Identification of skeletal myoblast progenitor cell lineages using CRISPR / CAS9-based transcriptional activators

Abstract

This invention provides gRNAs and DNA targeting systems that target Pax7, as well as compositions for activating the endogenous myogenic transcription factor Pax7 in cells. [Solution] Disclosed herein are methods and systems for increasing Pax7 expression, methods for activating the endogenous myogenic transcription factor Pax7 in cells, methods for differentiating stem cells into skeletal muscle progenitor cells, and compositions and methods for treating subjects requiring regenerated muscle progenitor cells. The compositions and methods may comprise a Cas9-based transcription activator protein and at least one guide RNA (gRNA) that targets Pax7.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 888,916, filed on August 19, 2019, and U.S. Provisional Patent Application No. 62 / 968,743, filed on January 31, 2020, each of which is incorporated herein by reference in its entirety. Statement Regarding Federally Sponsored Research This invention was made with government support under grants 1DP2 - OD008586 and 1R01DA036865 awarded by the National Institutes of Health. The government has certain rights in this invention. Field The present disclosure relates to compositions and methods for increasing the expression of Pax7 in stem cells, inducing the differentiation of stem cells into skeletal muscle progenitor cells, and using these skeletal muscle progenitor cells to regenerate damaged muscle tissue.

Background Art

[0002] Introduction Human pluripotent stem cells (hPSCs) are a promising cell source for regenerative medicine, disease modeling, and drug discovery in the pathology of muscle diseases. Targeted differentiation of hPSCs into skeletal muscle cells can be achieved by stepwise small molecule-based protocols or ectopic expression of transgenes. While having the advantage of being transgene-free, small molecule-based protocols tend to be relatively inefficient over the long term and lack the scalability required for cell therapy or drug screening applications. Transgene-based methods rely on the overexpression of major myogenic transcription factors, including Pax3, Pax7, and MyoD. These protocols are highly efficient in producing populations of myobiogenic cells, and they do so more rapidly than transgene-free methods. The creation of satellite cells, such as skeletal muscle stem cell populations, is particularly attractive for myobiogenic cell therapy. While satellite cells can robustly regenerate damaged muscle in vivo, they cannot be isolated or enlarged ex vivo without losing their stem cell characteristics, resulting in a loss of engraftment ability. Therefore, the creation of functional Pax7+ satellite cells from hPSCs has been attempted by pairing various differentiation protocols with exogenous Pax7 cDNA overexpression. There is a need for alternative methods to create populations of myobiogenic cells. [Overview of the project]

[0003] In one embodiment, the disclosure relates to a guide RNA (gRNA) molecule that targets Pax7 or the promoter or regulatory element of the Pax7 gene. The gRNA may comprise a polynucleotide sequence corresponding to at least one of sequence numbers 1-8 or 69-76, or a variant thereof. In a further embodiment, the disclosure relates to a DNA targeting system for increasing Pax7 expression. The DNA targeting system may include at least one gRNA that binds to and targets the Pax7 gene or a portion thereof. In some embodiments, the at least one gRNA includes a polynucleotide sequence corresponding to at least one of sequence numbers 1-8 or 69-76, or a variant thereof. In some embodiments, the DNA targeting system further comprises a clustered, regularly spaced, short palindromic repeat-associated (Cas) protein or fusion protein, the fusion protein comprising two heterologous polypeptide domains, the first polypeptide domain comprising a Cas protein, a zinc finger protein, or a TALE protein, and the second polypeptide domain having transcriptional activation activity. In some embodiments, the Cas protein comprises a Streptococcus pyogenes Cas9 molecule or a variant thereof. In some embodiments, the fusion protein is VP64-dCas9-VP64( VP64 dCas9 VP64 ) includes. In some embodiments, the Cas protein includes Cas9 that recognizes the protospacer adjacent motif (PAM) of NGG (SEQ ID NO: 31), NGA (SEQ ID NO: 32), NGAN (SEQ ID NO: 33), or NGNG (SEQ ID NO: 34). Another aspect of this disclosure provides an isolated polynucleotide sequence comprising the gRNA molecule disclosed herein. Another aspect of this disclosure provides isolated polynucleotide sequences encoding the DNA targeting systems disclosed herein. Another aspect of this disclosure provides a vector comprising an isolated polynucleotide sequence disclosed herein. Another aspect of this disclosure provides gRNA molecules and vectors encoding clustered, regularly spaced, short palindromic repeat-associated (Cas) proteins disclosed herein. Another aspect of this disclosure provides cells comprising a gRNA disclosed herein, a DNA targeting system disclosed herein, an isolated polynucleotide sequence disclosed herein, or a vector disclosed herein, or a combination thereof. Another aspect of this disclosure provides a pharmaceutical composition comprising a gRNA disclosed herein, a DNA targeting system disclosed herein, an isolated polynucleotide sequence disclosed herein, a vector disclosed herein, or a cell disclosed herein, or a combination thereof.

[0004] Another aspect of this disclosure provides a method for activating the endogenous myogenic transcription factor Pax7 in cells. The method may include administering to cells a gRNA disclosed herein, a DNA targeting system disclosed herein, an isolated polynucleotide sequence disclosed herein, or a vector disclosed herein. Another aspect of this disclosure provides a method for differentiating stem cells into skeletal muscle progenitor cells. The method may include administering a gRNA disclosed herein, a DNA targeting system disclosed herein, an isolated polynucleotide sequence disclosed herein, or a vector disclosed herein to stem cells.

[0005] In some embodiments, endogenous expression of Pax7 mRNA is increased in skeletal muscle progenitor cells. In some embodiments, expression of Myf5, MyoD, MyoG, or a combination thereof is increased in skeletal muscle progenitor cells. In some embodiments, stem cells are induced to myogenic differentiation. In some embodiments, skeletal muscle progenitor cells maintain Pax7 expression after at least about six passages. Another aspect of this disclosure provides a method for treating a subject in need thereof. The method may include the step of administering the cells disclosed herein to the subject. In some embodiments, the level of dystrophin+ fibers in the subject increases. In some embodiments, muscle regeneration in the subject increases. This disclosure provides other aspects and embodiments that will become apparent from the following detailed description and accompanying drawings. [Brief explanation of the drawing]

[0006] [Figure 1A-1G]This figure shows the generation of myogenic precursors from hPSCs by VP64-dCas9-VP64-mediated activation of endogenous PAX7. (Figure 1A) Schematic diagram of myogenic differentiation of hPSCs using small molecule and lentiviral activation of PAX7. (Figure 1B) Lentiviral constructs used for gRNA and inducible VP64-dCas9-VP64 and PAX7 cDNA expression. (Figure 1C) Representative phase-contrast image showing morphological changes during the first 10 days of differentiation. Scale bar = 200 μm. (Figure 1D) RNA was harvested on day 0 and day 2 for qRT-PCR analysis of mesoderm markers. Results are expressed as a doubling compared to day 0 (mean ± SEM, n = 3 independent copies). (Figure 1E) Representative FACS plot on day 14, sorted for growth of VP64-dCas9-VP64-2a-mCherry+ cells. (Figure 1F) Representative immunohistochemical staining of PAX7 5 days after sorting. Scale bar = 100 μm. (Figure 1G) Growth of purified myogenic precursors derived from iPSC differentiation during the post-sorting growth phase was monitored for 2 weeks. Doubling of growth over 2 weeks was significantly greater in VP64-dCas9-VP64 treated cells compared to PAX7 cDNA treated cells. P-values ​​were determined by one-way ANOVA followed by Tukey's post-hoc test (mean ± SEM, n = 3 independent replicas). [Figure 2A-2F]Figure 2A shows the characterization of myogenic precursors derived from iPSCs by VP64-dCas9-VP64 mediated activation of endogenous PAX7 or exogenous PAX7 cDNA expression. The relative amount of total PAX7 mRNA was determined by qRT-PCR using primers complementary to the sequence present in the gene itself. Figure 2B shows the detection of endogenous PAX7 mRNA using primers complementary to the sequence in the 3'UTR of either isoform PAX7-A or PAX7-B. Figure 2C shows the mRNA expression levels of myogenic markers MYF5, MYOD, and MYOG during the growth phase. Figure 2D shows the immunofluorescence staining of early and mature myogenic markers MYF5, MYOD, and MYOG, and myosin heavy chain (MHC). Figure 2E shows representative FACS analysis of CD29 and CD56 surface marker expression during the growth phase. Figure 2F shows the mean fluorescence intensity (MFI) of CD56 staining intensity over treatment. All p-values ​​were determined by one-way ANOVA followed by Tukey's post-hoc test (mean ± SEM, n=3 independent copies). [Figure 3A-3C] This figure demonstrates the in vivo regeneration potential of transplantation of myogenic precursors produced by VP64-dCas9-VP64 into immunodeficient mice. (Figure 3A) Detection of human-derived fibers in VP64-dCas9-VP64 treated cells one month after intramuscular injection of 5 × 10⁵ differentiated iPSCs into NSG mice pre-injected with BaCl2. Sections are stained with human-specific dystrophin and lamin A / C antibodies to mark donor-derived fibers and nuclei. Scale bar = 100 μm. (Figure 3B) Quantification of human dystrophin+ fibers in sections with the highest number of dystrophin+ fibers in each muscle. *p<0.05 was determined by Student's t-test compared to control (mean ± SEM, n=3 mice). (Figure 3C) Identification of donor-derived satellite cells expressing PAX7 and human-specific lamin A / C and located adjacent to the basement membrane as indicated by laminin staining. Scale bar = 25 μm. [Figure 4A-4D]This figure shows that induction of endogenous PAX7 expression persists after multiple passages and dox removal. (Figure 4A) Representative immunostaining of PAX7 and MHC in differentiated iPSCs after 4 passages in the presence of dox. Scale bar = 200 μm. (Figure 4B) Representative immunostaining of PAX7 and myosin heavy chain (MHC) after differentiation induction by 7 days of dox removal. Scale bar = 200 μm. (Figure 4C) Quantification of PAX7+ nuclei after 0 passages and an average of 4 additional passages, with or after dox removal (mean ± SEM, n=3 independent experiments). (Figure 4D) Representative immunostaining of the FLAG epitope against VP64-dCas9-VP64 after 7 days of dox removal. Scale bar = 100 μm. [Figures 5A-5D] Figure 5A shows that VP64-dCas9-VP64 leads to sustained PAX7 expression and stable chromatin remodeling at the target locus. Human genome track extending to the PAX7 TSS region, depicting H3K4me3 and H3K27ac enrichment in human skeletal muscle myoblasts (HSMMs). Data from ENCODE (GEO:GSM733637;GEO:GSM733755). Black bars indicate ChIP-qPCR target regions. Figure 5B shows targeted activation of endogenous PAX7, which induced significant enrichment of H3K4me3 and H3K27ac around the TSS in the presence of dox under growth conditions. Figure 5C shows that histone mark enrichment persists after 15 days in the absence of dox under growth conditions (mean ± SEM, n=3 independent replicas). (Figure 5D) Using the N-terminal FLAG epitope tag, we verified the depletion of VP64-dCas9-VP64 after 15 days without dox, which was associated with sustained PAX7 protein expression. [Figures 6A-6E]Figure 6A shows the identification of overall transcriptional changes induced by endogenous versus exogenous PAX7. Expression heatmap of inter-sample distances in a matrix using the entire gene expression profiles between the four groups and their replicas. Figure 6B shows the differential expression of the top 200 variable genes among all four groups after filtering for genes with low read counts. Colored bars indicate z-scores. Figure 6C shows Venn diagrams of overexpressed genes in each group compared to gRNA alone (duplicate > 2 and padj < 0.05). Figure 6D shows GO biological process terminology for co-genes among the three groups derived from the Venn diagram in Figure 4C. The term list was generated using Enrichr; P-values ​​were calculated using Fisher's exact test. Figure 6E shows expression profiles of selected premyogenic, myogenic, and satellite cell marker genes from RNA-seq data (mean ± SEM, n = 3 independent replicas). TPM: Transcripts per million. [Figure 7A-7C] Figures 1A-1G show the screening of gRNAs for PAX7 activation using VP64-dCas9-VP64. (Figure 7A) gRNA target sites relative to the genomic browser location of the human PAX7 gene. (Figure 7B) Cells expressing VP64-dCas9-VP64 were treated with CHIRON99021 for 2 days and lipofected with PAX7 target gRNAs. Cells were harvested after 6 days for qRT-PCR analysis. gRNAs 3, 4, 5, and 8 significantly upregulated PAX7 compared to mock transfection, but were not significantly differentiating from each other. (Figure 7C) Lentiviral transduction of gRNAs over 1 week in paraxial mesoderm cells expressing P64-dCas9-VP64 and gRNAs. gRNA 4 significantly outperformed the other gRNAs. The p-value was determined by one-way ANOVA followed by Tukey's post-hoc test; p<0.05 (mean ± SEM, n=3 independent copies). [Figure 8A-8J]Figures 2A-2F and 3A-3C illustrate the characterization and transplantation of myogenic precursors derived from H9 ESCs by VP64dCas9VP64-mediated activation of endogenous PAX7 or exogenous PAX7 cDNA expression. (Figure 8A) Representative immunostaining of PAX7 5 days after sorting. Scale bar = 100 μm. (Figure 8B) Growth curve of purified myogenic precursors during the growth phase after sorting was monitored for 2 weeks. (Figure 8C) The relative amount of total PAX7 mRNA was determined by qRT-PCR using primers complementary to the sequence present in the gene itself. (Figure 8D) Endogenous PAX7 mRNA was detected using primers complementary to the sequence (sequencing) at the 3'UTR of either the PAX7-A or PAX7-B isoform. (Figure 8E) mRNA expression levels of myogenic markers MYF5, MYOD, and MYOG during the growth phase. (Figure 8F) Representative FACS analysis of CD29 and CD56 surface marker expression during growth phase. (Figure 8G) Mean fluorescence intensity (MFI) of CD56 staining intensity over treatment. (Figure 8H) Representative immunostaining of PAX7 and MHC in differentiated H9 ESCs after 4 passages in the presence of dox. Scale bar = 200 μm. (Figure 8I) Detection of human-derived fibers in VP64dCas9VP64-treated cells 1 month after intramuscular injection of 5 × 10⁵ differentiated ESCs into BaCl2 pre-injected NSG mice. Sections are stained with human-specific dystrophin and lamin A / C antibodies to mark donor-derived fibers and nuclei. Scale bar = 100 μm. (Figure 8J) Identification of donor-derived satellite cells expressing PAX7 and human-specific lamin A / C. All P values ​​were determined by one-way ANOVA followed by Tukey's post-hoc test (mean ± SEM, n = 3 independent replicas). Scale bar = 25 μm. [Figures 9A-9E]Figures 6A-6E show the RNA-seq analysis related to the above. (Figure 9A) Multidimensional scaling (MDS) of the top 500 differentially expressed genes. (Figure 9B) Heatmap showing the differential expression of the top 50 variable genes among three PAX7 expression groups. Colored bars indicate z-scores. (Figure 9C) Expression profiles from selected genes overexpressed in response to cDNA encoding PAX7-A, from RNA-seq (mean ± SEM, n=3 independent copies). (Figure 9D) GO biological process terminology for genes specifically enriched in cells treated with VP64dCas9VP64+gRNA, PAX7-A cDNA, or PAX7-B cDNA, corresponding to the Venn diagram in Figure 4C. (Figure 9E) Additional expression profiles of known satellite cell surface markers. [Modes for carrying out the invention]

[0007] Detailed explanation Various DNA targeting systems and methods of use are disclosed herein, and may include, for example, DNA targeting systems using CRISPR / Cas, zinc fingers, or TALE.

