WO2023154011A2 - Compositions et méthodes pour édition génomique - Google Patents

Compositions et méthodes pour édition génomique Download PDF

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WO2023154011A2
WO2023154011A2 PCT/SG2023/050068 SG2023050068W WO2023154011A2 WO 2023154011 A2 WO2023154011 A2 WO 2023154011A2 SG 2023050068 W SG2023050068 W SG 2023050068W WO 2023154011 A2 WO2023154011 A2 WO 2023154011A2
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prime editor
polypeptide
prime
sequence
complex
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PCT/SG2023/050068
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WO2023154011A3 (fr
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Haojie YU
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National University Of Singapore
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • 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 RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07049—RNA-directed DNA polymerase (2.7.7.49), i.e. telomerase or reverse-transcriptase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor

Definitions

  • the invention relates generally to the field of genome editing.
  • the specification teaches a prime editor polypeptide that is capable of carrying out prime editing in the presence of a prime editing guide RNA (pegRNA).
  • pegRNA prime editing guide RNA
  • Prime editing is a remarkably versatile genome editing method with a variety of biotechnological and biomedical applications, including for the treatment of genetic diseases. It is adapted from the CRISPR system of genome editing, and involves the use of a nickase endonuclease and a reverse transcriptase (typically fused into a single recombinant protein), along with an extended guide RNA comprising a template for the reverse transcriptase that also contains a desired genetic edit.
  • Prime editing allows for sequence changes such as base substitutions, insertions, or deletions to be made in the genome without requiring a double-strand break (DSB) or donor DNA templates, or the use of homology directed repair (HDR) or non-homologous end joining (NHEJ) to fix DNA breaks.
  • DSB double-strand break
  • HDR homology directed repair
  • NHEJ non-homologous end joining
  • Prime editing system One potential way to introduce a prime editing system into animals and plants in vivo is to package the components into a viral capsid. The target cell or organism can then be transduced with the virus, and the components synthesised intracellularly.
  • viral vectors including the promising FDA-approved adeno-associated virus (AAV) vectors, have small packaging capacities and cannot accommodate multiple prime editing components, especially the large fusion protein of the nickase and reverse transcriptase.
  • AAV adeno-associated virus
  • a prime editor polypeptide comprising a nucleic acid programmable DN A binding protein (napDNAbp) domain and a reverse transcriptase polypeptide, wherein the reverse transcriptase polypeptide is modified to lack RNase H activity, and wherein the reverse transcriptase comprises or consists of an amino acid sequence having at least 70% sequence identity to positions X to Y of SEQ ID NO: 1, wherein X is position 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60, and wherein Y is position 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502
  • Prime editor complex comprising a prime editor polypeptide as defined herein and a prime editor guide RNA (pegRNA).
  • pegRNA prime editor guide RNA
  • Disclosed herein is one or more polynucleotides encoding the prime editor complex as defined herein.
  • a vector comprising the one or more polynucleotides as defined herein.
  • a cell comprising the vector as defined herein.
  • a cell comprising the prime editor complex as defined herein.
  • composition comprising i) a prime editor polypeptide, a prime editor complex, a polynucleotide or a vector as defined herein; and ii) a pharmaceutically acceptable excipient.
  • Disclosed herein is a method of installing a nucleotide change in a nucleic acid sequence, the method comprising contacting the nucleic acid sequence with a prime editor complex as defined herein for a sufficient time and under conditions to install the nucleotide change in the nucleic acid sequence.
  • Disclosed herein is a method of treating a disease or condition in a subject, the method comprising administering a prime editor complex, a vector or a pharmaceutical composition as defined herein to the subject.
  • a method of simultaneously editing both strands of a double-stranded DNA sequence at a target site to be edited wherein the system comprises a first prime editor complex and a second prime editor complex, wherein each of the first and second prime editor complexes comprises a prime editor polypeptide as defined herein and a prime editor guide RNA (pegRNA), wherein the pegRNA of the first prime editor complex encodes a first single stranded DNA sequence and the pegRNA of the second prime editor complex encodes a second single stranded DNA sequence, wherein the first single-stranded DNA sequence and the second single-stranded DNA sequence each comprises a region of complementarity to the other, and wherein the first single-stranded DNA sequence and the second single-stranded DNA sequence form a duplex comprising an edited portion
  • Figure 1 is a schematic representation of the original prime editor PE2 and optimised prime editor PE2Rminus without RNase H domain.
  • Figure 2 shows that PE2Rminus enhances prime editing efficiency.
  • A Diagram of the TAGTAG to GAGGAG transition required to convert stop codon to Glu to restore function of GFP in HEK293FT cells.
  • B Schematic representation of paired pegRNAs in trans encoding the same edits on both DNA strands simultaneously.
  • PBS stands for primer binding site;
  • RT template stands for reverse transcription template.
  • C and D Representative images of flow cytometry (C) and analysis (D) of the percentage of GFP positive cells after prime editing with different setups as indicated. *p ⁇ 0.05, **p ⁇ 0.01; bars in (D) indicate mean ⁇ s.d..
  • Figure 3 shows knockout of EDER in human HEK293FT cells by introducing a stop codon (TGOTGA) on exon 4.
  • Figure 4 shows that dual pegRNAs increases prime editing efficiency.
  • A Schematic presentation of positions of pegRNAs and nick sgRNAs on GFP locus.
