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WO2024193704A1 - Acides nucléiques guides ciblant la dmd et leurs utilisations - Google Patents

Acides nucléiques guides ciblant la dmd et leurs utilisations Download PDF

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Publication number
WO2024193704A1
WO2024193704A1 PCT/CN2024/083354 CN2024083354W WO2024193704A1 WO 2024193704 A1 WO2024193704 A1 WO 2024193704A1 CN 2024083354 W CN2024083354 W CN 2024083354W WO 2024193704 A1 WO2024193704 A1 WO 2024193704A1
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Prior art keywords
sequence
seq
guide
nucleic acid
dmd gene
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PCT/CN2024/083354
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English (en)
Inventor
Guoling LI
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Huidagene Therapeutics Co., Ltd.
Huidagene Therapeutics (Singapore) Pte. Ltd.
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Publication of WO2024193704A1 publication Critical patent/WO2024193704A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the disclosure contains a Sequence Listing XML file which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety.
  • Said XML copy created on March 21, 2024, by software “WIPO Sequence” according to WIPO Standard ST. 26, is named HGP029PCT. xml, and is 122, 529 bytes in size.
  • DMD Duchenne muscular dystrophy
  • DMD is an X-linked recessive disorder that primarily affects males, with symptoms typically appearing in early childhood. It would be desired to develop therapies to treat or even cure DMD.
  • the disclosure provides guide nucleic acid targeting DMD and systems and methods using the same, which efficiently guides gene editing protein to DMD gene, achieving efficient gene editing that leads to restoration of dystrophin expression and muscle performance.
  • the disclosure provides a guide nucleic acid comprising a guide sequence capable of hybridizing to a target sequence on a target strand of a DMD gene; wherein the protospacer sequence on the nontarget strand of the DMD gene corresponding to the target sequence is located at or within an exon of the DMD gene, or at or within a splice donor or a splice acceptor of the exon; wherein the exon is selected from the group consisting of Exon 43, Exon 44, Exon 45, Exon 46, Exon 51, Exon 53, Exon 55.
  • the protospacer sequence is immediately 3’ to a protospacer adjacent motif (PAM) of 5’-NTN-3’, wherein N is A, T, G, or C.
  • PAM protospacer adjacent motif
  • the guide sequence comprises (1) a sequence of any one of SEQ ID NOs: 36, 1-35, and 37-51 or a 5’ or 3’ end truncation thereof with 1, 2, 3, 4, 5, or 6, nucleotides truncated at the 5’ or 3’ end; or (2) a sequence having a sequence identity of at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100%to any one of SEQ ID NOs: 36, 1-35, and 37-51 or a 5’ or 3’ end truncation thereof with 1, 2, 3, 4, 5, or 6 nucleotides truncated at the 5’ or 3’ end; or (3) a sequence having at most 1, 2, 3, 4, 5, or 6 nucleotide differences, whether consecutive or not, compared to any one of SEQ ID NOs: 36, 1-35, and 37-51.
  • the guide nucleic acid comprises a scaffold sequence capable of forming a complex with a nucleic acid programmable binding protein (napBP) , and wherein the hybridization of the guide sequence to the target sequence guides the complex to the DMD gene.
  • napBP nucleic acid programmable binding protein
  • the scaffold sequence has substantially the same secondary structure as the secondary structure of the sequence of SEQ ID NO: 52 or 53; or wherein the scaffold sequence comprises (1) a sequence of SEQ ID NO: 52 or 53 or a 5’ or 3’ end truncation thereof with 1, 2, 3, 4, 5, or 6, nucleotides truncated at the 5’ or 3’ end; or (2) a sequence having a sequence identity of at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100%to SEQ ID NO: 52 or 53 or a 5’ or 3’ end truncation thereof with 1, 2, 3, 4, 5, or 6 nucleotides truncated at the 5’ or 3’ end; or (3) a sequence having at most 1, 2, 3, 4, 5, or 6 nucleotide differences, whether consecutive or not, compared to SEQ ID NO: 52 or 53.
  • the guide nucleic acid comprises a sequence having a sequence identity of at least about 80%(e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of any one of SEQ ID NOs: 71-125; or a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide differences, whether consecutive or not, compared to the sequence of any one of SEQ ID NOs: 71-125.
  • the disclosure provides a polynucleotide comprising or encoding one or more (e.g., two, three) copies of the guide nucleic acid of the disclosure.
  • the disclosure provides a system comprising:
  • napBP nucleic acid programmable binding protein
  • the napBP is capable of recognizing a protospacer adjacent motif (PAM) of 5’-NTN-3’ immediately 5’ to a protospacer sequence on the nontarget strand of the DMD gene, wherein N is A, T, G, or C.
  • PAM protospacer adjacent motif
  • the napBP is a nucleic acid programmable DNA endonuclease (napDNAn) .
  • the napDNAn is a Cas9 endonuclease, a Cas12 endonuclease, or an IscB endonuclease.
  • the napBP comprises a sequence having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NO: 55 or 57 or a N-terminal truncation thereof without the first N-terminal Methionine.
  • the napBP retains at least 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the guide sequence-specific DNA endonuclease activity of the sequence of SEQ ID NO: 55 or 57.
  • the disclosure provides a vector comprising the polynucleotide of the disclosure.
  • the vector is a plasmid, an adeno-associated virus (AAV) vector, a retroviral vector, an adenoviral vector, or a lentiviral vector.
  • AAV adeno-associated virus
  • the disclosure provides a recombinant adeno-associated virus (rAAV) vector genome comprising:
  • a first polynucleotide sequence comprising a sequence encoding a guide nucleic acid of the disclosure
  • napBP nucleic acid programmable binding protein
  • rAAV vector genome is adapted to be encapsulated into a rAAV particle.
  • the rAAV vector genome comprises a sequence having a sequence identity of at least about 80%to the sequence of SEQ ID NO: 69; or comprises the sequence of SEQ ID NO: 69.
  • the rAAV vector genome of the disclosure wherein the rAAV vector genome comprises, from the 5’ to 3’,
  • rAAV vector genome comprises a sequence having a sequence identity of at least about 80%to the sequence of SEQ ID NO: 69;
  • rAAV vector genome does not contain any nucleotide difference from the sequence of SEQ ID NO: 69 in any one of the components (1) to (18) ;
  • rAAV vector genome contains one or more nucleotide difference or does contain any nucleotide difference from the sequence of SEQ ID NO: 69 between any two adjacent components of the components (1) to (18) .
  • the disclosure provides a recombinant AAV (rAAV) particle comprising the rAAV vector genome of the disclosure.
  • rAAV recombinant AAV
  • the rAAV particle comprising a capsid with serotype of wild type AAV9.
  • the disclosure provides a method for production of the rAAV particle of the disclosure, comprising culturing in a host cell a transgene plasmid comprising the rAAV vector genome of the disclosure.
  • the disclosure provides a cell comprising a transgene plasmid comprising the rAAV vector genome of the disclosure for the production of a rAAV particle.
  • the disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising (1) the system of the disclosure or the rAAV particle of the disclosure, and (2) a pharmaceutically acceptable excipient.
  • the disclosure provides a method of modifying expression of a DMD gene, comprising contacting the DMD gene with the system of the disclosure, the rAAV particle of the disclosure, or the pharmaceutical composition of the disclosure.
  • the disclosure provides a cell or a progeny thereof comprising the guide nucleic acid of the disclosure, the system of the disclosure, or the rAAV particle of the disclosure.
  • the disclosure provides a cell or a progeny thereof comprising DMD gene modified by the system of the disclosure, the rAAV particle of the disclosure, or the method of the disclosure.
  • the disclosure provides a method for preventing, diagnosing, or treating a DMD associated disease in a subject in need thereof, comprising administering to the subject the system of the disclosure, the rAAV particle of the disclosure, or the pharmaceutical composition of the disclosure, wherein the napBP modifies DMD gene, and wherein the modification of the DMD gene treats the disease.
  • the DMD associated disease is Duchenne muscular dystrophy.
  • FIG. 1 is a schematic showing an exemplary target dsDNA, an exemplary guide RNA, and an exemplary napDNAbp.
  • FIG. 2 is another schematic showing an exemplary target dsDNA, an exemplary guide RNA, and an exemplary napDNAbp.
  • FIG. 3 is a schematic showing an exemplary target dsDNA, an exemplary transcript (target RNA) transcribed from the target dsDNA, an exemplary guide RNA, and an exemplary napRNAbp.
  • target RNA exemplary transcript transcribed from the target dsDNA
  • guide RNA exemplary guide RNA
  • napRNAbp an exemplary napRNAbp
  • FIG. 4 is another schematic showing an exemplary target dsDNA, an exemplary transcript (target RNA) transcribed from the target dsDNA, an exemplary guide RNA, and an exemplary napRNAbp, wherein the guide sequence contains a mismatch with the target sequence.
  • target RNA exemplary transcript transcribed from the target dsDNA
  • guide RNA exemplary guide RNA
  • napRNAbp an exemplary napRNAbp
  • FIG. 6 shows a single-cut gene editing strategy for treating Duchenne muscular dystrophy via a single AAV vector. Schematic of the therapeutic vector and single-cut strategy. The removal of one exon by hfCas12Max system (All-in-one AAV) editing applies to approximately 83%of all DMD patients.
  • One single gRNA is designed to target the vicinity of the intron-exon boundary and splice signal sequences, restoring the correct ORF by either exon skipping or exon reframing event.
  • FIG. 7 shows generation of novel humanized DMD mice (model) with human exon51.
  • FIG. 8 shows gRNA design and screening in vitro for editing of DMD gene.
  • FIG. 8A Flow diagram for detection of genome editing efficiency by transfected with plasmid encoding hfCas12Max and designed gRNAs, followed by FACS and NGS analysis.
  • FIG. 8B-8C Three guide sequence (G31-G33) targeting splicing acceptor (SA) and five guide sequence (G34-G38) targeting splicing donor (SD) of DMD Exon 51 were designed.
  • NGS sequencing of the DMD gene Exon51 shows genomic indel (%) (editing efficiency) after transfection with each of the 7 gRNAs in HEK293T (B) and iPSC (C) , respectively.
  • FIG. 8D shows genomic indel (%) (editing efficiency) after transfection with each of the 7 gRNAs in HEK293T (B) and iPSC (C) , respectively.
  • FIG. 8D shows genomic indel (%) (edit
  • FIG. 9 shows rescue of dystrophin expression by intramuscular (IM) injection in tibialis anterior (TA) muscle 4-week post-injection of the rAAV9 particles with gRNA configurations “Dg” , “DgD” , “DgDgD” , or “DgDgDg” into ⁇ mE51E52, hE51KI mouse model in a fixed dose of2.5E11 vg .
  • IM intramuscular
  • TA tibialis anterior
  • FIG. 10 shows rescue of dystrophin expression by systemic delivery of the rAAV particles with gRNA configuration “DgDgDg” .
  • FIG. 11 and FIG. 12 shows rescue of dystrophin expression by tail vein injection of the rAAV particles with gRNA configuration “DgDgDg” .
  • Nucleic acid programmable binding protein for example, nucleic acid programmable DNA binding protein, (napDNAbp) , such as Cas9, Cas12, IscB, nucleic acid programmable RNA binding protein (napRNAbp) , such as, Cas13, is capable of binding to a target nucleic acid (e.g., dsDNA, mRNA) as guided by a guide nucleic acid (e.g., a guide RNA) comprising a guide sequence targeting the target nucleic acid.
  • a target nucleic acid e.g., dsDNA, mRNA
  • a guide nucleic acid e.g., a guide RNA
  • the target nucleic acid is eukaryotic.
  • the guide nucleic acid comprises a scaffold sequence responsible for forming a complex with the napBP, and a guide sequence that is intentionally designed to be responsible for hybridizing to a target sequence of the target nucleic acid, thereby guiding the complex comprising the napBP and the guide nucleic acid to the target nucleic acid.
  • an exemplary target dsDNA (e.g., DMD gene) is depicted to comprise a 5’ to 3’ single DNA strand and a 3’ to 5’ single DNA strand.
  • An exemplary guide nucleic acid (e.g., a guide RNA) is depicted to comprise a guide sequence and a scaffold sequence.
  • the guide sequence is designed to hybridize to a part of the 3’ to 5’ single DNA strand, and so the guide sequence “targets” that part.
  • the 3’ to 5’ single DNA strand is referred to as a “target strand (TS) ” of the target dsDNA
  • the opposite 5’ to 3’ single DNA strand is referred to as a “nontarget strand (NTS) ” of the target dsDNA.
  • target sequence That part of the target strand based on which the guide sequence is designed and to which the guide sequence may hybridize is referred to as a “target sequence”
  • protospacer sequence the opposite part on the nontarget strand corresponding to that part is referred to as the “protospacer sequence” , which is 100% (fully) reversely complementary to the target sequence and is said to be “corresponding to” the target sequence in the disclosure.
  • an exemplary target dsDNA (e.g., DMD gene) is depicted to comprise a 5’ to 3’ single DNA strand and a 3’ to 5’ single DNA strand.
  • an exemplary target RNA (transcript, e.g., a pre-mRNA) may be transcribed using the 3’ to 5’ single DNA strand as a synthesis template, and thus the 3’ to 5’ single DNA strand is referred to as a “template strand” or a “antisense strand” .
  • the transcript so transcribed has the same primary sequence as the 5’ to 3’ single DNA strand except for the replacement of T with U, and thus the 5’ to 3’ single DNA strand is referred to as a “coding strand” or a “sense strand” .
  • An exemplary guide nucleic acid (e.g., a guide RNA) is depicted to comprise a guide sequence and a scaffold sequence.
  • the guide sequence is designed to hybridize to a part of the transcript (target RNA) , and so the guide sequence “targets” that part. And thus, that part of the target RNA based on which the guide sequence is designed and to which the guide sequence may hybridize is referred to as a “target sequence” .
  • the guide sequence is 100% (fully) reversely complementary to the target sequence.
  • the guide sequence is reversely complementary to the target sequence and contains a mismatch with the target sequence (as exemplified in FIG. 4) .
  • nucleic acid sequence e.g., a DNA sequence, an RNA sequence
  • a nucleic acid sequence is written in 5’ to 3’ direction /orientation unless explicitly indicated otherwise.
