Compositions and methods for allele-specific MYH7 gene editing

Abstract

The present technology relates to compositions and methods for allele-specific editing (e.g., knocking out, knocking down, or otherwise altering expression) of a gene, e.g., the myosin heavy chain 7 (MYH7) gene, carrying mutations linked to diseases including cardiomyopathies, e.g., hypertrophic cardiomyopathy (HCM) in particular. The present technology also provides compositions and methods for preventing and / or treating these diseases (e.g., cardiomyopathies) caused by mutations in the MYH7 gene.

Description

COMPOSITIONS AND METHODS FOR ALLELE-SPECIFIC MYH7GENE EDITINGCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 610,306 filed December 14, 2023, the entire disclosure of which is incorporated herein in its entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The present application is being filed electronically via USPTO Patent Center and includes an electronically submitted Sequence Listing in XML format. The name of the XML file containing the Sequence Listing is TENA_053_01WO_SeqList_ST26.xml. The XML file, created on December 13, 2024, is 297,694 bytes in size and is being submitted electronically via USPTO Patent Center. The contents of the electronic Sequence Listing are incorporated by reference herein in their entirety.TECHNICAL FIELD

[0003] The present technology relates to compositions and methods for allelespecific editing (e.g., knocking out or knocking down) of the myosin heavy chain 7 (MYH7) gene carrying disease-causing mutations, and for preventing and / or treating diseases (e.g., cardiomyopathies) caused by or associated with such mutations in the MYH7 gene.BACKGROUND

[0004] The MYH7 gene encodes a myosin heavy chain beta (MHC-[3) isoform expressed primarily in the heart and also in skeletal muscles (type I fibers). Distinct from the fast isoform of cardiac myosin heavy chain (referred to as MHC-a), MHC-[3 is the major protein making up the thick filament in cardiac muscles and plays a major role in “slow twitch” movement and cardiac muscle contraction.

[0005] Several mutations in the MYH7 gene have been associated with inherited cardiomyopathies including hypertrophic cardiomyopathy (HCM). A significant percentage (about 20%, see Marian and Brunwald, Circ. Res. (2017) 121(7)749-770) ofHCM is the result of an autosomal dominant condition, in which a single copy of the mutant MYH7 gene results in enlargement of the left ventricle of the heart. It can be caused by different dominant negative mutations across the entire MYH7 gene. 142 pathogenic and 284 likely pathogenic mutations across almost every exon of MYH7 have been reported to lead to HCM and are highly heterogenous across the patient population, rendering it unfeasible to design a therapeutic specific for each pathogenic mutation. Therefore, a more practical approach to treat MYH7-associated cardiomyopathies that would benefit larger portions of this genetically diverse patient population is needed.SUMMARY

[0006] The present technology relates to compositions and methods for allelespecific editing (e.g., knocking out, knocking down, or otherwise altering expression) of a gene, e.g., the MYH7 gene, carrying pathogenic mutations linked to diseases including cardiomyopathies, e.g., HCM.

[0007] In some aspects, provided is a method of allele-specific editing of a gene carrying a heterozygous mutation in a subject or a cell therefrom (e.g., wherein the subject carries a wild-type (WT) allele and a mutant allele of the gene), the method comprising: (a) identifying a heterozygous genetic variant within the gene locus, wherein the subject carries a reference allele and an alternate allele of the genetic variant; (b) determining which of the reference and alternate alleles resides on the same chromosome as the mutation; and (c) disrupting the expression via CRISPR / Cas- mediated knockout of the mutant allele by: (i) targeting the reference allele of the genetic variant if it is determined to be on the same chromosome as the mutation in step (b), or (ii) targeting the alternate allele of the genetic variant if it is determined to be on the same chromosome as the mutation in step (b). In some embodiments, the gene is MYH7. In some embodiments, the heterozygous mutation in the MYH7 gene is a dominant negative mutation.

[0008] In some embodiments, the heterozygous genetic variant comprises a singlenucleotide polymorphism (SNP). In some embodiments, the SNP has an at least 10%, at least 20%, at least 30, or at least 40% heterozygosity in the human population. In some embodiments, the SNP is selected from the group consisting of rs735711 , rs735712, rs2069540, rs2069542, rs2231126, and rs7157716.

[0009] In some embodiments, the determining of step (b) is by phased sequencing or long-read RNA sequencing.

[0010] In some embodiments, the disrupting of step (c) is by forming an indel or a frameshift mutation in the mutant allele. In some embodiments, the disrupting of step (c) is by introducing to the subject or the cell a CRISPR / Cas system comprising (i) a Cas nuclease or a polynucleotide encoding the same; and (ii) a guide RNA (gRNA) targeting the reference or the alternate allele of the heterozygous genetic variant. In some embodiments, the Cas nuclease is a Cas9 nuclease, e.g., SpCas9 or SaCas9. In some embodiments, the Cas nuclease is SaCas9 having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 2; and / or the polynucleotide encoding the Cas nuclease has a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 1 .

[0011] In some embodiments, the gRNA comprises a complementary region specific to the reference or the alternate allele of the heterozygous genetic variant. In some embodiments, the complementary region comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 12-23, 144-147, and 229-239. In some embodiments, (a) the SNP is rs735711 , and the complementary region comprises a nucleotide sequence of SEQ ID NO: 12, 13, 232, or 233; (b) the SNP is rs735712, and the complementary region comprises a nucleotide sequence of SEQ ID NO: 14, 15, 234, or 235; (c) the SNP is rs2069540, and the complementary region comprises a nucleotide sequence of SEQ ID NO: 16, 17, 229, or 230; (d) the SNP is rs2069542, and the complementary region comprises a nucleotide sequence of SEQ ID NO: 18, 19, 236, or 237; (e) the SNP is rs2231126, and the complementary region comprises a nucleotide sequence of SEQ ID NO: 20, 21 , 238, or 239; or (f) the SNP is rs7157716, and the complementary region comprises a nucleotide sequence of SEQ ID NO: 22, 23, 144, 145, 146, 147, or 231.

[0012] In some embodiments, the polynucleotide encoding the Cas nuclease and a polynucleotide encoding the gRNA are in a vector in a head-to-tail orientation. In some embodiments, the vector further comprises a protein expression-driving promoter operably linked to the polynucleotide encoding the Cas nuclease and an RNAexpression-driving promoter operably linked to the polynucleotide encoding the gRNA. In some embodiments, the protein expression-driving promoter is a human troponin T (TNNT2) promoter having a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 24-27. In some embodiments, the RNA expressiondriving promoter is a human U6 promoter having a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 31. In some embodiments, the vector comprises the following 5’ to 3’ arrangement of elements: 5’ - the protein expressiondriving promoter - the polynucleotide encoding the Cas nuclease - the RNA expressiondriving promoter - the polynucleotide encoding the gRNA - 3’.

[0013] In some embodiments, the vector further comprises a polyadenylation (poly(A)) sequence. In some embodiments, the poly(A) sequence is selected from the group consisting of a synthetic poly(A) sequence of SEQ ID NO: 43, a BGH poly(A) sequence of SEQ ID NO: 44, and a SV40 poly(A) sequence of SEQ ID NO: 45. In some embodiments, the poly(A) sequence is between the polynucleotide encoding the Cas nuclease and the RNA expression-driving promoter, or wherein the vector comprises the following 5’ to 3’ arrangement of elements: 5’ - the protein expression-driving promoter - the polynucleotide encoding the Cas nuclease -the poly(A) sequence - the RNA expression-driving promoter - the polynucleotide encoding the gRNA - 3’.

[0014] In some embodiments, the vector further comprises a self-inactivation site 5’ to the protein expression-driving promoter, within the protein expression-driving promoter, between the protein expression-driving promoter and the polynucleotide encoding the Cas nuclease, within the polynucleotide encoding the Cas nuclease, or 3’ to the polynucleotide encoding the Cas nuclease. In some embodiments, the selfinactivation site is within the polynucleotide encoding the Cas nuclease, optionally near the 5’ end of the polynucleotide encoding the Cas nuclease, further optionally after the start codon “ATG”. In some embodiments, the self-inactivation site comprises a gRNA target region having a nucleotide sequence identical or complement to the complementary region of the gRNA. In some embodiments, the self-inactivation site further comprises a less optimal PAM sequence for the Cas nuclease. In some embodiments, the Cas nuclease is SaCas9, and the less optimal PAM sequence is gcgagc, gcgaga, or gcgagg.

[0015] In some embodiments, the vector is an adeno-associated virus (AAV) vector, a retroviral vector, or a lentiviral vector. In some embodiments, the vector is an AAV vector, optionally wherein the AAV vector is an AAV9 vector. In some embodiments, the vector comprises any capsid protein described herein, optionally wherein the capsid protein is a wild-type AAV9 capsid protein or an engineered variant thereof.

[0016] In some embodiments, the method has an at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% editing efficiency of, or efficiency of forming an indel or a frameshift mutation in, the mutant allele of the gene. In some embodiments, the method does not, or substantially does not, edit or an indel or a frameshift mutation in the WT allele of the gene.

[0017] In some aspects, provided is a guide RNA (gRNA) for allele-specific editing of MYH7, wherein the gRNA comprises a complementary region specific to an SNP within the MYH7 gene locus. In some embodiments, the SNP is selected from the group consisting of rs735711 , rs735712, rs2069540, rs2069542, rs2231126, and rs7157716. In some embodiments, (a) the SNP is rs735711 , and the complementary region comprises a nucleotide sequence of SEQ ID NO: 12, 13, 232, or 233; (b) the SNP is rs735712, and the complementary region comprises a nucleotide sequence of SEQ ID NO: 14, 15, 234, or 235; (c) the SNP is rs2069540, and the complementary region comprises a nucleotide sequence of SEQ ID NO: 16, 17, 229, or 230; (d) the SNP is rs2069542, and the complementary region comprises a nucleotide sequence of SEQ ID NO: 18, 19, 236, or 237; (e) the SNP is rs2231126, and the complementary region comprises a nucleotide sequence of SEQ ID NO: 20, 21 , 238, or 239; or (f) the SNP is rs7157716, and the complementary region comprises a nucleotide sequence of SEQ ID NO: 22, 23, 144, 145, 146, 147, or 231.

[0018] In some aspects, provided is a composition for allele-specific editing of a myosin heavy chain 7 (MYH7) gene carrying a heterozygous mutation in a subject or a cell therefrom, the composition comprising: (i) a gRNA comprising a complementary region specific to a reference allele of a first SNP within the MYH7 gene locus; or (ii) a second gRNA comprising a complementary region specific to an alternate allele of the first SNP within the MYH7 gene locus, and (iii) optionally, a Cas nuclease or a polynucleotide encoding the same.

[0019] In some embodiments, the first SNP has an at least 10%, at least 20%, at least 30, or at least 40% heterozygosity in the human population. In some embodiments, the first SNP is selected from the group consisting of rs735711 , rs735712, rs2069540, rs2069542, rs2231126, and rs7157716. In some embodiments, (a) the SNP is rs735711 , the complementary region of the first gRNA comprises a nucleotide sequence of SEQ ID NO: 12 or 232, and / or the complementary region of the second gRNA comprises a nucleotide sequence of SEQ ID NO: 13 or 233; (b) the SNP is rs735712, the complementary region of the first gRNA comprises a nucleotide sequence of SEQ ID NO: 14, and / or the complementary region of the second gRNA comprises a nucleotide sequence of SEQ ID NO: 15; (c) the SNP is rs2069540, the complementary region of the first gRNA comprises a nucleotide sequence of SEQ ID NO: 16 or SEQ ID NO: 229, and / or the complementary region of the second gRNA comprises a nucleotide sequence of SEQ ID NO: 17 or SEQ ID NO: 230; (d) the SNP is rs2069542, the complementary region of the first gRNA comprises a nucleotide sequence of SEQ ID NO: 18, and / or the complementary region of the second gRNA comprises a nucleotide sequence of SEQ ID NO: 19; (e) the SNP is rs2231126, the complementary region of the first gRNA comprises a nucleotide sequence of SEQ ID NO: 20, and / or the complementary region of the second gRNA comprises a nucleotide sequence of SEQ ID NO: 21 ; or (f) the SNP is rs7157716, the complementary region of the first gRNA comprises a nucleotide sequence of SEQ ID NO: 22 or SEQ ID NO: 231 , and / or the complementary region of the second gRNA comprises a nucleotide sequence of SEQ ID NO: 23, 144, 145, 146, or 147. In some embodiments, the SNP is rs2069540, the complementary region of the first gRNA comprises a nucleotide sequence of SEQ ID NO: 229, and the complementary region of the second gRNA comprises a nucleotide sequence of SEQ ID NO: 230. In some embodiments, the SNP is rs7157716, the complementary region of the first gRNA comprises a nucleotide sequence of SEQ ID NO: 231 , and the complementary region of the second gRNA comprises a nucleotide sequence of SEQ ID NO: 146.

[0020] In some embodiments, the Cas nuclease is a Cas9 nuclease, e.g., SpCas9 or SaCas9. In some embodiments, the Cas nuclease is SaCas9 having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 2; and / or the polynucleotide encoding the Cas nuclease has a nucleotide sequence having at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 1 .

[0021] In some embodiments, the polynucleotide encoding the Cas nuclease and a polynucleotide encoding the first or second gRNA are in a vector in a head-to-tail orientation. In some embodiments, the vector further comprises a protein expressiondriving promoter operably linked to the polynucleotide encoding the Cas nuclease and an RNA expression-driving promoter operably linked to the polynucleotide encoding the first or second gRNA. In some embodiments, the protein expression-driving promoter is a human troponin T (TNNT2) promoter having a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 24-27. In some embodiments, the RNA expression-driving promoter is a human U6 promoter having a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 31. In some embodiments, the vector comprises the following 5’ to 3’ arrangement of elements: 5’ - the protein expression-driving promoter - the polynucleotide encoding the Cas nuclease- the RNA expression-driving promoter - the polynucleotide encoding the first or second gRNA - 3’.

[0022] In some embodiments, the vector further comprises a polyadenylation (poly(A)) sequence. In some embodiments, the poly(A) sequence is selected from the group consisting of a synthetic poly(A) sequence of SEQ ID NO: 43, a BGH poly(A) sequence of SEQ ID NO: 44, and a SV40 poly(A) sequence of SEQ ID NO: 45. In some embodiments, the poly(A) sequence is between the polynucleotide encoding the Cas nuclease and the RNA expression-driving promoter, or wherein the vector comprises the following 5’ to 3’ arrangement of elements: 5’ - the protein expression-driving promoter- the polynucleotide encoding the Cas nuclease -the poly(A) sequence - the RNA expression-driving promoter - the polynucleotide encoding the first or second gRNA - 3’.

[0023] In some embodiments, the vector further comprises a self-inactivation site 5’ to the protein expression-driving promoter, within the protein expression-driving promoter, between the protein expression-driving promoter and the polynucleotide encoding the Cas nuclease, within the polynucleotide encoding the Cas nuclease, or 3’ to the polynucleotide encoding the Cas nuclease. In some embodiments, the self-inactivation site is within the polynucleotide encoding the Cas nuclease, optionally near the 5’ end of the polynucleotide encoding the Cas nuclease, further optionally after the start codon “ATG”. In some embodiments, the self-inactivation site comprises a gRNA target region having a nucleotide sequence identical or complement to the complementary region of the first or second gRNA. In some embodiments, the selfinactivation site further comprises a less optimal PAM sequence for the Cas nuclease. In some embodiments, the Cas nuclease is SaCas9, and the less optimal PAM sequence is gcgagc, gcgaga, or gcgagg.

[0024] In some embodiments, the vector is an adeno-associated virus (AAV) vector, a retroviral vector, or a lentiviral vector. In some embodiments, the vector is an AAV vector, optionally wherein the AAV vector is an AAV9 vector. In some embodiments, the vector comprises any capsid protein described herein, optionally wherein the capsid protein is a wild-type AAV9 capsid protein or an engineered variant thereof.

[0025] In some embodiments, the composition further comprises: (iv) a third gRNA comprising a complementary region specific to a reference allele of a second SNP within the MYH7 gene locus; or (v) a fourth gRNA comprising a complementary region specific to an alternate allele of the second SNP within the MYH7 gene locus, wherein the second SNP is different from the first SNP. In some embodiments, the second SNP has an at least 10%, at least 20%, at least 30, or at least 40% heterozygosity in the human population. In some embodiments, the second SNP is selected from the group consisting of rs735711 , rs735712, rs2069540, rs2069542, rs2231126, and rs7157716.

[0026] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier, diluent, or excipient.

[0027] In some aspects, provided is a method of treating a disease caused by or associated with a heterozygous mutation in MYH7 in a subject in need thereof, wherein the subject carries a WT allele and a mutant allele of the MYH7 gene, the method comprising allele-specific editing the mutant allele of the MYH7 gene according to any of the method disclosed herein, or administering to the subject a composition according to various embodiments disclosed herein. In some embodiments, the disease or condition is a heart disease, for example, cardiomyopathy, e.g., HCM.

[0028] In some embodiments, the method improves one or more measures of cardiac function, increases ejection fraction, and / or reduces left ventricular internal dimension and / or left ventricular mass.

[0029] In some embodiments, the administering is systemic administration or local administration to the heart. In some embodiments, the systemic administration is intravenous administration. In some embodiments, the local administration is by direct injection into the heart or cardiac tissue, intracoronary administration, or retrograde coronary sinus infusion.BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG. 1 shows a schematic of thin and thick myofilament proteins (top) and a schematic of the MYH7 gene (bottom). MYH7 encodes a protein which is a component of the thick myofilament. Dominant negative mutations in MYH7 account for 20-30% of HCM diagnoses in the United States. MYH7 has 40 exons, and 142 pathogenic and 284 likely pathogenic mutations for cardiomyopathy have been reported across almost every exon of MYH7.

[0031] FIG. 2 shows a schematic of the MYH7 gene and its exons (top), wherein the diamond indicates that SNP rs2069540 is located in the first exon. The sequences of the reference and alternate alleles of the SNP is also shown (bottom), wherein the G (reference allele) to A (alternate allele) mutation does not affect the encoded amino acid (threonine, T).

[0032] FIG. 3 shows that the percentage of eligible patient population would increase by identifying two or more SNPs as indicated. For example, for SNPs rs2069540 and rs7157716, 65.6% of the population would be heterozygous for at least one of the two SNPs and would thus be suitable for treatment. Eligible patients would receive a drug product targeting the reference or alternate allele of the SNP for which they are heterozygous.

[0033] FIG. 4A is a schematic showing a design of a self-inactivation site for a selfinactivating Cas construct. The self-inactivation site comprises a gRNA target region and a less optimal PAM sequence. As a result, gene editing of the genome target site, which is usually designed to be near an optimal PAM sequence, is more efficient; gene editingof the AAV gnome, which leads to self-inactivation of the Cas construct, is less efficient.This is reflected in the first and second waves of editing shown in the right panel.

[0034] FIG. 4B is a diagram of a self-inactivating Cas cassette, which contains a coding sequence for a Cas9 nuclease and a gRNA, and the Cas9 encoding sequence contains a self-inactivation site. The gRNA directs Cas-dependent cutting of the target human genome site first and the expression cassette at the self-inactivation site second.

[0035] FIG. 5 shows a diagram of an exemplary approach to allele-specific editing of the MYH7 gene carrying a heterozygous pathogenic mutation in heterozygous carriers of the rs2069540 SNP. In this example, the SNP (marked by a diamond) is located on the paternal copy of chromosome ( / .e., the paternal chromosome carries the alternate allele of rs2069540, and the maternal chromosome carries the reference allele of rs2069540). If the MYH7 pathogenic mutation (marked by a cross) is located on the paternal copy of chromosome ( / .e., “in cis” with rs2069540) (top panel), then gRNA specific to the alternate allele of rs2069540 (e.g., “TGCCGAGACTGAGTATGGCAAGG” (SEQ ID NO: 17)) can be used to knock out the paternal, pathogenic allele of MYH7 while preserving the functional, maternal, wild-type allele. If the MYH7 pathogenic mutation is located on the maternal copy of chromosome ( / .e., “in trans” with rs2069540) (bottom panel), then gRNA specific to the reference allele of rs2069540 (e.g., “TGCCGAGACCGAGTATGGCAAGG” (SEQ ID NO: 16)) can be used to knock out the maternal, pathogenic allele of MYH7 while preserving the functional, paternal, wild-type allele.DETAILED DESCRIPTION

[0036] The present technology relates to compositions and methods for allelespecific editing (e.g., knocking out, knocking down, or otherwise altering expression) of a gene, e.g., the myosin heavy chain 7 (MYH7) gene, carrying mutations linked to diseases including cardiomyopathies, e.g., hypertrophic cardiomyopathy (HCM) in particular. The present technology also provides compositions and methods for preventing and / or treating these diseases (e.g., cardiomyopathies) caused by mutations in the MYH7 gene. As most disease-causing MYH7 mutations are dominant negative and are scattered over the entire coding region in a highly heterogenous fashion across the patient population, the present technology strives to provide a therapeutically practical approach for disease allele-specific gene editing of MYH7 by targetingheterogenous genetic variants (e.g., single-nucleotide polymorphisms (SNPs)) within the MYH7 locus that allow distinction between paternal versus maternal copies of chromosomes and linkage of disease-causing mutations to a particular copy of chromosome, thereby knocking down or knocking out the disease-causing allele, for example, via the CRSPR / Cas system, while leaving behind a wild-type (WT), functional copy of the MYH7 gene.

[0037] Diploid organisms like humans have two sets of chromosomes in their somatic cells, one copy of paternal chromosomes and one copy of maternal chromosomes inherited from each parent. As such, humans have two copies (also referred to as two alleles) of each gene, which can be the same as or different from each other in sequence. For autosomal dominant mutations, such as most mutations in the MYH7 gene associated with HCM, one copy or allele of the gene carries the diseasecausing mutation, and the other copy or allele is wild-type. But because the diseasecausing copy or allele acts in a dominant fashion, it results in a disease phenotype. There are usually other genetic variances between the two alleles of a gene ( / .e., heterozygous variances), such as SNPs, that are silent or do not cause a phenotypic difference. Such heterozygous variances allow one to distinguish the paternal copy versus the maternal copy of the chromosome. Accordingly, if one were to also know which copy of the chromosome carries the disease-causing mutations, for example, by linking an SNP to the disease-causing mutation on the same chromosome (e.g., haplotyping), one may selectively knock out, knock down, or otherwise inhibits expression of the diseasecausing gene allele by targeting that copy of the chromosome while leaving the other copy intact. For example, by using the CRISPR / Cas system, one can design guide RNAs (gRNAs) specific to the sequence or region containing the SNP, thereby knocking out the allele of the gene that carries both the SNP and the disease-causing mutations. This approach is not sensitive to the exact location or sequence of any individual pathogenic mutation. By identifying and utilizing common SNPs within that gene locus, one may design therapeutics to selectively target a disease-causing gene allele that are more generally applicable in a genetically diverse patient population.

[0038] While the present disclosure is capable of being embodied in various forms, the description below of several embodiments is made with the understanding that the present disclosure is to be considered as an exemplification of the invention and is not intended to limit the invention to the specific embodiments illustrated. Headings areprovided for convenience only and are not to be construed to limit the invention in any manner. Embodiments illustrated under any heading may be combined with embodiments illustrated under any other heading.

[0039] The use of numerical values in the various quantitative values specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges were both preceded by the word "about." It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term “about.” It is to be understood that such range format is used for convenience and brevity and should be understood flexibly to include numerical values explicitly specified as limits of a range, but also to include all individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly specified. For example, a ratio in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also to include individual ratios, such as about 2, about 3, and about 4, and sub-ranges, such as about 10 to about 50, about 20 to about 100, and so forth. It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0040] All publications disclosed herein are incorporated by reference in their entirety. To the extent any materials incorporated by reference conflict with the present disclosure, the present disclosure controls.Definitions

[0041] Generally, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise specified, each of the following terms has the meaning set forth in this section.

[0042] The indefinite articles “a” and “an” denote at least one of the associated nouns and are used interchangeably with the terms “at least one” and “one or more.” For example, the phrase “a module” means at least one module, or one or more modules.

[0043] The conjunctions “or” and “and / or” are used interchangeably.

[0044] The term “about,” as used herein when referring to a measurable value, suchas an amount or concentration and the like, is meant to encompass variations of 20%, 10%, 5%, 1 %, 0.5%, or even 0.1 % of the specified amount.

[0045] The term “AAV” is an abbreviation for adeno-associated virus. The term covers all subtypes of AAV, except where a subtype is indicated, and to both naturally occurring and recombinant forms. The abbreviation “rAAV” refers to recombinant adeno- associated virus. “AAV5” refers to AAV subtype 5. “AAV9” refers to AAV subtype 9. The genomic sequences of various serotypes of AAV, as well as the sequences of the native inverted terminal repeats (ITRs), Rep proteins, and capsid subunits may be found in the literature or in public databases such as GenBank. An “AAV vector” or “rAAV vector” is used in the art to refer either to the DNA packaged into in the rAAV virion or to the rAAV virion itself, depending on context. As used herein, unless otherwise apparent from context, rAAV vector refers to a nucleic acid (typically a plasmid) comprising a polynucleotide sequence capable of being packaged into an rAAV virion, but with the capsid or other proteins of the rAAV virion. Generally, an rAAV vector comprises a heterologous polynucleotide sequence ( / .e., a polynucleotide not of AAV origin) and one or two AAV ITRs flanking the heterologous polynucleotide sequence. An “AAV particle” refers to an extracellular viral particle including at least one viral capsid protein (e.g., VP1 ) and an encapsidated AAV vector (or fragment thereof), including the capsid proteins.

[0046] The term “administering” to a subject is a procedure by which one or more delivery agents, together or separately, are introduced into or applied onto a subject such that target cells which are present in the subject are eventually contacted with the agent.

[0047] For brevity and clarity, the disclosure refers to “capsid protein” or “capsid proteins” of AAV. Those skilled in the art understand that such references refer to VP1 , VP2, or VP3, or combinations thereof. As in wild-type AAV and most recombinant expression systems VP1 , VP2, and VP3 are expressed from the same open reading frame, engineering of the sequence that encodes VP3 inevitably alters the sequences of the C-terminal domain of VP1 and VP2. One may also express the capsid proteins from different open reading frames, in which case the capsid of the resulting rAAV virion could contain a mixture of wild-type and engineered capsid proteins, and mixtures of different engineered capsid proteins.

[0048] The term “cardiomyopathy” refers to the deterioration of the function of themyocardium ( / .e., the actual heart muscle) for any reason. Subjects with cardiomyopathy are often at risk of arrhythmia or sudden cardiac death or both. The term “hypertrophic cardiomyopathy” refers to a disease of the heart and myocardium in which a portion of the myocardium is hypertrophied. The term “familial hypertrophic cardiomyopathy” refers to a genetic disorder characterized by increased growth ( / .e., hypertrophy) in thickness of the wall of the left ventricle.

[0049] A “clinically effective amount,” “clinically effective concentration,” or “clinically effective dose” refers to a concentration or dose of a peptide, composition, or pharmaceutical composition that is shown to be effective in clinical trials or is predicted to be effective based on early phase or pre-clinical trials. In some embodiments, a “clinically effective amount” is the same as a “therapeutically effective amount.” In some embodiments, a “clinically effective amount” is higher or lower than a “therapeutically effective amount.” Further, the effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the subject’s response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and seventy of the condition may require an increase or decrease in the actual effective amount administered.

[0050] The term “construct” refers to any polynucleotide that contains a recombinant nucleic acid molecule. A construct may be present in a vector (e.g., a bacterial vector, a viral vector) or may be integrated into a genome. A “vector” is a nucleic acid molecule that is capable of introducing a specific nucleic acid sequence into a cell or into another nucleic acid sequence, or as a means of transporting another nucleic acid molecule. Vectors may be, for example, plasmids, cosmids, viruses, an RNA vector, or a linear or circular DNA or RNA molecule that may include chromosomal, non-chromosomal, semisynthetic, or synthetic nucleic acid molecules. Exemplary vectors are those capable of autonomous replication (episomal vector), capable of delivering a polynucleotide to a cell genome (e.g., viral vector), or capable of expressing nucleic acid molecules to which they are linked (expression vectors).

[0051] The term “delivery”, which is used interchangeably with “transduction,” refers to the process by which exogenous nucleic acid molecules are transferred into a cell such that they are located inside the cell. Delivery of nucleic acids is a distinct process fromexpression of nucleic acids.

[0052] The term “expression” refers to the process by which a polypeptide is produced based on the encoding sequence of a nucleic acid molecule, such as a gene. The process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof. An expressed nucleic acid molecule is typically operably linked to an expression control sequence (e.g., a promoter).

[0053] The term “expression cassette” or “expression construct” refers to a DNA polynucleotide sequence operably linked to a promoter.