  • B Flow cytometry analysis of the percentage of GFP positive cells after prime editing with different setups as indicated in A. *p ⁇ 0.05, **p ⁇ 0.01; bars in B indicate mean ⁇ s.d..
  • Figure 5 shows the deletion of a 5-bp DNA fragment in the LDLR gene in HEK293T cells.
  • Next generation sequencing analysis showed that the desired editing efficiency with PE2Rminus is significantly higher than PE2.
  • Figure 6 shows the editing efficiency of several C-terminally truncated PE2Rminus variants.
  • the same molar amounts of plasmid encoding PE2, PE2Rminus, and truncated PE2Rminus was transfected into HEK293T GFP reporter cells together with fixed amounts of plasmids encoding two pegRNAs. Cells were harvested for flow cytometry to measure the percentage of GPF GFP-positive cells 3 days post-transfection. The statistical analysis is done between PE2Rminus with all other constructs. *p ⁇ 0.05, **p ⁇ 0.01; bars indicate mean ⁇ s.d..
  • construct 1 positions 1-677 of SEQ ID NO: 1, construct 2: positions 1-498 of SEQ ID NO: 1, construct 3: positions 1-497 of SEQ ID NO: 1, construct 4, positions 1-496 of SEQ ID NO: 1, construct 5: positions 1-495 of SEQ ID NO: 1, construct 6: positions 1-494 of SEQ ID NO: 1, construct 7: positions 1-493 of SEQ ID NO: 1, construct 8: positions 1-488 of SEQ ID NO: 1, construct 9: positions 1-483 of SEQ ID NO: 1, construct 10: positions 1-478 of SEQ ID NO: 1 and construct 11: positions 1-473 of SEQ ID NO: 1).
  • the present specification teaches a prime editor polypeptide that is capable of carrying out prime editing in the presence of a prime editing guide RNA (pegRNA) to install a desired nucleotide change in a target sequence.
  • the prime editor polypeptide may comprise a nucleic acid programmable DNA binding protein (napDNAbp) domain connected to a reverse transcriptase polypeptide, wherein the reverse transcriptase polypeptide is modified to lack RNase H activity.
  • the reverse transcriptase polypeptide is modified to lack RNase H activity by deleting its RNase H domain.
  • the inventors have made the surprising discovery that removal of the RNase H domain from a prime editor can produce a prime editor with higher prime editing efficiency.
  • the inventors have also discovered that the use of a prime editor without RNase H activity in combination with a pair of pegRNAs encoding complementary sequences of a target genomic site can substantially increase editing efficiency.
  • the reverse transcriptase may comprise or consist of an amino acid sequence having at least 70% sequence identity to positions X to Y of SEQ ID NO: 1, wherein X is position 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60, and wherein Y is position 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502 or 503.
  • the reverse transcriptase may comprise or consist of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, or at least 99.8% sequence identity to positions X to Y of SEQ ID NO: 1, wherein X is position 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60, and wherein Y is position 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502 or 503.
  • Y is position 498, 499, 500, 501, 502 or 503 of SEQ ID NO: 1. In one embodiment, Y is position 498 to 503, 498 to 502, 498 to 501, 498 to 500 or 498 to 499 of SEQ ID NO: 1. In one embodiment, Y is position 498 of SEQ ID NO: 1.
  • the reverse transcriptase may comprise or consist of an amino acid sequence having at least 70% sequence identity to positions X to Y of SEQ ID NO: 1, wherein X is position 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, 30 or 31, and wherein Y is position 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502 or 503.
  • the reverse transcriptase may comprise or consist of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, or at least 99.8% sequence identity to positions X to Y of SEQ ID NO: 1, wherein X is position 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, 30 or 31, and wherein Y is position 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502 or 503.
  • X is position 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, 30 or 31, and Y is position 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502 or 503.
  • X is position 1, and Y is position 498, 499, 500, 501, 502 or 503 of SEQ ID NO: 1.
  • Y is position 498 to 503, 498 to 502, 498 to 501, 498 to 500 or 498 to 499 of SEQ ID NO: 1.
  • Y is position 498 of SEQ ID NO: 1.
  • the reverse transcriptase comprises or consists of an amino acid sequence having at least 70% sequence identity to positions 1 to 488, 1 to 489, 1 to 490, 1 to 491, 1 to 492, 1 to 493, 1 to 494, 1 to 495, 1 to 496, 1 to 497, 1 to 498, 1 to 499, 1 to 500, 1 to 501, 1 to 502 or 1 to 503 of SEQ ID NO: 1.
  • the reverse transcriptase comprises or consists of an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2 (i.e. positions 1 to 498 of SEQ ID NO: 1).
  • the reverse transcriptase may comprise or consist of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO:
  • the reverse transcriptase comprises or consists of an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 3 (i.e. positions 31 to 498 of SEQ ID NO: 1).
  • the reverse transcriptase may comprise or consist of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 sequence identity to SEQ ID NO:
  • the reverse transcriptase comprises or consists of an amino acid sequence having at least 70% sequence identity to positions 31 to 488, 31 to 489, 31 to 490, 31 to 491, 31 to 492, 31 to 493, 31 to 494, 31 to 495, 31 to 496, 31 to 497, 31 to 498, 31 to 499, 31 to 500, 31 to 501, 31 to 502 or 31 to 503 of SEQ ID NO: 1.
  • the prime editor polypeptide is capable of carrying out prime editing in the presence of a prime editing guide RNA (pegRNA) to install a desired nucleotide change in a target sequence.
