CRISPR / Cas-based base editing compositions for repairing dystrophin function

Abstract

Disclosed herein is a CRISPR / Cas-based base editing composition and a method for treating Duchenne muscular dystrophy by restoring dystrophin function.In one aspect, the present disclosure relates to a CRISPR / Cas-based base editing system for modifying the RNA splicing site coded in target genomic DNA.In some embodiments, modifying the RNA splicing site coded in genomic DNA results in the exclusion or inclusion of at least one exon sequence in RNA transcript.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 833,454, filed Apr. 12, 2019, which is hereby incorporated by reference in its entirety. Description of Government Sponsorship The invention was made with government support under Contract No. R01AR069085 awarded by the National Institutes of Health. The United States government has certain rights in the invention. This disclosure is directed to CRISPR / Cas - based base - editing compositions and methods of treating Duchenne muscular dystrophy by restoring dystrophin function.

Background Art

[0002] Introduction Duchenne muscular dystrophy (DMD) is typically caused by the deletion of one or more exons from the dystrophin gene, resulting in a disruption of the reading frame. Dystrophin protein expression can be repaired by correcting the reading frame by inducing the exclusion of one or more additional exons. Intron removal and selected exon inclusion during mRNA splicing are critical to normal gene function and are often misregulated in genetic disorders. These diseases can be studied and treated using techniques that modulate exon selection, such as mRNA processing and exon skipping techniques. Exon skipping aims to repair the correct reading frame or induce alternative splicing by blocking spliceosome recognition of the splicing sequence, resulting in the removal of a specific exon along with adjacent introns. Studies have shown that by targeting exon splice acceptors with Cas9, indels generated during DNA repair may disrupt the splicing site and exclude exons. However, the need remains to precisely modify the splicing sites in the dystrophin gene in order to completely and / or partially repair dystrophin function. [Overview of the project]

[0003] In one embodiment, the disclosure relates to a CRISPR / Cas-based base editing system for modifying an RNA splicing site encoded in the genomic DNA of a subject. In some embodiments, modifying the RNA splicing site encoded in the genomic DNA results in the exclusion or inclusion of at least one exon sequence in the RNA transcript. In one embodiment, the disclosure relates to a CRISPR / Cas-based base editing system for repairing dystrophin function in a subject. In some embodiments, the subject has a mutated dystrophin gene, and at least one guide RNA (gRNA) targets an RNA splicing site in the mutated dystrophin gene of the subject. In some embodiments, administration of the CRISPR / Cas-based base editing system to the subject results in the exclusion or inclusion of at least one exon sequence from the RNA transcript of the dystrophin gene of the subject, and the repair of the reading frame of the dystrophin gene in the subject. The CRISPR / Cas-based base editing system may comprise a fusion protein and at least one guide RNA (gRNA). In some embodiments, at least one gRNA binds to and targets the polynucleotide sequence corresponding to SEQ ID NO: 1. In some embodiments, the fusion protein includes a Cas protein and a base editing domain. In a further embodiment, the present disclosure relates to isolated polynucleotides encoding the CRISPR / Cas-based base editing system. Another aspect of this disclosure provides a vector comprising the isolated polynucleotide.

[0004] Another aspect of this disclosure provides cells comprising the isolated polynucleotide or the vector. Another aspect of this disclosure provides compositions for restoring dystrophin function in cells having a mutant dystrophin gene. In some embodiments, the compositions include the CRISPR / Cas-based base editing system. Another aspect of the present disclosure provides a kit comprising the CRISPR / Cas-based base editing system, the isolated polynucleotide, the vector, the cells, and / or the composition. Another aspect of this disclosure provides a method for restoring dystrophin function in cells or subjects having a mutant dystrophin gene. The method may involve contacting the cells or subject with the CRISPR / Cas-based base editing system. In some embodiments, the "AG" splice acceptor in exon 45 of the mutant dystrophin gene is converted to an "AA" sequence, and dystrophin function is restored by exon 45 skipping. [Brief explanation of the drawing]

[0005] [Figure 1A] This figure shows a CRISPR / Cas9-based base editor design (Komor et al., Nature (2016) 533(7603):420-4) in which Cas9 components can be derived from various species such as Streptococcus pyogenes and Staphylococcus aureus. In some embodiments, the base editor design includes cytidine deaminase, a linker, nCas9, and a uracil glycosylase inhibitor (UGI). Uracil DNA glycosylase catalyzes U:G → C:G reverse mutations. In some embodiments, the base editor design includes a cytidine deaminase such as rat cytidine deaminase, e.g., rAPOBEC1. In some embodiments, the base editor design includes an XTEN linker (16aa). In some embodiments, the base editor design includes nCas9 (RNA-guided and facilitated mismatch repair on strands with unedited Gs). In some embodiments, the base editor design includes a UGI such as a UGI derived from the Bacillus subtilis bacteriophage PBS1. [Figure 1B]This figure shows alternative CRISPR / Cas9-based base editor designs (Koblan et al., Nat. Biotechnol. (2018) 36(9):843-846). In the BE4max design, bifurcated localization signals were further added to the N and C-terminuses. Eight codon usage frequencies were tested. In the AncBE4max design, ancestral sequence reconstructions for APOBEC were used. In some embodiments, Cas9 components can be derived from various species, such as Streptococcus pyogenes and Staphylococcus aureus. [Figure 1C] This diagram shows C→T (or G→A) base editing within a 5bp window at protospacer positions 4-8. [Figure 1D] This diagram shows the mechanism of base excision repair. [Figure 2A] This figure shows a schematic diagram illustrating R-loop formation by a base editor and the interaction between the cytidine deaminase enzyme and ssDNA. [Figure 2B] This diagram shows a schematic representation of designing a gRNA for base editing of a splice acceptor, and the strict requirements for the "AG" splice acceptor to enter the editing window determined by the availability of PAM (which varies depending on the Cas9 species, where "Sp" is Streptococcus pyogenes and "Sa" is Staphylococcus aureus). [Figure 3A] This figure shows the splice acceptor design strategy for exons 44 and 45 (and many others), where g1 and G2 are targeted for base editing. [Figure 3B] This figure shows %G>A base editing (N=3) at the splice acceptor site of exon 44 using the exon 44 gRNA of 5'-CGCCTGCAGGTAAAAGCATA-3' (SEQ ID NO: 9). [Figure 3C] This figure shows %G>A base editing (N=3) at the splice acceptor site of exon 45 using the exon 45 gRNA corresponding to 5'-GTTCCTGTAAGATACCAAAA-3' (Sequence ID 1). [Figure 4A]This figure shows a schematic diagram of exons 41-50 of the dystrophin gene. [Figure 4B] This figure shows the predicted sequence of the dystrophin gene resulting from the deletion of exon 44. As a result, intron 43 will directly move into intron 44. [Figure 4C] This figure shows the sequence of the dystrophin gene with exon 44 deleted. The insertion or deletion may be located at the junction of intron 43 and intron 44 after the deletion of exon 44. [Figure 4D] This figure shows the confirmation of the deletion of exon 44 in the dystrophin gene in clone c11, compared to clone c2, which does not have a deletion in exon 44. [Figure 5] This figure shows a schematic diagram of myogenic differentiation of iPSCs. [Figure 6] This figure shows the myogenic differentiation of iPSCs in which the Δ44 mutation cleaves the dystrophin protein. [Figure 7] This diagram shows the outline of Δ44 iPSC editing. [Figure 8A] This figure shows %G>A base editing events in Δ44 iPSCs using BE4max. [Figure 8B] This figure shows all gVG03 d12 editing events in Δ44 iPSC using BE4max. [Figure 9A] This figure shows %G>A base editing events in Δ44 iPSCs using AncBE4max. [Figure 9B] This figure shows all gVG03 d12 editing events in Δ44 iPSC using AncBE4max. [Figure 10] This figure shows Δ44 iPSC editing after 12 days using BE4max and AncBE4max. [Figure 11] This figure shows the RT-PCR of MyoD differentiation of edited cells. [Figure 12]This figure shows % non-G base editing events in Δ44 iPSCs using AncBE4max delivered by lentivirus on day 7 (D7) and day 14 (D14). [Figure 13] This figure shows % non-G base editing events in Δ44 iPSCs using AncBE4max delivered by electroporation on day 7 (D7) and day 14 (D14). [Figure 14] This figure shows schematic diagrams of the wild-type (WT), Δ44, and Δ44-45 versions of the dystrophin gene (left), as well as a Western blot of MyoD-differentiated Δ44 iPSC cells edited with AncBE4max and exon 45 gRNA (right). [Modes for carrying out the invention]

[0006] The present disclosure provides CRISPR / Cas-based base editing compositions and methods of treating Duchenne muscular dystrophy (DMD) by restoring dystrophin function. DMD is typically caused by deletions in the dystrophin gene that disrupt the reading frame. Because internally truncated dystrophin proteins have been shown to be able to continue to be partially functional, many strategies for treating DMD aim to restore the reading frame by removing or skipping additional exons. In mammalian genes, there are conserved sequences that mark the boundaries between introns and exons. One important splicing site is the "AG" that precedes an exon and is called the splice acceptor. Complete nuclease Cas9 has been used to target and force skipping of the splice acceptor of the dystrophin exon, thereby relying on semi-random indels formed during the DNA repair process to cleave the splicing site. The CRISPR / Cas-based base editing system disclosed herein enables a more accurate base editing method to convert the "AG" splice acceptor to "AA" to promote exon skipping. In contrast to the semi-random indels generated by the conventional CRISPR-Cas9 system, base editing technologies have been developed for precise single base pair modifications without inducing double-stranded DNA breaks. Base editors can directly change C to T or G on the reverse strand to A, and these can target both the splice donor "GT" and acceptor "AG" of various exons to modulate mRNA splicing.

[0007] 1. Definitions As used herein, the terms "comprise(s)", "include(s)", "having", "has", "can", "contain(s)", and variations thereof are intended to be open transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Also, this disclosure contemplates other embodiments that "comprise", "consist of", and "consist essentially of" the embodiments or elements presented herein, whether or not explicitly recited. Regarding the recitation of numerical ranges herein, each intervening numerical value there between is specifically contemplated with the same degree of precision. For example, in the range of 6 - 9, the numerical values 7 and 8 are contemplated in addition to the numbers 6 and 9, and in the range of 6.0 - 7.0, the numerical values 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are specifically contemplated.

[0008] As used herein, the term "about" or "approximately" means within an acceptable error range for a particular value determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within three standard deviations, or more than three standard deviations, per the practice in the art. Alternatively, "about" can mean within up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or biological processes, the term can mean within up to 10 fold, preferably up to 5 fold, more preferably up to 2 fold of a value. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. In case of any conflict, this document, including its definitions, shall prevail. Preferred methods and materials are described below, but similar or equivalent methods and materials may be used in carrying out or testing the present invention. All publications, patent applications, patents, and other references referenced herein are incorporated herein by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to limit the scope of this invention.

[0009] In this specification, "adeno-associated virus" or "AAV," as used interchangeably, refers to a small virus belonging to the genus Dependovirus of the family Parvoviridae that infects humans and some other primate species. AAV is not currently known to cause disease, and therefore, it elicits a very mild immune response. As used herein, “amino acids” refers to naturally occurring and non-naturally occurring synthetic amino acids, as well as amino acid analogs and amino acid mimes that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids are encoded by the genetic code. Amino acids may be referred to herein either by their commonly known three-letter abbreviation or by the single-letter abbreviation recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Amino acids include side chains and polypeptide backbone portions.

[0010] As used herein, "binding region" refers to a region within a target region that is recognized and bound to by a CRISPR / Cas-based base editing system. As used herein, "chromatin" refers to the organized complex of chromosomal DNA associated with histones. As used herein for compatibility purposes, “Clustered Regularly Interspaced Short Palindromic Repeats” and “CRISPR” refer to loci containing multiple short direct repeats found in the genomes of approximately 40% of sequenced bacteria and 90% of sequenced archaea.

[0011] As used herein, “coding sequence” or “coding nucleic acid” means a nucleic acid (RNA or DNA molecule) containing a polynucleotide sequence that codes for a protein. The coding sequence may further include start and stop signals that can operably ligate to regulatory elements, including promoters and polyadenylation signals, and can direct expression in cells of an organism or mammal to which the nucleic acid is administered. The coding sequence may be codon-optimized. As used herein with respect to nucleic acids, “complement” or “complementary” means a nucleic acid capable of forming Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairings between nucleotides or nucleotide analogs of a nucleic acid molecule. “Complementarity” means a property shared between two nucleic acid sequences such that their nucleotide bases are complementary at their respective positions when they are aligned antiparallel to each other.

