[go: up one dir, main page]

WO2025128545A1 - Systèmes d'éléments génétiques mobiles modifiés - Google Patents

Systèmes d'éléments génétiques mobiles modifiés Download PDF

Info

Publication number
WO2025128545A1
WO2025128545A1 PCT/US2024/059345 US2024059345W WO2025128545A1 WO 2025128545 A1 WO2025128545 A1 WO 2025128545A1 US 2024059345 W US2024059345 W US 2024059345W WO 2025128545 A1 WO2025128545 A1 WO 2025128545A1
Authority
WO
WIPO (PCT)
Prior art keywords
editing system
genome editing
r2tg
genome
rna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2024/059345
Other languages
English (en)
Inventor
Max Atticus ENGLISH
Liyang Zhang
Sean BENLER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Aera Therapeutics Inc
Original Assignee
Aera Therapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Aera Therapeutics Inc filed Critical Aera Therapeutics Inc
Publication of WO2025128545A1 publication Critical patent/WO2025128545A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • 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
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • 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]
    • C12N9/222—Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes
    • C12N9/226—Class 2 CAS enzyme complex, e.g. single CAS protein
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/90—Vectors containing a transposable element

Definitions

  • XML copy created on December 9, 2024, is named 098791-000104WOPT_SL.xml and is 115,701bytes in size.
  • TECHNOLOGY FIELD [0003] The present disclosure relates to recombinant mobile element systems and uses thereof. Specifically, the recombinant mobile element systems of the present disclosure are derived from R2Tg, which is a non-long terminal repeat (nLTR) retrotransposon from the zebra finch Taeniopygia guttata.
  • nLTR non-long terminal repeat
  • BACKGROUND is a non-long terminal repeat (nLTR) retrotransposon from the zebra finch Taeniopygia guttata.
  • MGEs mobile genetic elements
  • ORF open reading frame
  • R2Tg RNA inserts itself via template-primed reverse transcription into the multicopy 28S rDNA locus.
  • the determinants of this specificity are poorly defined, and possibly derive from both the DNA binding domains of the protein and short homologous sequences outside of either (or both) of the UTR sequences.
  • a nLTR retrotransposon system must have a high full-length insertion efficiency at a target site.
  • this disclosure describes, in part, a recombinant mobile element system derived from R2Tg, which is a non-long terminal repeat (nLTR) retrotransposon from the zebra finch Taeniopygia guttata, having at least one mutation that increases the 3’ insertion, the 5’ insertion, or both.
  • nLTR non-long terminal repeat
  • an R2Tg enzyme comprises an amino acid sequence SEQ ID NO:1
  • an R2Tg enzyme comprises an amino acid sequence SEQ ID NO:1 (wt sequence) with a mutation selected from the group consisting of D944K, S870R, E638K, T1367R, G1056R, and combinations thereof;
  • a payload RNA wherein the payload RNA comprises an insertion template and optionally one or more of a 5’ homology arm, a 3’ homology arm, and a protein binding element, wherein the insertion template comprises a sequence for a nucleic acid insertion into the genome.
  • an R2Tg enzyme comprises an amino acid sequence SEQ ID NO:2 or comprises a variant of a protein effector comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 2; ii) a payload RNA, wherein the payload RNA comprises an insertion template and optionally one or more of a 5’ homology arm, a 3’ homology arm, and a protein binding element, wherein the insertion template comprises a sequence for a nucleic acid insertion into the genome.
  • the 5’ homology arm is less than or about 105 base pairs, is less than or about 50 base pairs, is less than or about 25 base pairs.
  • the 3’ homology arm is less than or about 50 base pairs, is less than or about 25 base pairs, is less than or about 10 base pairs.
  • the R2Tg element enzyme further comprises a targeting domain.
  • the targeting domain is a natural targeting domain or an engineered targeting domain
  • the targeting domain is a natural targeting domain.
  • the targeting domain is an engineered targeting domain.
  • the nucleic acid insertion into the genome is a DNA or RNA insertion template.
  • the R2Tg element enzyme is a modified R2Tg element enzyme.
  • the coding sequence of the R2Tg element enzyme is modified.
  • the modified R2Tg element enzyme further comprises a mutation selected from S645K, E638K, V661R, G257R, G236R, I558R, D555R, S870R, T934R, C587K, E638K, V661R, L388R, E471R, L243R, D923K, S763R, I308R, T1016R, T1023K, G1056R, N1103K, Y1242K, T1367R or combinations thereof.
  • the modified R2Tg element enzyme is modified by an N-terminal or C-terminal truncation of the R2Tg element enzyme sequence.
  • the modified R2Tg element enzyme is modified by an N-terminal truncation of the R2Tg element enzyme sequence.
  • the modified R2Tg element enzyme comprises SEQ ID. NO:3.
  • the genome editing system targets a genomic locus.
  • the genome editing system targets a genomic locus other than the 28S rRNA locus.