  • a prime editing guide RNA pegRNA
  • protein protein
  • peptide and “polypeptide” are used interchangeably herein, and refer to a polymer of amino acid residues linked together by peptide (amide) bonds.
  • the terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long.
  • a protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins.
  • One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a famesyl group, an isofamesyl group, a fatty acid group, a linker for conjugation, functionalisation, or other modification, etc.
  • a protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex.
  • a protein, peptide, or polypeptide may be naturally-occurring, recombinant, or synthetic, or any combination thereof.
  • a protein, peptide, or polypeptide may be chimeric, i.e., it may comprise one or more naturally- occurring or synthetic proteins or peptides, or one or more fragments of a naturally-occurring or synthetic protein or peptide, or combinations thereof.
  • Any of the proteins provided herein may be produced by any method known in the art.
  • the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference.
  • PE primary editor
  • PE system or “prime editor (PE)” or “PE system” or “PE editing system” refers to the compositions involved in the method of genome editing described herein, including, but not limited to the napDNAbps, reverse transcriptases, fusion proteins (e.g., comprising napDNAbps and reverse transcriptases), prime editing guide RNAs, and complexes comprising fusion proteins and prime editing guide RNAs, as well as accessory elements, such as second strand nicking components (e.g., second strand sgRNAs), 5' endogenous DNA flap removal endonucleases (e.g., FEN1), and inhibitors of the DNA mismatch repair pathway (e.g., dominant negative MLH1 protein) for helping to drive the prime editing process towards the edited product formation.
  • second strand nicking components e.g., second strand sgRNAs
  • FEN1 5' endogenous DNA flap removal endonucleases
  • inhibitors of the DNA mismatch repair pathway e.g.
  • primary editor or “prime editor polypeptide” refers to the herein described polypeptide constructs comprising a napDNAbp (e.g., Cas9 nickase) and a reverse transcriptase and is capable of carrying out prime editing on a target nucleotide sequence in the presence of a pegRNA (or "extended guide RNA”).
  • primary editor complex refers to the prime editor polypeptide complexed with a pegRNA, and/or further complexed with a second guide RNA (e.g., a second guide RNA capable of directing the second-site nicking step of the non-edited strand as described herein).
  • a “reverse transcriptase” or “RT” herein may be any naturally-occurring or engineered reverse transcriptase that is capable of synthesising a complementary DNA (cDNA) using an RNA template.
  • sources for naturally-occurring RTs include, but are not limited to, Moloney murine leukemia virus (M-MLV); human T-cell leukemia virus type 1 (HTLV-1); mouse mammary tumor virus (MMTV); bovine leukemia virus (BLV); xenotropic murine leukemia virus-related virus (XMRV); human immunodeficiency virus (HIV); human papilloma virus (HPV); equine infectious anemia virusa (EIAV); avian sarcoma leukosis virus (ASLV); Rous sarcoma virus (RSV); avian myeloblastosis virus (AMV); avian erythroblastosis virus helper virus; avian myelocytomatosis virus MC29 helper
  • An engineered RT may be a genetically-engineered variant of a wild-type RT comprising one or more mutations (e.g., point mutations, inversions, deletions, insertions or rearrangements) relative to a reference sequence (e.g., a reference wild-type sequence).
  • the reverse transcriptase is an engineered variant of Moloney murine leukemia virus RT (M-MLV RT ; SEQ ID NO: 1).
  • M-MLV RT Moloney murine leukemia virus RT
  • Reverse transcriptases typically have three enzymatic activities, including RNA- and DNA-dependent DNA polymerisation activity, and an RNase H activity that catalyses the cleavage of RNA in RNA-DNA hybrids. The inventors have found that the RNAse H activity of RTs is not essential for the prime editing methods described herein.
  • the reverse transcriptase polypeptide modified to lack RNase H activity may be one that has been modified to have substantially reduced RNase H activity. This means that the reverse transcriptase polypeptide has less than about 30%, less than about 25%, less than about 20%, more preferably less than about 15%, less than about 10%, less than about 7.5%, or less than about 5%, and most preferably less than about 5% or less than about 2% or 0%, of the RNase H activity of the corresponding wild-type reverse transcriptase polypeptide.
  • the RNase H activity of a reverse transcriptase polypeptide may be determined by a variety of assays that are well known in the art.
  • wild type is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms.
  • the reverse transcriptase polypeptide may additionally have a number of specific mutations to result in an increased thermostability, thermoactivity, increased resistance to inhibitors, and/or to confer other desired properties on reverse transcriptases as described.
  • Such mutations include point mutations, frame shift mutations, deletions and insertions.
  • one or more point mutations, resulting in one or more amino acid substitutions are used to produce reverse transcriptases having enhanced or increased thermostability and/or thermoreactivity or increased resistance to inhibitors.
  • nucleic acid programmable DNA binding protein refers to a protein which uses RNA:DNA hybridisation to target and bind to specific sequences in a DNA molecule.
  • Each napDNAbp is associated with at least one guide nucleic acid (e.g., a guide RNA), which localises the napDNAbp to a DNA sequence that comprises a DNA strand (i.e., a target strand) that is complementary to the guide nucleic acid or a portion thereof (e.g., the protospacer of a guide RNA).
  • the guide nucleic- acid "programs" the napDNAbp to localise and bind to a complementary sequence.
  • the napDNAbp includes one or more nuclease activities, which then cut the DNA, leaving various types of lesions.
  • the napDNAbp may comprise a nuclease activity that cuts the non-target strand at a first location, and/or cuts the target strand at a second location.