[0008] Advances in genome engineering techniques have led to the establishment of the Type II clustered, regularly spaced, short palindromic repeat (CRISPR) / Cas9 system as a programmable transcription factor capable of targeting and repressing endogenous genes. Mutations in the catalytic residues of the Cas9 protein result in nuclease-null Cas9 (dCas9), which can be fused to various effector domains that exert their functions at precise genomic loci defined by guide RNA (gRNA). For example, fusion of dCas9 to the transactivation domain VP64 can potently activate genes in their native chromosomal contexts if the gRNA is designed at the target gene promoter. In contrast to ectopic expression of transgenes, activation of endogenous genes promotes chromatin remodeling and the induction of autonomously maintained gene networks. Targeting endogenous genes can also capture the full complexity of transcript isoforms, mRNA localization, and other effects of non-coding regulatory elements, which can be critical for proper cellular reprogramming. Cell reprogramming can be achieved using CRISPR / Cas9-based transcriptional regulators, as well as somatic cell reprogramming and targeted differentiation of pluripotent stem cells into various cell types. However, prior to the work detailed herein, there have been no demonstrations of hPSC differentiation using CRISPR / Cas9-based transcriptional activators to produce cells capable of in vivo transplantation, engraftment, and tissue regeneration, nor have there been any attempts to produce myogenic progenitor cells by activation of the endogenous Pax7 gene.

[0009] Manipulated CRISPR / Cas9-based transcription activators can potently and specifically activate endogenous fate-determining genes to direct the differentiation of pluripotent stem cells. As detailed herein, in both human ES and iPS cells, VP64-dCas9-VP64 was used to activate the endogenous myogenic transcription factor Pax7 to directly reprogram human pluripotent stem cells and direct their differentiation into skeletal muscle precursors. Functional skeletal muscle precursor cells can be induced to differentiate in vitro and, when transplanted into mice, can participate in the regeneration of damaged muscle in vivo. Compared to exogenous overexpression of Pax7 cDNA, endogenous activation results in the creation of more proliferative myogenic precursors that can maintain Pax7 expression over multiple passages under serum-free conditions while maintaining the ability to undergo terminal myogenic differentiation. Transplantation of myogenic precursors derived from endogenous activation of Pax7 into immunodeficient mice resulted in a greater number of human dystrophin+ myofibrils compared to exogenous Pax7 overexpression. The results detailed herein also revealed functional differences between myogenic precursors produced by CRISPR-based endogenous activation of Pax7 and exogenous Pax7 cDNA overexpression. These investigations demonstrate the practical applications of CRISPR / Cas9-based transcription activators for myogenic progenitor cell differentiation, as well as their potential for cell therapy and musculoskeletal regenerative medicine. The methods of these investigations can be applied using any DNA-binding domain, such as zinc finger proteins or TALE proteins, as well as Cas proteins.

[0010] A system for increasing the expression of Pax7 is described herein, which may include a Cas9 protein such as VP64-dCas9-VP64, and at least one guide RNA (gRNA) targeting a promoter or regulatory element of the Pax7 or Pax7 gene. Methods for activating endogenous myogenic transcription factor Pax7 in cells, methods for differentiating stem cells into skeletal muscle progenitor cells, and methods for treating a subject in need thereof are further provided herein. The methods may include administering to the cell or subject a system for increasing the expression of Pax7, or administering cells transduced or transfected by the system.

[0011] 1. Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0012] As used herein, the terms “comprise,” “include,” “having,” “has,” “can,” “contain,” and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures. The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. The present disclosure contemplates other embodiments that “comprise,” “consist of,” and “consist essentially of” the embodiments or elements presented herein, whether explicitly described or not. Regarding the description of numerical ranges in this specification, with the same degree of precision, each intervening number therebetween is explicitly contemplated. For example, for the numerical range of 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the numerical range of 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0013] As applied to one or more values of interest, the term "about" or "approximately" as used in this specification refers to a value that is of the same degree as the stated reference value. In certain embodiments, the term "about" refers to a range of values that fall within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less (except where such numbers would exceed 100% of the possible values) in either direction (above or below) of the stated reference value, unless otherwise specified or otherwise apparent from the context. Alternatively, "about" can mean within 3 standard deviations or more than 3 standard deviations through practice in the relevant art. Alternatively, with respect to biological systems or processes, etc., the term "about" can mean within one order of magnitude, preferably within 5-fold, more preferably within 2-fold of the value. As used interchangeably herein, "adeno-associated virus" or "AAV" 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 as a result, the virus elicits a very mild immune response.

[0014] As used herein, “amino acids” refers to naturally occurring amino acids and non-natural 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 herein by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Amino acids include side chains and polypeptide backbone portions. As used herein, the term "binding region" refers to a region within a nuclease target region that is recognized and bound by the nuclease.

[0015] In this specification, the interchangeable terms "clustered, regularly spaced 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. As used herein, “coding sequence” or “coding nucleic acid” means a nucleic acid (RNA or DNA molecule) containing a nucleotide sequence that codes for a protein. The coding sequence may further include start and terminate signals operatively linked to regulatory elements, including a promoter and a polyadenylation signal, which can direct expression in the cells of an individual or mammal to which the nucleic acid is administered. The coding sequence may be codon-optimized.

[0016] As used herein, “complementary” or “complementary” means that nucleic acids may have Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairings between nucleotides or nucleotide analogs of a nucleic acid molecule. “Complementarity” refers to a property shared between two nucleic acid sequences such that the nucleotide bases at each position would be complementary if they were aligned antiparallel to each other.

[0017] The terms “control,” “reference level,” and “reference” are used interchangeably herein. The reference level may be a predetermined value or range of values ​​adopted as a criterion for assessing measured results against it. As used herein, “control group” refers to a group of subjects that are the control. The predetermined level may be a cutoff value from the control group. The predetermined level may be the mean from the control group. The cutoff value (or predetermined cutoff value) may be determined by adaptive indicator model (AIM) methodology. The cutoff value (or predetermined cutoff value) may be determined by receiver operating curve (ROC) analysis from a biological sample of the patient group. ROC analysis, commonly known in the field of biology, is a determination of the ability of a test to distinguish one condition from another, for example, to determine the performance of each marker in identifying patients with CRC. A description of ROC analysis is provided in PJHeagerty et al. (Biometrics 2000, 56, 337-44), the disclosure of which 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 in the 25th–75th percentile range, preferably a value corresponding to the 25th, 50th, or 75th percentile, more preferably a value corresponding to the 75th percentile. Such statistical analysis may be performed using any method known in the art and may be carried out through several commercially available software packages (e.g., Analyse-it Software Ltd., Leeds, UK; StataCorp LP, College Station, TX; SAS Institute Inc., Cary, NC.). Healthy or normal levels or ranges for target or protein activity may be defined according to standard practice. Controls may be subjects or cells without the systems detailed herein. Controls may be subjects or samples derived from subjects with a known disease state.The subjects or samples derived therefrom may be healthy, pathological, pathological before treatment, pathological during treatment, pathological after treatment, or a combination thereof. As used herein, "fusion protein" refers to a chimeric protein created through the translation of two or more conjugated genes that originally encode separate proteins. Translation of the fusion gene yields a single polypeptide with functional properties derived from each of the original separate proteins.

[0018] As used herein, “gene construct” refers to a DNA or RNA molecule containing a polynucleotide that codes for a protein. The coding sequence includes start and terminate signals operatively ligated to a regulatory element, including a promoter and a polyadenylation signal, which can direct the expression of the nucleic acid molecule in the cells of an individual to which it is administered. As used herein, the term “expressible form” refers to a gene construct containing the necessary regulatory elements operatively ligated to a protein-coding sequence, such that the coding sequence would be expressed if present in the cells of an individual. As used herein, “genome editing” or “gene editing” refers to altering a gene. Genome editing may include correcting or restoring a mutant gene. Genome editing may include knocking out a gene, such as a mutant gene or a normal gene. By altering a gene of interest using genome editing, diseases may be treated or muscle repair may be enhanced.

[0019] As used herein in the context of two or more nucleic acid or polypeptide sequences, “identical” or “same” means that the sequences have a specified percentage of residues that are the same across a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences across the specified region, determining the number of positions in both sequences where identical residues exist, determining 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 the alignment results in one or more staggered ends and the specified region of comparison contains only a single sequence, the residues of the single sequence are included in the denominator but not in the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) may be considered equivalent. Identity may be performed manually or by using a computer sequencing algorithm such as BLAST or BLAST 2.0.

[0020] As used interchangeably in this specification, “mutant gene” or “mutant gene” refers to a gene that has undergone a detectable mutation. A mutant gene has undergone changes such as loss, addition, or exchange of genetic material that affect the normal transmission and expression of the gene. As used in this specification, “broken gene” refers to a mutant gene that has a mutation that causes an immature stop codon. The broken gene product is shortened compared to the full-length, unbroken gene product. As used herein, "normal gene" refers to a gene that has not undergone any alteration such as loss, addition, or exchange of genetic material. Normal genes undergo normal gene transmission and gene expression. For example, a normal gene may be a wild-type gene.

[0021] As used herein, “nucleic acid,” “oligonucleotide,” or “polynucleotide” means at least two nucleotides linked together by a covalent bond. A single-stranded description also defines the sequence of the complementary strand. Therefore, a polynucleotide also includes the complementary strand of the described single-stranded sequence. Many variants of a polynucleotide can be used for the same purposes as a given polynucleotide. Therefore, a polynucleotide also includes substantially identical polynucleotides and their complements. A single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. Therefore, a polynucleotide also includes probes that hybridize under stringent hybridization conditions. A polynucleotide may be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. Polynucleotides can be nucleic acids, natural or synthetic, DNA, genomic DNA, 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. Polynucleotides can be obtained by chemical synthesis or by recombinant methods.

[0022] An "open reading frame" refers to a sequence of codons that begins with a start codon and ends with a stop codon. In eukaryotic genes with multiple exons, introns are removed, and then the exons are joined after transcription to produce the final mRNA for protein translation. An open reading frame can be a continuous sequence of codons. In some embodiments, open reading frames apply only to spliced ​​mRNA with respect to protein expression, and not to genomic DNA.

[0023] As used herein, “operationally 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 the art, variations in this distance can be accommodated without loss of promoter function. 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.

[0024] A "peptide" or "polypeptide" is a linked sequence of two or more amino acids linked by peptide bonds. Polypeptides may be natural, synthetic, or modified or combined natural and synthetic forms. Peptides and polypeptides include proteins such as binding proteins, receptors, and antibodies. The terms "polypeptide," "protein," and "peptide" are used interchangeably herein. "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 tail domains. A "domain" is a portion of a polypeptide that forms a small unit of the polypeptide and is typically 15 to 350 amino acids long. Exemplary domains include those with enzymatic or ligand-binding activity. Typical domains consist of a series of beta-sheets and lower-organizational compartments such as alpha-helices. "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. Motifs can be 2–20, 2–15, or 2–10 amino acids long. In some embodiments, a motif contains 3, 4, 5, 6, or 7 sequential amino acids. A domain may consist of a series of motifs of the same type.

[0025] In this specification, the terms “immature stop codon” or “out-of-frame stop codon” are interchangeable and refer to nonsense mutations in the DNA sequence that result in a stop codon at a location not typically found in wild-type genes. Immature stop codons can shorten or make proteins shorter compared to their full-length counterparts.

[0026] As used herein, “promoter” means a synthetic or naturally occurring molecule that can confer, activate, or enhance the expression of nucleic acids in cells. Promoters may include one or more specific transcriptional regulatory sequences to further enhance their expression and / or alter their spatial and / or temporal expression. Promoters may also include 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 constitutively or differentially regulate the expression of gene components in relation to the cell, tissue, or organ in which expression occurs, or to the developmental stage 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, human U6 (hU6) promoter, and CMV IE promoter.

[0027] The term "recombinant," when used for 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 such modified cells. For example, a recombinant cell may express genes not found in the cell's native (naturally occurring) form, or it may express a second copy of a native gene that would otherwise be normally or abnormally expressed, underexpressed, or not expressed at all.

[0028] As used herein, “sample” or “test sample” may mean any sample in which the presence and / or level of a target is to be detected or determined, or any sample containing a DNA targeting system or its components as detailed herein. A sample may include a liquid, solution, emulsion, or suspension. A sample may include a medical sample. A sample may include any biological fluid or tissue, such as blood, whole blood, plasma and serum fractions, muscle, interstitial fluid, sweat, saliva, urine, tears, synovial fluid, bone marrow, cerebrospinal fluid, nasal secretions, sputum, amniotic fluid, bronchoalveolar lavage fluid, gastric lavage, vomit, fecal matter, lung tissue, peripheral blood mononuclear cells, total leukocytes, lymph node cells, spleen cells, tonsillar cells, cancer cells, tumor cells, bile, digestive fluids, skin, or a combination thereof. In some embodiments, a sample may include an aliquot. In other embodiments, a sample may include a biological fluid. A sample may be obtained by any means known in the art. The sample may be used directly as if obtained from a patient, or it may be pretreated by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, etc., in order to modify the characteristics of the sample in any way as described herein or otherwise known in the art. In this specification, the interchangeable terms “spacer” and “spacer region” refer to a region within a TALE or zinc finger target region that lies between, but is not part of, the binding regions for two TALE or zinc finger proteins.

[0029] As used herein, “subject” or “patient” may mean an animal that desires or requires the composition or method described herein. The subject may be human or non-human. The subject may be any vertebrate. The subject may be a mammal. The mammal may be a primate or a non-primate. Mammals may be non-primates such as cattle, pigs, camels, llamas, hedgehogs, anteaters, platypuses, elephants, alpacas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, and mice. Mammals may be primates such as humans. Mammals may be non-human primates such as monkeys, cynomolgous monkeys, rhesus monkeys, chimpanzees, gorillas, orangutans, and gibbons. The subject may be of any age or developmental stage, such as adult, juvenile, or nymph. The subject may be male. The subject may be female. In some embodiments, the subject has a specific genetic marker. The subjects may be receiving other forms of treatment.

[0030] "Substantially identical" may mean that the first and second amino acid or polynucleotide sequences are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% across regions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or 1100 amino acids or nucleotides, respectively.