  • the modified R2Tg element is fused to a TALEN protein, zinc finger protein, argonaute, or meganuclease protein.
  • the genome editing system further comprises a guide RNA.
  • the 5’ homology arm, the 3’ homology arm, or both the 5’ and 3’ homology arm of the payload RNA is engineered to target a genomic locus other than the 28 S rRNA locus.
  • the 5’ homology arm, the 3’ homology arm, or both the 5’ and 3’ homology arm target an exogenously introduced landing sequence.
  • the insertion region is introduced into the genome of a specific cell type.
  • the specific cell type is a post-mitotic cell.
  • the genome editing system functions in post-mitotic cells.
  • the genome editing system functions independently from intrinsic nucleic acid repair systems.
  • the payload RNA template further comprises a 5’ untranslated region (UTR), a 3’ UTR, or both a 5’ UTR and a 3’ UTR.
  • the 5’ homology arm and the 3’ homology arm are located between the 5’ UTR and 3’ UTR. [0041] In some embodiments of any of the aspects, the 5’ homology arm and the 3’ homology arm are located outside the 5’ UTR and 3’ UTR. 4 4900-8642-4067.5 Attorney Docket No.: 098791-000104WOPT [0042] In some embodiments of any of the aspects, the payload RNA further comprises a 5’ untranslated region (UTR), a 3’ UTR, or both a 5’ and a 3’ UTR, wherein the UTRs are truncated.
  • UTR untranslated region
  • the payload RNA does not comprise a 5’ UTR. [0044] In some embodiments of any of the aspects, the payload RNA does not comprise a 3’ UTR. [0045] In some embodiments of any of the aspects, the payload RNA further comprises a nuclear retention element. [0046] In some embodiments of any of the aspects, the payload RNA further comprises a Cas9 or Cas12 guide RNA, and wherein the Cas9 or Cas12 guide RNA comprises an extension with a 5’ homology sequence, a 3’ homology sequence, a 5’ untranslated region (UTR), a 3’ UTR, an insertion template, or any combination thereof.
  • the nucleic acid insertion template is a sequence of greater than 1000 base pairs.
  • the R2Tg element enzyme comprises a nuclear localization signal (NLS).
  • the insertion region comprises a template for a reporter gene, a transcription factor gene, a transgene, an enzyme gene, or a therapeutic gene.
  • modified Cas9 protein is fused to an R2Tg element enzyme.
  • modified Cas9 fusion protein targets an endogenous landing site.
  • Cas9 fusion protein targets an exogenously introduced landing site in the genome of the post-mitotic cell.
  • described herein is a method of editing a genome comprising subjecting the cell to a genome editing system as described herein.
  • the components of the genome editing system are delivered as all RNA in lipid nanoparticles or another RNA delivery reagent.
  • the non-LTR site specific retrotransposon is delivered as mRNA.
  • the guide RNAs are delivered as synthetic RNA.
  • the payload is delivered as mRNA.
  • the genome editing system targets and edits the genome at more than one site.
  • FIGS.1A and 1B depict the insertion activities across the range of cargo sizes with different homology arms for R2Tg wild type sequence.
  • FIGS.2A and 2B illustrate the insertion activities across the range of cargo sizes with different homology arms for R2Tg sequence with 184 nucleotide truncations at the N- terminus.
  • SEQ ID NO: 2 comprises a mutation selected from S645K, E638K, V661R, G257R, G236R, I558R, D555R, S870R, T934R, C587K, E638K, V661R, L388R, E471R, L243R, D923K, S763R, I308R, T1016R, T1023K, G1056R, N1103K, Y1242K, T1367R or combinations thereof.
  • the invention disclosed herein is a genome editing system comprising: i) an R2Tg enzyme comprises an amino acid sequence SEQ ID NO:5, or comprises a variant of a protein effector comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:5; ii) a payload RNA, wherein the payload RNA comprises an insertion template and optionally one or more of a 5’ homology arm, a 3’ homology arm, and a protein binding element, wherein the insertion template comprises a sequence for a nucleic acid insertion into the genome.
  • the N-terminal truncation is at least 50 nucleotides, at least 100 nucleotides, at least 150 nucleotides, at least 180 nucleotides.
  • the invention disclosed herein is a genome editing system comprising: i) an R2Tg enzyme comprises an amino acid sequence SEQ ID NO:6, or comprises a variant of a protein effector comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to an amino acid sequence selected 8 4900-8642-4067.5 Attorney Docket No.: 098791-000104WOPT from the group consisting of SEQ ID NO: 6; ii) a payload RNA, wherein the payload RNA comprises an insertion template and optionally one or more of a 5’ homology arm, a 3’ homology arm, and a protein binding element, wherein the insertion template comprises a sequence for a nucleic acid insertion into the genome.
  • the invention disclosed herein is a genome editing system comprising: i) an R2Tg enzyme comprises an amino acid sequence SEQ ID NO:7, or comprises a variant of a protein effector comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 7; ii) a payload RNA, wherein the payload RNA comprises an insertion template and optionally one or more of a 5’ homology arm, a 3’ homology arm, and a protein binding element, wherein the insertion template comprises a sequence for a nucleic acid insertion into the genome.