  • the target DNA may be cut to form a "double-stranded break" (i.e., both strands are cut), or the target DNA may be cut at only a single site (i.e., the DNA is "nicked" on one strand).
  • Exemplary napDNAbp with different nuclease activities include "Cas9 nickase” (“nCas9”) and a deactivated Cas9 having no nuclease activities (“dead Cas9” or “dCas9”).
  • the napDNAbp domain is a Cas9 protein or variant thereof.
  • the term “Cas9” or “Cas9 nuclease” refers to a napDNAbp 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 trans-encoded small RNA
  • me endogenous ribonuclease 3
  • Cas9 domain The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA.
  • 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.
  • sgRNA single guide RNAs
  • Cas9 recognises a short motif in 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, J. J.
  • Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophiles.
  • Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems", RNA Biology 10:5, 726-737(2013); the entire contents of which are incorporated herein by reference.
  • a Cas9 nuclease comprises one or more mutations that partially impair or inactivate the DNA cleavage domain.
  • a nuclease-inactivated Cas9 domain may interchangeably be referred to as a "dCas9" protein (for nuclease-"dead” Cas9).
  • a Cas9 domain (or a fragment thereof) having an inactive DNA cleavage domain
  • Methods for generating a Cas9 domain (or a fragment thereof) having an inactive DNA cleavage domain are known (see, e.g., Jinek et al., Science, 337:816-821(2012); Qi et al., "Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression” Cell, 28; 152(5): 1173-83 (2013), the entire contents of each of which are incorporated herein by reference).
  • the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvCl subdomain.
  • the HNH subdomain cleaves the strand complementary to the gRNA, whereas the RuvCl subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the mutations D10A and H840A completely inactivate the nuclease activity of S. pyogenes Cas9 (Jinek et al., Science, 337:816-821(2012); Qi et al, Cell, 28; 152(5): 1173-83 (2013)).
  • proteins comprising fragments of Cas9 are provided.
  • a protein comprises one of two Cas9 domains: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavage domain of Cas9.
  • proteins comprising Cas9 or fragments thereof are referred to as "Cas9 variants".
  • a Cas9 variant shares homology to Cas9, or a fragment thereof.
  • a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, at least about 99.8% identical, or at least about 99.9% identical to wild-type Cas9.
  • the Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more amino acid changes compared to wild type Cas9.
  • the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild type Cas9).
  • a fragment of Cas9 e.g., a gRNA binding domain or a DNA-cleavage domain
  • the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild type Cas9.
  • the Cas9 protein or variant thereof has nickase activity.
  • nickase refers to a Cas9 with one of the two nuclease domains inactivated. This enzyme is capable of cleaving only one strand of a target DNA.
  • the Cas9 nickase has an inactivated RuvC domain.
  • the Cas9 protein is Streptococcus pyogenes Cas9 protein (SpCas9), or variant thereof, with nickase activity.
  • the Cas9 nickase is SpCas9(H840A), i.e., spCas9 with an inactivating H840A mutation in the RuvC domain.
  • wild-type is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms.
  • variant should be taken to mean the exhibition of qualities that have a pattern that deviates from what occurs in nature, e.g., a variant Cas9 protein is a Cas9 comprising one or more changes in amino acid residues as compared to a wild-type Cas9 amino acid sequence.
  • variant encompasses homologous proteins having at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% percent identity with a reference sequence and having the same or substantially the same functional activity or activities as the reference sequence.
  • the term also encompasses mutants, truncations, or domains of a reference sequence, and which display the same or substantially the same functional activity or activities as the reference sequence.
  • the prime editor polypeptide is a fusion protein between the napDNAbp domain and the reverse transcriptase polypeptide.
  • the napDNAbp domain and the reverse transcriptase polypeptide are joined by a linker.
  • 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.
  • Another example includes a fusion of Cas9 or variant thereof to a reverse transcriptase.
  • Any of the proteins provided herein may be produced by any method known in the art.
  • the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker.
  • Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference.
  • linker refers to a molecule linking two polypeptides.
  • the linker is an amino acid linker sequence, e.g. an amino acid linker connecting Cas9 and a reverse transcriptase.
  • the linker is an organic molecule, group, polymer, or chemical moiety.
  • the linker is 5-100 amino acids in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80- 90, 90-100 amino acids in length. Longer or shorter linkers are also contemplated.
  • the linker comprises or consists of an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 7.
  • the C terminus of the reverse transcriptase polypeptide is connected to a nuclear localisation sequence (NLS) polypeptide.
  • the N terminus of the napDNAbp domain is connected to an NLS polypeptide.
  • a nuclear localisation sequence (NLS) polypeptide is connected to both the C terminus of the reverse transcriptase polypeptide and the N terminus of the napDNAbp domain is connected to an NLS polypeptide.
  • nuclear localisation sequence refers to an amino acid sequence that promotes import of a protein into the cell nucleus, for example, by nuclear transport.
  • Nuclear localisation sequences are known in the art and would be apparent to the skilled person.
  • NLS sequences are described in Plank et al., PCT application PCT/EP2000/011690, filed November 23, 2000, published as WO/2001/038547 on May 31, 2001, the contents of which are incorporated herein by reference for its disclosure of exemplary nuclear localisation sequences.
  • the NLS comprises or consists of an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 6.
  • the prime editor polypeptide comprises or consists of an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 4.
  • the prime editor polypeptide may comprise or consist of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 4.
