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WO2025151814A1 - Utilisation d'édition primaire dans la correction de mutations de cftr - Google Patents

Utilisation d'édition primaire dans la correction de mutations de cftr

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Publication number
WO2025151814A1
WO2025151814A1 PCT/US2025/011233 US2025011233W WO2025151814A1 WO 2025151814 A1 WO2025151814 A1 WO 2025151814A1 US 2025011233 W US2025011233 W US 2025011233W WO 2025151814 A1 WO2025151814 A1 WO 2025151814A1
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seq
sequence
pegrna
cftr
ngrna
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David R. Liu
Alexander Anthony SOUSA
Colin Hemez
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Broad Institute Inc
Harvard University
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Broad Institute Inc
Harvard University
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4712—Cystic fibrosis
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3519—Fusion with another nucleic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/34—Allele or polymorphism specific uses

Definitions

  • Prime editing enables the replacement of targeted DNA nucleotides with any specified replacement of up to hundreds of nucleotides, thereby enabling a wide variety of substitutions, insertions, and deletions in the genomes of living systems 1–4 .
  • the mechanism of prime editing is inherently resistant both to bystander editing (unwanted editing outcomes at the target site) 1 and to off-target editing 1,3,5–17 .
  • Prime editors do not require the creation of double-stranded DNA breaks (DSBs), minimizing undesirable outcomes such as uncontrolled insertions and deletions (indels) 2–4,18 , large deletions 19,20 , p53 activation 21–23 , retrotransposon insertion 24 , and chromosomal defects 19,25–28 .
  • PE does not require co-delivery of donor DNA template, is active in mitotic and non-mitotic cells 1,5,29–34 , and has been successfully performed in vivo in mice 5,29,32–38 and in non-human primates 39 .
  • Prime editors combine a programmable nickase such as Streptococcus pyogenes Cas9 (SpCas9) H840A nickase with a reverse transcriptase (RT) such as an engineered Moloney murine leukemia virus RT 1 .
  • a PE guide RNA guides the prime editor protein to its spacer-specified genomic target and also contains a 3′ extension with a primer binding site (PBS) complementary to the nicked target DNA and an RT template (RTT) encoding the desired edited sequence 1,40 .
  • PBS primer binding site
  • RTT RT template
  • the 3′ flap of edited DNA can displace the original sequence and be ligated into the genome, creating a DNA heteroduplex of edited and unedited DNA strands.
  • This editing intermediate is then resolved into a permanent edit on both strands by DNA repair or replication 41 .
  • a nickase, reverse transcriptase, and pegRNA constitute a ‘PE2’ editing system.
  • a ‘PE3’ system adds an additional nicking guide RNA (ngRNA) that nicks the unedited strand of the DNA heteroduplex intermediate to enhance editing efficiency by directing mismatch repair to remake the unedited strand using the edited strand as a template 1 (FIG.1A). Additional prime editing strategies to correct pathogenic mutations and treat diseases are needed. SUMMARY OF THE INVENTION [0005] Prime editing strategies can be developed to correct pathogenic mutations, such as the 3-base pair CTT deletion (F508del) in the CFTR (cystic fibrosis transmembrane conductance regulator) gene that results in the loss of phenylalanine 508 in the CFTR protein.
  • F508del 3-base pair CTT deletion
  • CFTR cystic fibrosis transmembrane conductance regulator
  • CFTR cystic fibrosis
  • the present disclosure provides prime editing guide RNAs (pegRNAs) comprising a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence: TCTGTATCTATATTCATCAT (SEQ ID NO: 1), ACCATTAAAGAAAATATCAT (SEQ ID NO: 2), ATTATGCCTGGCACCATTAA (SEQ ID NO: 3), CTGTATCTATATTCATCATA (SEQ ID NO: 4), TTCATCATAGGAAACACCAA (SEQ ID NO: 5), CAGTTTTCCTGGATTATGCC (SEQ ID NO: 6), CATTCTGTTCTCAGTTTTCC (SEQ ID NO: 7), or CTGTAT
  • pegRNAs prime editing guide RNAs
  • the pegRNA comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence of GTCTGTATCTATATTCATCATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCT AGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTGGCACCATTAAAGA AAATATCATCTTTGGTGTTTCCTATGATGAATATAGATACGCGGTTCTATCTAGTTAC GCGTTAAACCAACTAGAA (SEQ ID NO: 250), TCTGTATCTATATTCATCATCATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC TAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTGGCACCATTAAAG AAAATATCATCTTTGGTGTGAGTTACGATGAATATAGATACGCGGTTCTATCTAG T
  • ngRNAs nicking guide RNAs
  • a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 3/370 B1195.70196WO00 13363760.2 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence TTCACTTCTAATGGTGATTA (SEQ ID NO: 745), TCACTTCTAATGGTGATTAT (SEQ ID NO: 746), AATGGTGATTATGGGAGAAC (SEQ ID NO: 747), GGGAGAACTGGAGCCTTCAG (SEQ ID NO: 748), GGAGAACTGGAGCCTTCAGA (SEQ ID NO: 749), GAGGGTAAAATTAAGCACAG (SEQ ID NO: 750), CATTCTGTTCTCAGTTTTCC (SEQ ID NO: 7), CAGTTTTCCTGGATTATGCC
  • the present disclosure provides dead single guide RNAs (dsgRNAs) comprising a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence CTCCCTCCAAGTTGTCCACG (SEQ ID NO: 764), CTGGAGCCTTCAGA (SEQ ID NO: 765), ATTTTACCCTCTGA (SEQ ID NO: 766), GCCAGGCATAATCC (SEQ ID NO: 767), TCCTGGATTATGCC (SEQ ID NO: 768), TCTTTAATGGTGCC (SEQ ID NO: 769), TTACCTCTTCTAGT (SEQ ID NO: 770), ATGCCAACTAGAAG (SEQ ID NO: 771), GCCAAATATATAATT (SEQ ID NO: 772), or CCGCGCGCGCGCGCGC
  • the step of contacting results in the insertion of the sequence 5′-CTT-3′ into the CFTR gene.