  • SEQ ID NO: 6 comprises a mutation selected from D944K, S645K, V661R, G257R, G236R, I558R, D555R, S870R, T934R, C587K, E638K, V661R, L388R, E471R, L243R, D923K, S763R, I308R, T1016R, T1023K, G1056R, N1103K, Y1242K, T1367R or combinations thereof.
  • SEQ ID NO: 6 comprises a mutation selected from D944K, S645K, V661R, G257R, G236R, I558R, D555R, S870R, T934R, C587K, E638K, V661R, L388R, E471R, L243R, D923K, S763R, I308R, T1016R, T1023K, G1056R, N1103K, Y1242K, T1367R or combinations thereof; and wherein SEQ ID NO:6 is modified by an N- terminal truncation.
  • the N-terminal truncation is at least 50 nucleotides, at least 100 nucleotides, at least 150 nucleotides, at least 180 nucleotides.
  • the invention disclosed herein is a genome editing system comprising: i) an R2Tg enzyme comprises an amino acid sequence SEQ ID NO:8, or comprises a variant of a protein effector comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 8; ii) a payload RNA, wherein the payload RNA comprises an insertion template and optionally one or more of a 5’ homology arm, a 3’ homology arm, and a protein binding element, wherein the insertion template comprises a sequence for a nucleic acid insertion into the genome.
  • the invention disclosed herein is a genome editing system comprising: i) an R2Tg enzyme comprises an amino acid sequence SEQ ID NO:9, or comprises a variant of a protein effector comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to an amino acid sequence selected 9 4900-8642-4067.5 Attorney Docket No.: 098791-000104WOPT from the group consisting of SEQ ID NO: 9; ii) a payload RNA, wherein the payload RNA comprises an insertion template and optionally one or more of a 5’ homology arm, a 3’ homology arm, and a protein binding element, wherein the insertion template comprises a sequence for a nucleic acid insertion into the genome.
  • SEQ ID NO: 8 comprises a mutation selected from D944K, S645K, E638K, V661R, G257R, G236R, I558R, D555R, S870R, T934R, C587K, E638K, V661R, L388R, E471R, L243R, D923K, S763R, I308R, T1016R, T1023K, G1056R, N1103K, Y1242K or combinations thereof.
  • SEQ ID NO: 8 comprises a mutation selected from D944K, S645K, E638K, V661R, G257R, G236R, I558R, D555R, S870R, T934R, C587K, E638K, V661R, L388R, E471R, L243R, D923K, S763R, I308R, T1016R, T1023K, G1056R, N1103K, Y1242K or combinations thereof; and wherein SEQ ID NO:8 is modified by an N- terminal truncation.
  • the N-terminal truncation is at least 50 nucleotides, at least 100 nucleotides, at least 150 nucleotides, at least 180 nucleotides.
  • the invention disclosed herein is a genome editing system comprising: i) an R2Tg enzyme comprises an amino acid sequence SEQ ID NO:10, or comprises a variant of a protein effector comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 10; ii) a payload RNA, wherein the payload RNA comprises an insertion template and optionally one or more of a 5’ homology arm, a 3’ homology arm, and a protein binding element, wherein the insertion template comprises a sequence for a nucleic acid insertion into the genome.
  • the invention disclosed herein is a genome editing system comprising: i) an R2Tg enzyme comprises an amino acid sequence SEQ ID NO:11, or comprises a variant of a protein effector comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 11; ii) a payload RNA, wherein the payload RNA comprises an insertion template and optionally one or more of a 5’ homology arm, a 3’ homology arm, and a protein binding element, wherein the insertion template comprises a sequence for a nucleic acid insertion into the genome.
  • SEQ ID NO: 10 comprises a mutation selected from D944K, S645K, E638K, V661R, G257R, G236R, I558R, D555R, S870R, T934R, C587K, E638K, V661R, L388R, E471R, L243R, D923K, S763R, I308R, T1016R, T1023K, N1103K, Y1242K, T1367R or combinations thereof.
  • SEQ ID NO: 10 comprises a mutation selected from D944K, S645K, E638K, V661R, G257R, G236R, I558R, D555R, S870R, T934R, C587K, E638K, V661R, L388R, E471R, L243R, D923K, S763R, I308R, T1016R, T1023K, N1103K, Y1242K, T1367R or combinations thereof; and wherein SEQ ID NO:10 is modified by an N- terminal truncation.
  • the N-terminal truncation is at least 50 nucleotides, at least 100 nucleotides, at least 150 nucleotides, at least 180 nucleotides.
  • each of the verbs, “comprise,” “include” and “have” and conjugates thereof, are used to indicate that the object or objects of the verb are not necessarily a complete listing of components, elements or parts of the subject or subjects of the verb.
  • Other terms as used herein are meant to be defined by their well-known meanings in the art.
  • targeting sequence refers a nucleotide sequence or molecule comprising a nucleotide sequence that is capable of hybridizing to a specific target sequence, e.g., the targeting sequence has a nucleotide sequence which is at least partially complementary to the sequence being targeted along the 22 4900-8642-4067.5 Attorney Docket No.: 098791-000104WOPT length of the targeting sequence.
  • the targeting sequence or targeting molecule may be part of a RNA guide molecule that can form a complex with a CRISPR nuclease.