[0031] A "transcription activator-like effector" or "TALE" refers to a protein structure that recognizes and binds to a specific DNA sequence. A "TALE DNA-binding domain" refers to a DNA-binding domain containing an array of 33-35 amino acid repeats in tandem, also known as an RVD module, each of which specifically recognizes a single base pair of DNA. The RVD modules can be arranged in any order to associate arrays that recognize a given sequence. The binding specificity of the TALE DNA-binding domain is determined by the RVD array followed by a single truncated repeat of 20 amino acids. A "repeat variable diresidue" or "RVD" refers to a pair of adjacent amino acid residues within a 33-35 amino acid DNA recognition motif (also known as an "RVD module") of the TALE DNA-binding domain. The RVD determines the nucleotide specificity of the RVD module. RVD modules can be combined to produce an RVD array. As used herein, "RVD array length" refers to the number of RVD modules corresponding to the length of the nucleotide sequence within the TALEN target region, i.e., the binding region, recognized by the TALEN. A TALE DNA-binding domain may have 12 to 27 RVD modules, each containing an RVD that recognizes a single base pair of DNA. Specific RVDs recognizing each of the four possible DNA nucleotides (A, T, C, and G) have been identified. Because the TALE DNA-binding domain is modular, repeats recognizing the four different DNA nucleotides can be linked together to recognize any specific DNA sequence. These target DNA-binding domains can then be combined with catalytic domains to create functional enzymes, including artificial transcription factors, methyltransferases, integrases, nucleases, and recombinases.

[0032] As used herein, “target gene” refers to any nucleotide sequence that encodes a known or putative gene product. A target gene may be a mutant gene involved in a genetic disorder. In certain embodiments, the target gene is Pax7, or a transcription factor for Pax7, or a regulatory element for Pax7. As used herein, "target region" refers to a region of a target gene that is designed to be bound by a CRISPR / Cas9-based gene editing system.

[0033] As used herein, “transgene” refers to a gene or genetic material containing a gene sequence isolated from one organism and introduced into another organism. This non-natural segment of DNA may retain the ability to produce RNA or protein in the transgenic organism, or it may alter the normal function of the genetic code of the transgenic organism. The introduction of a transgene has the potential to alter the phenotype of the organism. "Treatment" or "treating" means, when referring to the protection of a subject from a disease, suppressing, inhibiting, improving, or completely eliminating the disease. Preventing a disease involves administering the composition of the present invention to a subject before the onset of the disease. Suppressing a disease involves administering the composition of the present invention to a subject after the induction of the disease but before its clinical manifestation. Inhibiting or improving a disease involves administering the composition of the present invention to a subject after the clinical manifestation of the disease.

[0034] As used herein with respect to polynucleotides, “variant” means (i) a portion or fragment of a reference nucleotide sequence; (ii) a complement of a reference nucleotide sequence or a portion thereof; (iii) a nucleic acid substantially identical to a reference nucleic acid or its complement; or (iv) a nucleic acid that, under stringent conditions, hybridizes to a reference nucleic acid, its complement, or a sequence substantially identical thereto.

[0035] A “variant” of a peptide or polypeptide is one in which the amino acid sequence differs due to an insertion, deletion, or conservative substitution of amino acids, but retains at least one biological activity. A variant may also mean a protein having an amino acid sequence substantially identical to a reference protein that has an amino acid sequence retaining at least one biological activity. Typical examples of “biological activity” include the ability to be bound by a specific antibody or polypeptide, or to induce an immune response. A variant may also mean a functional fragment of a polypeptide. A variant may also mean multiple copies of a polypeptide. Multiple copies can exist in a tandem state or be separated by a linker. Conservative substitution of amino acids, i.e., replacing an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree and distribution of charged regions), is recognized in the art as typically involving only minor changes. These minor changes can, in part, be identified by considering the hydropathic index of the amino acid, as understood in the art (Kyte et al., J. Mol. Biol. 1982, 157, 105-132). The hydropathic index of an amino acid is based on consideration of its hydrophobicity and charge. It is known in the art that amino acids with similar hydropathic indexes can be substituted while still retaining protein function. In one embodiment, amino acids with a hydropathic index of ±2 are substituted. The hydrophilicity of amino acids can also be used to identify substitutions that would result in a protein that retains biological function. Considering the hydrophilicity of amino acids in the context of a peptide makes it possible to calculate the maximum local mean hydrophilicity of that peptide. Substitutions can be carried out using amino acids with hydrophilicity values ​​within ±2 of each other. Both the hydrophobic index and hydrophilicity value of an amino acid are influenced by the specific side chain of that amino acid. Consistent with these observations, it is understood that amino acid substitutions that are compatible with biological function depend on the relative similarity of amino acids, and in particular on the side chains of such amino acids, as revealed by their hydrophobicity, hydrophilicity, charge, size, and other properties.

[0036] 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 the Cas9 protein and at least one gRNA molecule. As used herein, "zinc finger" refers to a protein that recognizes and binds to DNA sequences. Zinc finger domains are the most common DNA-binding motifs in the human proteome. A single zinc finger contains approximately 30 amino acids, and the domain typically functions by binding to three consecutive base pairs of DNA via the interaction of a single amino acid side chain per base pair.

[0037] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have meanings commonly understood by those skilled in the art. For example, any nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. The meaning and scope of terms should be clear; however, in any potentially ambiguous matters, the definitions provided herein shall precede any dictionary or supplementary definitions. Furthermore, unless otherwise required by context, singular terms shall include plural forms, and plural terms shall include singular forms.

[0038] 2. Pax7 Pax7 (paired-box gene 7) is a protein that acts as a myogenic transcription factor. Pax7 may be a factor in the expression of neural crest markers such as Slug, Sox9, Sox10, and HNK-1. Pax7 can be expressed in the palatine shelf of the maxilla, Meckel's cartilage, midbrain, nasal cavity, nasal epithelium, nasal aponeurosis, and pons. Pax7 can bind to DNA as a heterodimer with Pax3. Pax7 can also interact with PAXBP1 and / or DAXX.

[0039] Pax7 is a transcription factor that plays a role in myogenesis by regulating muscle progenitor cell proliferation. Skeletal muscle growth and regeneration are attributed to satellite cells, which are muscle stem cells located beneath the basement membrane surrounding each myofibrils. Quiet satellite cells express the transcription factor Pax7, and when activated, they may co-express Pax7 and MyoD. Most cells can then proliferate, downregulate Pax7, and differentiate. In contrast, other cells may maintain Pax7 expression but lose MyoD expression and return to a quiescent state. When Pax7 is expressed or activated in stem cells, the stem cells can differentiate into skeletal muscle progenitor cells. Stem cells can be, for example, induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). Stem cells can be induced to differentiate into myogenic cells. In some embodiments, Pax7 expression or activation results in the expression of Myf5, MyoD, MyoG, or a combination thereof. In some embodiments, Pax7 expression or activation results in muscle regeneration. In some embodiments, Pax7 expression or activation results in an increase in muscle stem cells that can contribute to dystrophin+ fibers.

[0040] 3. CRISPR / Cas-based gene editing systems Gene constructs for genome editing, genome modification, or altering the gene expression of a gene, such as the gene encoding Pax7, are provided herein. Each gene construct comprises at least one gRNA that targets a gene sequence. The disclosed gRNAs may be included in a CRISPR / Cas9-based gene editing system that targets a region in the Pax7 gene or the promoter or regulatory element of the Pax7 gene, thereby activating the endogenous expression of Pax7.

[0041] CRISPR / Cas-based gene editing systems may be specific to the Pax7 gene or its promoter or regulatory element. CRISPR / Cas-based gene editing systems may also be CRISPR / Cas9-based gene editing systems specific to the Pax7 gene or its promoter or regulatory element. As used interchangeably herein, “clustered, regularly spaced 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. The CRISPR system is a microbial nuclease system involved in defense against invading phages and plasmids, providing a form of adaptive immunity. CRISPR loci in microbial hosts contain a combination of CRISPR-related (Cas) genes and non-coding RNA elements that can program the specificity of CRISPR-mediated nucleic acid cleavage. Short segments of foreign DNA, called spacers, are incorporated into the genome between CRISPR repeats and act as “memories” of past exposures. Cas proteins, such as the Cas9 protein, form a complex with the 3' end of an sgRNA (also interchangeably referred to herein as "gRNA"), and the protein-RNA pair recognizes its genomic target by complementary base pairing between the 5' end of the sgRNA sequence and a predetermined 20bp DNA sequence known as a protospacer. This complex is directed to homologous loci in pathogen DNA via the encoded region within the crRNA, i.e., the protospacer, and the protospacer adjacent motif (PAM) in the pathogen genome. A non-coding CRISPR array is transcribed and cleaved in direct repeats into short crRNAs containing individual spacer sequences, which direct the Cas nuclease to the target site (protospacer). By simply replacing the 20bp recognition sequence of the expressed sgRNA, the Cas9 nuclease can be directed to a new genomic target. Using CRISPR spacers, exogenous gene elements are recognized and silenced in a manner similar to RNAi in eukaryotes.

[0042] Three classes of CRISPR systems (Type I, II, and III effector systems) are known. The Type II effector system uses a single effector enzyme, such as Cas9, to cleave dsDNA by performing double-strand breaks on target DNA in four sequential steps. Compared to the Type I and III effector systems, which require multiple individual effectors acting as a complex, the Type II effector system can function in alternative backgrounds such as eukaryotic cells. The Type II effector system consists of a long pre-crRNA transcribed from a spacer-containing CRISPR locus, the Cas9 protein, and tracrRNA involved in pre-crRNA processing. The tracrRNA hybridizes to the repeat region that separates the spacer of the pre-crRNA, thus initiating dsRNA cleavage by endogenous RNase III. Following this cleavage, a second cleavage event by Cas9 occurs within each spacer, producing mature crRNA that remains bound to tracrRNA and Cas9, forming a Cas9:crRNA-tracrRNA complex.

[0043] The Cas9:crRNA-tracrRNA complex unwinds the DNA double helix and cleaves it, searching for a sequence that matches the crRNA. Target recognition occurs when complementarity is detected between the “protospacer” sequence in the target DNA and the residual spacer sequence in the crRNA. Cas9 mediates the cleavage of the target DNA if the correct protospacer adjacency motif (PAM) is also present at the 3' end of the protospacer. For protospacer targeting, the sequence must be immediately followed by the protospacer adjacency motif (PAM), which is a short sequence recognized by the Cas9 nuclease required for DNA cleavage. Different type II systems have different PAM requirements. The Streptococcus pyogenes CRISPR system may have a PAM sequence for this Cas9 (SpCas9) such as 5'-NRG-3' where R is either A or G, which characterized the specificity of this system in human cells. The inherent capability of CRISPR / Cas9-based gene editing systems lies in their simple ability to simultaneously target multiple distinct genomic loci by co-expressing a single Cas9 protein with two or more sgRNAs. For example, while the S. pyogenes type II system naturally prefers the use of "NGG" sequences where "N" can be any nucleotide, engineered systems also accept other PAM sequences such as "NAG" (Hsu et al., Nature Biotechnology 2013 doi:10.1038 / nbt.2647). Similarly, Cas9 derived from Neisseria meningitidis (NmCas9), which normally has the natural PAM NNNNGATT, exhibits activity across a variety of PAMs, including the highly degenerate NNNNGNNN PAM (Esvelt et al. Nature Methods 2013 doi:10.1038 / nmeth.2681).

[0044] The Cas9 molecule of S. aureus recognizes the sequence motif NNGRR(R=A or G) (SEQ ID NO: 38) and directs the cleavage of a target nucleic acid sequence located 1 to 10 bp upstream, for example, 3 to 5 bp from that sequence. In certain embodiments, the Cas9 molecule of S. aureus recognizes the sequence motif NNGRRN(R=A or G) (SEQ ID NO: 39) and directs the cleavage of a target nucleic acid sequence located 1 to 10 bp upstream, for example, 3 to 5 bp from that sequence. In certain embodiments, the Cas9 molecule of S. aureus recognizes the sequence motif NNGRRT(R=A or G) (SEQ ID NO: 40) and directs the cleavage of a target nucleic acid sequence located 1 to 10 bp upstream, for example, 3 to 5 bp from that sequence. In certain embodiments, the Cas9 molecule of S. aureus recognizes the sequence motif NNGRRV(R=A or G) (SEQ ID NO: 41) and directs the cleavage of a target nucleic acid sequence located 1 to 10 bp upstream, for example, 3 to 5 bp from that sequence. In the embodiments described above, N may be any nucleotide residue, such as A, G, C, or T. The Cas9 molecule may be manipulated to alter the PAM specificity of the Cas9 molecule.

[0045] An engineered form of S. pyogenes' type II effector system has been shown to function in human cells with respect to genome editing. In this system, the Cas9 protein is directed to a genomic target site by a synthetically reconstituted “guide RNA” (“gRNA”, also used herein interchangeably as a chimeric single-stranded guide RNA (“sgRNA”)), which is typically a crRNA-tracrRNA fusion that eliminates the need for RNase III and crRNA processing. CRISPR / Cas9-based engineered systems for use in genome editing and treating genetic diseases are provided herein. CRISPR / Cas9-based engineered systems can be designed to target any gene, including genes involved in genetic diseases, aging, tissue regeneration, or wound healing. A CRISPR / Cas9-based gene editing system may comprise a Cas9 protein or Cas9 fusion protein and at least one gRNA. In certain embodiments, the system comprises two gRNA molecules. Cas9 fusion proteins may contain domains with different endogenous activities toward Cas9, such as a transactivation domain.

[0046] The target gene (e.g., the Pax7 gene, or a regulatory element of the Pax7 gene) may be involved in cell differentiation or any other process in which gene activation may be desired or which may have mutations such as frameshift mutations or nonsense mutations. In some embodiments, the target or target gene includes a regulatory element of the Pax7 gene. CRISPR / Cas9-based gene editing systems may or may not mediate off-target changes to protein-coding regions of the genome. CRISPR / Cas9-based gene editing systems may or may not bind to and recognize target regions. The target gene may be the Pax7 gene.

[0047] a. Cas protein CRISPR / Cas-based gene editing systems may include Cas proteins or Cas fusion proteins. In some embodiments, the Cas protein is a Cas12 protein (also referred to as Cpf1), such as the Cas12a protein. The Cas12 protein may be derived from any bacterial or archaeal species, including but not limited to Francisella novicida, Acidaminococcus species, Lachnospiraceae species, and Prevotella species. In some embodiments, the Cas protein is a Cas9 protein. The Cas9 protein is an endonuclease capable of cleaving nucleic acids, encoded by a CRISPR locus, and involved in the type II CRISPR system. The Cas9 protein is found in Streptococcus pyogenes, Staphylococcus aureus (S. aureus), Acidovorax avenae, Actinobacillus pleuropneumoniae, Actinobacillus succinogenes, Actinobacillus suis, Actinomyces species, and Cycliphilus denitrificans.

[0048] Aminomonas paucivorans, Bacillus cereus, Bacillus smithii, Bacillus thuringiensis, Bacteroides species, Blastopirellula marina, Bradyrhizobium species, Brevibacillus laterosporus, Campylobacter coli, Campylobacter jejuni, Campylobacter lari, Candidatus punseispirillum Puniceispirillum), Clostridium cellulolyticum, Clostridium perfringens, Corynebacterium accolens, Corynebacterium diphtheria, Corynebacterium matruchotii, Dinoroseobacter shibae, Eubacterium dolichum, gamma proteobacterium, Gluconacetobacter diazotrophicus, Haemophilus parainfluenzae, Haemophilus sputum Helicobacter sputorum), Helicobacter canadensis, Helicobacter cinaedi, Helicobacter mustelae(mustelae), Ilyobacter polytropus, Kingella kingae, Lactobacillus crispatus, Listeria ivanovii, Listeria monocytogenes, Listeriaceae bacterium, Methylocystis species, Methylosinus trichosporium, Mobiluncus mulieris, Neisseria bacilliformis, Neisseria cinerea, Neisseria flavescens, Neisseria lactamica (Neisseria Lactamica, Neisseria species, Neisseria wadsworthii, Nitrosomonas species, Parvibaculum lavamentivorans, Pasteurella multocida, Phascolarctobacterium succinatutens, Ralstonia syzygii, Rhodopseudomonas palustris, Rhodovulum species, Simonsiella muelleri, Sphingomonas species, Sporolactobacillus vinea (vineae), Staphylococcus lugdunensis, Streptococcus species, Subdoligranulum species, Tistrella mobilisThe Cas9 molecule may be derived from any bacterial or archaeal species, including but not limited to *Streptococcus mobilis*, *Treponema* species, or *Verminephrobacter eiseniae*. In certain embodiments, the Cas9 molecule is the *Streptococcus pyogenes* Cas9 molecule (also referred to herein as "SpCas9"). In certain embodiments, the Cas9 molecule is the *Staphylococcus aureus* Cas9 molecule (also referred to herein as "SaCas9").