  • a DNA sequence of ATGC it is usually understood as 5’-ATGC-3’ unless otherwise indicated. Its reverse sequence is 5’-CGTA-3’. Its fully complementary sequence is 5’-TACG-3’. Its fully reverse complementary sequence is 5’-GCAT-3’. Note that the fully complementary sequence usually does not have the ability to base-pair /hybridize with the original sequence.
  • the double-strand sequence of a dsDNA may be represented with the sequence of its 5’ to 3’ single DNA strand conventionally written in 5’ to 3’ direction /orientation unless otherwise indicated.
  • the dsDNA may be simply represented as 5’-ATGC-3’.
  • either the 5’ to 3’ single DNA strand or the 3’ to 5’ single DNA strand of a dsDNA can be a nontarget strand from which a protospacer sequence is selected.
  • the 5’ to 3’ single DNA strand is the sense strand of the gene
  • the 3’ to 5’ single DNA strand is the antisense strand of the gene.
  • the sense strand or the antisense strand of a gene can be a nontarget strand from which a protospacer sequence is selected.
  • the transcript (target RNA) transcribed from the dsDNA then has a (target) sequence of 5’-AUGC-3’.
  • the guide sequence of a guide nucleic acid is designed to have a sequence of 5’-AUGC-3’ that is fully reversely complementary to the 3’ to 5’ strand of the target dsRNA, which would be set forth in ATGC in the electric sequence listing butmarked as an RNA sequence; and in another embodiment, the guide sequence of a guide nucleic acid is designed to have a sequence of 5’-GCAU-3’ that is fully reversely complementary to the 5’ to 3’ strand of the target dsRNA, which would be set forth in GCAT in the electric sequence listing but marked as an RNA sequence.
  • the guide sequence of a guide nucleic acid is fully reversely complementary to the target sequence and the target sequence is fully reversely complementary to the protospacer sequence
  • the guide sequence is identical to the protospacer sequence except for the U in the guide sequence due to its RNA nature and correspondingly the T in the protospacer sequence due to its DNA nature.
  • symbol “t” is used to denote both T in DNA and U in RNA (See “Table 1: List of nucleotides symbols” , the definition of symbol “t” is “thymine in DNA/uracil in RNA (t/u) ” ) .
  • such a guide sequence could be set forth in the same sequence as a corresponding protospacer sequence.
  • a single SEQ ID NO in the electronic sequence listing can be used to denote both such guide sequence and protospacer sequence, regardless whether such a single SEQ ID NO is marked as DNA or RNA in the electronic sequence listing.
  • a reference is made to such a SEQ ID NO that sets forth a protospacer /guide sequence it refers to either a protospacer sequence that is a DNA sequence or a guide sequence that is an RNA sequence depending on the context, no matter whether it is marked as a DNA or an RNA in the electronic sequence listing.
  • the guide sequence of a guide nucleic acid is designed to have a sequence of 5’-GCAU-3’ that is fully reversely complementary to the (target) sequence of the target RNA, which would be set forth in GCAT in the electric sequence listing but marked as an RNA sequence.
  • the term “activity” refers to a biological activity.
  • the activity includes enzymatic activity, e.g., catalytic ability of an effector.
  • the activity can include nuclease activity, e.g., dsDNA endonuclease activity, RNA endonuclease activity.
  • nucleic acid programmable binding protein napBP
  • nucleic acid programmable binding domain napBD
  • a programmable nucleic acid e.g., DNA or RNA
  • gRNA guide nucleic acid
  • the napBP may be indirectly associated with (e.g., bound to) the target nucleic acid via the interaction (e.g., binding) between the napBP and the programmable nucleic acid (e.g., scaffold sequence of the programmable nucleic acid) and the interaction (e.g., hybridization) between the programmable nucleic acid (e.g., the guide sequence of the programmable nucleic acid) and the target nucleic acid (e.g., the target sequence of the target nucleic acid) .
  • the napBP is a nucleic acid programmable DNA binding protein (napDNAbp) .
  • the napBP is a nucleic acid programmable RNA binding protein (napRNAbp) .
  • the term “complex” refers to a grouping of two or more molecules.
  • the complex comprises a polypeptide and a nucleic acid interacting with (e.g., binding to, coming into contact with, adhering to) one another.
  • the term “complex” can refer to a grouping of a guide nucleic acid and a polypeptide (e.g., a napBP) .
  • the term “complex” can refer to a grouping of a guide nucleic acid, a polypeptide (e.g., a napBP) , and a target nucleic acid.
  • the term “protospacer adjacent motif’ or “PAM” refers to a short DNA sequence (or a DNA motif) adjacent to a protospacer sequence on the nontarget strand of a dsDNA.
  • adjacent includes instances wherein there is no nucleotide between the protospacer sequence and the PAM and also instances wherein there are a small number (e.g., 1, 2, 3, 4, or 5) of nucleotides between the protospacer sequence and the PAM.
  • a “immediately adjacent (to) ” B, A “immediately 5’ to” B, and A “immediately 3’ to” B mean that there is no nucleotide between A and B.
  • the PAM is immediately 5’ to a protospacer sequence. In some embodiments, the PAM is immediately 3’ to a protospacer sequence.
  • the term “guide nucleic acid” refers to any nucleic acid that facilitates the targeting of a napBP to a target nucleic acid.
  • the guide nucleic acid may be designed to include a guide sequence capable of hybridizing to a specific sequence of a target nucleic acid, and the guide nucleic acid may also comprise a scaffold sequence facilitating the guiding of a napBP to the target nucleic acid.
  • the guide nucleic acid is a guide RNA.
  • the guide nucleic acid is a nucleic acid encoding a guide RNA.
  • nucleic acid As used herein, the terms “nucleic acid” , “polynucleotide” , and “nucleotide sequence” are used interchangeably to refer to a polymeric formof nucleotides of any length, including deoxyribonucleotides, ribonucleotides, combinations thereof, and analogs or modifications thereof.
  • guide RNA is used interchangeably with the term “CRISPR RNA (crRNA) ” , “single guide RNA (sgRNA) ” , or “RNA guide”
  • guide sequence is used interchangeably with the term “spacer sequence”
  • sinaffold sequence is used interchangeably with the term “direct repeat sequence” .
  • the guide sequence is so designed to be capable of hybridizing to a target sequence.
  • the term “hybridize” , “hybridizing” , or “hybridization” refers to a reaction in which one or more polynucleotide sequences react to forma complex that is stabilized via hydrogen bonding between the bases of the polynucleotide sequences. The hydrogen bonding may occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence specific manner.
  • Apolynucleotide sequence capable of hybridizing to a given polynucleotide sequence is referred to as the “complement” of the given polynucleotide sequence.
  • the hybridization of a guide sequence and a target sequence is so stabilized to permit an effector polypeptide (e.g., a napBP) that is complexed with a nucleic acid comprising the guide sequence or a function domain associated (e.g., fused) with the effector polypeptide to act (e.g., cleave, deaminize) on the target sequence or its complement or nearby sequence.
  • an effector polypeptide e.g., a napBP
  • a nucleic acid comprising the guide sequence or a function domain associated (e.g., fused) with the effector polypeptide to act (e.g., cleave, deaminize) on the target sequence or its complement or nearby sequence.
  • the guide sequence is reversely complementary to a target sequence.
  • reverse complementary refers to the ability of nucleobases of a first polynucleotide sequence, such as a guide sequence, to base pair with nucleobases of a second polynucleotide sequence, such as a target sequence, by traditional Watson-Crick base-pairing. Two reverse complementary polynucleotide sequences are able to non-covalently bind under appropriate temperature and solution ionic strength conditions.
  • a first polynucleotide sequence (e.g., a guide sequence) comprises 100% (fully) reverse complementarity to a second nucleic acid (e.g., a target sequence) .
  • a first polynucleotide sequence (e.g., a guide sequence) is reverse complementary to a second polynucleotide sequence (e.g., a target sequence) if the first polynucleotide sequence comprises at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%complementarity to the second nucleic acid (i.e., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
  • the term “substantially complementary” refers to a first polynucleotide sequence (e.g., a guide sequence) that has a certain level of complementarity to a second polynucleotide sequence (e.g., a target sequence) (e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%of the nucleotides of the first polynucleotide sequence can base-pair with the nucleotides of the second polynucleotide sequence, or at most 1, 2, 3, 4, or 5 contiguous or non-contiguous nucleotides of the first polynucleotide sequence mismatch the nucleotides of the second polynucleotide sequence) .
  • the level of complementarity is such that the first polynucleotide sequence (e.g., a guide sequence) can hybridize to the second polynucleotide sequence (e.g., a target sequence) with sufficient affinity to permit an effector polypeptide (e.g., a napBP) that is complexed with a nucleic acid comprising the first polynucleotide sequence or a function domain associated (e.g., fused) with the effector polypeptide to act (e.g., cleave, deaminize) on the target sequence or its complement or nearby sequence.
  • a guide sequence that is substantially complementary to a target sequence has less than 100%complementarity to the target sequence.
  • a guide sequence that is substantially complementary to a target sequence has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementarity to the target sequence, and/or has at most 1, 2, 3, 4, or 5 contiguous or non-contiguous nucleotide mismatches from the target sequence.
  • sequence identity is related to sequence homology. Homology comparisons may be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs may calculate percentage sequence identity (%) between two or more sequences (polypeptide or polynucleotide sequences) . Sequence homologies may be generated by any of a number of computer programs known in the art, for example, BLAST, FASTA. Asuitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A; Devereux et al., 1984, Nucleic Acids Research 12: 387) .
  • Examples of other software than may perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 ibid-Chapter 18) , FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) , and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60) .
  • Acommonly used online tool to calculate percentage sequence identity between two or more sequences is available on the website of EMBL's European Bioinformatics Institute (www dot ebi dot ac dot uk slash jdispatcher slash) , allowing fast online calculation of percentage sequence identity by global alignment or local alignment.
  • polypeptide and “peptide” are used interchangeably herein to refer to polymers of amino acids of any length.
  • Aprotein may have one or more polypeptides.
  • An amino acid polymer can also be modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.
  • a “variant” is interpreted to mean a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, respectively, but retains essential properties, e.g., binding property of a napBP.
  • Atypical variant of a polynucleotide differs in nucleic acid sequence from another reference polynucleotide. Achange in the nucleic acid sequence of the polynucleotide variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide.
  • a change in the nucleic acid sequence of the polynucleotide variant may result in an amino acid substitution, addition, and/or deletion in the polypeptide encoded by the reference polynucleotide.
  • Atypical variant of a polypeptide differs in amino acid sequence from another reference polypeptide. Generally, the difference is limited so that the sequences of the reference polypeptide and the polypeptide variant are closely similar overall and, in many regions, identical.
  • the polypeptide variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, and/or deletions in any combination.
  • Avariant of a polynucleotide or polypeptide may be naturally occurring, such as, an allelic variant, or it may be a variant that is not known to occur naturally.
  • Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans.
  • the terms “upstream” and “downstream” refer to the relative positions of two or more elements within a nucleic acid in 5’ to 3’ direction.
  • Afirst sequence is upstream of a second sequence when the 3’ end of the first sequence is present at the left side of the 5’ end of the second sequence.
  • Afirst sequence is downstream of a second sequence when the 5’ end of the first sequence is present at the right side of the 3’ end of the second sequence.
  • the PAM is upstream of a napBP-induced indel, and a napBP-induced indel is downstream of the PAM.
  • the PAM is downstream of a napBP-induced indel, and a napBP-induced indel is upstream of the PAM.
  • wild type has the meaning commonly understood by those skilled in the art to mean a typical form of an organism, a strain, a gene, or a feature that distinguishes it from a mutant or variant when it exists in nature. It can be isolated from sources in nature and not intentionally modified.
  • nucleic acid or polypeptide As used herein, the terms “non-naturally occurring” and “engineered” are used interchangeably and refer to artificial participation. When these terms are used to describe a nucleic acid or a polypeptide, it is meant that the nucleic acid or polypeptide is at least substantially freed from at least one other component of its association in nature or as found in nature.
  • regulatory element is intended to include promoters, enhancers, internal ribosome entry sites (IRES) , and other expression control elements (e.g., transcription termination signals, such as, polyadenylation signals and poly-U sequences) .
  • IRES internal ribosome entry sites
  • regulatory elements e.g., transcription termination signals, such as, polyadenylation signals and poly-U sequences
  • Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences) .
  • Regulatory elements may also direct expression in a time-dependent manner, e.g., in a cell cycle-dependent or developmental stage-dependent manner, which may or may not be tissue or cell type specific.
  • the term “cell” is understood to refer not only to a particular individual cell, but to the progeny or potential progeny of the cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term.
  • in vivo refers to inside the body of an organism
  • ex vivo or “in vitro” means outside the body of an organism.
  • the term “treat” , “treatment” , or “treating” is an approach for obtaining beneficial or desired results including clinical results.
  • the beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from a disease, diminishing the extent of a disease, stabilizing a disease (e.g., delaying the worsening of a disease) , delaying the spread (e.g., metastasis) of a disease, delaying the recurrence of a disease, reducing recurrence rate of a disease, delay or slowing the progression of a disease, ameliorating a disease state, providing a remission (partial or total) of a disease, decreasing the dose of one or more other medications required to treat a disease, delaying the progression of a disease, increasing the quality of life, and/or prolonging survival.
  • treatment is a reduction of pathological consequence of a disease (such as cancer)
  • disease includes the terms “disorder” and “condition” and is not limited to those have been specifically medically defined.
  • reference to “not” a value or parameter generally means and describes “other than” a value or parameter.
  • the method is not used to treat cancer of type X means the method may be used to treat cancer of types other than X.
  • the term “and/or” in a phrase such as “A and/or B” is intended to include both A and B; A or B; A (alone) ; and B (alone) .
  • the term “and/or” in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C;A and B; B and C; A (alone) ; B (alone) ; and C (alone) .
  • DMD Duchenne muscular dystrophy
  • DMD is an X-linked recessive disorder that primarily affects males, with symptoms typically appearing in early childhood. It would be desired to develop therapies to treat or even cure DMD.
  • the disclosure provides tools and methods for treating DMD associated diseases, such as, Duchenne muscular dystrophy.