  • the RNA guide molecule When the RNA guide molecule comprising the targeting sequence is present contemporaneously with the CRISPR nuclease, the RNA guide molecule is capable of targeting the CRISPR nuclease to the specific target sequence.
  • the targeting sequence or targeting molecule may comprise a homology arm that targets a RNA template molecule to a target site for insertion or copying of an insert template sequence at the target site.
  • Each possibility represents a separate embodiment.
  • “cell-specific,” or “cell-type specific,” would be understood by one of skill in the art to mean occurring or being expressed at a higher frequency or existing at an increased level in one cell type in contrast to other cell types.
  • NLS is intended to encompass not only the nuclear localization sequence of a particular peptide, but also derivatives thereof that are capable of directing translocation of a cytoplasmic 24 4900-8642-4067.5 Attorney Docket No.: 098791-000104WOPT polypeptide across the nuclear envelope barrier.
  • NLSs are capable of directing nuclear translocation of a polypeptide when attached to the N-terminus, the C-terminus, or both the N- and C-termini of the polypeptide.
  • a polypeptide having an NLS coupled by its N-terminus or C-terminus to amino acid side chains located randomly along the amino acid sequence of the polypeptide will be translocated.
  • the R2Tg element naturally targets the 28S rRNA locus.
  • the instant disclosure contemplates the insertion of payloads into either the 28 S rRNA locus or into other genomic loci.
  • the insertion site is a targeted genomic insertion site.
  • the insertion site is targeted by a targeting domain in a fusion protein.
  • the insertion site is targeted by a targeting domain in a landing pad.
  • the insertion site has been exogenously introduced to the genome (i.e. via lentivirus, or recombinase, and the like).
  • promoters include one or more pol III promoter (e.g., 1, 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof.
  • pol III promoters include, but are not limited to, U6 and HI promoters.
  • Non-viral vehicles examples include lipid nanoparticles, cell- penetrating peptides (CPPs), DNA nanoclews, gold nanoparticles, streptolysin O, multifunctional envelope-type nanodevices (MENDs), lipid-coated mesoporous silica particles, and other inorganic nanoparticles.
  • Lipid particles [0166]
  • the delivery vehicles may comprise lipid particles, e.g., lipid nanoparticles (LNPs) and liposomes.
  • LNPs may encapsulate nucleic acids within cationic lipid particles (e.g., liposomes), and may be delivered to cells with relative ease.
  • lipid nanoparticles do not contain any viral components, which helps minimize safety and immunogenicity concerns.
  • Lipid particles may be used for in vitro, ex vivo , and in vivo deliveries. Lipid particles may be used for various scales of cell populations. 38 4900-8642-4067.5 Attorney Docket No.: 098791-000104WOPT [0168] In some examples.
  • LNPs may be used for delivering DNA molecules and/or RNA molecules. In certain cases, LNPs may be use for delivering RNP complexes.
  • Components in LNPs may comprise cationic lipids 1,2- dilineoyl-3- dimethylammonium -propane (DLinDAP), l,2-dilinoleyloxy-3-N,N- dimethylaminopropane (DLinDMA), l,2-dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA), 1,2- dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLinKC2-DMA), (3- o-[2"- (methoxypolyethyleneglycol 2000) succinoyl]-l,2-dimyristoyl-sn-glycol (PEG-S-DMG), R-3- [( ⁇ -methoxy-poly(ethylene glycol)2000) carbamoyl]-l,2-dimyristyloxlpropyl-3-amine (P
  • lipid particle may be liposome.
  • Liposomes are spherical vesicle structures composed of a uni- or multilamellar lipid bilayer surrounding internal aqueous compartments and a relatively impermeable outer lipophilic phospholipid bilayer.
  • liposomes are biocompatible, nontoxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their load across biological membranes and the blood brain barrier (BBB).
  • BBB blood brain barrier
  • Liposomes can be made from several different types of lipids, e.g., phospholipids.
  • a liposome may comprise natural phospholipids and lipids such as 1,2-distearoryl-sn- glycero-3 -phosphatidyl choline (DSPC), sphingomyelin, egg phosphatidylcholines, monosialoganglioside, or any combination thereof.
  • DSPC 1,2-distearoryl-sn- glycero-3 -phosphatidyl choline
  • sphingomyelin sphingomyelin
  • egg phosphatidylcholines monosialoganglioside, or any combination thereof.
  • monosialoganglioside monosialoganglioside
  • liposomes may further comprise cholesterol, sphingomyelin, and/or l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), e.g., to increase stability and/or to prevent the leakage of the liposomal inner cargo.
  • Stable nucleic-acid-lipid particles SNALPs
  • 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 neutral helper lipid, cholesterol, a diffusible polyethylene glycol (PEG)-lipid, or any combination thereof.
  • DLinDMA ionizable lipid
  • PEG diffusible polyethylene glycol
  • SNALPs may comprise synthetic cholesterol, l,2-distearoyl-sn-glycero-3- phosphocholine, PEG- cDMA, and l,2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane (DLinDMA).