[0049] Cas molecules or Cas fusion proteins may interact with one or more gRNA molecules and, in cooperation with the gRNA molecules, may localize to a target domain and, in certain embodiments, a site containing a PAM sequence. The ability of a Cas molecule or Cas fusion protein to recognize a PAM sequence can be determined, for example, using transformation assays known in the art.

[0050] In certain embodiments, the ability of a Cas molecule or Cas fusion protein to interact with and cleave a target nucleic acid is dependent on a protospacer-adjacent motif (PAM) sequence. 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. Cas molecules from various bacterial species may recognize different sequence motifs (e.g., PAM sequences). In certain embodiments, the Cas12 molecule from Francisella nobicida recognizes a sequence motif called TTTN (SEQ ID NO: 56). In certain embodiments, the Cas9 molecule from S. piogenes recognizes a sequence motif called NGG and directs cleavage of a target nucleic acid sequence 1-10 bp, e.g., 3-5 bp upstream of that sequence. In certain embodiments, the Cas9 molecule of S. thermophilus recognizes the sequence motifs NGGNG (SEQ ID NO: 35) and / or NNAGAAW (W=A or T) (SEQ ID NO: 36) and directs the cleavage of a target nucleic acid sequence located 1 to 10 bp upstream, for example, 3 to 5 bp, from these sequences. In certain embodiments, the Cas9 molecule of S. mutans recognizes the sequence motifs NGG (SEQ ID NO: 31) and / or NAAR (R=A or G) (SEQ ID NO: 37) and directs the cleavage of a target nucleic acid sequence located 1 to 10 bp upstream, for example, 3 to 5 bp, from this sequence. In certain embodiments, the Cas9 molecule of S. aureus recognizes the sequence motif NNGRR (R=A or G) (SEQ ID NO: 38) and directs the cleavage of a target nucleic acid sequence located 1 to 10 bp upstream, for example, 3 to 5 bp, from this sequence. In certain embodiments, the Cas9 molecule of S. aureus recognizes a sequence motif called NNGRRN(R=A or G) (SEQ ID NO: 39) and directs the cleavage of a target nucleic acid sequence located 1 to 10 bp upstream, for example, 3 to 5 bp from that sequence. In certain embodiments, the Cas9 molecule of S. aureus recognizes a sequence motif called NNGRRT(R=A or G) (SEQ ID NO: 40) and directs the cleavage of a target nucleic acid sequence located 1 to 10 bp upstream, for example, 3 to 5 bp from that sequence.In a particular embodiment, the Cas9 molecule of S. aureus recognizes a sequence motif called NNGRRV(R=A or G;V=A or C or G) (SEQ ID NO: 41) and directs the cleavage of a target nucleic acid sequence located 1 to 10 bp upstream, for example, 3 to 5 bp, from that sequence. In the above embodiment, N can be any nucleotide residue, e.g., A, G, C, or T. The Cas9 molecule can be manipulated to alter the PAM specificity of the Cas9 molecule.

[0051] In certain embodiments, the vector encodes at least one Cas9 molecule that recognizes either NNGRRT (SEQ ID NO: 40) or NNGRRV (SEQ ID NO: 41) protospacer adjacent motif (PAM). In certain embodiments, the at least one Cas9 molecule is a S. aureus Cas9 molecule. In certain embodiments, the at least one Cas9 molecule is a S. aureus Cas9 molecule variant.

[0052] The Cas protein can be mutated to inactivate its nuclease activity. Inactivated Cas9 proteins that lack endonuclease activity (also referred to as "iCas9" or "dCas9") are targeted to genes in bacteria, yeast, and human cells by gRNA to silence gene expression through steric hindrance. Exemplary mutations in the S. pyogenes Cas9 sequence include D10A, E762A, H840A, N854A, N863A, and / or D986A. Exemplary mutations in the S. aureus Cas9 sequence include D10A and N580A. In certain embodiments, the Cas9 molecule is a variant of the S. aureus Cas9 molecule. In some embodiments, dCas9 is a Cas9 molecule containing at least two mutations selected from D10A, E762A, H840A, N854A, N863A, and / or D986A with respect to the S. pyogenes Cas9 sequence. In some embodiments, the Cas protein is the dCas9 protein. In some embodiments, the Cas protein is the dCas12 protein. In certain embodiments, the S. aureus Cas9 molecular variant contains the D10A mutation. The nucleotide sequence encoding this S. aureus Cas9 variant is described in SEQ ID NO: 50. In certain embodiments, the S. aureus Cas9 molecular variant contains the N580A mutation. The nucleotide sequence encoding this S. aureus Cas9 molecular variant is described in SEQ ID NO: 51.

[0053] The polynucleotide encoding the Cas molecule may be a synthetic polynucleotide. For example, a synthetic polynucleotide may be chemically modified. A synthetic polynucleotide may be codon-optimized, for example, by replacing at least one uncommon or less common codon with a common codon. For example, a synthetic polynucleotide may direct the synthesis of an optimized messenger mRNA, such as one described herein, optimized for expression in a mammalian expression system. Additionally or alternatively, nucleic acids encoding Cas molecules or Cas polypeptides may include a nuclear localization sequence (NLS). Nuclear localization sequences are known in the art. An exemplary codon-optimized nucleic acid sequence encoding the Cas9 molecule of S. pyogenes is described in SEQ ID NO: 42. The corresponding amino acid sequence of the S. pyogenes Cas9 molecule is described in SEQ ID NO: 43. Exemplary codon-optimized nucleic acid sequences encoding the Cas9 molecule of S. aureus and optionally containing a nuclear localization sequence (NLS) are described in SEQ ID NOs. 44-48, 52, and 53, provided below. Another exemplary codon-optimized nucleic acid sequence encoding the Cas9 molecule of S. aureus includes nucleotides 1293-4451 of SEQ ID NO. 55. The amino acid sequence of the S. aureus Cas9 molecule is described in SEQ ID NO. 49. The amino acid sequence of Streptococcus pyogenes Cas9 (with the D10A, H849A mutation) is described in SEQ ID NO. 54.

[0054] b. Fusion protein Alternatively or additionally, CRISPR / Cas-based gene editing systems may include fusion proteins. A fusion protein may comprise two heterologous polypeptide domains, where the first polypeptide domain comprises a DNA-binding protein such as a Cas protein, zinc finger protein, or TALE protein, and the second polypeptide domain has activities such as transcriptional activation, transcriptional repression, transcription release factor activity, histone modification activity, nuclease activity, nucleic acid association activity, methylase activity, or demethylase activity. The fusion protein may comprise a first polypeptide domain, such as a Cas9 protein or a mutant Cas9 protein, fused to a second polypeptide domain having activities such as transcriptional activation, transcriptional repression, transcription release factor activity, histone modification activity, nuclease activity, nucleic acid association activity, methylase activity, or demethylase activity. In some embodiments, the second polypeptide domain has transcriptional activation activity. In some embodiments, the second polypeptide domain comprises a synthetic transcription factor. The fusion protein may comprise one second polypeptide domain. The fusion protein may contain two second polypeptide domains. For example, the fusion protein may contain a second polypeptide domain at the N-terminus of the first polypeptide domain and a second polypeptide domain at the C-terminus of the first polypeptide domain. In other embodiments, the fusion protein may contain a single first polypeptide domain and more than one (e.g., two or three) second polypeptide domains in tandem.

[0055] i) Transcriptional activation activity The second polypeptide domain may have transcriptional activation activity, i.e., a transactivation domain. For example, gene expression of endogenous mammalian genes, such as human genes, can be achieved by targeting a mammalian promoter with a fusion protein of a first polypeptide domain, such as dCas9 or dCas12, and a transactivation domain, using a combination of gRNAs. The transactivation domain may include the VP16 protein, multiple VP16 proteins, e.g., the VP48 domain or VP64 domain, the p65 domain of NF kappa B transcription activator activity, or p300. For example, the fusion protein may be dCas9-VP64. In other embodiments, the Cas9 protein may be VP64-dCas9-VP64 (sequence number 57 encoded by sequence number 58). In other embodiments, the transcription-activating fusion protein may be dCas9-p300. In some embodiments, p300 may include the polypeptide of sequence number 59 or sequence number 60.

[0056] ii) Transcriptional repressive activity The second polypeptide domain may possess transcriptional repressive activity. The second polypeptide domain may also possess Kruppel-associated box activity such as a KRAB domain, ERF repressor domain activity, Mxil repressor domain activity, SID4X repressor domain activity, Mad-SID repressor domain activity, or TATA box-binding protein activity. For example, the fusion protein may be dCas9-KRAB.

[0057] iii) Transcription termination factor activity The second polypeptide domain may possess transcription termination factor activity. The second polypeptide domain may possess eukaryotic termination factor 1 (ERF1) activity or eukaryotic termination factor 3 (ERF3) activity.

[0058] iv) Histone modification activity The second polypeptide domain may have histone modification activity. The second polypeptide domain may have histone deacetylase, histone acetyltransferase, histone demethylase, or histone methyltransferase activity. The histone acetyltransferase may be p300 or a CREB-binding protein (CBP) protein, or a fragment thereof. For example, the fusion protein may be dCas9-p300. In some embodiments, p300 may include the polypeptide of SEQ ID NO: 59 or SEQ ID NO: 60.

[0059] v) Nuclease activity The second polypeptide domain may have nuclease activity different from that of the Cas9 protein. A nuclease, or a protein with nuclease activity, is an enzyme that can cleave phosphodiester bonds between nucleotide subunits of nucleic acids. Nucleases are usually further divided into endonucleases and exonucleases, although some enzymes may fall into both categories. Well-known nucleases include deoxyribonucleases and ribonucleases.

[0060] vi) Nucleic acid associated activity The second polypeptide domain may have nucleic acid association activity or a nucleic acid-binding protein-DNA binding domain (DBD). A DBD is an independently folding protein domain containing at least one motif that recognizes double-stranded or single-stranded DNA. A DBD may recognize a specific DNA sequence (recognition sequence) or have general affinity for DNA. The nucleic acid association region may be selected from helix-turn-helix regions, leucine zipper regions, winged helix regions, winged helix-turn-helix regions, helix-loop-helix regions, immunoglobulin folds, B3 domains, zinc fingers, HMG boxes, Wor3 domains, and TAL effector DNA binding domains. vii) Methylase activity The second polypeptide domain may have methylase activity involving the transfer of methyl groups to DNA, RNA, proteins, small molecules, cytosine, or adenine. In some embodiments, the second polypeptide domain includes a DNA methyltransferase.

[0061] viii) Demethylase activity The second polypeptide domain may possess demethylase activity. The second polypeptide domain may include enzymes that remove methyl (CH3-) groups from nucleic acids, proteins (particularly histones), and other molecules. Alternatively, the second polypeptide may convert methyl groups to hydroxymethylcytosine in a mechanism for demethylating DNA. The second polypeptide may catalyze this reaction. For example, Tet1 could be a second polypeptide that catalyzes this reaction.

[0062] c.gRNA A CRISPR / Cas-based gene editing system includes at least one gRNA molecule. For example, a CRISPR / Cas-based gene editing system may include two gRNA molecules. The gRNA provides targeting for the CRISPR / Cas-based gene editing system. The gRNA is a fusion of two non-coding RNAs: crRNA and tracrRNA. In some embodiments, the polynucleotide includes crRNA and / or tracrRNA. The sgRNA can target any desired DNA sequence by replacing the sequence encoding a 20bp protospacer that confers targeting specificity through complementary base pairing with the desired DNA target. The gRNA mimics the naturally occurring crRNA:tracrRNA double helix involved in the type II effector system. This double helix, which may include, for example, a 42-nucleotide crRNA and a 75-nucleotide tracrRNA, acts as a guide for Cas9 to cleave the target nucleic acid. A “target region,” “target sequence,” or “protospacer” refers to a region of a target gene (e.g., the Pax7 gene) that a CRISPR / Cas9-based gene editing system targets and binds to. The portion of gRNA that targets a target sequence in the genome may be called a “targeting sequence,” “targeting region,” or “targeting domain.” A “protospacer” or “gRNA spacer” may refer to a region of a target gene that a CRISPR / Cas9-based gene editing system targets and binds to; a “protospacer” or “gRNA spacer” may also refer to a portion of gRNA that is complementary to the target sequence in the genome. A gRNA may include a gRNA scaffold. A gRNA scaffold may promote Cas9 binding to the gRNA and enhance endonuclease activity. The gRNA scaffold is a polynucleotide sequence that follows the gRNA portion, corresponding to the sequence that the gRNA targets. Together, the gRNA targeting region and the gRNA scaffold form a single polynucleotide. The scaffold may include the polynucleotide sequence of Sequence ID No. 85. CRISPR / Cas9-based gene editing systems may include at least one gRNA, which targets various DNA sequences. These target DNA sequences may overlap.At the 3' end of a protospacer in the genome, the PAM sequence follows the target sequence or protospacer. Different type II systems have different PAM requirements. For example, the Streptococcus pyogenes type II system uses an "NGG" sequence where "N" can be any nucleotide. In some embodiments, the PAM sequence may be "NGG" where "N" can be any nucleotide. In some embodiments, the PAM sequence may be NNGRRT (SEQ ID NO: 40) or NNGRRV (SEQ ID NO: 41).