  • the present disclosure provides compositions comprising any of the pegRNAs, ngRNAs, and/or dsgRNAs described herein. In some embodiments, the composition further comprises a prime editor.
  • the present disclosure provides systems comprising any of the pegRNAs, ngRNAs, and/or dsgRNAs described herein. In some embodiments, the system further comprises a prime editor.
  • the present disclosure provides pharmaceutical compositions comprising any of the pegRNAs, ngRNAs, dsgRNAs, or compositions described herein.
  • the present disclosure provides for the use of any of the pegRNAs, ngRNAs, dsgRNAs, compositions, polynucleotides, vectors, pharmaceutical compositions, and/or cells disclosed herein in the manufacture of a medicament for the treatment of a disease or disorder (e.g., cystic fibrosis).
  • a disease or disorder e.g., cystic fibrosis
  • any of the pegRNAs, ngRNAs, dsgRNAs, compositions, polynucleotides, vectors, pharmaceutical compositions, and/or cells disclosed herein are for use in medicine.
  • FIGs.2A-2E show that prime editing enhancements synergistically enhance correction of CFTR F508del.
  • FIG.2A shows a schematic of the prime editing system with enhancements that improve F508del correction. Enhancements include (5) epegRNA 3′ structured RNA motifs, (6) co-expression of MLH1dn, (7) translationally silent edits, and (8) engineered and evolved prime editors (PEmax and PE6).
  • FIG.2D shows PE5max correction of F508del in HEK293T cells with the NGG2 PBS13 RTT41 epegRNA encoded with SE0-SE4.
  • FIG.2E shows a comparison of F508del correction with PEmax and PE6 variants a-g in HEK293T cells. All conditions use the NGG2 PBS13 RTT41 SE2 epegRNA, MLH1dn, and the +104 ngRNA.
  • data and error bars represent mean and standard deviation, respectively, of three independent biological replicates (shown as black dots).
  • FIG.5 shows optimization of PE2 strategy to install the CFTR F508del mutation in HEK293T cells.
  • a panel of 18 pegRNAs with a PBS length of 14 nt and variable RTT lengths was transfected into HEK293T cells (top data plot).
  • FIG.9 shows the correction of CFTR F508del in HEK293T cells with several ngRNAs using PE5max and an epegRNA.
  • a PE5max experiment was performed against a panel of ngRNAs to identify the most efficient strategy to correct CFTR F508del.
  • X-axis labels identify different ngRNAs by their nicking position relative to the epegRNA nick (in base pairs).
  • the PE4max x-axis label specifies an editing condition with no ngRNA. Data and error bars represent mean and standard deviation, respectively, collected from three independent biological replicates (shown as black dots).
  • FIG.10 shows that silent edits co-installed with the F508del corrective CTT insertion disrupt NGG2’s PAM and recode several F508del mutation proximal codons.
  • Schematics of the silent edits shown as boxed base pairs
  • co-installed with the F508del corrective CTT insertion shown as underlined base pairs
  • the undisrupted PAM of NGG2 is shown as double-underlined base pairs in the SE0 schematic. Sequences shown correspond to the SEQ ID NOs indicated in the figure.
  • FIG.14A-14C show analysis of off-target editing in primary CF patient airway epithelial cells.
  • FIG.14A shows indel and substitution quantification at the top 32 human genomic sites identified by CIRCLE-seq for epegRNA NGG2 PBS13 RTT41 SE2.
  • FIG.15B shows screening of ngRNA using PE6c, MLH1dn, epegRNA NGG2 PBS13 RTT41 SE2, and the -40 dsgRNA.
  • FIG.15C shows screening of petRNA using SE2 silent edits, PE6c, MLH1dn, the +104 ngRNA, and the -40 dsgRNA.
  • FIG.15D shows editing efficiencies using petRNAs at previously reported positive control target sites. For FIG.15C and FIG.15D, an nCas9 (based on PEmax) and an MCP-RT (using a PE6c-based reverse transcriptase) were used for petRNA editing, as previously described 94 .
  • FIG.16A DeepPrime without the co-installation of silent edits (SE0)
  • FIG.16B PRIDICT without the co-installation of silent edits
  • FIG.16C DeepPrime with the co-installation of SE2 silent edits
  • FIG.16D PRIDICT with the co-installation of SE2 silent edits. All conditions use PE6c, MLH1dn, the +104 10/370 B1195.70196WO00 13363760.2 ngRNA, and the -40 dsgRNA is shown.
  • FIG.18 shows CFTR.F508 ⁇ screen A: Screen of 72 different silent edit strategies (here shown with all PBS13/RTT41 and all 19 bp protospacer). Top-performing pegRNA (OP253) shows 1.56x-fold higher editing than SE2. [0040] FIG.19 shows CFTR.F508 ⁇ screen A: Improved silent edit strategies can recode codons both before and after the position of the therapeutic edit (F at position 508).
  • CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA).
  • crRNA CRISPR RNA
  • type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and a Cas9 domain.
  • the tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA.