  • the lipid particles may also comprise one or more other types of lipids, e.g., cationic lipids, such as amino lipid 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]- dioxolane (DLin-KC2-DMA), DLin-KC2-DMA4, C12- 200 and colipids disteroylphosphatidyl choline, cholesterol, and PEG-DMG.
  • DLin-KC2-DMA amino lipid 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]- dioxolane
  • DLin-KC2-DMA DLin-KC2-DMA4-dimethylaminoethyl-[l,3]- dioxolane
  • DLin-KC2-DMA DLin-KC2-DMA4-dimethylaminoethyl-[l,3]- dioxolane
  • DLin-KC2-DMA
  • lipoplexes may be complexes comprising lipid(s) and non-lipid components.
  • lipoplexes and polyplexes include FuGENE-6 reagent, a non-liposomal solution containing lipids and other components, zwitterionic amino lipids (ZALs), Ca2J) (e.g., forming DNA/Ca 2+ microcomplexes), polyethylenimine (PEI) (e.g., branched PEI), and poly(L-lysine) (PLL).
  • ZALs zwitterionic amino lipids
  • Ca2J zwitterionic amino lipids
  • Ca2J zwitterionic amino lipids
  • Ca2J zwitterionic amino lipids
  • Ca2J zwitterionic amino lipids
  • Ca2J zwitterionic amino lipids
  • Ca2J zwitterionic amino lipids
  • Ca2J zwitterionic amino lipids
  • CPPs are short peptides that facilitate cellular uptake of various molecular cargo (e.g., from nanosized particles to small chemical molecules and large fragments of DNA).
  • CPPs may be of different sizes, amino acid sequences, and charges.
  • CPPs can translocate the plasma membrane and facilitate the delivery of various molecular cargoes to the cytoplasm or an organelle.
  • CPPs may be introduced into cells via different mechanisms, e.g., direct penetration in the membrane, endocytosis-mediated entry, and translocation through the formation of a transitory structure.
  • CPPs may have an amino acid composition that either contains a high relative abundance of positively charged amino acids such as lysine or arginine or has sequences that contain an alternating pattern of polar/charged amino acids and non-polar, hydrophobic amino acids. These two types of structures are referred to as polycationic or amphipathic, respectively.
  • a third class of CPPs are the hydrophobic peptides, containing only apolar residues, with low net charge or have hydrophobic amino acid groups that are crucial for 40 4900-8642-4067.5 Attorney Docket No.: 098791-000104WOPT cellular uptake.
  • CPPs trans-activating transcriptional activator
  • Tat Human Immunodeficiency Virus 1
  • HAV-1 Human Immunodeficiency Virus 1
  • CPPs include to Penetratin, Tat (48-60), Transportan, and (R-AhX-R4) (Ahx refers to aminohexanoyl), Kaposi fibroblast growth factor (FGF) signal peptide sequence, integrin b3 signal peptide sequence, polyarginine peptide Args sequence, Guanine rich-molecular transporters, and sweet arrow peptide.
  • FGF Kaposi fibroblast growth factor
  • FGF Kaposi fibroblast growth factor
  • integrin b3 signal peptide sequence
  • polyarginine peptide Args sequence sequence
  • Guanine rich-molecular transporters and sweet arrow peptide.
  • sweet arrow peptide sweet arrow peptide
  • CPPs can be used for in vitro and ex vivo work quite readily, and extensive optimization for each cargo and cell type is usually required.
  • CPPs may be covalently attached to the engineered protein directly, which is then complexed with the gRNA and delivered to cells.
  • CPP may also be used to delivery RNPs.
  • CPPs may be used to deliver the compositions and systems to plants.
  • CPPs may be used to deliver the components to plant protoplasts, which are then regenerated to plant cells and further to plants.
  • DNA nanoclews [0181] In some embodiments, the delivery vehicles comprise DNA nanoclews.
  • a DNA nanoclew refers to a sphere-like structure of DNA (e.g., with a shape of a ball of yarn).
  • the nanoclew may be synthesized by rolling circle amplification with palindromic sequences that aide in the self-assembly of the structure. The sphere may then be loaded with a payload.
  • An example of DNA nanoclew is described in Sun W et al, J Am Chem Soc.2014 Oct 22; 136(42): 14722-5; and Sun W et al, Angew Chem Int Ed Engl.2015 Oct 5;54(41):12029-33.
  • a DNA nanoclew may be coated, e.g., coated with PEI to induce endosomal escape.
  • the delivery vehicles comprise gold nanoparticles (also referred to AuNPs or colloidal gold).
  • Gold nanoparticles may form complex with cargos.
  • Gold nanoparticles may be coated, e.g., coated in a silicate and an endosomal disruptive polymer, PAsp(DET).
  • PAsp(DET) an endosomal disruptive polymer
  • gold nanoparticles include AuraSense Therapeutics' Spherical Nucleic Acid (SNATM) constructs, and those described in Mout R, et al. (2017). ACS Nano 11:2452-8; Lee K, et al. (2017). Nat Biomed Eng 1:889-901.
  • the delivery vehicles comprise iTOP.
  • iTOP refers to a combination of small molecules drives the highly efficient intracellular delivery of native proteins, independent of any transduction peptide.
  • iTOP may be used for induced transduction by osmocytosis and propanebetaine, using NaCl-mediated hyperosmolality together with a transduction compound (propanebetaine) to trigger macropinocytotic uptake into cells of extracellular macromolecules.