[0054] The term “gene therapy” involves the transfer of heterologous DNA to cells of a mammal, particularly a human, with a disorder or conditions for which therapy or diagnosis is sought. The DNA is introduced into the selected target cells in a manner such that the heterologous DNA is expressed, and a therapeutic product encoded thereby is produced. Alternatively, the heterologous DNA may in some manner mediate expression of DNA that encodes the therapeutic product; it may encode a product, such as a peptide or RNA that in some manner mediates, directly or indirectly, expression of a therapeutic product. Gene therapy may also be used to deliver nucleic acid encoding a gene product to replace a defective gene or supplement a gene product produced by the mammal or the cell in which it is introduced. The introduced nucleic acid may encode a therapeutic gene product that is not normally produced in the mammalian host or that is not produced in therapeutically effective amounts or at a therapeutically useful time. The heterologous DNA encoding the therapeutic product may be modified prior to introduction into the cells of the afflicted host to enhance or otherwise alter the product or expression thereof.

[0055] The term “host cell” as used herein refers to a cell or microorganism targeted for genetic modification by introduction of a construct or vector carrying a nucleotide sequence for expression of a protein or polypeptide of interest.

[0056] The term “modified” refers to a substance or compound (e.g., a cell, a polynucleotide sequence, and / or a polypeptide sequence) that has been altered or changed as compared to the corresponding unmodified substance or compound.

[0057] The term “nucleic acid” or “polynucleotide” refers to a polymeric compoundincluding covalently linked nucleotides comprising natural subunits (e.g., purine or pyrimidine bases). Purine bases include adenine and guanine, and pyrimidine bases include uracil, thymine, and cytosine. Nucleic acid molecules include polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), which includes cDNA, genomic DNA, and synthetic DNA, either of which may be single- or double-stranded. A nucleic acid molecule encoding an amino acid sequence includes all nucleotide sequences that encode the same amino acid sequence.

[0058] The term “operably linked” or “operatively linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a polynucleotide sequence if the promoter affects the transcription or expression of the polynucleotide sequence.

[0059] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length, though a number of amino acid residues may be specified. Polypeptides may include amino acid residues including natural and / or non-natural amino acid residues. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. In some embodiments, the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.

[0060] The term “promoter” as used herein refers a polynucleotide sequence that has one or more recognition site(s) to which an RNA polymerase binds, such that in a host or target cell, an RNA polymerase may initiate and transcribe a polynucleotide sequence “downstream” of the promoter into an RNA. Similarly stated, a “promoter” is operably linked or operatively linked to a polynucleotide sequence if in a host or target cell in which the promoter is active, an RNA polymerase initiates transcription of the polynucleotide at a transcription state site. Promoters operative in mammalian cells generally comprise an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated and / or another sequence found 70 to 80 bases upstream from the start of transcription, a CNCAAT region where N may be anynucleotide.

[0061] The term “recombinant” as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature, or that the polynucleotide is assembled from synthetic oligonucleotides. A “recombinant” protein is a protein produced from a recombinant polypeptide. A recombinant virion is a virion that comprises a recombinant polynucleotide and / or a recombinant protein, e.g., a recombinant capsid protein.

[0062] The term “sequence identity” or “identity” when referring to a polynucleotide or polypeptide sequence refers to the percentage of bases or amino acids between two polynucleotide or polypeptide sequences that are the same, and in the same relative position. As such one polynucleotide or polypeptide sequence has a certain percentage of sequence identity compared to another polynucleotide or polypeptide sequence. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. Methods of sequence alignment for comparison and determination of percent sequence identity is well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat’l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wl), by manual alignment and visual inspection (see, e.g., Brent et al., Current Protocols in Molecular Biology (2003)), by use of algorithms know in the art including the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977); and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. In some embodiments, the determination of the percentage of sequence identity may take place after a local alignment. Such alignments are well known in the art, for instance, the service EMBOSS Matcher identifies local similarities between two sequences using an algorithm based on the LALIGN application, version 2.0u4. In an example, the identity between two nucleic acid sequences may be calculated using the service Matcher (EMBOSS) set to the default parameters, e.g., matrix (DNAfull), gap open (16), gap extend (4), alternativematches (1 ).

[0063] The term “subject” refers to a mammalian subject, preferably a human. A “subject in need thereof” refers to a subject who has been diagnosed with a cardiac disease (e.g., cardiomyopathy) or is at an elevated risk of developing the disease. The phrases “subject” and “patient” are used interchangeably herein.

[0064] A “therapeutically effective amount” as used herein is an amount that produces a desired effect in a subject for an indication, condition, disease, or disorder. In certain embodiments, the therapeutically effective amount is an amount that yields maximum therapeutic effect. In other embodiments, the therapeutically effective amount yields a therapeutic effect that is less than the maximum therapeutic effect. For example, a therapeutically effective amount may be an amount that produces a therapeutic effect while avoiding one or more side effects associated with a dosage that yields maximum therapeutic effect. A therapeutically effective amount for a particular composition will vary based on a variety of factors, including, but not limited to, the characteristics of the therapeutic composition (e.g., activity, pharmacokinetics, pharmacodynamics, and bioavailability); the physiological condition of the subject (e.g., age, body weight, sex, disease type and stage, medical history, general physical condition, responsiveness to a given dosage, and other present medications); the nature of any pharmaceutically acceptable carriers, excipients, and preservatives in the composition; and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, namely, by monitoring a subject’s response to administration of the therapeutic composition and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy, 21st Edition, Univ, of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, PA, 2005.

[0065] The term “transgene” refers to a nucleic acid sequence encoding a protein or RNA (e.g., a therapeutic protein), which is partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal’s genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout). A transgenecan include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of a selected nucleic acid.

[0066] The terms “treat,” “treating,” and “treatment,” as used herein with regard to cancer, refers to alleviating the cancer partially or entirely, inhibiting cancer cell growth, reducing the number of cancer cells, preventing the cancer, decreasing the likelihood of occurrence or recurrence of the cancer, slowing the progression or development of the cancer, or eliminating, reducing, or slowing the development of one or more symptoms associated with the cancer. For example, “treating” may refer to preventing or slowing the existing tumor from growing larger, preventing or slowing the formation or metastasis of cancer, and / or slowing the development of certain symptoms of the cancer. In some embodiments, the term “treat,” “treating,” or “treatment” means that the subject has a reduced number or size of tumor compared to a subject not being administered the treatment. In some embodiments, the term “treat,” “treating,” or “treatment” means that one or more symptoms of the cancer are alleviated in a subject receiving the pharmaceutical compositions as disclosed and described herein, compared to a subject who does not receive such treatment.

[0067] The term “upstream” refers to a portion of a polynucleotide that is, with reference to a transcription start site (TSS), 5’ to the TSS on the sense strand (or coding strand) of the polynucleotide; and 3’ to the TSS on the antisense strand of the polynucleotide. The term “downstream” refers to a portion of a polynucleotide that is, with reference to a TSS, 3’ to TSS on the sense strand (or coding strand) of the polynucleotide; and 5’ to the TSS on the antisense strand of the polynucleotide. Thus, a deletion from the upstream end of a promoter is a deletion of one or more base pairs in the non-transcribed region of the polynucleotide, 5’ to the TSS on the sense strand (or equivalently, 3’ to the TSS on the antisense strand). A deletion from the downstream end of a promoter is a deletion of one or more base pairs in the transcribed region of the polynucleotide, 3’ to the TSS on the sense strand (or equivalently, 5’ to the TSS on the antisense strand).

[0068] The term “variant” refers to a protein or nucleic acid having one or more genetic changes (e.g., insertions, deletions, substitutions, or the like) that returns all or substantially all of the functions of the reference protein or nucleic acid. For example, a variant of a therapeutic protein retains the same or substantially the same activity and / orprovides the same or substantially the same therapeutic benefit to a subject in need thereof. A variant of a promoter sequence retains the ability to initiate transcription at the same or substantially the same level as the reference promoter, and retains the same or substantially the same cell type specificity. In particular embodiments, polynucleotides variants have at least or about 50%, 55%, 60%, 65%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a reference sequence. In particular embodiments, protein variants have at least or about 50%, 55%, 60%, 65%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a reference sequence.

[0069] A “vector” refers to a DNA construct containing a nucleic acid molecule that is operably linked to a suitable control sequence capable of effecting the expression of the nucleic acid molecule in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control termination of transcription and translation. The vector may be a plasmid, a phage particle, a virus, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself.

[0070] The term “wild-type” or “WT” refers to the naturally-occurring polynucleotide sequence encoding a protein, or a portion thereof, or protein sequence, or portion thereof, respectively, as it normally exists in vivo in a normal or healthy subject.Methods for Allele-specific Gene Editing

[0071] In some aspects, provided are methods for allele-specific gene editing, e.g., editing an allele of a gene (e.g., the MYH7 gene) which carries a heterozygous mutation, in a subject or a cell therefrom. In some embodiments, the subject carries a wild-type (WT) allele and a mutant allele ( / .e., an allele that carries the mutation or the mutationcarrying allele) of the gene (e.g., the MYH7 gene). The heterozygous mutation may be a dominant negative mutation and / or a disease-associated mutation, in which the subject carries a WT allele and a mutant / disease-causing allele of the gene (e.g., the MYH7 gene). The editing may include knocking down, knocking out, or otherwise altering theexpression of the gene, for example, through the CRSIPR / Cas system. As used herein, “knock out” includes deleting all or a portion of the target nucleotide sequence in a way that interferes with the function of the target gene. For example, a knockout can be achieved by altering a target nucleotide sequence by inducing an indel in a functional domain of the target gene, or one that causes a frameshift mutation thereby disrupting the expression of the target gene. “Knock down” refers to genetic modifications that result in reduced expression of the edited gene. As used herein, “indel” refers to a mutation resulting from an insertion, deletion, or a combination thereof, of nucleotide bases in the genome. Thus, an indel typically inserts or deletes nucleotides from a sequence. As will be appreciated by those skilled in the art, an indel in a coding region of a genomic sequence will result in a frameshift mutation, unless the length of the indel is a multiple of three. A gene editing system, e.g., the CRISPR / Cas system, of the present disclosure can be used to induce an indel of any length in a target polynucleotide sequence.

[0072] In some embodiments, the methods comprise (a) identifying a heterozygous genetic variant within the target gene locus (e.g., the MYH7 gene locus), wherein the subject carries a reference allele and an alternate allele of the genetic variant; (b) determining which of the reference and alternate alleles resides on the same chromosome as the mutation (e.g., haplotyping); and (c) disrupting the expression of the mutant allele or the mutation-carrying allele by targeting whichever one of the reference and the alternate alleles of the genetic variant determined to be on the same chromosome as the mutation in step (b), for example, via CRISPR / Cas-mediated knockout of the mutant allele. For example, if the reference allele is determined to be on the same chromosome as the mutation, then the mutant allele or the mutation-carrying allele can be knocked out by targeting the sequence / region containing the reference allele of the genetic variant; alternatively, if the alternate allele is determined to be on the same chromosome as the mutation, then the mutant allele or the mutation-carrying allele can be knocked out by targeting the sequence / region containing the alternate allele of the genetic variant.MYH7 as a target for allele-specific editing

[0073] In some embodiments, the gene to be allele-specific edited using the present technology is MYH7, which encodes the [3-myosin heavy chain ([3-MHC) protein that actsas a molecular motor to drive active contraction during cardiac systole. More than 400 pathogenic and likely pathogenic mutations, many of which dominant negative, have been identified in MYH7 and linked to HCM pathology, and these mutations are distributed throughout the gene (FIG. 1).

[0074] In some embodiments, the heterozygous genetic variant within the target gene locus (e.g., the MYH7 gene locus) in step (a) of the present technology comprises a SNP. There are many synonymous SNPs that are heterozygous within the MYH7 locus, making them a good candidate to be using for linking (pathogenic) mutations in the MYH7 gene to a particular chromosome and thereby target the mutations in an allelespecific fashion. In some embodiments, the SNP is common in the human population, for example, has an at least 10%, at least 20%, at least 30, or at least 40% heterozygosity in the human population, i.e., at least 10%, at least 20%, at least 30, or at least 40% of the human population is heterozygous for that SNP. In some embodiments, the SNP is common in a particular patient population (e.g., a particular ethnic group or a subtype of cardiomyopathy or HCM), for example, has an at least 10%, at least 20%, at least 30, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% heterozygosity in the patient population, i.e., at least 10%, at least 20%, at least 30, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% of the patient population is heterozygous for that SNP.

[0075] In some embodiments, the SNP is located in a region within a coding sequence (CDS), an exon, an intron, a sequence spanning a portion of an exon and a portion of an adjacent intron, or a regulatory region of the MYH7 gene. In some embodiments, the SNP is located with an exon of the MYH7 gene, so that one can design a gRNA specific to the region / sequence containing the SNP for purposes of knocking out the MYH7 gene allele through CRISPR / Cas.

[0076] Exemplary SNPs within the MYH7 gene locus are summarized in Table 1A below. The table shows six exemplary synonymous SNPs in the MYH7 gene, their positions on human genome build 38 (position Hg38), their internationally recognized RSID numbers, the SNP ID numbers in a chromosome-position-reference-alternate format (chr-pos-ref-alt), and their impact upon the proteins in human genome variation nomenclature (HGVSp). Additionally, the percent of heterozygous individuals who would be eligible for a gene therapy targeting the reference or alternate allele calculated usingthe 1000 genomes reference panel (phase 3), an ethnically and geographically diverse group of 2557 individuals used for the human genome variation project (https: / / www.internationalgenome.org / category / reference / ), is shown. The presence of each allele in every sampled ethnic group confirmed in a separate database of genomic variation (https: / / gnomad.broadinstitute.org / ) comprising more than 110,000 individuals of diverse ancestry is also shown. Each of these SNPs is present at a significant allele frequency in almost every ethnic group with the highest and lowest minor allele frequency shown in the gnomAD column.Table 1A. Exemplary SNPs in the MYH7 gene

[0077] In some embodiments, the SNP is selected from the group consisting of rs735711 , rs735712, rs2069540, rs2069542, rs2231126, and rs7157716. In some embodiments, the SNP is rs735711. In some embodiments, the SNP is rs735712. In some embodiments, the SNP is rs2069540. In some embodiments, the SNP is rs2069542. In some embodiments, the SNP is rs2231126. In some embodiments, the SNP is rs7157716.

[0078] In some embodiments, the SNP has two or more alternative bases or alleles, one of which may be referred to as a reference allele, and the others alternate alleles. It should be noted that the term “reference allele” simply refers to the base that is found in the reference genome, and it is not necessarily the major or most dominant allele. In contrast, the alternative allele refers to any base, other than the reference, that is found at that locus. The alternative allele is not necessarily the minor allele, and it may or maynot be linked to a phenotype. There can be more than one alternative allele per variant. For example, rs2069540 is a SNP located in the first coding exon of MYH7 with the reference allele being a “G” and the alternate allele being an “A” (FIG. 2). The SNP, however, is synonymous as it does not change the amino acid being coded (threonine, T).

[0079] In some embodiments, the method of the present technology can be expanded to identify two or more heterozygous genetic variants within the MYH7 gene locus, for example, two or more SNPs, in step (a). As shown in FIG. 3, by identifying multiple SNPs (and for each developing two therapeutic products such as gRNAs, one covering the reference allele and the other covering the alternate allele), there is a possibility of increasing the eligible patient population ( / .e., people who are heterozygous for at least one of the targeted SNPs) that could benefit from the method of the present technology. For example, by designing a combination therapy for both rs2069540 and rs7157716, 65.6% of the population would be at least heterozygous for one of those SNPs and would thus be suitable for a drug product targeting the reference or alternate alleles of at least one of rs2069540 and rs7157716.Haplotyping

[0080] In some embodiments, techniques of haplotyping may be used in step (b) of the present technology to determine which of the reference and alternate alleles of the genetic variant (e.g., SNP) resides on the same chromosome as the mutation. Techniques known to a person skilled in the art to identify haplotype information include genome phasing or phased sequencing, which utilizes DNA sequence technologies (e.g., long-read sequencing, highly accurate long-read sequencing (HiFi), Hi-C, strand-seq) to produce phased assemblies and determine which variants are from the same copy of a chromosome. Alternatively, if the genetic variant (e.g., SNP) and the mutation both reside in a coding region (e.g., exons), long-read RNA sequencing (RNA-seq) techniques can be used to sequence the full-length transcript and determine which allele of the genetic variant (SNP) is from the same copy of chromosome that is transcribed with the gene.Gene editing by CRISPR / Cas system

[0081] In some embodiments, the CRISPR / Cas system is used to knock down, knock out, or otherwise disrupt the expression of the mutant allele in step (c) of thepresent technology. CRISPR / Cas refers to clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins (also interchangeably referred to as “Cas nucleases” or “Cas endonucleases” in this disclosure), and the CRISPR / Cas system refers collectively to transcripts and other elements involved in the expression of or directing the activity of Cas genes, including sequences encoding a Cas nuclease, a guide RNA (gRNA) sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), and / or other sequences and transcripts from a CRISPR locus. It was originally discovered in prokaryotic organisms (e.g., bacteria and archaea) as a system involved in defense against invading phages and plasmids that provides a form of acquired immunity. Now it has been adapted and used as a popular gene editing tool in research and clinical applications.

[0082] CRISPR / Cas systems generally comprise at least two components: one or more guide RNAs (gRNAs), which sequence-specifically bind to DNA and a Cas protein, which has nuclease functionality. The Cas nuclease can introduce a double-strand break (DSB) into the target site, followed by disruptions or alterations, such as insertions and / or deletions (indels), as discussed herein. CRISPR-Cas systems fall into two major classes: class 1 systems use a complex of multiple Cas nucleases to degrade nucleic acids; class 2 systems use a single large Cas nuclease for the same purpose. Class 1 is divided into types I, III, and IV; class 2 is divided into types II, V, and VI. Different Cas nucleases adapted for gene editing applications include, but are not limited to, Cas1 , Cas1 B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), Casi o, CasX, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof. The most widely used Cas9 is a type II Cas nuclease and is described herein as illustrative. These Cas nucleases may be originated from different source species. For example, Cas9 can be derived from S. pyogenes (SpCas9) or S. aureus (SaCas9).

[0083] In the original microbial genome, the type II CRISPR system incorporates sequences from invading DNA between CRISPR repeat sequences encoded as arrays within the host genome. Transcripts from the CRISPR repeat arrays are processed into CRISPR RNAs (crRNAs) each harboring a variable sequence transcribed from the invading DNA, known as the “protospacer” sequence, as well as part of the CRISPRrepeat. Each crRNA hybridizes with a second transactivating CRISPR RNA (tracrRNA), and these two RNAs form a complex with the Cas9 nuclease. The protospacer-encoded portion of the crRNA directs the Cas9 complex to cleave complementary target DNA sequences, provided that they are adjacent and 5’ to short sequences known as “protospacer adjacent motifs” (PAMs).

[0084] While the foregoing description has focused on Cas9 nuclease, it should be appreciated that other RNA-guided nucleases exist which utilize gRNAs that differ in some ways from those described to this point. For instance, Cpf1 (also known as Cas12a) is an RNA-guided nuclease that only requires a crRNA and does not need a tracrRNA to function.

[0085] Since its discovery, the CRISPR system has been adapted for inducing sequence specific DSBs and targeted genome editing in a wide range of cells and organisms spanning from bacteria to eukaryotic cells including human cells. Eukaryotic cells repair DSBs by two primary repair pathways: non-homologous end-joining (NHEJ) and homology-directed repair (HDR). HDR typically occurs during late S phase or G2 phase, when a sister chromatid is available to serve as a repair template. NHEJ is more common and can occur during any phase of the cell cycle, but it is more error prone. In gene editing, NHEJ is generally used to produce insertion / deletion mutations (indels), which can produce targeted loss of function in a target gene by shifting the open reading frame (ORF) and producing alterations in the coding region or an associated regulatory region. HDR, on the other hand, is a preferred pathway for producing targeted knock- ins, knockouts, or insertions of specific mutations in the presence of a repair template with homologous sequences. Several methods are known to a skilled artisan to improve HDR efficiency, including, for example, chemical modulation (e.g., treating cells with inhibitors of key enzymes in the NHEJ pathway); timed delivery of the gene editing system at S and G2 phases of the cell cycle; cell cycle arrest at S and G2 phases; and introduction of repair templates with homology sequences.

[0086] In its use in gene editing applications, artificially designed, synthetic gRNAs have replaced the original crRNA:tracrRNA complexes, including in certain embodiments via a single gRNA. For example, the gRNAs can be single guide RNAs (sgRNAs) composed of a crRNA, a tetraloop, and a tracrRNA. The crRNA usually comprises a complementary region (also called a spacer, usually about 20 nucleotides in length) thatis user-designed to recognize a target DNA of interest. The tracrRNA sequence comprises a scaffold region for Cas nuclease binding. The crRNA sequence and the tracrRNA sequence are linked by the tetraloop and each have a short repeat sequence for hybridization with each other, thus generating a chimeric sgRNA. One can change the genomic target of the Cas nuclease by simply changing the spacer or complementary region sequence present in the gRNA. The complementary region will direct the Cas nuclease to the target DNA site through standard RNA-DNA complementary base pairing rules. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence, gene, or locus. Typically, “target sequence” generally refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between the target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex.

[0087] For the Cas nuclease to function, there must be a PAM immediately downstream of (3’ to) the target sequence in the genomic DNA. Recognition of the PAM by the Cas nuclease is thought to destabilize the adjacent genomic sequence, allowing interrogation of the sequence by the gRNA and resulting in gRNA-DNA pairing when a matching sequence is present. The specific sequence of PAM varies depending on the species of the Cas gene. For example, the SpCas recognizes a PAM sequence of 5’- NGG-3’ or, at less efficient rates, 5’-NAG-3’, where N can be any nucleotide. For another example, the SaCas9 PAM sequence is NNGRR or for optimal on-target cutting is NNGRRT, wherein N can be any nucleotide, and R can be guanine or adenine. Other Cas nuclease variants with alternative PAMs have also been characterized and successfully used for genome editing. Therefore, the PAM sequence adjacent to the target sequence is an essential targeting component for the design of CRISPR / Cas9- mediated gene editing, and when designing gRNAs targeting a specific genomic locus, one skilled in the art needs to consider the availability and / or location of PAM sequences optimal for the Cas nuclease of choice at the target locus and, if necessary, select a different Cas nuclease / PAM sequence pair based on the DNA sequence at the target locus. PAMs for use with different Cas endonucleases are known in the art. Illustrative examples of Cas enzymes that can be used as described herein and PAMs for use with their respective Cas endonucleases are shown in Table 2 below (where M is adenine orcytosine; N is any nucleotide; R is guanine or adenine; V is guanine, cytosine, or adenine; W is adenine or thymine; and Y is cytosine or thymine).Table 2. Exemplary Cas proteins and their corresponding PAM sequences

[0088] In some embodiments, the Cas nuclease is any Cas nuclease (e.g., any Cas9 nuclease) which is encoded by a gene equal to or less than 3.3 kb in size, equal to or less than 3.2 kb in size, equal to or less than 3.1 kb in size, equal to or less than 3 kb in size, equal to or less than 2.9 kb in size, or equal to or less than 2.8 kb in size. Insome embodiments, the Cas nuclease is any Cas nuclease (e.g., any Cas9 nuclease) which has the protein size of equal to or less than 1 ,100 amino acids, equal to or less than 1 ,075 amino acids, equal to or less than 1 ,060 amino acids, equal to or less than 1 ,050 amino acids, equal to or less than 1 ,000 amino acids, equal to or less than 950 amino acids, or equal to or less than 900 amino acids.

[0089] In some embodiments, the Cas nuclease is a Cas9 nuclease. In some embodiments, the Cas9 is derived from S. aureus, S. pyogenes, F. novicida, N. meningitidis, S. thermophilus, Acidaminococcus sp., G. stearothermophilus, N. mucosa, S. canis, Lachnospiraceae bacterium., S. sanguinis, N. subflava, S. epidermidis, S. agalactiae, L monocytogenes, Actinomyces sp., S. anginosus, S. dysgalactiae, C. jejuni, B. thuringiensis, C. difficile, S. cristatus, S. mutans, or S. pneumonia. In some embodiments, Cas9 endonuclease is a S. aureus Cas9 (SaCas9) or a variant thereof. In some embodiments, Cas9 endonuclease is a S. pyogenes Cas9 (SpCas9) or a variant thereof.

[0090] In some embodiments, the Cas nuclease is a Cas12a nuclease. In some embodiments, the Cas12a is derived from Acidaminococcus sp., M. bovoculi, A. cellulolyticus, Prevotella sp., S. mutans, Acidovorax sp., A. acidocaldarius, P. aeruginosa, L. crispatus, M. osloensis, or K. oxytoca.

[0091] In some embodiments, Cas nuclease is a Cas12b nuclease. In some embodiments, the Cas12b is derived from Lachnospiraceae bacterium, Ruminococcus sp., P. gingivalis, P. intermedia, Enterobacter sp., S. pneumoniae, Acidobacterium sp., B. fragilis, S. marcescens, B. uniformis, or B. thuringiensis.

[0092] In some embodiments, the Cas nuclease is a CasX nuclease. In some embodiments, the CasX is derived from Ruminococcus sp., Pseudomonas sp., or S. epidermidis.

[0093] In some embodiments, Cas nucleases may comprise one or more mutations to alter their activity, specificity, recognition, and / or other characteristics. For example, the Cas nuclease may have one or more mutations that alter its fidelity to mitigate off- target effects (e.g., eSpCas9, SpCas9-HF1 , HypaSpCas9, HeFSpCas9, and evoSpCas9 high-fidelity variants of SpCas9). For another example, the Cas nuclease may have one or more mutations that alter its PAM specificity.

[0094] In some embodiments, a coding sequence encoding a Cas nuclease can be codon optimized for expression in particular cells, such as eukaryotic cells. The eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human primate. In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization.

[0095] In some embodiments, a polynucleotide encoding a Cas9 nuclease comprises or consists of a sequence that shares at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 1. In some embodiments, the Cas9 nuclease used as described herein or encoded by the polynucleotides described herein comprises an amino acid sequence that shares at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 2.Table 3. Example Cas nuclease sequencesGuide RNAs (QRNAS) for targeting genetic variants in MYH7

[0096] In some embodiments, a gRNA specific to the genetic variant is used with the CRISPR / Cas system to knock down, knock out, or otherwise disrupt the expression of the mutant allele in step (c) of the present technology. The gRNA may comprise a crRNA sequence, which in turn comprises a complementary region (also called a spacer) that recognizes and binds a complementary target sequence of interest. The length of the spacer or complementary region is generally between 15 and 30 nucleotides, usually about 20 nucleotides in length, although will vary based on the requirements of the specific CRISPR / Cas system. In certain embodiments, the spacer or complementary region is fully complementary to the target sequence. In other embodiments, the spacer is partially complementary to the target sequence, for example at least 80%, 85%, 90%, 95%, 98%, or 99% complementary.

[0097] In some embodiments, the gRNA comprises a complementary region that binds to a target sequence in the MYH7 gene. In some embodiments, the complementary region binds to a target sequence comprising about 20, 21 , or 22 consecutive nucleotides in any one of SEQ ID NOs: 241 -251 . Exemplary target sequences are presented in Table 4A below.Table 4A. Exemplary target sequences

[0098] In certain embodiments, the gRNA further comprises a tracrRNA sequence, which comprises a scaffold region for binding to a nuclease. The length and / or sequence of the tracrRNA may vary depending on the specific nuclease being used for editing. In certain embodiments, nuclease binding by the gRNA does not require a tracrRNA sequence. In those embodiments where the gRNA comprises a tracrRNA, the crRNA sequence may further comprise a repeat region for hybridization with complementary sequences of the tracrRNA. Exemplary gRNAs (e.g., sgRNAs) for use with various common Cas nucleases that require both a crRNA and tracrRNA, including Cas9 andCas12b (C2c1 ), are provided in Table 4B below (wherein n is any nucleotide; s is guanine or cytosine). For each exemplary gRNA, sequences for different portions of the gRNA, including the crRNA repeat region, tetraloop, and tracrRNA, are shown. In some embodiments, the gRNA comprises two or more gRNA molecules, for example, a crRNA and a tracrRNA, as two separate molecules. In other embodiments, the gRNA is a single guide RNA (sgRNA), including those comprising a crRNA and a tracrRNA on a single RNA molecule. In certain of these embodiments, the crRNA and tracrRNA are linked by an intervening tetraloop.Table 4B. Example gRNA sequences for CRISPR / Cas

[0099] In some embodiments, the gRNA comprises a complementary region specific to any genetic variant within the MYH7 locus disclosed herein, for example, any of the SNPs provided in Table 1. Exemplary complementary region sequences for use in the present technology are provided in Table 4C below. In some embodiments, the gRNA complementary region sequence is described by its encoding DNA sequence. For example, the gRNA “gagaucaucgccaagcugacca” (SEQ ID NO: 227) is described herein and in Table 4C below by its encoding DNA sequence “gagatcatcgccaagctgacca” (SEQ ID NO: 146). Because each genetic variant (e.g., SNP) has more than one allele, for example, a reference allele and an alternate allele, exemplary complementary regionsequences specific to each allele are provided. For some SNPs, and for some alleles thereof, more than one complementary region is provided. The nucleotide corresponding to the SNP in each gRNA sequence is highlighted (bold and underlined). One may select the sequence specific to a particular allele to use in the present technology, depending on which copy of chromosome is targeted.Table 4C. Exemplary gRNA complementary region sequences

[0100] In some embodiments, the gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in any one of SEQ ID NOs: 12-23, 144-147, or 229-239or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 12-23, 144-147, or 229- 239.