  • Cas9/crRNA/tracrRNA endonucleolytically cleaves a linear or circular dsDNA target complementary to the spacer.
  • the strand in the target DNA not complementary to crRNA is first cut endonucleolytically, then trimmed 3′-5′ exonucleolytically.
  • RNA-binding and cleavage typically requires protein and both RNAs.
  • single guide RNAs (“sgRNA”, or simply “gRNA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species.
  • sgRNA single guide RNAs
  • Cas9 recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus non-self.
  • Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, and such 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” (2013) RNA Biology 10:5, 726-737; 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).
  • 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.
  • 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 Cas9 protein fused to a polymerase such as a reverse transcriptase (i.e., a prime editor).
  • a polymerase such as a reverse transcriptase (i.e., a prime editor).
  • 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.
  • the Cas protein equivalents may include other napDNAbps from any type of CRISPR system (e.g., type II, V, VI), including Cpf1 (a type-V CRISPR-Cas system), C2c1 (a type V CRISPR-Cas system), C2c2 (a type VI CRISPR-Cas system), and C2c3 (a type V CRISPR-Cas system).
  • Cpf1 a type-V CRISPR-Cas system
  • C2c1 a type V CRISPR-Cas system
  • C2c2 a type VI CRISPR-Cas system
  • C2c3 a type V CRISPR-Cas system
  • guide RNAs associate with a Cas protein, directing (or programming) the Cas protein to a specific sequence in a DNA molecule that includes a sequence complementary to the protospacer sequence for the guide RNA.
  • a gRNA is a component of 16/370 B1195.70196WO00 13363760.2 the CRISPR/Cas system.
  • the sequence specificity of a Cas DNA-binding protein is determined by gRNAs, which have nucleotide base-pairing complementarity to target DNA sequences.
  • the native gRNA comprises a 20 nucleotide (nt) Specificity Determining Sequence (SDS), or spacer, which specifies the DNA sequence to be targeted, and is immediately followed by an 80 nt scaffold sequence, which associates the gRNA with the Cas protein.
  • an SDS of the present disclosure has a length of 15 to 100 nucleotides, or more.
  • an SDS may have a length of 15 to 90, 15 to 85, 15 to 80, 15 to 75, 15 to 70, 15 to 65, 15 to 60, 15 to 55, 15 to 50, 15 to 45, 15 to 40, 15 to 35, 15 to 30, or 15 to 20 nucleotides.
  • the SDS is 20 nucleotides long.
  • a targeting sequence may be 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% complementary to its target sequence.
  • the SDS of template DNA or target DNA may differ from a complementary region of a gRNA by 1, 2, 3, 4, or 5 nucleotides.
  • the guide RNA is about 15-120 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence (e.g., a target sequence in CFTR).
  • the guide RNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 nucleotides
  • a napDNAbp e.g., a Cas9 protein
  • a polymerase e.g., a reverse transcriptase
  • the linker can also be a nucleotide sequence in the case of joining two nucleotide sequences together (e.g., in a gRNA).
  • the linker is a non-peptidic linker.
  • the linker is an organic molecule, group, polymer, or chemical moiety.
  • the nickase is a Cas9 that comprises one or more mutations in an HNH-like domain relative to a wild type Cas9 sequence or to an equivalent amino acid position in other Cas9 variants or Cas9 equivalents.
  • the nickase is a Cas9 that comprises an aspartate-to-alanine substitution (D10A) in the RuvC1 catalytic domain of Cas9 relative to a canonical SpCas9 sequence or to an equivalent amino acid position in other Cas9 variants or Cas9 equivalents.
  • the nickase is a Cas9 that comprises an H840A, N854A, and/or N863A mutation relative to a canonical SpCas9 sequence, or to an equivalent amino acid position in other Cas9 variants or Cas9 equivalents.
  • the term “Cas9 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 nickase is a Cas protein that is not a Cas9 nickase.
  • the napDNAbp of a prime editor is a Cas9 nickase (nCas9) that nicks only a single strand.
  • the napDNAbp can be selected from the group consisting of: Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas12b2, Cas13a, Cas12c, Cas12d, Cas12e, Cas12h, Cas12i, Cas12g, Cas12f (Cas14), Cas12f1, Cas12j (Cas ⁇ ), and Argonaute and optionally has a nickase activity such that only one strand is cut.
  • the pegRNAs have a 3 ⁇ extension arm, a spacer, and a gRNA core.
  • the 3 ⁇ extension arm further comprises in the 5 ⁇ to 3 ⁇ direction a DNA synthesis template, a primer binding site, and a linker.
  • the DNA synthesis template may also be referred to more broadly as the “DNA synthesis template” where the polymerase of a prime editor described herein is not an RT, but another type of polymerase.
  • the pegRNAs have a 5 ⁇ extension arm, a spacer, and a gRNA core.
  • the 5 ⁇ extension further comprises in the 5 ⁇ to 3 ⁇ direction a DNA synthesis template, a primer binding site, and a linker.
  • the spacer sequence of the pegRNA is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 nucleotides in length. In certain 22/370 B1195.70196WO00 13363760.2 embodiments, the spacer sequence of the pegRNA is about 20 nucleotides in length. In some embodiments, the primer binding site is about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, or about 17 nucleotides in length. In certain embodiments, the primer binding site is about 9, about 10, about 11, about 12, about 13, about 14, or about 15 nucleotides in length.
  • the 3′ structured motif in the pegRNAs disclosed herein comprises the sequence CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 248), or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 248).
  • pegRNAs are further described, e.g., in International Patent Application No. PCT/US2020/023721, filed March 19, 2020, which published as WO 2020/191239; International Patent Application No.