  • Examples of iTOP methods and reagents include those described in D'Astolfo DS, Pagliero RJ, Pras A, et al. (2015).
  • the delivery vehicles may 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 shRNA, 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.
  • the delivery vehicles may be streptolysin O (SLO).
  • SLO is a toxin produced by Group A streptococci that works by creating pores in mammalian cell membranes. SLO may act in a reversible manner, which allows for the delivery of proteins (e.g., up to 100 kDa) to the cytosol of cells without compromising overall viability. Examples of SLO include those described in Sierig G, et al. (2003).
  • the delivery vehicles may comprise multifunctional envelope-type nanodevice (MENDs).
  • MENDs may comprise condensed plasmid DNA, a PLL core, and a lipid film shell.
  • a MEND may further comprise cell-penetrating peptide (e.g., stearyl octaarginine). The cell penetrating peptide may be in the lipid shell.
  • the lipid envelope may be modified 42 4900-8642-4067.5 Attorney Docket No.: 098791-000104WOPT with one or more functional components, e.g., one or more of: polyethylene glycol (e.g., to increase vascular circulation time), ligands for targeting of specific tissues/cells, additional cell- penetrating peptides (e.g., for greater cellular delivery), lipids to enhance endosomal escape, and nuclear delivery tags.
  • the MEND may be a tetra-lamellar MEND (T- MEND), which may target the cellular nucleus and mitochondria.
  • a MEND may be a PEG-peptide-DOPE-conjugated MEND (PPD-MEND), which may target bladder cancer cells.
  • PPD-MEND PEG-peptide-DOPE-conjugated MEND
  • MENDs include those described in Kogure K, et al. (2004). J Control Release 98:317-23; Nakamura T, et al. (2012). Acc Chem Res 45:1113-21.
  • Lipid-coated mesoporous silica particles [0187]
  • the delivery vehicles may comprise lipid-coated mesoporous silica particles.
  • Lipid-coated mesoporous silica particles may comprise a mesoporous silica nanoparticle core and a lipid membrane shell.
  • the silica core may have a large internal surface area, leading to high cargo loading capacities.
  • pore sizes, pore chemistry, and overall particle sizes may be modified for loading different types of cargos.
  • the lipid coating of the particle may also be modified to maximize cargo loading, increase circulation times, and provide precise targeting and cargo release. Examples of lipid-coated mesoporous silica particles include those described in Du X, et al. (2014). Biomaterials 35:5580-90; Durfee PN, et al. (2016). ACS Nano 10:8325-45.
  • the delivery vehicles may comprise inorganic nanoparticles.
  • inorganic nanoparticles examples include carbon nanotubes (CNTs) (e.g., as described in Bates K and Kostarelos K. (2013). Adv Drug Deliv Rev 65:2023-33.), bare mesoporous silica nanoparticles (MSNPs) (e.g., as described in Luo GF, et al. (2014). Sci Rep 4:6064), and dense silica nanoparticles (SiNPs) (as described in Luo D and Saltzman WM. (2000). Nat Biotechnol 18:893-5).
  • CNTs carbon nanotubes
  • MSNPs bare mesoporous silica nanoparticles
  • SiNPs dense silica nanoparticles
  • 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).
  • examples of exosomes include those described in Schroeder A, et al., J 43 4900-8642-4067.5 Attorney Docket No.: 098791-000104WOPT 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 Gene Ther.
  • Embodiment 16 The genome editing system of Embodiment 1 or 2, wherein the genome editing system targets a genomic locus other than the 28S rRNA locus. 17. Embodiment 17. The genome editing system of Embodiment 16, wherein an N- terminal zinc finger domain of the R2Tg element enzyme is modified to target a genomic locus other than the 28S rRNA locus. 18. Embodiment 18. The genome editing system of Embodiment 16, wherein the genomic locus is selected from the target site listed the in Tables 2 and 3, e.g., SEQ ID NO:12-86. 19. Embodiment 19.
  • Embodiment 9 The genome editing system of Embodiment 9, wherein the modified R2Tg element is fused to a Cas12 protein that is fully active, catalytically dead, or functioning as a nickase. 46 4900-8642-4067.5 Attorney Docket No.: 098791-000104WOPT 23.
  • Embodiment 23 The genome editing system of Embodiment 21, further comprising a guide RNA.
  • the genome editing system of Embodiment 9, wherein the modified R2Tg element is fused to a TALEN protein, zinc finger protein, argonaute, or meganuclease protein. 25.
  • Embodiment 25 The genome editing system of Embodiment 22, further comprising a guide RNA. 26.
  • UTR untranslated region
  • Embodiment 34 The genome editing system of Embodiment 32, wherein the 5’ homology arm and the 3’ homology arm are located between the 5’ UTR and 3’ UTR. 47 4900-8642-4067.5 Attorney Docket No.: 098791-000104WOPT 34.
  • Embodiment 34 The genome editing system of Embodiment 32, wherein the 5’ homology arm and the 3’ homology arm are located outside the 5’ UTR and 3’ UTR. 35.
  • Embodiment 35 The genome editing system of Embodiment 1 or 2, wherein the payload RNA further comprises a 5’ untranslated region (UTR), a 3’ UTR, or both a 5’ and a 3’ UTR, wherein the UTRs are truncated. 36.
  • UTR untranslated region