  • DMD associated diseases includes diseases that are associated with DMD gene, the transcription (including transcript; e.g., increased or decreased transcription, nonfunctional or dysfunctional transcript) thereof, and/or the expression (including expression and expression product; e.g., increased or decreased expression, nonfunctional or dysfunctional expression product) thereof, including those that are directly or indirectly caused by the transcription (including transcription and transcript, e.g., DMD mRNA) of DMD gene, and those that are directly or indirectly caused by the expression (including expression product, e.g., dystrophin) of DMD gene.
  • the term “DMD associated disease” refers to a disease that is caused by abnormal expression of DMD gene.
  • the term “DMD associated disease” refers to a disease that is caused by the deficiency of a functional expression product of DMD gene.
  • transcript includes any transcription product by transcription from a gene, including mRNA, non-coding RNA, and any variants, derivatives, or ancestors thereof, for example, pre-mRNA, and any transcripts or isoforms produced from the gene or the pre-mRNA by, e.g., alternative promoter usage, alternative splicing, alternative initiation, and any naturally occurring variants thereof or processed products therefrom.
  • the transcript of the DMD gene is also termed as DMD transcript in the disclosure.
  • the DMD gene is human DMD gene.
  • Human wild type DMD gene has NCBI Gene ID: 1756.
  • the transcript is human DMD mRNA.
  • the expression product is human dystrophin.
  • Human wild type dystrophin has NCBI Accession No.: XP_054182593.1 for isoform X1.
  • a nonsense mutation of human DMD gene may lead to abnormal expression of DMD gene that fails to generate functional dystrophin and therefore causes DMD associated diseases.
  • the disclosure provides, in some embodiments, tools for modifying the expression of such a mutated DMD gene by cleaving (e.g., relying on the function of Cas endonuclease) and repairing (e.g., relying on the self-repairing mechanism of cells) the mutated DMD gene, thereby incorporating an indel mutation into the mutated DMD gene that leads to the expression of a functional dystrophin, which may be a dystrophin mutant that is sufficiently functional as wild type dystrophin.
  • the tools can be delivered, for example, by rAAV vectors to subjects in need.
  • the tools include guide nucleic acids comprising a guide sequence designed to be capable of hybridizing to the mutated DMD gene, thereby guiding a complex comprising the guide nucleic acid and a napBP to the mutated DMD gene and modifying the mutated DMD gene, leading to the generation of functional dystrophin from the modified DMD gene.
  • the syndromes e.g., syndromes of Duchenne muscular dystrophy
  • the disclosure provides a guide nucleic acid comprising a guide sequence capable of hybridizing to a target sequence on a target strand of a DMD gene or a target sequence on a transcript of a DMD gene.
  • the guide nucleic acid comprises a scaffold sequence capable of forming a complex with a nucleic acid programmable binding protein (napBP) , and wherein the hybridization of the guide sequence to the target sequence guides the complex to the DMD gene or the transcript.
  • napBP nucleic acid programmable binding protein
  • the disclosure provides a system comprising:
  • a guide nucleic acid or a polynucleotide (e.g., a DNA, an RNA, a DNA/RNA mixture) encoding the guide nucleic acid, comprising:
  • napBP nucleic acid programmable binding protein
  • the system is a complex comprising the napBP complexed with the guide nucleic acid.
  • the complex further comprises the DMD gene or transcript thereof hybridized with the guide sequence.
  • the system is a composition comprising the component (1) and the component (2) .
  • the disclosure provides various delivery of the system of the disclosure, for example, delivery via a rAAV vector.
  • the disclosure provides a recombinant adeno-associated virus (rAAV) vector genome encoding or comprising the system of the disclosure.
  • the rAAV vector genome is a DNA (e.g., a ssDNA, a dsDNA) or an RNA.
  • a recombinant adeno-associated virus (rAAV) vector genome e.g., a DNA rAAV vector genome, an RNA rAAV vector genome
  • rAAV adeno-associated virus
  • a first polynucleotide sequence comprising a sequence encoding a guide nucleic acid comprising:
  • napBP nucleic acid programmable binding protein
  • rAAV vector genome is adapted to be encapsulated into a rAAV particle (e.g., a DNA-encapsulated rAAV particle, an RNA-encapsulated rAAV particle) .
  • a rAAV particle e.g., a DNA-encapsulated rAAV particle, an RNA-encapsulated rAAV particle
  • the disclosure provides a method of modifying expression of a DMD gene, comprising contacting the DMD gene or transcript thereof with a system comprising:
  • a guide nucleic acid or a polynucleotide (e.g., a DNA, an RNA, a DNA/RNA mixture) encoding the guide nucleic acid, comprising:
  • napBP nucleic acid programmable binding protein
  • the modification of the expression of the DMD gene treats, detects, or diagnose a DMD associated disease. In some embodiments, the modification of the expression of the DMD gene detects or diagnose the presence, progress, or development of a DMD associated disease.
  • the disclosure provides various mechanisms for modifying expression of a DMD gene for various purposes, including, but not limited to, treatment or diagnosis of DMD associated diseases.
  • the modification could be on gene level, transcription level, transcript level, or translation level.
  • the modification leads to expression of a functional expression product from the DMD gene so as to treat DMD associated diseases.
  • modifying expression of a DMD gene includes, but not limited to, modifications of any one or more stages of the transcription and translation procedure starting from the DMD gene to an expression product of the DMD gene, e.g., dystrophin.
  • modifications include:
  • modifying the DMD gene such as, cleaving the DMD gene (and thereby introducing an indel mutation into the DMD gene) , changing (e.g., adding, deleting, substituting) any one or more nucleotides of the DMD gene by, for example, base editing, prime editing;
  • modifying the transcript of the DMD gene such as, a DMD mRNA, for example, cleaving (and typically degrading) the transcript, changing (e.g., adding, deleting, substituting) any one or more nucleotides of the transcript by, for example, base editing; and
  • the translation of the transcript e.g., a DMD mRNA
  • the DMD gene e.g., a DMD mRNA
  • modifying the translation of the transcript e.g., a DMD mRNA of the DMD gene, such as, increasing or decreasing the translation of the transcript by, for example, epigenetic modification.
  • the DMD gene comprises a mutation.
  • the DMD gene is a human DMD gene comprising a mutation.
  • Various DMD mutation that causes a DMD associated disease is known in the art.
  • the DMD gene comprises a nonsense mutation.
  • the napBP is a nucleic acid programmable DNA binding protein (napDNAbp) .
  • the napDNAbp is a nucleic acid programmable dsDNA endonuclease (napDNAn) .
  • guiding the complex to the DMD gene enables the napDNAn to specifically cleave the DMD gene in a guide sequence-specific manner.
  • the specific cleavage of the DMD gene leads to incorporation of an insertion and/or deletion (an indel) mutation into the DMD gene.
  • the specific cleavage of the DMD gene or the insertion and/or deletion mutation generates an in-frame stop codon in the DMD gene.
  • an in-frame stop codon in a gene and/or in the transcript transcribed from a gene typically stops the translation of a protein from the transcript.
  • stop codon is TAG, TAA, or TGA in DNA, and UAG, UAA, or UGA in RNA.
  • the specific cleavage of the DMD gene or the insertion and/or deletion mutation generates a 3n+1 frameshift mutation, a 3n+2 frameshift mutation, a 3n-1 frameshift mutation, or a 3n-2 frameshift mutation in the DMD gene, wherein n is 0 or a positive integer (e.g., 1, 2, 3) .
  • the frameshift mutation decreases or eliminates transcription of the DMD gene and/or translation of a transcript (e.g., mRNA) of the DMD gene. In some embodiments, the frameshift mutation generates an in-frame stop codon in the DMD gene.
  • the frameshift mutation leads to expression of a mutant of dystrophin. In some embodiments, the mutant is functional. In some embodiments, the mutant is non-functional.
  • the frameshift mutation leads to expression of a functional mutant of dystrophin.
  • the term “functional mutant” of a protein refers to a mutant of the protein that substantially has one or more functions (e.g., all known functions) of the protein, for example, a mutant having at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%of the function of the protein or having about 100%function of the protein.
  • the frameshift mutation leads to expression of a non-functional mutant of dystrophin.
  • non-functional mutant of a protein refers to a mutant of the protein that substantially lacks one or more functions (e.g., all known functions) of the protein, for example, a mutant having at most about 35%, 30%, 20%, 15%, 10%, 5%, or 1%of the function of the protein or having about 0%function of the protein.
  • the frameshift mutation leads to expression of a non-pathogenic mutant of dystrophin.
  • non-pathogenic mutant of a protein refers to a mutant of the protein that is not pathogenic or has not been reported to cause any disease.
  • the frameshift mutation does not lead to expression of a pathogenic mutant of dystrophin.
  • pathogenic mutant of a protein refers to a mutant of the protein that is pathogenic or has been reported to cause any disease.
  • the system of disclosure is combined with a supplement to deliver the wild type expression product of the DMD gene.
  • a cDNA encoding the wild type expression product of DMD gene is delivered in combination with the system of disclosure delivered in rAAV particles in the same or separate rAAV particles.
  • Also provided in the disclosure are tools and methods using DNA base editing to modify the expression of DMD gene.
  • the napDNAbp is a nickase and further comprises a base editing domain.
  • nickase means that it substantially lacks dsDNA endonuclease activity (i.e., it is substantially incapable of cleaving both strands of a dsDNA) and is substantially capable of nicking one strand of a dsDNA.
  • the one strand is the target strand of the dsDNA.
  • the one strand is the sense strand or the antisense strand of the dsDNA.
  • the napDNAbp is an endonuclease deficient napDNAbp and further comprises a base editing domain.
  • the term “endonuclease deficient napDNAbp” is used interchangeably with “endonuclease inactive napDNAbp” or “dead napDNAbp” , meaning that it substantially lacks dsDNA endonuclease activity (i.e., it is substantially incapable of cleaving double strands of a dsDNA) , and it substantially lacks nickase activity (i.e., it is substantially incapable of nicking either strand of a dsDNA) .
  • guiding the complex to the DMD gene enables the napDNAbp that is a nickase or dead napDNAbp and further comprises the base editing domain to specifically base edit the DMD gene in a guide sequence-specific manner.
  • base edit means editing the base of a nucleotide (e.g., a ribonucleotide, a deoxyribonucleotide) , including but not limited to inserting (adding) , deleting (excising) , and substituting the base.
  • a nucleotide e.g., a ribonucleotide, a deoxyribonucleotide
  • the base editing of the DMD gene substitutes a nucleotide of the DMD gene to a different nucleotide. In some embodiments, the base editing of the DMD gene substitutes one nucleotide of the DMD gene to one different nucleotide, which is also known as single base editing. In some embodiments, the base editing of the DMD gene substitutes two or three or more nucleotides, whether consecutive or not, of the DMD gene to two or three or more different nucleotides, which is also known as multiple or multiplexed base editing.
  • codon includes 61 non-stop codons encoding 22 amino acids and three stop codons (i.e., TAA, TAG, TGA) .
  • Each codon is composed of three nucleotides.
  • a nucleotide change in a non-stop codon may convert the non-stop codon to another non-stop codon encoding the same or different amino acid, or to a stop codon.
  • Anucleotide change in a stop codon may convert the stop codon or another stop codon, or to a non-stop codon.
  • the codon in the disclosure e.g., a non-stop codon, a stop codon, a start codon, is an in-frame codon.
  • the base editing substitutes A in a codon of the DMD gene with T, C, or G.
  • base editing substitutes T in a codon of the DMD gene with A, C, or G.
  • base editing substitutes C in a codon of the DMD gene with A, T, or G.
  • base editing substitutes G in a codon of the DMD gene with A, T, or C.
  • the base editing substitutes a stop codon of the DMD gene with a non-stop codon. In some embodiments, base editing substitutes a non-stop codon of the DMD gene with a different non-stop codon. In some embodiments, base editing substitutes a non-stop codon of the DMD gene with a stop codon.
  • the DMD gene may contain a pathogenic mutation, which may be a pathogenic codon substitution where a wild type codon of the DMD gene is substituted with a pathogenic codon.
  • the wild type codon is a stop codon
  • the pathogenic codon is a non-stop codon.
  • the pathogenic codon substitution of a wild type stop codon to a non-stop codon may lead to the expression of a mutant of the expression product of the DMD gene containing additional C-terminal amino acids, which mutant may be pathogenic (e.g., non-functional) .
  • the wild type codon is a non-stop codon
  • the pathogenic codon is a stop codon or a non-stop codon different from the wild type non-stop codon.
  • the pathogenic codon substitution of a wild type non-stop codon with a stop codon may leads to prematurely terminated (partial) translation of the transcript of the DMD gene, which may generate a N-terminal truncation of the expression product of the DMD gene or generate no expression product.
  • the N-terminal truncation of the expression product may be pathogenic (e.g., non-functional) and may be stable or unstable (e.g., quickly degraded by cellular enzyme) .
  • the pathogenic codon substitution of a wild type non-stop codon with a non-stop codon different from the wild type non-stop codon may lead to the expression of a mutant of the expression product of the DMD gene, which mutant may be pathogenic (e.g., non-functional) , and which mutant may be stable or unstable (e.g., quickly degraded by cellular enzyme) .
  • the base editing of the DMD gene decreases or eliminates transcription of the DMD gene and/or translation of a transcript (e.g., mRNA) of the DMD gene.
  • a transcript e.g., mRNA
  • the base editing of the DMD gene eliminates full expression of the DMD gene by substituting a non-stop codon in the DMD gene with a stop codon, leaving no translation or prematurely terminated (partial) translation of the transcript of the DMD gene.
  • the base editing of the DMD gene generates an in-frame stop codon in the DMD gene.
  • the base editing of the DMD gene leads to expression of a functional expression product of the DMD gene, e.g., a functional mutant of dystrophin.
  • the base editing of the DMD gene leads to expression of a mutant of dystrophin, for example, by substituting a non-wild type stop codon with a non-wild type, non-stop codon or substituting a non-wild type, non-stop codon with a different non-wild type, non-stop codon.
  • the mutant of dystrophin is functional. In some embodiments, the mutant of dystrophin is non-functional.
  • the base editing of the DMD gene leads to expression of a non-functional mutant of dystrophin, for example, by substituting a non-wild type stop codon with a non-wild type, non-stop codon or substituting a non-wild type, non-stop codon with a different non-wild type, non-stop codon, and the resulting expression product of the base edited DMD gene is a non-functional mutant of dystrophin.