  • PE6 refers to a suite of prime editors (PE6a, PE6b, PE6c, PE6d, PE6e, PE6f, and PE6g) comprising improved reverse transcriptase and/or Cas9 variants.
  • the improved reverse transcriptase and Cas9 domains of the PE6 variants can also be combined with each other to offer cumulative benefits.
  • a PE6 prime editor comprising an improved reverse transcriptase variant of PE6a and an improved Cas9 variant of PE6e is referred to herein as the prime editor “PE6a-e” (or “PE6e-a”).
  • Prime editing is described in Anzalone, A. V. et al., Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019), which is incorporated herein by reference. See also International PCT Application, PCT/US2020/023721, filed March 19, 2020, and published as WO 2020/191239, which is incorporated herein by reference.
  • the replacement strand containing the desired edit (e.g., a single nucleobase substitution) shares the same sequence as the endogenous strand (or is homologous to it) immediately downstream of the nick site of the target site to be edited (with the exception that it includes the desired edit).
  • the endogenous strand downstream of the nick site is replaced by the newly synthesized 33/370 B1195.70196WO00 13363760.2 replacement strand containing the desired edit.
  • Cas protein-reverse transcriptase fusions or related systems are used to target a specific DNA sequence with a guide RNA, generate a single strand nick at the target site, and use the nicked DNA as a primer for reverse transcription of an engineered DNA synthesis template that is integrated with the guide RNA.
  • the prime editors described herein are not limited to reverse transcriptases but may include the use of virtually any DNA polymerase. Indeed, while the application throughout may refer to prime editors with “reverse transcriptases,” it is set forth here that reverse transcriptases are only one type of DNA polymerase that may work with prime editing.
  • the pegRNA also contains new genetic information in the form of an extension that encodes a replacement strand of DNA containing a desired nucleotide change which is used to replace a corresponding endogenous DNA strand at the target site.
  • the mechanism of prime editing involves nicking the target site in one strand of the DNA to expose a 3′-hydroxyl group. The exposed 3′-hydroxyl group can then be used to prime the DNA polymerization of the edit- encoding extension on pegRNA directly into the target site.
  • the extension which provides the template for polymerization of the replacement strand containing the edit—can be formed from RNA or DNA.
  • the newly synthesized (or replacement) strand of DNA may also be referred to as a single strand DNA flap, which would compete for hybridization with the complementary homologous endogenous DNA strand, thereby displacing the corresponding endogenous strand.
  • Resolution of the hybridized intermediate also referred to as a heteroduplex, comprising the single strand DNA flap synthesized by the reverse transcriptase hybridized to the endogenous DNA strand with the exception of mismatches at positions where desired nucleotide edits are installed in the edit strand
  • prime editing operates by contacting a target DNA molecule (for which a change in the nucleotide sequence is desired to be introduced) with a nucleic acid programmable DNA binding protein (napDNAbp) complexed with a prime editing guide RNA (pegRNA).
  • a target DNA molecule for which a change in the nucleotide sequence is desired to be introduced
  • napDNAbp nucleic acid programmable DNA binding protein
  • pegRNA prime editing guide RNA
  • the prime editing guide RNA comprises an extension at the 3′ or 5′ end of the guide RNA, or at an intramolecular location in the guide RNA, and encodes the desired nucleotide change (e.g., single nucleotide substitution, insertion, or deletion).
  • the napDNAbp/extended gRNA complex contacts the DNA molecule, and the extended gRNA guides the napDNAbp to bind to a target locus.
  • a nick in one of the strands of DNA of the target locus is introduced (e.g., by a nuclease or chemical agent), thereby creating an available 3′ end in one of the strands of the target locus.
  • the nick is created in the strand of DNA that corresponds to the R-loop strand, i.e., the strand that is not hybridized to the guide RNA sequence, i.e., the “non-target strand.”
  • the nick could be introduced in either of the strands.
  • the nick could be introduced into the R-loop “target strand” (i.e., the strand hybridized to the protospacer of the extended gRNA) or the “non-target strand” (i.e., the strand forming the single-stranded portion of the R-loop and which is complementary to the target strand).
  • target strand i.e., the strand hybridized to the protospacer of the extended gRNA
  • the “non-target strand” i.e., the strand forming the single-stranded portion of the R-loop and which is complementary to the target strand.
  • the 3′ end of the DNA strand formed by the nick
  • interacts with the extended portion of the guide RNA in order to prime reverse transcription i.e., “target-primed RT”.
  • the 3′ end DNA strand hybridizes to a specific RT priming sequence on the extended portion of the guide RNA, i.e., the “reverse transcriptase priming sequence” or “primer binding site” on the pegRNA.
  • a reverse transcriptase (or other suitable DNA polymerase) is introduced that synthesizes a single strand of DNA from the 3′ end of the primed site towards the 5′ end of the prime editing guide RNA.
  • the DNA polymerase e.g., reverse transcriptase
  • the cell s endogenous DNA repair and replication processes resolve the mismatched DNA to incorporate the nucleotide change(s) to form the desired altered product.
  • the process can also be driven towards product formation with “second strand nicking.” This process may introduce at least one or more of the following genetic changes: transversions, transitions, deletions, and insertions.
  • Prime editor refers to the polypeptide or polypeptide components involved in prime editing as described herein.
  • 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 farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, 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 just a fragment of a naturally occurring protein or peptide.
  • a protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. Any of the proteins provided herein may be produced by any method known in the art. For example, 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 37/370 B1195.70196WO00 13363760.2 Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the contents of which are incorporated herein by reference.