[0063] The number of gRNA molecules encoded by a gene construct (e.g., an AAV vector) may be at least 1 type of gRNA, at least 2 different gRNAs, at least 3 different gRNAs, at least 4 different gRNAs, at least 5 different gRNAs, at least 6 different gRNAs, at least 7 different gRNAs, at least 8 different gRNAs, at least 9 different gRNAs, at least 10 different gRNAs, at least 11 different gRNAs, at least 12 different gRNAs, at least 13 different gRNAs, at least 14 different gRNAs, at least 15 different gRNAs, at least 16 different gRNAs, at least 17 different gRNAs, at least 18 different gRNAs, at least 18 different gRNAs, at least 20 different gRNAs, at least 25 different gRNAs, at least 30 different gRNAs, at least 35 different gRNAs, at least 40 different gRNAs, at least 45 different gRNAs, or at least 50 different gRNAs.The number of gRNAs encoded by the vectors currently disclosed is: at least 1 gRNA to at least 50 different gRNAs, at least 1 gRNA to at least 45 different gRNAs, at least 1 gRNA to at least 40 different gRNAs, at least 1 gRNA to at least 35 different gRNAs, at least 1 gRNA to at least 30 different gRNAs, at least 1 gRNA to at least 25 different gRNAs, at least 1 gRNA to at least 20 different gRNAs, at least 1 gRNA to at least 16 different gRNAs, at least 1 gRNA to at least 12 different gRNAs, at least 1 gRNA to at least 8 different gRNAs, at least 1 gRNA to at least 4 different gRNAs, at least 4 gRNAs to at least 50 different gRNAs, at least 4 different gRNAs to at least 45 different gRNAs, at least 4 different gRNAs to at least 40 different gRNAs, at least 4 different gRNAs to at least 35 different gRNAs, It could be at least 4 different gRNAs to at least 30 different gRNAs, at least 4 different gRNAs to at least 25 different gRNAs, at least 4 different gRNAs to at least 20 different gRNAs, at least 4 different gRNAs to at least 16 different gRNAs, at least 4 different gRNAs to at least 12 different gRNAs, at least 4 different gRNAs to at least 8 different gRNAs, at least 8 different gRNAs to at least 50 different gRNAs, at least 8 different gRNAs to at least 45 different gRNAs, at least 8 different gRNAs to at least 40 different gRNAs, at least 8 different gRNAs to at least 35 different gRNAs, 8 different gRNAs to at least 30 different gRNAs, at least 8 different gRNAs to at least 25 different gRNAs, 8 different gRNAs to at least 20 different gRNAs, at least 8 different gRNAs to at least 16 different gRNAs, or 8 different gRNAs to at least 12 different gRNAs.In a particular embodiment, a gene construct (e.g., an AAV vector) encodes one gRNA molecule, i.e., a first gRNA molecule, and optionally a Cas9 molecule. In a particular embodiment, a first gene construct (e.g., a first AAV vector) encodes one gRNA molecule, i.e., a first gRNA molecule, and optionally a Cas9 molecule, and a second gene construct (e.g., a second AAV vector) encodes one gRNA molecule, i.e., a second gRNA molecule, and optionally a Cas9 molecule.

[0064] The gRNA molecule contains a targeting domain, which is a polynucleotide sequence complementary to the target DNA sequence that follows the PAM sequence. The gRNA may contain a "G" at the 5' end of the targeting domain or the complementary polynucleotide sequence. The targeting domain of the gRNA molecule may contain a complementary polynucleotide sequence of 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, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, or at least 35 base pairs of the target DNA sequence that follows the PAM sequence. In certain embodiments, the targeting domain of the gRNA molecule has a length of 19–25 nucleotides. In certain embodiments, the targeting domain of the gRNA molecule has a length of 20 nucleotides. In certain embodiments, the targeting domain of the gRNA molecule is 21 nucleotides long. In certain embodiments, the targeting domain of the gRNA molecule is 22 nucleotides long. In certain embodiments, the targeting domain of the gRNA molecule is 23 nucleotides long.

[0065] The gRNA may target regions within or near the Pax7 gene, or within or near the regulatory elements or promoter of the Pax7 gene. In certain embodiments, the gRNA may target at least one of the exons, introns, promoter regions, enhancer regions, or transcription regions of the gene. The gRNA may target Pax7 or the promoter or regulatory elements of the Pax7 gene. In some embodiments, the gRNA targets the Pax7 promoter. The gRNA may contain a targeting domain comprising a polynucleotide sequence corresponding to at least one of the sequence numbers 1-8, 69-76, or 77-84 shown in Table 1, or their complements or variants. In some embodiments, the gRNA targets a polynucleotide sequence comprising at least one complement of sequence numbers 1-8. In some embodiments, the gRNA is encoded by a polynucleotide sequence comprising at least one of sequence numbers 1-8. In some embodiments, the gRNA comprises a polynucleotide sequence selected from sequence numbers 69-76. In some embodiments, the gRNA binds to and targets polynucleotides containing sequences selected from SEQ ID NOs. 77-84 in Table 4.

[0066] [Table 1] [Table 2]

[0067] Single or multiplexed gRNAs can be designed to activate Pax7 expression, thereby differentiating stem cells into skeletal muscle progenitor cells. After treatment with the constructs or systems detailed herein, stem cells can differentiate into skeletal muscle progenitor cells. Genetically modified stem cells or patient cells can be transplanted into a subject.

[0068] d. DNA targeting systems DNA targeting systems or compositions comprising such gene constructs are further provided herein. The DNA targeting composition comprises at least one gRNA molecule (e.g., two gRNA molecules) that targets a gene, as described above. At least one gRNA molecule is capable of binding to and recognizing a target region. In some embodiments, the DNA targeting composition comprises a first gRNA and a second gRNA. In some embodiments, the first gRNA molecule and the second gRNA molecule contain different targeting domains.

[0069] The DNA-targeting composition may further comprise at least one Cas molecule or fusion protein. In some embodiments detailed above, the DNA-targeting composition further comprises at least one dCas9 protein or fusion protein. In some embodiments, the Cas9 molecule or fusion protein recognizes either NNGRRT (SEQ ID NO: 40) or NNGRRV (SEQ ID NO: 41) PAM. In some embodiments, the DNA-targeting composition comprises the nucleotide sequence described in SEQ ID NO: 55. In certain embodiments, the vector is configured to form first and second double-strand breaks in or near the Pax7 gene. The DNA-targeting composition may further contain donor DNA or a transgene.

[0070] 4. Genetic constructs A DNA targeting system or one or more of its components may be encoded by or contained within a gene construct. A gene construct may include polynucleotides such as vectors and plasmids. The construct may be recombinant. In some embodiments, the gene construct includes a promoter operatively ligated to a polynucleotide encoding at least one gRNA molecule and / or a Cas molecule or fusion protein. In some embodiments, the gene construct includes a promoter operatively ligated to a polynucleotide encoding at least one gRNA molecule and / or a dCas molecule or fusion protein. In some embodiments, the gene construct includes a promoter operatively ligated to a polynucleotide encoding at least one gRNA molecule and / or a Cas9 molecule or fusion protein. In some embodiments, the promoter is operatively ligated to a polynucleotide encoding a first gRNA molecule, a second gRNA molecule, and / or a Cas9 molecule or fusion protein. The gene construct may exist intracellularly as a functional extrachromosomal molecule. The gene construct may be a centromere, telomere, or a linear minichromosome including a plasmid or cosmid. The gene construct may be transformed or transduced into a cell. Gene constructs can be formulated into any suitable type of delivery vehicle, including, for example, viral vectors, lentiviral expression, mRNA electroporation, and lipid-mediated transfection. Cells transformed or transduced using the DNA targeting systems or their components as detailed herein are further provided herein. The cells may be, for example, stem cells or fibroblasts. In some embodiments, the stem cells are pluripotent stem cells. In some embodiments, the fibroblasts are cutaneous fibroblasts.

[0071] Viral delivery systems are further provided herein. In some embodiments, the vector is an adeno-associated virus (AAV) vector. AAV vectors are small viruses belonging to the Dependvirus genus of the Parvoviridae family that infect humans and some other primate species. Using AAV vectors, CRISPR / Cas9-based gene editing systems can be delivered using various construct configurations. For example, an AAV vector can deliver Cas9 and gRNA expression cassettes in separate vectors or in the same vector. Alternatively, when a small Cas9 protein derived from a species such as Staphylococcus aureus or Neisseria meningitidis is used, both Cas9 and up to two gRNA expression cassettes can be combined in a single AAV vector within a 4.7kb packaging limit.

[0072] In some embodiments, the AAV vector is a modified AAV vector. Modified AAV vectors may have enhanced cardiomyocyte and / or skeletal muscle tissue tropism. Modified AAV vectors may have the ability to deliver and express CRISPR / Cas9-based gene editing systems in mammalian cells. For example, a modified AAV vector may be an AAV-SASTG vector (Piacentino et al. Human Gene Therapy 2012, 23, 635-646). Modified AAV vectors 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 via systemic and local delivery (Seto et al. Current Gene Therapy 2012, 12, 139-151). Modified AAV vectors can also be AAV2i8G9 (Shen et al. J. Biol. Chem. 2013, 288, 28814-28823).

[0073] 5. Pharmaceutical Compositions Pharmaceutical compositions comprising the gene constructs or DNA targeting systems described herein are further provided herein. DNA targeting systems or at least one component thereof, as detailed herein, can be formulated into pharmaceutical compositions according to standard techniques well known to those skilled in the art of pharmaceuticals. Pharmaceutical compositions can be formulated according to the mode of administration to be used. If the pharmaceutical compositions are injectable, they are sterile, pyrogen-free, and particle-free. Isotonic formulations are preferably used. Generally, additives for isotonicity may include sodium chloride, dextrose, mannitol, sorbitol, and lactose. In some cases, isotonic solutions such as phosphate-buffered saline are preferred. Stabilizers include gelatin and albumin. In some embodiments, vasoconstrictors are added to the formulation.

[0074] The composition may further contain pharmaceutically acceptable excipients. pharmaceutically acceptable excipients may be functional molecules such as vehicles, adjuvants, carriers, or diluents. The term "pharmaceutically acceptable carrier" may be a non-toxic, inert, solid, semi-solid, or liquid filler, diluent, encapsulating material, or any type of formulation aid. Examples of pharmaceutically acceptable carriers include diluents, lubricants, binders, disintegrants, colorants, flavorings, sweeteners, antioxidants, preservatives, lubricants, solvents, suspending agents, wetting agents, surfactants, emollients, propellants, humectants, powders, pH adjusters, and combinations thereof. Pharmaceutically acceptable excipients may be transfection promoters, including 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 transfection promoters.

[0075] The transfection promoter may be a polyanion, polycation, or lipid, including poly-L-glutamate (LGS). The transfection promoter is poly-L-glutamate, and more preferably, poly-L-glutamate is present in the composition for genome editing in skeletal muscle or cardiac muscle at a concentration of less than 6 mg / mL. Transfection 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 used and administered together with the gene construct. In some embodiments, the DNA vector coding composition may also include liposomes, such as lipids, lecithin liposomes as a DNA-liposome mixture or other liposomes known in the art (see, for example, International Patent Publication W09324640), calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or transfection accelerators such as other known transfection accelerators. In some embodiments, the transfection accelerator is a polyanion, polycation, or lipid, including poly-L-glutamate (LGS).

[0076] 6. Administration DNA targeting systems or at least one component thereof, or pharmaceutical compositions containing the same, as detailed herein, can be administered to a subject. Such compositions may be administered in dosages and techniques well known to those skilled in the art of medicine, taking into account factors such as the age, sex, weight, and medical condition of a particular subject, as well as the route of administration. The DNA targeting systems or at least one component thereof, gene constructs, or compositions containing the same that are disclosed herein may be administered to a subject by a variety of routes, including orally, parenterally, sublingually, percutaneously, rectally, transmucosally, topically, intranasally, intravaginally, by inhalation, oral administration, intrapleurally, intravenously, intra-arterially, intraperitoneally, subcutaneously, intradermally, epidermally, intramuscularly, intranasally, intrathecally, intracranially, and intra-articularly, or combinations thereof. In certain embodiments, the DNA targeting system, gene construct, or composition containing the same is administered to a subject intramuscularly, intravenously, or in combination thereof. For veterinary use, DNA targeting systems, gene constructs, or compositions containing the same may be administered as formulations that are appropriately acceptable in accordance with normal veterinary practice. A veterinarian can readily determine the most appropriate drug regimen and route of administration for a particular animal. DNA targeting systems, gene constructs, or compositions containing the same may be administered by traditional syringes, needle-free injection devices, "microprojectile bombardment gone guns," or by other physical methods such as electroporation ("EP"), "hydrodynamic methods," or ultrasound.

[0077] DNA targeting systems, gene constructs, or compositions containing the same can be delivered to a target by several techniques, including DNA injection (also called DNA inoculation) with or without in vivo electroporation, liposome-mediated delivery, nanoparticle-enhanced delivery, and recombinant vectors such as recombinant lentiviruses, recombinant adenoviruses, and recombinant adenovirus-associated viruses. The composition may be injected into skeletal muscle or cardiac muscle. For example, the composition may be injected into the tibialis anterior muscle or tail.

[0078] In some embodiments, the DNA targeting system, gene construct, or composition containing the same is administered by 1) tail vein injection (systemic) into adult mice; 2) intramuscular injection, for example, local injection into muscles such as the TA or gastrocnemius muscle in adult mice; 3) intraperitoneal injection into P2 mice; or 4) facial vein injection (systemic) into P2 mice. In some embodiments, the DNA targeting system, gene construct, or composition containing the same is administered to humans by intravenous or intramuscular injection. When a system or gene construct detailed herein, or at least one component thereof, or a pharmaceutical composition comprising the same, and a vector are delivered onto it toward the target cells, the transfected cells may express gRNA molecules and Cas9 molecules or fusion proteins. In some embodiments, Cas9 is dCas9 or a fusion protein.

[0079] Any of the delivery methods and / or routes of administration detailed herein may be used with a multitude of cell types currently under investigation for cell-based therapies, including, but not limited to, immortalized myoblasts such as wild-type and patient-derived strains, primary dermal fibroblasts, stem cells such as induced pluripotent stem cells, bone marrow-derived precursors, skeletal muscle precursors, patient-derived human skeletal myoblasts, CD133+ cells, mesoangioblasts, cardiomyocytes, hepatocytes, chondrocytes, mesenchymal progenitor cells, hematopoietic stem cells, smooth muscle cells, and MyoD or Pax7 transdextrins, or other myogenic progenitor cells. Stem cells may be human pluripotent stem cells. Stem cells may be induced pluripotent stem cells (iPSCs). Stem cells may be embryonic stem cells (ESCs).

[0080] 7. Method a. Methods for activating the endogenous myogenic transcription factor Pax7 Methods for activating the endogenous myogenic transcription factor Pax7 in cells are provided herein. The methods may include administering to cells a DNA targeting system as detailed herein, an isolated polynucleotide sequence as detailed herein, a vector as detailed herein, cells as detailed herein, or a combination thereof. In some embodiments, endogenous expression of Pax7 mRNA is increased in skeletal muscle progenitor cells. In some embodiments, expression of Myf5, MyoD, MyoG, or a combination thereof is increased in skeletal muscle progenitor cells. In some embodiments, stem cells are induced to myogenic differentiation. In some embodiments, skeletal muscle progenitor cells maintain Pax7 expression after at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, or at least about 15 passages.

[0081] b. Methods for differentiating stem cells into skeletal muscle progenitor cells A method for differentiating stem cells into skeletal muscle progenitor cells is provided herein. The method may include the step of administering to cells a DNA targeting system as detailed herein, an isolated polynucleotide sequence as detailed herein, a vector as detailed herein, cells as detailed herein, or a combination thereof. In some embodiments, endogenous expression of Pax7 mRNA is increased in skeletal muscle progenitor cells. In some embodiments, expression of Myf5, MyoD, MyoG, or a combination thereof is increased in skeletal muscle progenitor cells. In some embodiments, stem cells are induced to myogenic differentiation. In some embodiments, skeletal muscle progenitor cells maintain Pax7 expression after at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, or at least about 15 passages.