[0101] In some embodiments, the gRNA comprises a complementary region that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 17. In some embodiments, the gRNA comprises a complementary region that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 22. In some embodiments, the gRNA comprises a complementary region that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 144. In some embodiments, the gRNA comprises a complementary region that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 145. In some embodiments, the gRNA comprises a complementary region that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 146. In some embodiments, the gRNA comprises a complementary region that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 147. In some embodiments, the gRNA comprises a complementary region that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 229. In some embodiments, the gRNA comprises acomplementary region that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 230. In some embodiments, the gRNA comprises a complementary region that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 231 .

[0102] In some embodiments, the gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in any one of SEQ ID NOs: 12-23, 144-147, and 229-231 , with up to one, two, three, or four nucleotide mismatches, substitutions, deletions, or additions. In some embodiments, the gRNA comprises a complementary region comprising SEQ ID NO: 17, with up to one, two, three, or four nucleotide mismatches, substitutions, deletions, or additions. In some embodiments, the gRNA comprises a complementary region comprising SEQ ID NO: 22, with up to one, two, three, or four nucleotide mismatches, substitutions, deletions, or additions. In some embodiments, the gRNA comprises a complementary region comprising SEQ ID NO: 144, with up to one, two, three, or four nucleotide mismatches, substitutions, deletions, or additions. In some embodiments, the gRNA comprises a complementary region comprising SEQ ID NO: 145, with up to one, two, three, or four nucleotide mismatches, substitutions, deletions, or additions. In some embodiments, the gRNA comprises a complementary region comprising SEQ ID NO: 146, with up to one, two, three, or four nucleotide mismatches, substitutions, deletions, or additions. In some embodiments, the gRNA comprises a complementary region comprising SEQ ID NO: 147, with up to one, two, three, or four nucleotide mismatches, substitutions, deletions, or additions. In some embodiments, the gRNA comprises a complementary region comprising SEQ ID NO: 229, with up to one, two, three, or four nucleotide mismatches, substitutions, deletions, or additions. In some embodiments, the gRNA comprises a complementary region comprising SEQ ID NO: 230, with up to one, two, three, or four nucleotide mismatches, substitutions, deletions, or additions. In some embodiments, the gRNA comprises a complementary region comprising SEQ ID NO: 231 , with up to one, two, three, or four nucleotide mismatches, substitutions, deletions, or additions.

[0103] In some embodiments, the gRNA comprises a complementary region comprising, consisting of, or consisting essentially of SEQ ID NO: 12. In some embodiments, the gRNA comprises a complementary region comprising, consisting of, or consisting essentially of SEQ ID NO: 13. In some embodiments, the gRNA comprisesa complementary region comprising, consisting of, or consisting essentially of SEQ ID NO: 14. In some embodiments, the gRNA comprises a complementary region comprising, consisting of, or consisting essentially of SEQ ID NO: 15. In some embodiments, the gRNA comprises a complementary region comprising, consisting of, or consisting essentially of SEQ ID NO: 16. In some embodiments, the gRNA comprises a complementary region comprising, consisting of, or consisting essentially of SEQ ID NO: 17. In some embodiments, the gRNA comprises a complementary region comprising, consisting of, or consisting essentially of SEQ ID NO: 18. In some embodiments, the gRNA comprises a complementary region comprising, consisting of, or consisting essentially of SEQ ID NO: 19. In some embodiments, the gRNA comprises a complementary region comprising, consisting of, or consisting essentially of SEQ ID NO: 20. In some embodiments, the gRNA comprises a complementary region comprising, consisting of, or consisting essentially of SEQ ID NO: 21. In some embodiments, the gRNA comprises a complementary region comprising, consisting of, or consisting essentially of SEQ ID NO: 22. In some embodiments, the gRNA comprises a complementary region comprising, consisting of, or consisting essentially of SEQ ID NO: 144. In some embodiments, the gRNA comprises a complementary region comprising, consisting of, or consisting essentially of SEQ ID NO: 145. In some embodiments, the gRNA comprises a complementary region comprising, consisting of, or consisting essentially of SEQ ID NO: 146. In some embodiments, the gRNA comprises a complementary region comprising, consisting of, or consisting essentially of SEQ ID NO: 147. In some embodiments, the gRNA comprises a complementary region comprising, consisting of, or consisting essentially of SEQ ID NO: 229. In some embodiments, the gRNA comprises a complementary region comprising, consisting of, or consisting essentially of SEQ ID NO: 230. In some embodiments, the gRNA comprises a complementary region comprising, consisting of, or consisting essentially of SEQ ID NO: 231. In some embodiments, the gRNA comprises a complementary region comprising, consisting of, or consisting essentially of SEQ ID NO: 232. In some embodiments, the gRNA comprises a complementary region comprising, consisting of, or consisting essentially of SEQ ID NO: 233. In some embodiments, the gRNA comprises a complementary region comprising, consisting of, or consisting essentially of SEQ ID NO: 234. In some embodiments, the gRNA comprises a complementary region comprising, consisting of, or consisting essentially of SEQ ID NO: 235. In someembodiments, the gRNA comprises a complementary region comprising, consisting of, or consisting essentially of SEQ ID NO: 236. In some embodiments, the gRNA comprises a complementary region comprising, consisting of, or consisting essentially of SEQ ID NO: 237. In some embodiments, the gRNA comprises a complementary region comprising, consisting of, or consisting essentially of SEQ ID NO: 238. In some embodiments, the gRNA comprises a complementary region comprising, consisting of, or consisting essentially of SEQ ID NO: 239.

[0104] In some embodiments, the gRNA specific to SNP rs735711 comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 12 or 232 (corresponding to the reference allele) or SEQ ID NO: 13 or 233 (corresponding to the alternate allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 12, 13, 232, or 233. In some embodiments, the gRNA specific to SNP rs735712 comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 14 or 234 (corresponding to the reference allele) or SEQ ID NO: 15 or 235 (corresponding to the alternate allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 14, 15, 234, or 235. In some embodiments, the gRNA specific to SNP rs2069540 comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 16 or 229 (corresponding to the reference allele) or SEQ ID NO: 17 or 230 (corresponding to the alternate allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 16, 17, 229, or 230. In some embodiments, the gRNA specific to SNP rs2069540 comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 230 (corresponding to the alternate allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 230. In some embodiments, the gRNA specific to SNP rs2069542 comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 18 or 236 (corresponding to the reference allele) or SEQ ID NO: 19 or 237 (corresponding to thealternate allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 18, 19, 236, or 237. In some embodiments, the gRNA specific to SNP rs2231126 comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 20 or 238 (corresponding to the reference allele) or SEQ ID NO: 21 or 239 (corresponding to the alternate allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 20, 21 , 238, or 239.

[0105] In some embodiments, the gRNA specific to SNP rs7157716 comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 22 or 231 (corresponding to the reference allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 22 or 231. In some embodiments, the gRNA specific to SNP rs7157716 comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 23, 144, 145, 146, or 147 (corresponding to the alternate allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 23, 144, 145, 146, or 147. In some embodiments, the gRNA specific to SNP rs7157716 comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 146 (corresponding to the alternate allele) or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 146.

[0106] In some embodiments, administration of Cas nuclease and / or gRNA, or vectors comprising the same, as described herein to a cell with a heterozygous mutation in the MYH7 gene results in an editing efficiency (e.g., knocking down, knocking out, or otherwise altering the expression of the mutant allele) of, or an efficiency of indel formation (e.g., forming a frameshift mutation) in, the mutant allele of the MYH7 gene of at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%. In some embodiments, administrationof the Cas nuclease and / or gRNA, or vectors comprising the same, as described herein to a cell with a heterozygous mutation in the MYH7 gene does not, or substantially does not edit, or form indels in, the wild-type allele of the MYH7 gene.Delivery of CRISPR / Cas system to cells

[0107] In some embodiments, the CRISPR / Cas system is delivered to a cell to knock down, knock out, or otherwise disrupt the expression of the mutant allele in step (c) of the present technology. The components of a CRISPR / Cas system can be implemented in any suitable manner, meaning that the components of such systems including the Cas nuclease and the gRNA can be delivered, formulated, or administered in any suitable form to the cells. For example, the Cas nuclease may be delivered to a cell complexed with the gRNA (e.g., as a ribonucleoprotein (RNP) complex), the Cas nuclease may be delivered to a cell separate (e.g., uncomplexed) to the gRNA, the Cas nuclease may be delivered to a cell as a polynucleotide (e.g., DNA or RNA) encoding the nuclease that is separate from the gRNA, or both the Cas nuclease and the gRNA molecule may be delivered as polynucleotides encoding each component.

[0108] In some embodiments, one or more vectors driving expression of one or more components of the CRISPR / Cas system can be introduced into the cell such that expression of the components of the CRISPR / Cas system direct formation of the CRISPR complex at one or more target sites. Components can also be delivered to cells as ribonucleoprotein complexes, proteins, DNA, and / or RNA. For example, a Cas enzyme, an RNA guide sequence linked to a tracr-mate sequence, and a tracrRNA sequence could each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the components expressed from the same or different regulatory elements, may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR / Cas system not included in the first vector. The vector may comprise one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a “cloning site”). In some embodiments, one or more insertion sites are located upstream and / or downstream of one or more sequence elements of one or more vectors. In addition, a nucleic acid encoding the Cas nuclease may be delivered with one or more gRNAs.

[0109] In some embodiments, polynucleotides encoding a Cas nuclease and a gRNA are delivered to the cell in one or more vectors. The polynucleotide encoding theCas protein and the polynucleotide encoding the gRNA can be in the same vector, or in separate vectors. When in the same vector, the polynucleotide encoding the Cas protein and the polynucleotide encoding the gRNA can be in a head-to-head, head-to-tail, or tai I- to-tail orientation.

[0110] In some embodiments, the vector comprises an expression cassette comprising (i) a first polynucleotide encoding a Cas nuclease (e.g., a Cas9 such as SaCas9) operably linked to a protein-expression driving promoter, and / or (ii) a second polynucleotide encoding a gRNA specific to a genetic variant (e.g., a SNP) within the MYH7 locus operably linked to an RNA expression-driving promoter. In some embodiments, the first polynucleotide and the second polynucleotide have a head-to- head orientation in the same vector. In some embodiments, the first polynucleotide and the second polynucleotide have a head-to-tail orientation in the same vector. In some embodiments, the first polynucleotide and the second polynucleotide have a tail-to-tail orientation in the same vector. In some embodiments, the first polynucleotide and the second polynucleotide are in separate vectors.

[0111] The polynucleotides, expression cassettes, and / or vectors contemplated herein may be combined with other sequences, such as promoters, enhancers, untranslated regions (UTRs), introns, signal sequences, Kozak sequences, polyadenylation (poly(A)) signals, post-transcriptional regulatory elements, additional restriction enzyme sites, multiple cloning sites, internal ribosomal entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), termination codons, transcriptional termination signals, polynucleotides encoding self-cleaving polypeptides, epitope tags, and / or any other regulatory elements as disclosed elsewhere herein or as known in the art. In some embodiments, the polynucleotides, expression cassettes, and / or vectors described herein may also contain a ribosome binding site for translation initiation, a transcription terminator, and / or polynucleotide sequences for amplifying expression. The expression cassette may be flanked by one or more inverted terminal repeats (ITRs). The ITRs in an expression cassette serve as markers used for viral packaging of the expression cassette. The expression cassette can be integrated into the host cell genome, thereby expressing the transgene within a host cell.

[0112] As used herein, the term “regulatory element” refers to those non-translated regions of the vector (e.g., origin of replication, selection cassettes, promoters,enhancers, translation initiation signals (Kozak sequence), introns, poly(A) sequences, 5' and 3' untranslated regions) which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. The transcriptional regulatory element may be functional in either a eukaryotic cell (e.g., a mammalian cell) or a prokaryotic cell (e.g., bacterial or archaeal cell). In some embodiments, a polynucleotide sequence described herein is operably linked to multiple control elements that allow expression of the polynucleotide in both prokaryotic and eukaryotic cells.Promoters

[0113] The term “promoter” as used herein refers to a DNA sequence that directs the binding of RNA polymerase and thereby promotes RNA synthesis. Promoters and corresponding protein or polypeptide expression may be ubiquitous, meaning strongly active in a wide range of cells, tissues, and species or cell-type specific, tissue-specific, or species specific. Examples of ubiquitous promoters include the CAG promoter and CMV promoter. Promoters may be “constitutive,” meaning continually active, or “inducible,” meaning the promoter can be activated or deactivated by the presence or absence of biotic or abiotic factors. Also included in the nucleic acid constructs or vectors of the invention are enhancer sequences that may or may not be contiguous with the promoter sequence. Enhancer sequences influence promoter-dependent gene expression and may be located in the 5' or 3' regions of the native gene.

[0114] In some embodiments, the first promoter driving expression of the Cas nuclease is any promoter suitable for protein expression described herein or known in the art. In some embodiments, the first promoter is a constitutive promoter. As used herein, a “constitutive promoter” is one wherein the level of expression does not vary from one cell type compared to a different cell type. Suitable constitutive promoters include elongation factor 1 alpha (EF1 a) promoter, cytomegalovirus (CMV) immediate- early promoter, simian vacuolating virus 40 (SV40) early promoter, spleen focus-forming virus (SFFV) promoter, phosphoglycerate kinase (PGK) promoter, human beta actin promoter, polyubiquitin C gene (UBC) promoter, and CAG promoter.

[0115] In some embodiments, the first promoter is a muscle cell-specific promoter or a cardiac-specific promoter described herein or known in the art. In some embodiments, the first promoter is a cardiomyocyte-specific promoter. A “cardiac-specific” or “cardiomyocyte-specific” promoter as used herein specifies a promoter whose activity in cardiac cells or cardiomyocytes is at least 2-fold higher than in any other noncardiac cell type. Preferably, a cardiac-specific or cardiomyocyte-specific promoter suitable for the present technology has an activity in cardiac cells or cardiomyocytes which is at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, or at least 50-fold higher compared to its activity in a non-cardiac cell type. Examples of cardiac-specific or cardiomyocyte-specific promoters include, but are not limited to, alpha myosin heavy chain promoter, myosin light chain 2v promoter, alpha-cardiac actin promoter, alpha-tropomyosin promoter, cardiac troponin C promoter, cardiac troponin I promoter, cardiac myosin-binding protein C promoter, and sarco / endoplasmic reticulum Ca2+ATPase (SERCA) promoter (e.g., isoform 2 of SERCA2).Table 5A. Exemplary cardiac-specific promoter sequences

[0116] In some embodiments, the first promoter is a cardiac troponin T (TNNT2) promoter, for example, a human TNNT2 promoter, a chicken TNNT2 promoter, a mouse TNNT2 promoter, or a chimeric or variant of any of the foregoing. In some embodiments, the TNNT2 promoter is modified from the corresponding wild-type promoter, e.g., by deletion, insertion, or substitution of polynucleotides. In some embodiments, the TNNT2 promoter is a modified human TNNT2 promoter (e.g., a truncated human TNNT2 promoter) as described herein. Illustrative polynucleotide sequences of TNNT2 promoter are shown in Table 5A. The transcription start site (TSS) of certain TNNT2 promoters are bolded and underlined. As used herein, the term “transcription start site” or “TSS” refers to the first base pair transcribed by an RNA polymerase when the RNA polymerase initiates transcription. A TSS is different from the start codon (canonically, ATG), which must be downstream of the TSS in the transcribed region of the polynucleotide. The location of a TSS can be determined experimentally or by prediction using any of various prediction algorithms. Annotated TSSs are available from the Eukaryotic Promoter Database and the LICSC Genome Browser. As used herein, the TSS for TNNT2 is defined to be the sequence identified by the C at the 5’ end of the motif identified by dbTSS: CTCCATC.

[0117] Additional examples of TNNT2 promoters can be found in WO2021 / 163357, WO2023 / 283649, and / or U.S. Patent No. 11 ,129,908, the entire contents of each of which are incorporated by reference herein.

[0118] In some embodiments, the first promoter is a human troponin T (HuTNNT2) promoter or a chimeric or variant thereof. In some embodiments, the first promoter comprises, consists of, or consists essentially of a nucleotide sequence set forth in any one of SEQ ID NOs: 24-27, or a nucleotide sequence that shares at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%,at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 24-27.

[0119] In some embodiments, the first promoter is a modified TNNT2 promoter, e.g., a promoter that comprises a polynucleotide sequence of at least 200 base pairs that comprises one or more continuous or discontinuous polynucleotide segments each sharing at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a corresponding segment of the HuTNNT2 600 segment provided in Table 5A as SEQ ID NO: 24. As it is a “promoter,” the modified TNNT2 promoter must be capable of promoting initiation of transcription by an RNA polymerase in a host or target cell at or near a TSS within the promoter ( / .e., at or near the TTS of TNNT2 as defined herein) or, if the endogenous TSS of TNNT2 is not present in the modified TNNT2 promoter then at a heterologous TSS at most 100 base pairs downstream (3’ on the sense strand) to the downstream (3’) end of the modified TNNT2 promoter. Similarly stated, the modified TNNT2 promoter may comprise only sequences upstream of the TSS of HuTNNT2 600 or more comprise the TSS of TNNT2-600.

[0120] In some embodiments, the first promoter is a modified TNNT2 promoter comprising a polynucleotide sequence of between about 300 to about 500 base pairs, between about 350 to about 500 base pairs, between about 350 to about 450 bp, between about 400 to about 450 base pairs, or between about 375 to about 425 base pairs in length. In some embodiments, the modified TNNT2 promoter comprises a polynucleotide sequence of between about 350 base pairs to about 450 base pairs, between about 375 base pairs to about 425 base pairs, between about 375 base pairs to about 400 base pairs, between about 375 base pairs to about 425 base pairs, between about 400 base pairs to about 425 base pairs, or between about 400 base pairs to about 450 base pairs. In some embodiments, the modified TNNT2 promoter comprises a polynucleotide sequence of about 400 base pairs.

[0121] In some embodiments, the first promoter has the same cell-type specificity as a native troponin T promoter of about 600 bp, for example, the reference HuTNNT2 600 promoter (SEQ ID NO: 24) having about 600 base pairs.

[0122] In some embodiments, the first promoter is a modified TNNT2 promoter comprising between 300 bp and 500 bp of SEQ ID NO: 24. For instance, the modified TNNT2 promoter may comprise SEQ ID NO: 25, 26, or 27. In some examples, the 300 bp-500 bp sequence may be linked to further polynucleotide sequences but may not belinked to additional sequences derived from SEQ ID NO: 24. For example, in an embodiment, the modified TNNT2 promoter may include nog more than 500 bp of SEQ ID NO: 24 but may include additional unrelated polynucleotide sequences. In another example, the modified TNNT2 promoter may include SEQ ID NO: 25, 26, or 27 and no additional sequences derived from SEQ ID NO: 24 but may include additional unrelated polynucleotide sequences.

[0123] In some embodiments, the TNNT2 promoter is modified by deletion of polynucleotides. A modification may include one, two, three or more internal deletions. Each deletion may be a deletion of 1 base pair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs, 10 base pairs, 15 base pairs, 20 base pairs, 25 base pairs, 30 base pairs, 40 base pairs, 50 base pairs, 60 base pairs, 70 base pairs, 80 base pairs, 90 base pairs, 100 base pairs, 125 base pairs, 150 base pairs, 175 base pairs, 200 base pairs, 225 base pairs, 250 base pairs, 275 base pairs, or 300 base pairs with respect to the reference HuTNNT2 600 promoter (SEQ ID NO: 24) having about 600 base pairs.

[0124] In some embodiments, the TNNT2 promoter is modified by the deletion of polynucleotides from the upstream end of the promoter with respect to the reference HuTNNT2 600 promoter (SEQ ID NO: 24) having about 600 base pairs. A modification may include the deletion of 1 base pair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs, 10 base pairs, 15 base pairs, 20 base pairs, 25 base pairs, 30 base pairs, 40 base pairs, 50 base pairs, 60 base pairs, 70 base pairs, 80 base pairs, 90 base pairs, 100 base pairs, 125 base pairs, 150 base pairs, 175 base pairs, 200 base pairs, 225 base pairs, 250 base pairs, 275 base pairs, or 300 base pairs from the upstream end of the promoter with respect to the reference HuTNNT2 600 promoter (SEQ ID NO: 24) having about 600 base pairs. In some embodiments, the modification is a 200 base pair deletion from the upstream end of the promoter with respect to the reference HuTNNT2 600 promoter (SEQ ID NO: 24) having about 600 base pairs.

[0125] In some embodiments, the TNNT2 promoter is modified by the deletion of polynucleotides from the downstream end of the promoter with respect to the reference HuTNNT2 600 promoter (SEQ ID NO: 24) having about 600 base pairs. A modification may include the deletion of 1 base pair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs, 10 base pairs, 15 base pairs, 20 base pairs, 25 base pairs, 30 base pairs, 40 base pairs, 50 base pairs, 60 base pairs, 70 base pairs, 80 base pairs, 90 base pairs, 100 basepairs, 125 base pairs, 150 base pairs, 175 base pairs, 200 base pairs, 225 base pairs, 250 base pairs, 275 base pairs, or 300 base pairs from the downstream end of the promoter with respect to the reference HuTNNT2 600 promoter (SEQ ID NO: 24) having about 600 base pairs.

[0126] In some embodiments, the TNNT2 promoter is modified by an internal deletion of polynucleotides. A modification may include the internal deletion of 1 base pair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs, 10 base pairs, 15 base pairs, 20 base pairs, 30 base pairs, 40 base pairs, 50 base pairs, 60 base pairs, 70 base pairs, 80 base pairs, 90 base pairs, 100 base pairs, 125 base pairs, 150 base pairs, 175 base pairs, 200 base pairs, 225 base pairs, 250 base pairs, 275 base pairs, or 300 base pairs with respect to the reference HuTNNT2 600 promoter (SEQ ID NO: 24) having about 600 base pairs.

[0127] In some embodiments, the TNNT2 promoter is modified by the insertion of polynucleotides. A modification may include the insertion of 1 base pair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs, 10 base pairs, 15 base pairs, 20 base pairs, 25 base pairs, 30 base pairs, 35 base pairs, 40 base pairs, 45 base pairs, 50 base pairs, 55 base pairs, 60 base pairs, 65 base pairs, 70 base pairs, 75 base pairs, 80, base pairs, 85 base pairs, 90 base pairs, 100 base pairs, 125 base pairs, 150 base pairs, 175 base pairs, 200 base pairs, 225 base pairs, 250 base pairs, 275 base pairs, or 300 base pairs with respect to the reference HuTNNT2 600 promoter (SEQ ID NO: 24) having about 600 base pairs.

[0128] In some embodiments, the TNNT2 promoter is modified by the substitution of polynucleotides. A modification may include the substitution of 1 base pair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs, 6 base pairs, 7 base pairs, 8 base pairs, 9 base pairs, or 10 base pairs with respect to the reference HuTNNT2 600 promoter (SEQ ID NO: 24) having about 600 base pairs.

[0129] In some embodiments, the first promoter is a mouse troponin T (MsTNNT2) promoter or a chimeric or variant thereof. In some embodiments, the first promoter comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 124, or a nucleotide sequence that shares at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 124.

[0130] In some embodiments, the first promoter is a chicken troponin T (ChTNNT2) promoter or a chimeric or variant thereof. In some embodiments, the first promoter comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 125, or a nucleotide sequence that shares at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 125.

[0131] In some embodiments, the first promoter is a chimeric variant derived from the human, mouse, or chicken TNNT2 promoter. A chimeric promoter can be generated by combining fragments from the TNNT2 promoter to sequences from other cardiacspecific promoters or ubiquitous or core promoters. In some embodiments, the first promoter comprises, consists of, or consists essentially of a nucleotide sequence set forth in any one of SEQ ID NOs: 126-131 , or a nucleotide sequence that shares at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 126-131.

[0132] In some embodiments, the second promoter driving expression of the gRNA is any promoter suitable for RNA expression described herein or known in the art. In some embodiments, the second promoter is an RNA polymerase III (Pol III) promoter, for example, a human Pol III promoter. Exemplary Pol III promoters for driving expression of gRNAs are provided in Table 5B below.Table 5B. Exemplary Pol III promoter sequences

[0133] In some embodiments, the second promoter is a human H1 promoter. In some embodiments, the second promoter comprises, consists of, or consists essentially of a nucleotide sequence set forth in any one of SEQ ID NOs: 28-30, or a nucleotide sequence that shares at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 28-30.

[0134] In some embodiments, the second promoter is a human H1 promoter. In some embodiments, the second promoter comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 28 or 29, or a nucleotide sequence that shares at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 28 or 29.

[0135] In some embodiments, the second promoter is a U6 promoter, for example, a human U6 promoter or a U6 promoter from other species such as mouse, C. elegans, and drosophila. In some embodiments, the second promoter comprises, consists of, or consists essentially of a nucleotide sequence set forth in any one of SEQ ID NOs: 31-37, or a nucleotide sequence that shares at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 31 -37.

[0136] In some embodiments, the second promoter is a human U6 promoter. In some embodiments, the second promoter comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 31 , or a nucleotide sequence that shares at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 31.Enhancers

[0137] The term “enhancer” refers to a segment of DNA which contains sequences capable of providing enhanced transcription and in some instances can function independent of their orientation relative to another control sequence. An enhancer can function cooperatively or additively with promoters and / or other enhancer elements. The term “enhancer” further refers to a DNA sequence that directs the binding of transcriptional regulatory proteins (e.g., transcriptional machinery) and RNA polymerase, and thereby promotes RNA synthesis. An enhancer may overlap with a promoter or be upstream or downstream of the promoter.

[0138] In some embodiments, the vector and / or expression cassette further comprises one or more enhancers. The one or more enhancers can be operably linked to the first and / or second promoter and modulate the expression of a transgene operably linked to the promoter. The presence of an enhancer can modulate transgene expression by, for example, increasing expression or decreasing expression. An enhancer can modulate transgene expression by, for example, increasing expression levels in a desired cell type, for example, a cardiac cell. An enhancer can modulate transgene expression by, for example, decreasing expression levels in an “off-target” cell type, or a cell type in which expression is not desired. In some embodiments, the first promoter is a TNNT2 promoter and comprises one or more enhancers. For example, a ACTC1 cardiac enhancer can be linked to a human TNNT2 promoter. In some embodiments, the first promoter is a TNNT2 promoter and comprises no enhancer. In some embodiments, the vector comprises an enhancer that is operably linked to another enhancer. For example, a ACTC1 cardiac enhancer can be operably linked to an aMHC enhancer. In some embodiments, the expression cassette comprises an enhancer that is operably linked to a promoter and operably linked to another enhancer. Exemplary enhancers are provided in Table 5C below.Table 5C. Exemplary enhancer sequences

[0139] In some embodiments, the enhancer comprises an ACTC1 cardiac enhancer (ACTCIe). In some embodiments, the enhancer comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 38, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 38.

[0140] In some embodiments, the enhancer comprises an aMHC enhancer (aMHCe). In some embodiments, the enhancer comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 39, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 39.Introns

[0141] In some embodiments, the vector and / or expression cassette further comprises one or more intron sequences, for example, a synthetic or chimeric intron sequence. The intron sequence can be used to adjust the length ( / .e., size) of the expression cassette for improving recombinant AAV packaging. The intron sequence can also be used to improve the efficiency of transgene expression ( / .e., mRNA production or transcription) in a host cell containing the expression cassette from the vector. Exemplary intron sequences are provided in Table 5D below.Table 5D. Exemplary intron sequences

[0142] In some embodiments, the intron comprises an CMV intron (CMVint). In some embodiments, the intron comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 40, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 40.