[0012] The terms “control,” “reference level,” and “reference” are used interchangeably within this specification. A reference level may be a predetermined value or range used as a benchmark for evaluating measured results against it. As used herein, “control group” means a group of control subjects. A predetermined level may be a cutoff value from the control group. A predetermined level may be the mean from the control group. The cutoff value (or predetermined cutoff value) may be determined by an adaptive indicator model (AIM) method. The cutoff value (or predetermined cutoff value) may be determined by receiver operating curve (ROC) analysis from biological samples of a patient group. ROC analysis, commonly known in the biological field, is, for example, determining the ability of a test to distinguish one condition from another, to determine the performance of each marker in identifying patients with CRC. A description of ROC analysis is provided in PJ Heagerty et al. (Biometrics 2000, 56, 337-44), whose disclosure is incorporated herein by reference in its entirety. Alternatively, the cutoff value may be determined by quartile analysis of biological samples from the patient group. For example, the cutoff value may be determined by selecting a value corresponding to any value within the range of the 25th to 75th percentiles, preferably the 25th, 50th, or 75th percentile, more preferably the 75th percentile. Such statistical analysis may be performed using any method known in the art and can be implemented by any number of commercially available software packages (e.g., those of Analyse-it Software Ltd., Leeds, UK; StataCorp LP, College Station, Texas; SAS Institute Inc.; and Cary, North Carolina). Healthy or normal levels or ranges for targets or protein activity may be defined according to standard practices. Controls may be subjects or cells that do not have the constructs or systems detailed herein. Controls may be subjects or samples derived from subjects whose disease status is known.The subject, or a sample derived therefrom, may be healthy, in a diseased state, in a diseased state before treatment, in a diseased state during treatment, in a diseased state after treatment, or a combination thereof.

[0013] As used interchangeably in this specification, “Duchenne muscular dystrophy” or “DMD” refers to a recessive, fatal X-linked disorder that leads to muscle degeneration and ultimately death. DMD is a common hereditary monogenic disorder that occurs in 1 in 3,500 men. DMD is the result of hereditary or spontaneous mutations that cause nonsense or frameshift mutations in the dystrophin gene. The vast majority of dystrophin mutations that cause DMD are exon deletions that disrupt the leading frame and cause immature translation termination of the dystrophin gene. Individuals with DMD typically lose the ability to physically support themselves during childhood, become progressively fragile during their teenage years, and die in their twenties. As used herein, “dystrophin” refers to a rod-shaped cytoplasmic protein that is part of a protein complex that connects the cytoskeleton of muscle fibers to the cell membrane via the surrounding extracellular matrix. Dystrophin provides structural stability to the dystroglycan complex of the cell membrane, which is responsible for regulating the integrity and function of muscle cells. The dystrophin gene, or “DMD gene,” as used herein for interchangeability, is 2.2 megabases long at locus Xp21. The primary transcript measures approximately 2,400 kb, and the mature mRNA is approximately 14 kb. The 79 exons encode a protein of more than 3,500 amino acids.

[0014] As used herein, “exon 45” refers to exon 45 of the dystrophin gene. Exon 45 is often adjacent to frame-disrupting deletions in DMD patients and has been targeted in clinical trials for oligonucleotide-based exon skipping. As used herein, “enhancer” refers to a non-coding DNA sequence containing multiple activator and repressor binding sites. Enhancers range in length from 200 bp to 1 kb and may be proximal, i.e., 5' upstream of the promoter or within the first intron of the gene being regulated, or distal, i.e., in an intergeneric region considerably distant from the intron or locus of an adjacent gene. Through DNA looping, active enhancers come into contact with the promoter in a manner dependent on the specificity of the core DNA-binding motif promoter. Four to five enhancers may interact with a single promoter. Similarly, enhancers may regulate multiple genes without linkage limitations and may “skip” adjacent genes to regulate more distal ones. Transcriptional regulation may involve elements located on different chromosomes than those where the promoter resides. Proximal enhancers or promoters of adjacent genes may act as platforms for recruiting more distal elements.

[0015] As used herein, "functional" and "fully functional" refer to proteins that possess biological activity. A "functional gene" is a gene that is transcribed into mRNA, which is then translated into a functional protein. As used herein, "fusion protein" refers to a chimeric protein created by combining two or more genes that originally encoded separate proteins. Translation of the fusion gene yields a single polypeptide with functional properties derived from each of the original proteins. As used herein, “gene construct” means a DNA or RNA molecule containing a polynucleotide sequence that codes for a protein. The coding sequence includes start and stop signals that can direct expression in the cells of an individual to which the nucleic acid molecule is administered, and are operably ligated with regulatory elements, including a promoter and a polyadenylation signal. As used herein, “expressible form” means a gene construct containing the necessary regulatory elements, which are operably ligated with a protein-coding sequence, so that the coding sequence is expressed when present in the cells of an individual.

[0016] As used herein, “genome editing” means altering a gene. Genome editing may include repairing or restoring a mutated gene. Genome editing may include base editing to alter splice acceptor sites. Genome editing, such as base editing, may be used to treat a disease or enhance muscle repair by altering a target gene. As used herein, the term "heterogeneous" refers to a nucleic acid containing two or more subsequences that are not found in nature in the same relation to one another. For example, recombinant nucleic acids typically have two or more sequences from unrelated genes that are synthetically arranged to create a new functional nucleic acid, such as a promoter from one source and a coding region from another source. Thus, the two nucleic acids are heterogeneous in this context. When added to a cell, recombinant nucleic acids will also be heterogeneous with respect to the cell's endogenous genes. Therefore, within a chromosome, heterogeneous nucleic acids will include non-native (not naturally occurring) nucleic acids incorporated into the chromosome, or non-native (not naturally occurring) extrachromosomal nucleic acids. Similarly, heterogeneous proteins indicate that a protein contains two or more subsequences that are not found in nature in the same relation to one another (for example, a "fusion protein" in which two subsequences are encoded by a single nucleic acid sequence).

[0017] As used herein, “identical” or “sameness” in the context of two or more nucleic acid or polypeptide sequences means that the sequences have a specified percentage of identical residues across a specified region. The percentage can be calculated by optimally aligning the two sequences, comparing them across a specified region, determining the number of positions where identical residues exist in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the specified region, and multiplying the result by 100 to obtain the percentage of sequence identity. If the two sequences are of different lengths, or if alignment results in one or more twisted ends and the specified comparison region contains only a single sequence, the residues of the single sequence are included in the denominator but not the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) may be considered equivalents. Identity can be performed manually or by using computer sequencing algorithms such as BLAST or BLAST 2.0.

[0018] As used herein for compatibility purposes, “mutant gene” or “mutated gene” means a gene that has undergone a detectable mutation. A mutant gene has undergone changes such as loss, acquisition, or exchange of genetic material that affect the normal transmission and expression of the gene. As used herein, “disrupted gene” means a mutant gene that has a mutation that results in an immature stop codon. A disrupted gene product is cleaved compared to a full-length, undisrupted gene product. As used herein, "normal gene" refers to a gene that has not undergone any alteration such as loss, acquisition, or exchange of genetic material. Normal genes undergo normal gene transmission and gene expression. As used herein, “nucleic acid,” “oligonucleotide,” or “polynucleotide” means at least two nucleotides covalently linked together. A description of a single strand also defines the sequence of its complementary strand. Thus, a nucleic acid also encompasses the complementary strand of the single strand shown. Many variants of a nucleic acid can be used as a given nucleic acid for the same purpose. Thus, a nucleic acid also encompasses substantially identical nucleic acids and their complements. A single strand provides a probe that can hybridize with a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses probes that hybridize under stringent hybridization conditions.

[0019] Nucleic acids may be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. Nucleic acids may be DNA (both genomic and cDNA), RNA, or hybrids, and may contain combinations of deoxyribonucleotides and ribonucleotides, as well as combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acids may be obtained by chemical synthesis or by recombinant methods. As used herein, “operably linked” means that the expression of a gene is under the control of a promoter to which it is spatially connected. The promoter may be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene may be approximately the same as the distance between the promoter and the gene it controls in the gene from which the promoter originates. As is known in this art, variations in this distance can be adapted without loss of promoter function.

[0020] Nucleic acids or amino acid sequences are “operatably linked” (or “operatably linked”) when they are arranged in a functional relationship with one another. For example, a promoter or enhancer is operatably linked to a coding sequence if it regulates or modulates the transcription of the coding sequence. Operatally linked DNA sequences are typically continuous, and operatably linked amino acid sequences are typically continuous and within the same reading frame. However, some polynucleotide elements may be operatably linked but not continuous, as enhancers generally function when separated from the promoter by several kilobases or more, and intron sequences may be of variable length. Similarly, certain amino acid sequences that are discontinuous in a primary polypeptide sequence may still be operatably linked, for example, thanks to polypeptide chain folding. With respect to fusion polypeptides, the terms “operatably linked” and “operatably linked” can mean that each component is linked to the other components and performs the same function as if it were not so linked. As used herein, "partially functional" refers to a protein encoded by a mutant gene that has lower biological activity than a functional protein but higher biological activity than a non-functional protein.

[0021] A "peptide" or "polypeptide" is a linked sequence of two or more amino acids linked by peptide bonds. Polypeptides can be native, synthetic, modified, or a combination of native and synthetic. Examples of peptides and polypeptides include proteins such as binding proteins, receptors, and antibodies. The terms "polypeptide," "protein," and "peptide" are used interchangeably in this specification. "Primary structure" refers to the amino acid sequence of a particular peptide. "Secondary structure" refers to the locally ordered three-dimensional structure within a polypeptide. These structures are commonly known as domains, such as enzyme domains, extracellular domains, transmembrane domains, pore domains, and cytoplasmic terminal domains. A "domain" is a portion of a polypeptide that forms a compact polypeptide unit and is typically 15 to 350 amino acids long. Exemplary domains include those with enzymatic or ligand-binding activity. Typical domains consist of less organized sections, such as beta-sheet and alpha-helix segments. "Tertiary structure" refers to the complete three-dimensional structure of a polypeptide monomer. A "quaternary structure" refers to a three-dimensional structure formed by the non-covalent association of independent tertiary units. A "motif" is a portion of a polypeptide sequence containing at least two amino acids. A motif can be, for example, 2-20, 2-15, or 2-10 amino acids long. In some embodiments, a motif contains 3, 4, 5, 6, or 7 consecutive amino acids. A domain can consist of a series of motifs of the same type.

[0022] As used herein for compatibility purposes, “immature stop codon” or “out-of-frame stop codon” refers to a nonsense mutation in the DNA sequence that results in a stop codon at a location not normally found in wild-type genes. Immature stop codons can cause proteins to be cleaved or shortened compared to their full-length version. As used herein, “promoter” means a synthetic or naturally occurring molecule that can give, activate, or enhance the expression of nucleic acids in a cell. A promoter may contain one or more specific transcriptional regulatory sequences to further enhance expression and / or to alter its spatial and / or temporal expression. A promoter may also contain distal enhancer or repressor elements, which may be located thousands of base pairs away from the transcription start site. Promoters may be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. Promoters may modulate the expression of gene components constitutively, differentially with respect to the developmental stage in which expression occurs with respect to the cell, tissue, or organ in which expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducers. Typical examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter, or SV40 late promoter, and the CMV IE promoter.

[0023] For example, the term “recombinant” used in reference to cells, nucleic acids, proteins, or vectors indicates that the cells, nucleic acids, proteins, or vectors have been modified by the introduction of heterologous nucleic acids or proteins or by alteration of native nucleic acids or proteins, or that the cells originate from cells that have been thus modified. Thus, for example, recombinant cells express genes not found in the cell’s native (naturally occurring) form, or express a second copy of a native gene that would otherwise be normally or abnormally expressed, underexpressed, or not expressed at all. As used herein, “skeletal muscle” refers to a type of striated muscle that is under the control of the somatic nervous system and is attached to bone by bundles of collagen fibers known as tendons. Skeletal muscle consists of individual components known as muscle cells, or “muscle cells,” and sometimes colloquially called “muscle fibers.” Muscle cells are formed from the fusion of developing myoblasts (a type of embryonic progenitor cell that gives rise to muscle cells) in a process known as myogenesis. These long, cylindrical, multinucleated cells are also called myofibrils.

[0024] As used herein, "skeletal muscle condition" refers to a condition related to skeletal muscle, such as muscular dystrophy, aging, muscle degeneration, wound healing, and muscle weakness or atrophy. As used interchangeably herein, “subject” and “patient” refer to any vertebrate, including, but not limited to, mammals (e.g., cattle, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, and mice, non-human primates (e.g., cynomolgus or rhesus monkeys, chimpanzees, and other monkeys), and humans). In some embodiments, the subject may be human or non-human. The subject or patient may be undergoing other forms of treatment. The subject may be of any age or developmental stage, such as adult, adolescent, or infant. In some embodiments, the subject may have specific genetic markers.

