quantification of RNA encapsulation efficiency.
  • LNPs containing mRNA were delivered by retro-orbital injection into B6 wild type female mice (three doses spaced one week apart). Fresh LNPs were formulated for each dose. Mice were sacrificed one week after the final dose and the liver was harvested. Three liver punches from each mouse liver (one from each lobe) were taken for gDNA extraction. The editing rates were then determined by targeted amplicon deep sequencing.
  • Genomic DNA was isolated for prime editing analysis from treated cells, zebrafish embryos or mouse liver. Genomic loci spanning each target site were PCR amplified with locus-specific primers carrying tails complementary to the Truseq adapters. 200 ng of genomic DNA was used for the 1 st PCR using Phusion master mix (Thermo) with locus specific primers that contain i5 and i7 adaptor tails. PCR products from the 1 st PCR were used for the 2 nd PCR with i5 primers and i7 primers to complete the adaptors and include the i5 and i7 indices. All primers used for the amplicon sequencing are listed in the Supplementary table.
  • PCR products were purified with Ampure beads (0.9X reaction volume), eluted with 25ul of TE buffer, and quantified by Qubit. Equal molar ratios of each amplicon were pooled and sequenced using Illumina Miniseq. Amplicon sequencing data was analyzed with CRISPResso2. Briefly, demultiplexing and base calling were both performed using bcl2fastq Conversion Software v2.18 (Illumina, Inc.), allowing 1 barcode mismatch with a minimum trimmed read length of 75. Alignment of sequencing reads to each amplicon sequence was performed using CRISPResso2. Since there are multiple base changes or an insertion or deletion in our prime editing samples, we analyze precise editing and indels separately.
  • samples were analyzed by CRISPResso2 in regular batch mode using the following parameters: “-q 30”, discard indel reads TRUE”, and “-qwc (or — quantification window coordinates)” and “-- expected hdr amplicon seq” providing the desired edited amplicon sequence for each target site.
  • the qwc value specifies the quantification window for indel analysis, including the entire sequence between pegRNA and nicking sgRNA-directed Cas9 nicking sites (when TWIN -PE or PE3 is employed), as well as an additional 10 bp beyond both cut sites.
  • Intended PE editing efficiency was calculated as the percentage of reads with the desired edit without indels (“-discard indel reads TRUE.” mode) out of the total number of reads ((number of desired edit-containing reads) / (number of reference-aligned reads)).
  • Unintended editing frequency was calculated as the number of discarded reads divided by the total number of reads ((number of indel-containing reads) / (number of reference-aligned reads)).
  • the desired prime editing outcome comprises multiple base changes or an insertion or deletion
  • samples were analyzed by CRISPResso2 in regular batch mode using the following parameters: “-q 30”, discard indel reads TRUE”, “-qwc” and “— expected hdr amplicon seq” providing the desired edited amplicon sequence for each target site.
  • the intended edit was calculated by (the number of HDR- aligned reads) / (number of reference-aligned reads).
  • Unintended edits were calculated as the number of discarded reads divided by the total number of reads ((number of reads “Discarded” reads from the reference-aligned sequences + number of the “Discarded” reads from the HDR-aligned sequences)/(number of reference-aligned reads)).
  • the analysis of scaffold sequences incorporated within the unintended edited sequence population was determined using the scaffold sequence analysis pipeline previously described 11 , where incorporation of at least 2 bases of the scaffold sequence were required to assign the read as containing a scaffold insertion.
  • SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase. Nature 480, 379-382 (2011). Laguette, N. et al. SAMHDl is the dendritic- and myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx. Nature 474, 654-657 (2011). Deutschmann, J. & Gramberg, T. SAMHD1 ... and Viral Ways around It. Viruses 13 (2021). Bergamaschi, A. et al.
  • the human immunodeficiency virus type 2 Vpx protein usurps the CUL4A-DDB1 DCAF1 ubiquitin ligase to overcome a postentry block in macrophage infection.
  • Hrecka, K. et al. Vpx relieves inhibition of HIV-1 infection of macrophages mediated by the SAMHD1 protein. Nature 474, 658-661 (2011). Korin, Y.D.
  • a prime editor protein comprising a Cas9 nickase fused to a Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase domain, wherein the M- MLV reverse transcriptase domain comprises a V223M and a L435K mutation.
  • M-MLV Moloney Murine Leukemia Virus
  • a programmable prime editing system for modification of a double- stranded target DNA sequence comprising a target strand and a complementary non-target strand comprising: the prime editor protein according to potential claim Pl; and a prime editing guide RNA (pegRNA) comprising a spacer sequence having a region of complementarity to the target strand of the double-stranded target DNA sequence.
  • pegRNA prime editing guide RNA
  • the programmable prime editing system according to potential claims 3, wherein the programmable prime editing system further comprises an inhibitor of SAMHDl.
  • P6 The prime editing system according to any one of potential claims P3-P5, wherein the programmable prime editing system comprises a nicking sgRNA.
  • P7 A method of modifying a double- stranded target DNA sequence, the method comprising contacting the double-stranded target DNA sequence with the prime editor protein and the pegRNA according to any one of potential claims P3-P6.
  • a method of modifying a double-stranded target DNA sequence in a cell comprising: sequentially introducing into the cell, in the following order:
  • a programmable prime editing system comprising (i) a prime editor protein comprising a Cas9 nickase fused to a M- MLV reverse transcriptase domain and (ii) a pegRNA comprising a spacer sequence having a region of complementarity to a target strand of the double-stranded target DNA sequence; and
  • M-MLV reverse transcriptase domain comprises a V223M and a L435K mutation.
  • Pl 1 The method of potential claim PIO, wherein the inhibitor of SAMHD1 is selected from the group consisting of Vpx, Vpx (S13E), EBV BGLF4, HHV-6/7 U69, HCMV UL97, and KSHV ORF36.
  • P13 The method according to any one of potential claims P8-P12, wherein the nicking sgRNA is introduced into the cell 6-24 hours after introducing the programmable prime editing system into the cell.
  • P14 The method according to any one of potential claims P8-P13, wherein the programmable prime editing system is introduced into the cell by a first method selected from the group consisting of electroporation, transfection, viral delivery, virus-like particle delivery, and lipid nanoparticle delivery.
  • nicking sgRNA is introduced into the cell by a second method selected from the group consisting of electroporation, transfection, viral delivery, virus-like particle delivery, and lipid nanoparticle delivery.

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L'invention concerne des systèmes d'édition de base présentant une efficacité d'édition de base améliorée, et des procédés d'utilisation de ceux-ci.
PCT/US2024/049905 2023-10-06 2024-10-04 Éditeurs de base à efficacité d'édition de base améliorée Pending WO2025076306A1 (fr)

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