[0143] In some embodiments, the intron comprises a chimeric intron (Chimint). In some embodiments, the intron comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 41 , or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 41 .WPRE sequences and other post-transcriptional elements

[0144] In some embodiments, the vector and / or expression cassette further comprises one or more post-transcriptional regulatory elements, for example, a woodchuck hepatitis virus post-transcriptional element (WPRE). The WPRE sequence can be inserted, for example, proximal to on the 3’ end of a transgene in a viral vector to, for example, optimize gene expression in a viral vector. In some embodiments, the WPRE comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 42, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 42.Table 5E. Exemplary WPRE sequencesPoly(A) sequences

[0145] In some embodiments, the vector and / or expression cassette further comprises one or more poly(A) sequences. The term “poly(A) sequence” as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase II. Polyadenylation sequences can promote mRNA stability by addition of a poly(A) tail to the 3' end of the coding sequence and thus, contribute to increased translational efficiency. Cleavage and polyadenylation are directed by a poly(A) sequence in the RNA. The core poly(A) sequence for mammalian pre-mRNAs has two recognition elements flanking a cleavage-polyadenylation site. Typically, an almost invariant AAUAAA hexamer lies 20-50 nucleotides upstream of a more variable element rich in U or Gil residues. Cleavage of the nascent transcript occurs between these two elements and is coupled to the addition of up to 250 adenosines to the 5’ cleavage product. In someembodiments, the core poly(A) sequence is an ideal poly(A) sequence (e.g., AATAAA, ATTAAA, AGTAAA). Non-limiting examples of poly(A) sequences include SV40 poly(A) sequence, bovine growth hormone (BGH) poly(A) sequence, rabbit [3-globin poly(A) sequence (r|3gpA), variants thereof, and other suitable heterologous or endogenous poly(A) sequences known in the art. Exemplary poly(A) sequences are provided in Table 5F below.Table 5F. Exemplary poly(A) sequences

[0146] In some embodiments, the poly(A) sequence comprises a synthetic poly(A) sequence. In some embodiments, the poly(A) sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 43, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 43.

[0147] In some embodiments, the poly(A) sequence comprises a BGH poly(A) sequence. In some embodiments, the poly(A) sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 44, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 44.

[0148] In some embodiments, the poly(A) sequence comprises a SV40 poly(A) sequence. In some embodiments, the poly(A) sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 45, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 45.Additional elements

[0149] In some embodiments, the vector and / or expression cassette further comprises one or more additional elements, for example, a Kozak sequence and a nuclear localization sequence (NLS). Exemplary additional regulatory sequences are provided in Table 5G below.Table 5G. Exemplary additional regulatory sequences

[0150] In some embodiments, the vector and / or expression cassette comprises a Kozak sequence comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 46, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 46.

[0151] In some embodiments, the vector and / or expression cassette comprises a SV40 NLS comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 47, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 47.

[0152] In some embodiments, the vector and / or expression cassette comprises a nucleoplasmin NLS comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 48, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 48.

[0153] In some embodiments, the vector and / or expression cassette further comprises a transcription termination signal. Elements directing the efficient termination and polyadenylation of the heterologous nucleic acid transcripts increase heterologous gene expression. Transcription termination signals are generally found downstream of the polyadenylation signal.

[0154] In some embodiments, the vector and / or expression cassette further comprises a termination signal for producing the gRNA. In some embodiments, the polynucleotide encoding the gRNA described herein, and / or the expression cassette encoding the gRNA described herein, comprise a pol III termination signal. In some embodiments, the pol III termination signal is TTTTTG.ITRs

[0155] In some embodiments, the expression cassette is flanked by AAV inverted terminal repeats (ITRs) at the 5’ and 3’ ends. ITRs function as recognition sites for replication and markers used for viral packaging of the expression cassette. ITRs form T-shaped secondary structures by two adjacent inverted repeats separated by an unpaired nucleotide. ITRs are required for packaging the expression cassette into an rAAV virion, which provide the function of expressing the transgene after a host cell is targeted by the rAAV virion. The ITRs contain tetranucleotide repeat motifs called Repbinding elements (RBE) that act as contact points for the Rep68 / 78 proteins encoded by the rep gene. The ITRs also contain a packaging signal for genome encapsidation, which directs 3’ genomic transport into preassembled capsids by Rep proteins. Any naturally occurring or synthetically derived ITRs described herein or known in the art can be used.

[0156] In some embodiments, the ITRs flanking the expression cassette are ITRs of the same AAV serotype as the Rep protein used in making the virions described herein. For example, where a Rep protein from AAV9 is used, the transgene expression cassette used in the expression system comprises ITRs from AAV9 as well. In another example, where a Rep protein from AAV2 is used, the transgene expression cassetteused in the expression system comprises ITRs from AAV2 as well. In another example, where a Rep protein from AAV5 is used, the transgene expression cassette used in the expression system comprises ITRs from AAV5 as well. The ITRs may be of the same or different serotype as the capsid protein used in packaging the virion described herein.

[0157] In some embodiments, the ITRs comprise, consist of, or consist essentially of a nucleotide sequence set forth in SEQ ID NO: 49 or 50, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 49 or 50, as shown in Table 5H below.Table 5H. Exemplary ITR sequences

[0158] In some embodiments, the expression cassette is flanked by one or both of a 5’ ITR and a 3’ ITR. In some embodiments, the 5’ ITR comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 49, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 49. In some embodiments, the 3’ ITR comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 50, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 50.Illustrative Expression Cassettes

[0159] Illustrative, non-limiting arrangements and orientations of the elements in the vector and / or expression cassette are provided below, with a 5’ to 3’ arrangement of elements of:

[0160] 5’-first promoter-Cas nuclease-poly(A)-second promoter-gRNA-3’;

[0161] 5’-muscle cell-specific or cardiac (e.g., cardiomyocyte)-specific promoter- Cas nuclease-poly(A)-pol III promoter (e.g., U6 promoter)-gRNA-3’;

[0162] 5’-constitutive promoter-Cas nuclease-poly(A)-pol III promoter (e.g., U6 promoter)-gRNA-3’;

[0163] 5’-human TNNT2 promoter-Cas nuclease-poly(A)-pol III promoter (e.g., U6 promoter)-gRNA-3’.

[0164] In some embodiments, the first polynucleotide encoding the Cas nuclease is oriented in a head-to tail orientation relative to the second polynucleotide encoding the gRNA. In some embodiments, such an arrangement exhibits improved performance (e.g., as measured by gene editing efficiency) relative to expression cassettes wherein the polynucleotide encoding the Cas nuclease is oriented in a head-to-head or a tail-to- tail orientation relative to the polynucleotide encoding the gRNA. Additionally, the illustrative orientations of the expression cassette can include flanking ITR sequences on the 5’ and 3’ ends of the expression cassette.Self-inactivating Cas constructs

[0165] In some embodiments, the vector and / or expression cassette comprising (i) a first polynucleotide encoding a Cas nuclease (e.g., a Cas9 such as SaCas9) operably linked to a protein-expression driving promoter, and / or (ii) a second polynucleotide encoding a gRNA specific to a genetic variant (e.g., a SNP) within the MYH7 locus operably linked to an RNA expression-driving promoter is self-inactivating, and Cas9 expression can be self-terminated after expression of the Cas9 protein, resulting in transient expression of Cas9. Self-inactivating Cas constructs can be useful where longterm or constitutive expression of Cas proteins (e.g., when introduced in a gene therapy) is undesirable due to potential off-target effects, immune responses, and unintended genomic modifications. Because Cas9 is a prokaryotic protein, it will be highly immunogenic in eukaryotic organisms such as human. Thus, and especially in gene therapies for human subjects, limiting Cas expression after the intended genomic editing of target genes is complete may offer several advantages, including increased safety profile, reduced immunogenicity by Cas proteins, and / or reduced off-target effects.

[0166] In some embodiments, the self-inactivating vector and / or expression cassette comprises a self-inactivation site which, upon recognition and cleavage by theCas protein expressed by the expression cassette, can terminate transcription / translation of the first polynucleotide encoding the Cas nuclease, e.g., through a negative feedback loop. The self-inactivation site may be placed anywhere in the expression cassette, e.g., within the first promoter driving the expression of the Cas nuclease, within the first polynucleotide encoding the Cas nuclease, in between the first polynucleotide and the first promoter, or after the first polynucleotide encoding the Cas nuclease (e.g., before or within a poly(A) sequence). If the self-inactivation site is placed within the first polynucleotide encoding the Cas nuclease, it can be near the 5’ end (e.g., after the start codon “ATG”), near the 3’ end, or somewhere in between.

[0167] In some embodiments, the self-inactivation site comprises a gRNA target region. Upon recognition by a gRNA, the gRNA target region can be cleaved by a Cas nuclease (e.g., the Cas nuclease encoded by the first polynucleotide of the expression cassette), resulting in termination of the Cas transcript. The gRNA that recognizes and / or directs Cas-dependent cleaving of this target sequence can be different from the gRNA encoded by the second polynucleotide of the expression cassette or can be the same as the gRNA encoded by the second polynucleotide of the expression cassette. If the former, then the expression cassette may comprise a third polynucleotide encoding a second gRNA operably linked to a third promoter, and the second gRNA may upon expression direct the Cas nuclease to cut at the gRNA target region, thereby terminating the Cas expression.

[0168] In some embodiments, the self-inactivation site comprises a gRNA target region recognized by the same gRNA encoded by the second polynucleotide of the expression cassette (see FIG. 4A). For example, the gRNA target region can comprise the same sequence as the gRNA encoded by the second polynucleotide of the expression cassette or a portion thereof (e.g., the complementary region or spacer portion), or a sequence that is reverse complement to the gRNA encoded by the second polynucleotide of the expression cassette or a portion thereof (e.g., the complementary region or spacer portion). In some embodiments, the gRNA target region comprises a nucleotide sequence corresponding to any gRNA sequence described herein or a portion thereof (e.g., the complementary region or spacer portion) including, for example, those provided in Table 4C. In some embodiments, the gRNA target region comprises, consists of, or consists essentially of a nucleotide sequence set forth in any one of SEQ ID NOs: 12-23, 144-147, and 229-239, or a nucleotide sequence that shares at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 12-23, 144-147, and 229-239. In some embodiments, the gRNA target region comprises, consists of, or consists essentially of a nucleotide sequence reverse complement to any one of SEQ ID NOs: 12- 23, 144-147, and 229-239, or a reverse complement to a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 12-23, 144-147, and 229-239.

[0169] In certain of these embodiments, the self-inactivation site further comprises a less optimal PAM sequence of the particular Cas nuclease encoded by the first polynucleotide of the expression cassette (see FIG. 4A). The PAM sequence can be 5’ or 3’ to the gRNA target region. For a non-limiting example, if the Cas nuclease is SaCas9, the less optimal PAM sequence placed 3’ to the gRNA target region can be NNGRRC, NNGRRG, or NNGRRA, wherein N is any nucleotide, and R is guanine or adenine (see FIG. 4A). By incorporating a less optimal PAM sequence adjacent to the gRNA garget region, Cas-dependent cleaving at the self-inactivation site is less efficient compared to cutting near an optimal PAM sequence. Because the genomic target site intended by the expression cassette is usually adjacent to an optimal PAM sequence (e.g., in the case of SaCas9, it is NNGRRT), upon expression of the Cas nuclease and gRNA encoded by the expression cassette, the cleavage at the intended genomic target site would be more effective and would thus take place more efficiently. The cleavage directed by the same Cas nuclease and gRNA at the self-inactivation site would be less effective and take place less efficiently due to the less optimal PAM sequence (FIGS. 3A- 3B). Accordingly, the self-inactivating expression cassette would achieve the sequential cutting of (1 ) its intended genomic target site, thereby to complete the genomic gene editing; and (2) the self-inactivation site of the expression cassette, thereby to terminate the expression of the Cas protein after the genomic gene editing is complete. In some embodiments, the self-inactivating expression cassette is the same or similar to that depicted in FIG. 4B, including the relative location and / or orientation of elements as presented therein.Vectors

[0170] In some embodiments, polynucleotides encoding a Cas nuclease and a gRNA for use in the present technology are in the form of one or more vectors. The vector can be any viral vector or non-viral vector known in the art or described herein. In some embodiments, the vector is a viral vector. In some embodiments the viral vector is an adeno-associated virus vector (AAV), an adenoviral vector, a lentiviral vector, a retroviral vector, a herpes simplex virus vector (HSV), or a poxvirus vector.

[0171] As used herein, the term “retrovirus” or “retroviral” refers an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into a host genome. Retrovirus vectors are a common tool for gene delivery. Once the virus is integrated into the host genome, it is referred to as a “provirus.” The provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules encoded by the virus. In some embodiments, a retroviral vector is altered so that it does not integrate into the host cell genome. Illustrative retroviruses include, but are not limited to, (1 ) genus gammaretrovirus, such as, Moloney murine leukemia virus (M-MuLV or MMLV), Moloney murine sarcoma virus (MoMSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), and feline leukemia virus (FLV); (2) genus spumavirus, such as, simian foamy virus; and (3) genus lentivirus, such as, human immunodeficiency virus-1 and simian immunodeficiency virus.

[0172] As used herein, the term “lentiviral” or “lentivirus” refers to a group (or genus) of complex retroviruses. Illustrative lentiviruses include but are not limited to, human immunodeficiency virus (HIV), including HIV type 1 , and HIV type 2; visna-maedi virus (VMV) virus; caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0173] In some embodiments, the viral vector is an adenoviral vector. The genetic organization of adenovirus includes an approximate 36 kb, linear, double-stranded DNA virus, which allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kb.

[0174] In some embodiments, the viral vector is an AW vector, such as an AAV vector selected from the group consisting of serotype 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12,rh.10, rh.20, rh.74, and a variant or chimeric AAV derived thereof. In some embodiments, the AAV expression vector is pseudotyped to enhance targeting. A pseudotyping strategy can promote gene transfer and sustain expression in a target cell type. For example, the AAV2 genome can be packaged into the capsid of another AAV serotype such as AAV5, AAV7, or AAV8, producing pseudotyped vectors such as AAV2 / 5, AAV2 / 7, and AAV2 / 8 respectively, as described in Balaji et al., J. Surg. Res. Sep. (2013) 184(1 ):691 -698. In some embodiments, an AAV9 may be used to target expression in myofibroblast-like lineages, as described in Piras et al., Gene Therapy (2016) 23:469- 478. In some embodiments, AAV1 , AAV6, or AAV9 is used, and in some embodiments, the AAV is engineered, as described in Asokari et al., Hum. Gene Then Nov. (2013) 24(11 ):906-913; Pozsgai et al., Mol. Then (2017) 25(4): 855-869; Kotterman, M.A. and D.V. Schaffer, Nature Reviews Genetics (2014) 15:445-451 ; and US20160340393A1 to Schaffer et al. In some embodiments, the viral vector is AAV engineered to increase target cell infectivity as described in US20180066285A1 . In some embodiments, the vector is an AAV9 vector.

[0175] In some embodiments, the vector is a non-viral vector. In some embodiments, the non-viral vector is a naked DNA (e.g., a DNA plasmid). In some embodiments, the non-viral vector is a plasmid. In some embodiments, the non-viral vector is a liposome or lipid vector comprising plasmid DNA and a lipid solution.

[0176] In some embodiments, the vector is a recombinant vector.

[0177] In some aspects of the disclosure, a vector is used to deliver the expression cassette described herein to cardiac cells of a subject, e.g., to treat cardiomyopathy.

[0178] In some embodiments, the viral vectors described herein are replication incompetent, in that it cannot independently further replicate and package its genome. For example, when a cardiac cell is targeted with a virion, the transgene is expressed in the targeted cardiac cell, however, since the targeted cardiac cell lacks packaging and accessory function genes, the virion is not able to replicate. In some embodiments, the viral vectors described herein are replication competent.

[0179] In some embodiments, the vectors described herein are capable of being delivered to both dividing and non-dividing cells. In some embodiments, the vectors described herein are capable of being delivered to non-dividing cells. In someembodiments, the vectors described herein are capable of being delivered to dividing cells.

[0180] In some embodiments, the vectors comprising the expression cassettes described herein lead to cardiac cell-specific expression of the coding sequence(s). In some embodiments, the vectors comprising the expression cassettes described herein lead to cardiomyocyte-specific expression of the coding sequence(s). In some embodiments, the vectors comprising the expression cassettes described herein allow high expression of the coding sequence(s) in a cardiac cell (e.g., a cardiomyocyte) and low or no expression in other cells (e.g., low or no expression in liver cells, low or no expression in muscle cells except for muscle cells of the heart, low or no expression in cardiac fibroblasts). In some embodiments, the vectors comprising the expression cassettes described herein allow high expression of the coding sequence(s) in heart tissue of a subject (e.g., in human heart). In some embodiments, the vectors comprising the expression cassettes described herein allow no or low expression of the coding sequence(s) in tissues of a subject other than the heart (e.g., in liver or in muscles except those of the heart). “High” and “low” can be relative to each other, for example, the expression of a transgene in cardiac cells (e.g., cardiomyocytes) and / or heart tissue can be at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 50-fold, 100-fold, 150-fold, or 200-fold higher than its expression in other cells and tissues (e.g., liver, muscle except for the heart).

[0181] In some embodiments, the vector genome has a size of less than 6 kilobases. In some embodiments, the vector genome has a size of less than 5.6 kilobases. In some embodiments, the vector genome has a size of about, at most or less than 4.0 kilobases, 4.5 kilobases, 4.6 kilobases, 4.7 kilobases, 4.8 kilobases, 4.9 kilobases, 5 kilobases, 5.1 kilobases, 5.2 kilobases, 5.3 kilobases, 5.4 kilobases, or 5.5 kilobases. In some embodiments, the vector genome has a size of 4 kilobases to 5.2 kilobases. In some embodiments, the vector genome has a size of 4 kilobases to 5 kilobases. In some embodiments, the vector genome has a size of 4 kilobases to 4.8 kilobases. In some embodiments, the vector genome has a size of equal to or less than 4.9 kilobases. In some embodiments, the vector genome has a size of equal to or less than 4.8 kilobases. In some embodiments, the vector genome has a size of equal to or less than 4.7 kilobases. In some of these embodiments, the vector is an AAV vector, e.g., an AAV9 vector.

[0182] In some of these embodiments, the vector is an AAV vector or a variant thereof. In some of these embodiments, the vector is an AAV9 vector or a variant thereof. In some of these embodiments, the vector is an AAV5 vector or a variant thereof. In some of these embodiments, the vector is an AAV2 vector or a variant thereof.

[0183] The capsid proteins of AAV largely determine the immunogenicity and tropism of AAV vectors. In some embodiments, the AAV is an AAV subtype 9 (AAV9). In some embodiments, AAV9 is a preferred AAV vector due to its ability to transduce the heart following systemic delivery. While AAV9 can achieve moderate transduction of the heart, the majority of vector traffics to the liver. Moreover, in order to achieve therapeutic levels of transduction in the heart, relatively high systemic doses are required, potentially leading to systemic inflammation and in turn, toxicity.

[0184] Methods of introducing polynucleotides into a host cell are known in the art, and any known method can be used to introduce the polynucleotides described herein into a cell. Suitable methods include e.g., viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle-mediated nucleic acid delivery, microfluidics delivery methods, and the like.Recombinant AAV virions

[0185] In some embodiments, polynucleotides encoding a Cas nuclease and a gRNA for use in the present technology are in the form of a recombinant AAV (rAAV) virus or virion, for example, to deliver the expression cassettes described herein to cardiac cells.

[0186] In some embodiments, the AAV is any AAV known in the art or described herein. In some embodiments, the AAV is an AAV selected from the group consisting of serotype 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, rh.1O, rh.20, rh.74, or a chimeric or variant AAV derived therefrom. In some embodiments, the AAV is AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12, AAVrh.10, AAVrh.20, AAVrh.74, or a variant thereof.

[0187] In some embodiments, the rAAV virus or virion comprises an AAV capsid protein and an expression cassette as described herein. Capsid proteins are structural proteins that make up the assembled icosahedral packaging of the rAAV virion that contains the expression cassette. Capsid proteins are classified by the serotype. Wildtype capsid serotypes in rAAV virions can be, for example, AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12, AAVrh.10, AAVrh.20, or AAVrh.74. Engineered capsid types include chimeric capsids and mosaic capsids. Capsids are selected for rAAV virions based on their ability to transduce specific tissue or cell types.

[0188] Any capsid protein that can facilitate rAAV virion transduction into cardiac cells for delivery of a transgene, as described herein, can be used. Capsid proteins used in rAAV virions for transgene delivery to cardiac cells that result in high expression can be, for example, AAV4, AAV6, AAV7, AAV8, and AAV9. In some embodiments, the AAV capsid protein described herein is a wild-type AAV capsid protein from AAV serotype 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, rh.1O, rh.20, rh.74, or a variant thereof. In some embodiments, the AAV is AAV9 or a variant thereof. In some embodiments, the AAV is AAV5 or a variant thereof. In some embodiments, the AAV is AAV2 or a variant thereof.

[0189] Artificial capsids, such as chimeric capsids generated through combinatorial libraries, can also be used for transgene delivery to cardiac cells that results in high expression. Other capsid proteins with various features can also be used in the rAAV virions of the disclosure. AAV vectors and capsids are provided in U.S. Pat. Pub. Nos. US10011640B2; US7892809B2, US8632764B2, US8889641 B2, US9475845B2, US10889833B2, US10480011 B2, and US10894949B2, the entire contents of each of which are incorporated by reference herein; and Int’l Pat. Pub. Nos. WO2020198737A1 , W02019028306A2, WO2016054554A1 , WO2018152333A1 , WO2017106236A1 , WO2008124724A1 , W02017212019A1 , W02020117898A1 , WO2017192750A1 , W02020191300A1 , and W02017100671A1 , the entire contents of each of which are incorporated by reference herein.

[0190] In some embodiments, the rAAV virus or virion comprises a wild-type AAV9 capsid protein or a variant thereof. Wild-type AAV9 VP1 has the amino acid sequence of SEQ ID NO: 51 ; wild-type AAV9 VP2 has the amino acid sequence of SEQ ID NO: 52; wild-type AAV9 VP3 has the amino acid sequence of SEQ ID NO: 53, as shown belowand provided in Table 6A. The N-terminal residue of VP1 , VP2, and VP3, the VR sites (e.g., VR-I, VR-II, VR-IV, VR-V, VR-VII, and VR-VIII), as well as the last 35 amino acid positions,, are indicated (in bold, and underlined) in the sequence of full-length VP1 (SEQ ID NO: 51 ). In some embodiments, the capsid protein comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to any one of SEQ ID NOs: 51 -53.VPl — > ( SEQ ID NO : 51 )MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEP VNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLG LVVP2 — > ( SEQ ID NO : 52 )EEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPS GVVPS — > ( SEQ ID NO : 53 )GSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTVR- IVRI ISGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSF YCVR- IVLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQT LKVR-VFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMAS HKVR-VI I VR-VI I IEGEDRFFPLSGSLI FGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADP PTAFNKDKLNS FI TQYS TGQVSVE IEWELQKENSKRWNPE I QYTSNYYKSNNVEFAVNTEGVYSEP RPIGTRYLTRNL

[0191] As labeled in AAV9 VP1 (SEQ ID NO: 51 ) above, the VR-I site is between amino acids 262 and 269 in the parental sequence (“NSTSGGSS”, SEQ ID NO: 124): the VR- site is between amino acids 327 and 332 in the parental sequence (“DNNGVK”, SEQ ID NO: 125): the VR-IV site is between amino acids 448 and 462 in the parental sequence (“SKTINGSGQNQQTLK”, SEQ ID NO: 54); the VR-V site is between amino acids 491 and 504 in the parental sequence (“TTVTQNNNSEFAWP”, SEQ ID NO: 55); the VR-VII site is between amino acids 547 and 553 in the parental sequence (“GTGRDNVDADK”, SEQ ID NO: 56); the VR-VIII site is between amino acids 581 and 595 in the parental sequence (“ATNHQSAQAQAQTGW”, SEQ ID NO: 57); the last 35 amino acid positions are between amino acids 702 to 736 in the parental sequence (“TSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL”, SEQ ID NO: 126. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 51 , excluding the VR-I, VR-II, VR-IV, VR-V, VR-VII, VR-VIII, and / or the last 35 amino acid positions. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 51 , excluding the VR-IV and / or VR- VIII site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 51 , excluding the VR-IV site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 51 , excluding the VR-VIII site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 52, excluding the VR-I, VR-II, VR- IV, VR-V, VR-VII, VR-VIII site, and / or the last 35 amino acid positions. In some embodiments, the capsid protein comprises a sequence that shares at least about 80%(e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 52, excluding the VR-IV and / or VR-VIII site. In some embodiments, the engineered AAV9 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 51 , excluding the VR-II site and / or the last 35 amino acid positions. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 52, excluding the VR-IV site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 52, excluding the VR-VIII site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 52, excluding the VR-II site and / or the last 35 amino acid positions. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 53, excluding the VR-I, VR-II, VR-IV, VR-V, VR- VII, VR-VIII site, and / or the last 35 amino acid positions. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 53, excluding the VR-IV and / or VR-VIII site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 53, excluding the VR-IV site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 53, excluding the VR-VIII site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 53, excluding the VR-II site and / or the last 35 amino acid positions

[0192] In some embodiments, the rAAV virus or virion comprises a wild-type AAV5 capsid protein or a variant thereof. Wild-type AAV5 VP1 has the amino acid sequence of SEQ ID NO: 58; wild-type AAV5 VP2 has the amino acid sequence of SEQ ID NO: 59; wild-type AAV5 VP3 has the amino acid sequence of SEQ ID NO: 60, as shown below and provided in Table 6A. The N-terminal residue of VP1 , VP2, and VP3, the VR sites(e.g., VR-I, VR-II, VR-IV, VR-V, VR-VII, VR-VIII, as well as the last 35 amino acid positions), are indicated (in bold, and underlined) in the sequence of full-length VP1 (SEQ ID NO: 58). In some embodiments, the capsid protein comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to any one of SEQ ID NOs: 58-60.VPl — > ( SEQ ID NO : 58 )MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYNYLGPGNGLDRGEPV NRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGGNLGKAVFQAKKRVLEPFGLVEVP2 — > ( SEQ ID NO : 59 ) VPS — > ( SEQ ID NO :60 ) EGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGSQQLQIPAQPASSLGADTMSAG GGVR- IGPLGDNNQGADGVGNASGDWHCDSTWMGDRWTKSTRTWVLPSYNNHQYREIKSGSVDGSNANA YEVR- I IGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVKI FNIQVKEVTVQDSTTTIANNLT STVQVFTDDDYQLPYWGNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLVR- IVRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANT YKVR-VNWFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIVR-VI I VR-VI I IFNS QPANPGTTATYLEGNML ITSESETQ P VNRVAYNVGGQMATNNQSSTTAPATGT YNL QE I VP GSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL

[0193] As labeled in AAV5 VP1 (SEQ ID NO: 58) above, the VR-I site is between amino acids 252 and 256 in the parental sequence (“SGSVD”, SEQ ID NO: 127); the VR- II site is between amino acids 316 and 321 in the parental sequence (“VQDSTT”, SEQ ID NO: 128); the VR-IV site is between amino acids 437 and 461 in the parental sequence (“RFVSTNNTGGVQFNKNLAGRYANTY”, SEQ ID NO: 61 ); the VR-V site is between amino acids 477 and 490 in the parental sequence (“LGSGVNRASVSAFA”, SEQ ID NO: 62); the VR-VII site is between amino acids 533 and 546 in the parental sequence (“PANPGTTATYLEGN”, SEQ ID NO: 63); the VR-VIII site is between amino acids 570 and 584 in the parental sequence (“ATNNQSSTTAPATGT”, SEQ ID NO: 64); the last 35 amino acid positions are between amino acids 690 and 724 in the parental sequence (“TNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL”, SEQ ID NO: 129). In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 58, excluding the VR-IV, VR-V, VR-VII, and / or VR-VIII site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 58, excluding the VR-IV and / or VR-VIII site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 58, excluding the VR-IV site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 58, excluding the VR-VIII site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 58, excluding the VR-II site and / or the last 35 amino acid positions. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 59, excluding the VR-IV, VR-V, VR-VII, and / or VR-VIII site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 59, excluding the VR-IV and / or VR-VIII site. In some embodiments, thecapsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 59, excluding the VR-IV site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 59, excluding the VR-VIII site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 59, excluding the VR-II site and / or the last 35 amino acid positions. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 60, excluding the VR-IV, VR-V, VR-VII, and / or VR-VIII site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 60, excluding the VR-IV and / or VR-VIII site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 60, excluding the VR-IV site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 60, excluding the VR-VIII site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 60, excluding the VR-II site and / or the last 35 amino acid positions.Table 6A. Exemplary wild-type AAV capsid protein sequences

[0194] In some embodiments, the engineered capsid protein is an engineered AAVrh.10 capsid protein comprising one or more amino acid substitutions and / or insertions compared to the wild-type AAVrh.10 capsid protein described herein. In some embodiments, the engineered capsid protein is an engineered AAVrh.10 capsid protein comprising an insertion peptide sequence or insertion motif compared to the wild-type AAVrh.10 capsid protein.