[0025] "To treat," "to treat," or "treatment" are used interchangeably herein to describe reversing, mitigating, or inhibiting the progression of a disease or one or more symptoms of such a disease to which such terms apply. Depending on the condition of the subject, this term also means preventing the disease, including preventing the onset of the disease or preventing symptoms associated with the disease. Treatment may be carried out in either an acute or chronic form. This term also means reducing the severity of the disease or symptoms associated with such a disease before the disease develops. Prevention or reduction of the severity of such a disease before the disease develops means administering the antibody or pharmaceutical composition of the present invention to a subject who is not suffering from the disease at the time of administration. "To prevent" also means preventing the recurrence of the disease or one or more symptoms associated with such a disease. "Treatment" and "therapeutic" mean the act of treating, as defined above.

[0026] With respect to nucleic acids, the term “mutant” as used herein means (i) a portion or fragment of a referenced polynucleotide sequence, (ii) a complement of a referenced polynucleotide sequence or a portion thereof, (iii) a nucleic acid substantially identical to a referenced nucleic acid or its complement, or (iv) a nucleic acid that, under stringent conditions, hybridizes with a referenced nucleic acid, its complement, or a sequence substantially identical thereto. A "mutant" of a peptide or polypeptide is defined as having a different amino acid sequence due to an insertion, deletion, or conservative substitution of amino acids, but retaining at least one biological activity. A mutant can also refer to a protein having an amino acid sequence substantially identical to a reference protein, while retaining at least one biological activity. Conservative substitution of amino acids, i.e., replacing an amino acid with an amino acid having similar properties (e.g., hydrophilicity, degree and distribution of charged regions), is typically recognized in this field as involving minor changes. These minor changes can be partially identified by considering the hydrophobicity / hydrophilicity index of amino acids, as understood in this field. (Kyte et al., J. Mol. Biol. 157:105-132 (1982)). The hydrophobicity / hydrophilicity index of amino acids is based on consideration of their hydrophobicity and charge. It is known in this field that substituting amino acids with similar hydrophobicity / hydrophilicity indexes may still retain protein function. In one embodiment, amino acids with ±2 hydrophobicity / hydrophilicity indexes are substituted. Furthermore, the hydrophilicity of amino acids can be used to identify substitutions that result in proteins that retain biological function. Considering the hydrophilicity of amino acids in the context of peptides allows for the calculation of the peptide's maximum local mean hydrophilicity. Substitutions can be made using amino acids with hydrophilicity values ​​within ±2 of each other. Both the hydrophobicity index and hydrophilicity value of amino acids are influenced by the specific side chains of those amino acids. Consistent with this observation, it is understood that amino acid substitutions that are compatible with biological function depend on the relative similarity of amino acids, specifically their side chains, as revealed by their hydrophobicity, hydrophilicity, charge, size, and other properties.

[0027] As used herein, “vector” means a nucleic acid sequence containing an origin of replication. A vector may be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. A vector may be a DNA or RNA vector. A vector may be a self-replicating extrachromosomal vector, preferably a DNA plasmid. For example, a vector may encode a CRISPR / Cas-based base editing system as described herein, comprising a polynucleotide sequence encoding a fusion protein, such as at least one gRNA polynucleotide sequence of SEQ ID NO: 7 or SEQ ID NO: 8 and / or SEQ ID NO: 1.

[0028] 2. A CRISPR / Cas-based base editing system for repairing dystrophin A CRISPR / Cas-based base editing system is provided herein. A CRISPR / Cas-based base editing system can be used to modify an RNA splicing site encoded in a target genomic DNA. A CRISPR / Cas-based base editing system may be used in the repair of dystrophin gene function. A CRISPR / Cas-based base editing system may comprise a fusion protein and at least one guide RNA (gRNA). In some embodiments, at least one gRNA binds to and targets a polynucleotide sequence corresponding to SEQ ID NO: 1. In some embodiments, at least one gRNA is encoded by the polynucleotide sequence of SEQ ID NO: 1. The fusion protein may contain two heterologous polypeptide domains. In some embodiments, the fusion protein comprises a Cas protein and a base editing domain. In some embodiments, at least one gRNA binds to and targets a) a fragment of SEQ ID NO: 1, b) a complement or fragment thereof of SEQ ID NO: 1, c) a nucleic acid substantially identical to SEQ ID NO: 1 or its complement, or d) a nucleic acid, its complement, or a polynucleotide sequence substantially identical thereto that hybridizes with SEQ ID NO: 1 under stringent conditions. In some embodiments, at least one gRNA comprises a polynucleotide sequence corresponding to SEQ ID NO: 1 or a variant thereof.

[0029] a) Dystrophin gene Dystrophin is a rod-shaped cytoplasmic protein that is part of a protein complex that connects the cytoskeleton of muscle fibers to the cell membrane via the surrounding extracellular matrix. Dystrophin provides structural stability to the dystroglycan complex of the cell membrane. The dystrophin gene is 2.2 megabases long at locus Xp21. The primary transcript is measured at approximately 2,400 kb, and the mature mRNA is approximately 14 kb. The 79 exons encode a protein of more than 3,500 amino acids. Normal skeletal muscle tissue contains only small amounts of dystrophin, but the absence of dystrophin due to abnormal expression leads to the development of severe and incurable symptoms. Certain mutations in the dystrophin gene result in the production of deficient dystrophin and a severe dystrophy phenotype in affected individuals. Certain mutations in the dystrophin gene result in a partially functional dystrophin protein and a much milder dystrophy phenotype in affected individuals.

[0030] DMD is the result of hereditary or spontaneous mutations that cause nonsense or frameshift mutations in the dystrophin gene. Naturally occurring mutations and their consequences are relatively well understood in DMD. In-frame deletions occurring in the exon 45-55 region contained within the rod domain can produce a highly functional dystrophin protein, and many carriers are known to be asymptomatic or exhibit mild symptoms. Furthermore, more than 60% of patients can theoretically be treated by targeting exons in this region of the dystrophin gene. Efforts have been made to repair the disrupted dystrophin reading frame in DMD patients by skipping non-essential exons during mRNA splicing (e.g., exon 45 skipping) to produce an internally deleted but functional dystrophin protein. Deletion of an internal dystrophin exon (e.g., exon 45 deletion) can produce a dystrophin protein that retains the proper reading frame and is internally cleaved but partially functional. Deletion of dystrophin between exons 45 and 55 results in a much milder phenotype compared to DMD.

[0031] In certain embodiments, excision of exon 45 to repair the leading frame leads to phenotypic remission in DMD subjects, including DMD subjects with deletion mutations. In certain embodiments, exon 45 of the dystrophin gene refers to the 45th exon of the dystrophin gene. Exon 45 is often adjacent to frame-disrupting deletions in DMD patients and has been targeted in clinical trials for oligonucleotide-based exon skipping.

[0032] The CRISPR / Cas-based base editing systems detailed herein can be used to modify RNA splicing sites encoded in the genomic DNA of a subject. In some embodiments, modifying RNA splicing sites encoded in genomic DNA results in the exclusion or inclusion of at least one exon sequence in the RNA transcript. The CRISPR / Cas-based base editing systems detailed herein can be used to repair dystrophin function in a subject. In some embodiments, the subject has a mutated dystrophin gene, and at least one guide RNA (gRNA) targets an RNA splicing site in the mutated dystrophin gene of the subject. In some embodiments, administration of the CRISPR / Cas-based base editing system to the subject results in the exclusion or inclusion of at least one exon sequence from the RNA transcript of the dystrophin gene of the subject, and the repair of the reading frame of the dystrophin gene in the subject.

[0033] The systems and vectors disclosed herein can modify the splice acceptor site at exon 45 in the dystrophin gene, for example, the human dystrophin gene. Modification of the splice acceptor site may result in the deletion of exon 45 from the dystrophin protein product (i.e., exon 45 skipping), which may increase the function or activity of the encoded dystrophin protein or lead to improvement of the condition in question. In certain embodiments, exon 45 skipping can repair the dystrophin reading frame. In some embodiments, the splice acceptor site at exon 45 is located within a sequence containing the polynucleotide sequence of SEQ ID NO: 1. The systems or gene constructs (e.g., vectors) disclosed herein can mediate highly efficient exon 45 skipping of the dystrophin gene (e.g., the human dystrophin gene). The systems or gene constructs (e.g., vectors) disclosed herein can repair dystrophin protein expression in cells derived from DMD patients. Exon 45 is often adjacent to deletions that disrupt the frame in DMD. Approximately 8% of all DMD patients can be treated using exon skipping to remove exon 45 from the dystrophin transcript. The systems or gene constructs (e.g., vectors) disclosed herein can be transfected into human DMD cells to mediate efficient gene modification and conversion to the correct reading frame. Protein repair may occur concurrently with frame repair and can be detected in a bulk population of cells treated with a CRISPR / Cas-based base editing system.

[0034] b) Fusion protein A CRISPR / Cas-based base editing system includes a fusion protein or a nucleic acid sequence encoding a fusion protein. The fusion protein includes a Cas protein and a base editing domain. In some embodiments, the nucleic acid sequence encoding the fusion protein is DNA. In some embodiments, the nucleic acid sequence encoding the fusion protein is RNA. i) Cas protein The Cas protein forms a complex with the 3' end of the gRNA. The specificity of CRISPR-based systems depends on two factors: the targeting sequence and the protospacer-adjacent motif (PAM). The targeting or recognition sequence is located at the 5' end of the gRNA and is the correct DNA sequence known as the protospacer, designed to pair with a base pair on the host DNA (target nucleic acid or target DNA). By simply replacing the recognition sequence of the gRNA, the Cas protein can be directed to a new genomic target. The PAM sequence is located on the DNA to be modified and is recognized by the Cas protein. The PAM recognition sequence of the Cas protein can be species-specific.

[0035] In some embodiments, the CRISPR / Cas-based base editing system may include a Cas9 protein, such as catalytically dead dCas9. The Cas9 protein is an endonuclease that cleaves nucleic acids, is encoded by a CRISPR locus, and is involved in the type II CRISPR system. The Cas9 molecule can interact with one or more gRNA molecules and, in cooperation with the gRNA molecules, localize to a target domain, in certain embodiments, a site containing a PAM sequence. The ability of the Cas9 molecule to recognize a PAM sequence can be determined, for example, using a previously described transformation assay (Jinek 2012). In some embodiments, the Cas9 protein is derived from Streptococcus pyogenes. In some embodiments, the Cas9 protein contains the polypeptide sequence of SEQ ID NO: 2. In some embodiments, the Cas9 protein is derived from Staphylococcus aureus. In some embodiments, the Cas9 protein contains the polypeptide sequence of SEQ ID NO: 3.

[0036] In some embodiments, the Cas9 protein may be mutated to reduce or inactivate its nuclease activity. To silence gene expression by steric hindrance, an inactivated Cas9 protein lacking endonuclease activity (also called "iCas9" or "dCas9") may be targeted to genes in bacteria, yeast, and human cells via gRNA. Exemplary mutations for reducing or inactivating nuclease activity, referencing the Streptococcus pyogenes Cas9 sequence, include D10A, E762A, H840A, N854A, N863A, and / or D986A. Exemplary mutations for inactivating nuclease activity, referencing the Staphylococcus aureus Cas9 sequence, include D10A and N580A. In some embodiments, an inactivated Cas9 protein derived from Streptococcus pyogenes (also called iCas9 or "dCas9", Sequence ID No. 5) may be used. As used herein, both "iCas9" and "dCas9" may refer to Cas9 proteins having amino acid substitutions D10A and H840A and whose nuclease activity has been inactivated. In some embodiments, the Cas protein may be a mutant Cas9 protein having the amino acid substitution D10A (referred to as "nCas9" and possessing nickase activity, e.g., SEQ ID NO: 4).

[0037] The Cas9 protein or mutant Cas9 protein may be derived from any bacterial or archaeal species, such as Streptococcus pyogenes, Staphylococcus aureus, Streptococcus thermophilus, or Neisseria meningitides. In some embodiments, the Cas protein or mutant Cas9 protein may be derived from the genera Streptococcus, Staphylococcus, Brevibacillus, Corynebacter, Sutterella, Legionella, Francisella, Treponema, Filifactor, Eubacterium, Lactobacillus, Bacteroides. Cas9 proteins are derived from bacterial genera such as ides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma, or Campylobacter.In some embodiments, the Cas9 protein or mutant Cas9 protein may be used to protect against, but is not limited to, Streptococcus pyogenes, Francisella novicida, Staphylococcus aureus, Neisseria meningitidis, Streptococcus thermophilus, Treponema denticola, Brevibacillus laterosporus, Campylobacter jejuni, Corynebacterium diphtheria, Eubacterium ventriosum, Streptococcus pasteurianus, Lactobacillus farciminis, Sphaerochaeta globus The group is selected from those including *Campylobacter globus*, the genus *Azospirillum*, *Gluconacetobacter diazotrophicus*, *Neisseria cinerea*, *Roseburia intestinalis*, *Parvibaculum lavamentivorans*, *Nitratifractor salsuginis*, and *Campylobacter lari*.