[0082] c. Methods for treating the target Methods for activating the endogenous myogenic transcription factor Pax7 in cells are provided herein. The methods may include administering a DNA targeting system, an isolated polynucleotide sequence, a vector, cells, or a combination thereof, to cells as detailed herein. In some embodiments, endogenous expression of Pax7 mRNA is increased in the subject. In some embodiments, expression of Myf5, MyoD, MyoG, or a combination thereof is increased in the subject. In some embodiments, cells in the subject are induced to myogenic differentiation. In some embodiments, levels of dystrophin+ fibers in the subject are increased. In some embodiments, muscle regeneration in the subject is increased.

[0083] 8. Examples (Example 1) material and method gRNA design, transfection, and plasmid construction. Pax7 promoter-targeted gRNAs were designed using crispr.mit.edu and cloned into a gRNA vector (Addgene plasmid 41824). Candidate Pax7 gRNAs were transiently transfected with Lipofectamine 3000 on day 2 of CHIRON99021-induced differentiation of H9 ESCs constitutively expressing VP64-dCas9-VP64. Cells were harvested at day 6 for Pax7 qRT-PCR analysis. For doxycycline (dox)-induced expression of VP64-dCas9-VP64, the pLV-hUBC-VP64dCas9VP64-T2A-GFP plasmid (Addgene plasmid 59791) served as a source vector for generating pLV-tightTRE-VP64dCas9VP64-T2A-mCherry. Pax7 gRNA was cloned into pLV-hU6-gRNA-PGK-rtTA3-Blast, which was created using pLV-CMV-rtTA3-Blast (Addgene plasmid 26429) as the source vector. Pax7 cDNA (DNASU plasmid HsCD00443491) was cloned into a lentiviral construct to create the pLV-tightTRE-Pax7-P2A-mCherry construct. The PAX7-A sequence was confirmed to be the same as the PAX7 sequence used in a previous paper on directional differentiation. The PAX7-B sequence was obtained by PCR of mRNA isolated from cells treated with VP64dCas9VP64+gRNA and cloned into the lentiviral tightTRE-PAX7-B-P2A-mCherry construct. The target sequence of the gRNA is shown in Table 2. The primers used are shown in Table 3.

[0084] [Table 3]

[0085] [Table 4] JPEG2026108644000006.jpg178162

[0086] Lentivirus production. HEK293T cells were obtained from the American Tissue Collection Center (ATCC) and purchased through Duke University Cancer Center Facilities. They were cultured at 37°C with 5% CO2 in Dulbecco's modified Eagle medium (Invitrogen) supplemented with 10% FBS (Sigma) and 1% penicillin / streptomycin (Invitrogen). Approximately 3.5 million cells were seeded in 10 cm TCPS dishes. After 24 hours, the cells were transfected with the second-generation envelope and packaging plasmids pMD2.G (Addgene #12259) and psPAX2 (Addgene #12260) using calcium phosphate precipitation. The medium was changed 12 hours after transfection, and the viral supernatant was harvested 24 and 48 hours after this medium change. The viral supernatant was pooled, centrifuged at 500g for 5 minutes, filtered through a 0.45μm filter, and concentrated to 20× using a Lenti-X Concentrator (Clontech) according to the manufacturer's protocol. Undifferentiated hPSCs were transduced with pLV-hU6-gRNA-PGK-rtTA3-Blast, and cells were selected using 2μg / mL blastosidine (Thermo) to create a homogeneous population of stably transduced cells. Immediately before differentiation, the hPSCs were resuspended and seeded with lentiviruses encoding inducible VP64-dCas9-VP64 or Pax7 cDNA.

[0087] Cell culture. H9 ESCs (obtained from the WiCell Stem Cell Bank) and DU11 iPSCs were used for these studies. DU11 iPSCs were generated by episomal reprogramming of BJ fibroblasts (ATCC cell line, CRL-2522) derived from healthy male neonatal cells at the Duke iPSC Shared Resource Facility. Cell stability, correct karyotype, and pluripotency were confirmed. hPSCs were maintained in mTeSR (Stem Cell Technologies) and seeded on tissue culture-treated plates coated with ES-compatible Matrigel (Corning). For differentiation, hPSCs were dissociated into single cells using Accutase (Stem Cell Technologies) and seeded in mTeSR medium supplemented with 10 μM Y27632 (Stem Cell Technologies) on Matrigel-coated plates at a size of 2.3–3.3 × 10⁶ cells. 4 / cm 2 The cells were seeded. The following day, the mTeSR medium was replaced with E6 medium supplemented with 10 μM CHIR99021 (Sigma) to initiate mesoderm differentiation. Two days later, CHIR99021 was removed, and the cells were maintained in E6 medium containing 10 ng / mL FGF2 (Sigma) and 1 μg / mL doxycycline (dox) (Sigma).

[0088] Fluorescence-activated cell sorting and augmentation of sorted cells. On day 14 after differentiation induction, cells were dissociated using 0.25% trypsin-EDTA (Thermo) and washed with neutralizing medium (10% FBS in DMEM / F12). Cells were pelletized by centrifugation and resuspended in flow medium (5% FBS in PBS). Cells were sorted for mCherry expression, pelletized, resuspended in growth medium (E6 supplemented with 10 ng / mL FGF2 and 1 μg / mL dox), and plated on Matrigel-coated plates. Cells were subculturified every 3-4 days at approximately 80% confluence. Terminal differentiation was induced by removing dox from the medium in 100% confluent cultures.

[0089] Flow cytometry analysis. For flow cytometry analysis of surface markers, cells were harvested during the proliferation phase on day 20 of differentiation. Cells were dissociated using 0.25% trypsin-EDTA, washed with PBS, and then resuspended in flow buffer (PBS with 5% FBS). Cells were treated with 0.25 μg / 10 6 Individual cells were incubated with one of the following conjugate antibodies: IgG1-K isotype control-FITC (eBioscience 11-4714-41), CD56-FITC (eBioscience 11-0566-41), or CD29-FITC (eBioscience 11-0299-41). Cells were analyzed using a SONY SH800 flow cytometer.

[0090] Cell transplantation into immunodeficient mice. All animal experiments were conducted under protocols approved by the Duke Institutional Animal Care and Use Committee. Seven-week-old female NOD.SCID. gamma mice (Duke CCIF Breeding Core) were used for these in vivo studies. Prior to intramuscular cell transplantation, mice were pre-injected with 30 μL of 1.2% BaCl2 (Sigma). 24 hours later, differentiated iPSC or ESC-derived MPCs were injected into the tibialis anterior (TA) muscle (5 × 10⁻¹⁴). 5 Individual cells (15 μL Hanks equilibrium salt solution). Four weeks after injection, the mice were euthanized and TA muscle tissue was harvested.

[0091] Immunofluorescence staining of cultured cells and tissue sections. Cultured cells were seeded on autoclaved glass coverslips (1 mm, Thermo) coated with Matrigel for immunofluorescence staining during the proliferation phase. For differentiation, cells were grown to confluence and differentiated on 24-well tissue culture plates coated with Matrigel, and immunofluorescence staining was performed directly in the wells. Cells were fixed with 4% PFA for 15 minutes and permeabilized in blocking buffer (PBS supplemented with 3% BSA and 0.2% Triton X-100) at room temperature for 1 hour. Samples were incubated overnight at 4°C with the following antibodies: Pax7 (1:20, Developmental Studies Hybridoma Bank), myosin heavy chain MF20 (1:200, DSHB), Myf5 (1:200, Santa Cruz sc-302), and MyoD 5.8A (1:200, Santa Cruz sc-32758). Samples were washed with PBS for 15 minutes and incubated with a suitable 1:500 diluted secondary antibody and DAPI from Invitrogen at room temperature for 1 hour. Samples were washed with PBS for 15 minutes, coverslips were mounted using ProLong Gold Antifade Reagent (Invitrogen), or the wells were kept in PBS and imaged using conventional fluorescence microscopy. Harvested TA muscle was mounted and frozen in an optimal cutting temperature (OCT) compound cooled in liquid nitrogen. Continuous 10 μm frozen sections were collected. Frozen sections were fixed with 2% PFA for 5 minutes and permeabilized with PBS + 0.2% Triton-X for 10 minutes. Blocking buffer (PBS supplemented with 5% goat serum, 2% BSA, and 0.1% Triton X-100) was applied at room temperature for 1 hour. The samples were incubated overnight at 4°C with one of the following antibody combinations: human-specific MANDYS106 (1:200, Sigma MABT827), human-specific lamin A / C (1:100, Thermo MA31000), Pax7 (1:10, Developmental Studies Hybridoma Bank), or lamin (1:200, Sigma L9393).The samples were washed with PBS for 15 minutes and incubated with a suitable 1:500 diluted secondary antibody and DAPI from Invitrogen at room temperature for 1 hour. The samples were washed with PBS for 15 minutes, the slides were mounted using ProLong Gold Antifade Reagent (Invitrogen), and imaged using conventional fluorescence microscopy.

[0092] Quantitative reverse transcription PCR. RNA was isolated using the RNeasy Plus RNA isolation kit (Qiagen). cDNA was synthesized using the SuperScript VILO cDNA synthesis kit (Invitrogen). Real-time PCR using PerfeCTa SYBR Green FastMix (Quanta Biosciences) was performed using the CFX96 real-time PCR detection system (Bio-Rad). The results are expressed as a doubling of the expression of the gene of interest normalized to GAPDH expression using the ΔΔCt method.

[0093] Chromatin immunoprecipitation (ChIP) qPCR. ChIP was performed using the EpiQuik ChIP kit (EpiGentek) according to the manufacturer's instructions. Soluble chromatin was immunoprecipitated with antibodies against H3K27ac and H3K4me3 (abcam), and gDNA was purified for qPCR analysis. All sequences for ChIP-qPCR primers can be found in Table 3. qPCR was performed using PerfeCTa SYBR Green FastMix (Quanta BioSciences), and the data are presented as double-variable gDNA relative to the negative control (gRNA alone) and normalized to the GAPDH locus region.

[0094] RNA-Seq. RNA was extracted from newly sorted cells on day 14 of differentiation using the Total RNA Purification Plus Micro Kit (Norgen). Library preparation and sequencing were performed using GENEWIZ on an Illumina HiSeq in a 2×150bp sequencing configuration. All RNA-seq samples were first validated for consistent quality using FastQC v0.11.2 (Babraham Institute). Raw reads were trimmed using Trimmomatic v0.32 (Bolger et al. Bioinformatics 2014, 30, 2114-2120) with a 4bp sliding window (SLIDINGWINDOW:4:20) to remove adapters and bases with a mean quality score (Q) <20 (Phred33). Subsequently, the trimmed reads were aligned to the primary assembly of the GRCh38 human genome using STAR v2.4.1a (Dobin et al. Bioinformatics 2013, 29, 15-21) to remove alignments containing non-standard splice junctions. The aligned reads were assigned to genes in the GENCODE v19 comprehensive gene annotation (Harrow et al. Genome Res. 2012, 22, 1760-1774) using the featureCounts command (v1.4.6-p4) (Liao et al. Nucleic Acids Res. 2013, 41, e108-e108) in a subread package with default settings. After filtering out genes that were not sufficiently quantified, the subsequent counts were normalized for each replica using the R package DESeq2, and the normalized values ​​were used for analysis. A heatmap was created using the pheatmap package in the R software.Biological processes and pathways were generated using the web-based online tool Enrichr (Chen et al. BMC Bioinformatics 2013, 14, 128). For estimating transcript and gene abundances, total transcripts per million (TPM) were calculated using the rsem-calculate-expression function in the RSEM v1.2.21 package (Li and Dewey. BMC Bioinformatics 2011, 12, 323).

[0095] (Example 2) Developmental conditions for VP64-dCas9-VP64-mediated endogenous Pax7 activation in hPSCs During embryonic differentiation, PAX7 and its paralog PAX3 identify myofibrinoplasms within the paraxial mesoderm. Differentiation of hPSCs into paraxial mesodermal cells can be initiated by the GSK3 inhibitor CHIR99021 (Tan et al. Stem Cells Dev. 2013, 22, 1893-1906). Two human pluripotent stem cell lines, H9 ESCs and DU11 iPSCs, were used for differentiation studies. For target gene activation, the inventors used dCas9 (VP64-dCas9-VP64), which the inventors previously showed to be approximately 10 times more potent than a single VP64 fusion, with VP64 domains fused to both the N and C terminals. To test the efficacy of VP64-dCas9-VP64-mediated activation of PAX7, we designed eight gRNAs ranging from -490 to +158 base pairs relative to the transcription start site of the human PAX7 gene (Figure 7A). H9 ESCs stably expressing VP64-dCas9-VP64 differentiated into paraxial mesodermal cells upon 2 days of CHIR99021 addition in E6 medium, as previously described (Shelton et al. Stem Cell Rep. 2014, 3, 516-529). Individual gRNAs were transfected into the cells, and samples were harvested after 6 days for gene expression analysis using qRT-PCR. Four of the eight gRNAs significantly upregulated PAX7 compared to cells transfected with mocks (Figure 7B). In the second screening, the inventors packaged four individual gRNAs that demonstrated the best performance in transfection experiments with lentiviruses to achieve more stable and robust expression. Cells were harvested 8 days after transduction. gRNA #4 was identified as the most potent gRNA and used for subsequent investigations (Figure 7C).

[0096] (Example 3) VP64-dCas9-VP64-mediated differentiation of hPSCs into myogenic progenitor cells Next, the inventors tested the hypothesis that endogenous PAX7 activation in paraxial mesoderm cells would be sufficient to generate myogenic progenitor cells (MPCs) with the potential to differentiate into myotubes in vitro (Figure 1A). Prior to differentiation, hPSCs were transduced with a lentivirus expressing PAX7 promoter-targeted gRNA, reverse tetracycline trans-activator (rtTA), and a blastosidine resistance gene. Cells were selected using blastosidine for stable vector expression, and then transduced with an additional lentivirus encoding either doxycycline (dox)-inducible VP64-dCas9-VP64 or PAX7 cDNA, which also included a co-transcribed mCherry reporter gene (Figure 1B). hPSCs were differentiated using CHIR99021 for 2 days and then maintained in E6 medium with dox and FGF2 to support MPC proliferation (Figure 1C) (Pawlikowski et al. Dev. Dyn. 2017, 246, 359-367). Addition of CHIR99021 induced paraxial mesoderm differentiation, as indicated by high levels of the panmesoderm marker Brachyury (T) at the mRNA level, the paraxial mesoderm markers MSGN1 and TBX6, and the premyogenic mesoderm marker PAX3 (Figure 1D). Transduced cells were sorted based on mCherry expression after 2 weeks of growth (Figure 1E). mCherry+ cells accounted for approximately 20% of VP64-dCas9-VP64 transduced cells, compared to approximately 50% for PAX7 cDNA transduced cells. This is likely due to the larger size of the VP64-dCas9-VP64 vector compared to the PAX7 cDNA vector (7.9kb vs. 4.9kb between LTRs), resulting in a decrease in lentiviral titer. These purified MPCs were maintained in serum-free E6 medium supplemented with dox and FGF2 and passaged when the cells reached approximately 80% confluence. The sorted cells demonstrated high purity PAX7+ cells in both endogenous activated cells and exogenous cDNA-expressing cells when protein expression was assessed by immunofluorescence staining 5 days after sorting (Figure 1F and Figure 8A).Both iPSCs and ESCs treated with VP64-dCas9-VP64 demonstrated remarkable growth potential, resulting in an average increase of 85-fold and 95-fold cell number, respectively, over two weeks after purification. Furthermore, the growth potential of these cells surpassed that of PAX7 cDNA overexpressing cells (Figure 1G, Figure 8B).