[0195] The wild-type AAVrh.10 VP1 has the amino acid sequence of SEQ ID NO: 130; the wild-type AAVrh.10 VP2 has the amino acid sequence of SEQ ID NO: 131 ; the wild-type AAVrh.10 VP3 has the amino acid sequence of SEQ ID NO: 132, as shown below and provided in Table 6A. The N-terminal residue of VP1 , VP2, and VP3, the variable region (VR) sites (e.g., VR-I, VR-II, VR-IV, VR-V, VR-VII and VR-VIII), as well as the last 35 amino acid positions, are indicated in bold and underlined in the sequence of full-length VP1 (SEQ ID NO: 130). In some embodiments, the engineered AAVrh.10 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 130. In some embodiments, the engineered AAVrh.10 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 131. In some embodiments, the engineered AAVrh.10 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 132.VPl — > ( SEQ ID NO : 130 )MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEP VNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLVP2 — > ( SEQ ID NO : 131 )EPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPIVP3 — > ( SEQ ID NO : 132 )GEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTVR- IYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLN VR- I IFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYG YLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYQFEDVPFHSSYAHSQSLDRLMNPLIVR- IV VR-VDQYLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNFA VR-VI IWTGAT K YHLNGRD S L VNP GVAMAT HKDDE E R F FP S S GVLM FGKQGAGKDNVDYSS VML T S E E E VR-VI I IIKTTNPVATEQYGWADNLQQQNAAPIVGAVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNF HPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFSQAKLASFITQYSTGQVSVEIEWELQKENSK RWNPE I QYTSNYYKSTNVDFAVNTDGTYSEPRPIGTRYLTRNL

[0196] As labeled in AAVrh.10 VP1 (SEQ ID NO: 130) above, the VR-I site is between amino acids 263 and 267 in the parental sequence (“NGTSG”, SEQ ID NO: 133); the VR-II site is between amino acids 328 and 333 in the parental sequence (“QNEGTK”, SEQ ID NO: 134); the VR-IV site is between amino acids 449 and 464 in the parental sequence (“SRTQSTGGTAGTQQLL”, SEQ ID NO: 135); the VR-V site is between amino acids 493 and 506 in the parental sequence (“TTLSQNNNSNFAWT”, SEQ ID NO: 136); the VR-VII site is between amino acids 549 and 559 in the parental sequence (“GAGKDNVDYSS”, SEQ ID NO: 137); the VR-VIII site is between amino acids 583 and 597 in the parental sequence (“ADNLQQQNAAPIVGA”, SEQ ID NO: 138); the last 35 amino acid positions are between amino acids 704 and 738 in the parental sequence (“TSNYYKSTNVDFAVNTDGTYSEPRPIGTRYLTRNL”, SEQ ID NO: 139). In some embodiments, the engineered AAVrh.10 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 130, excluding the VR-I, VR-II, VR-IV, VR-V, VR-VII, VR-VIII, and / or the last 35 amino acid positions. In some embodiments, the engineered AAVrh.10 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 130, excluding the VR-IV and / or VR-VIII site. In some embodiments, the engineeredAAVrh.10 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 130, excluding the VR-II site and / or the last 35 amino acid positions. In some embodiments, the engineered AAVrh.10 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 131 , excluding the VR-l, VR-II, VR-IV, VR-V, VR-VII, VR-VIII, and / or the last 35 amino acid positions. In some embodiments, the engineered AAVrh.10 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 131 , excluding the VR-VI and / or VR-VIII site. In some embodiments, the engineered AAVrh.10 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 131 , excluding the VR-II site and / or the last 35 amino acid positions. In some embodiments, the engineered AAVrh.10 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 132, excluding the VR-I, VR-II, VR-IV, VR-V, VR-VII, VR-VIII, and / or the last 35 amino acid positions. In some embodiments, the engineered AAVrh.10 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 132, excluding the VR-IV and / or VR-VIII site. In some embodiments, the engineered AAVrh.10 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 132, excluding the VR-II site and / or the last 35 amino acid positions.

[0197] In some embodiments, the engineered capsid protein is an engineered AAVrh.74 capsid protein comprising one or more amino acid substitutions and / or insertions compared to the wild-type AAVrh.74 capsid protein described herein. In some embodiments, the engineered capsid protein is an engineered AAVrh.74 capsid protein comprising an insertion peptide sequence or insertion motif compared to the wild-type AAVrh.74 capsid protein.

[0198] The wild-type AAVrh.74 VP1 has the amino acid sequence of SEQ ID NO: 140; the wild-type AAVrh.74 VP2 has the amino acid sequence of SEQ ID NO: 141 ; the wild-type AAVrh.74 VP3 has the amino acid sequence of SEQ ID NO: 142, as shown below and provided in Table 6A. The N-terminal residue of VP1 , VP2, and VP3, thevariable region (VR) sites (e.g., VR-I, VR-II, VR-IV, VR-V, VR-VII and VR-VIII) , as well as the last 35 amino acid positions, are indicated in bold and underlined in the sequence of full-length VP1 (SEQ ID NO: 140). In some embodiments, the engineered AAVrh.74 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 140. In some embodiments, the engineered AAVrh.74 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 141. In some embodiments, the engineered AAVrh.74 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 142.VPl — > ( SEQ ID NO : 140 )MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLDKGEP VNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLVP2 — > ( SEQ ID NO : 141 )EPLGLVESPVKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPI VPS — > ( SEQ ID NO : 142 )GEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPT VR- IYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLN VR- I IFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYG YLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYNFEDVPFHSSYAHSQSLDRLMNPLIVR- IV VR-VDQYLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNFA VR-VI IWTGAT K YHLNGRD S L VNP GVAMAT HKDDE E R F FP S S GVLM FGKQGAGKDNVDYSS VML T S E E E VR-VI I IIKTTNPVATEQYGWADNLQQQNAAPIVGAVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNF HPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQAKLASFITQYSTGQVSVEIEWELQKENSK RWNPE I QYTSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL

[0199] As labeled in AAVrh.74 VP1 (SEQ ID NO: 140) above, the VR-I site is between amino acids 263 and 267 in the parental sequence (“NGTSG”, SEQ ID NO:133); the VR-II site is between amino acids 328 and 333 in the parental sequence (“QNEGTK”, SEQ ID NO: 134); the VR-IV site is between amino acids 449 and 464 in the parental sequence (“SRTQSTGGTAGTQQLL”, SEQ ID NO: 135); the VR-V site is between amino acids 493 and 506 in the parental sequence (“TTLSQNNNSNFAWT”, SEQ ID NO: 136); the VR-VII site is between amino acids 549 and 559 in the parental sequence (“GAGKDNVDYSS”, SEQ ID NO: 137); the VR-VIII site is between amino acids 583 and 597 in the parental sequence (“ADNLQQQNAAPIVGA”, SEQ ID NO: 138); the last 35 amino acid positions are between amino acids 704 and 738 in the parental sequence (“TSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL”, SEQ ID NO: 139). In some embodiments, the engineered AAVrh.74 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 140, excluding the VR-I, VR-II, VR-IV, VR-V, VR-VII, VR-VIII, and / or the last 35 amino acid positions. In some embodiments, the engineered AAVrh.74 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 140, excluding the VR-IV and / or VR-VIII site. In some embodiments, the engineered AAVrh.74 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 140, excluding the VR-II site and / or the last 35 amino acid positions. In some embodiments, the engineered AAVrh.74 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 141 , excluding the VR-I, VR-II, VR-IV, VR-V, VR-VII, VR-VIII, and / or the last 35 amino acid positions. In some embodiments, the engineered AAVrh.74 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 141 , excluding the VR-IV and / or VR-VIII site. In some embodiments, the engineered AAVrh.74 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 141 , excluding the VR-II and / or the last 35 amino acid positions. In some embodiments, the engineered AAVrh.74 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 30, excluding the VR-I, VR-II, VR-IV, VR-V, VR-VII, VR-VIII, and / or the last 35 amino acid positions. In some embodiments, the engineered AAVrh.74 capsid protein comprises a sequence thatshares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 30, excluding the VR-IV and / or VR-VIII site. In some embodiments, the engineered AAVrh.74 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 30, excluding the VR-II site and / or the last 35 amino acid positions.

[0200] In some embodiments, the rAAV virus or virion comprises a chimeric capsid protein, for example, an AAV5 / AAV9 chimeric capsid protein. In some embodiments, the AAV5 / AAV9 chimeric capsid protein comprises at least 1 , 2, 3, 4, 5 or more polypeptide segments each that are derived from AAV9 capsid protein and from AAV5 capsid protein. In some embodiments, at least one polypeptide segment is derived from the AAV5 capsid protein and at least one polypeptide segment is derived from the AAV9 capsid protein.

[0201] In some embodiments, the rAAV virus or virion comprises a combinatory capsid protein. As used herein, “combinatory capsid protein” refers to a AAV5 / AAV9 chimeric capsid protein, which further comprises amino acid variations with respect to the chimeric parental sequence at one or more sites. In some embodiments, the one or more sites of the chimeric parental sequence are selected from those equivalent to the VR-IV site, the VR-V site, the VR-VII site, and the VR-VIII site of the corresponding wildtype capsid protein.

[0202] In some embodiments, the rAAV virus or virion comprises an engineered capsid protein. Engineered capsid proteins can be derived from a parental, e.g., wildtype, capsid and include, for example, a variant polypeptide sequence with respect to a parental capsid sequence at one or more sites. For example, variant polypeptide sequences of the parental capsid can occur at the VR-IV site, VR-V site, VR-VII site and / or VR-VIII site. In some embodiments, the engineered capsid protein has a substitution or insertion at the VR-IV region of the capsid protein (e.g., of wild-type AAV9). In some embodiments, the engineered capsid protein has a substitution or insertion at the VR-V region of the capsid protein (e.g., of wild-type AAV9). In some embodiments, the engineered capsid protein has a substitution or insertion at the VR-VII region of the capsid protein (e.g., of wild-type AAV9). In some embodiments, the engineered capsid protein has a substitution or insertion at the VR-VIII region of the capsid protein (e.g., of wild-type AAV9). In some embodiments, the engineered capsid protein has a substitutionor insertion at the VR-IV region and the VR-VIII region of the capsid protein (e.g., of wildtype AAV9). Exemplary variant AAV9 capsid proteins (e.g., comprising one or more substitutions or insertions) are described in WO2021 / 163357, WO2021 / 216456, and U.S. Patent No. 11 ,129,908, the entire contents of each of which are incorporated by reference herein. In some embodiments, the present disclosure provides a rAAV capsid protein disclosed in WO2021 / 163357 as CR9-01 , and this disclosure is specifically incorporated by reference herein in its entirety.

[0203] In some embodiments, the rAAV virus or virion comprises an engineered AAV9 capsid protein comprising a variant polypeptide sequence. In some embodiments, the variant polypeptide sequence comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen substitutions at the VR-VIII site relative to the parental sequence at the VR-VIII site. In some embodiments, the variant polypeptide sequence comprises at least six substitutions at the VR-VIII site relative to the parental sequence at the VR-VIII site. In some embodiments, the variant polypeptide sequence comprises at least eight substitutions at the VR-VIII site relative to the parental sequence at the VR-VIII site.

[0204] In some embodiments, the parental sequence is the AAV9 VP1 parental sequence (SEQ ID NO: 51 ). In some embodiments, the parental sequence is the AAV5 VP1 parental sequence (SEQ ID NO: 58). In some embodiments, the parental sequence is the AAVrh.10 VP1 parental sequence (SEQ ID NO: 130). In some embodiments, the parental sequence is the AAVrh.74 VP1 parental sequence (SEQ ID NO: 140). In some embodiments, the variant polypeptide sequence occurs only at the VR-VIII site. In some embodiments, the engineered AAV9 capsid protein comprises, relative to reference SEQ ID NO: 51 , one, two, three, four, five, or more substitutions at positions from 584 to 590 in the VR-VIII site, or one, two, three, four, five, or more substitutions at positions from 585 to 590 in the VR-VIII site.Substitution motifs

[0205] In some embodiments, the engineered capsid proteins described herein comprise a variant polypeptide comprising one or more amino acid substitutions relative to a wild-type or parental capsid protein described herein (referred to herein as a “substitution motif”). In some embodiments, the substitution motif does not comprise amino acid insertions.

[0206] In some embodiments, the engineered capsid protein comprises a substitution motif comprising an amino acid substitution at one, two, three, four, five, six, seven, eight, nine, or ten positions relative to a wild-type AAV capsid protein sequence. In some embodiments, the engineered capsid protein comprises a substitution motif comprising an amino acid substitution at two positions relative to a wild-type AAV capsid protein sequence. In some embodiments, the engineered capsid protein comprises a substitution motif comprising an amino acid substitution at three positions relative to a wild-type AAV capsid protein sequence. In some embodiments, the engineered capsid protein comprises a substitution motif comprising an amino acid substitution at four positions relative to a wild-type AAV capsid protein sequence. In some embodiments, the engineered capsid protein comprises a substitution motif comprising an amino acid substitution at five positions relative to a wild-type AAV capsid protein sequence. In some embodiments, the engineered capsid protein comprises a substitution motif comprising an amino acid substitution at six positions relative to a wild-type AAV capsid protein sequence. In some embodiments, the engineered capsid protein comprises a substitution motif comprising an amino acid substitution at seven positions relative to a wild-type AAV capsid protein sequence. In some embodiments, the engineered capsid protein comprises a substitution motif comprising an amino acid substitution at eight positions relative to a wild-type AAV capsid protein sequence. In some embodiments, the engineered capsid protein comprises a substitution motif comprising an amino acid substitution at one, two, three, four, five, six, seven, or eight of the following positions relative to a wild-type AAV9 capsid protein sequence.

[0207] In some embodiments, the engineered capsid protein comprises a variant polypeptide in the VR-VIII site. In some embodiments, the engineered capsid protein comprises a variant polypeptide at positions 581-595 of the capsid protein, wherein the amino acid numbering is according to the AAV9 VP1 sequence of SEQ ID NO: 51. A person of skill in the art will recognize the equivalent positions in other AAV serotype capsid proteins, e.g., positions 570-583 of the AAV5 VP1 capsid sequence of SEQ ID NO: 58, positions 583-596 of the AAVrh.10 VP1 capsid sequence of SEQ ID NO: 130, and positions 583-596 of the AAVrh.74 VP1 capsid sequence of SEQ ID NO: 140. The variant polypeptide may comprise substitutions across the entire VR-VIII site (e.g., between positions 581 -595) or may comprise substitutions in a portion of the VR-VIII site (e.g., between positions 582-591 ).

[0208] In some embodiments, the engineered capsid protein comprises the variant amino acid sequence of X1X2X3X4X5K at a specified site. In some embodiments, the engineered capsid protein comprises a variant amino acid sequence in the VR-II site. In some embodiments, the engineered capsid protein comprises the variant amino acid sequence X1X2X3X4X5K in a VR-II site of an AAV VP capsid polypeptide sequence. In some embodiments, the engineered capsid protein comprises the variant amino acid sequence X1X2X3X4X5K at amino acid positions 327 to 332 according to the amino acid numbering of SEQ ID NO: 51 (WT AAV9 VP1 protein).

[0209] In some embodiments, the site is between amino acids 316 and 333 according to an AAV VP1 amino acid sequence. In some embodiments, the site is at positions 327 to 332, wherein the amino acid numbering is according to SEQ ID NO: 51. In some embodiments, the site is at positions 328 to 333, wherein the amino acid numbering is according to SEQ ID NO: 130. In some embodiments, the site is at positions 328 to 333, wherein the amino acid numbering is according to SEQ ID NO: 140.

[0210] In some embodiments, the variant amino acid sequence X1X2X3X4X5K is QTDGVK (SEQ ID NO: 148) In some embodiments, the variant amino acid sequence X1X2X3X4X5K is QQDGTK (SEQ ID NO: 149).

[0211] In some embodiments, the engineered capsid protein comprises a variant amino acid sequence within the last 35 amino acid positions of the capsid protein. In some embodiments, the engineered capsid protein comprises the variant amino acid sequence X1X2X3GX4 within the last 35 amino acid positions of an AAV VP capsid polypeptide sequence. In some embodiments, the engineered capsid protein comprises the variant amino acid sequence X1X2X3GX4 at amino acid positions 716 to 720 according to the amino acid numbering of SEQ ID NO: 51 .

[0212] In some embodiments, the capsid protein comprises the variant amino acid sequence of X1X2X3GX4 at a specified site. In some embodiments, the site is at amino acid positions 716 to 720 wherein the amino acid numbering is according to SEQ ID NO: 51. In some embodiments, the site is at positions 718 to 722, wherein the amino acid numbering is according to SEQ ID NO: 58. In some embodiments, the site is at positions 718 to 722, wherein the amino acid numbering is according to SEQ ID NO: 130. In some embodiments, the site is at positions 704 to 708, wherein the amino acid numbering is according to SEQ ID NO: 140.

[0213] In some embodiments, the variant amino acid sequence X1X2X3GX4 is selected from the group consisting of: NQYGV (SEQ ID NO: 150), NVHGV (SEQ ID NO: 151 ), NTHGV (SEQ ID NO: 152), and NTRGE (SEQ ID NO: 153). In some embodiments, the variant amino acid sequence X1X2X3GX4 is NQYGV (SEQ ID NO: 150). In some embodiments, the variant amino acid sequence X1X2X3GX4 is NTRGE (SEQ ID NO: 153).In some embodiments, the variant polypeptide comprises one or more substitutions (e.g., a substitution motif). In some embodiments, the non-naturally occurring amino acid motif comprises a substitution motif and does not comprise an insertion motif.

[0214] In some embodiments, the engineered AAV9 capsid protein comprises an amino acid sequence of X1DVQX2X3PGFX4X5X6X7X8 (SEQ ID NO: 65) at the VR-VIII site (e.g., of a wild-type AAV9 capsid protein or a variant thereof), wherein each of Xi , X2, X3, X4, Xs, Xs, X7, and Xs is any amino acid.

[0215] In some embodiments, Xi is alanine (A).

[0216] In some embodiments, X2 is glutamine (Q).

[0217] In some embodiments, X7 is threonine (T).

[0218] In some embodiments, X5 is alanine (A) or proline (P).

[0219] In some embodiments, Xe is glutamine (Q) or glutamic acid (E).

[0220] In some embodiments, X4 is glutamine (Q), glycine (G), arginine (R), asparagine (N), histidine (H), methionine (M), proline (P), or serine (S).

[0221] In some embodiments, Xs is glutamic acid (E), methionine (M), glutamine (Q), aspartic acid (D), leucine (L), alanine (A), cysteine (C), histidine (H), phenylalanine (F), tyrosine (Y), threonine (T), valine (V), isoleucine (I), serine (S), or asparagine (N). In some embodiments, Xs is glutamic acid (E).

[0222] In some embodiments, X3 is leucine (L), histidine (H), valine (V), cysteine (C), glutamine (Q), glycine (G), isoleucine (I), methionine (M), phenylalanine (F), proline (P), threonine (T), or tyrosine (Y).

[0223] In some embodiments, Xi is A, X2 is Q, X7 is T, and / or the capsid protein comprises in the VR-VIII site an amino acid sequence of ADVQQX3PGFX4X5X6TX8 (SEQ ID NO: 66), wherein each of X3, X4, X5, Xs, and Xs is any amino acid.

[0224] In some embodiments, Xs is A or P, and Xs is Q or E, and / or the capsid protein comprises in the VR-VIII site an amino acid sequence of X1 DVQX2X3PGFX4AQX7X8 (SEQ ID NO: 67), X1 DVQX2X3PGFX4AEX7X8 (SEQ ID NO: 68), X1DVQX2X3PGFX4PQX7X8 (SEQ ID NO: 69), X1DVQX2X3PGFX4PEX7X8 (SEQ ID NO: 70), ADVQQX3PGFX4AQTX8 (SEQ ID NO: 71 ), ADVQQX3PGFX4AETX8 (SEQ ID NO: 72), ADVQQX3PGFX4PQTX8 (SEQ ID NO: 73), or ADVQQX3PGFX4PETX8 (SEQ ID NO: 74), wherein each of Xi , X2, X3, X4, X7, and Xs is any amino acid.

[0225] In some embodiments, X3 is selected from L, H, V, C, Q, G, I, M, F, P, T, or Y, X4 is selected from Q, G, R, N, H, M, P, or S, and Xs is selected from E, M, Q, D, L, A, C, H, F, Y, T, V, I, S, or N. In some embodiments, X3 is L and Xs is E.

[0226] In some embodiments, X4 is Q, X5 is A, and Xs is Q, and / or the capsid protein comprises in the VR-VIII site an amino acid sequence of X1DVQX2X3PGFQAQX7X8 (SEQ ID NO: 75) or ADVQQXsPGFQAQTXs (SEQ ID NO: 76), wherein each of Xi , X2, X3, X7, and Xs is any amino acid.

[0227] In some embodiments, X3 is L, and Xs is E, and / or the capsid protein comprises in the VR-VIII site an amino acid sequence ofXi DVQX2LPGFX4X5X6X7E (SEQ ID NO: 77)or ADVQQLPGFX4X5X6TE (SEQ ID NO: 78), wherein each of Xi, X2, X4, X5, Xs, and X7 is any amino acid. In some embodiments, X4 is Q.

[0228] In some embodiments, X4 is Q, and / or the capsid protein comprises in the VR-VIII site an amino acid sequence of X1DVQX2X3PGFQX5X6X7X8 (SEQ ID NO: 79) or ADVQQXsPGFQXsXsTXs (SEQ ID NO: 80), wherein each of Xi, X2, X3, X5, X6, X7, and Xs is any amino acid.

[0229] In some embodiments, the engineered AAV capsid protein comprises in the VR-VIII site an amino acid sequence of X1 DVQX2X3PGFX4AX6X7X8, wherein each of Xi , X2, X3, X4, Xs, X7, and Xs is any amino acid (SEQ ID NO: 154).

[0230] In some embodiments, the engineered AAV capsid comprises in the VR-VIII site an amino acid sequence of X1 DVQX2X3PGFX4X5QX7X8, wherein each of Xi, X2, X3, X4, Xs, X7, and Xs is any amino acid (SEQ ID NO: 155).

[0231] In some embodiments, the engineered AAV capsid protein comprises in the VR-VIII site an amino acid sequence of ADVQQX3PGFX4PETX8, wherein each of X3, X4 and / or Xs is any amino acid (SEQ ID NO: 156). In some embodiments, Xs is L, H, S, V,C, Q, G, I, M, F, P, T, or Y. In some embodiments, X4 is Q, G, R, N, H, M, P, or S. In some embodiments, Xs is E, G, M, Q, D, L, A, C, H, F, Y, T, V, I, S, or N.

[0232] In some embodiments, the engineered AAV capsid protein comprises in the VR-VIII site an amino acid sequence of ADVQQX3PGFX4AQTX8, wherein each of X3, X4 and / or Xs is any amino acid (SEQ ID NO: 157). In some embodiments, Xs is L, H, S, V, C, Q, G, I, M, F, P, T, or Y. In some embodiments, X4 is Q, G, R, N, H, M, P, or S. In some embodiments, Xs is E, G, M, Q, D, L, A, C, H, F, Y, T, V, I, S, or N.

[0233] In some embodiments, the engineered AAV capsid protein comprises in the VR-VIII site an amino acid sequence of ADVQQX3PGFQAQTE, wherein X3 is any amino acid (SEQ ID NO: 158).

[0234] In some embodiments, the engineered AAV capsid protein comprises in the VR-VIII site an amino acid sequence of ADVQQX3PGFX4X5X6X7X8, wherein each of X3, X4, Xs, Xs, X7, and Xs is any amino acid (SEQ ID NO: 159).

[0235] In some embodiments, the engineered AAV capsid protein comprises in the VR-VIII site an amino acid sequence of ADVQQX3PGFX4X5X6X7E, wherein each of, X3, X4, Xs, Xs, and X7 is any amino acid (SEQ ID NO: 160).

[0236] In some embodiments, the engineered AAV capsid protein comprises in the VR-VIII site an amino acid sequence of ADVQQHPGFX4X5X6TE, wherein each of X4, Xs, and Xs is any amino acid (SEQ ID NO: 161 ).

[0237] In some embodiments, the engineered AAV9 capsid protein comprises, consists essentially of, or consists of a sequence having at least about 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100%) identity to any one of the following sequences at the VR-VIII site (positions 581-595 relative to reference sequence SEQ ID NO: 51 ), with up to 1 , 2, or 3 substitutions: ADVQQLPGFQAQTEW (SEQ ID NO: 81 ), ADVQQHPGFQAQTEW (SEQ ID NO: 82), ADVQQVPGFQAQTMW (SEQ ID NO: 83), ADVQQVPGFQAQTQW (SEQ ID NO: 84), ADVQQLPGFGAQTEW (SEQ ID NO: 85), ADVQQLPGFRPETEW(SEQ ID NO: 86), and ADVQQLPGFNAQTEW(SEQ ID NO: 87).

[0238] In some embodiments, the engineered AAV9 capsid protein comprises in the VR-VIII site an amino acid sequence selected from ADVQQLPGFQAQTEW (SEQ ID NO: 81 ), ADVQQHPGFQAQTEW (SEQ ID NO: 82), ADVQQVPGFQAQTMW (SEQ ID NO:83), ADVQQVPGFQAQTQW (SEQ ID NO: 84), ADVQQLPGFGAQTEW (SEQ ID NO:85), ADVQQLPGFRPETEW(SEQ ID NO: 86), and ADVQQLPGFNAQTEW(SEQ ID NO: 87).

[0239] In some embodiments, the engineered AAV9 capsid protein comprises in the VR-VIII site an amino acid sequence selected from ADVQQLPGFQAQTEW (SEQ ID NO: 81 ) and ADVQQHPGFQAQTEW (SEQ ID NO: 82).

[0240] In some embodiments, the engineered AAV9 capsid protein comprises one or more amino acid substitutions selected from the group consisting of A581 R, A581 N, A581 D, A581 C, A581 Q, A581 E, A581 G, A581 H, A5811, A581 L, A581 K, A581 M, A581 F, A581 P, A581 O, A581 S, A581T, A581W, A581Y, A581V, T582D, N583V, H584Q, Q585T, Q585C, Q585V, Q585L, Q585N, Q585S, Q585P, Q585A, Q585M, Q585E, Q585Y, Q585G, Q585H, Q585I, Q585R, Q585D, Q585K, Q585F, Q585O, Q585, S586D, S586T, S586G, S586K, S586M, S586N, S586I, S586Q, S586L, S586P, S586F, S586R, S586A, S586C, S586E, S586H, S586O, S586W, S586Y, S586V, A587P, A587S, A587N,Q588G, Q588R, Q588V, A589F, A589T, Q590I, Q590S, Q590N, Q590G, Q590D,Q590R, Q590H, Q590T, Q590M, Q590F, Q590Y, Q590L, Q590A, Q590C, Q590E,Q590K, Q590P, Q590O, Q590W, Q590V, A591 I, A591 R, A591 N, A591 D, A591 C,A591 Q, A591 E, A591 G, A591 H, A591 L, A591 K, A591 M, A591 F, A591 P, A5910, A591 S, A591T, A591W, A591Y, A591V, Q592I, Q592R, Q592N, Q592D, Q592C, Q592A, Q592E, Q592G, Q592H, Q592 L, Q592K, Q592M, Q592F, Q592P, Q592O, Q592S, Q592T, Q592W, Q592Y, Q592V, T593I, T593R, T593N, T593D, T593C, T593A, T593E, T593G, T593H, T593L, T593K, T593M, T593F, T593P, T593O, T593S, T593Q, T593W, T593Y, T593V, G594I, G594R, G594N, G594D, G594C, G594A, G594E, G594Q, G594H, G594 L, G594K, G594M, G594F, G594P, G594O, G594S, G594T, G594W, G594Y, and G594V.

[0241] In some embodiments, the engineered capsid protein may comprise one or more (e.g., three, four, five, six, seven or eight) amino acid substitutions selected from the group consisting of S586L, T582D, N583V, H584Q, A587P, Q588G, A589F, and G594E, relative to reference sequence SEQ ID NO: 51.

[0242] In some embodiments, the engineered capsid protein may comprise one or more (e.g., three, four, five, six, seven or eight) amino acid substitutions selected fromthe group consisting of S586H, T582D, N583V, H584Q, A587P, Q588G, A589F, and G594E, relative to reference sequence SEQ ID NO: 51.

[0243] In some embodiments, the engineered capsid protein comprises one or more amino acid substitutions selected from the group consisting of: D327Q, D327V, N328T, N328Q, N328S, N329D, G330N, and V331T. In some embodiments, engineered the capsid protein comprises the amino acid substitutions D327Q, N328T, and N329D. In some embodiments, the engineered capsid protein comprises the amino acid substitutions D327Q, N328Q, N329D, V331T.