[0038] In certain embodiments, the ability of a Cas9 molecule or mutant Cas9 protein to interact with and cleave a target nucleic acid is PAM sequence-dependent. The PAM sequence is a sequence in the target nucleic acid. In certain embodiments, cleavage of the target nucleic acid occurs upstream of the PAM sequence. Cas9 molecules from various bacterial species can recognize various sequence motifs (e.g., PAM sequences). In certain embodiments, the Cas9 molecule from Streptococcus pyogenes recognizes the sequence motif NGG (SEQ ID NO: 10), which directs cleavage of the target nucleic acid sequence 1-10 bp upstream of that sequence, for example, 3-5 bp (see, e.g., Mali 2013). In certain embodiments, the Cas9 molecule from Staphylococcus aureus recognizes the sequence motif NNGRR (R=A or G) (SEQ ID NO: 12), which directs cleavage of the target nucleic acid sequence 1-10 bp upstream of that sequence, for example, 3-5 bp. In certain embodiments, the Cas9 molecule of Staphylococcus aureus recognizes the sequence motif NNGRRN(R=A or G) (SEQ ID NO: 13), which directs the cleavage of a target nucleic acid sequence 1 to 10 bp upstream, for example, 3 to 5 bp from the sequence. In certain embodiments, the Cas9 molecule of Staphylococcus aureus recognizes the sequence motif NNGRRT(R=A or G) (SEQ ID NO: 14), which directs the cleavage of a target nucleic acid sequence 1 to 10 bp upstream, for example, 3 to 5 bp from the sequence. In certain embodiments, the Cas9 molecule of Staphylococcus aureus recognizes the sequence motif NNGRRV(R=A or G, V=A or C or G) (SEQ ID NO: 15), which directs the cleavage of a target nucleic acid sequence 1 to 10 bp upstream, for example, 3 to 5 bp from the sequence. In the embodiments described above, N can be any nucleotide residue, for example, any of A, G, C, or T. The Cas9 molecule can be manipulated to change the PAM specificity of the Cas9 molecule.

[0039] In some embodiments, the Cas9 protein or mutant Cas9 protein can recognize the PAM sequence NGG (SEQ ID NO: 10) or NGA (SEQ ID NO: 19). In some embodiments, the Cas9 protein or mutant Cas9 protein can recognize the PAM sequence NNNRRT (SEQ ID NO: 11). In some embodiments, the Cas9 protein or mutant Cas9 protein is a Staphylococcus aureus Cas9 protein and recognizes the sequence motifs NNGRR(R=A or G) (SEQ ID NO: 12), NNGRRN(R=A or G) (SEQ ID NO: 13), NNGRRT(R=A or G) (SEQ ID NO: 14), or NNGRRV(R=A or G) (SEQ ID NO: 15). In the embodiments described above, N can be any nucleotide residue, for example, any of A, G, C, or T. The Cas9 molecule can be manipulated to change its PAM specificity. In addition, or instead, the nucleic acid encoding the Cas9 molecule or Cas9 polypeptide may contain a nuclear localization sequence (NLS). Nuclear localization sequences are known in this field.

[0040] ii) Base editing domain The fusion protein contains a Cas protein and a base editing domain. Base editing enables direct, irreversible conversion of a specific DNA base to another at a target genomic locus without requiring double-strand DNA breaks (DSBs). Figure 1D shows the design process of one base editor. In some embodiments, the base editing domain includes (i) a cytidine deaminase domain and (ii) at least one uracil glycosylase inhibitor (UGI) domain. The cytidine deaminase domain can convert the DNA base cytosine to uracil (see Figure 1C). In some embodiments, the cytidine deaminase domain may include apolipoprotein B mRNA editing enzymes, catalytic polypeptide-like (APOBEC) family deaminases. In some embodiments, the cytidine deaminase domain may include APOBEC1 deaminase, APOBEC2 deaminase, APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3F deaminase, APOBEC3G deaminase, APOBEC3H deaminase, or a combination thereof. In some embodiments, the cytidine deaminase domain includes APOBEC1 deaminase. In some embodiments, the cytidine deaminase domain includes rat APOBEC1 deaminase. In some embodiments, a cytidine deaminase enzyme (e.g., rAPOBEC1) can be fused with the N-terminus of a dCas to produce a base editing enzyme named BE1.

[0041] In some embodiments, at least one UGI domain includes a domain capable of inhibiting uracil-DNA glycosylase (UDG) activity. UDG activity may include removing uracil from nucleic acids by cleaving N-glycosidic bonds. UDG activity can initiate the base excision repair (BER) pathway. UGI domains capable of inhibiting UDG activity can prevent subsequent U:G mismatches from being repaired back to C:G base pairs, and thus can manipulate the cellular DNA repair process to increase the yield of desired results (e.g., T:A base pairs). In some embodiments, at least one UGI domain includes a polypeptide having the amino acid sequence of SEQ ID NO: 20. In some embodiments, at least one UGI domain includes an amino acid sequence encoded by the polynucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 18. In some embodiments, the base editing domain includes one or two UGI domains. When multiple UGI domains are present in the base editing domain, slightly different or mutated sequences of the UGI domains may be used to avoid the tendency for two identical sequences to recombine when they are adjacent to each other on the same construct. In some embodiments, a UGI can be fused with a cytidine deaminase enzyme (e.g., rAPOBEC1) fused to the N-terminus of a dCas to produce a base editing enzyme named BE2. In some embodiments, two UGIs can be fused with a cytidine deaminase enzyme (e.g., rAPOBEC1) fused to the N-terminus of a dCas to produce a base editing enzyme named BE4.

[0042] In some embodiments, the fusion protein may have the following structure:NH2-[cytidine deaminase domain]-[Cas protein]-[UGI domain]-COOH (wherein each "-" symbol (instance) includes an optional linker). The linker may be any amino acid sequence. The linker may be, for example, approximately 2-10, approximately 5-10, approximately 5-20, or approximately 10-25 amino acids in length. The linker may be at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acids in length. The linker may have a length of less than 30, less than 29, less than 28, less than 27, less than 26, less than 25, less than 24, less than 23, less than 22, less than 21, less than 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 12, less than 11, or less than 10 amino acids. In some embodiments, the linker includes an XTEN linker (16 amino acids). In some embodiments, the fusion protein may include the following structure: NH2-[cytidine deaminase domain]-[Cas protein]-[UGI domain]-[UGI domain]-COOH (wherein each "-" symbol includes an optional linker). In some embodiments, the fusion protein may further include a nuclear localization sequence (NLS). In some embodiments, the fusion protein comprises the following structure: NH2-[cytidine deaminase domain]-[Cas9 protein]-[UGI domain]-[NLS]-COOH (wherein each "-" symbol includes an optional linker). In some embodiments, the fusion protein may include the amino acid sequence encoded by or corresponding to SEQ ID NO: 7 or SEQ ID NO: 8.

[0043] c) gRNA A CRISPR / Cas-based base editing system may contain at least one gRNA. The gRNA may target the dystrophin gene. The gRNA may bind to and target a portion of the dystrophin gene. The gRNA may target RNA splicing sites within the dystrophin gene. The gRNA may target RNA splicing sites within a mutated dystrophin gene. At least one gRNA may target a nucleic acid sequence containing Sequence ID No. 1. In some embodiments, at least one gRNA is encoded by a nucleic acid sequence containing Sequence ID No. 1. The gRNA results in targeting by the CRISPR / Cas-based base editing system. The gRNA is a fusion of two non-coding RNAs, namely crRNA and tracrRNA. The sgRNA can target any desired DNA sequence by complementary base pairing, exchanging a sequence encoding a 20 bp protospacer that provides targeting specificity with the desired DNA target. The gRNA mimics the naturally occurring crRNA:tracrRNA double helix involved in the type II effector system. For example, this double helix, which may contain a crRNA of 42 nucleotides and a tracrRNA of 75 nucleotides, acts as a guide for Cas9.

[0044] In some embodiments, at least one gRNA can target and bind to a target region. In some embodiments, 1 to 20 gRNAs can be used to modify a target gene, for example, to modify a splice acceptor site. For example, 1 to 20 gRNAs, 1 to 15 gRNAs, 1 to 10 gRNAs, 1 to 5 gRNAs, 2 to 20 gRNAs, 2 to 15 gRNAs, 2 to 10 gRNAs, 2 to 5 gRNAs, 5 to 20 gRNAs, 5 to 15 gRNAs, or 5 to 10 gRNAs can be included in a CRISPR / Cas-based base editing system and used to modify a splice acceptor site. In some embodiments, at least one gRNA, at least two gRNAs, at least three gRNAs, at least four gRNAs, at least five gRNAs, at least six gRNAs, at least seven gRNAs, at least eight gRNAs, at least nine gRNAs, at least ten gRNAs, at least eleven gRNAs, at least twelve gRNAs, at least thirteen gRNAs, at least fourteen gRNAs, at least fifteen gRNAs, or at least twenty gRNAs may be included in a CRISPR / Cas-based base editing system and used to modify splice acceptor sites. In some embodiments, fewer than 20 gRNAs, fewer than 19 gRNAs, fewer than 18 gRNAs, fewer than 17 gRNAs, fewer than 16 gRNAs, fewer than 15 gRNAs, fewer than 14 gRNAs, fewer than 13 gRNAs, fewer than 12 gRNAs, fewer than 11 gRNAs, fewer than 10 gRNAs, fewer than 9 gRNAs, fewer than 8 gRNAs, fewer than 7 gRNAs, fewer than 6 gRNAs, fewer than 5 gRNAs, fewer than 4 gRNAs, or fewer than 3 gRNAs may be included in a CRISPR / Cas-based base editing system and used to modify splice acceptor sites.

[0045] CRISPR / Cas-based base editing systems can use gRNAs of various sequences and lengths. The gRNA may contain a complementary polynucleotide sequence to the target DNA sequence, such as the target sequence containing SEQ ID NO: 1, or a complementary polynucleotide sequence to the target sequence containing SEQ ID NO: 1, followed by an NGG. The gRNA may contain a "G" at the 5' end of the complementary polynucleotide sequence. The gRNA may contain a complementary polynucleotide sequence to the target DNA sequence, followed by an NGG, of 5–40 base pairs, 5–35 base pairs, 5–30 base pairs, 10–35 base pairs, or 10–30 base pairs. The gRNA may contain a complementary polynucleotide sequence of the target DNA sequence, followed by an NGG, comprising at least 10 base pairs, at least 11 base pairs, at least 12 base pairs, at least 13 base pairs, at least 14 base pairs, at least 15 base pairs, at least 16 base pairs, at least 17 base pairs, at least 18 base pairs, at least 19 base pairs, at least 20 base pairs, at least 21 base pairs, at least 22 base pairs, at least 23 base pairs, at least 24 base pairs, at least 25 base pairs, at least 30 base pairs, or at least 35 base pairs. The gRNA may contain a complementary polynucleotide sequence of the target DNA sequence, followed by an NGG, comprising less than 40 base pairs, less than 35 base pairs, less than 30 base pairs, less than 25 base pairs, less than 24 base pairs, less than 23 base pairs, less than 22 base pairs, less than 21 base pairs, less than 20 base pairs, less than 19 base pairs, less than 18 base pairs, less than 17 base pairs, less than 16 base pairs, or less than 15 base pairs. The gRNA may target at least one of the promoter region, enhancer region, or transcribed region of the target gene. The gRNA may contain a nucleic acid sequence corresponding to at least one of the following: SEQ ID NO: 1, its complement, its variant, or a fragment thereof.

[0046] 3. Compositions for restoring dystrophin function The present invention is directed toward compositions for restoring dystrophin function by modifying or removing the splice acceptor site of exon 45. The compositions may include the above-described CRISPR / Cas-based base editing system. The compositions may also include a viral delivery system. For example, the viral delivery system may include an adeno-associated viral vector or a modified lentiviral vector. Methods for introducing nucleic acids into host cells are known in this field, and nucleic acids (e.g., expression constructs) can be introduced into cells using any known method. Suitable methods include, for example, viral or bacteriophage infection, translocation, conjugation, protoplast fusion, polycation or lipid:nucleic acid conjugate, lipofection, electroporation, nucleofection, immunoliposomes, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated translocation, DEAE-dextran-mediated translocation, liposome-mediated translocation, particle gun technology, calcium phosphate precipitation, direct macroinjection, and nanoparticle-mediated nucleic acid delivery. In some embodiments, compositions may be delivered by mRNA delivery and ribonucleoprotein (RNP) complex delivery.