  • Embodiment 44 The method of Embodiment 43, wherein the Cas9 fusion protein comprises a Cas9 portion and an R2Tg element portion. 45. Embodiment 45. The method of Embodiment 44, wherein the Cas9 fusion protein comprises a targeting domain, a reverse transcriptase domain, and a nickase domain. 46. Embodiment 46. The method of Embodiment 43, wherein the Cas12 fusion protein comprises a Cas12 portion and an R2Tg element portion. 47. Embodiment 47.
  • a method of inserting an exogenous nucleic acid into the genome of a post-mitotic cell comprising subjecting the genome of the post- mitotic cell to a modified Cas9 protein that inserts the exogenous nucleic acid into the genome of the postmitotic cell.
  • a modified Cas9 protein that inserts the exogenous nucleic acid into the genome of the postmitotic cell.
  • Embodiment 51 A method of editing a genome comprising subjecting the cell to the genome editing system of Embodiment 1 or 2. 52.
  • Embodiment 52. A method of correcting a genetic mutation related to disease or human pathology, wherein the method comprises making small nucleotide changes or small nucleotide insertions (1-100 bp) in a human genome using the genome editing system of Embodiment 1 or 2. 53.
  • Embodiment 52 or Embodiment 53 or the genome editing system of any one of Embodiments above, wherein the components of the genome editing system are delivered as all RNA in lipid nanoparticles or another RNA delivery reagent.
  • Embodiment 55 The method of Embodiment 54, wherein the non-LTR site specific retrotransposon is delivered as mRNA.
  • Embodiment 56 The method of any Embodiments above, wherein the guide RNAs are delivered as synthetic RNA. 57.
  • Embodiment 57 The method of any Embodiments above, wherein the payload is delivered as mRNA. 58. Embodiment 58.
  • Embodiment 1 or 2 wherein the genome editing system targets and edits the genome at more than one site.
  • the technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.
  • EXPERIMENTAL DESIGN [0193] Developed a workflow for arrayed mutant screening that captures both the initiation of insertion at the 3’ of the cargo and its completion. [0194] Developed an evolutionary approach for semi-rational R2Tg protein mutagenesis based on amino acid usage across related orthologs. [0195] Identified mutant variants with either increased initial 3’ insertion, full-length 5’ insertion, or both.
  • Example 1 Identification of starting R2Tg variant and donor sequence [0196] In AlphaFold-predictions of the structure of R2Tg, a significant stretch of the N- terminal region appears to be completely unstructured. We reasoned that this may relate to 50 4900-8642-4067.5 Attorney Docket No.: 098791-000104WOPT non-canonical translation initiation playing a role in the wild-type R2Tg; the observed start codon (ATG) may not be the true start of the polypeptide and so the true N-terminal start position may exclude these unstructured residues. We therefore designed experiments comparing full-length R2Tg to an N-terminally truncated variant in which the residues up to and including E184 were removed.
  • the R2Tg protein was delivered as an mRNA with a consensus Kozak sequence and AUG start codon, both of which drive canonical translation initiation mechanisms.
  • mRNA were produced in vitro and consisted of a 5’ 7-methyl guanosine (m7G) cap structure, a stretch of RNA derived from the plasmid vector backbone, a homology arm sequence (each of 25, 50, or 105bp) complementary to the human 28S rDNA sequence upstream of the canonical R2 target site, the 5’ UTR, the cargo, the 3’ UTR, a homology arm sequence (10bp) complementary to the human 28S rDNA sequence downstream of the canonical R2 target site, and a hard-coded poly(A) tail.
  • m7G 7-methyl guanosine
  • R2Tg mutagenesis screen we selected an mRNA donor of intermediate length containing a synthetic antisense expression cassette to represent an artificial (i.e. non- R2 ORF) sequence.
  • the total insertion length would therefore be 1,598bp including 5’ (175bp) and 3’ (325bp) UTRs, with a 50bp homology arm upstream of the 5’ UTR.
  • junction ddPCR primer-probe sets To quantify the frequency of both 3’ UTR insertion (indicative of insertion initiation) and 5’ UTR insertion (indicative of complete, full-length insertion), we developed corresponding junction ddPCR primer-probe sets. Junction amplicon detection was normalized to a reference primer-probe set targeting an established autosomal reference gene (ApoB), allowing us to calculate the relative copy number of the R2Tg cargo insertions. To reduce the possibility for non-specific primer amplification and facilitate gDNA loading into ddPCR droplets, we pre-digested gDNA samples with two parallel restriction enzyme sets corresponding to the 5’ UTR (NcoI & BamHI) and 3’ UTR (BbsI & BsrGI) detection assays.
  • R2Tg ⁇ 184N appears to have higher full-length insertion activity across a range of cargo sizes compared to wild type (wt) R2Tg as shown in FIGS.1 and 2.
  • Point mutants of R2Tg ⁇ 184N have increased 3’ insertion frequencies, 5’ insertion frequencies, or both relative to “wild-type” R2Tg ⁇ 184N as shown in FIGS.3 and 4.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Mycology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