[0097] (Example 4) Characterization of myogenic progenitor cells derived from endogenous or exogenous PAX7 expression PAX7 mRNA levels were assessed by qRT-PCR during the growth phase, 5 days after selection. PAX7 mRNA derived from endogenous chromosomal loci can be distinguished from total PAX7 mRNA produced from either lentivirus or endogenous chromosomal loci using distinct primer pairs. While overexpression of PAX7 cDNA yields more total PAX7 mRNA (Figures 2A and 8C), robust detection of any endogenous PAX7 isoform was observed only in VP64-dCas9-VP64 treated cells (Figures 2B and 8D). The human PAX7 gene encodes multiple isoforms whose differential sequences have been identified, but whose intrinsic biological functions remain unknown. Differential transcription termination in either exon 8 or exon 9 produces the PAX7-A and PAX7-B isoforms, respectively. The 3'-terminus differences of these transcripts allow for differential detection using specific qRT-PCR primers.

[0098] Downstream myogenic regulators MYF5, MYOD, and MYOG were also detected at the mRNA level by qRT-PCR (Figure 2C, Figure 8E). At the protein level, the majority of cells in both endogenous and exogenous PAX7-expressing cells co-expressed the activated satellite cell marker MYF5 (>90%). The myoblast marker MYOD was expressed in 15.9% and 6.8%, respectively, and was more highly expressed in cells expressing endogenous PAX7 compared to exogenous PAX7 cDNA. The mature myogenic markers MYOG and myosin heavy chain (MHC) were low in some cells (Figure 2D).

[0099] Human satellite cells co-express PAX7 along with the CD29 and CD56 surface markers. Approximately 10 days after sorting, the inventors assessed their MPCs for CD29 and CD56 expression and found that 100% of cells in all groups expressed CD29 independently of PAX7 expression. The inventors found that CD56 expression was more dependent on PAX7 expression, with only 27.4% of cells expressing CD56 in the gRNA-only group compared to 69.2% and 87.5% of cells in the PAX7 cDNA and VP64-dCas9-VP64 treated groups, respectively (Figures 2E and 8F). Assessment of mean fluorescence intensity (MFI) of CD56 staining also revealed that the mean CD56 expression level per cell was significantly higher in the VP64-dCas9-VP64 treated group (Figures 2F and 8G).

[0100] (Example 5) Transplantation of myogenic precursors generated by VP64-dCas9-VP64 into immunodeficient mice demonstrates in vivo regenerative potential. The inventors then determined whether MPCs derived from VP64-dCas9-VP64-mediated PAX7 activation possess in vivo regeneration potential. Cells that had been enlarged after sorting and passaged three times were transplanted into the tibialis anterior muscle (TA) of immunodeficient NOD.SCID.gamma (NSG) mice pre-injected with barium chloride (BaCl2) to create a regenerative microenvironment (Hall et al. Sci. Transl. Med. 2010, 2, 57ra83-57ra83). 24 hours after injury, mice were injected with 500,000 cells treated with either gRNA alone, PAX7 cDNA overexpression, or VP64-dCas9-VP64-mediated endogenous PAX7 activation. One month after transplantation, the muscle was harvested and engraftment was evaluated by immunostaining using human-specific dystrophin and lamin A / C antibodies. Human nuclei were detected by lamin A / C staining under all three conditions; however, only the endogenous PAX7-activated group demonstrated the consistent presence of human dystrophin (Figure 3A and Figure 8I). The number of human dystrophin+ fibers was quantified across three mice per condition by counting the section with the most abundant human dystrophin+ fibers in each sample (Figure 3B). We also investigated whether the transplanted cells could seed into the satellite cell niche. Immunostaining for PAX7, human lamin A / C, and laminin was performed to define the boundaries of human-derived satellite cells. Subbasement-membrane PAX7 and human lamin A / C double-positive cells were identified only in muscle transplanted with VP64dCas9VP64-activated MPC (Figure 3C, Figure 8J).

[0101] (Example 6) Induction of endogenous PAX7 expression persists after multiple passages and dox removal. During the growth of the selected cells, the inventors noticed a significant decrease in PAX7+ cells in the cDNA overexpression group after an average of four passages over an average of 32 days in three independent experiments. The initial number of cells expressing the PAX7 protein was >90% at day 5 after selection, but quantification of PAX7+ nuclei after approximately four passages after the initial flow selection revealed that only a small number of cells (35.8%) expressed the PAX7 protein despite maintenance in dox during the growth phase. Conversely, the vast majority (93%) of endogenously activated PAX7 cells retained PAX7 protein expression over multiple passages without premature differentiation (Figures 4A and 4C). The depletion of PAX7+ cells in the cDNA overexpression group did not correspond to the adoption of a myogenic fate, as indicated by the absence of MHC+ cells (Figure 4A). The inventors hypothesized that this could be due to high levels of PAX7 protein that inhibit cell proliferation, allowing cells that silence the promoter or contaminating cells through sorting to outnumber the cell population. Consistent with this possibility, Pax7 cDNA overexpression has so far been involved in inducing cell cycle exit without committing to myogenic differentiation. Interestingly, a previously published study also observed this phenomenon of PAX7 loss over multiple passages when using a tet-inducible PAX7 cDNA overexpression system. That study required modifying the serum-free differentiation protocol to a culture medium containing highly pro-mitotic 20% fetal bovine serum in order to improve the retention of PAX7 protein expression in cDNA-overexpressing cells.

[0102] Premyogenic cell differentiation was induced by withdrawing dox when the cells reached 100% confluence. Abundant MHC+ myofibrils were observed in VP64-dCas9-VP64 treated cells (Figure 4B, Figure 8H). Interestingly, in contrast to PAX7 cDNA treated cells, where 5.2% were PAX7+ one week after dox withdrawal, 50% of these cells remained PAX7+, and the endogenous gene was still activated even one week after dox withdrawal (Figure 4C). Staining for the FLAG epitope confirmed the absence of VP64-dCas9-VP64 in differentiated cells at this point (Figure 4D).

[0103] (Example 7) VP64-dCas9-VP64 leads to sustained PAX7 expression and stable chromatin remodeling at the target locus. The inventors hypothesized that epigenetic remodeling of the endogenous PAX7 promoter autonomously upregulates PAX7 in cells without the continued presence of VP64-dCas9-VP64. To investigate this, the inventors performed chromatin immunoprecipitation (ChIP)-qPCR on cells during dox administration and 15 days after dox withdrawal. Cells were analyzed at 30 days of differentiation under +dox conditions, then augmented and passaged more than three times over 15 days in the absence of dox. Using ChIP-seq data generated as part of the Encyclopedia of DNA Elements (ENCODE) project, the inventors identified transcriptionally active PAX7-enriched histone modifications in human skeletal muscle myoblasts (HSMMs), including H3K4me3 and H3K27ac (Figure 5A). Four qPCR primers were designed to tile the region from -731 bp to +926 bp relative to the PAX7 transcription start site (TSS). ChIP qPCR under dox conditions demonstrated significant enrichment of H3K4me3 and H3K27ac only at the endogenous PAX7 locus in response to VP64-dCas9-VP64 treatment (Figure 5B). Furthermore, these histone modifications were maintained for 15 days after dox removal (Figure 5C). To ensure that there was no leakage expression of VP64-dCas9-VP64 after dox removal, we performed Western blotting against the FLAG epitope tag and could not detect VP64-dCas9-VP64 15 days after dox removal (Figure 5D). Conversely, PAX7 remained detectable by Western blotting in the absence of VP64-dCas9-VP64, corresponding to ChIP-qPCR enrichment of the active histone mark.

[0104] (Example 8) Identification of global transcriptional changes induced by endogenous versus exogenous PAX7 To evaluate transcriptome-wide gene expression changes induced by endogenous activation of PAX7 compared to exogenous cDNA overexpression, the inventors performed RNA sequencing (RNA-seq) analysis. Differentiated cells treated with gRNA alone, VP64-dCas9-VP64 with gRNA, cDNA encoding the PAX7-A isoform, or cDNA encoding the PAX7-B isoform were selected for mCherry expression on day 14, and RNA was extracted for sequencing. The inventors included PAX7-B because it is highly expressed in VP64-dCas9-VP64-treated cells (Figure 2B), although little is known about its relationship with PAX7-A. To accurately measure differences between samples, the inventors created a sample distance matrix of RNA-seq data (Figure 6A). This revealed clear differences between the four treatments, and the four distinct clusters were readily apparent despite the commonality of induced PAX7 expression in three of the four groups. Multidimensional scaling (MDS) of the top 500 differentially expressed genes also showed branched clustering of sample groups with PAX7 cDNA overexpression that contributed most to the variability between transcriptome profiles (Figure 9A). We considered the top 200 most variable genes across the four groups and proposed a list of gene clusters evident in heatmaps for GO terminology analysis (Figure 6B). These analyses revealed overall developmental pathways, including mesodermal development, and WNT signaling pathway genes overexpressed in the gRNA-only group. Additionally, this group overexpressed genes involved in cardiac development, such as HAND1 and HAND2, showing a slightly higher tendency in this group to differentiate into cardiac cell lineages. Consistent with these observations, CHIR99021 is also used as an initiator for the differentiation of hPSCs into cardiomyocytes.

[0105] GO analysis of genes differentiated in the VP64-dCas9-VP64 group revealed a strong association with myogenesis (Figures 6B and 9B). Genes represented in this group included the embryonic myoblast marker HOXC12, the embryonic myosin heavy chain MYH3, and other myogenic regulators MYOD and MYOG.

[0106] Genes enriched after treatment with PAX7-A were associated with CNS development and the NOTCH1 signaling pathway. Interestingly, one of the most distinctly upregulated genes in this group was DLK1, which is required for normal emphysoskeletal muscle development (Figures 9B and 9C). However, in vitro overexpression of DLK1 inhibits satellite cell proliferation and induces cell cycle departure and premature differentiation. Conversely, DLK1 knockout increases Pax7+ myogenic progenitor cell proliferation in vitro and enhances postnatal muscle regeneration in vivo. This suggests that DLK1 is involved in maintaining the equilibrium between satellite cell quiescence and activation. Furthermore, the specific upregulation of both DLK1 and DIO3 in these cells (Figures 9B and 9C) suggests the activity of a DLK1-DIO3 gene cluster. The DLK1-DIO3 locus encodes the largest mammalian megacluster of microRNAs (miRNAs), which are strongly expressed in newly isolated satellite cells and strongly decelerated in proliferating satellite cells. This deceleration of DLK1-DIO3 is associated with the upregulation of muscle-specific miRNAs, including miR-1, which target the PAX7 3'UTR to fine-tune its expression and control satellite cell differentiation. Therefore, it is feasible that overexpression of only the PAX7-A isoform leads to negative feedback and expression of genes and miRNAs that regulate quiescence.

[0107] Genes specifically overexpressed in response to PAX7-B included the brain development genes VIT and OTP, as well as other PAX genes involved in kidney development, PAX2 and PAX8. Although PAX7 is not involved in kidney development, CHIR99021 has been used to differentiate hPSCs into the renal lineage.

[0108] Next, the inventors compared each of the three PAX7 expression groups with the gRNA-only groups, filtered out genes with low read counts, and extracted a list of genes with changes greater than 2x and padj < 0.05. The inventors compared the genes on these lists and found that 56 genes shared across all three groups were enriched with GO terms involved in skeletal muscle development (Figures 6C and 6D). This suggests that all three groups were more effective in directing hPSCs to the skeletal myogenic program than gRNA-only treatment and 14 days of CHIR-mediated differentiation, compared to small molecule protocols alone. However, when examining individual genes, the VP64-dCas9-VP64 group outperformed the other groups in terms of pre-myogenic and myogenic gene expression (Figure 6E). Many known satellite cell surface markers and genes were also more highly expressed in the VP64-dCas9-VP64 group compared to other groups, demonstrating a more specific and robust commitment to myogenesis and satellite cell differentiation (Figures 6E and 9D).

[0109] (Example 9) Consideration The practical applications of a CRISPR / Cas9-based transcription activator for the differentiation of hPSCs into myogenic progenitor cells via targeted activation of the endogenous PAX7 gene are detailed herein. This method can serve as an alternative to the transgene overexpression models previously used for myogenic progenitor cell differentiation. Using a minimal small molecule differentiation protocol with initial paraxial mesoderm differentiation using CHIR99021 and maintenance with FGF2 under serum-free culture conditions, targeted activation of the endogenous PAX7 gene was demonstrated to produce a myogenic progenitor cell population that could pass at least 6 times while maintaining PAX7 expression. These cells readily differentiated upon dox removal and subsequent loss of dCas9 activator expression, engrafted in mouse muscle, produced human dystrophin+ fibers, and simultaneously occupied satellite cell niches. Targeting the endogenous PAX7 promoter resulted in enrichment of H3K4me3 and H3K27ac histone modifications, which were demonstrated to persist for 15 days after dox removal. The enrichment of these chromatin marks was not observed during PAX7 cDNA overexpression. Although PAX7 cDNA overexpression from hPSCs has previously resulted in varying degrees of engraftment in NSG mice, we have not achieved similarly good engraftment results using PAX7 cDNA overexpression under the conditions used herein. However, previous studies have used differentiation protocols different from those used in this study, such as creating embryoid bodies, incorporating additional small molecules, or including animal serum in the culture medium. It is detailed herein that activation of endogenous PAX7 rather than exogenous PAX7 cDNA overexpression increases the efficacy of hPSC differentiation into myogenic progenitor cells with robust growth and differentiation potential, while simultaneously preserving post-transplant regenerative properties.

[0110] Previous studies using exogenous PAX7 cDNA rely on the overexpression of the PAX7-A isoform alone. However, differential RNA cleavage and polyadenylation produce PAX7-B, which contains a highly conserved paired tail domain and is considered to be the standard sequence. Both isoforms are expressed in human myogonia, and orthologues of these PAX7 protein variants are also present in mouse muscle, indicating the biological significance of both isoforms. Although the individual functions of these protein variants are not yet elucidated, they may play a differential role in myogenesis, which may be necessary for proper satellite stem cell function and myogenic differentiation. RNA-seq analysis demonstrated overlapping myogenic functions in cells produced by endogenous activation of either isoform of VP64-dCas9-VP64 or overexpression of PAX7 cDNA; however, the VP64-dCas9-VP64 group shared more commonly upregulated genes using PAX7-B than PAX7-A (89 and 30 genes, respectively) and showed a higher degree of similarity, as also depicted in the sample distance matrix. The dissimilarity between the overexpression of the two cDNAs indicated that they have distinct functions and can influence overall gene expression in different ways. For example, PAX7-B more effectively upregulated the premyogenic genes PAX3 and DMRT2, as well as the satellite cell genes CXCR4 and HEY1, than PAX7-A. Conversely, expression of the DLK1-DIO3 locus, which is involved in satellite cell quiescence, is more robust in response to PAX7-A than to PAX7-B. Therefore, VP64-dCas9-VP64-mediated PAX7 induction can enable the expression of both isoforms, potentially leading to proper induction of myogenesis at expression levels likely within the physiological range. Furthermore, endogenous activation of PAX7 can preserve its 3'UTR, which is a binding target for many muscle-specific miRNAs that play a role in organizing proper muscle development and regeneration.