[0047] a) Constructs and plasmids The compositions described above may include a gene construct encoding a CRISPR / Cas-based base editing system disclosed herein. A gene construct, such as a plasmid or expression vector, may include at least one nucleic acid and / or gRNA encoding a CRISPR / Cas-based base editing system. The compositions described above may include a gene construct encoding a modified adeno-associated virus (AAV) vector and a nucleic acid sequence encoding a CRISPR / Cas-based base editing system disclosed herein. In some embodiments, the compositions described above may include a gene construct encoding a modified adenovirus vector and a nucleic acid sequence encoding a CRISPR / Cas-based base editing system disclosed herein. A gene construct, such as a plasmid, may include a nucleic acid encoding a CRISPR / Cas-based base editing system. The compositions described above may include a gene construct encoding a modified lentiviral vector. A gene construct, such as a plasmid, may include a nucleic acid encoding a fusion protein and at least one gRNA. The gene construct may exist in cells as a functional extrachromosomal molecule. A gene construct can be a linear minichromosome containing centromeres, telomeres, plasmids, or cosmids.

[0048] Furthermore, a gene construct may be part of the genome of a recombinant viral vector, including recombinant lentiviruses, recombinant adenoviruses, and recombinant adenovirus-associated viruses. A gene construct may be part of the genetic material in a weakened living microorganism or a recombinant microbial vector living in a cell. A gene construct may contain regulatory elements for gene expression of a nucleic acid coding sequence. These regulatory elements may be promoters, enhancers, start codons, stop codons, or polyadenylation signals.

[0049] Nucleic acid sequences can constitute a gene construct that may be a vector. A vector can express fusion proteins, such as CRISPR / Cas-based base editing systems, in mammalian cells. A vector may be recombinant. A vector may contain heterologous nucleic acids encoding fusion proteins, such as CRISPR / Cas-based base editing systems. A vector may be a plasmid. A vector may be useful for transposing cells with nucleic acids encoding a CRISPR / Cas-based base editing system, and the transformed host cells are cultured and maintained under conditions that allow for the expression of the CRISPR / Cas-based base editing system. The coding sequence can be optimized for stability and high expression levels. In some cases, codons are selected to reduce RNA secondary structure formation, such as those caused by intramolecular binding.

[0050] The vector may contain heterologous nucleic acid encoding a CRISPR / Cas-based base editing system, and may further contain a start codon that may be upstream of the coding sequence of the CRISPR / Cas-based base editing system, and a stop codon that may be downstream of the coding sequence of the CRISPR / Cas-based base editing system. The start and stop codons may be in-frame with the coding sequence of the CRISPR / Cas-based base editing system. The vector may also contain a promoter operably linked to the coding sequence of the CRISPR / Cas-based base editing system. The CRISPR / Cas-based base editing system may be under photo-inducible or chemical-inducible control to enable dynamic control of base editing in space and time. Promoters operably linked to the coding sequence of a CRISPR / Cas-based base editing system may include human immunodeficiency virus (HIV) promoters such as Simian virus 40 (SV40) promoter, mouse mammary cancer virus (MMTV) promoter, and bovine immunodeficiency virus (BIV) terminal repeat (LTR) promoter; cytomegalovirus (CMV) promoters such as Moloney virus promoter, aconite leukemia virus (ALV) promoter, and CMV pre-early promoter; Epstein-Barr virus (EBV) promoter; or Roussarcoma virus (RSV) promoter. Alternatively, promoters may be human gene-derived promoters such as human ubiquitin C (hUbC), human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein. Furthermore, promoters may be tissue-specific promoters, such as natural or synthetic muscle or skin-specific promoters. Examples of such promoters are described in U.S. Patent Application Publication US20040175727, the contents of which are incorporated herein in their entirety.

[0051] The vector may also include polyadenylation signals that may be downstream of CRISPR / Cas-based base editing systems. These polyadenylation signals may be SV40 polyadenylation signals, LTR polyadenylation signals, bovine growth hormone (bGH) polyadenylation signals, human growth hormone (hGH) polyadenylation signals, or human β-globin polyadenylation signals. The SV40 polyadenylation signal may be a polyadenylation signal from the pCEP4 vector (Invitrogen, San Diego, California). The vector may also include a CRISPR / Cas-based base editing system or an enhancer upstream of the sgRNA. The enhancer may be required for DNA expression. The enhancer may be human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer such as those derived from CMV, HA, RSV, or EBV. Polynucleotide functional enhancers are described in U.S. Patents 5,593,972, 5,962,428, and WO94 / 016737, the contents of which are fully incorporated for reference. The vector may also include a mammalian origin of replication to maintain the vector outside the chromosome and generate multiple copies of the vector in the cell. The vector may also include regulatory sequences that may be well-suited for gene expression in mammalian or human cells administered with the vector. The vector may also include a reporter gene such as green fluorescent protein ("GFP") and / or a selection marker such as hygromycin ("Hygro").

[0052] The vector may be an expression vector or system for producing a protein by routine techniques and readily available starting materials, including Sambrook et al., Molecular Cloning and Laboratory Manual, Second Ed., Cold Spring Harbor (1989), which is fully incorporated for reference. In some embodiments, the vector may include a nucleic acid sequence encoding a CRISPR / Cas-based base editing system, which includes a nucleic acid sequence encoding a fusion protein and a nucleic acid sequence encoding at least one gRNA, including the nucleic acid sequence of Sequence ID No. 1, its complement, its variant, or a fragment thereof. In some embodiments, the composition is delivered by mRNA and protein / RNA complexes (ribonucleoproteins (RNPs)). For example, a purified fusion protein can be combined with a guide RNA to form an RNP complex.

[0053] b) Modified lentiviral vectors A composition for modifying the splice acceptor site of exon 45 may include a modified lentiviral vector. The modified lentiviral vector includes a first polynucleotide sequence encoding a fusion protein and a second polynucleotide sequence encoding at least one gRNA. The first polynucleotide sequence may be operably ligated to a promoter. The promoter may be a constitutive promoter, an inductive promoter, an inhibitory promoter, or a regulatory promoter. The second polynucleotide sequence codes for at least one gRNA. For example, the second polynucleotide sequence may code for 1 to 20 gRNAs, 1 to 15 gRNAs, 1 to 10 gRNAs, 1 to 5 gRNAs, 2 to 20 gRNAs, 2 to 15 gRNAs, 2 to 10 gRNAs, 2 to 5 gRNAs, 5 to 20 gRNAs, 5 to 15 gRNAs, or 5 to 10 gRNAs. The second polynucleotide sequence may encode at least 1 gRNA, at least 2 gRNAs, at least 3 gRNAs, at least 4 gRNAs, at least 5 gRNAs, at least 6 gRNAs, at least 7 gRNAs, at least 8 gRNAs, at least 9 gRNAs, at least 10 gRNAs, at least 11 gRNAs, at least 12 gRNAs, at least 13 gRNAs, at least 14 gRNAs, at least 15 gRNAs, at least 16 gRNAs, at least 17 gRNAs, at least 18 gRNAs, at least 19 gRNAs, or at least 20 gRNAs. The second polynucleotide sequence may encode fewer than 20 gRNAs, fewer than 19 gRNAs, fewer than 18 gRNAs, fewer than 17 gRNAs, fewer than 16 gRNAs, fewer than 15 gRNAs, fewer than 14 gRNAs, fewer than 13 gRNAs, fewer than 12 gRNAs, fewer than 11 gRNAs, fewer than 10 gRNAs, fewer than 9 gRNAs, fewer than 8 gRNAs, fewer than 7 gRNAs, fewer than 6 gRNAs, fewer than 5 gRNAs, fewer than 4 gRNAs, or fewer than 3 gRNAs. The second polynucleotide sequence may be operably ligated to a promoter. The promoter may be a constitutive promoter, an inductive promoter, an inhibitory promoter, or a regulatory promoter. At least one gRNA may bind to a target gene or locus, such as a target region containing the splice acceptor site of exon 45.

[0054] c) Adeno-associated virus vector AAV can be used to deliver compositions to cells using various construct configurations. For example, AAV can deliver fusion proteins and gRNA expression cassettes on separate vectors. Alternatively, both fusion proteins and up to two gRNA expression cassettes can be combined in a single AAV vector within a 4.7 kb packaging limit.

[0055] The above-described composition includes a modified adeno-associated virus (AAV) vector. The modified AAV vector may be capable of delivering and expressing site-specific nucleases in mammalian cells. For example, the modified AAV vector may be an AAV-SASTG vector (Piacentino et al. (2012) Human Gene Therapy 23:635-646). The modified AAV vector may be based on one or more of several capsid types, including AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9. Modified AAV vectors can be based on AAV2 pseudotypes with alternative muscle-tropic AAV capsids such as AAV2 / 1, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV2 / 9, AAV2.5, and AAV / SASTG vectors, which efficiently transduce skeletal or cardiac muscle through systemic and local delivery (Seto et al. Current Gene Therapy (2012) 12:139-151).

[0056] 4. Method for repairing dystrophin function in subjects with mutant dystrophin genes. This specification provides a method for restoring dystrophin function (e.g., a mutant dystrophin gene, e.g., a mutant human dystrophin gene) in cells and / or subjects suffering from DMD and / or having a mutant dystrophin gene. Also provided herein is a method for treating Duchenne muscular dystrophy in subjects requiring such treatment. Furthermore, this specification provides a method for altering RNA splicing sites encoded in the genomic DNA of a subject. This method may include administering a CRISPR / Cas-based gene editing system, a polynucleotide or vector encoding the CRISPR / Cas-based gene editing system, or a composition of the CRISPR / Cas9-based gene editing system to cells or subjects or their cells, as detailed herein. In some embodiments, the subjects suffer from Duchenne muscular dystrophy.

[0057] The method may include administering a gene construct (e.g., a vector) or a composition containing the same as disclosed herein to cells or a subject. The method may also include administering a gene construct (e.g., a vector) or a composition containing the same as disclosed herein for genome editing, e.g., base editing, in skeletal muscle or cardiac muscle of a subject. The use of a gene construct (e.g., a vector) or a composition containing the same disclosed herein for delivering a CRISPR / Cas-based gene editing system to skeletal muscle or cardiac muscle may restore fully or partially functional protein expression. CRISPR / Cas-based gene editing systems have the advantages of advanced genome editing due to their high success rate and efficient gene modification. The method may include administering a CRISPR / Cas-based gene editing system, such as administering a fusion protein, a polynucleotide sequence encoding the fusion protein, and / or at least one gRNA comprising, encoding, or corresponding to, SEQ ID NO: 1, its complement, its variant, or a fragment thereof.

[0058] 5. Pharmaceutical Compositions A CRISPR / Cas-based base editing system may be present in a pharmaceutical composition. The pharmaceutical composition may contain approximately 1 ng to 10 mg of DNA encoding a CRISPR / Cas-based base editing system. The pharmaceutical composition according to the present invention is formulated according to the mode of administration to be used. If the pharmaceutical composition is for injection, it is sterile, pyrogenic, and particle-free. Isotonic formulations are preferably used. Generally, additives for isotonicity include sodium chloride, dextrose, mannitol, sorbitol, and lactose. In some cases, isotonic solutions such as phosphate-buffered saline are preferred. Gelatin and albumin are examples of stabilizers. In some embodiments, vasoconstrictors are added to the formulation. Pharmaceutical compositions containing a CRISPR / Cas-based base editing system may further contain pharmaceutically acceptable excipients. These pharmaceutically acceptable excipients may be functional molecules such as vehicles, adjuvants, carriers, or diluents. Examples of pharmaceutically acceptable excipients include surfactants such as immunostimulatory complexes (ISCOMS), Freund's incomplete adjuvants, LPS analogs including monophosphoryl lipid A, muramil peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known pharmaceutically acceptable excipients.

[0059] Translocation promoters include polyanions, polycations including poly-L-glutamic acid (LGS), or lipids. The translocation promoter is poly-L-glutamic acid, and more preferably, poly-L-glutamic acid is present in a pharmaceutical composition containing a CRISPR / Cas-based base editing system at a concentration of less than 6 mg / ml. Translocation promoters may also include surfactants such as immunostimulatory complexes (ISCOMS), Freund's incomplete adjuvants, LPS analogs including monophosphoryl lipid A, muramil peptides, quinone analogs, and vesicles such as squalene and squalene, and hyaluronic acid may also be administered in combination with the gene construct. In some embodiments, the DNA vector encoding a CRISPR / Cas-based base editing system may include liposomes such as lipids, lecithin liposomes, or other liposomes known in the art as a DNA-liposome mixture (see, for example, W09324640), as well as translocation accelerators such as calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known translocation accelerators. Preferably, the translocation accelerator is a polyanion, a polycation including poly-L-glutamic acid (LGS), or a lipid.