  • the base editing of the DMD gene leads to expression of wild type dystrophin, for example, by substituting a non-wild type stop codon or a non-wild type, non-stop codon with a wild type non-stop codon, and the resulting expression product of the base edited DMD gene is wild type dystrophin.
  • the base editing of the DMD gene leads to expression of a functional mutant of dystrophin, for example, by substituting a non-wild type stop codon with a non-wild type, non-stop codon or substituting a non-wild type, non-stop codon with a different non-wild type, non-stop codon, and the resulting expression product of the base edited DMD gene is a functional mutant of dystrophin.
  • the base editing of the DMD gene leads to expression of a non-pathogenic mutant of dystrophin, for example, by substituting a non-wild type stop codon with a non-wild type, non-stop codon or substituting a non-wild type, non-stop codon with a different non-wild type, non-stop codon, and the resulting expression product of the base edited DMD gene is non-pathogenic.
  • the base editing of the DMD gene does not lead to expression of a pathogenic mutant of dystrophin.
  • Also provided in the disclosure are tools and methods using DNA prime editing to modify the expression of DMD gene.
  • the napDNAbp is a nickase and further comprises a reverse transcriptase domain
  • the guide nucleic acid is a prime editing guide nucleic acid (e.g., pegRNA) .
  • nickase means that it substantially lacks dsDNA endonuclease activity (i.e., it is substantially incapable of cleaving both strands of a dsDNA) and is substantially capable of nicking one strand of a dsDNA.
  • the one strand is the nontarget strand of the dsDNA.
  • the one strand is the sense strand or the antisense strand of the dsDNA.
  • guiding the complex to the DMD gene enables the napDNAbp that is a nickase and further comprises a reverse transcriptase domain to specifically prime edit the DMD gene in a guide sequence-specific manner.
  • Prime Editing Adding Precision and Flexibility to CRISPR Editing” (blog dot addgene dot org slash prime-editing-crisp-cas-reverse-transcriptase) .
  • Prime editing may allow insertion (addition) , deletion, or substitution of one or more nucleotides of the DMD gene.
  • the prime editing of the DMD gene decreases or eliminates transcription of the DMD gene and/or translation of a transcript (e.g., mRNA) of the DMD gene.
  • a transcript e.g., mRNA
  • the prime editing of the DMD gene leads to expression of a functional expression product of the DMD gene, e.g., a functional mutant of dystrophin.
  • the prime editing of the DMD gene generates an in-frame stop codon in the DMD gene, for example, by inserting a stop codon, by deleting one or more nucleotides to generate a frameshift mutation that further generates a stop codon, or substituting a non-stop codon with a stop codon.
  • the prime editing of the DMD gene leads to expression of a mutant of dystrophin, for example, by substituting a non-wild type stop codon with a non-wild type, non-stop codon or substituting a non-wild type, non-stop codon with a different non-wild type, non-stop codon.
  • the mutant of dystrophin is functional. In some embodiments, the mutant of dystrophin is non-functional.
  • the prime editing of the DMD gene leads to expression of a non-functional mutant of dystrophin, for example, by substituting a non-wild type stop codon with a non-wild type, non-stop codon or substituting a non-wild type, non-stop codon with a different non-wild type, non-stop codon, and the resulting expression product of the prime edited DMD gene is a non-functional mutant of dystrophin.
  • the prime editing of the DMD gene leads to expression of wild type dystrophin, for example, by substituting a non-wild type stop codon or a non-wild type, non-stop codon with a wild type non-stop codon, and the resulting expression product of the prime edited DMD gene is wild type dystrophin.
  • the prime editing of the DMD gene leads to expression of a functional mutant of dystrophin, for example, by substituting a non-wild type stop codon with a non-wild type, non-stop codon or substituting a non-wild type, non-stop codon with a different non-wild type, non-stop codon, and the resulting expression product of the prime edited DMD gene is a functional mutant of dystrophin.
  • the prime editing of the DMD gene leads to expression of a non-pathogenic mutant of dystrophin, for example, by substituting a non-wild type stop codon with a non-wild type, non-stop codon or substituting a non-wild type, non-stop codon with a different non-wild type, non-stop codon, and the resulting expression product of the prime edited DMD gene is non-pathogenic.
  • the prime editing of the DMD gene does not lead to expression of a pathogenic mutant of dystrophin.
  • the napDNAbp is an endonuclease deficient napDNAbp and further comprises epigenomic modification domain, e.g., methylation domain, demethylation domain.
  • epigenomic modification domain e.g., methylation domain, demethylation domain.
  • the term “endonuclease deficient napDNAbp” is used interchangeably with “endonuclease inactive napDNAbp” or “dead napDNAbp” , meaning that it substantially lacks dsDNA endonuclease activity (i.e., it is substantially incapable of cleaving double strands of a dsDNA) , and it substantially lacks nickase activity (i.e., it is substantially incapable of nicking either strand of a dsDNA) .
  • guiding the complex to the DMD gene enables the napDNAbp that is an endonuclease deficient napDNAbp and further comprises epigenomic modification domain, e.g., methylation domain, demethylation domain, to specifically epigenetically modify the DMD gene in a guide sequence-specific manner.
  • epigenomic modification domain e.g., methylation domain, demethylation domain
  • the epigenomic modification of the DMD gene decreases or eliminates transcription of the DMD gene and/or translation of a transcript (e.g., mRNA) of the DMD gene.
  • a transcript e.g., mRNA
  • the epigenomic modification of the DMD gene increases transcription of the DMD gene and/or translation of a transcript (e.g., mRNA) of the DMD gene.
  • a transcript e.g., mRNA
  • the transcript of the DMD gene is a DMD pre-mRNA or a DMD mRNA.
  • the transcript comprises a mutation.
  • the transcript is a transcript of human DMD gene comprising a mutation.
  • the DMD gene comprises a nonsense mutation.
  • the transcript comprises a nonsense mutation.
  • the napBP is a nucleic acid programmable RNA binding protein (napRNAbp) .
  • RNA cleavage to modify the expression of DMD gene.
  • the napRNAbp is a nucleic acid programmable RNA endonuclease (napRNAn) .
  • guiding the complex to the transcript of the DMD gene enables the napRNAn to specifically cleave the transcript of the DMD gene in a guide sequence-specific manner.
  • the cleavage of a transcript by an RNA endonuclease leads to degradation of the transcript.
  • RNA base editing to modify the expression of DMD gene.
  • the napRNAbp is an endonuclease deficient napRNAbp and further comprises a base editing domain.
  • the term “endonuclease deficient napRNAbp” is used interchangeably with “endonuclease inactive napRNAbp” or “dead napRNAbp” , meaning that it substantially lacks RNA endonuclease activity (i.e., it is substantially incapable of cleaving an RNA) .
  • guiding the complex to the transcript of the DMD gene enables the napRNAbp that is an endonuclease deficient napRNAbp and further comprises the base editing domain to specifically base edit the transcript in a guide sequence-specific manner.
  • the base editing of the transcript substitutes a nucleotide of the transcript to a different nucleotide. In some embodiments, the base editing of the transcript substitutes one nucleotide of the transcript to one different nucleotide, which is also known as single base editing. In some embodiments, the base editing of the transcript substitutes two or three or more nucleotides, whether consecutive or not, of the transcript to two or three or more different nucleotides, which is also known as multiple or multiplexed base editing.
  • the base editing substitutes A in a codon of the transcript with U, C, or G; substitutes U in a codon of the transcript with A, C, or G; substitutes C in a codon of the transcript with A, U, or G; or substitutes G in a codon of the transcript with A, U, or C.
  • the base editing substitutes a stop codon of the transcript with a non-stop codon; substitutes a non-stop codon of the transcript with a different non-stop codon; or substitutes a non-stop codon of the transcript with a stop codon.
  • the transcript may contain a pathogenic mutation, which may be a pathogenic codon substitution where a wild type codon of the transcript is substituted with a pathogenic codon.
  • the wild type codon is a stop codon
  • the pathogenic codon is a non-stop codon.
  • the pathogenic codon substitution of a wild type stop codon to a non-stop codon may lead to the translation of a mutant of the expression product of the DMD gene containing additional C-terminal amino acids, which mutant may be pathogenic (e.g., non-functional) .
  • the wild type codon is a non-stop codon
  • the pathogenic codon is a stop codon or a non-stop codon different from the wild type non-stop codon.
  • the pathogenic codon substitution of a wild type non-stop codon with a stop codon may leads to prematurely terminated (partial) translation of the transcript, which may generate a N-terminal truncation of the expression product of the DMD gene or generate no expression product.
  • the N-terminal truncation of the expression product may be pathogenic (e.g., non-functional) and may be stable or unstable (e.g., quickly degraded by cellular enzyme) .
  • the pathogenic codon substitution of a wild type non-stop codon with a non-stop codon different from the wild type non-stop codon may lead to the translation of a mutant of the expression product of the DMD gene, which mutant may be pathogenic (e.g., non-functional) , and which mutant may be stable or unstable (e.g., quickly degraded by cellular enzyme) .
  • the base editing of the transcript decreases or eliminates translation of the transcript.
  • the base editing of the transcript eliminates full translation of the transcript by substituting a non-stop codon in the transcript with a stop codon, leaving no translation or prematurely terminated (partial) translation of the transcript.
  • the base editing of the transcript generates an in-frame stop codon in the transcript.
  • the base editing of the transcript leads to translation of a functional expression product of the transcript, e.g., a functional mutant of dystrophin.
  • the base editing of the transcript leads to expression of a mutant of dystrophin, for example, by substituting a non-wild type stop codon with a non-wild type, non-stop codon or substituting a non-wild type, non-stop codon with a different non-wild type, non-stop codon.
  • the mutant of dystrophin is functional. In some embodiments, the mutant of dystrophin is non-functional.
  • the base editing of the transcript leads to expression of a non-functional mutant of dystrophin, for example, by substituting a non-wild type stop codon with a non-wild type, non-stop codon or substituting a non-wild type, non-stop codon with a different non-wild type, non-stop codon, and the resulting expression product of the transcript is a non-functional mutant of dystrophin.
  • the base editing of the transcript leads to expression of wild type dystrophin, for example, by substituting a non-wild type stop codon or a non-wild type, non-stop codon with a wild type non-stop codon, and the resulting expression product of the transcript is wild type dystrophin.
  • the base editing of the transcript leads to expression of a functional mutant of dystrophin, for example, by substituting a non-wild type stop codon with a non-wild type, non-stop codon or substituting a non-wild type, non-stop codon with a different non-wild type, non-stop codon, and the resulting expression product of the transcript is a functional mutant of dystrophin.
  • the base editing of the transcript leads to expression of a non-pathogenic mutant of dystrophin, for example, by substituting a non-wild type stop codon with a non-wild type, non-stop codon or substituting a non-wild type, non-stop codon with a different non-wild type, non-stop codon, and the resulting expression product of the transcript is non-pathogenic.
  • the base editing of the transcript gene does not lead to expression of a pathogenic mutant of dystrophin.
  • the napBP is capable of forming a complex with the guide nucleic acid of the disclosure by complexing with the scaffold sequence of the guide nucleic acid and is thereby guided to the DMD gene or transcript thereof via the hybridization of the guide sequence of the guide nucleic acid to the target sequence of the DMD gene or transcript.
  • the activity of the napBP or the functional domain associated with (e.g., bound to) the napBP
  • any such napBP can be used with the guide nucleic acid of the disclosure, and when a napBP is selected, the scaffold sequence compatible to the napBP for complexing with the napBP can also be selected accordingly.
  • the scaffold sequence is generally conserved.
  • the napBP (e.g., napDNAbp) is capable of recognizing a protospacer adjacent motif (PAM) on the nontarget strand of the DMD gene, wherein the PAM is immediately 5’ or 3’ to a protospacer sequence on the nontarget strand of the DMD gene, and wherein the protospacer sequence is fully reversely complementary to the target sequence.
  • PAM protospacer adjacent motif
  • the PAM comprises sequence 5’-NN-3’, 5’-NNN-3’, 5’-NNNN-3’, 5’-NNNNN-3’, or 5’-NNNNNN-3’, wherein N is A, T, G, or C.
  • Non-limiting examples of the napBP include CRISPR-associated (Cas) protein, IscB, TAL nuclease, meganuclease, and zinc-finger nuclease.
  • CRISPR-associated (Cas) protein include Cas9 (e.g., dCas9 and nCas9) , Cas12a/Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12f/Cas14, Cas12g, Cas12h, Cas12i, and Cas12k.
  • Cas protein include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csn1 or Csx12) , Cas10, Cas10d, Cas12a/Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12f/Cas14, Cas12g, Cas12h, Cas12i, Cas12k, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Cs
  • napDNAbp are also within the scope of this disclosure, e.g., IscB, IsrB, although they may not be specifically listed in this disclosure. See, e.g., Makarova et al. “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here? ” CRISPR J. 2018 October; 1: 325-336. doi: 10.1089/crispr. 2018.0033; Yan et al., “Functionally diverse type V CRISPR-Cas systems” Science. 2019 Jan. 4; 363 (6422) : 88-91. doi: 10.1126/science. aav7271, the entire contents of each are hereby incorporated by reference.
  • the Cas protein is an endonuclease, a nickase, or a dead Cas.
  • the PAM comprises sequence 5’-NTN-3’, wherein N is A, T, G, or C, and wherein the PAM is immediately 5’ to the protospacer sequence.
  • the PAM comprises sequence 5’-TTN-3’, wherein N is A, T, G, or C.
  • the napBP is a Class 2, Type V CRISPR-associated protein (Cas12) .
  • the Cas12 is Cas12a (Cpf1) , Cas12b (C2c1) , Cas12c (C2c3) , Cas12d (CasY) , Cas12e (CasX) , Cas12f (Cas14) , Cas12i, or Cas12k (C2c10, C2C7) , e.g., Cas12i1, Cas12i1, Cas12i3, Cas12i4, xCas12i (SiCas12i) , Cas12Max, hfCas12Max, or a mutant thereof.
  • the PAM comprises sequence 5’-NGG-3’, wherein N is A, T, G, or C, and wherein the PAM is immediately 3’ to the protospacer sequence.
  • the napBP is a Class 2, Type II CRISPR-associated protein (Cas9) , e.g., SaCas9, SpCas9, or a mutant thereof.
  • the PAM comprises sequence 5’-NNNGAN-3’, wherein N is A, T, G, or C, and wherein the PAM is immediately 3’ to the protospacer sequence.