[0244] In some embodiments, the engineered capsid protein comprises one or more amino acid substitutions selected from the group consisting of: N716D, T717Q, T717M, T717V, T717A, T717P, T717I, E718Y, E718R, E718H, E718N, E718M, E718Q, V720E, V720Q, V720I, and V720A. In some embodiments, the capsid protein comprises the amino acid substitutions T717Q and E718Y. In some embodiments, the capsid protein comprises the amino acid substitutions E718R and V720E.

[0245] In some embodiments, the engineered AAV9 capsid protein comprises any substitution and / or insertion motif described herein. In some embodiments, the engineered capsid protein comprises a substitution motif having at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any substitution motif described herein. In some embodiments, the engineered capsid protein comprises an insertion motif having at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any insertion motif described herein.

[0246] It should be noted that the above modified VR-VIII motifs are described in the context of AAV9 capsid proteins for illustrative purposes only and are not meant to be limited to AAV9 capsid proteins. Instead, any modified VR-VIII motif described herein can be applied to other AAV capsid proteins of a different serotype (e.g., AAV5, AAVrh.10, or AAVrh.74), for example, by replacing the wild-type sequence at the VR-VIII site of the corresponding capsid protein (e.g., amino acid positions 570 to 583 of wildtype AAV5 VP1 capsid protein sequence according to SEQ ID NO: 58, amino acid positions 583 to 596 of wild-type AAVrh.10 VP1 capsid protein sequence, or amino acidpositions 583 to 596 of wild-type AAVrh.74 VP1 capsid protein sequence) with any of the modified VR-VIII motifs described herein to generate a variant of the capsid protein of a particular serotype. In some embodiments, the engineered capsid protein is a variant of an AAV5, AAV9, AAVth.10, or AAVrh.74 capsid protein.

[0247] In some embodiments, the engineered AAV9 capsid protein comprises one, two, three, four, five, or more insertions in the VR-VIII site. In some embodiments, the engineered AAV9 capsid protein comprises, relative to reference SEQ ID NO: 51 , one, two, three, four, five, or more insertions at positions from 584 to 590 in the VR-VIII site, or one, two, three, four, five, or more insertions at positions from 585 to 590 in the VR- VIII site.

[0248] In some embodiments, the engineered capsid protein comprises an insertion polypeptide or insertion motif compared to the wild-type or parental capsid protein. In some embodiments, the engineered capsid protein additionally comprises one or more amino acid substitutions in the amino acid sequence of the wild-type or parental capsid protein sequence from which it is derived. In some embodiments, the insertion motif is inserted at a surface loop region of the capsid protein, for example, at a VR-IV, VR-V, VR-VII and / or VR-VIII site, as described.

[0249] In some embodiments, the insertion motif comprises or consists of the amino acid sequence RGDXKGL, wherein X can be any amino acid. In some embodiments, the insertion motif comprises or consists of an amino acid sequence of “RGDAARL” (SEQ ID NO: 88); and / or the engineered capsid protein comprises an amino acid sequence of “RGDAARL” (SEQ ID NO: 88).

[0250] In some embodiments, the insertion motif comprises or consists of an amino acid sequence of “SHVRGDL” (SEQ ID NO: 89); and / or the engineered capsid protein comprises an amino acid sequence of “SHVRGDL” (SEQ ID NO: 89).

[0251] In some embodiments, the insertion motif comprises or consists of an amino acid sequence of “VVSSGAR” (SEQ ID NO: 90); and / or the engineered capsid protein comprises an amino acid sequence of “VVSSGAR” (SEQ ID NO: 90).

[0252] In some embodiments, the insertion motif comprises or consists of an amino acid sequence of “VRGD” (SEQ ID NO: 91 ); and / or the engineered capsid protein comprises an amino acid sequence of “VRGD” (SEQ ID NO: 91 ).

[0253] In some embodiments, the insertion motif comprises or consists of an amino acid sequence of “RTDLKGL” (SEQ ID NO: 92); and / or the engineered capsid protein comprises an amino acid sequence of “RTDLKGL” (SEQ ID NO: 92).

[0254] In some embodiments, the insertion motif comprises or consists of an amino acid sequence of “RGDTKGL” (SEQ ID NO: 163); and / or the engineered capsid protein comprises an amino acid sequence of “RGDTKGL” (SEQ ID NO: 163).

[0255] In some embodiments, the insertion motif comprises or consists of an amino acid sequence of “RGDAKGL” (SEQ ID NO: 164); and / or the engineered capsid protein comprises an amino acid sequence of “RGDAKGL” (SEQ ID NO: 164).

[0256] In some embodiments, the insertion motif comprises or consists of an amino acid sequence of “RGDVKGL” (SEQ ID NO: 165); and / or the engineered capsid protein comprises an amino acid sequence of “RGDVKGL” (SEQ ID NO: 165).

[0257] In some embodiments, the insertion motif comprises or consists of an amino acid sequence of “RGDVKGL” (SEQ ID NO: 165); and / or the engineered capsid protein comprises an amino acid sequence of “RGDLVST” (SEQ ID NO: 212).

[0258] In some embodiments, the insertion motif comprises or consists of an amino acid sequence of “RGDVKGL” (SEQ ID NO: 165); and / or the engineered capsid protein comprises an amino acid sequence of “RGDGGVL” (SEQ ID NO: 213).

[0259] In some embodiments, the insertion motif comprises or consists of an amino acid sequence of “RGDVKGL” (SEQ ID NO: 165); and / or the engineered capsid protein comprises an amino acid sequence of “RGDHASW” (SEQ ID NO: 214).

[0260] The insertion motif can occur (e.g., be inserted) at any position of the capsid protein, for example, at a surface or an exposed region of the capsid protein. In some embodiments, the engineered AAV capsid protein comprises an insertion motif as described herein inserted at a surface loop region of the capsid protein, e.g., the VR-I, VR-II, VR-IV, VR-V, VR-VII, and / or VR-VIII site of the capsid protein. In some embodiments, the engineered capsid protein comprises an insertion motif as described inserted at the VR-IV and / or the VR-VIII site of the capsid protein. In certain of these embodiments, the engineered capsid protein additionally comprises one or more amino acid substitutions in the same VR site as the insertion or at a different location from the insertion.

[0261] In some embodiments, the engineered capsid protein is an engineered AAV9 capsid protein that comprises an insertion polypeptide or insertion motif at the VR-VIII site, e.g., between amino acids 588 (glutamine (Q)) and 589 (alanine (A)) within the VR- VIII site in reference to the wild-type full-length AAV9 capsid protein of SEQ ID NO: 51. In some embodiments, the engineered AAV9 capsid protein further comprises one or more amino acid substitutions within the VR-VIII site, including, for example, at one or more of amino acid positions 587-590 in reference to the wild-type full-length AAV9 capsid protein of SEQ ID NO: 51. In certain of these embodiments, the insertion motif comprises an amino acid sequence of RGDAARL (SEQ ID NO: 88), RTDLKGL (SEQ ID NO: 92), YPSTGSG (SEQ ID NO: 93), FAGSLTRA (SEQ ID NO: 94), DRTLTTR (SEQ ID NO: 95), RIAGRDV (SEQ ID NO: 96), SLGSGVR (SEQ ID NO: 97), RGDTKGL (SEQ ID NO: 163), RGDAKGL (SEQ ID NO: 164), RGDVKGL (SEQ ID NO: 165), RGDLVST (SEQ ID NO: 212), RGDGGVL (SEQ ID NO: 213), or RGDHASW” (SEQ ID NO: 214).

[0262] In some embodiments, provided herein is an engineered capsid protein comprising an amino acid sequence “ATNHQSX1X2X3X4AQTGW” (SEQ ID NO: 175) in the VR-VIII site, wherein Xi , X2, X3, and X4 can individually be any amino acid, and an insertion motif is inserted between X2 and X3 (for example, between amino acid positions 588 and 589 of wild-type AAV9 VP1 capsid protein sequence). In some embodiments, provided herein is an engineered capsid protein comprising an amino acid sequence “ATNHQSX1X2X3X4AQTEW’ (SEQ ID NO: 176) wherein Xi, X2, X3, and X4can individually be any amino acid, and an insertion motif is inserted between X2 and Xs (for example, between amino acid positions 588 and 589 of wild-type AAV9 VP1 capsid protein sequence). In some embodiments, provided herein is an engineered capsid protein comprising an amino acid sequence “ATNHQLX1X2X3X4AQTGW” (SEQ ID NO: 177) in the VR-VIII site, wherein Xi , X2, X3, and X4 can individually be any amino acid, and an insertion motif is inserted between X2 and X3 (for example, between amino acid positions 588 and 589 of wild-type AAV9 VP1 capsid protein sequence). In some embodiments, provided herein is an engineered capsid protein comprising an amino acid sequence “ATNHQLX1X2X3X4AQTEW” (SEQ ID NO: 178) in the VR-VIII site, wherein Xi, X2, X3, and X4 can individually be any amino acid, and an insertion motif is inserted between X2 and X3 (for example, between amino acid positions 588 and 589 of wild-type AAV9 VP1 capsid protein sequence).

[0263] In some embodiments, the engineered capsid protein is an engineered AAV9 capsid protein that comprises an insertion polypeptide or insertion motif at the VR-IV site, e.g., between amino acids 453 (glycine (G)) and 454 (serine (S)), and / or between amino acids 456 (glutamine (Q)) and 457 (asparagine (N)), within the VR-IV site in reference to the wild-type full-length AAV9 capsid protein of SEQ ID NO: 51. In certain of these embodiments, the insertion motif comprises an amino acid sequence of SHVRGDL (SEQ ID NO: 89), WSSGAR (SEQ ID NO: 90), PQYGRGG (SEQ ID NO: 98), LQVSRVS (SEQ ID NO: 99), VRSYSSN (SEQ ID NO: 100), “TMRVGSL” (SEQ ID NO: 101 ), GAYSRGV (SEQ ID NO: 102), LRGGSLG (SEQ ID NO: 103), or“VYGTGVR” (SEQ ID NO: 104).

[0264] In some embodiments, the engineered capsid protein is an engineered AAV5 capsid protein that comprises an insertion polypeptide or insertion motif at the VR-VIII site, e.g., between amino acids 574 (glutamine (Q)) and 575 (serine (S)) within the VR- VIII site in reference to the wild-type full-length AAV5 capsid protein of SEQ ID NO: 58. In certain of these embodiments, the insertion motif comprises an amino acid sequence of DKLIIVS (SEQ ID NO: 105), AEDRTKL (SEQ ID NO: 106), LSASASL (SEQ ID NO: 107), LADQTKL (SEQ ID NO: 108), LLLKLQE (SEQ ID NO: 109), ELPVKTG (SEQ ID NO: 110), LDLKWG (SEQ ID NO: 111 ), or RDAVL (SEQ ID NO: 112).

[0265] In some embodiments, the engineered AAV9 capsid protein comprises an insertion peptide sequence or insertion motif at the VR-VIII site, e.g., between amino acids 588 (glutamine (Q)) and 589 (alanine (A)) within the VR-VIII site in reference to the wild-type full-length AAV9 capsid protein of SEQ ID NO: 51. The insertion motif can be any motif as described herein, including those provided in Table 6B below. In some embodiments, the engineered AAV9 capsid protein further comprises one or more amino acid substitutions within the VR-VIII site, including, for example, at one or more of amino acid positions 587-590 in reference to the wild-type full-length AAV9 capsid protein of SEQ ID NO: 51.

[0266] In some embodiments, the engineered AAV9 capsid protein further comprises one or more amino acid substitutions within the VR-VIII site, including, for example, at one or more of amino acid positions 586 and / or 594 in reference to the wildtype full-length AAV9 capsid protein of SEQ ID NO: 51 . In certain of these embodiments, the engineered AAV9 capsid protein comprises a sequence of “ATNHQSX1X2X3X4AQTEW’ (SEQ ID NO: 176) at the VR-VIII site, where Xi , X2, X3, andX4 can individually be any amino acid, and an insertion motif is inserted between X2 and X3 (i.e., between amino acid positions 588 and 589). In certain of these embodiments, the engineered AAV9 capsid protein comprises a sequence of “ATNHQLX1X2X3X4AQTGW’ (SEQ ID NO: 177) at the VR-VIII site, where Xi , X2, X3, and X4 can individually be any amino acid, and an insertion motif is inserted between X2 and X3 (i.e., between amino acid positions 588 and 589). In certain of these embodiments, the engineered AAV9 capsid protein comprises a sequence of “ATNHQLX1X2X3X4AQTEW’ (SEQ ID NO: 178) at the VR-VIII site, where Xi , X2, X3, and X4 can individually be any amino acid, and an insertion motif is inserted between X2 and X3 (i.e., between amino acid positions 588 and 589).Table 6B. Exemplary engineered AAV9 capsid protein VR-VIII sequences with superior performance in nonhuman primates.

[0267] In some embodiments, the engineered AAV9 capsid protein comprises a “substitution + insertion motif”, wherein the substitution + insertion motif comprises: an insertion peptide sequence or insertion motif at the VR-VIII site, e.g., between amino acids 588 (glutamine (Q)) and 589 (alanine (A)) within the VR-VIII site in reference to the wild-type full-length AAV9 capsid protein of SEQ ID NO: 51 , and (ii) one or more amino acid substitutions within the VR-VIII site, including, for example, at one or more of amino acid positions 586, 587, 588, 590, and 594 in reference to the wild-type full-length AAV9 capsid protein of SEQ ID NO: 51 . In some embodiments, the substitution + insertion motif comprises amino acids 586 to 594, including the insertion motif, within the VR-VIII site in reference to the wild-type full-length AAV9 capsid protein of SEQ ID NO: 51. In some embodiments, the substitution + insertion motif can be any disclosed herein, including those provided in Table 6C below.Table 6C. Exemplary substitution + insertion motifs

[0268] In some embodiments, the engineered AAV9 capsid protein, at the VR-VIII site, comprises an insertion motif comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 88, 92, 163-165, and 212-214. In some embodiments, the engineered AAV9 capsid protein, at the VR-VIII site, comprises an insertion motif comprising or consisting of an amino acid sequence of any one of SEQ ID NO: 163. In some embodiments, the engineered AAV9 capsid protein, at the VR-VIII site, comprises an insertion motif comprising or consisting of an amino acid sequence of any one of SEQ ID NO: 164. In some embodiments, the engineered AAV9 capsid protein, at the VR-VIII site, comprises an insertion motif comprising or consisting of an amino acid sequence of any one of SEQ ID NO: 212. In some embodiments, the engineered AAV9 capsid protein, at the VR-VIII site, comprises an insertion motif comprising or consisting of an amino acid sequence of any one of SEQ ID NO: 213. In some embodiments, the engineered AAV9 capsid protein, at the VR-VIII site, comprises an insertion motif comprising or consisting of an amino acid sequence of any one of SEQ ID NO: 214.

[0269] In some embodiments, the engineered AAV9 capsid protein, at the VR-VIII site, comprises an insertion motif comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 88, 92, 163-165, and 212-214, with up to 1 , 2, or 3 amino acid substitutions. In some embodiments, the engineered AAV9 capsid protein, at the VR-VIII site, comprises an insertion motif comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 163, 164, and 212-214, with up to 1 , 2, or 3 amino acid substitutions.

[0270] In some embodiments, the engineered AAV9 capsid protein, at the VR-VIII site, comprises, consists essentially of, or consists of an amino acid sequence that shares at least about 70% or 80% (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 166, or the amino acid sequence set forth SEQ ID NO: 166. In some embodiments, the engineered AAV9 capsid protein, at the VR- VIII site, comprises, consists essentially of, or consists of an amino acid sequence that shares at least about 70% or 80% (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 168, or the amino acid sequence set forth in SEQ ID NO: 168. In some embodiments, the engineered AAV9 capsid protein, at the VR-VIII site, comprises, consists essentially of, or consists of an amino acid sequence that shares at least about 70% or 80% (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NOs: 215, or the amino acid sequence set forth in SEQ ID NO: 215. In some embodiments, the engineered AAV9 capsid protein, at the VR-VIII site, comprises, consists essentially of, or consists of an amino acid sequence that shares at least about 70% or 80% (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 216, or the amino acid sequence set forth in SEQ ID NOs: 216. In some embodiments, the engineered AAV9 capsid protein, at the VR-VIII site, comprises, consists essentially of, or consists of an amino acid sequence that shares at least about 70% or 80% (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 217, or the amino acid sequence set forth in SEQ ID NO: 217.

[0271] In some embodiments, the engineered AAV9 capsid protein comprises at least 90% or at least 95% amino acid sequence identity to AAV9 VP3 SEQ ID NO: 53, and comprises the amino acid sequence of ATNHQSSVRGDTKGLAGAQTGW (SEQ ID NO: 166) replacing the natural amino acid sequence at amino acid positions 581 to 595, wherein the amino acid numbering is according to AAV9 VP1 SEQ ID NO: 51 .

[0272] In some embodiments, the engineered AAV9 capsid protein comprises at least 90% or at least 95% amino acid sequence identity to AAV9 VP3 SEQ ID NO: 53, and comprises the amino acid sequence of ATNHQSSVRTDLKGLAGAQTGW (SEQ ID NO: 168) replacing the natural amino acid sequence at amino acid positions 581 to 595, wherein the amino acid numbering is according to AAV9 VP1 SEQ ID NO: 51 .

[0273] In some embodiments, the engineered AAV9 capsid protein comprises at least 90% or at least 95% amino acid sequence identity to AAV9 VP3 SEQ ID NO: 53, and comprises the amino acid sequence of ATNHQENRRGDLVSTTQAQTGW (SEQ ID NO: 215) replacing the natural amino acid sequence at amino acid positions 581 to 595, wherein the amino acid numbering is according to AAV9 VP1 SEQ ID NO: 51 .

[0274] In some embodiments, the engineered AAV9 capsid protein comprises at least 90% or at least 95% amino acid sequence identity to AAV9 VP3 SEQ ID NO: 53, and comprises the amino acid sequence of ATNHQENRRGDGGVLAQAQTGW (SEQ ID NO: 216) replacing the natural amino acid sequence at amino acid positions 581 to 595, wherein the amino acid numbering is according to AAV9 VP1 SEQ ID NO: 51 .

[0275] In some embodiments, the engineered AAV9 capsid protein comprises at least 90% or at least 95% amino acid sequence identity to AAV9 VP3 SEQ ID NO: 53, and comprises the amino acid sequence of ATNHQSSVRGDHASWAQAQTGW(SEQ ID NO: 217) replacing the natural amino acid sequence at amino acid positions 581 to 595, wherein the amino acid numbering is according to AAV9 VP1 SEQ ID NO: 51 .

[0276] Exemplary engineered capsid protein sequences are provided in Table 6D below. In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of an amino acid sequence set forth in any one of SEQ ID NOs: 113- 123 and 198-211 , or an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 113-123 and 198-211 .

[0277] In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 113 or SEQ ID NO: 114. In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of SEQ ID NO: 113. In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of SEQ ID NO: 114.

[0278] In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 198 or 200. In some embodiments, the engineeredcapsid protein comprises, consists of, or consists essentially of SEQ ID NO: 198. In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of SEQ ID NO: 200.

[0279] In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 208- 211. In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of SEQ ID NO: 208. In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of SEQ ID NO: 209. In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of SEQ ID NO: 210. In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of SEQ ID NO: 211 .

[0280] In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 224-226. In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of SEQ ID NO: 224. In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of SEQ ID NO: 2025. In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of SEQ ID NO: 226.Table 6D. Exemplary engineered AAV capsid protein sequences

[0281] In some embodiments, the rAAV virus or virion comprises a wild-type AAVrh.10 capsid protein or a variant thereof as known in the art. In some embodiments, the rAAV virus or virion comprises a wild-type AAVrh.74 capsid protein or a variant thereof as known in the art. In some embodiments, the rAAV virus or virion comprises an AAV-SLB101 capsid protein or a variant thereof as known in the art or described in, e.g. , WO 2021 / 072197, which is incorporated by reference herein in its entirety. In some embodiments, the rAAV virus or virion comprises an AAVmod capsid protein or a variant thereof as known in the art or described in, e.g. , WO 2022 / 173847 or in Olivieri et al. (2021 ) 24th Annual Meeting of the American Society of Gene & Cell Therapy available at https: / / www.affiniatx.com / pdf / asgct_2021_olivieri.pdf, both of which are incorporated by reference herein in their entireties. In some embodiments, the rAAV virus or virion comprises an AAVmut1 dec1 , AAVdecol , and / or AAVmutl capsid protein or a variant thereof as known in the art or described in, e.g. , WO 2022 / 173847, which is incorporated by reference herein in its entirety. In some embodiments, the rAAV virus or virion comprises an AAVcc.47 capsid protein or a variant thereof as known in the art or described in, e.g., Gonzalez et al. Nature Communications 13:5947 (2022), which is incorporated by reference herein in its entirety. In some embodiments, the rAAV virus or virion comprises an AAVHSC16 capsid protein or a variant thereof as known in the art or described in, e.g., Smith et al. Molecular Therapy Methods & Clinical Development 26:224-238 (2022), which is incorporated by reference herein in its entirety. In some embodiments, the rAAV virus or virion comprises a MyoAAV capsid protein or variant thereof as known in the art or described in, e.g., Tabebordbar et al. Cell 184(19):4919- 4938. (2021 ), which is incorporated by reference herein in its entirety. In some embodiments, the rAAV virus or virion comprises a MyoAAV-4E, MyoAAV-3F, MyoAAV- 4A, or MyoAAV-4D capsid protein or variant thereof as known in the art or described in, e.g. , Tabebordbar et al, which is incorporated by reference herein in its entirety. In some embodiments, the rAAV virus or virion comprises a 4D-C102 or C102 capsid protein or avariant thereof as known in the art or described in, e.g., US2021 / 0380643, which is incorporated by reference herein in its entirety.

[0282] In some embodiments, the rAAV virus or virion comprises a capsid protein (such as any described herein) and a vector genome, and the vector genome comprises an expression cassette flanked by ITRs. In some embodiments, the rAAV virus or virion specifically transduces heart cells and / or cardiomyocytes. In some embodiments, the rAAV virus or virion traffics to the heart. In some embodiments, the rAAV virus or virion traffics to at least one organ other than the liver.

[0283] In some embodiments, the rAAV virus or virion exhibits a higher transduction efficiency (e.g., a higher heart transduction efficiency) than an rAAV virus or virion having a wild-type AAV9 VP1 capsid protein of SEQ ID NO: 51. In some embodiments, the rAAV virus or virion exhibits a higher transduction efficiency (e.g., a higher heart transduction efficiency) in a primate or as assessed in a primate than an rAAV virus or virion having a wild-type AAV9 VP1 capsid protein of SEQ ID NO: 51 .

[0284] In some embodiments, administration of the rAAV virus or virion to a subject leads to a lower liver viral load than administration of an rAAV virus or virion having a wild-type AAV9 VP1 capsid protein of SEQ ID NO: 51. In some embodiments, administration of the rAAV virus or virion to a subject leads to at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times lower liver viral load than administration of an rAAV virus or virion having a wild-type AAV9 VP1 capsid protein of SEQ ID NO: 51. In some embodiments, administration of the rAAV virus or virion to a subject leads to a lower liver viral load in a primate or as assessed in a primate than administration of an rAAV virus or virion having a wild-type AAV9 VP1 capsid protein of SEQ ID NO: 51. In some embodiments, administration of the rAAV virus or virion to a subject leads to at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times lower liver viral load in a primate or as assessed in a primate than administration of an rAAV virus or virion having a wild-type AAV9 VP1 capsid protein of SEQ ID NO: 51.

[0285] In some embodiments, the rAAV virus or virion exhibits a higher heart-to- liver transduction ratio than an rAAV virus or virion having a wild-type AAV9 VP1 capsid protein of SEQ ID NO: 51. In some embodiments, the rAAV virion exhibits a heart-to- liver transduction ratio which is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times higher than an rAAV virus or virion having a wild-type AAV9 VP1 capsid protein of SEQ ID NO: 51 . Insome embodiments, the rAAV virus or virion exhibits a higher heart-to-liver transduction ratio in a primate or as assessed in a primate than an rAAV virus or virion having a wildtype AAV9 VP1 capsid protein of SEQ ID NO: 51 . In some embodiments, the rAAV virion exhibits a heart-to-liver transduction ratio which is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times higher in a primate or as assessed in a primate than an rAAV virus or virion having a wild-type AAV9 VP1 capsid protein of SEQ ID NO: 51 .

[0286] In some embodiments, the rAAV virus or virion is replication defective, in that the rAAV virus or virion cannot independently further replicate and package its genome. For example, when a cardiac cell is targeted with rAAV virions, the transgene is expressed in the targeted cardiac cell, however, due to the fact that the targeted cardiac cell lacks AAV rep and cap genes and accessory function genes, the rAAV is not able to replicate.

[0287] In some embodiments, rAAV virus or virion encapsulating the expression cassettes as described herein can be produced using helper-free production. rAAVs are replication-deficient viruses and normally require components from a live helper virus, such as adenovirus, in a host cell for packaging of infectious rAAV virions. rAAV helper- free production systems allow the production of infectious rAAV virions without the use of a live helper virus. In the helper-free system, a host packaging cell line is cotransfected with three plasmids. A first plasmid may contain adenovirus gene products (e.g., E2A, E4, and VA RNA genes) needed for the packaging of rAAV virions. A second plasmid may contain required AAV genes (e.g., REP and CAP genes). A third plasmid contains the polynucleotide sequence encoding the transgene of interest and a promoter flanked by ITRs. A host packaging cell line can be, for example, AAV-293 host cells. Suitable host cells contain additional components required for packaging infectious rAAV virions that are not supplied by the plasmids. In some embodiments, the CAP genes can encode, for example, AAV capsid proteins as described herein.Pharmaceutical Compositions for Allele-specific Gene Editing

[0288] In some embodiments, provided are pharmaceutical compositions for allelespecific gene editing, e.g. , editing an allele of a gene (e.g. , the MYH7 gene) which carries a heterozygous mutation, in a subject or a cell therefrom. In some embodiments, the pharmaceutical composition is for allele-specific gene editing of the MYH7 gene carrying a heterozygous mutation in a subject or a cell therefrom, the pharmaceutical compositioncomprising: (i) a first gRNA for specifically targeting a reference allele of a SNP within the MYH7 gene locus; or (ii) a second gRNA for specifically targeting an alternate allele of the SNP within the MYH7 gene locus. In some embodiments, the pharmaceutical composition further comprises, a Cas nuclease or a nucleic acid encoding the same. The heterozygous mutation may be a dominant negative mutation and / or a disease- associated mutation, in which the subject carries a WT allele and a mutant / disease- causing allele of the gene (e.g., the MYH7 gene).

[0289] In some embodiments, the first and / or second gRNAs can be any gRNA described herein, for example, those specific to a SNP (e.g., any of the SNPs provided in Table 1) within the MYH7 gene locus for use with the CRISPR / Cas system to knock down, knock out, or otherwise disrupt the expression of the mutant allele of the MYH7 gene. In some embodiments, the first and / or second gRNAs comprise a complementary region specific to any genetic variant within the MYH7 locus disclosed herein, for example, any of the SNPs provided in Table 1. In some embodiments, the first and / or second gRNAs comprise a complementary region specific to a reference or an alternate allele of any of the SNPs provided in Table 4C. Depending on which of the reference or alternate alleles to target for gene editing, one skilled in the art may select the gRNA specific to that allele to use in the present technology.

[0290] In some embodiments, the first and / or second gRNAs comprise a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in any one of SEQ ID NOs: 12-23, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 12-23. In some embodiments, the first and / or second gRNAs comprise a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in any one of SEQ ID NOs: 12-23, with up to one, two, three, or four nucleotide mismatches, substitutions, deletions, or additions.

[0291] In some embodiments, the SNP targeted by the first and second gRNAs is rs735711. In certain of these embodiments, the first gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 12 or 232 (corresponding to the reference allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 12 or 232; and / or the second gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 13 or 233(corresponding to the alternate allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 13 or 233.

[0292] In some embodiments, the SNP targeted by the first and second gRNAs is rs735712. In certain of these embodiments, the first gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 14 or 234 (corresponding to the reference allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 14 or 234; and / or the second gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 15 or 235 (corresponding to the alternate allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 15 or 235.

[0293] In some embodiments, the SNP targeted by the first and second gRNAs is rs2069540. In certain of these embodiments, the first gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 16 (corresponding to the reference allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 16; and / or the second gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 17 (corresponding to the alternate allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 17.

[0294] In some embodiments, the SNP targeted by the first and second gRNAs is rs2069540. In certain of these embodiments, the first gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 229 (corresponding to the reference allele), or a nucleotide sequencethat shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 229; and / or the second gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 230 (corresponding to the alternate allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 230.

[0295] In some embodiments, the SNP targeted by the first and second gRNAs is rs2069542. In certain of these embodiments, the first gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 18 or 236 (corresponding to the reference allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 18 or 236; and / or the second gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 19 or 237 (corresponding to the alternate allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 19 or 237.