  • nucleic acid refers to a polymer of nucleotides.
  • the polymer may include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogues (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C5-bromouridine, C5- fluorouridine, C5-iodouridine, C5-propynyl uridine, C5-propynyl cytidine, C5- methylcytidine, 7-deazaadenosine, 7-deazaguanos
  • Prime editor complex comprising a prime editor polypeptide disclosed herein and a prime editor guide RNA (pegRNA).
  • pegRNA prime editor guide RNA
  • a pair of prime editor complexes comprising a first prime editor complex and a second prime editor complex
  • each of the first and second prime editor complexes comprises a prime editor polypeptide disclosed herein and a prime editor guide RNA (pegRNA)
  • pegRNA prime editor guide RNA
  • the pegRNA of the first prime editor complex encodes a first single-stranded DNA sequence
  • the pegRNA of the second prime editor complex encodes a second single- stranded DNA sequence
  • each of the first and second single-stranded DNA sequences comprises a region of complementarity to the other, and wherein the region of complementarity comprises an edited duplex DNA sequence which integrates into the target site to be edited.
  • guide RNA is a particular type of guide nucleic acid which is mostly commonly associated with a Cas protein of a CRISPR-Cas9 system and which associates with Cas9, directing the Cas9 protein to a specific sequence in a DNA molecule that includes complementarity to protospacer sequence of the guide RNA.
  • this term also embraces the equivalent guide nucleic acid molecules that associate with Cas9 equivalents, homologues, orthologues, or paralogues, whether naturally occurring or non- naturally occurring (e.g., engineered or recombinant), and which otherwise program the Cas9 equivalent to localise to a specific target nucleotide sequence.
  • the Cas9 equivalents may include other napDNAbp from any type of CRISPR system (e.g., type II, V, VI), including Cpfl (a type-V CRISPR-Cas systems), C2cl (a type V CRISPR-Cas system), C2c2 (a type VI CRISPR-Cas system) and C2c3 (a type V CRISPR-Cas system).
  • Cpfl a type-V CRISPR-Cas systems
  • C2cl a type V CRISPR-Cas system
  • C2c2 a type VI CRISPR-Cas system
  • C2c3 a type V CRISPR-Cas system
  • guide RNA may also be referred to as a “traditional guide RNA” to contrast it with the modified forms of guide RNA termed "prime editing guide RNAs" (or “pegRNAs”) which have been invented for the prime editing methods and composition disclosed herein.
  • primary editing guide RNAs or “pegRNAs”
  • the term "protospacer” refers to the sequence in the target DNA adjacent to the PAM (protospacer adjacent motif) sequence ( ⁇ 20 bp).
  • the protospacer shares the same sequence as the spacer sequence of the guide RNA.
  • the guide RNA anneals to the complement of the protospacer sequence on the target DNA (specifically, one strand thereof, i.e., the "target strand” versus the "non-target strand” of the target DNA sequence).
  • PAM protospacer adjacent motif
  • protospacer as the ⁇ 20-nt target- specific guide sequence on the guide RNA itself, rather than referring to it as a "spacer”.
  • protospacer as used herein may be used interchangeably with the term “spacer”.
  • spacer The context of the description surrounding the appearance of either "protospacer” or “spacer” will help inform the reader as to whether the term is in reference to the gRNA or the DNA target.
  • the term "protospacer adjacent sequence” or "PAM” refers to an approximately 2-6 base pair DNA sequence that is an important targeting component of a Cas9 nuclease.
  • the PAM sequence is on either strand, and is downstream in the 5' to 3' direction of Cas9 cut site.
  • the canonical PAM sequence i.e., the PAM sequence that is associated with the Cas9 nuclease of Streptococcus pyogenes or SpCas9
  • N is any nucleobase followed by two guanine (“G”) nucleobases.
  • Different PAM sequences can be associated with different Cas9 nucleases or equivalent proteins from different organisms.
  • any given Cas9 nuclease e.g., SpCas9, may be modified to alter the PAM specificity of the nuclease such that the nuclease recognises alternative PAM sequence.
  • primary editing guide RNA or “pegRNA” or “extended guide RNA” refer to a specialised form of a guide RNA that has been modified to include one or more additional sequences for implementing the prime editing methods and compositions described herein.
  • the pegRNA comprises a guide RNA and at least one nucleic acid extension arm.
  • the extension arms may comprise single-stranded RNA or DNA.
  • the extension arm occurs at the 3' end of a traditional gRNA.
  • the extension arm occurs at the 5' end of a traditional gRNA.
  • the extension arm occurs at an intramolecular region of the traditional gRNA, e.g., in the gRNA core region which associates and/or binds to the napDNAbp.
  • the extension arm comprises (i) a DNA synthesis template sequence comprising a desired nucleotide change, and (ii) a primer binding site.
  • the DNA synthesis template may encode 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.
  • the "primer binding site” or “PBS” comprises a sequence on the pegRNA that hybridises to a single-strand DNA sequence having a 3' end generated from the nicked DNA of the R-loop.
  • a 3'-ended single- stranded DNA (ssDNA) flap is formed, which serves as a primer sequence that anneals to the primer binding site on the pegRNA to prime reverse transcription.
  • An extension arm of a pegRNA may further comprise other functional sequence elements, e.g., a "spacer or linker” sequence, a transcription terminator sequence, or other structural elements, e.g., aptamers, stem loops, hairpins, toe loops (e.g., a 3' toeloop), or an RNA- protein recruitment domain (e.g., MS2 hairpin).