[0060] 6.Delivery method This specification provides a method for delivering pharmaceutical formulations of CRISPR / Cas-based base editing systems in order to provide gene constructs and / or proteins of CRISPR / Cas-based base editing systems. Delivery of the CRISPR / Cas-based base editing system may be translocation or electroporation of the CRISPR / Cas-based base editing system as one or more nucleic acid molecules expressed in cells and delivered to the cell surface. CRISPR / Cas-based base editing system proteins may be delivered to cells. Nucleic acid molecules may be electroporated using a BioRad Gene Pulser Xcell or Amaxa Nucleofector IIb instrument or other electroporation device. Several different buffers may be used, including BioRad electroporation solution, Sigma phosphate-buffered saline product number D8537 (PBS), Invitrogen OptiMEM I (OM), or Amaxa Nucleofector solution V (NV). Translocation may involve a translocation reagent such as Lipofectamine 2000.

[0061] Vectors encoding CRISPR / Cas-based base-editing system proteins can be delivered to mammals by in vivo electroporation, liposome-mediated delivery, nanoparticle-assisted delivery, and / or DNA injection (also known as DNA vaccination) with or without recombinant vectors. Recombinant vectors can be delivered by any viral mode, which may be recombinant lentivirus, recombinant adenovirus, and / or recombinant adeno-associated virus. Gene expression of a target gene can be induced by introducing polynucleotides encoding a CRISPR / Cas-based base editing system protein into cells. For example, one or more polynucleotide sequences encoding a CRISPR / Cas-based base editing system directed towards a target gene can be introduced into mammalian cells. When the CRISPR / Cas-based base editing system is delivered to cells, and the resulting vector is delivered to mammalian cells, the transfected cells express the CRISPR / Cas-based base editing system. Gene expression of a target gene can be induced or modulated in mammals by administering a CRISPR / Cas-based base editing system to mammals. Mammals may include humans, non-human primates, cattle, pigs, sheep, goats, antelopes, bison, buffalo, bovids, deer, hedgehogs, elephants, llamas, alpacas, mice, rats, or chickens, preferably humans, cattle, pigs, or chickens.

[0062] When the gene constructs or compositions disclosed herein are delivered to tissue, and the resulting vectors to mammalian cells, the translocated cells express gRNA molecules and Cas9 molecules. The gene constructs or compositions can be administered to mammals to alter gene expression or remanipulate or modify the genome. For example, the gene constructs or compositions can be administered to mammals to repair dystrophin function. Mammals may be humans, non-human primates, cattle, pigs, sheep, goats, antelopes, bison, buffalo, bovids, deer, hedgehogs, elephants, llamas, alpacas, mice, rats, or chickens, preferably humans, cattle, pigs, or chickens. Genetic constructs (e.g., vectors) encoding gRNA and Cas9 molecules can be delivered to mammals by in vivo electroporation, liposome-mediated delivery, nanoparticle-enhanced delivery, and / or DNA injection (also known as DNA vaccination) with or without recombinant vectors. Recombinant vectors can be delivered by any viral mode. Viral modes can be recombinant lentiviruses, recombinant adenoviruses, and / or recombinant adeno-associated viruses.

[0063] Genetic repair of dystrophin function in the dystrophin gene (e.g., human dystrophin gene) can be achieved by introducing a gene construct (e.g., a vector) or a composition containing the same into cells. In certain embodiments, the gene construct (e.g., a vector) or a composition containing the same disclosed herein is introduced into myoblasts derived from DMD patients. In certain embodiments, the gene construct (e.g., a vector) or a composition containing the same is introduced into fibroblasts derived from DMD patients, and the genetically corrected fibroblasts can be treated with MyoD to induce differentiation into myoblasts, which can then be transplanted into a subject, such as damaged muscle of the subject, to verify whether the corrected dystrophin protein is functional and / or to treat the subject. Modified cells include induced pluripotent stem cells, bone marrow-derived precursors, skeletal muscle precursors, human skeletal myoblasts derived from DMD patients, and CD133 + These can be stem cells, such as medium angioblasts, and cells transduced with MyoD or Pax7, or other myogenic progenitor cells. For example, CRISPR / Cas-based gene editing systems can induce neural or myogenic differentiation of induced pluripotent stem cells.

[0064] 7. Route of administration CRISPR / Cas-based base editing systems and their compositions can be administered to subjects by a variety of routes, including oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, inhalation, buccal administration, intrapleural, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal, subarachnoid, and intra-articular, or combinations thereof. For veterinary use, the compositions can be administered as appropriately acceptable formulations in accordance with normal veterinary practices. Veterinarians can easily determine the most appropriate drug regimen and route of administration for a particular animal. CRISPR / Cas-based base editing systems and their compositions can be administered by conventional syringes, needleless infusion devices, "microparticle impact gene guns," or by other physical methods such as electroporation ("EP"), "hydrodynamic methods," or ultrasound. The composition can be delivered to mammals by several techniques, including in vivo electroporation, liposome-mediated delivery, nanoparticle-enhanced delivery, and DNA injection (also known as DNA vaccination) with or without recombinant vectors such as recombinant lentiviruses, recombinant adenoviruses, and recombinant adenovirus-associated viruses.

[0065] The gene constructs (e.g., vectors) or compositions containing them disclosed herein may be administered to a subject by a variety of routes, including oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, inhalation, buccal administration, intrapleural, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal, subarachnoid, and intraarticular, or combinations thereof. In certain embodiments, the gene constructs (e.g., vectors) or compositions disclosed herein are administered to a subject (e.g., a subject suffering from DMD) intramuscularly, intravenously, or in combination thereof. For veterinary use, the gene constructs (e.g., vectors) or compositions disclosed herein may be administered as appropriately acceptable formulations in accordance with normal veterinary practices. A veterinarian can easily determine the most appropriate dosage regimen and route of administration for a particular animal. The compositions may be administered by conventional syringes, needleless infusion devices, "microparticle impact gene guns," or by other physical methods such as electroporation ("EP"), "hydrodynamic methods," or ultrasound.

[0066] The gene constructs (e.g., vectors) or compositions disclosed herein may be delivered to mammals by several techniques, including in vivo electroporation, liposome-mediated, nanoparticle-enhanced, recombinant lentivirus, recombinant adenovirus, and recombinant adenovirus-associated virus-assisted DNA injection (also known as DNA vaccination), with or without recombinant vectors. The compositions may be injected into skeletal muscle or cardiac muscle. For example, the compositions may be injected into the tibialis anterior muscle or tail. In some embodiments, the gene constructs (e.g., vectors) or compositions disclosed herein are administered by 1) tail vein injection into adult mice (systemic), 2) intramuscular injection into adult mice, such as local injection into the TA or gastrocnemius muscle, 3) intraperitoneal injection into P2 mice, or 4) facial vein injection into P2 mice (systemic).

[0067] 8.Cell type Any of these delivery methods and / or routes of administration can be used to deliver the (descibed) base editing systems described herein to a multitude of cell types. For example, cell types include, but are not limited to, immortalized myoblasts such as wild-type and DMD patient-derived strains, primary DMD dermal fibroblasts, induced pluripotent stem cells, bone marrow-derived precursors, skeletal muscle precursors, human skeletal myoblasts derived from DMD patients, and CD133 +Examples include cells, medium angioblasts, cardiomyocytes, hepatocytes, chondrocytes, mesenchymal progenitor cells, hematopoietic stem cells, smooth muscle cells, and cells transduced with MyoD or Pax7, or other myogenic progenitor cells. Immortalization of human myobiocytes can be used for clonal induction of genetically corrected myobiocytes. Cells can be modified ex vivo to isolate and expand clonal populations of immortalized DMD myoblasts that contain a genetically corrected or repaired dystrophin gene in the protein-coding region of the genome and are free from mutations introduced by other nucleases. Alternatively, transient in vivo delivery of CRISPR / Cas-based systems by non-viral or non-integrated viral gene transfer, or by direct delivery of gRNAs containing purified proteins and cell-permeable motifs, may enable highly specific in situ correction and / or repair with minimal or no risk of exogenous DNA integration.

[0068] 9. Kit Kits that can be used to correct mutated dystrophin genes and / or repair dystrophin function are provided herein. The kits include at least one gRNA that conjugates to, targets, encodes, or corresponds to, the polynucleotide sequence of SEQ ID NO: 1, its complement, its variant, or a fragment thereof, and instructions for using a CRISPR / Cas-based editing system for repairing dystrophin function. Kits that can be used for base editing of the dystrophin gene in skeletal muscle or cardiac muscle are also provided herein. The kits include a gene construct (e.g., a vector) or a composition containing the same for genome editing in skeletal muscle or cardiac muscle as described above, for example, base editing, and instructions for using the composition.

[0069] Instructions included in the kit may be affixed to the packaging material or included as accompanying documentation. Instructions are typically, but not limited to, documents or printed objects. Any medium capable of storing such instructions and communicating them to the end user is contemplated in this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips) and optical media (e.g., CD-ROMs). As used herein, the term “instructions” may refer to the address of an internet site providing the instructions. A gene construct (e.g., a vector) or composition containing the same for repairing dystrophin function in skeletal muscle or cardiac muscle may include a modified AAV vector containing the aforementioned gRNA molecule and fusion protein that specifically bind to and cleave a region of the dystrophin gene. The aforementioned CRISPR / Cas-based gene editing system may be included in the kit to specifically bind to and target a specific region in the mutated dystrophin gene, such as the exon 45 splice acceptor region. [Examples]

[0070] 10. Examples The foregoing description may be better understood by referring to the following examples, which are provided for illustrative purposes and are not intended to limit the scope of the invention. The invention has multiple embodiments, as illustrated by the following non-limiting examples.

[0071] (Example 1) Based on the availability of PAM, gRNAs were designed to base-edit splice acceptors (see Figures 2A and 2B). A DNA base editor system containing both Streptococcus pyogenes and Staphylococcus aureus Cas9 proteins (Figures 1A and 1B) was designed to target human dystrophin exons in the DMD gene deletion hotspot between exons 45–55. The BE4max (Addgene#112093) and AncBE4max (Addgene#112094) designs, described in Figure 1B, performed better at lower concentrations than the design in Figure 1A, where expression levels were limited. The BE4max and AncBE4max designs performed similarly. Since the gRNAs bind to a Cas9 moiety that is constant across all designs, the same gRNA can be used through multiple generations of base editors (as long as the Cas9 species remains the same).

[0072] Splice acceptor G>A base editing was assayed in various dystrophin exons by plasmid translocation (Lipofectamine 2000) of human HEK293T cells using 400 ng of gRNA plasmid and 400 ng of BE4max or AncBE4max plasmid. Deep sequencing of target sites using the MiSeq system (Illumina) was performed to determine %G>A base editing. See Table 1. While some exons showed poor editing efficiency (i.e., <0.1% editing), 7–8% of alleles were observed to be edited at exon 45 using the exon 45 gRNA sequence 5'-GTTCCTGTAAGATACCAAAA-3' (SEQ ID NO: 1). Exon 45 is the dystrophin exon whose removal may treat the second largest group of DMD patients (approximately 8%) (Aartsma-Rus et al. Human Mutation (2009) 30(3):293-9).

[0073] [Table 1]

[0074] Splice acceptor G>A base editing was assayed in exons 44 and 45 by plasmid translocation (Lipofectamine 2000) of human HEK293T cells using 400 ng of gRNA plasmid and 400 ng or 1000 ng of BE4max plasmid. Deep sequencing of target sites using the MiSeq system (Illumina) was performed to determine %G>A base editing. Translocation conditions were optimized by increasing the amount of BE3max plasmid to increase base editing. Base editing increased to 7–8% in exon 45 gRNA, as shown in Figures 3B and 3C. Appropriate exon skipping may be provided by editing both G1 and G2, as shown in Figure 3A.

[0075] To test the effect of disrupting splicing sites on exon skipping, we created a human induced pluripotent stem cell (iPSC) line containing a deletion of dystrophin exon 44. See Figures 4A–4D. This pluripotent cell line models a hereditary DMD mutation with a disrupted leading frame of the DMD gene, which can be corrected by removing exon 45. Since iPSCs do not express dystrophin, it is difficult to determine whether the edited exon is skipped. Dystrophin was expressed using MyoD overexpression in iPSCs, and RNA and protein levels were analyzed (Figure 5).