  • the napBP is a IscB protein, e.g., OgeuIscB or a mutant thereof.
  • the recognizing ability of the napBP to a target nucleic acid may not be limited to any specific PAM, which means that the napBP can recognize any PAM, such that the PAM is not a substantial restriction on the selection of a protospacer sequence or a target sequence.
  • a napBP is called “PAMless” , for example, a PAMless SpCas9 mutant.
  • the napBP is PAMless.
  • the napBP is a Class 2, Type VI CRISPR-associated protein (Cas13) .
  • Cas13 can be targeted to RNA by a guide nucleic acid. Cas13 is particularly useful since there is no PAM restriction for eukaryotic transcripts when Cas13 is used as the napBP.
  • the Cas13 is Cas13a (C2c2) , Cas13b (such as, Cas13b1, Cas13b2) , Cas13c, Cas13d, Cas13e (Cas13X) , Cas13f (Cas13Y) , or a mutant thereof.
  • a Cas protein e.g., Cas9, Cas12, Cas13
  • a CRISPR RNA crRNA
  • the crRNA also comprises a scaffold sequence capable of complexing with the Cas protein.
  • the napBP comprises a sequence having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NO: 55 or 57 or a N-terminal truncation thereof without the first N-terminal Methionine.
  • the napBP retains at least 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the guide sequence-specific DNA endonuclease activity of the sequence of SEQ ID NO: 55 or 57.
  • the napBP comprises a sequence of SEQ ID NO: 57.
  • the sequence encoding the napBP comprises a sequence having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NO: 63 or a 5’ end truncation thereof without the first 5’ ATG codon.
  • the napBP encoded by the sequence retains at least 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the guide sequence-specific DNA endonuclease activity of the sequence of SEQ ID NO: 57.
  • the sequence encoding the napBP comprises a sequence of SEQ ID NO: 63.
  • the napBP further comprises a base editing domain.
  • the base editing domain is capable of substituting a base of a nucleotide with a different base.
  • the base editing domain is capable of deaminating a base of a nucleotide.
  • the base editing domain comprises a deaminase domain capable of deaminating a base (e.g., an adenine, a guanine, a cytosine, a thymine, an uracil) of a nucleotide.
  • a base e.g., an adenine, a guanine, a cytosine, a thymine, an uracil
  • the deaminase domain is capable of deaminating an adenine (A) to a hypoxanthine (I) .
  • the deamination of the adenine to the hypoxanthine converts the adenosine (A) or deoxyadenosine (dA) containing the adenine to a guanosine (G) or deoxyguanosine (dG) .
  • the deaminase domain is capable of deaminating a cytosine (C) to an uracil (U) .
  • the deamination of the cytosine to the uracil converts the cytidine (C) or deoxycytidine (dC) containing the cytosine to a uridine (U) or a deoxythymidine (dT) .
  • the base editing domain is capable of excising a base (e.g., an adenine, a guanine, a cytosine, a thymine, an uracil) of a nucleotide.
  • a base e.g., an adenine, a guanine, a cytosine, a thymine, an uracil
  • the base editing domain comprises a base excising domain capable of excising a base of a nucleotide.
  • the base editing domain comprises a deaminase domain and a base excising domain.
  • the deaminase domain is a double-stranded RNA-specific adenosine deaminase (ADAR) , such as, ADAR1, ADAR2 (ADARB1) , ADAR3 (ADARB2) , or the deaminase domain thereof, or a mutant thereof.
  • ADAR RNA-specific adenosine deaminase
  • the ADAR is human ADAR or ADAR of non-human species. In some embodiments, the ADAR is octopus ADAR.
  • the deaminase domain is human ADAR2 deaminase domain (hADAR2DD) .
  • the deaminase domain is a mutant of hADAR2DD, e.g., hADAR2DD-E488Q, hADAR2DD-E488Q+T375G, hADAR2DD-E488Q+V351G+S486A+T375S+S370C+P462A+N597I+L332I+I398V+K350I+M383L+D619G+S582 T+V440I+S495N+K418E+S661T, hADAR2DD-E488Q+V351G+S486A+T375A+S370C+P462A+N597I+L332I+I398V+K350I+M383L+D619G+S582 T+V440I+S495N+K418E+S661T.
  • the deaminase domain is apolipoprotein B mRNA editing enzyme, catalytic polypeptide (APOBEC) , such as, APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3E, APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, or the deaminase domain thereof, or a mutant thereof.
  • APOBEC catalytic polypeptide
  • the deaminase domain is activation-induced cytidine deaminase (AID, AICDA, single-stranded DNA cytosine deaminase) , or the deaminase domain thereof, or a mutant thereof.
  • AID activation-induced cytidine deaminase
  • AICDA single-stranded DNA cytosine deaminase
  • the napBP further comprises a transcription inhibiting domain (e.g., KRAB domain or SID domain) . In some embodiments, the napBP further comprises a KRAB domain.
  • a transcription inhibiting domain e.g., KRAB domain or SID domain
  • the napBP further comprises a KRAB domain.
  • the napBP further comprises a DNA methyltransferase, such as, DNMT3l, DNMT3a.
  • the napBP further comprises a DNMT3l domain and a DNMT3a domain.
  • the napBP further comprises a DNMT3l domain, a DNMT3a domain, and a KRAB domain.
  • the napBP, the KRAB domain, the DNMT3l domain, and the DNMT3a domain are arranged from N-terminal to C-terminal.
  • the protospacer sequence is a stretch of about, at least about, or at most about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, or more contiguous nucleotides on the nontarget strand of the DMD gene, or a stretch of contiguous nucleotides on the nontarget strand of the DMD gene in a numerical range between any two of the preceding values, e.g., a stretch of fromabout 16 to about 50 contiguous nucleotides.
  • the protospacer sequence is a stretch of about 20, 30, or 50 contiguous nucleotides on the nontarget strand of the DMD gene
  • the protospacer sequence on the nontarget strand of the DMD gene corresponding to the target sequence is immediately 3’ to PAM of 5’-NTN-3’, wherein N is A, T, G, or C.
  • the protospacer sequence on the nontarget strand of the DMD gene corresponding to the target sequence is immediately 5’ to PAM of 5’-NGG-3’, wherein N is A, T, G, or C.
  • the protospacer sequence on the nontarget strand of the DMD gene corresponding to the target sequence is immediately 5’ to PAM of 5’-NNNGAN-3’, wherein N is A, T, G, or C.
  • the protospacer sequence is a stretch of about 20, 30, or 50 contiguous nucleotides of the nontarget strand of the DMD gene immediately 3’ to PAM of 5’-NTN-3’, wherein N is A, T, G, or C.
  • the protospacer sequence is a stretch of about 20, 30, or 50 contiguous nucleotides of the nontarget strand of the DMD gene immediately 5’ to PAM of 5’-NGG-3’, wherein N is A, T, G, or C.
  • the protospacer sequence is a stretch of about 20, 30, or 50 contiguous nucleotides of the nontarget strand of the DMD gene immediately 5’ to PAM of 5’-NNNGAN-3’, wherein N is A, T, G, or C.
  • the target sequence is a stretch of contiguous nucleotides identified from the target strand of the DMD gene or from the transcript thereof.
  • the target sequence is a stretch of about, at least about, or at most about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, or more contiguous nucleotides on the target strand of the DMD gene or on the transcript thereof, or a stretch of contiguous nucleotides on the target strand of the DMD gene or on the transcript thereof in a numerical range between any two of the preceding values, e.g., a stretch of from about 16 to about 50 contiguous nucleotides.
  • the target sequence is a stretch of about 20, 30, or 50 contiguous nucleotides on the target strand of the
  • the nontarget strand is the sense strand of the DMD gene.
  • the nontarget strand is the antisense strand of the DMD gene.
  • the target strand is the sense strand of the DMD gene.
  • the target strand is the antisense strand of the DMD gene.
  • the protospacer sequence on the nontarget strand of the DMD gene corresponding to the target sequence or target sequence on the target strand of the DMD gene is located at or within an exon of the DMD gene or transcript thereof, or at or within a splice donor or a splice acceptor of the exon.
  • the exon is selected from the group consisting of Exon 43, Exon 44, Exon 45, Exon 46, Exon 51, Exon 53, Exon 55.
  • the DMD gene is human DMD gene, non-human primate DMD gene, or mouse DMD gene.
  • the DMD gene is in a eukaryotic cell, for example, a human cell, a non-human primate cell, or a mouse cell.
  • the DMD gene is in a muscle cell, such as, a myocardial cell, a diaphragm muscle, a tibialis anterior muscle cell.
  • the guide sequence is in a length of about, at least about, or at most about 14 nucleotides, e.g., about, at least about, or at most about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, or more nucleotides, or in a length of nucleotides in a numerical range between any two of the preceding values, e.g., in a length of from about 16 to about 50 nucleotides. In some embodiments, the guide sequence is in a length of about 20, 30, or 50 nucleotides.
  • the guide sequence is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (fully) , optionally about 100% (fully) , reversely complementary to the target sequence; (2) the guide sequence contains no more than 5, 4, 3, 2, or 1 mismatch or contains no mismatch with the target sequence; or (3) the guide sequence comprises no mismatch with the target sequence in the first 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, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 nucleotides at the 5’ end of the guide sequence when the PAM is immediately 5’ to the protospacer sequence or at the
  • the guide sequence contains 1 mismatch with the target sequence. In some embodiments, the guide sequence is about98%reversely complementary to the target sequence. In some embodiments, the 1 mismatch in the guide sequence is at a position corresponding the nucleotide of the target sequence that is intended to be substituted.
  • the protospacer sequence, the target sequence, or the guide sequence is selected according to the editing efficiency of the napDNAn guided to the DMD gene or transcript by the guide sequence.
  • the editing efficiency can be represented by indels %and measured by sequencing of the edited DMD gene. The higher the editing efficiency is, the more suitable the guide sequence is.
  • the protospacer sequence, the target sequence, or the guide sequence is selected according to the level of transcription of the DMD gene and/or level of translation of a transcript (e.g., mRNA) of the DMD gene (level of expression of the DMD gene) in in vivo animal model.
  • a transcript e.g., mRNA
  • level of expression of the DMD gene level of expression of the DMD gene
  • the level of the transcript (e.g., mRNA) of the DMD gene is increased in a cell model (e.g., HEK293T cell model) or an animal model (e.g., a mouse model, a non-human primate model) by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or more, upon administration of the systemof the disclosure to the cell model or the animal model, compared to the level of the transcript (e.g., mRNA) of the DMD gene in the same cell model or animal model that does not receive the administration.
  • a cell model e.g., HEK293T cell model
  • an animal model e.g., a mouse model, a non-human primate model
  • the level of the expression product (e.g., dystrophin) of the DMD gene is increased in a cell model (e.g., HEK293T cell model) or an animal model (e.g., a mouse model, a non-human primate model) by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or more, upon administration of the system of the disclosure to the cell or the animal model, compared to the level of the expression product (e.g., dystrophin) of the DMD gene in the same cell model or animal model that does not receive the administration.
  • the expression product is a functional mutant of the expression product (e.g., dystrophin) of the DMD gene.
  • the guide nucleic acid comprises a scaffold sequence 5’ to a guide sequence. In some embodiments, the guide nucleic acid comprises a scaffold sequence 3’ to a guide sequence.
  • the guide nucleic acid comprises one scaffold sequence and one guide sequence.
  • the guide nucleic acid comprises one scaffold sequence 5’ to one guide sequence. In some embodiments, the guide nucleic acid comprises one scaffold sequence 3’ to one guide sequence.
  • the guide nucleic acid comprises one or more scaffold sequence and/or one or more guide sequence, provided that the guide nucleic acid does not comprise one scaffold sequence and one guide sequence.
  • the guide nucleic acid comprises, from 5’ to 3’, one scaffold sequence, one guide sequence, and one scaffold sequence, wherein scaffold sequences are the same or different.
  • the guide nucleic acid comprises, from 5’ to 3’, one guide sequence, one scaffold sequence, and one guide sequence, wherein guide sequences are the same or different.
  • the guide nucleic acid comprises, from 5’ to 3’, one scaffold sequence, one guide sequence, one scaffold sequence, and one guide sequence, wherein scaffold sequences are the same or different, and wherein guide sequences are the same or different.
  • the guide nucleic acid comprises, from 5’ to 3’, one guide sequence, one scaffold sequence, one guide sequence, and one scaffold sequence, wherein scaffold sequences are the same or different, and wherein guide sequences are the same or different.
  • the guide nucleic acid comprises, from 5’ to 3’, one scaffold sequence, one guide sequence, one scaffold sequence, one guide sequence, and one scaffold sequence, wherein scaffold sequences are the same or different, and wherein guide sequences are the same or different.
  • the guide nucleic acid comprises, from 5’ to 3’, one guide sequence, one scaffold sequence, one guide sequence, one scaffold sequence, and one guide sequence, wherein scaffold sequences are the same or different, and wherein guide sequences are the same or different.
  • the guide nucleic acid comprises, from 5’ to 3’, one scaffold sequence, one guide sequence, one scaffold sequence, one guide sequence, one scaffold sequence, and one guide sequence, wherein scaffold sequences are the same or different, and wherein guide sequences are the same or different.
  • the guide nucleic acid comprises, from 5’ to 3’, one guide sequence, one scaffold sequence, one guide sequence, one scaffold sequence, one guide sequence, and one scaffold sequence, wherein scaffold sequences are the same or different, and wherein guide sequences are the same or different.
  • the guide nucleic acid comprises a linker or no linker between any adjacent scaffold sequence and guide sequence. In some embodiments, the guide nucleic acid comprises no linker between any adjacent scaffold sequence and guide sequence.
  • the systemor rAAV vector genome of the disclosure may comprise or encode one guide nucleic acid or comprise or encode multiple (e.g., 2, 3, 4, or more) guide nucleic acids, e.g., for the purpose of improving the editing efficiency of the system.
  • the system further comprises one or more additional guide nucleic acids, or the first polynucleotide sequence further comprises one or more additional sequences encoding one or more additional guide nucleic acids, each of the additional guide nucleic acids comprising:
  • an additional guide sequence capable of hybridizing to an additional target sequence on a target strand of the DMD gene or an additional target sequence on the transcript thereof, thereby guiding the complex to the DMD gene or the transcript.
  • the additional protospacer sequence is on the same strand as the protospacer sequence.