[0296] In some embodiments, the SNP targeted by the first and second gRNAs is rs2231126. In certain of these embodiments, the first gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 20 or 238 (corresponding to the reference allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 20 or 238; and / or the second gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 21 or 239(corresponding to the alternate allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 21 or 239.

[0297] In some embodiments, the SNP targeted by the first and second gRNAs is rs7157716. In certain of these embodiments, the first gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence setforth in SEQ ID NO: 22 (corresponding to the reference allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 22; and / or the second gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 23 (corresponding to the alternate allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 23.

[0298] In some embodiments, the SNP targeted by the first and second gRNAs is rs7157716. In certain of these embodiments, the first gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 22 (corresponding to the reference allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 22; and / or the second gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 23 (corresponding to the alternate allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 23.

[0299] In some embodiments, the SNP targeted by the first and second gRNAs is rs7157716. In certain of these embodiments, the first gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 22 (corresponding to the reference allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 22; and / or the second gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 144 (corresponding to the alternate allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 144.

[0300] In some embodiments, the SNP targeted by the first and second gRNAs is rs7157716. In certain of these embodiments, the first gRNA comprises a complementaryregion comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 22 (corresponding to the reference allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 22; and / or the second gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 145 (corresponding to the alternate allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 145.

[0301] In some embodiments, the SNP targeted by the first and second gRNAs is rs7157716. In certain of these embodiments, the first gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 22 (corresponding to the reference allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 22; and / or the second gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 146 (corresponding to the alternate allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 146.

[0302] In some embodiments, the SNP targeted by the first and second gRNAs is rs7157716. In certain of these embodiments, the first gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 22 (corresponding to the reference allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 22; and / or the second gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 147 (corresponding to the alternate allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 147.

[0303] In some embodiments, the SNP targeted by the first and second gRNAs is rs7157716. In certain of these embodiments, the first gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 22 (corresponding to the reference allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 22; and / or the second gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 23 (corresponding to the alternate allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 23.

[0304] In some embodiments, the SNP targeted by the first and second gRNAs is rs7157716. In certain of these embodiments, the first gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 231 (corresponding to the reference allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 231 ; and / or the second gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 23 (corresponding to the alternate allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 23.

[0305] In some embodiments, the SNP targeted by the first and second gRNAs is rs7157716. In certain of these embodiments, the first gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 231 (corresponding to the reference allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 231 ; and / or the second gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 144 (corresponding to the alternate allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 144.

[0306] In some embodiments, the SNP targeted by the first and second gRNAs is rs7157716. In certain of these embodiments, the first gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 231 (corresponding to the reference allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 231 ; and / or the second gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 145 (corresponding to the alternate allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 145.

[0307] In some embodiments, the SNP targeted by the first and second gRNAs is rs7157716. In certain of these embodiments, the first gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 231 (corresponding to the reference allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 231 ; and / or the second gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 146 (corresponding to the alternate allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 146.

[0308] In some embodiments, the SNP targeted by the first and second gRNAs is rs7157716. In certain of these embodiments, the first gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 231 (corresponding to the reference allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 231 ; and / or the second gRNA comprises a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 147 (corresponding to the alternate allele), or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 147.

[0309] In some embodiments, the pharmaceutical composition further comprises a Cas nuclease or a nucleic acid encoding the same, for use with the gRNAs to knock down, knock out, or otherwise disrupt the expression of the mutant allele of the MYH7 gene. The Cas nuclease can be any Cas disclosed herein, including, for example, a Cas9 nuclease (e.g., SpCas9 or SaCas9). In some embodiments, the nucleic acid encoding a Cas9 nuclease comprises or consists of a sequence that shares at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 1. In some embodiments, the Cas9 nuclease in the pharmaceutical composition or encoded by the nucleic acid in the pharmaceutical composition comprises an amino acid sequence that shares at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 2.

[0310] In some embodiments, the nucleic acid encoding a Cas nuclease and the first or second gRNA are in the form of one or more vectors as described herein. A polynucleotide encoding the Cas nuclease and a polynucleotide encoding the first or second gRNA can be in the same vector, or in separate vectors. For example, the polynucleotide encoding the Cas nuclease and the polynucleotide encoding the first gRNA can be in the same vector; and separately, the polynucleotide encoding the Cas nuclease and the polynucleotide encoding the second gRNA can be in the same vector. When in the same vector, the polynucleotide encoding the Cas protein and the polynucleotide encoding the first (or second) gRNA can be in a head-to-head, head-to- tail, or tail-to-tail orientation. In some embodiments, the polynucleotide encoding the Cas nuclease and the polynucleotide encoding the first (or second) gRNA are in the same vector, in a head-to-tail orientation.

[0311] The polynucleotide encoding the Cas nuclease can be operably linked to a protein-expression driving promoter as described herein. Additionally, the polynucleotide encoding the first (or second) gRNA can be operably linked to an RNA expression-driving promoter as described herein. The vectors comprising the polynucleotide encoding the Cas nuclease and the polynucleotide encoding the first (or second) gRNA may further comprises one or more regulatory elements described herein, including, for example, enhancers, introns, WPRE sequences and other post-transcriptional elements, poly(A) sequences, Kozak sequences, nuclear localization sequences, and / or ITRs.

[0312] In some embodiments, the polynucleotide encoding a Cas nuclease is a selfinactivating construct described herein, i.e., Cas expression can be self-terminated in a negative-feedback loop.

[0313] In some embodiments, the vectors comprising the polynucleotide encoding the Cas nuclease and the polynucleotide encoding the first (or second) gRNA can be any viral vector or non-viral vector known in the art or described herein, including, for example, AAVs, adenoviral vectors, lentiviral vectors, retroviral vectors, HSVs, and poxvirus vectors. In some embodiments, the vectors are AAV vectors, viruses or virions, including any AAV described herein, for example, an AAV comprising any wild-type or engineered capsid protein as described herein.

[0314] In some embodiments, the pharmaceutical composition may comprise gRNAs for specifically targeting the reference and alternate alleles of one or more additional SNPs. In some embodiments, the pharmaceutical composition further comprises a third gRNA for specifically targeting a reference allele of a second SNP within the MYH7 gene locus; and a fourth gRNA for specifically targeting an alternate allele of the second SNP within the MYH7 gene locus. The second SNP may be selected from the SNPs provided in Table 1 but is different from the first SNP. As shown in FIG. 3, by identifying multiple SNPs (and for each developing two therapeutic products such as gRNAs, one covering the reference allele and the other covering the alternate allele), there is a possibility of increasing the eligible patient population (i.e., people who are heterozygous for at least one of the identified SNPs) that could benefit from the composition of the present technology. Eligible patients that are heterozygous for at least one of the identified SNPs can receive a drug product from the pharmaceutical composition that targets the reference or alternate allele of the SNP for which they are heterozygous.

[0315] In some embodiments, the third and fourth gRNAs comprise a complementary region selected from those provided in Table 4C that correspond to the target SNP. In some embodiments, the third and fourth gRNAs comprise a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in any one of SEQ ID NOs: 12-23, 144-147, and 229-239, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one ofSEQ ID NOs: 12-23, 144-147, and 229-239. In some embodiments, the third and fourth gRNAs comprise a complementary region comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in any one of SEQ ID NOs: 12-23, 144- 147, and 229-239, with up to one, two, three, or four nucleotide mismatches, substitutions, deletions, or additions.

[0316] In some embodiments, as with the first or second gRNAs, the third and fourth gRNAs may be in the form of one or more vectors as described herein. A polynucleotide encoding the Cas nuclease and a polynucleotide encoding the third or fourth gRNA can be in the same vector, or in separate vectors. For example, the polynucleotide encoding the Cas nuclease and the polynucleotide encoding the third gRNA can be in the same vector; and separately, the polynucleotide encoding the Cas nuclease and the polynucleotide encoding the fourth gRNA can be in the same vector. When in the same vector, the polynucleotide encoding the Cas protein and the polynucleotide encoding the third (or fourth) gRNA can be in a head-to-head, head-to-tail, or tail-to-tail orientation. In some embodiments, the polynucleotide encoding the Cas nuclease and the polynucleotide encoding the third (or fourth) gRNA are in the same vector, in a head-to- tail orientation.

[0317] In some embodiments, the polynucleotide encoding the third (or fourth) gRNA can be operably linked to an RNA expression-driving promoter as described herein. The vectors comprising the polynucleotide encoding the Cas nuclease and the polynucleotide encoding the third (or fourth) gRNA may further comprises one or more regulatory elements described herein, including, for example, enhancers, introns, WPRE sequences and other post-transcriptional elements, poly(A) sequences, Kozak sequences, nuclear localization sequences, and / or ITRs.

[0318] In some embodiments, the vectors comprising the polynucleotide encoding the Cas nuclease and the polynucleotide encoding the third (or fourth) gRNA can be any viral vector or non-viral vector known in the art or described herein, including, for example, AAVs, adenoviral vectors, lentiviral vectors, retroviral vectors, HSVs, and poxvirus vectors. In some embodiments, the vectors are AAV vectors, viruses or virions, including any AAV described herein, for example, an AAV comprising any wild-type or engineered capsid protein as described herein.

[0319] In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, or excipients for parenteral delivery. Pharmaceutically acceptable carriers, diluents, or excipients can include vehicles that are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. Illustrative pharmaceutical forms suitable for injectable use include, e.g., sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.

[0320] In some embodiments, the pharmaceutical composition comprises about 1 X108genome copies per milliliter (GC / mL), about 5X108GC / mL, about 1 xio9GC / mL, about 5x109GC / mL, about 1 x10GC / mL, about 5x101° GC / mL, about 1 X101 1GC / mL, about 5x1011GC / mL, about 1 xi o12GC / mL, about 5x1012GC / mL, about 5x1013GC / mL, about 1 xi o14GC / mL, or about 5x1014GC / mL of the vector (e.g., rAAV virus or virion).

[0321] In some embodiments, the pharmaceutical composition comprises about 1 x8viral genomes per milliliter (vg / mL), about 5X108vg / mL, about 1 xi o9vg / mL, about 5X109vg / mL, about 1 xi o10vg / mL, about 5X101° vg / mL, about 1 xio11vg / mL, about 5x1011vg / mL, about 1 xi o12vg / mL, about 5x1012vg / mL, about 5x1013vg / mL, about 1 x14vg / mL, or about 5x1014vg / mL of the vector (e.g., rAAV virus or virion).

[0322] In some embodiments, the pharmaceutical composition comprises less than about 1 xi o15viral genomes per milliliter (vg / mL), less than about 5x1014vg / mL, less than about 1 x14vg / mL, less than about 5x1013vg / mL, less than about 1 xi o13vg / mL, less than about 5x1012vg / mL, less than about 1 xi o12vg / mL, less than about 5x1011vg / mL, or less than about 1 x1 o11vg / mL of the vector (e.g., rAAV virus or virion).

[0323] In some embodiments, the pharmaceutical composition comprises less than about 1 xi o14viral genomes per milliliter (vg / mL) or less than about 1 xi o13vg / mL of the vector (e.g., rAAV virus or virion).

[0324] In some embodiments, the pharmaceutical composition comprises from about 1 X1011viral genomes per milliliter (vg / mL) to about 1 xi o15vg / mL of the vector(e.g., rAAV virus or virion). In some embodiments, the pharmaceutical composition comprises from about 1 X101 1viral genomes per milliliter (vg / mL) to about 1 X1014vg / mL of the vector (e.g., rAAV virus or virion). In some embodiments, the pharmaceutical composition comprises from about 1 xi o12viral genomes per milliliter (vg / mL) to about 1 X1014vg / mL of the vector (e.g., rAAV virus or virion). In some embodiments, the pharmaceutical composition comprises from about 1 xi o12viral genomes per milliliter (vg / mL) to about 1 xi o13vg / mL of the vector (e.g., rAAV virus or virion). In some embodiments, the pharmaceutical composition comprises from about 1 xio12viral genomes per milliliter (vg / mL) to about 6x1013vg / mL of the vector (e.g., rAAV virus or virion).

[0325] In some embodiments, the pharmaceutical composition comprises any amount or concentration range of the vector (e.g., rAAV virus or virion) between the values referenced herein.

[0326] In some embodiments, the pharmaceutical composition is administered in a total volume of about 1 mL, about 5 mL, about 10 mL, about 20 mL, about 25mL, about 30 mL, about 35 mL, about 40 mL, about 45 mL, about 50 mL, about 55 mL, about 60 mL, about 65 mL, about 70 mL, about 75 mL, about 80 mL, about 85 mL, about 90 mL, about 95 mL, about 100 mL, about 105 mL, about 110 mL, about 115 mL, about 120 mL, about 125 mL, about 130 mL, about 135 mL, about 140 mL, about 145 mL, about 150 mL, about 155 mL, about 160 mL, about 165 mL, about 170 mL, about 175 mL, about 180 mL, about 185 mL, about 190 mL, about 200 mL, about 205 mL, about 210 mL, about 215 mL, or about 220 mL.

[0327] In some embodiments, the pharmaceutical composition can be formulated (e.g., injectable, lyophilized, liquid formulations, or oral formulations) to be compatible with its intended route of administration. Examples of routes of administration include oral administration, extracorporeal administration, parenteral administration, intravenous administration, subcutaneous administration, intralesional administration (e.g., injection into tumors), and by administration into biological spaces infiltrated by tumors (e.g., intraspinal administration, intracerebel lar administration, intraperitoneal administration, intralymphatic administration, intranodal administration, and / or pleural administration). For example, a pharmaceutical composition provided herein can be administered systemically by oral administration or by intravenous administration (e.g., injection orinfusion). Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0328] In some embodiments, the pharmaceutical composition can be coformulated in the same dosage unit or can be individually formulated in separate dosage units. The term “dosage unit” herein refers to a portion of a pharmaceutical composition that contains an amount of a therapeutic agent suitable for a single administration to provide a therapeutic effect. Such dosage units may be administered one to a plurality (e.g., 1 to about 10, 1 to 8, 1 to 6, 1 to 4, or 1 to 2) of times per day, or as many times as needed to elicit a therapeutic response.Kits

[0329] In some embodiments, provided are kits comprising a container housing a pharmaceutical composition as described herein.

[0330] The kit can include any of compositions described herein, either mixed together or individually packaged, and in dry or hydrated form. The rAAV virions and / or other agents described herein can be packaged separately into discrete vials, bottles or other containers. Alternatively, any of the rAAV virions and / or agents described herein can be packaged together as a single composition, or as two or more compositions that can be used together or separately. The compounds and / or agents described herein can be packaged in appropriate ratios and / or amounts to facilitate conversion of selected cells across differentiation boundaries to form cardiac progenitor cells and / or cardiomyocytes.

[0331] The kit can include instructions for administering those compositions, compounds and / or agents. Such instructions can provide the information described throughout this application. The rAAV virion or pharmaceutical composition can beprovided within any of the kits in the form of a delivery device. Alternatively, a delivery device can be separately included in the kits, and the instructions can describe how to assemble the delivery device prior to administration to a subject.

[0332] Any of the kits can also include syringes, catheters, scalpels, sterile containers for sample or cell collection, diluents, pharmaceutically acceptable carriers, and the like. The kits can provide other factors such as any of the supplementary factors or drugs described herein for the compositions in the preceding section or other parts of the application.Cells

[0333] In some embodiments, provided are isolated cells or populations of cells comprising one or more vectors (e.g., rAAV viruses or virions) described herein.

[0334] In some embodiments, the cell is a cardiac cell. As used herein the term “cardiac cell” refers to any cell present in the heart that provides a cardiac function, such as heart contraction or blood supply, or otherwise serves to maintain the structure of the heart. Cardiac cells as used herein encompass cells that exist in the epicardium, myocardium or endocardium of the heart. Cardiac cells also include, for example, cardiac muscle cells or cardiomyocytes, and cells of the cardiac vasculatures, such as cells of a coronary artery or vein. Other non-limiting examples of cardiac cells include epithelial cells, endothelial cells, fibroblasts, cardiac stem or progenitor cells, cardiac conducting cells and cardiac pacemaking cells that constitute the cardiac muscle, blood vessels and cardiac cell supporting structure. Cardiac cells may be derived from stem cells, including, for example, embryonic stem cells or induced pluripotent stem cells.

[0335] In some embodiments, the cell is a cardiomyocyte.

[0336] In some embodiments, the cell is an induced pluripotent stem cell (iPSC). In some embodiments, a cell is an iPSC-derived cardiomyocyte.

[0337] In some embodiments, provided are methods of contacting the cell with any vector (e.g., an rAAV virus or virion) described herein. In some embodiments, the cell is a cardiac cell. In some embodiments, the cell is a cardiomyocyte. In some embodiments, the contacting is in vitro. In some embodiments, the contacting is in vivo.

[0338] In some embodiments, provided are methods of contacting a tissue with any vector (e.g., an rAAV virus or virion) described herein. In some embodiments, the tissueis cardiac tissue. In some embodiments, the contacting is in vitro. In some embodiments, the contacting is in vivo.

[0339] In some embodiments, provided are methods of contacting an organ with any vector (e.g., an rAAV virus or virion) described herein. In some embodiments, the organ is heart. In some embodiments, the heart is diseased or at risk of disease. In some embodiments, the heart has borderline or reduced ejection fraction. In some embodiments, the heart has a normal ejection fraction. In some embodiments, the heart comprises a genetic mutation in the MYH7 gene associated with a heart disease. In some embodiments, the genetic mutation is a deleterious or dominant negative mutation in MYH7. In some embodiments, the contacting is in vitro. In some embodiments, the contacting is in vivo.

[0340] In some embodiments, provided are cell therapy compositions comprising any cell described herein.

[0341] In some embodiments, provided are methods for allele-specific editing of a gene (e.g., the MYH7 gene) in a cell. The method may comprise, for example, transducing a target cell with the rAAV viruses or virions, rAAV vector genomes, or expression cassettes described herein. A target cell can be, for example and without limitation, a cardiac cell, a muscle cell, a cardiomyocyte, an iPSC, or an iPSC-derived cardiomyocyte (iPSC-CM). In some embodiments, the cell is an MYH7 mutant cell, e.g., comprises a genetic mutation in the MYH7 gene. In one embodiment, the mutation in the MYH7 gene causes or is known to cause cardiomyopathy, e.g., HCM.Therapeutic Methods

[0342] In some aspects, provided are methods for preventing and / or treating a disease or condition in a subject in need thereof. In some embodiments, the disease or condition is caused by or associated with a mutation (e.g., a dominant negative mutation) in the MYH7 gene. Subjects who are suitable for the compositions and / or methods of the present technology include individuals (e.g., mammalian subjects, such as humans, non-human primates, domestic mammals, experimental non-human mammalian subjects such as mice, rats, etc.) having a heterozygous mutation (e.g., a dominant negative mutation) in the MYH7 gene. In some embodiments, the subject is a human.

[0343] In some embodiments, the method for preventing and / or treating a disease or condition caused by or associated with a mutation (e.g., a dominant negative mutation) in the MYH7 gene in a subject in need thereof comprises allele-specific editing of the MYH7 gene, e.g., knocking down, knocking out, or otherwise altering the expression of the mutation-carrying allele of the MYH7 gene, for example, through the CRSIPR / Cas system using the method described herein in the subject, or the method comprises administering to the subject a pharmaceutical composition for allele-specific editing of the MYH7 gene as described herein. In certain of these embodiments, the subjects suitable for the methods and / or compositions of the present technology are those having one or more heterozygous genetic variants (e.g., one or more SNPs) within the MYH7 gene locus, including those who are heterozygous for one or more SNPs selected from Table 1 , that allow distinction between the paternal and maternal copies of chromosomes for allele-specific editing.

[0344] In some embodiments, the disease or condition caused by or associated with a mutation (e.g., a dominant negative mutation) in the MYH7 gene is a heart disease. In some embodiments, the heart disease is cardiomyopathy, including, for example, hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), idiopathic DCM, arrhythmogenic cardiomyopathy (ACM), and arrhythmogenic right ventricular cardiomyopathy (ARVC). In some embodiments, the heart disease is HCM. In some embodiments, the heart disease is heart failure, including, for example, heart failure with reduced ejection fraction and ischemic heart failure. In some embodiments, the heart disease is arrhythmia, including, for example, atrial and / or ventricular arrhythmia and malignant ventricular arrhythmia. In some embodiments, the heart disease is cardiomyopathy associated with a pulmonary embolus, venous thrombosis, myocardial infarction, transient ischemic attack, peripheral vascular disorder, atherosclerosis, ischemic cardiac disease, other myocardial injury or vascular disease, and / or cardiac diseases associated with myocardial tissue hypercontractility, such as heart failure related to left ventricular hypercontractility. In some embodiments, the subject has or is at risk of having a heart disease (e.g., cardiomyopathy or HCM) caused by or associated with a mutation (e.g., a dominant negative mutation) in the MYH7 gene.

[0345] In some embodiments, the pharmaceutical composition for use in the method for preventing and / or treating a disease or condition caused by or associated with a mutation (e.g., a dominant negative mutation) in the MYH7 gene (e.g., rAAVvectors, viruses, or virions) can be administered to the subject by systemic application (such as parenteral application), for example, by intravenous (e.g., by IV infusion), intraarterial, or intraperitoneal delivery. In some embodiments, the pharmaceutical composition (e.g., rAAV vectors, viruses, or virions) can be delivered by direct administration to the heart tissue.

[0346] In some embodiments, the pharmaceutical composition (e.g., rAAV vectors, viruses, or virions) can be delivered by intracoronary administration. In some embodiments, the administration is by antegrade epicardial coronary artery infusion, e.g., a single infusion over a 10-minute period in a cardiac catheterization laboratory after angiography (percutaneous intracoronary delivery without vessel balloon occlusion) with the use of standard 5F or 6F guide or diagnostic catheters.

[0347] In some embodiments, the pharmaceutical composition (e.g., rAAV vectors, viruses, or virions) can be delivered by direct injection into the heart or cardiac catheterization, or by intracardiac catheter delivery via retrograde coronary sinus infusion (RCSI).

[0348] When direct injection is used, it may be performed either by open-heart surgery or by minimally invasive surgery. In some cases, the pharmaceutical composition (e.g., rAAV vectors, viruses, or virions) can be delivered to the pericardial space by injection or infusion.

[0349] In some embodiments, the amount, concentration, and volume of the pharmaceutical composition that modulates contractile function in myocardial tissue administered to a subject can be controlled and / or optimized to substantially improve the functional parameters of the heart while mitigating adverse side effects.

[0350] The amount of the composition that modulates contractile function administered to myocardial tissue can also be an amount required to result in the detectable expression of a therapeutic protein or nucleic acid (e.g., Cas9 or gRNA) in the heart; preserve and / or improve contractile function; delay the emergence of cardiomyopathy or reverse the pathological course of the disease; increase myocyte viability; improve myofilament function; inhibit left ventricular hypertrophy; cardiac hypertrophy regression, normalize systolic and diastolic function in heart; and restore normal cross-bridge behavior at the myofilament level.

[0351] In some embodiments, the compositions and / or methods disclosed herein result in reduced expression of the mutant MYH7 gene and / or restoration of function of the MYH7 gene and gene product in a cardiac cell or tissue (such as heart) of the subject being treated.

[0352] In some embodiments, the method comprises administering an rAAV vector, virus, or virion at a dose of about 1 X108genome copies per milliliter (GC / mL), about 5x108GC / mL, about 1 x9GC / mL, about 5x109GC / mL, about 1 xio10GC / mL, about 5x101° GC / mL, about 1 x11GC / mL, about 5x1011GC / mL, about 1 x12GC / mL, about 5x1012GC / mL, about 5x1013GC / mL, about 1 x14GC / mL, or about 5x1014GC / mL of the rAAV vector, virus, or virion.

[0353] In some embodiments, the method comprises intravenously administering an rAAV vector, virus, or virion at a dose of about 3X1012GC / mL, about 3X1013GC / mL, about 1 x14GC / mL, or about 3x1014GC / mL of the rAAV vector, virus, or virion.

[0354] In some embodiments, the method comprises administering, by localized delivery to the heart, an rAAV vector, virus, or virion at a dose of about 3X101 1GC / mL, about 3x1012GC / mL, about 1 xi o13GC / mL, or about 3x1013GC / mL of the rAAV vector, virus, or virion.

[0355] In some embodiments, the method comprises administering an rAAV vector, virus, or virion at a dose of about 1 xi o8viral genomes per milliliter (vg / mL), about 5x108vg / mL, about 1 xio9vg / mL, about 5x109vg / mL, about 1 xi o10vg / mL, about 5x101° vg / mL, about 1 X1011vg / mL, about 5x1011vg / mL, about 1 xi o12vg / mL, about 5x1012vg / mL, about 5X1013vg / mL, about 1 xi o14vg / mL, or about 5X1014vg / mL of the rAAV vector, virus, or virion.

[0356] In some embodiments, the method comprises administering an rAAV vector, virus, or virion at a dose of less than about 1 xi o15viral genomes per milliliter (vg / mL), less than about 5X1014vg / mL, less than about 1 xio14vg / mL, less than about 5X1013vg / mL, less than about 1 xi o13vg / mL, less than about 5x1012vg / mL, less than about 1 X1012vg / mL, less than about 5x1011vg / mL, or less than about 1 xi o11vg / mL of the rAAV vector, virus, or virion.

[0357] In some embodiments, the method comprises administering an rAAV vector, virus, or virion at a dose of less than about 1 xi o14viral genomes per milliliter (vg / mL) or less than about 1 xi o13vg / mL of the rAAV vector, virus, or virion.

[0358] In some embodiments, the method comprises administering an rAAV vector, virus, or virion at a dose of from about 1 xi o11viral genomes per milliliter (vg / mL) to about 1 xi o15vg / mL, from about 1 xi o11vg / mL to about 1 xl 014vg / mL, from about 1 xl012vg / mL to about 1 xl 014vg / mL, from about 1 xi o12vg / mL to about 1 xl013vg / mL, or from about 1 xi o12vg / mL to about 6X1013vg / mL of the rAAV vector, virus, or virion.

[0359] In some embodiments, the method comprises administering an rAAV vector, virus, or virion at any dose or dose range of the disclosure between the values referenced herein.

[0360] In some embodiments, the method comprises intravenously administering an rAAV vector, virus, or virion at a dose of about 1 xl012vg / mL, about 3xlO12vg / mL, about 6x1012vg / mL, or about 9x1012vg / mL of the rAAV vector, virus, or virion.

[0361] In some embodiments, the method comprises administering, by localized delivery to the heart, an rAAV vector, virus, or virion at a dose of about 1 xl012vg / mL, about 3x1012vg / mL, about 6X1012vg / mL, or about 9x1012vg / mL of the rAAV vector, virus, or virion.

[0362] Genome copies per milliliter can be determined by quantitative polymerase change reaction (qPCR) using a standard curve generated with a reference sample having a known concentration of the polynucleotide genome of the virus. For AAV, the reference sample used is often the transfer plasmid used in generation of the rAAV virion, but other reference samples may be used.

[0363] Alternatively, the concentration of a viral vector can be determined by measuring the titer of the vector on a cell line. Viral titer is typically expressed as viral particles (vp) per unit volume (e.g., vp / mL). In various embodiments, the pharmaceutical composition comprises about 1 xi o8viral particles per milliliter (vp / mL), about 5X108vp / mL, about 1 xio9vp / mL, about 5x109vp / mL, about 1 xi o10vp / mL, about 5x101° vp / mL, about 1 X1011vp / mL, about 5x1011vp / mL, about 1 xi o12vp / mL, about 5x1012vp / mL, about 5X1013vp / mL, about 1 xio14vp / mL, or about 5X1014of the rAAV vector, virus, or virion.

[0364] The vector, virus, or virion administered to the subject can be traced by a variety of methods. For example, recombinant viruses labeled with or expressing a marker (such as green fluorescent protein, or beta-galactosidase) can readily be detected. The recombinant viruses may be engineered to cause the target cell to express a marker protein, such as a surface-expressed protein or a fluorescent protein. Alternatively, the infection of target cells with recombinant viruses can be detected by their expression of a cell marker that is not expressed by the animal employed for testing (for example, a human-specific antigen when injecting cells into an experimental animal). The presence and phenotype of the target cells can be assessed by fluorescence microscopy (e.g., for green fluorescent protein, or beta-galactosidase), by immunohistochemistry (e.g., using an antibody against a human antigen), by ELISA (using an antibody against a human antigen), or by RT-PCR analysis using primers and hybridization conditions that cause amplification to be specific for RNA indicative of a cardiac phenotype.