  • Protospacer refers to the sequence ( ⁇ 20 bp) in DNA adjacent to the PAM (protospacer adjacent motif) sequence.
  • 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).
  • the canonical PAM sequence (i.e., the PAM sequence that is associated with the Cas9 nuclease of Streptococcus pyogenes or SpCas9) is 5 ⁇ -NGG-3 ⁇ , wherein “N” is any nucleobase followed by two guanine (“G”) nucleobases.
  • SpCas9 can also recognize additional non-canonical PAMs (e.g., NAG and NGA).
  • Different PAM sequences can be associated with different Cas9 nucleases or equivalent proteins from different organisms.
  • any given Cas9 nuclease e.g., SpCas9
  • Reverse transcriptase describes a class of polymerases characterized as RNA-dependent DNA polymerases. All known reverse transcriptases require a primer to synthesize a DNA transcript from an RNA template. Historically, reverse transcriptase has been used primarily to transcribe mRNA into cDNA, which can then be cloned into a vector for further manipulation.
  • silica refers to a mutation in a nucleic acid molecule that does not have an effect on the phenotype of the nucleic acid molecule, or the protein it produces if it encodes a protein.
  • Synonymous mutations refer to substitutions of one base for another in a gene such that the corresponding amino acid residue of the protein produced by the gene is not modified. This is due to the redundancy of the genetic code, allowing for multiple different codons to encode for the same amino acid in a particular organism.
  • a silent mutation is in a noncoding region or a junction of a coding region and a non-coding region (e.g., an intron/exon junction), it may be in a region that does not impact any biological properties of the nucleic acid molecule (e.g., splicing, 39/370 B1195.70196WO00 13363760.2 gene regulation, RNA lifetime, etc.).
  • the spacer sequence anneals to the complement of the protospacer sequence to form a ssRNA/ssDNA hybrid structure at the target site and a corresponding R loop ssDNA structure of the endogenous DNA strand.
  • Subject refers to an individual organism, for example, an individual mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, a goat, a cattle, a cat, or a dog.
  • the target site further refers to 40/370 B1195.70196WO00 13363760.2 the sequence within a nucleic acid molecule (e.g., a nucleic acid molecule comprising CFTR) to which a complex of, for example, a prime editor and a pegRNA binds.
  • treatment refers to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder (e.g., cystic fibrosis), or one or more symptoms thereof, as described herein.
  • treatment refers to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder (e.g., cystic fibrosis), or one or more symptoms thereof, as described herein.
  • treatment may be administered after one or more symptoms have developed and/or after a disease has been diagnosed.
  • treatment may be administered in the absence of symptoms, e.g., to prevent or delay onset of a symptom or inhibit onset or progression of a disease (e.g., cystic fibrosis).
  • 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.
  • variant Cas9 is a Cas9 comprising one or more changes in amino acid residues (i.e., “substitutions”) as compared to a wild type Cas9 amino acid sequence.
  • vector refers to a nucleic acid that can be modified to encode a gene of interest and that is able to enter a host cell, mutate, and replicate within the host cell, and then transfer a replicated form of the vector into another host cell.
  • the pegRNA comprises a backbone scaffold comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA AAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 17) or GTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAA AAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 18), or GTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTTAT CAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 20).
  • the pegRNA comprises an RTT comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the 43/370 B1195.70196WO00 13363760.2 sequence AGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 21), TAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 22), CATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 23), CACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 24), TGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 25), GCCTGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 26),
  • the pegRNA comprises a PBS comprising the sequence: ATGAATATAGA (SEQ ID NO: 205), ATGAATATAGAT (SEQ ID NO: 206), ATGAATATAGATA (SEQ ID NO: 207), ATGAATATAGATAC (SEQ ID NO: 208), ATATTTTCTTT (SEQ ID NO: 209), ATGAATATAG (SEQ ID NO: 210), ATGAATATAGATACA (SEQ ID NO: 211), ATGAATATAGATACAG (SEQ ID NO: 212), ATGAATATAGATACAGA (SEQ ID NO: 213), ATATTTTCTTTA (SEQ ID NO: 214), ATATTTTCTTTAA (SEQ ID NO: 215), ATATTTTCTTTAAT (SEQ ID NO: 216), ATGGTGCCAG (SEQ ID NO: 217), ATGGTGCCAGGC (SEQ ID NO: 218), ATGGTGCCAGGCA (SEQ ID NO: 219), ATGGTGCCAGGCAT
  • the ngRNA comprises a backbone scaffold comprising the sequence GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA AAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 17) or GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA AAAGTGGCACCGAGTCGGTGCTTTT (SEQ ID NO: 761).
  • the dsgRNA comprises a backbone scaffold comprising the sequence GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA AAAGTGGCACCGAGTCGGTGCTTT (SEQ ID NO: 782), GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA AAAGTGGCACCGAGTCGGTGCTTTT (SEQ ID NO: 761), or GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA AAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 17).
  • the dsgRNA comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence of any of the dsgRNAs in Table 1.
  • the dsgRNA comprises a sequence of any of the dsgRNAs in Table 1.
  • a pegRNA, ngRNA, or dsgRNA spacer is any RNA sequence having sufficient complementarity with a target polynucleotide sequence (e.g., CFTR) to hybridize with the target sequence and direct sequence-specific binding of a napDNAbp (e.g., Cas9, which may be part of a prime editor) to the target sequence.
  • a target polynucleotide sequence e.g., CFTR
  • a napDNAbp e.g., Cas9, which may be part of a prime editor
  • Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith- Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows- Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).