  • the additional protospacer sequence is on the different strand from the protospacer sequence.
  • the additional protospacer sequence is the same or different from the protospacer sequence.
  • the additional target sequence is the same or different from the target sequence.
  • the additional guide sequence is the same or different from the guide sequence.
  • the additional scaffold sequence is the same or different from the scaffold sequence.
  • the scaffold sequences of the multiple guide nucleic acids may be the same or different (e.g., different by no more than 5, 4, 3, 2, or 1 nucleotide) to be compatible to the same napBP.
  • the systemcomprises different napBP e.g., a Cas12i and a Cas9
  • the scaffold sequences of the multiple guide nucleic acids may be different to be compatible to the different napBP, respectively.
  • the additional guide nucleic acid and the guide nucleic acid are operably linked to or under the regulation of the same regulatory element (e.g., promoter) or separate regulatory elements (e.g., separate promoters) .
  • the guide nucleic acid (e.g., the guide nucleic acid, the additional guide nucleic acid) is an RNA, i.e., a guide RNA (gRNA) .
  • the guide nucleic acid is an unmodified guide RNA.
  • the guide nucleic acid is a modified guide RNA.
  • the guide nucleic acid comprises a modification.
  • the guide nucleic acid is a modified RNA containing a modified ribonucleotide.
  • the guide nucleic acid is a modified RNA containing a deoxyribonucleotide.
  • the guide nucleic acid is a modified RNA containing a modified deoxyribonucleotide. In some embodiments, the guide nucleic acid comprises a modified or unmodified deoxyribonucleotide and a modified or unmodified ribonucleotide.
  • the guide sequence or the additional guide sequence comprises (1) a sequence of any one of SEQ ID NOs: 1-51 or a 5’ or 3’ end truncation thereof with 1, 2, 3, 4, 5, or 6, nucleotides truncated at the 5’ or 3’ end; or (2) a sequence having a sequence identity of at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100%to any one of SEQ ID NOs: 1-51 or a 5’ or 3’ end truncation thereof with 1, 2, 3, 4, 5, or 6 nucleotides truncated at the 5’ or 3’ end; or (3) a sequence having at most 1, 2, 3, 4, 5, or 6 nucleotide differences, whether consecutive or not, compared to any one of SEQ ID NOs: 1-51.
  • the guide sequence or the additional guide sequence comprises a sequence of SEQ ID NO: 36.
  • the scaffold sequence is compatible with the napBP of the disclosure and is capable of complexing with the napBP.
  • the scaffold sequence may be a naturally occurring scaffold sequence identified along with the napBP, or a variant thereof maintaining the ability to complex with the napBP.
  • the ability to complex with the napBP is maintained as long as the secondary structure of the variant is substantially identical to the secondary structure of the naturally occurring scaffold sequence.
  • a nucleotide deletion, insertion, or substitution in the primary sequence of the scaffold sequence may not necessarily change the secondary structure of the scaffold sequence (e.g., the relative locations and/or sizes of the stems, bulges, and loops of the scaffold sequence do not significantly deviate from that of the original stems, bulges, and loops) .
  • nucleotide deletion, insertion, or substitution may be in a bulge or loop region of the scaffold sequence so that the overall symmetry of the bulge and hence the secondary structure remains largely the same.
  • the nucleotide deletion, insertion, or substitution may also be in the stems of the scaffold sequence so that the lengths of the stems do not significantly deviate from that of the original stems (e.g., adding or deleting one base pair in each of two stems correspond to 4 total base changes) .
  • the scaffold sequence or the additional scaffold sequence has substantially the same secondary structure as the secondary structure of the sequence of SEQ ID NO: 52 or 53.
  • the scaffold sequence or the additional scaffold sequence comprises (1) a sequence of SEQ ID NO: 52 or 53 or a 5’ or 3’ end truncation thereof with 1, 2, 3, 4, 5, or 6, nucleotides truncated at the 5’ or 3’ end; or (2) a sequence having a sequence identity of at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100%to SEQ ID NO: 52 or 53 or a 5’ or 3’ end truncation thereof with 1, 2, 3, 4, 5, or 6 nucleotides truncated at the 5’ or 3’ end; or (3) a sequence having at most 1, 2, 3, 4, 5, or 6 nucleotide differences, whether consecutive or not, compared to SEQ ID NO: 52 or 53.
  • the scaffold sequence or the additional scaffold sequence comprises the sequence of SEQ ID NO:53.
  • the guide nucleic acid comprises a sequence having a sequence identity of at least about 80%(e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of any one of SEQ ID NOs: 71-125; or a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide differences, whether consecutive or not, compared to the sequence of any one of SEQ ID NOs: 71-125.
  • the guide nucleic acid comprises a sequence of any one of SEQ ID NOs: 71-74.
  • Also provided in the disclosure is a polynucleotide comprising or encoding the guide nucleic acid.
  • the polynucleotide comprising or encoding the guide nucleic acid is a DNA, a RNA, or a DNA/RNA mixture.
  • DNA/RNA mixture it refers to a nucleic acid comprising both one or more modified or unmodified ribonucleotides and one or more modified or unmodified deoxyribonucleotides, whether consecutive or not.
  • DNA or RNA it may also refer to a DNA containing one or more modified or unmodified ribonucleotides, whether consecutive or not, or an RNA containing one or more modified or unmodified deoxyribonucleotides, whether consecutive or not.
  • the guide nucleic acid is operably linked to or under the regulation of a promoter.
  • the promoter comprises a sequence having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NO: 59; or a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide differences, whether consecutive or not, compared to the sequence of SEQ ID NO: 59.
  • the polynucleotide encoding the napBP is a DNA, a RNA, or a DNA/RNA mixture.
  • DNA/RNA mixture it refers to a nucleic acid comprising both one or more modified or unmodified ribonucleotides and one or more modified or unmodified deoxyribonucleotides, whether consecutive or not.
  • DNA or RNA it may also refer to a DNA containing one or more modified or unmodified ribonucleotides, whether consecutive or not, or an RNA containing one or more modified or unmodified deoxyribonucleotides, whether consecutive or not.
  • the polynucleotide encoding the napBP is operably linked to or under the regulation of a promoter.
  • the second polynucleotide sequence comprises a promoter operably linked to the sequence encoding the napBP.
  • Suitable promoters include, for example, a Cbh promoter, a Cba promoter, a pol I promoter, a pol II promoter, a pol III promoter, a T7 promoter, a U6 promoter, a H1 promoter, a retroviral Rous sarcoma virus LTR promoter, a cytomegalovirus (CMV) promoter, a SV40 promoter, a dihydrofolate reductase promoter, a ⁇ -actin promoter, an elongation factor 1 ⁇ short (EFS) promoter, a ⁇ glucuronidase (GUSB) promoter, a cytomegalovirus (CMV) immediate-early (Ie) enhancer and/or promoter, a chicken ⁇ -actin (CBA) promoter or derivative thereof such as a CAG promoter, CB promoter, a (human) elongation factor 1 ⁇ -subunit (EF1 ⁇
  • the promoter is a muscle specific promoter.
  • the promoter is a EFS promoter.
  • the second polynucleotide sequence comprises a Kozak sequence (gccacc) 5’ to the sequence encoding the napBP.
  • the second polynucleotide sequence comprises a sequence encoding a nuclear localization signal (NLS) 5’ a nd/or 3’ to the sequence encoding the napBP.
  • NLS nuclear localization signal
  • the second polynucleotide sequence comprises a sequence encoding a nuclear export signal (NES) 5’ a nd/or 3’ to the sequence encoding the napBP.
  • NES nuclear export signal
  • the second polynucleotide sequence comprises a sequence encoding a first NLS 5’ to the sequence encoding the napBP and a second sequence encoding a second NLS 3’ to the sequence encoding the napBP.
  • the NLS, the first NLS, and/or the second NLS is a SV40 NLS, a bpSV40 NLS (SEQ ID NO: 62) , or a Nucleoplasmin NLS (npNLS) (SEQ ID NO: 65) .
  • the napBP comprises a bpSV40 NLS at the N-terminal of the napBP and a npNLS at the C-terminal of the napBP.
  • the second polynucleotide sequence comprises a WPRE sequence downstream of the sequence encoding the napBP.
  • the WPRE sequence is selected from the group consisting of Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE) , WPRE3 (ashortened WPRE) , and a functional variant (e.g., a functional truncation) thereof.
  • WPRE Woodchuck Hepatitis Virus
  • WPRE3 ashortened WPRE
  • a functional variant e.g., a functional truncation
  • the second polynucleotide sequence comprises a sequence encoding a polyadenylation (polyA) signal downstream of the sequence encoding the napBP.
  • polyA polyadenylation
  • the second polynucleotide sequence comprises, downstream of the sequence encoding the napBP, a WPRE sequence followed by a sequence encoding a polyadenylation (polyA) signal.
  • polyA polyadenylation
  • the polyAsignal is selected from a group consisting of a bovine growth hormone polyadenylation (bGH polyA) signal, a small polyA (SPA) signal, a human growth hormone polyadenylation (hGH polyA) signal, a SV40 polyA (SV40 polyA) signal, a rabbit beta globin polyA (rBG polyA) signal, a combination of SV40 late polyadenylation signal upstream element and SV40 late polyadenylation signal, and a functional variant (e.g., a functional truncation) thereof.
  • bGH polyA bovine growth hormone polyadenylation
  • SPA small polyA
  • hGH polyA human growth hormone polyadenylation
  • SV40 polyA SV40 polyA
  • rBG polyA rabbit beta globin polyA
  • the polyA signal is a combination of SV40 late polyadenylation signal upstream element and SV40 late polyadenylation signal.
  • the second polynucleotide sequence comprises, from 5’ to 3’, the promoter, the Kozak sequence, the first sequence encoding the first NLS, the sequence encoding the napBP, the second sequence encoding the second NLS, the WPRE sequence, and the sequence encoding the polyA signal.
  • the promoter comprises a sequence encoding a polypeptide having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NO: 60; or a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide differences, whether consecutive or not, compared to the sequence of SEQ ID NO: 60.
  • the Kozak sequence is gccacc.
  • the NLS, the first NLS, and/or the second NLS comprises a sequence encoding a polypeptide having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NO: 62 or 65; or a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid differences, whether consecutive or not, compared to the sequence of SEQ ID NO: 62 or 65.
  • the sequence encoding the NLS, the first sequence encoding the first NLS, and/or the second sequence encoding the second NLS comprises a sequence encoding a polypeptide having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NO: 61 or 64; or a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide differences, whether consecutive or not, compared to the sequence of SEQ ID NO: 61 or 64.
  • the WPRE sequence comprises a sequence encoding a polypeptide having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NO: 66; or a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide differences, whether consecutive or not, compared to the sequence of SEQ ID NO: 66.
  • the sequence encoding the polyA signal comprises a sequence encoding a polypeptide having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NO: 67; or a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide differences, whether consecutive or not, compared to the sequence of SEQ ID NO: 67.
  • the second polynucleotide sequence comprises, from 5’ to 3’, the promoter of SEQ ID NO: 60, the Kozak sequence of gccacc, an in-frame start codon ATG, the first sequence encoding the first NLS of SEQ ID NO: 62, the sequence encoding the napBP of SEQ ID NO: 57, the second sequence encoding the second NLS of SEQ ID NO: 65, an in-frame stop codon, the WPRE sequence of SEQ ID NO: 66, and the sequence encoding the polyA signal of SEQ ID NO: 67.
  • the second polynucleotide sequence comprises, from 5’ to 3’, the promoter of SEQ ID NO: 60, the Kozak sequence of gccacc, an in-frame start codon ATG, the first sequence of SEQ ID NO: 61 encoding the first NLS, the sequence of SEQ ID NO: 63 encoding the napBP, the second sequence of SEQ ID NO: 64 encoding the second NLS, an in-frame stop codon, the WPRE sequence of SEQ ID NO: 66, and the sequence encoding the polyA signal of SEQ ID NO: 67.
  • the rAAV vector genome comprises a 5’ inverted terminal repeat (ITR) sequence and a 3’ ITR sequence.
  • the 5’ ITR sequence and the 3’ ITR sequence are both wild-type ITR sequences from AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-DJ, AAV PHP. eB, or a member of the Clade to which any of the AAV1-AAV13 belong, or a functional variant (e.g., a functional truncation) thereof.
  • the 5’ ITR sequence comprises a sequence having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NO: 58.
  • the 3’ ITR sequence comprises a sequence having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NO: 68.
  • the rAAV vector genome comprises, from 5’ to 3’, the first polynucleotide sequence, and the second polynucleotide sequence.
  • the rAAV vector genome comprises, from 5’ to 3’, the second polynucleotide sequence, and the first polynucleotide sequence.
  • the rAAV vector genome comprises, from the 5’ to 3’,
  • the rAAV vector genome comprises a sequence having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the sequence of SEQ ID NO: 69.
  • the rAAV vector genome comprises, from the 5’ to 3’,
  • the rAAV vector genome comprises a sequence having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the sequence of SEQ ID NO: 69;
  • rAAV vector genome does not contain any nucleotide difference from the sequence of SEQ ID NO: 69 in any one of the components (1) to (18) ;
  • rAAV vector genome contains one or more nucleotide difference or does contain any nucleotide difference from the sequence of SEQ ID NO: 69 between any two adjacent components of the components (1) to (18) .
  • the rAAV vector genome comprises the sequence of SEQ ID NO: 69.
  • the rAAV vector genome of the disclosure is a DNA rAAV vector genome or an RNA rAAV vector genome.
  • DNA rAAV vector genome it means that the rAAV vector genome is a DNA that can be encapsulated into a rAAV particle.
  • RNA rAAV vector genome it means that the rAAV vector genome is an RNA that can be encapsulated into a rAAV particle.
  • the disclosure provides a recombinant AAV (rAAV) particle comprising the rAAV vector genome of the disclosure.
  • a simple introduction of AAV for delivery may refer to “Adeno-associated Virus (AAV) Guide” (addgene. org/guides/aav/) .
  • Adeno-associated virus when engineered to delivery, e.g., a protein-encoding sequence of interest, may be termed as a (r) AAV vector, a (r) AAV vector particle, or a (r) AAV particle, where “r” stands for “recombinant” .
  • the genome packaged in AAV vectors for delivery may be termed as a (r) AAV vector genome, vector genome, or vg for short, while viral genome may refer to the original viral genome of natural AAVs.