  • a spacer or linker sequence e.g., a "spacer or linker” sequence, a transcription terminator sequence, or other structural elements, e.g., aptamers, stem loops, hairpins, toe loops (e.g., a 3' toeloop), or an RNA- protein recruitment domain (e.g., MS2 hairpin).
  • target DNA site refers to a sequence within a nucleic acid molecule that is edited by a prime editor (PE) polypeptide disclosed herein.
  • the target site further refers to the sequence within a nucleic acid molecule to which a complex of the prime editor (PE) polypeptide and gRNA binds.
  • PE prime editor
  • the pegRNA comprises, or consists essentially of, from 5' to 3' end, (a) a spacer sequence (which binds to the protospacer in the target DNA); (b) a gRNA scaffold that is responsible for Cas9 binding; (c) an extension arm comprising (i) a DNA synthesis template sequence encoding a desired genetic change, and (ii) a primer binding site.
  • PEI refers to a prime editor (PE) complex comprising a desired PEgRNA and a fusion protein comprising Cas9(H840A) and a wild type MMLV RT.
  • PE2 refers to a PE complex comprising a desired PEgRNA and a fusion protein comprising Cas9(H840A) and a variant MMLV RT with five amino acid substitutions (D200N, T330P, L603W, T306K and W313F).
  • PE3 refers to PE2 plus a second-strand nicking guide RNA 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.
  • PE3b refers to PE3 but wherein the second-strand nicking guide RNA is designed for temporal control such that the second strand nick is not introduced until after the installation of the desired edit. This is achieved by designing a gRNA with a spacer sequence that matches only the edited strand, but not the original allele. Using this strategy (i.e., PE3b), mismatches between the protospacer and the unedited allele should disfavour nicking by the sgRNA until after the editing event on the PAM strand takes place.
  • Disclosed herein is one or more polynucleotides encoding a prime editor complex as defined herein. Disclosed herein is also a vector comprising the one or more polynucleotides as defined herein.
  • vector refers to a nucleic acid that can be modified to encode a gene of interest and that is able to enter into a host cell, mutate and replicate within the host cell, and then transfer a replicated form of the vector into another host cell.
  • exemplary suitable vectors include viral vectors, such as adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, bacteriophages, filamentous phages, and conjugative plasmids.
  • host cell refers to a cell that can host, replicate, and express a vector described herein, e.g., a vector comprising a nucleic acid molecule encoding a fusion protein comprising a Cas9 or Cas9 equivalent and a reverse transcriptase.
  • the polynucleotide encoding the gRNA or pegRNA and the polynucleotide encoding the prime editor polypeptide are located on the same vector. In some embodiments, the polynucleotide encoding the gRNA or pegRNA and the polynucleotide encoding the prime editor polypeptide are located on different vectors. In some embodiments the polynucleotide encoding each pegRNA used in the methods herein is located on a different vector. In some embodiments the polynucleotides encoding some of the pegRNAs used in the methods herein tire located on the same vector.
  • the vectors used herein may also encode any of the components of the prime editing methods herein, e.g., accessory gRNAs for second strand nicking, accessory endonucleases for 5' endogenous DNA flap removal, or accessory protein inhibitors of the DNA mismatch repair pathway.
  • the vector of the present disclosure may comprise one or more regulatory elements (e.g., promoters, transcriptional terminators and/or other regulatory elements) that are operably linked to a gene of interest to control the expression of tire gene of interest.
  • regulatory elements e.g., promoters, transcriptional terminators and/or other regulatory elements
  • a promoter operably linked to a gene of interest may be constitutively active, meaning that the promoter is always active in a given cellular context.
  • constitutive promoters include cytomegalovirus immediate early promoter (CMV), simian virus (SV40) promoter, adenovirus major late (MLP) promoter, Rous sarcoma virus (RSV) promoter, mouse mammary tumor virus (MMTV) promoter, phosphoglycerate kinase (PGK) promoter, elongation factor-alpha (EFla) promoter, ubiquitin promoters, actin promoters, tubulin promoters, immunoglobulin promoters, a functional fragment thereof, or a combination of any of the foregoing.
  • CMV cytomegalovirus immediate early promoter
  • MLP adenovirus major late
  • RSV Rous sarcoma virus
  • MMTV mouse mammary tumor virus
  • PGK phosphoglycerate kinas
  • a promoter may also be conditionally active, meaning that the promoter is only active in the presence of a specific condition (e.g., heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohol).
  • a conditional promoter may only be active in the presence of a specific protein that connects a protein associated with a regulatory element in the promoter to the basic transcriptional machinery, or only in the absence of an inhibitory molecule.
  • a subclass of conditionally-active promoters are inducible promoters that require the presence of a small molecule "inducer" for activity.
  • inducible promoters include, but are not limited to, arabinose- inducible promoters, Tet-On® promoters, and tamoxifen-inducible promoters.
  • a promoter may be wild-type, or it may be modified for more efficient or efficacious expression.
  • constitutive, conditional, and inducible promoters are well known to the skilled person, and the skilled person will be able to ascertain a variety of such promoters useful in carrying out the instant invention, which is not limited in this respect.
  • the nucleotide sequence encoding the pegRNA may be operably linked to at least one promoter that is recognised by RNA polymerase III (Pol III).
  • Pol III promoters include U6, Hl and tRNA promoters.