[0111] Conditional expression of PAX7 in hPSCs via lentiviral transduction may be the most promising method for creating a homogeneous population of engraftable MPCs; however, to avoid the undesirable consequence of viral vector genomic integration, non-integration reprogramming may ultimately be used. VP64-dCas9-VP64 has been demonstrated to rapidly remodel the epigenetic signature of the target locus to achieve neural differentiation when the gRNA is transiently delivered. The epigenetic signature was shown to be stably maintained in the absence of VP64-dCas9-VP64, as demonstrated herein. Transient delivery of these target transcription activators via transfection, electroporation, or nonviral nanoparticle delivery of mRNA / gRNA or purified ribonucleoprotein complexes may provide alternatives to the integration-prone methods.

[0112] The extensive CRISPR genomics toolbox offers numerous possibilities for manipulating cell fate, improving our understanding of the molecular differences between myoblasts, satellite cells, and MPCs derived from hPSCs. Forced cell fate transitions, largely dependent on stochastic factors including the activation of endogenous networks (though largely obscure), can create stable new identities while counteracting the epigenetic memory of old identities. Further investigation into tissue-specific progenitor cell differentiation from pluripotent cells could reveal fundamental guidelines that could inform revised models for creating well-defined populations of cells capable of regenerating and thriving in the progenitor cell niche over the long term. The results detailed herein describe a novel method for the differentiation and augmentation of myogenic precursors from hPSCs through deterministic editing of transcriptional regulation using new genome manipulation tools, which may enable new disease modeling and cell therapies in disorders of skeletal muscle regeneration.

[0113] The foregoing description of specific embodiments will very well illustrate the general nature of the invention, which means that others may readily modify and / or adapt such specific embodiments to various applications without excessive experimentation and without departing from the general concept of the disclosure, by applying knowledge within the scope of their skills in the art. Therefore, such adaptations and modifications are intended to fall within the meaning and scope of equivalents of the disclosed embodiments, based on the teachings and guidance provided herein. The terms and phrases herein are for illustrative purposes only and not for limiting purposes, and it should be understood that they are to be interpreted by those skilled in the art in light of the teachings and guidance provided herein. The breadth and scope of this disclosure should not be limited by any of the exemplary embodiments described above, but should be defined solely by the following claims and their equivalents.

[0114] All publications, patents, patent applications, and / or other documents cited in this application are incorporated by reference as a whole for all purposes to the same extent as each individual publication, patent, patent application, and / or other document is individually indicated to be incorporated by reference for all purposes. For completeness, various aspects of the present invention are described in the following numbered clauses. Clause 1. A guide RNA (gRNA) molecule that targets Pax7, comprising a polynucleotide sequence corresponding to at least one of sequence numbers 1-8 or 69-76, or a variant thereof. Clause 2. gRNA as described in Clause 1, including crRNA, tracrRNA, or a combination thereof. Clause 3. A DNA targeting system for increasing Pax7 expression, comprising at least one gRNA that binds to and targets the Pax7 gene, the regulatory region of the Pax7 gene, the promoter region of the Pax7 gene, or a portion thereof. Clause 4. The DNA targeting system according to Clause 3, comprising at least one gRNA containing a polynucleotide sequence corresponding to at least one of sequence numbers 1-8 or 69-76, or a variant thereof. Clause 5. The DNA targeting system described in Clause 3 or 4 includes gRNA, crRNA, tracrRNA, or a combination thereof.

[0115] Clause 6. A DNA targeting system according to any one of Clauses 3 to 5, further comprising a clustered, regularly spaced short palindromic repeat-associated (Cas) protein or fusion protein, wherein the fusion protein comprises two heterologous polypeptide domains, the first polypeptide domain comprising a Cas protein, a zinc finger protein, or a TALE protein, and the second polypeptide domain having transcriptional activation activity. Clause 7. The Cas protein comprises the Streptococcus pyogenes Cas9 molecule or a variant thereof, as described in Clause 6 of the DNA targeting system. Clause 8. The fusion protein comprises VP64-dCas9-VP64, as described in Clause 6 of the DNA targeting system.

[0116] Clause 9. The DNA targeting system according to Clause 6, comprising Cas9, which recognizes the protospacer adjacent motif (PAM) of NGG (SEQ ID NO: 31), NGA (SEQ ID NO: 32), NGAN (SEQ ID NO: 33), or NGNG (SEQ ID NO: 34). Clause 10. An isolated polynucleotide sequence containing the gRNA molecule described in Clause 1 or 2. Clause 11. An isolated polynucleotide sequence encoding a DNA targeting system as described in any one of Clauses 3 to 9. Clause 12. A vector comprising an isolated polynucleotide sequence as described in Clause 10 or 11. Clause 13. A vector encoding a gRNA molecule as described in Clause 1 or 2, and a clustered, regularly spaced, short palindromic repeat-associated (Cas) protein. Clause 14. Cells comprising gRNA as described in Clause 1 or 2, a DNA targeting system as described in any one of Clauses 3 to 9, an isolated polynucleotide sequence as described in Clause 10 or 11, or a vector as described in Clause 12 or 13, or a combination thereof.

[0117] Clause 15. A pharmaceutical composition comprising gRNA as described in Clause 1 or 2, a DNA targeting system as described in any one of Clauses 3 to 9, an isolated polynucleotide sequence as described in Clause 10 or 11, a vector as described in Clause 12 or 13, or cells as described in Clause 14, or a combination thereof. Clause 16. A method for activating the endogenous myogenic transcription factor Pax7 in cells, comprising the step of administering to cells a gRNA described in Clause 1 or 2, a DNA targeting system described in any one of Clauses 3 to 9, an isolated polynucleotide sequence described in Clause 10 or 11, or a vector described in Clause 12 or 13. Clause 17. A method for differentiating stem cells into skeletal muscle progenitor cells, comprising the step of administering to stem cells a gRNA as described in Clause 1 or 2, a DNA targeting system as described in any one of Clauses 3 to 9, an isolated polynucleotide sequence as described in Clause 10 or 11, or a vector as described in Clause 12 or 13. Clause 18. Endogenous expression of Pax7 mRNA is increased in skeletal muscle progenitor cells, as described in Clause 17. Clause 19. The method according to any one of Clauses 17-18, wherein the expression of Myf5, MyoD, MyoG, or a combination thereof is increased in skeletal muscle progenitor cells. Clause 20. Stem cells are induced to undergo myogenic differentiation, as described in any one of Clauses 17-19. Clause 21. The method according to any one of Clauses 17-20, wherein skeletal muscle progenitor cells maintain Pax7 expression after at least about 6 passages. Clause 22. A method for treating a subject in need thereof, comprising the step of administering the cells described in Clause 14 to the subject. Clause 23. The level of dystrophin+ fibers in the subject increases as described in Clause 22. Clause 24. Muscle regeneration in the subject is increased by the method described in Clause 22.

[0118] array [Table 5]

[0119] [Table 6]

[0120] [Table 7]

[0121] Sequence ID 31 ngg Sequence ID 32 nga Sequence ID 33 ngan Sequence ID 34 ngng Sequence ID 35 nggng Sequence ID 36 nnagaaw(W=A or T) Sequence ID 37 naar(R=A or G) Sequence ID 38 nngrr (R=A or G; N is any nucleotide residue, e.g., A, G, C, or T) Sequence ID 39 nngrrn (R=A or G; N is any nucleotide residue, e.g., A, G, C, or T) Sequence ID 40 nngrrt(R=A or G; N is any nucleotide residue, e.g., A, G, C, or T) Sequence ID 41 nngrrv (R=A or G; N is any nucleotide residue, e.g., A, G, C, or T)

[0122] Sequence ID 42 S. pyogenes Cas9-encoding codon-optimized polynucleotide JPEG2026108644000010.jpg77150 JPEG2026108644000011.jpg218150

[0123] Sequence ID 43 Amino acid sequence of Streptococcus pyogenes Cas9 JPEG2026108644000012.jpg88150

[0124] Sequence ID 44 Codon-optimized nucleic acid sequence encoding S. aureus Cas9 JPEG2026108644000013.jpg134150 JPEG2026108644000014.jpg95150

[0125] Sequence ID 45 Codon-optimized nucleic acid sequence encoding S. aureus Cas9 JPEG2026108644000015.jpg130150 JPEG2026108644000016.jpg100150

[0126] Sequence ID 46 Codon-optimized nucleic acid sequence encoding S. aureus Cas9 JPEG2026108644000017.jpg125150 JPEG2026108644000018.jpg104150

[0127] Sequence ID 47 Codon-optimized nucleic acid sequence encoding S. aureus Cas9 JPEG2026108644000019.jpg116150 JPEG2026108644000020.jpg83150

[0128] Sequence ID 48 Codon-optimized nucleic acid sequence encoding S. aureus Cas9 JPEG2026108644000021.jpg137150 JPEG2026108644000022.jpg98150

[0129] Sequence ID 49 Amino acid sequence of Staphylococcus aureus Cas9 JPEG2026108644000023.jpg67150

[0130] Sequence ID 50 Nucleic acid sequence encoding the D10A variant of S. aureus Cas9 JPEG2026108644000024.jpg43150 JPEG2026108644000025.jpg185150

[0131] Sequence ID 51 Nucleic acid sequence encoding the N580A variant of S. aureus Cas9 JPEG2026108644000026.jpg39150 JPEG2026108644000027.jpg189150

[0132] Sequence ID 52 Codon-optimized nucleic acid sequence encoding S. aureus Cas9 JPEG2026108644000028.jpg34150 JPEG2026108644000029.jpg164150

[0133] Sequence ID 53 Codon-optimized nucleic acid sequence encoding S. aureus Cas9 JPEG2026108644000030.jpg50150 JPEG2026108644000031.jpg144150

[0134] Sequence ID 54 Streptococcus pyogenes Cas9 (possessing D10A and H849A) JPEG2026108644000032.jpg71150 JPEG2026108644000033.jpg18150

[0135] Sequence ID 55 Vector (pDO242) encoding a codon-optimized nucleic acid sequence that codes for S. aureus Cas9. JPEG2026108644000034.jpg193150 JPEG2026108644000035.jpg226150 JPEG2026108644000036.jpg10150

[0136] Sequence ID 56 tttn(N can be any nucleotide residue, e.g., A, G, C, or T)

[0137] Sequence ID 57 VP64-dCas9-VP64 protein JPEG2026108644000037.jpg95150 Sequence ID 58 VP64-dCas9-VP64 DNA JPEG2026108644000038.jpg87150 JPEG2026108644000039.jpg189150

[0138] Sequence ID 59 Human p300 protein (with L553M mutation) JPEG2026108644000040.jpg26150 JPEG2026108644000041.jpg124150

[0139] Sequence ID 60 Human p300 core effector protein (sequence number 59, aa1048~1664) JPEG2026108644000042.jpg42150

[0140] Sequence ID 85 Polynucleotide sequences of gRNA scaffolds JPEG2026108644000043.jpg10150

Claims

1. A guide RNA (gRNA) molecule that targets Pax7, comprising a polynucleotide sequence corresponding to at least one of sequence numbers 1-8 or 69-76, or a variant thereof.

2. The gRNA according to claim 1, comprising crRNA, tracrRNA, or a combination thereof.

3. A DNA targeting system for increasing Pax7 expression, comprising the Pax7 gene, the regulatory region of the Pax7 gene, the promoter region of the Pax7 gene, or at least one gRNA that binds to and targets a portion thereof.

4. The DNA targeting system according to claim 3, wherein at least one gRNA comprises a polynucleotide sequence corresponding to at least one of sequence numbers 1-8 or 69-76, or a variant thereof.

5. The DNA targeting system according to claim 3 or 4, wherein the gRNA comprises crRNA, tracrRNA, or a combination thereof.

6. A DNA targeting system according to any one of claims 3 to 5, further comprising clustered, regularly spaced short palindromic repeat-associated (Cas) proteins or fusion proteins, The fusion protein comprises two heterologous polypeptide domains: the first polypeptide domain contains a Cas protein, zinc finger protein, or TALE protein, and the second polypeptide domain has transcriptional activation activity, forming a DNA targeting system.

7. The DNA targeting system according to claim 6, wherein the Cas protein comprises the Streptococcus pyogenes Cas9 molecule or a variant thereof.

8. The DNA targeting system according to claim 6, wherein the fusion protein comprises VP64-dCas9-VP64.

9. The DNA targeting system according to claim 6, comprising Cas9, a Cas protein that recognizes the protospacer adjacent motif (PAM) of NGG (SEQ ID NO: 31), NGA (SEQ ID NO: 32), NGAN (SEQ ID NO: 33), or NGNG (SEQ ID NO: 34).

10. An isolated polynucleotide sequence comprising the gRNA molecule described in claim 1 or 2.

11. An isolated polynucleotide sequence encoding the DNA targeting system according to any one of claims 3 to 9.

12. A vector comprising the isolated polynucleotide sequence described in claim 10 or 11.

13. A vector encoding a gRNA molecule according to claim 1 or 2, and a clustered, regularly spaced, short palindromic repeat-associated (Cas) protein.

14. A cell comprising the gRNA according to claim 1 or 2, the DNA targeting system according to any one of claims 3 to 9, the isolated polynucleotide sequence according to claim 10 or 11, or the vector according to claim 12 or 13, or a combination thereof.

15. A pharmaceutical composition comprising the gRNA according to claim 1 or 2, the DNA targeting system according to any one of claims 3 to 9, the isolated polynucleotide sequence according to claim 10 or 11, the vector according to claim 12 or 13, or the cell according to claim 14, or a combination thereof.

16. A method for activating the endogenous myogenic transcription factor Pax7 in cells, comprising the step of administering to cells the gRNA described in claim 1 or 2, the DNA targeting system described in any one of claims 3 to 9, the isolated polynucleotide sequence described in claim 10 or 11, or the vector described in claim 12 or 13.

17. A method for differentiating stem cells into skeletal muscle progenitor cells, comprising the step of administering to stem cells the gRNA described in claim 1 or 2, the DNA targeting system described in any one of claims 3 to 9, the isolated polynucleotide sequence described in claim 10 or 11, or the vector described in claim 12 or 13.

18. The method according to claim 17, wherein endogenous expression of Pax7 mRNA is increased in skeletal muscle progenitor cells.

19. The method according to any one of claims 17 to 18, wherein the expression of Myf5, MyoD, MyoG, or a combination thereof is increased in skeletal muscle progenitor cells.

20. The method according to any one of claims 17 to 19, wherein stem cells are induced to undergo myogenic differentiation.

21. The method according to any one of claims 17 to 20, wherein skeletal muscle progenitor cells maintain Pax7 expression after at least about six passages.

22. A method for treating a subject in need thereof, comprising the step of administering the cells described in claim 14 to the subject.

23. The method according to claim 22, wherein the level of dystrophin+ fibers in the subject increases.

24. The method according to claim 22 or 23, wherein muscle regeneration in the subject is increased.

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