  • the serotypes of the capsids of rAAV particles can be matched to the types of target cells.
  • Table 2 of WO2018002719A1 lists exemplary cell types that can be transduced by the indicated AAV serotypes (incorporated herein by reference) .
  • the rAAV particle comprising a capsid with a serotype suitable for delivery into muscle cells.
  • the rAAV particle comprising a capsid with a serotype of AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-DJ, or AAV. PHP. eB, a member of the Clade to which any of the AAV1-AAV13 belong, or a functional variant (e.g., a functional truncation) thereof, encapsidating the rAAV vector genome.
  • the serotype of the capsid is wild type AAV9 or a functional variant thereof.
  • rAAV particles may be produced using the triple transfection method (described in detail in U.S. Pat. No. 6,001,650) .
  • the vector titers are usually expressed as vector genomes per ml (vg/ml) .
  • the vector titer is above 1 ⁇ 10 9 , above 5 ⁇ 10 10 , above 1 ⁇ 10 11 , above 5 ⁇ 10 11 , above 1 ⁇ 10 12 , above 5 ⁇ 10 12 , or above 1 ⁇ 10 13 vg/ml.
  • RNA sequence as a vector genome into a rAAV particle
  • systems and methods of packaging an RNA sequence as a vector genome into a rAAV particle is recently developed and applicable herein. See PCT/CN2022/075366, which is incorporated herein by reference in its entirety.
  • sequence elements described herein for DNA vector genomes when present in RNA vector genomes, should generally be considered to be applicable for the RNA vector genomes except that the deoxyribonucleotides in the DNA sequence are the corresponding ribonucleotides in the RNA sequence (e.g., dT is equivalent to U, and dA is equivalent to A) and/or the element in the DNA sequence is replaced with the corresponding element with a corresponding function in the RNA sequence or omitted because its function is unnecessary in the RNA sequence and/or an additional element necessary for the RNA vector genome is introduced.
  • dT is equivalent to U
  • dA is equivalent to A
  • a coding sequence e.g., as a sequence element of rAAV vector genomes herein, is construed, understood, and considered as covering and covers both a DNA coding sequence and an RNA coding sequence.
  • an RNA sequence can be transcribed from the DNA coding sequence, and optionally further a protein can be translated from the transcribed RNA sequence as necessary.
  • the RNA coding sequence per se can be a functional RNA sequence for use, or an RNA sequence can be produced from the RNA coding sequence, e.g., by RNA processing, or a protein can be translated from the RNA coding sequence.
  • a Cas13 coding sequence encoding a Cas13 polypeptide covers either a Cas13 DNA coding sequence from which a Cas13 polypeptide is expressed (indirectly via transcription and translation) or a Cas13 RNA coding sequence from which a Cas13 polypeptide is translated (directly) .
  • a gRNA coding sequence encoding a gRNA covers either a gRNA DNA coding sequence from which a gRNA is transcribed or a gRNA RNA coding sequence (1) which per se is the functional gRNA for use, or (2) from which a gRNA is produced, e.g., by RNA processing.
  • 5’-ITR and/or 3’-ITR as DNA packaging signals may be unnecessary and can be omitted at least partly, while RNA packaging signals can be introduced.
  • a promoter to drive transcription of DNA sequences may be unnecessary and can be omitted at least partly.
  • a sequence encoding a polyAsignal may be unnecessary and can be omitted at least partly, while a polyAtail can be introduced.
  • DNA elements of rAAV DNA vector genomes can be either omitted or replaced with corresponding RNA elements and/or additional RNA elements can be introduced, in order to adapt to the strategy of delivering an RNA vector genome by rAAV particles.
  • the disclosure provides a method for production of the rAAV particle of the disclosure, comprising culturing in a host cell a transgene plasmid comprising the rAAV vector genome of the disclosure. In yet another aspect, the disclosure provides a cell comprising a transgene plasmid comprising the rAAV vector genome of the disclosure.
  • the disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising (1) the system of the disclosure or the rAAV particle of the disclosure and (2) a pharmaceutically acceptable excipient.
  • the pharmaceutical composition comprises the rAAV particle in a concentration selected from the group consisting of about 1 ⁇ 10 10 vg/mL, 2 ⁇ 10 10 vg/mL, 3 ⁇ 10 10 vg/mL, 4 ⁇ 10 10 vg/mL, 5 ⁇ 10 10 vg/mL, 6 ⁇ 10 10 vg/mL, 7 ⁇ 10 10 vg/mL, 8 ⁇ 10 10 vg/mL, 9 ⁇ 10 10 vg/mL, 1 ⁇ 10 11 vg/mL, 2 ⁇ 10 11 vg/mL, 3 ⁇ 10 11 vg/mL, 4 ⁇ 10 11 vg/mL, 5 ⁇ 10 11 vg/mL, 6 ⁇ 10 11 vg/mL, 7 ⁇ 10 11 vg/mL, 8 ⁇ 10 11 vg/mL, 9 ⁇ 10 11 vg/mL, 1 ⁇ 10 12 vg/mL, 2 ⁇ 10 12 vg/mL, 3 ⁇ 10 12 vg/
  • the pharmaceutical composition is an injection formulation, such as i. v. injection formulation.
  • the volume of the injection is selected from the group consisting of about 1 microliter, 10 microliters, 50 microliters, 100 microliters, 150 microliters, 200 microliters, 250 microliters, 300 microliters, 350 microliters, 400 microliters, 450 microliters, 500 microliters, 550 microliters, 600 microliters, 650 microliters, 700 microliters, 750 microliters, 800 microliters, 850 microliters, 900 microliters, 950 microliters, 1000 microliters, and a volume of a numerical range between any of two preceding values, e.g., in a concentration of from about 10 microliters to about 750 microliters.
  • the disclosure provides a cell or a progeny thereof comprising the guide nucleic acid of the disclosure, the system of the disclosure, or the rAAV particle of the disclosure.
  • the cell is a eukaryote.
  • the cell is a human cell.
  • the cell is a human muscle cell.
  • the disclosure provides a cell or a progeny thereof comprising DMD gene or transcript thereof modified by the system of the disclosure, the rAAV particle of the disclosure, or the method of the disclosure.
  • the cell is a eukaryote.
  • the cell is a human cell.
  • the cell is a human muscle cell.
  • the cell is not within the body of an organism, such as, human or animal. In some embodiments, the cell is not a human embryonic stem cell. In some embodiments, the cell is not a human germ cell.
  • the disclosure provides a method for preventing, diagnosing, and/or treating a DMD associated disease in a subject in need thereof, comprising administering to the subject the system of the disclosure, the rAAV particle of the disclosure, or the pharmaceutical composition of the disclosure, wherein the napBP modifies DMD gene or transcript thereof, and wherein the modification of the DMD gene or transcript thereof treats the disease.
  • the DMD associated disease is Duchenne muscular dystrophy.
  • DMD gene is in a eukaryotic cell, for example, a human cell, a non-human primate cell, or a mouse cell, such as, a human muscle cell.
  • the administrating comprises local administration or systemic administration.
  • the administrating comprises intrathecal administration, intramuscular administration, intravenous administration, transdermal administration, intranasal administration, oral administration, mucosal administration, intraperitoneal administration, intracranial administration, intracerebroventricular administration, or stereotaxic administration.
  • the administration is injection or infusion.
  • the subject is a human, a non-human primate, or a mouse.
  • the level of the transcript (e.g., mRNA) of the DMD gene is increased in the subject by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or more compared to the level of the transcript (e.g., mRNA) of the DMD gene in the subject prior to the administration.
  • the level of the expression product (e.g., dystrophin) of the DMD gene is increased in the subject by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or more compared to the level of the expression product (e.g., dystrophin) of the DMD gene in the subject prior to the administration.
  • the expression product is a functional mutant of the expression product of the DMD gene.
  • the median survival of the subject suffering from the disease but receiving the administration is 5 days, 10 days, 20 days, 30 days, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1.5 year, 2 years, 2.5 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or more longer than that of a subject or a population of subjects suffering from the disease and not receiving the administration.
  • the dose of the rAAV particle for treatment of the DMD associated diseases may be either via a single dose, or multiple doses.
  • the actual dose may vary greatly depending upon a variety of factors, such as the vector choices, the target cells, organisms, tissues, the general conditions of the subject to be treated, the degrees of transformation/modification sought, the administration routes, the administration modes, the types of transformation/modification sought, etc.
  • the rAAV particle is administrated in a therapeutically effective dose.
  • the therapeutically effective dose of the rAAV particle may be about 1.0E+8 (1.0 ⁇ 10 8 ) , 2.0E+8, 3.0E+8, 4.0E+8, 6.0E+8, 8.0E+8, 1.0E+9, 2.0E+9, 3.0E+9, 4.0E+9, 6.0E+9, 8.0E+9, 1.0E+10, 2.0E+10, 3.0E+10, 4.0E+10, 6.0E+10, 8.0E+10, 1.0E+11, 2.0E+11, 3.0E+11, 4.0E+11, 6.0E+11, 8.0E+11, 1.0E+12, 2.0E+12, 3.0E+12, 4.0E+12, 6.0E+12, 8.0E+12, 1.0E+13, 2.0E+13, 3.0E+13, 4.0E+13, 6.0E+13, 8.0E+13, 1.0E+14, 2.0E+14, 3.
  • the disclosure provides a kit comprising the system of the disclosure, the rAAV particle of the disclosure, or the pharmaceutical composition of the disclosure, or any one, two, or all components of the same.
  • the kit further comprises an instruction to use the component (s) contained therein, and/or instructions for combining with additional component (s) that may be available or necessary elsewhere.
  • the kit further comprises one or more buffers that may be used to dissolve any of the component (s) contained therein, and/or to provide suitable reaction conditions for one or more of the component (s) .
  • buffers may include one or more of PBS, HEPES, Tris, MOPS, Na 2 CO 3 , NaHCO 3 , NaB, or combinations thereof.
  • the reaction condition includes a proper pH, such as a basic pH. In some embodiments, the pH is between 7-10.
  • any one or more of the kit components may be stored in a suitable container or at a suitable temperature, e.g., 4 degree Celsius.
  • mice All applicable institutional and/or national guidelines for the care and use of animals were followed. All experimental procedures on the mice were approved by the institute committee of Animal Care and Use (IACUC) of HuidaGene Therapeutics. All mice were housed in a constant temperature (24-26°C) and humidity (40-60%) room with a 12-hour light-dark cycle, and fed with the standard food.
  • IACUC Animal Care and Use
  • the HEK293T cells from American Type Culture Collection were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (#11965092, Gibco) supplemented with 10%fetal bovine serum (#04-001-1ACS, Biological Industries) and 1%penicillin/streptomycin (#15140122, Thermo Fisher Scientific) .
  • DMEM Dulbecco’s modified Eagle’s medium
  • fetal bovine serum #04-001-1ACS, Biological Industries
  • penicillin/streptomycin #15140122, Thermo Fisher Scientific
  • HEK293T cells were seeded on 12-well culture plates (#3513, Corning) at the same amount.
  • the expressing plasmid was then transfected using the polyethylenimine (PEI) reagent (#101000029, Polyplus) .
  • PEI polyethylenimine
  • Human DMD fibroblasts were reprogrammed into DMD iPSCs using the CytoTune-iPS Sendai Reprogramming Kit (#A16517, Thermo Fisher Scientific) .
  • Human DMD iPSCs were plated in cell-culture dishes coated with matrigel (#354277, Corning) and grew in the ncTarget medium (#RP01020, Nuwacell Biotechnologies) at 37 °C, 5%CO 2 .
  • the iPSCs were passaged at 80%confluence using hPSC Dissociation Buffer (#RP01007, Nuwacell Biotechnologies) .
  • human DMD iPSCs were treated with 10 mM ROCK inhibitor Y-27632 (#10005583, Cayman) .
  • the iPSCs were dissociated into single cells with Accutase (#7920, STEMCELL Technologies) .
  • nucleofection buffer #RP01005, Nuwacell Biotechnologies
  • the nucleofection process was performed with Lonza 2B Nucleofector, employing program B016.48 hours later, fluorescence-positive cells were sorted out by BD FACSAria TM III Sorter.
  • gRNA screening 5 ⁇ 10 3 cells were collected, and their lysis was amplified with different primer sets (Table S2) .
  • Table S2 For obtaining single iPSC clone, the cells were immediately seeded on matrigel-coated 100-mm culture dish (#430167, Corning) and maintained in the ncTarget medium with 10 mM Y-27632. After seven days, single cell colony was picked and transferred to the 12-well culture plate. After being subjected to genome sequencing, the desired cells were expanded in the ncTarget medium.
  • the AAV9 particles were produced by PackGene Biotech (Guangzhou, China) .
  • the pHelper, pRepCap, and transgene (GOI) plasmids at the ratio 2: 1: 1 was co-transfected into host cells when the confluency was between 70–90%.
  • the iodixanol density gradient centrifugation was used to purify AAV9 particles.
  • mice were anesthetized and their TA muscles were injected with AAV9 particles at the dose of 2.5 ⁇ 10 11 vg/leg/AAV or with the same volume saline solution.
  • mice were administrated with AAV9 particles at the dose of 2.5 ⁇ 10 13 vg/kg or with saline solution.
  • AAV9 particles For tail vein injection, 2-weeks old mice were administrated with AAV9 particles at the dose of 5 ⁇ 10 13 vg/kg or with saline solution.
  • Mouse HE (heart) , DI (diaphragm) , and TA (tibialis anterior) muscles were isolated at indicated time points and then cut into small pieces for further experiments.
  • Total mRNAs were isolated from mouse tissues by TRIzol reagent (#15596-018, Ambion) according to the manufacturer’s instructions. The mRNAs were reverse transcribed into cDNAs with HiScript II One Step RT-PCR Kit (#P611-01, Vazyme) . The cDNAs were amplified using Phanta max super-fidelity DNA polymerase and performed with Sanger and deep sequencing to analyze editing efficiency.