  • the same promoter may control the expression of more than one genes of interest, e.g. the genes for one or more gRNAs or pegRNAs.
  • a protein of interest refers to a gene that encodes a biomolecule of interest (e.g., a protein or an RNA molecule).
  • a protein of interest can include any intracellular protein, membrane protein, or extracellular protein, e.g., a nuclear protein, transcription factor, nuclear membrane transporter, intracellular organelle associated protein, a membrane receptor, a catalytic protein, and enzyme, a therapeutic protein, a membrane protein, a membrane transport protein, a signal transduction protein, or an immunological protein (e.g., an IgG or other antibody protein), etc.
  • the gene of interest may also encode an RNA molecule, including, but not limited to, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), antisense RNA, guide RNA, prime editing guide RNA (pegRNA), microRNA (miRNA), small interfering RNA (siRNA), and cell-free RNA (cfRNA).
  • mRNA messenger RNA
  • tRNA transfer RNA
  • rRNA ribosomal RNA
  • small nuclear RNA snRNA
  • antisense RNA small nuclear RNA
  • guide RNA prime editing guide RNA
  • pegRNA prime editing guide RNA
  • miRNA microRNA
  • siRNA small interfering RNA
  • cfRNA cell-free RNA
  • the cell may be a prokaryotic cell, e.g., a bacterial cell.
  • the cell may be a eukaryotic cell, e.g., a yeast, plant, insect, or mammalian cell.
  • the eukaryotic cell may be a mammalian cell.
  • the eukaryotic cell may be a rodent cell.
  • the eukaryotic cell may be a human cell. Suitable promoters to drive expression in different types of cells are known in the art.
  • composition comprising i) a prime editor polypeptide, a prime editor complex, a polynucleotide or a vector as defined herein; and ii) a pharmaceutically acceptable excipient.
  • the term "pharmaceutically acceptable excipient” means a pharmaceutically- acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the prime editor polypeptide, prime editor complex, polynucleotide or vector from one site (e.g., the delivery site) of the body, to another site (e.g., organ, tissue or portion of the body).
  • manufacturing aid e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid
  • solvent encapsulating material involved in carrying or transporting the prime editor polypeptide, prime editor complex, polynucleotide or vector from one site (e.g., the delivery site) of the body, to another site (e.g., organ, tissue or portion of the body).
  • a pharmaceutically acceptable excipient is "acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the tissue of the subject (e.g., physiologically compatible, sterile, physiologic pH, etc.).
  • materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glyco
  • a method of installing a nucleotide change in a nucleic acid sequence comprising contacting the nucleic acid sequence with a prime editor complex as defined herein for a sufficient time and under conditions to install the nucleotide change in the nucleic acid sequence.
  • the method may be carried in vitro or in vivo, preferably within a cell. This may take about 48 to 72 hours.
  • Disclosed herein is a method of treating a disease or condition in a subject, the method comprising administering a prime editor complex, a vector or a pharmaceutical composition as defined herein to the subject.
  • a prime editor complex a vector or a pharmaceutical composition as defined herein in the manufacture of a medicament for treating a disease or condition in a subject.
  • the term "subject”, as used herein, refers to an individual organism, for example, an individual mammal.
  • the subject is a human.
  • the subject is a non-human mammal.
  • the subject is a non-human primate.
  • the subject is a rodent.
  • the subject is a sheep, a goat, a cattle, a cat, or a dog.
  • the subject is a vertebrate, an amphibian, a reptile, a fish, an insect, a fly, or a nematode.
  • the subject is a research animal.
  • the subject is genetically engineered, e.g., a genetically engineered non-human subject.
  • the subject may be of either sex and at any stage of development.
  • treatment refers to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof.
  • treatment may be administered after one or more symptoms have developed and/or after a disease has been diagnosed. In other embodiments, treatment may be administered in the absence of symptoms, e.g., to prevent or delay onset of a symptom or inhibit onset or progression of a disease.
  • treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their recurrence.
  • the method corrects a disease-associated gene.
  • the disease-associated gene can be associated with a monogenetic disorder selected from the group consisting of: Adenosine Deaminase (ADA) Deficiency; Alpha- 1 Antitrypsin Deficiency; Cystic Fibrosis; Duchenne Muscular Dystrophy; Galactosemia; Hemochromatosis; Huntington’s Disease; Maple Syrup Urine Disease; Marfan Syndrome; Neurofibromatosis Type 1; Pachyonychia Congenita; Phenylkeotnuria; Severe Combined Immunodeficiency; Sickle Cell Disease; Smith-Lemli-Opitz Syndrome; and Tay-Sachs Disease.
  • the disease- associated gene can be associated with a polygenic disorder selected from the group consisting of: heart disease; high blood pressure; Alzheimer’s disease; arthritis; diabetes; cancer; and obesity.
  • an effective amount refers to an amount of a biologically active agent that is sufficient to elicit a desired biological response.
  • an effective amount of a prime editor may refer to the amount of the editor that is sufficient to edit a target site nucleotide sequence, e.g., a genome.
  • an effective amount of a prime editor (PE) provided herein may refer to the amount of the prime editor that is sufficient to induce editing of a target site specifically bound and edited by the fusion protein.
  • the effective amount of an editor may vary depending on various factors as, for example, on the desired biological response, e.g., on the specific allele, genome, or target site to be edited, on the cell or tissue being targeted, and on the agent being used.