La présente divulgation concerne des systèmes et des procédés d'insertion ciblée. Les systèmes de ciblage d'acide nucléique modifiés comprennent des composants d'éléments de rétrotransposon non LTR. Plus particulièrement, les systèmes d'éléments mobiles recombinants de la présente divulgation sont dérivés de R2Tg, un rétrotransposon à répétition terminale non longue (nLTR) à partir du diamant mandarin Taeniopygia guttata.
PCT/US2024/059345 2023-12-11 2024-12-10 Systèmes d'éléments génétiques mobiles modifiés Pending WO2025128545A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363608489P 2023-12-11 2023-12-11
US63/608,489 2023-12-11

Publications (1)

Publication Number Publication Date
WO2025128545A1 true WO2025128545A1 (fr) 2025-06-19

Family

ID=96058436

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/059345 Pending WO2025128545A1 (fr) 2023-12-11 2024-12-10 Systèmes d'éléments génétiques mobiles modifiés

Country Status (1)

Country Link
WO (1) WO2025128545A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200109398A1 (en) * 2018-08-28 2020-04-09 Flagship Pioneering, Inc. Methods and compositions for modulating a genome
US20230272434A1 (en) * 2021-10-19 2023-08-31 Massachusetts Institute Of Technology Genomic editing with site-specific retrotransposons

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200109398A1 (en) * 2018-08-28 2020-04-09 Flagship Pioneering, Inc. Methods and compositions for modulating a genome
US20230272434A1 (en) * 2021-10-19 2023-08-31 Massachusetts Institute Of Technology Genomic editing with site-specific retrotransposons

Similar Documents

Publication Publication Date Title
US12351815B2 (en) Engineered human-endogenous virus-like particles and methods of use thereof for delivery to cells
US12319938B2 (en) Enhanced virus-like particles and methods of use thereof for delivery to cells
US8569065B2 (en) Compositions and methods for the delivery of biologically active RNAs
AU2012358181A1 (en) Compositions and methods for the delivery of biologically active RNAs
CA2331384A1 (fr) Phage de transfert d'acide nucleique
WO2020186237A1 (fr) Progéniteurs microgliaux pour la régénération de la microglie fonctionnelle dans le système nerveux central et leurs utilisations thérapeutiques
US20240425832A1 (en) Compositions and methods of genome editing
WO2025128545A1 (fr) Systèmes d'éléments génétiques mobiles modifiés
WO2002087594A1 (fr) Vecteur vesiculaire non viral pour le transfert de gene cardiaque specifique
US20250066772A1 (en) Method of modulating vegf and uses thereof
JP2025520547A (ja) ＣＲＩＳＰＲ活性を調節するための操作Ａｃｒタンパク質
Owen IV et al. Approaches and limitations to gene therapy for mitochondrial diseases
Weber-Lotfi et al. Targeting Therapeutic Nucleic Acids into Mitochondria: A Long Challenge
WO2025245094A2 (fr) Séquences peptidiques pour l'administration améliorée de protéines dans un noyau
AU2024250590A1 (en) Fusion proteins for improved gene editing
WO2025261493A1 (fr) Procédé de régulation ciblée de pcsk9 au moyen d'une édition épigénétique et son utilisation
WO2025011467A1 (fr) Système d'écriture de gènes de mammifères

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24904721

Country of ref document: EP

Kind code of ref document: A1