  • dystrophin #D8168, Sigma
  • vinculin #13901S, Cell Signaling Technology
  • mouse tissues were embedded in the O. C. T. compound and then snap-frozen in liquid nitrogen-cooled isopentane. They were cut into 10 ⁇ m sections with a microtome and transferred onto the slides. The sections were fixed in 4%paraformaldehyde for 2 hours and permeabilized with 0.4% Triton-X/PBS for 30 mins at RT. After being blocked in 10%goat serum/PBS, the sections were probed with primary antibody against dystrophin (#ab15277, Abcam) at 4°C overnight. Following three washes, the sections were stained with secondary antibodies and DAPI for 3 hours at RT. After the wash in PBS, the coverslips were sealed with permanent synthetic mounting media. All pictures were observed and captured under an inverted Olympus FV3000 microscope.
  • mice were trained daily for a week prior to the experiment. Three mice were put simultaneously on the rotarod (Ugo Basile Inc. ) with an accelerating speed from 4 to 40 rpm over 30 seconds. When the mice fell off and onto the lever, the test was stopped and the time was recorded. Each mouse was tested five times and the average value of these five times was used for further comparison. The mice were trained daily for a week prior to the experiment. Three mice were put simultaneously on the rodent treadmill (Shanghai TOW Intelligent Technology Co., Ltd, AT-5MR) with an accelerating speed from 0 to 15 m/sover 30 seconds and recorded the running time before first falling.
  • rodent treadmill Shanghai TOW Intelligent Technology Co., Ltd, AT-5MR
  • mice were removed from the cage, weighed and held from the tip of the tail, causing the forelimbs to grasp the pull-bar assembly connected to the 47200-grip strength meter. The mouse was drawn along a straight line leading away from the sensor until the mouse could no longer grasp the gridiron and the peak amount of force in grams was recorded. The assessment was repeated 7-10 times with 10-second intervals between.
  • CK levels were measured in an Eppendorf tube via cardiac puncture under ketamine anesthesia prior to euthanasia. Samples were centrifuged at 3,000 ⁇ g for 10 min and then the serum was collected. CK activity was measured with creatine kinase (CK10) reagent (Pointe Scientific, 23-666-208) according to the manufacturer’s instructions.
  • CK10 creatine kinase
  • Quantitative data were derived from at least three independent experiments. The statistical significance of group differences was calculated by the unpaired Student's t-test between two groups among multiple groups. Differences in means were considered statistically significant when they reached P ⁇ 0.05. Significance levels are: *P ⁇ 0.05. **P ⁇ 0.01. ***P ⁇ 0.001.
  • Example 1 Screening of human DMD gene-targeting guide sequences by in vitro evaluation
  • This Example demonstrates the screening of human DMD gene-targeting guide sequences by in vitro evaluation of editing efficiency of a Cas12 endonuclease directed by such guide sequences to human DMD gene.
  • the expression plasmid comprised, from 5’ to 3’, a DMD-targeting gRNA expression cassette, a Cas12 expression cassette, and a mCherry expression cassette.
  • the DMD-targeting gRNA expression cassette comprised U6 promoter and a sequence encoding a DMD-targeting gRNA (one of SEQ ID NOs: 75-125; see Table 1) under the regulation of the U6 promoter.
  • the Cas12 expression cassette comprised, from 5’ to 3’, CBApromoter, Kozak sequence (gccacc) , start codon ATG, a sequence encoding 3xFlag tag, a sequence encoding SV40 NLS, a sequence encoding a Cas12 endonuclease (SEQ ID NO: 57; also named as “hfCas12Max” ) under the regulation of the CBA promoter, a sequence encoding npNLS (SEQ ID NO: 65) , and a sequence encoding bGH polyA signal.
  • the mCherry expression cassette comprised, from 5’ to 3’, CMV enhancer, CMV promoter, and a sequence encoding mCherry under the regulation of the CMV enhancer and the CMV promoter.
  • the Cas12 endonuclease and the DMD-targeting gRNA composed a CRISPR-Cas12 system targeting human DMD gene.
  • the DMD-targeting gRNA (one of SEQ ID NOs: 75-125; see Table 1) was composed of a scaffold sequence (direct repeat sequence) (SEQ ID NO: 52) capable of forming a complex with the Cas12 endonuclease (SEQ ID NO: 57) and a guide sequence (one of G1-G51 (SEQ ID NOs: 1-51; see Table 1) ) (3’ to the scaffold sequence) designed to be capable of hybridizing to human DMD gene.
  • the target site on the human DMD gene for each of the guide sequences G1-G51 is listed in Table 2.
  • SEQ ID NOs: 1-51 and 75-125 are denoted as RNA in the electronic sequencing of the disclosure, they denote both (1) the protospacer sequences on human DMD gene corresponding to the guide sequences and the gRNA coding sequences as DNA, respectively; and (2) the guide sequences and the gRNAs as RNA, respectively, in which case “t” denotes “u” .
  • a non-targeting guide sequence incapable of hybridizing to human DMD gene was used in place of the DMD-targeting guide sequence in the expression plasmid.
  • the experiment results show that for most of the DMD-targeting guide sequences G1-G51, high editing efficiency (indels %) (e.g., above 20%, 30%, or 40%) in vitro was achieved, making thempromising candidates for the development of DMD gene therapy.
  • the DMD-targeting guide sequence G36 (SEQ ID NO: 36) targeting SD of Exon 51 of human DMD gene that achieved significantly high editing efficiency (FIG. 8B and 8C) and low off-target editing (FIG. 8D and 8E) was selected for further testing in the subsequence Examples.
  • This Example demonstrates the in vivo editing efficiency of AAV-delivered CRISPR-Cas12 system containing the DMD-targeting guide sequence G36 (SEQ ID NO: 36) .
  • a rAAV transgene plasmid for packaging into wild type AAV9 capsid to prepare all-in-one rAAV9 particles was designed to comprise, from 5’ to 3’, the elements in Table 9 below, especially the three consecutive copies of gRNA (SEQ ID NO: 71) composed of scaffold sequence (SEQ ID NO: 53) 5’ to guide sequence G36 (SEQ ID NO: 36) .
  • the three consecutive copies of gRNA (SEQ ID NO: 71) may also be regarded as a single gRNA (SEQ ID NO: 74) with gRNA configuration of “DgDgDg” , wherein “D” denotes a direct repeat sequence (or a scaffold sequence) and “g” denotes a guide sequence.
  • the full length of the 5’ ITR-to-3’ ITR sequence of the transgene plasmid is set forth in SEQ ID NO: 69.
  • FIG. 7 shows the generation of the mouse model.
  • FIG. 7A shows sirius red staining and HE staining of TA, DI, and heart muscle of WT and ⁇ mE51E52, hE51KI mice.
  • FIG. 7B shows dystrophin immunohistochemistry from indicated muscles of WT and ⁇ mE51E52, hE51KI mice. Dystrophin and spectrin are shown in green and magenta, respectively.
  • the expression of dystrophin (FIG. 7B) in the ⁇ mE51E52, hE51KI mouse model was clearly lower than in the WT, and so was the significantly lower muscle performance (FIG. 7C) .
  • FIG. 9A shows overview for the in vivo intramuscular (IM) injection of rAAV particles into the TA muscle. Left leg was injected with saline as a control. Black arrows indicate time points for tissue collection after injection.
  • FIG. 9B-9C shows that dystrophin expression was rescued by intramuscular (IM) injection in tibialis anterior (TA) muscle 4-week post-injection of the rAAV9 particles with gRNA configurations “Dg” , “DgD” , “DgDgD” , or “DgDgDg” into the ⁇ mE51E52, hE51KI mouse model in a fixed dose of 2.5E11 vg (FIG. 9) .
  • FIG. 9A shows overview for the in vivo intramuscular (IM) injection of rAAV particles into the TA muscle. Left leg was injected with saline as a control. Black arrows indicate time points for tissue collection after injection.
  • FIG. 9B-9C shows that
  • Different rAAV particles with various gRNA configurations Dg, DgD, DgDgD, and DgDgDg having different combinations of Direct repeat (D) and guide sequence (g) ) were tested, showing that the rAAV particles with gRNA configuration “DgDgDg” achieved the best editing efficiency on both DNA and RNA editing levels.
  • “Productive editing” denotes that the introduction of indel mutation into DMD gene leads to expression of functional dystrophin (mutant) as needed.
  • Nonproductive editing denotes that the introduction of indel mutation into DMD gene does not lead to expression of functional dystrophin (mutant) as needed.
  • “Exon framed” denotes that the introduction of indel mutation into DMD gene generates frameshift mutation in the RNA transcribed from DMD gene that leads to expression of functional dystrophin (mutant) as needed.
  • “Exon skipping” denotes that the introduction of indel mutation into DMD gene generates a mutation in the RNA transcribed from DMD gene that leads to exon skipping of Exon 51 that leads to expression of functional dystrophin (mutant) as needed.
  • “Out of frame” denotes that the introduction of indel mutation into DMD gene generates nonsense mutation (stop codon) in the RNA transcribed fromDMD gene that pre-terminates translation of the transcribed RNA.
  • FIG. 9D shows western blot analysis of dystrophin and vinculin expression in TA muscles 4 weeks after injection with rAAV particles or saline, showing that the rAAV particles with gRNA configuration “DgDgDg” achieved the highest expression of dystrophin.
  • FIG. 9E shows comparison of dystrophin expression of different rAAV particles by immunofluorescence. Dystrophin is shown in green. Scale bar, 200 ⁇ m. Data are represented as mean ⁇ SEM.
  • FIG. 10A shows schematic of intraperitoneal injection of the rAAV particles into postnatal-day-3 (P3) ⁇ mE51E52, hE51KI mice in a dose of 2.5E13 vg/kg. Saline was injected as mock-treated controls. Black arrows indicate time points for tissue collection.
  • FIG. 10D shows western blot analysis, exhibiting restoration of dystrophin expression in the TA, DI, and heart of ⁇ mE51E52, hE51KI mice 4 weeks, 8 weeks, and 16 weeks after injection. Dilutions of protein extract from WT mice were used to standardize dystrophin expression (10%, 25%, and 50%) .
  • FIG. 10E shows immunohistochemistry for dystrophin in TA, DI, and heart of ⁇ mE51E52, hE51KI mice performed at indicated time points. Dystrophin is shown in green. Scale bar, 200 ⁇ m.
  • Dots and bars represent biological replicates and are mean ⁇ SEM. Different asterisks represent statistical significance (P ⁇ 0.01) using unpaired two-tailed Student’s t test.
  • TA tibialis anterior muscle.
  • DI diaphragm muscle.
  • the saline-injected ⁇ mE51E52, hE51KI mice (control) exhibited significantly lower muscle performance compared with wild type mice, whereas the treated ⁇ mE51E52, hE51KI mice exhibited muscle performance that was NOT significantly different from wild type mice, indicating that by the systemic administration of the rAAV particles, the muscle performance of ⁇ mE51E52, hE51KI mice was restored to the normal level of wild type mice.
  • FIG. 11A shows schematic of tail vein injection of the rAAV particles into 2-weeks old ⁇ mE51E52, hE51KI mice in a dose of 5E13 vg/kg. Saline was injected as mock-treated controls. Black arrows indicate time points for tissue collection.
  • FIG. 11A shows schematic of tail vein injection of the rAAV particles into 2-weeks old ⁇ mE51E52, hE51KI mice in a dose of 5E13 vg/kg. Saline was injected as mock-treated controls. Black arrows indicate time points for tissue collection.
  • FIG. 11D shows immunohistochemistry for dystrophin in TA, DI, and heart of ⁇ mE51E52, hE51KI mice performed at indicated time points. Dystrophin is shown in green. Scale bar, 200 ⁇ m.
  • DMD Duchenne muscular dystrophy

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Abstract

L'invention concerne des acides nucléiques guides ciblant la DMD, des systèmes les comprenant, et des procédés les utilisant pour traiter des maladies associées à la DMD.
PCT/CN2024/083354 2023-03-22 2024-03-22 Acides nucléiques guides ciblant la dmd et leurs utilisations WO2024193704A1 (fr)

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Citations (6)

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Publication number Priority date Publication date Assignee Title
AU2012200761A1 (en) * 2000-09-21 2012-03-01 Academisch Ziekenhuis Leiden Induction of exon skipping in eukaryotic cells
CN105793425A (zh) * 2013-06-17 2016-07-20 布罗德研究所有限公司 使用病毒组分靶向障碍和疾病的crispr-cas系统和组合物的递送、用途和治疗应用
WO2016164356A1 (fr) * 2015-04-06 2016-10-13 The Board Of Trustees Of The Leland Stanford Junior University Arn guides chimiquement modifiés pour la régulation génétique médiée par crispr/cas
WO2017095967A2 (fr) * 2015-11-30 2017-06-08 Duke University Cibles thérapeutiques pour la correction du gène de la dystrophine humaine par l'édition de gènes et procédés d'utilisation
CN113785063A (zh) * 2019-04-14 2021-12-10 杜克大学 用于治疗杜氏肌营养不良症的aav载体介导的大规模突变热点缺失
WO2023208003A1 (fr) * 2022-04-25 2023-11-02 Huidagene Therapeutics Co., Ltd. Nouveaux systèmes crispr-cas12i et leurs utilisations

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2012200761A1 (en) * 2000-09-21 2012-03-01 Academisch Ziekenhuis Leiden Induction of exon skipping in eukaryotic cells
CN105793425A (zh) * 2013-06-17 2016-07-20 布罗德研究所有限公司 使用病毒组分靶向障碍和疾病的crispr-cas系统和组合物的递送、用途和治疗应用
WO2016164356A1 (fr) * 2015-04-06 2016-10-13 The Board Of Trustees Of The Leland Stanford Junior University Arn guides chimiquement modifiés pour la régulation génétique médiée par crispr/cas
WO2017095967A2 (fr) * 2015-11-30 2017-06-08 Duke University Cibles thérapeutiques pour la correction du gène de la dystrophine humaine par l'édition de gènes et procédés d'utilisation
CN113785063A (zh) * 2019-04-14 2021-12-10 杜克大学 用于治疗杜氏肌营养不良症的aav载体介导的大规模突变热点缺失
WO2023208003A1 (fr) * 2022-04-25 2023-11-02 Huidagene Therapeutics Co., Ltd. Nouveaux systèmes crispr-cas12i et leurs utilisations

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Title
CHEMELLO, F. ET AL.: "Precise correction of Duchenne muscular dystrophy exon deletion mutations by base and prime editing", SCIENCE ADVANCES, vol. 7, 30 April 2021 (2021-04-30), XP093109681, DOI: 10.1126/sciadv.abg4910 *

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