  • a method of simultaneously editing both strands of a double-stranded DNA sequence at a target site to be edited wherein the system comprises a first prime editor complex and a second prime editor complex, wherein each of the first and second prime editor complexes comprises a prime editor polypeptide as defined herein and a prime editor guide RNA (pegRNA), wherein the pegRNA of the first prime editor complex encodes a first single stranded DNA sequence and the pegRNA of the second prime editor complex encodes a second single stranded DNA sequence, wherein the first single-stranded DNA sequence and the second single-stranded DNA sequence each comprises a region of complementarity to the other, and wherein the first single-stranded DNA sequence and the second single-stranded DNA sequence form a duplex comprising an edited portion as compared to the DNA sequence at the target site to be edited, which integrates into the target site to be edited.
  • pegRNA prime editor guide RNA
  • an agent includes a plurality of agents, including mixtures thereof.
  • M-MLV reverse transcriptase possesses DNA polymerase and ribonuclease H (RNase H) activity within a single polypeptide. It has been reported that the RT variants without the RNase H activity perform better in cDNA synthesis, likely due to increased DNA:RNA complex stability. To interrogate whether deleting RNase H domain from M-MLV RT would improve prime editing efficiency, the PE2 variant PE2Rminus was constructed by deleting the RNase H domain from PE2 ( Figure 1).
  • pCMV-PE2 (Addgene) plasmid as a template
  • DNA encoding 498 amino acids of M-MLV RT was amplified with Kpnl (GGTACC) at its 5’, and EcoRI (GAATTC) at its 3’.
  • the backbone pCMV-PE2 was digested with Kpnl and EcoRI followed by insertion of the PCR- amplified DNA fragment encoding 498 amino acids of M-MLV RT.
  • the constructed plasmid was named pCMV-PE2Rminus.
  • the full-length M-MLV reverse transcriptase (RT) in PE2 is 677 amino acids (the RNase H domain is underlined):
  • TETPDTSTLLIENSSP SEQ ID NO: 1
  • N-terminus of the RT may be truncated by up to 30 amino acids.
  • sequence for an N-terminal 30-amino acid truncation is given in SEQ ID NO: 3.
  • the amino acid sequence for PE2Rminus including both the SpCas9 nickase (H840A) and truncated RT is given in SEQ ID NO: 4.
  • the H840A domain is in bold.
  • the C-terminally truncated RT domain is in bold and underlined.
  • KRTADGSEFESPKKKRKV (SEQ ID NO: 5)
  • HEK293T cells were maintained in DMEM, 10% Fetal bovine serum (FBS), 1% pen-strep. IxlO 5 of HEK293T cells were plated in each well of 24 well plate 24 hours before transfection. On the day of transection, the cells were co-transfected with 500 ng of prime editor plasmid (PE2 or PE2Rminus), 150 ng of pegRNA-1 plasmid with or without 50 ng of pegRNA-2 plasmid or nicking sgRNA plasmid. Lipofectamine 3000 (Invitrogen) was used for the transfection according to the manufacturer’s instructions. FACS analysis was performed 3 days after transfection in GFP reporter cells. To detect editing efficiency in endogenous genomic loci, HEK293T cells were cultured for 3 days after transfection, and genomic DNA was isolated using DNeasy Blood & Tissue Kit (QIAGEN) according to the manufacturer’s instructions.
  • FBS Fetal bovine serum
  • Prime Editing analysis The alignment of the amplicon sequences to the reference sequence was performed using the well-established tool CRISPResso2.
  • CRISPResso2 was run in HDR mode using the provided desired allele as the expected allele with ‘discard_indel_reads’ on.
  • the prime editing efficiency was calculated as: percentage of [number of reads with the desired editing that does not contain indels within the quantification window] divided by [number of total reads].
  • the indel percentage was calculated as: percentage of [number of discarded reads (from either original or desired edited allele)] divided by [number of total reads].
  • HEK293FT GFP reporter cell line was generated with two premature TAG stop codons (TAGTAG) in the GFP coding sequence that prevent translation of a functional GFP protein ( Figure 2A).
  • TAGTAG premature TAG stop codons
  • PE2 PE2 editor with one pegRNA
  • PE3 PE2 editor with one pegRNA and an additional sgRNA to nick the non-edited DNA strand to encourage the edited strand to be utilized as a repair template by DNA repair machinery, thus increasing the desired editing efficiency
  • PE3B similar to PE3 except it employs a sequential nicking strategy in which the additional sgRNA can only cause the nick of the non-edited DNA strand upon recognizing the pegRNA-edited DNA strand).
  • PE2Rminus variants were constructed by deleting 1, 2, 3, 4, 5, 10, 15, 20, and 25 amino acids from the C-terminus of PE2Rminus. This produced prime editors that ranged from 473 to 497 amino acids in length ( Figure 6). The variants were tested for editing efficiency along with PE2 and PE2Rminus using the GFP reporter cell line. It was found that PE2Rminus showed the highest editing efficiency among all the constructs tested ( Figure 6).
  • pegRNAs used in this disclosure xxxx: CRISPR spacer xxxx: single guide RNA scaffold xxxx: reverse transcriptase template xxxx: primer binding site

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Abstract

L'invention concerne de manière générale le domaine de l'édition génomique. En particulier, la spécification concerne un polypeptide éditeur primaire pouvant réaliser une édition primaire en présence d'un ARN guide d'édition primaire (ARNpeg).
PCT/SG2023/050068 2022-02-08 2023-02-08 Compositions et méthodes pour édition génomique WO2023154011A2 (fr)

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