  • the ability of a pegRNA, ngRNA, or dsgRNA to direct sequence-specific binding of a prime editor to a target sequence may also be assessed by any suitable assay.
  • a prime editor and pegRNA may be provided to a host cell having the corresponding target sequence (e.g., CFTR, or a portion thereof), such as by transfection with vectors encoding the prime editor and pegRNA or by transfection of a ribonucleoprotein (RNP) complex, followed by an assessment of preferential cleavage, nicking, or editing within the target sequence.
  • cleavage or editing of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, prime editor, and pegRNA to be tested and a control pegRNA different from the test pegRNA, and comparing binding or rate of cleavage or editing at the target sequence between the test and control guide sequence reactions.
  • a pegRNA, ngRNA, or dsgRNA is about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 75, about 100, or more nucleotides in length. In some embodiments, a gRNA is about 50-150, about 60-140, about 70-130, about 80-120, or about 90-110 nucleotides in length.
  • the spacer sequence of a pegRNA, ngRNA, or dsgRNA is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 nucleotides in length.
  • a pegRNA, ngRNA, or dsgRNA comprises an optional linker sequence.
  • the gRNAs provided herein may comprise an optional linker sequence between the spacer and the backbone scaffold sequences.
  • the optional linker sequence is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 61/370 B1195.70196WO00 13363760.2 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, or at least 50 nucleotides in length.
  • the prime editor comprises a nucleic acid-programmable DNA-binding protein (napDNAbp) and a polymerase.
  • the prime editor comprises a napDNAbp (e.g., a Cas9 protein, such as SpCas9, or a variant thereof, such as nCas9 or dCas9) and a polymerase (e.g., a reverse transcriptase, such as an MMLV reverse transcriptase, a Tf1 reverse transcriptase, or a variant thereof).
  • the prime editor comprises a PE6 Cas9 variant and/or a PE6 reverse transcriptase.
  • the prime editor is a PE6c prime editor.
  • the one or more silent edits installed into the CFTR gene comprise changing the codon encoding CFTR F508 from TTT to TTC. In some embodiments, the one or more silent edits installed into the CFTR gene comprise changing the codon encoding CFTR G509 from GGT to GGA. In some embodiments, the one or more silent edits installed into the CFTR gene comprise changing the codon encoding CFTR V510 from GTT to GTG. In some embodiments, the one or more silent edits installed into the CFTR gene comprise changing the codon encoding CFTR V510 from GTT to GTC.
  • the one or more silent edits installed into the CFTR gene comprise changing the codon encoding CFTR S511 from TCC to AGC. In some embodiments, the one or more silent edits installed into the CFTR gene comprise changing the codon encoding CFTR S511 from TCC to TCT. In some embodiments, the one or more silent edits installed into the CFTR gene comprise changing the codon encoding CFTR Y512 from TAT to TAC. [0138] In some embodiments, the contacting step comprises delivering one or more polynucleotides encoding the pegRNA and the prime editor (and optionally a ngRNA and/or dsgRNA) to the nucleic acid sequence encoding the CFTR gene.
  • the contacting step is performed in a cell, such as a human or non-human airway epithelial sell. 63/370 B1195.70196WO00 13363760.2
  • the cell is in a tissue selected from the group consisting of lung tissue, pancreatic tissue, liver tissue, kidney tissue, or intestinal tissue.
  • the contacting step is performed in vitro.
  • the contacting step is performed in vivo.
  • the contacting step is performed in a subject.
  • a subject may have been diagnosed with a disease, or be at risk for having a disease.
  • the method is a method for treating a disease in a subject.
  • the disease is cystic fibrosis.
  • the present disclosure contemplates use of any of the pegRNAs, ngRNAs, dsgRNAs, compositions, polynucleotides, vectors, pharmaceutical compositions, and/or cells disclosed herein in the manufacture of a medicament for the treatment of a disease or disorder (e.g., cystic fibrosis).
  • a disease or disorder e.g., cystic fibrosis
  • any of the pegRNAs, ngRNAs, dsgRNAs, compositions, polynucleotides, vectors, pharmaceutical compositions, and/or cells disclosed herein are for use in medicine.
  • materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl o
  • PE6c for this edit is consistent with the prior findings that Tf1-based PE variants (PE6b and PE6c) frequently outperform alternatives for epegRNAs with predicted secondary structure folding energies smaller in magnitude (less stable) than -23 kcal/mol 34 .
  • the CFTR F508del correction strategy developed above installs silent edits—most notably, converting a serine codon from TCC to AGT—to evade cellular mismatch repair mechanisms. Partial incorporation of these silent edits could lead to nonsynonymous changes in protein sequence.
  • CIRCLE- seq 72 was used to identify sites within the human genome that are cleaved in vitro by Cas9 guided with the epegRNA, ngRNA, or dsgRNA used in the CFTR F508del prime editing strategy. Editing outcomes were assessed in primary CF airway epithelial cells from three separate donors at the top 32 candidate sites for each of the three guide RNAs, representing 96 total CIRCLE-seq-nominated off-target candidate sites investigated.
  • the final site (ngRNA-OT18) is situated within an intronic region of RUNX1 73 . Because ngRNA-OT18 is located ⁇ 60 kilobases from the nearest exon and showed a low mean indel formation rate of 0.048% in treated primary cells, this off-target site is not anticipated to have significant physiological consequences. No potential off-target sites were detected from the dsgRNA. These observations collectively suggest that the PE CFTR F508del correction strategy induces few off-target edits in the human genome under the tested conditions. Discussion [0176] By systematically refining a PE strategy to correct CFTR F508del, large improvements in editing efficiency were achieved. The initial PE3 experiments only resulted in up to 0.42% mean F508del correction.