[0076] Myogenic differentiation of this Δ44 iPSC strain by lentiviral transduction of MyoD cDNA confirms that the mutation removes dystrophin protein expression. See Figure 6. AncBE4max and gRNA cassettes based on Streptococcus pyogenes dCas9 were delivered to these cells by lentiviral transduction. Figure 7 shows an outline of the procedure. 200 μL of 20× virus was used for transduction of BE4max and AncBE4max. Figures 8A and 9A show the %G>A base editing events of BE4max and AncBE4max, respectively. Figures 8B and 9B show all gVG03 d12 editing events of BE4max and AncBE4max, respectively. The APOBEC enzyme during construct design should convert G>A, but in some cases G>T or G>C events may also occur. Any of these events resulting in G removal should disrupt the splicing, and therefore the sum of “non-G” events gives the effective editing rate. Figure 10 shows Δ44 iPSC editing (percentage of reads with G edited to any other base) after 12 days using BE4max and AncBE4max. Deep sequencing showed that 22% of splice acceptors were destroyed after 12 days. Figure 12 shows % non-G base editing events in Δ44 iPSCs using AncBE4max delivered by lentivirus (lentivrus). Figure 13 shows % non-G base editing events in Δ44 iPSCs using AncBE4max delivered by electroporation. Cells were collected after treatment with gRNA lentivirus for 7 days (D7) and 14 days (D14).

[0077] Overexpression of MyoD in this edited Δ44 iPSC strain, followed by RT-PCR, confirmed that splice acceptor base editing resulted in exon 45 skipping, which repaired the dystrophin reading frame. Because AncBE4max showed higher editing, these edited cells were differentiated with MyoD, and RNA was collected to search for skipping. Figure 11 shows the RT-PCR results after 35 amplification cycles using the following primers: 5'-CTACAACAAAGCTCAGGTCG-3' (SEQ ID NO: 16) and 5'-TTCTCAGGTAAAGCTCTGGAAAC-3' (SEQ ID NO: 17). Robust exon 45 skipping was observed in cells treated with exon 45 gRNA, but not in the control without gRNA. Overexpression of MyoD in this edited Δ44 iPSC strain, followed by Western blot analysis, further confirmed that splice acceptor base editing resulted in exon 45 skipping, which repaired the dystrophin reading frame. Δ44 iPSC cells transduced with AncBE4max lentivirus and gRNA lentivirus, or WT iPSCs, were differentiated with MyoD as shown in Figure 11 above. Cell lysates were collected and Western blotting was performed using antibodies against dystrophin protein and GAPDH. Western blot (Figure 14) shows that untreated Δ44 iPSC cells had significantly reduced dystrophin protein expression, especially in the largest isoform, but base editing (with gRNA) was able to repair some dystrophin protein expression.

[0078] For the sake of completeness, various aspects of the present invention are described in the following numbered clauses: Clause 1. A CRISPR / Cas-based base editing system for modifying an RNA splicing site encoded in target genomic DNA, comprising a fusion protein and at least one guide RNA (gRNA), wherein the fusion protein comprises a Cas protein and a base editing domain. Clause 2. A CRISPR / Cas-based base editing system as described in Clause 1, wherein altering an RNA splicing site encoded in genomic DNA results in the exclusion or inclusion of at least one exon sequence in an RNA transcript. Clause 3. A CRISPR / Cas-based base-editing system for repairing dystrophin function in a target, comprising a fusion protein and at least one guide RNA (gRNA), wherein the fusion protein comprises a Cas protein and a base-editing domain. Clause 4. A CRISPR / Cas-based base editing system as described in Clause 3, wherein the subject has a mutated dystrophin gene, and at least one guide RNA (gRNA) targets an RNA splicing site in the subject's mutated dystrophin gene.

[0079] Clause 5. A CRISPR / Cas-based base editing system as described in Clause 4, wherein administration of the CRISPR / Cas-based base editing system to a subject results in the exclusion or inclusion of at least one exon sequence from or within the RNA transcript of the dystrophin gene of the subject, and the repair of the reading frame of the dystrophin gene in the subject. Clause 6. A CRISPR / Cas-based base editing system described in any one of Clauses 1 to 5, wherein at least one guide RNA (gRNA) binds to and targets a polynucleotide sequence corresponding to Sequence ID No. 1. Clause 7. A CRISPR / Cas-based base editing system as described in Clause 6, wherein at least one gRNA binds to and targets a) a fragment of SEQ ID NO: 1, b) a complement or fragment thereof of SEQ ID NO: 1, c) a nucleic acid or its complement substantially identical to SEQ ID NO: 1, or d) a polynucleotide sequence corresponding to a nucleic acid, its complement, or a sequence substantially identical thereto that hybridizes with SEQ ID NO: 1 under stringent conditions. Clause 8. A CRISPR / Cas-based base editing system as described in Clause 6, wherein at least one gRNA comprises a polynucleotide sequence corresponding to SEQ ID NO: 1, or a variant thereof.

[0080] Clause 9. A CRISPR / Cas-based base editing system according to any one of Clauses 1 to 8, wherein the Cas protein comprises Cas9, and Cas9 comprises at least one amino acid mutation that removes the nuclease activity of Cas9. Clause 10. A CRISPR / Cas-based base editing system as described in Clause 9, wherein at least one amino acid mutation is at least one of D10A, H840A, or a combination thereof in the amino acid sequence corresponding to SEQ ID NO: 2 or 3. Clause 11. A CRISPR / Cas-based base editing system as described in any one of Clauses 1 to 10, wherein the Cas protein is the Streptococcus pyogenes Cas9 protein or the Staphylococcus aureus Cas9 protein. Clause 12. A CRISPR / Cas-based base editing system as described in any one of Clauses 1 to 11, wherein the Cas protein contains the amino acid sequence of SEQ ID NO: 4 or 5. Clause 13. A CRISPR / Cas-based base editing system as described in any one of Clauses 1 to 12, wherein the base editing domain comprises (i) a cytidine deaminase domain and (ii) at least one uracil glycosylase inhibitor (UGI) domain.

[0081] Clause 14. A CRISPR / Cas-based base editing system as described in Clause 13, wherein the cytidine deaminase domain comprises an apolipoprotein B mRNA editing enzyme, a catalytic polypeptide-like (APOBEC) deaminase. Clause 15. A CRISPR / Cas-based base editing system as described in Clause 13 or 14, wherein the cytidine deaminase domain contains APOBEC1 deaminase. Clause 16. A CRISPR / Cas-based base editing system as described in any one of Clauses 13-15, wherein the cytidine deaminase domain contains rat APOBEC1 deaminase. Clause 17. A CRISPR / Cas-based base editing system as described in any one of Clauses 13 to 16, wherein at least one UGI domain includes a domain capable of inhibiting UDG activity. Clause 18. A CRISPR / Cas-based base editing system as described in Clause 17, wherein at least one UGI domain comprises an amino acid sequence encoded by the amino acid sequence of SEQ ID NO: 20, or the polynucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 18.

[0082] Clause 19. A CRISPR / Cas-based base editing system as described in any one of Clauses 1 to 18, wherein the base editing domain includes one UGI domain or two UGI domains. Clause 20. A CRISPR / Cas-based base editing system described in any one of Clauses 1 to 19, wherein the fusion protein comprises the following structure: NH2-[cytidine deaminase domain]-[Cas protein]-[UGI domain]-COOH (wherein each "-" symbol includes an optional linker). Clause 21. A CRISPR / Cas-based base editing system as described in any one of Clauses 1 to 20, wherein the fusion protein comprises the following structure: NH2-[cytidine deaminase domain]-[Cas protein]-[UGI domain]-[UGI domain]-COOH (wherein each "-" symbol includes an optional linker). Clause 22. A CRISPR / Cas-based base editing system as described in Clause 21, wherein the fusion protein further comprises a nuclear localization sequence (NLS). Clause 23. A CRISPR / Cas-based base editing system as described in Clause 22, wherein the fusion protein comprises the following structure: NH2-[cytidine deaminase domain]-[Cas9 protein]-[UGI domain]-[NLS]-COOH (wherein each "-" symbol includes an optional linker).

[0083] Clause 24. A CRISPR / Cas-based base editing system as described in any one of Clauses 1 to 23, wherein the fusion protein comprises an amino acid sequence encoded by a polynucleotide corresponding to SEQ ID NO: 7 or SEQ ID NO: 8. Clause 25. An isolated polynucleotide encoding a CRISPR / Cas-based base editing system as described in any one of Clauses 1 to 24. Clause 26. An isolated polynucleotide as described in Clause 25, comprising a first polynucleotide encoding a fusion protein and a second polynucleotide encoding a gRNA. Clause 27. A vector comprising isolated polynucleotides as described in Clause 25 or 26. Clause 28. The vector according to Clause 27, wherein the vector comprises a heterologous promoter that drives the expression of an isolated polynucleotide. Clause 29. Cells containing isolated polynucleotides as described in Clause 25 or 26 or vectors as described in Clause 27 or 28.

[0084] Clause 30. A composition for repairing dystrophin function in cells having a mutant dystrophin gene, comprising a CRISPR / Cas-based base editing system as described in any one of Clauses 1 to 24. Clause 31. A kit comprising a CRISPR / Cas-based base editing system as described in any one of Clauses 1 to 24, an isolated polynucleotide as described in Clause 25 or 26, a vector as described in Clause 27 or 28, a cell as described in Clause 29, or a composition as described in Clause 30. Clause 32. A method for restoring dystrophin function in cells or subjects having a mutant dystrophin gene, comprising contacting cells or subjects with a CRISPR / Cas-based base editing system described in any one of Clauses 1 to 24. Clause 33. The method according to Clause 32, wherein the "AG" splice acceptor in exon 45 of the mutant dystrophin gene is converted to an "AA" sequence, and dystrophin function is restored by exon 45 skipping. Clause 34. The method described in Clause 32 or 33, wherein the subject has Duchenne muscular dystrophy. Another aspect of the present invention may be as follows: [1] A CRISPR / Cas-based base editing system for modifying an RNA splicing site encoded in target genomic DNA, comprising a fusion protein and at least one guide RNA (gRNA), wherein the fusion protein comprises a Cas protein and a base editing domain. [2] A CRISPR / Cas-based base editing system according to [1], wherein modifying an RNA splicing site encoded in genomic DNA results in the exclusion or inclusion of at least one exon sequence in an RNA transcript. [3] A CRISPR / Cas-based base editing system for repairing dystrophin function in a target, comprising a fusion protein and at least one guide RNA (gRNA), wherein the fusion protein contains a Cas protein and a base editing domain. [4] A CRISPR / Cas-based base editing system as described in [3] above, wherein the subject has a mutated dystrophin gene, and at least one guide RNA (gRNA) targets an RNA splicing site in the subject's mutated dystrophin gene. [5] The CRISPR / Cas-based base editing system described in [4], wherein administration of the CRISPR / Cas-based base editing system to a target results in the exclusion or inclusion of at least one exon sequence from or within the RNA transcript of the target dystrophin gene, and the repair of the reading frame of the dystrophin gene in the target. [6] A CRISPR / Cas-based base editing system according to any one of items [1] to [5] above, wherein at least one guide RNA (gRNA) binds to and targets a polynucleotide sequence corresponding to Sequence ID No. 1. [7] At least one gRNA, a) Fragment of Sequence ID 1, b) The complement or fragment thereof of Sequence ID No. 1, c) A nucleic acid or its complement that is substantially identical to Sequence ID No. 1, or d) A nucleic acid that hybridizes with sequence number 1 under stringent conditions, its complement, or a sequence substantially identical thereto. A CRISPR / Cas-based base editing system described in [6] above, which binds to and targets a corresponding polynucleotide sequence. [8] A CRISPR / Cas-based base editing system according to [6], wherein at least one gRNA comprises a polynucleotide sequence corresponding to Sequence ID No. 1, or a variant thereof. [9] A CRISPR / Cas-based base editing system according to any one of items [1] to [8] above, wherein the Cas protein contains Cas9, and Cas9 contains at least one amino acid mutation that removes the nuclease activity of Cas9.

[10] A CRISPR / Cas-based base editing system according to [9], wherein at least one amino acid mutation is at least one of D10A, H840A, or a combination thereof in the amino acid sequence corresponding to SEQ ID NO: 2 or 3.

[11] A CRISPR / Cas-based base editing system according to any one of items [1] to

[10] above, wherein the Cas protein is the Streptococcus pyogenes Cas9 protein or the Staphylococcus aureus Cas9 protein.

[12] A CRISPR / Cas-based base editing system according to any one of items [1] to

[11] above, wherein the Cas protein comprises the amino acid sequence of SEQ ID NO: 4 or 5.

[13] A CRISPR / Cas-based base editing system according to any one of items [1] to

[12] above, wherein the base editing domain comprises (i) a cytidine deaminase domain and (ii) at least one uracil glycosylase inhibitor (UGI) domain.

[14] A CRISPR / Cas-based base editing system as described in

[13] , wherein the cytidine deaminase domain comprises an apolipoprotein B mRNA editing enzyme and a catalytic polypeptide-like (APOBEC) deaminase.