  • the PE F508del correction strategy is DNA-free and takes advantage of transient RNA reagents that do not require DSBs.
  • the DSB-independent nature of PE may offer fewer undesired and uncharacterized outcomes, and also obviate the need for donor DNA template delivery, likely simplifying delivery and reducing cellular toxicity of correction. Consistent with the inherent resistance of the mechanism of PE to off-target editing, only very low frequencies ( ⁇ 0.1% at four sites) of potential off-target editing in primary CF patient airway epithelial cells following treatment with the optimized PE correction system was observed.
  • the enhanced edit-to-indel ratio of the PE strategies developed in this study results in unedited alleles predominantly encoding unmodified F508del CFTR protein that remains druggable by CF small-molecule correctors and potentiators, unlike the frameshifted CFTR alleles from the much higher frequency of nuclease-mediated indel byproduct formation that likely generate truncated CFTR proteins that cannot be rescued by small-molecule drugs.
  • RNA expression plasmids (pegRNAs, epegRNAs, ngRNAs, dsgRNAs, and sgRNAs) were cloned as previously described using isothermal assembly and synthetic gene fragments 61 .
  • Guide RNA sequences are provided in Table 1.
  • Guide RNA expression plasmids were purified using PureYield Plasmid Miniprep kits (Promega).
  • Prime editor and MLH1dn plasmids were purified using Qiagen Plasmid Plus Midi kit (Qiagen).
  • DNA PCR amplification was completed using Phusion U Green Multiplex PCR master mix (Thermo Fisher Scientific). Primers and gene fragments were ordered from Integrated DNA Technologies.
  • Synthetic guide RNA generation [0179] Synthetic pegRNAs and epegRNAs were ordered from Agilent Research Labs and contained 2′-O-methyl modifications at the first three nucleotides, 2′-O-methyl modifications at the third-to-last and second-to-last nucleotides, 3′-phosphorothioate linkages between the first three nucleotides, and 3′-phosphonoacetate linkages between the last two nucleotides.
  • Synthetic ngRNAs were ordered from Synthego and contained 2′-O-methyl modifications at 79/370 B1195.70196WO00 13363760.2 the first three and last three nucleotides and 3′-phosphorothioate linkages between the first three and last 2 nucleotides.
  • Synthetic dsgRNAs were ordered from Integrated DNA Technologies and contained 2′-O-methyl modifications at the first three and last three nucleotides and phosphorothioate linkages between the three first and last three nucleotides.
  • Guide RNA sequences are provided in Table 1.
  • Prime editor, adenine base editor, cytosine base editor, and MLH1dn-encoded mRNA were generated using in vitro transcription as previously described 61 . Briefly, the prime editor, base editor, or MLH1dn transcripts, containing a 5′ untranslated region (UTR), Kozak sequence, prime editor or MLH1dn open reading frame, and 3′ UTR were PCR amplified from a template plasmid containing an inactive T7 (dT7) promoter. PCR primers repair this dT7 promoter and also install a 119 nt poly(A) tail.
  • UTR 5′ untranslated region
  • PCR primers repair this dT7 promoter and also install a 119 nt poly(A) tail.
  • the purified PCR dsDNA amplicon was used as an in vitro transcription template using the HiScribe T7 high-yield RNA synthesis kit (NEB). In vitro transcription followed the manufacturer's optional protocol to include CleanCap reagent AG (Trilink) and substitute N1-Methylpseudouridine-5′-triphosphate (Trilink) for uridine triphosphate. Lithium chloride precipitation was used to purify mRNA from complete in vitro transcription reactions and mRNA transcripts were reconstituted in nuclease-free water.
  • Coating solution composition was LHC-8 basal medium (Thermo Fisher Scientific) 80/370 B1195.70196WO00 13363760.2 supplemented with 1.34 ⁇ l/ml Bovine serum albumin 7.5% (Thermo Fisher Scientific), 10 ⁇ l/ml Bovine collagen solution Type 1 (Advanced BioMatrix), and 10 ⁇ l/ml Fibronectin from human plasma (Thermo Fisher Scientific).
  • LHC-8 basal medium Thermo Fisher Scientific 80/370 B1195.70196WO00 13363760.2 supplemented with 1.34 ⁇ l/ml Bovine serum albumin 7.5% (Thermo Fisher Scientific), 10 ⁇ l/ml Bovine collagen solution Type 1 (Advanced BioMatrix), and 10 ⁇ l/ml Fibronectin from human plasma (Thermo Fisher Scientific).
  • Primary airway epithelial cells from non-CF donors were isolated from trachea or bronchi from postmortem lungs unsuitable for transplant. Primary CF airway epit
  • Cells were expanded on culture plates coated with human collagen IV (Sigma) in PneumaCult-Ex Plus medium (Ex-Plus medium, STEMCELL Technologies).
  • PneumaCult-Ex Plus medium Ex-Plus medium, STEMCELL Technologies.
  • ALI air-liquid interface
  • HEK293T transfections were conducted as previously described 61 . Briefly, approximately 16,000 HEK293T cells were plated per well of a 96 well plate in complete culture media. After 18-24 hours, cells were transfected at 70-80% confluency with variable amounts of plasmid DNA and 0.5 ⁇ L of Lipofectamine 2000 diluted in Opti-MEM (Thermo Fisher Scientific), following manufacturer’s instructions.