[15] A CRISPR / Cas-based base editing system according to

[13] or

[14] , wherein the cytidine deaminase domain contains APOBEC1 deaminase.

[16] A CRISPR / Cas-based base editing system according to any one of items

[13] to

[15] , wherein the cytidine deaminase domain contains rat APOBEC1 deaminase.

[17] A CRISPR / Cas-based base editing system according to any one of items

[13] to

[16] , wherein at least one UGI domain comprises a domain capable of inhibiting UDG activity.

[18] A CRISPR / Cas-based base editing system according to

[17] , wherein at least one UGI domain comprises an amino acid sequence encoded by the amino acid sequence of SEQ ID NO: 20, or the polynucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 18.

[19] A CRISPR / Cas-based base editing system according to any one of items [1] to

[18] above, wherein the base editing domain comprises one UGI domain or two UGI domains.

[20] The fusion protein has the following structure: NH 2 A CRISPR / Cas-based base editing system according to any one of items [1] to

[19] above, comprising -[cytidine deaminase domain]-[Cas protein]-[UGI domain]-COOH (wherein each "-" symbol includes an optional linker).

[21] The fusion protein has the following structure: NH 2 A CRISPR / Cas-based base editing system according to any one of items [1] to

[20] above, comprising -[cytidine deaminase domain]-[Cas protein]-[UGI domain]-[UGI domain]-COOH (wherein each "-" symbol includes an optional linker).

[22] A CRISPR / Cas-based base editing system according to

[21] , wherein the fusion protein further comprises a nuclear localization sequence (NLS).

[23] The fusion protein has the following structure: NH 2 A CRISPR / Cas-based base editing system as described in

[22] above, comprising -[cytidine deaminase domain]-[Cas9 protein]-[UGI domain]-[NLS]-COOH (wherein each of the "-" symbols includes an optional linker).

[24] A CRISPR / Cas-based base editing system according to any one of items [1] to

[23] , wherein the fusion protein comprises an amino acid sequence encoded by a polynucleotide corresponding to SEQ ID NO: 7 or SEQ ID NO: 8.

[25] An isolated polynucleotide encoding a CRISPR / Cas-based base editing system as described in any one of items [1] to

[24] above.

[26] The isolated polynucleotide according to

[25] , wherein the polynucleotide comprises a first polynucleotide encoding a fusion protein and a second polynucleotide encoding a gRNA.

[27] A vector comprising the isolated polynucleotide described in

[25] or

[26] above.

[28] The vector according to

[27] , wherein the vector comprises a heterologous promoter that drives the expression of an isolated polynucleotide.

[29] Cells containing the isolated polynucleotide described in

[25] or

[26] above, or the vector described in

[27] or

[28] above.

[30] A composition for repairing dystrophin function in cells having a mutant dystrophin gene, comprising a CRISPR / Cas-based base editing system as described in any one of items [1] to

[24] above.

[31] A kit comprising a CRISPR / Cas-based base editing system as described in any one of items [1] to

[24] above, an isolated polynucleotide as described in

[25] or

[26] above, a vector as described in

[27] or

[28] above, a cell as described in

[29] above, or a composition as described in

[30] above.

[32] A method for restoring dystrophin function in cells or subjects having a mutant dystrophin gene, comprising contacting cells or subjects with a CRISPR / Cas-based base editing system described in any one of items [1] to

[24] above.

[33] The method according to

[32] , wherein the "AG" splice acceptor in exon 45 of the mutant dystrophin gene is converted to an "AA" sequence, and dystrophin function is repaired by exon 45 skipping.

[34] The method according to

[32] or

[33] , wherein the subject has Duchenne muscular dystrophy.

[0085] array Exon 45 gRNA target sequence (SEQ ID NO: 1) GTTCCTGTAAGATACCAAAA Streptococcus pyogenes Cas9 (SEQ ID NO: 2)

[0086] Staphylococcus aureus Cas9 molecule (SEQ ID NO: 3)

[0087] Streptococcus pyogenes Cas9 (possessing D10A) (SEQ ID NO: 4)

[0088] Streptococcus pyogenes Cas9 (possessing D10A and H849A) (SEQ ID NO: 5)

[0089] The polynucleotide encoding UGI-1 (SEQ ID NO: 6) ACTAATCTGAGCGACATCATTGAGAAGGAGACTGGGAAACAGCTGGTCATTCAGGAGTCCATCCTGATGCTGCCTGAGGAGGTGGAGGAAGTGATCGGCAACAAGCCAGAGTCTGACATCCTGG TGCACACCGCCTACGACGAGTCCACAGATGAGAATGTGATGCTGCTGACCTCTGACGCCCCCGAGTATAAGCCTTGGGCCCTGGTCATCCAGGATTCTAACGGCGAGAATAAGATCAAGATGCTG

[0090] pCMV_BE4max sequence (sequence number 7)

[0091] pCMV_AncBE4max sequence (sequence number 8)

[0092] Exon 44 gRNA target sequence (SEQ ID NO: 9) CGCCTGCAGGTAAAAGCATA PAM (Sequence ID 10) NGG PAM (Sequence ID 11) NNNRRT PAM (Sequence ID 12) NNGRR (R=A or G)

[0093] PAM (Sequence ID 13) NNGRRN(R=A or G) PAM (Sequence ID 14) NNGRRT(R=A or G) PAM (Sequence ID 15) NNGRRV(R=A or G, V=A, C, or G) RT-PCR primer (SEQ ID NO: 16) CTACAACAAAGCTCAGGTCG RT-PCR primer (SEQ ID NO: 17) TTCTCAGGTAAAGCTCTGGAAAC

[0094] Polynucleotide encoding UGI-2 (SEQ ID NO: 18) ACCAACCTGTCTGACATCATCGAGAAGGAGACAGGCAAGCAGCTGGTCATCCAGGAGAGCATCCTGATGCTGCCCGAAGAAGTCGAAGAAGTGATCGGAAACAAGCCTGAGAGCGATATCCTGGTCCATACCGCCTACGAGAGAGTACCGACGAAAATGTGATGCTGCTGACATCCGACGCCCCAGAGTATAAGCCCTGGGCTCTGGTCATCCAGGATTCCAACGGAGAGAACAAAATCAAAATGCTG PAM (Sequence ID 19) NGA UGI polypeptide (SEQ ID NO: 20) TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML

Claims

1. A CRISPR / Cas-based base editing system for modifying RNA splicing sites encoded in target genomic DNA, wherein the CRISPR / Cas-based base editing system is as follows: A fusion protein or a polynucleotide encoding a fusion protein, wherein the fusion protein comprises Cas9, a cytidine deaminase domain containing rat APOBEC1 deaminase, and at least one uracilglycosylase inhibitor (UGI) domain containing the amino acid sequence of SEQ ID NO: 20, and At least one guide RNA (gRNA) or a polynucleotide encoding a gRNA, wherein the gRNA targets an RNA splicing site containing the polynucleotide sequence of SEQ ID NO: 1 or the complement of SEQ ID NO: 1, and the "AG" splice acceptor in exon 45 of the mutant dystrophin gene is converted to the "AA" sequence. A base editing system based on the aforementioned CRISPR / Cas, including the above.

2. A CRISPR / Cas-based base editing system according to claim 1, wherein the CRISPR / Cas-based base editing system modifies the splice acceptor of the RNA splicing site, and the gRNA targets the splice acceptor.

3. A CRISPR / Cas-based base editing system according to claim 1, wherein altering an RNA splicing site encoded in genomic DNA results in the exclusion or inclusion of at least one exon sequence in an RNA transcript.

4. The CRISPR / Cas-based base editing system according to claim 1, wherein the RNA splicing site is located in a mutant dystrophin gene in the target genomic DNA, and one or more exons of the mutant dystrophin gene are skipped by the CRISPR / Cas-based base editing system, thereby repairing the reading frame of the dystrophin gene.

5. A CRISPR / Cas-based base editing system according to claim 3, wherein altering an RNA splicing site encoded in genomic DNA results in the elimination of at least one exon sequence in the RNA transcript of the dystrophin gene in the target genomic DNA, wherein the at least one exon sequence includes exon 45.

6. A CRISPR / Cas-based base editing system according to claim 4, wherein dystrophin function is repaired by exon 45 skipping.

7. A CRISPR / Cas-based base editing system for repairing dystrophin function in a target, comprising a fusion protein and at least one guide RNA (gRNA), wherein the fusion protein comprises a Cas9 protein, a cytidine deaminase domain including rat APOBEC1 deaminase, and at least one uracilglycosylase inhibitor (UGI) domain including the amino acid sequence of SEQ ID NO: 20, and the at least one gRNA targets an RNA splicing site including the polynucleotide of SEQ ID NO: 1 or the complement of SEQ ID NO: 1, and converts the "AG" splice acceptor in exon 45 of the mutant dystrophin gene to an "AA" sequence.

8. A CRISPR / Cas-based base editing system according to claim 7, wherein the target has a mutated dystrophin gene, and at least one guide RNA (gRNA) targets an RNA splicing site in the mutated dystrophin gene of the target.

9. The CRISPR / Cas-based base editing system according to claim 8, wherein administration of the CRISPR / Cas-based base editing system to a target results in the exclusion or inclusion of at least one exon sequence from or within the RNA transcript of the target dystrophin gene, and the repair of the reading frame of the dystrophin gene in the target.

10. A CRISPR / Cas-based base editing system according to any one of claims 1 to 9, wherein Cas9 comprises at least one amino acid mutation that removes the nuclease activity of Cas9.

11. The CRISPR / Cas-based base editing system according to claim 10, wherein at least one amino acid mutation is at least one of D10A, H840A, or a combination thereof in the amino acid sequence of SEQ ID NO:

2.

12. A CRISPR / Cas-based base editing system according to any one of claims 1 to 11, wherein the Cas protein is the Cas9 protein of Streptococcus pyogenes or the Cas9 protein of Staphylococcus aureus.

13. A CRISPR / Cas-based base editing system according to any one of claims 1 to 12, wherein the Cas protein comprises the amino acid sequence of SEQ ID NO: 4 or 5.

14. A CRISPR / Cas-based base editing system according to any one of claims 1 to 13, wherein the fusion protein comprises one UGI domain or two UGI domains.

15. The fusion protein has the following structure: NH 2 A CRISPR / Cas-based base editing system according to any one of claims 1 to 14, comprising -[cytidine deaminase domain]-[Cas protein]-[UGI domain]-COOH (wherein each of the "-" symbols includes an optional linker).

16. The fusion protein has the following structure: NH 2 A CRISPR / Cas-based base editing system according to any one of claims 1 to 15, comprising -[cytidine deaminase domain]-[Cas protein]-[UGI domain]-[UGI domain]-COOH (wherein each of the "-" symbols includes an optional linker).

17. The CRISPR / Cas-based base editing system according to claim 16, wherein the fusion protein further comprises a nuclear localization sequence (NLS).

18. The fusion protein has the following structure: NH 2 A CRISPR / Cas-based base editing system according to claim 17, comprising -[cytidine deaminase domain]-[Cas9 protein]-[UGI domain]-[NLS]-COOH (wherein each of the "-" symbols includes an optional linker).

19. A CRISPR / Cas-based base editing system according to any one of claims 1 to 18, wherein the fusion protein comprises an amino acid sequence encoded by the polynucleotide sequence of SEQ ID NO: 7 or SEQ ID NO:

8.

20. An isolated polynucleotide encoding a CRISPR / Cas-based base editing system according to any one of claims 1 to 19.

21. The isolated polynucleotide according to claim 20, wherein the polynucleotide comprises a first polynucleotide encoding a fusion protein and a second polynucleotide encoding a gRNA.

22. A vector comprising an isolated polynucleotide according to claim 20 or 21.

23. The vector according to claim 22, wherein the vector comprises a heterologous promoter that drives the expression of an isolated polynucleotide.

24. A cell comprising an isolated polynucleotide according to claim 20 or 21, or a vector according to claim 22 or 23.

25. Use of a composition comprising a CRISPR / Cas-based base editing system according to any one of claims 1 to 19 in the manufacture of a drug for repairing dystrophin function in cells having a mutant dystrophin gene.

26. A kit comprising a CRISPR / Cas-based base editing system according to any one of claims 1 to 19, an isolated polynucleotide according to claim 20 or 21, a vector according to claim 22 or 23, a cell according to claim 24, or a composition according to claim 25.

27. A composition for repairing dystrophin function in cells or subjects having a mutant dystrophin gene, comprising a CRISPR / Cas-based base editing system according to any one of claims 1 to 19.

28. The composition according to claim 27, wherein dystrophin function is restored by exon 45 skipping.

29. The composition according to claim 27 or 28, wherein the subject is suffering from Duchenne muscular dystrophy.

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