  • HEK293T cells for fluorescence-activated cell sorting (FACS) [0191] Briefly, for a single confluent well of HEK293T cells in a 96 well plate: 72 hours after transfection, media was carefully removed and 30 ⁇ L TrypLE Express (ThermoFisher) was used to coat well cell monolayer, which was then incubated at 37 °C with 5% CO 2 for 5 minutes. Following incubation, 70 ⁇ L complete HEK293T cell growth media was added, and cells were resuspended by pipetting.
  • FACS fluorescence-activated cell sorting
  • High-throughput sequencing and data analysis [0192] High throughput sequencing (HTS) of genomic loci was completed as previously described 61 .
  • PCR1 Phusion U Green Multiplex PCR master mix
  • PCR1 Phusion U Green Multiplex PCR master mix
  • PCR1 approximately 150 ng of genomic DNA was used to template a PCR with primers containing Illumina adapter overhangs and cycled under the following conditions: 95 °C for 3 minutes; 27–30 cycles of 95 °C for 10 seconds, 58-61 °C (corresponding to the experimentally optimized Tm) for 20 seconds and 72 °C for 30 seconds, followed by 72 °C for 5 minutes.
  • PCR2 In the second round of PCR (PCR2), combinations of Illumina-barcoded forward and reverse primers were used to uniquely identify each sample and, with 1-2 ⁇ L of PCR1 as a template, were amplified under the following conditions: 95 °C for 3 minutes; 7 cycles of 95 °C for 10 seconds, 61 °C for 20 seconds, and 72 °C for 30 seconds, followed by 72 °C for 5 minutes.
  • PCR2 reactions from the same genomic locus were pooled and gel extracted with QIAquick gel extraction kit (Qiagen) to eliminate primer 83/370 B1195.70196WO00 13363760.2 dimers.
  • CFTR F508del HEK293T cell line The monoclonal CFTR F508del HEK293T cell line was isolated by limiting dilution. Briefly, after identifying the most efficient PE2 strategy to install the CFTR F508del CTT deletion into the endogenous CFTR gene of HEK293T cells (FIG.5), HEK293T cells were transfected with this PE2 strategy following methods listed above. After 72 hours, transfected cells were dissociated from adherent culture, counted, and plated across 1096 well plates in 100 ⁇ L of media per well at a concentration of 0.5 cells per each well.
  • the 24 pegRNA sequences with the highest predicted editing efficiency were synthesized as epegRNAs and tested experimentally.
  • SE2 epegRNAs were designed by incorporating the SE2 silent edits into the SE0 pegRNA sequences generated by each model.
  • 84/370 B1195.70196WO00 13363760.2 Ussing chamber assay [0196] Three weeks following nucleofection, primary airway epithelial cell cultures were treated for 24 hours with 10 ⁇ M forskolin (Cayman) and 100 ⁇ M 3-isobutyl-1- methylxanthine (IBMX, Sigma) prior to bioelectric studies.
  • Epithelial cell cultures were mounted in Ussing chambers (Physiologic Instruments, Inc., San Diego, CA) in symmetrical Cl- buffered Ringers solutions consisting of (mM): 135 NaCl, 5 HEPES, 0.6 KH2PO4, 2.4 K2HPO4, 1.2 MgCl2, 1.2 CaCl2, 5 dextrose, pH titrated to 7.40 at 37° C with NaOH.
  • the command voltage was set to 0 mV, and short-circuit current (I sc ) was monitored.
  • Off target amplicon sequencing was performed using genomic DNA from three prime edited primary CF airway epithelial cell cultures as well as three unedited controls.
  • Indels and substitutions at nominated off-target sites were quantified from high- throughput sequencing data using CRISPResso2 92 . Indels were quantified within a window from 10 bp 5′ upstream of the end of the epegRNA homology template to 10 bp 3′ downstream of the Cas9 nick position; this was achieved by setting the parameter WC to ‘18’ and the parameter W to ‘31’.
  • epegRNA following the screening described in Example 2 was CFTR-F508del-NGG2-PAM19-SCF-OPO253-PBS13-RTT45: (Spacer in bold, scaffold-F in italics, RTT in underline, PBS in bold italics, linker in bold underline, tevopreQ1 motif in italics underline) CTGTATCTATATTCATCATGTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAG TCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTGCCTGGCACCATTAAAGA AAATATCATCTTTGGTGTGTCATACGATGAATATAGATACGCGGTTCTATCTAGTTA CGCGTTAAACCAACTAGAA (SEQ ID NO: 251).
  • Shuttle peptide delivers base editor RNPs to rhesus monkey airway epithelial cells in vivo. Nat. Commun.14, 8051 (2023).
  • the invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process.
  • the invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
  • the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim.
  • any claim that is dependent on another claim can be modified to include one or more limitations found in any other claims that is dependent on the same base claim.
  • elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group.

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Abstract

La présente divulgation concerne des procédés d'édition CFTR à l'aide d'un éditeur primaire (par exemple, pour corriger une mutation F508del dans une protéine CFTR) et un pegARN.<i /> De tels procédés peuvent être utiles pour traiter la fibrose kystique. La présente divulgation concerne également des pegARN, des ngARN, des dsgARN, des systèmes et des compositions pour l'édition de CFTR et le traitement de la fibrose kystique. L'invention concerne également des polynucléotides, des vecteurs, des cellules et des kits pour l'édition de CFTR et le traitement de la fibrose kystique.
PCT/US2025/011233 2024-01-10 2025-01-10 Utilisation d'édition primaire dans la correction de mutations de cftr Pending WO2025151814A1 (fr)

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