[0365] In some embodiments, the vector, virus, or virion, or a pharmaceutical composition containing the same, is administered to the subject once a day, twice a day, three times a day, or four times a day for a period of about 1 day, about 2 days, about 3 days, about 5 days, about 7 days, about 10 days, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, or more than about 5 years. In some embodiments, the vector, virus, or virion, or a pharmaceutical composition containing the same, is administered every day, every other day, 3 times a week, every third day, weekly, biweekly ( / .e., every other week), every third week, monthly, every other month, every third month, every fourth month, every fifth month, every sixth month, every ninth month, every year, every 18 months, every 2 years, every 5 years, every 10 years, or every 20 years. In some embodiments, the dose regimens listed above could be repeated after a period of about 1 week, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, or more than about 5 years. In some embodiments, the schedule of administration is a hybrid of these periods, for example, the vector, virus, or virion, or apharmaceutical composition containing the same, is administered a number of times a week and that pattern is repeated a number of times a month every month or every second, third, fourth, fifth, or sixth month, and treatment according to that pattern is continued for part of a year to several years, as set out above. In some embodiments, the vector, virus, or virion, or a pharmaceutical composition containing the same, is administered in a cycle of a number or administrations over a week or two weeks, and the cycle is repeated at spaced intervals over a number of months or years, as set out above. In some embodiments treatment is continued until disease is eliminated, until no further improvement is achieved, or as long as the disease does not progress. In some embodiments, a disease or condition within a subject to be treated can be monitored to evaluate the effectiveness of the treatment using any appropriate method known to a skilled artisan.

[0366] In some embodiments, the vector, virus, or virion, or a pharmaceutical composition containing the same, is administered over a predetermined time period. Alternatively, the vector, virus, or virion, or a pharmaceutical composition containing the same, is administered until a particular therapeutic benchmark is reached. In some embodiments, the methods provided herein further include a step of evaluating one or more therapeutic benchmarks in the subject to determine whether to continue administration of the treatment.

[0367] In some embodiments, the method results in an editing efficiency (e.g., knocking down, knocking out, or otherwise altering the expression of the mutant allele) of, or an efficiency of indel formation (e.g., forming a frameshift mutation) in, the mutant allele of the MYH7 gene of at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%. In some embodiments, the method does not, or substantially does not, edit, or form indels in, the wild-type allele of the MYH7 gene.

[0368] In some embodiments, the method restores or improves cardiac function in the subject, and / or restores or improves contractile function of the heart in the subject.

[0369] In some embodiments, the method improves ejection fraction in the subject, for example, increases the ejection fraction by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200% in the subject with an MYH7 mutation.

[0370] In some embodiments, the method reduces left ventricular hypertrophy, left ventricular mass, and / or left ventricular wall thickness in the subject. In some embodiments, the method improves left ventricular relaxation and / or left ventricular filling pressure in the subject.

[0371] In some embodiments, the method decreases or prevents an increase in left ventricle internal dimension (LVID) in the subject, for example, decreases or prevents an increase in LVID (mm) by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200% in the subject with an MYH7 mutation.

[0372] In some embodiments, the method decreases or prevents an increase in left ventricle (LV) mass in the subject, for example, decreases or prevents an increase in LV mass (as measured in mg / g of body weight) by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200% in the subject with an MYH7 mutation.

[0373] In some embodiments, the method increases or prevents a decrease in stroke volume in the subject, for example, increases or prevents a decrease in stroke volume by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200% in the subject with an MYH7 mutation.

[0374] In some embodiments, the method increases or prevents a decrease in R amplitude in the subject, for example, increases or prevents a decrease in R amplitude (mV) by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200% in the subject with an MYH7 mutation.

[0375] In some embodiments, the method decreases or prevents an increase in Q wave, R wave, and S wave (QRS) interval in the subject, for example, decreases or prevents an increase in QRS interval by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200% in the subject with an MYH7 mutation.

[0376] In some embodiments, the method increases life span or prevents an increase in mortality over time in the subject with an MYH7 mutation.EXAMPLESExample 1. Allele-specific editing of MYH7 by CRISPR / Cas in heterozygous carriers of SNP rs2069540

[0377] This study shows an exemplary approach to allele-specific editing of the MYH7 gene carrying a heterologous mutation using CRISPR / Cas by targeting rs2069540 or rs7157716, synonymous SNPs located in the first exon of MYH7 or exon 24, respectively, that are heterozygous in many different population ancestries.

[0378] Dominant negative mutations in the MYH7 gene are the most common cause of hypertrophic cardiomyopathy (HCM) and occur across the entire gene, making it unpractical to design therapeutics specific to each pathogenic mutation. For example, rs2069540 is a synonymous SNP located in the first exon of MYH7 — the G to A mutation in the genetic sequence results in a synonymous change in the codon from “acc” to “act” both of which translate to threonine (see FIG. 2). Rs2069540 is also heterozygous in many different genetic ancestry groups (see Table 7 below), allowing distinction between parental and maternal copies of chromosomes and therefore allele-specific editing (e.g., knocking out) of the MYH7 gene. For example, as high as 70% Amish are heterozygous carriers of rs2069540; even in the lowest percentage group, about 39% of East Asians are heterozygous for rs2069540. An estimated 51 % of the total population is heterozygous for rs2069540.Table 7. Heterozygosity frequency of rs2069540 in different ancestry groups

[0379] rs7157716 is a synonymous SNP located in exon 24 of MYH7 — the T to C mutation in the genetic sequence results in a synonymous change in the codon from “att” to “ate” both of which translate to isoleucine. rs7157716 is also heterozygous in many different genetic ancestry groups, allowing distinction between parental and maternal copies of chromosomes and therefore allele-specific editing (e.g., knocking out) of the MYH7 gene. For example, as high as 71 % of Africans are heterozygous carriers of rs7157716 and about 16% of East Asians are heterozygous for rs7157716. An estimated 38.5% of the total population is heterozygous for rs7157716. Thus, therapeutics (e.g., gRNA for use with CRISPR / Cas gene editing systems) designed for rs7157716 can apply to over one-third of the population.

[0380] Thus, therapeutics (e.g., gRNA for use with CRISPR / Cas gene editing systems) designed for rs2069540 or rs7157716 can apply to about half of the population. By linking the pathogenic MYH7 mutation to the reference (or alternate) allele of the rs2069540 or rs7157716 SNP, one can knock out the disease allele of the MYH7 gene by targeting the same copy of the chromosome carrying both the pathogenic mutation and the reference (or alternate) allele of the SNP, while leaving behind the wild-type, functional allele of the MYH7 gene (see FIG. 5).

[0381] gRNAs specific to the reference and alternate alleles of rs2069540 andrs7157716 were designed (see FIG. 5). “TGCCGAGACTGAGTATGGCAAGG” (SEQ ID NO: 17) and “GCCGAGACTGAGTATGGCAAG” (SEQ ID NO: 230) are specific to the alternate allele of rs2069540 and can be used if the pathogenic MYH7 mutation is located on the same copy of chromosome as rs2069540 to knock out the pathogenic allele of MYH7 while preserving the functional, wild-type allele; alternatively, “TGCCGAGACCGAGTATGGCAAGG” (SEQ ID NO: 16) and“GCCGAGACCGAGTATGGCAAG” (SEQ ID NO: 229) are specific to the reference allele of rs2069540 and can be used if the pathogenic MYH7 mutation is located on the opposite copy of chromosome to rs2069540 to knock out the pathogenic allele of MYH7 while preserving the functional, wild-type allele. “GAGATCATTGCCAAGCTGACCA” (SEQ ID NO: 231 ) is specific to the reference allele of rs7157716 and can be used if the pathogenic MYH7 mutation is located on the opposite copy of chromosome as rs7157716 to knock out the pathogenic allele of MYH7 while preserving the functional, wild-type allele. Alternatively, “GAGATCATCGCCAAGCTGACC” (SEQ ID NO: 144), “GATCATCGCCAAGCTGACCAAG” (SEQ ID NO: 145),“GAGATCATCGCCAAGCTGACCA” (SEQ ID NO: 146, and“CATCGCCAAGCTGACCAAGGAG” (SEQ ID NO: 147 are specific to the alternate allele of rs7157716 and can be used if the pathogenic MYH7 mutation is located on the same copy of chromosome to rs7157716 to knock out the pathogenic allele of MYH7 while preserving the functional, wild-type allele.Differentiation of hiPSCs into Ventricular Cardiomyocytes

[0382] Cardiomyocytes were differentiated using a Wnt-modulating protocol. In brief, hiPSCs were induced with RPMI+B27(-insulin) + 7 pM CHIR990921. Two days post-induction, cells were placed in RPMI+B27(-insulin) for 24 hrs, then in RPMI+B27(- insulin) supplemented with 5 pM IWP-2 for 48 hours. Cells were fed at 7 days postinduction with RPMI+B27, and at 9 days post-induction with RPMI+B27 + 1 pM blasticidin to select for differentiated cardiomyocytes, with feeds lasting for 9 days with cells fed on alternate days. Purified cells were frozen down in CryoStor . Only cardiomyocytes >90% cTnT+ as verified by flow cytometry were used in this study.Single Nucleotide Polymorphism (SNP) Genotyping of hiPSC-CMs

[0383] DNA was collected from differentiated cardiomyocytes and genotyped via quantitative polymerase chain reaction (qPCR) with fluorescent probes specific for the target MYH7 SNPs. Raw data was collected and converted into allelic discrimination plots using the QuantStudio instrument and software suite (Applied Biosystems). SNP genotyping revealed that the hiPSC-CM cell line was homozygous for the alternate allele of rs2069540, and heterozygous for both alleles of rs7157716. This genotype allowed testing of gRNAs targeting both the reference and alternate allele of rs7157716, but only the alternate allele of rs2069540.Transfection of hiPSC-CMs with CRISPR Constructs

[0384] WTC-BSD15 cardiomyocytes (a cell line with wild-type MYH7) were transfected with a plasmid comprising polynucleotides encoding a Staphylococcus aureus (Sa) Cas9 nuclease and a gRNA using ViaFect (Promega) according to the manufacturer’s protocol at a 4:1 ViaFect: DNA ratio. After 24 hours, cells were washed and cultured in fresh media for an additional 7 or 14 days.

[0385] Confirmation of transfection was conducted using a microscope at 20x magnification with a filter set for 488 nm light and a white-light high intensity LED light source. An increase in green fluorescent signal was observed in cells transfected with a GFP control construct.Assessment of SaCas9 Expression in hiPSC-CMs with Western Blotting

[0386] Protein was extracted from hiPSC-CMs, quantified by BCA Protein Assay and SaCas9 was quantified using a SaCas9 primary antibody with a species-specific secondary antibody. A significant increase in SaCas9 protein expression was observed in cells transfected with CRISPR constructs, while SaCas9 expression was negligible in cells transfected with a GFP control. These results suggest that not only was the delivery of CRISPR constructs into cells successful, but that the constructs initiated the proper transcription and subsequent expression of the Cas9 protein.Assessment of Editing Efficiency with Next Generation Sequencing

[0387] DNA from transfected cells was collected and PCR-amplified using primers that produce a 400-450 bp fragment containing the SNP targeted for editing. PCRproducts were gel-purified and analyzed by next generation sequencing (NGS) based on a full amplicon library preparation. All reads produced by the sequencing were downloaded and analyzed for mismatches in the target allele sequence between cells transfected with CRISPR constructs and those transfected with a GFP control.

[0388] Results are provided below in Table 8 for gRNA JT301 which targets rs2069540, and for gRNAs JT343, JT344, JT345, and JT346, which target rs7157716. Enumeration indicates the minimum editing efficiency of the construct as measured by NGS. 11 mer and 16 mer each refer to an exact match for the SNP of interest + / - a certain number of base pairs upstream or downstream. 11 mer means the SNP of interest + / - 5 base pairs upstream or downstream, and 16 mer means the SNP of interest + / - 8 base pairs upstream or downstream).Table 8. Summary of NGS results indicating gRNA editing efficiency

[0389] Of the constructs comprising gRNAs targeting rs7157716, pJT345 produced the greatest degree of editing at an approximate minimum efficiency of 2.9%. Editing of the reference allele by pJT345 was less than 0.1 %. The construct pJT301 , which targeted rs2069540, produced a minimum editing efficiency of approximately 2.2%. Target specificity was high (estimated to be 34-fold higher editing of the targeted allele compared to the non-targeted allele). gRNAs that targeted the alternate allele did not induce editing of the reference allele and vice versa. Furthermore, most, if not all, of the detected editing occurred at the correct cut site.

[0390] Immortalized lymphoblasts (LCL) from the 2552 participants in the 1000 genomes project are available for commercial distribution by the Coriell Institute. The efficacy and allele-specificity for each of the proposed gRNAs will be examined in LCLs which are known to be heterozygous for each of the defined SNPs. Along with a Cas9enzyme, gRNAs will be co-transfected by a standard mechanism (e.g., through AAV vectors, lentiviral vectors, or other transfection reagents such as lipid nanoparticles). The efficacy of the allele-specific disruption will be measured by one or more of the multiple methods including quantitative PCR, digital droplet PCR, or next generation sequencing of cloned PCR fragments of the locus. Whole genome sequencing may also be performed to assess for the presence of off-target effects.

[0391] From the foregoing, it will be appreciated that specific embodiments of the invention have been described herein for purposes of illustration, but that various modifications may be made without deviating from the scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims

CLAIMSI / We claim:1 . A method of allele-specific editing of a gene carrying a heterozygous mutation in a subject or a cell therefrom, the method comprising:(a) identifying a heterozygous genetic variant within the gene locus, wherein the subject carries a reference allele and an alternate allele of the genetic variant;(b) determining which of the reference and alternate alleles resides on the same chromosome as the mutation; and(c) disrupting the expression via CRISPR / Cas-mediated knockout of the mutation-carrying allele by:(i) targeting the reference allele of the genetic variant if it is determined to be on the same chromosome as the mutation in step (b), or(ii) targeting the alternate allele of the genetic variant if it is determined to be on the same chromosome as the mutation in step (b).

2. The method of claim 1 , wherein the gene is myosin heavy chain 7 (MYH7).

3. The method of claim 2, wherein the heterozygous mutation in the MYH7 gene is a dominant negative mutation.

4. The method of any one of claims 1-3, wherein the heterozygous genetic variant comprises a single-nucleotide polymorphism (SNP).

5. The method of claim 4, wherein the SNP has an at least 10%, at least 20%, at least 30, or at least 40% heterozygosity in the human population.

6. The method of claim 5, wherein the SNP is selected from the group consisting of rs735711, rs735712, rs2069540, rs2069542, rs2231126, and rs7157716.

7. The method of claim 5, wherein the SNP is rs2069540.

8. The method of claim 5, wherein the SNP is rs7157716.

9. The method of any one of claims 1-6, wherein the determining of step (b) is by phased sequencing or long-read RNA sequencing.

10. The method of any one of claims 1 -6, wherein the disrupting of step (c) is by forming an indel or a frameshift mutation in the mutant allele.

11. The method of any one of claims 1 -10, wherein the disrupting of step (c) is by introducing to the subject or the cell a CRISPR / Cas system comprising (i) a Cas nuclease or a polynucleotide encoding the same; and (ii) a guide RNA (gRNA) targeting the reference or the alternate allele of the heterozygous genetic variant.

12. The method of claim 11 , wherein the Cas nuclease is a Cas9 nuclease.

13. The method of claim 12, wherein the Cas9 nuclease is SpCas9 or SaCas9.

14. The method of any one of claims 11 -13, wherein the Cas nuclease is SaCas9 having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 2; and / or the polynucleotide encoding the Cas nuclease has a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 1.

15. The method of any one of claims 11-14, wherein the gRNA comprises a complementary region specific to the reference or the alternate allele of the heterozygous genetic variant.

16. The method of claim 15, wherein the complementary region comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 12-23, 144-147, and 229-239.

17. The method of claim 15, wherein the complementary region comprises a nucleotide sequence set forth in SEQ ID NO: 230.

18. The method of claim 15, wherein the complementary region comprises a nucleotide sequence set forth in SEQ ID NO: 146.

19. The method of claim 16, wherein:(a) the SNP is rs735711 , and the complementary region comprises a nucleotide sequence of SEQ ID NO: 12, 13, 232, or 233;(b) the SNP is rs735712, and the complementary region comprises a nucleotide sequence of SEQ ID NO: 14, 15, 234, or 235;(c) the SNP is rs2069540, and the complementary region comprises a nucleotide sequence of SEQ ID NO: 16, 17, 229, or 230;(d) the SNP is rs2069542, and the complementary region comprises a nucleotide sequence of SEQ ID NO: 18, 19, 236, or 237;(e) the SNP is rs2231126, and the complementary region comprises a nucleotide sequence of SEQ ID NO: 20, 21 , 238, or 239; or(f) the SNP is rs7157716, and the complementary region comprises a nucleotide sequence of SEQ ID NO: 22, 23, 144, 145, 146, 147, or 231 .

20. The method of any one of claims 11 -19, wherein the polynucleotide encoding the Cas nuclease and a polynucleotide encoding the gRNA are in a vector in a head- to-tail orientation.

21. The method of claim 20, wherein the vector further comprises a protein expression-driving promoter operably linked to the polynucleotide encoding the Cas nuclease and an RNA expression-driving promoter operably linked to the polynucleotide encoding the gRNA.

22. The method of claim 21 , wherein the protein expression-driving promoter is a human troponin T (TNNT2) promoter having a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 24-27.

23. The method of claim 21 , wherein the RNA expression-driving promoter is a human U6 promoter having a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 31.

24. The method of any one of claims 21 -23, wherein the vector comprises the following 5’ to 3’ arrangement of elements: 5’ - the protein expression-driving promoter - the polynucleotide encoding the Cas nuclease - the RNA expressiondriving promoter - the polynucleotide encoding the gRNA - 3’.

25. The method of any one of claims 21 -24, wherein the vector further comprises apolyadenylation (poly(A)) sequence.

26. The method of claim 25, wherein the poly(A) sequence is selected from the group consisting of a synthetic poly(A) sequence of SEQ ID NO: 43, a BGH poly(A) sequence of SEQ ID NO: 44, and a SV40 poly(A) sequence of SEQ ID NO: 45.

27. The method of claim 25 or 26, wherein the poly(A) sequence is between the polynucleotide encoding the Cas nuclease and the RNA expression-driving promoter, or wherein the vector comprises the following 5’ to 3’ arrangement of elements: 5’ - the protein expression-driving promoter - the polynucleotide encoding the Cas nuclease -the poly(A) sequence - the RNA expression-driving promoter - the polynucleotide encoding the gRNA - 3’.

28. The method of any one of claims 21 -27, wherein the vector further comprises a self-inactivation site 5’ to the protein expression-driving promoter, within the protein expression-driving promoter, between the protein expression-driving promoter and the polynucleotide encoding the Cas nuclease, within the polynucleotide encoding the Cas nuclease, or 3’ to the polynucleotide encoding the Cas nuclease.

29. The method of claim 28, wherein the self-inactivation site is within the polynucleotide encoding the Cas nuclease, optionally near the 5’ end of the polynucleotide encoding the Cas nuclease, further optionally after the start codon “ATG”.

30. The method of claim 28 or 29, wherein the self-inactivation site comprises a gRNA target region having a nucleotide sequence identical or complement to the complementary region of the gRNA.31 . The method of any one of claims 24-26, wherein the self-inactivation site further comprises a less optimal PAM sequence for the Cas nuclease.

32. The method of claim 31 , wherein the Cas nuclease is SaCas9, and the less optimal PAM sequence is gcgagc, gcgaga, or gcgagg.

33. The method of any one of claims 20-32, wherein the vector is an adeno-associated virus (AAV) vector, a retroviral vector, or a lenti viral vector.

34. The method of claim 33, wherein the vector is an AAV vector, optionally wherein the AAV vector is an AAV9 vector.

35. The method of claim 34, wherein the vector comprises any capsid protein described herein, optionally wherein the capsid protein is a wild-type AAV9 capsid protein or an engineered variant thereof.

36. The method of any one of claims 1 -35, wherein the method has an at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% editing efficiency of, or efficiency of forming an indel or a frameshift mutation in, the mutant allele of the gene.

37. The method of any one of claims 1-36, wherein the method does not, or substantially does not, edit or an indel or a frameshift mutation in the WT allele of the gene.

38. A guide RNA (gRNA) for allele-specific editing of myosin heavy chain 7 (MYH7), wherein the gRNA comprises a complementary region specific to a singlenucleotide polymorphism (SNP) within the MYH7 gene locus.

39. The gRNA of claim 38, wherein the SNP is selected from the group consisting of rs735711 , rs735712, rs2069540, rs2069542, rs2231126, and rs7157716.

40. The gRNA of claim 38, wherein the SNP is rs2069540.41 . The gRNA of claim 38, wherein the SNP is rs7157716.

42. The gRNA of claim 39, wherein:(a) the SNP is rs735711 , and the complementary region comprises a nucleotide sequence of SEQ ID NO: 12, 13, 232, or 233;(b) the SNP is rs735712, and the complementary region comprises a nucleotide sequence of SEQ ID NO: 14, 15, 234, or 235;(c) the SNP is rs2069540, and the complementary region comprises a nucleotide sequence of SEQ ID NO: 16, 17, 229, or 230;(d) the SNP is rs2069542, and the complementary region comprises a nucleotide sequence of SEQ ID NO: 18, 19, 236, or 237;(e) the SNP is rs2231126, and the complementary region comprises a nucleotide sequence of SEQ ID NO: 20, 21 , 238, or 239; or(f) the SNP is rs7157716, and the complementary region comprises a nucleotide sequence of SEQ ID NO: 22, 23, 144, 145, 146, 147, or 231 .

43. A composition for allele-specific editing of a myosin heavy chain 7 (MYH7) gene carrying a heterozygous mutation in a subject or a cell therefrom, the composition comprising:(i) a first guide RNA (gRNA) comprising a complementary region specific to a reference allele of a first single-nucleotide polymorphism (SNP) within the MYH7 gene locus; or(ii) a second gRNA comprising a complementary region specific to an alternate allele of the first SNP within the MYH7 gene locus, and(iii) optionally, a Cas nuclease or a polynucleotide encoding the same.

44. The composition of claim 43, wherein the first SNP has an at least 10%, at least 20%, at least 30, or at least 40% heterozygosity in the human population.

45. The composition of claim 44, wherein the first SNP is selected from the group consisting of rs735711 , rs735712, rs2069540, rs2069542, rs2231126, and rs7157716.

46. The composition of claim 44, wherein the first SNP is rs2069540.

47. The composition of claim 44, wherein the first SNP is rs7157716.

48. The composition of claim 45, wherein:(a) the SNP is rs735711 , the complementary region of the first gRNA comprises a nucleotide sequence of SEQ ID NO: 12, and / or the complementary region of the second gRNA comprises a nucleotide sequence of SEQ ID NO: 13;(b) the SNP is rs735712, the complementary region of the first gRNA comprises a nucleotide sequence of SEQ ID NO: 14, and / or the complementary region of the second gRNA comprises a nucleotide sequence of SEQ ID NO: 15;(c) the SNP is rs2069540, the complementary region of the first gRNA comprises a nucleotide sequence of SEQ ID NO: 16 or 229, and / or the complementary region of the second gRNA comprises a nucleotide sequence of SEQ ID NO: 17 or 230;(d) the SNP is rs2069542, the complementary region of the first gRNA comprises a nucleotide sequence of SEQ ID NO: 18, and / or the complementary region of the second gRNA comprises a nucleotide sequence of SEQ ID NO: 19;(e) the SNP is rs2231126, the complementary region of the first gRNA comprises a nucleotide sequence of SEQ ID NO: 20, and / or the complementary region of the second gRNA comprises a nucleotide sequence of SEQ ID NO: 21 ; or(f) the SNP is rs7157716, the complementary region of the first gRNA comprises a nucleotide sequence of SEQ ID NO: 22 or 231 , and / or the complementary region of the second gRNA comprises a nucleotide sequence of SEQ ID NO: 23, 144, 145, 146, or 147.

49. The composition of claim 45, wherein the SNP is rs2069540, the complementary region of the first gRNA comprises a nucleotide sequence of SEQ ID NO: 229, and the complementary region of the second gRNA comprises a nucleotide sequence of SEQ ID NO: 230.

50. The composition of claim 45, wherein the SNP is rs7157716, the complementary region of the first gRNA comprises a nucleotide sequence of SEQ ID NO: 231 , and the complementary region of the second gRNA comprises a nucleotide sequence of SEQ ID NO: 146.51 . The composition of any one of claims 43-48, wherein the Cas nuclease is a Cas9 nuclease.

52. The composition of claim 51 , wherein the Cas9 nuclease is SpCas9 or SaCas9.

53. The composition of claim 51 or 52, wherein the Cas nuclease is SaCas9 having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 2; and / or the polynucleotide encoding the Cas nuclease has a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 1.

54. The composition of any one of claims 43-53, wherein the polynucleotide encoding the Cas nuclease and a polynucleotide encoding the first or second gRNA are in a vector in a head-to-tail orientation.

55. The composition of claim 54, wherein the vector further comprises a protein expression-driving promoter operably linked to the polynucleotide encoding the Cas nuclease and an RNA expression-driving promoter operably linked to the polynucleotide encoding the first or second gRNA.

56. The composition of claim 55, wherein the protein expression-driving promoter is a human troponin T (TNNT2) promoter having a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 24-27.

57. The composition of claim 55, wherein the RNA expression-driving promoter is a human U6 promoter having a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 31.

58. The composition of any one of claims 55-57, wherein the vector comprises the following 5’ to 3’ arrangement of elements: 5’ - the protein expression-driving promoter - the polynucleotide encoding the Cas nuclease - the RNA expressiondriving promoter - the polynucleotide encoding the first or second gRNA - 3’.

59. The composition of any one of claims 55-58, wherein the vector further comprises a polyadenylation (poly(A)) sequence.

60. The composition of claim 59, wherein the poly(A) sequence is selected from the group consisting of a synthetic poly(A) sequence of SEQ ID NO: 43, a BGH poly(A) sequence of SEQ ID NO: 44, and a SV40 poly(A) sequence of SEQ ID NO: 45.61 . The composition of claim 59 or 60, wherein the poly(A) sequence is between the polynucleotide encoding the Cas nuclease and the RNA expression-driving promoter, or wherein the vector comprises the following 5’ to 3’ arrangement of elements: 5’ - the protein expression-driving promoter - the polynucleotide encoding the Cas nuclease -the poly(A) sequence - the RNA expression-driving promoter - the polynucleotide encoding the first or second gRNA - 3’.

62. The composition of any one of claims 55-61 , wherein the vector further comprises a self-inactivation site 5’ to the protein expression-driving promoter, within the protein expression-driving promoter, between the protein expression-driving promoter and the polynucleotide encoding the Cas nuclease, within the polynucleotide encoding the Cas nuclease, or 3’ to the polynucleotide encoding the Cas nuclease.

63. The composition of claim 62, wherein the self-inactivation site is within the polynucleotide encoding the Cas nuclease, optionally near the 5’ end of the polynucleotide encoding the Cas nuclease, further optionally after the start codon “ATG”.

64. The composition of claim 62 or 63, wherein the self-inactivation site comprises a gRNA target region having a nucleotide sequence identical or complement to the complementary region of the first or second gRNA.

65. The composition of any one of claims 62-64, wherein the self-inactivation site further comprises a less optimal PAM sequence for the Cas nuclease.

66. The composition of claim 65, wherein the Cas nuclease is SaCas9, and the less optimal PAM sequence is gcgagc, gcgaga, or gcgagg.

67. The composition of any one of claims 54-66, wherein the vector is an adeno- associated virus (AAV) vector, a retroviral vector, or a lenti viral vector.

68. The composition of claim 67, wherein the vector is an AAV vector, optionally wherein the AAV vector is an AAV9 vector.

69. The composition of claim 68, wherein the vector comprises any capsid protein described herein, optionally wherein the capsid protein is a wild-type AAV9 capsidprotein or an engineered variant thereof.

70. The composition of any one of claims 43-69, wherein the composition further comprises:(iv) a third gRNA comprising a complementary region specific to a reference allele of a second SNP within the MYH7 gene locus; or(v) a fourth gRNA comprising a complementary region specific to an alternate allele of the second SNP within the MYH7 gene locus, wherein the second SNP is different from the first SNP.

71. The composition of claim 70, wherein the second SNP has an at least 10%, at least 20%, at least 30, or at least 40% heterozygosity in the human population.

72. The composition of claim 71 , wherein the second SNP is selected from the group consisting of rs735711 , rs735712, rs2069540, rs2069542, rs2231126, and rs7157716.

73. The composition of any one of claims 43-72, further comprising a pharmaceutically acceptable carrier, diluent, or excipient.

74. A method of treating a disease caused by or associated with a heterozygous mutation in a myosin heavy chain 7 (MYH7) gene in a subject in need thereof, wherein the subject carries a wild-type (WT) allele and a mutant allele of the MYH7 gene, the method comprising allele-specific editing the mutant allele of the MYH7 gene according to the method of any one of claims 1 -37, or administering to the subject the composition of any one of claims 43-73.

75. The method of claim 74, wherein the disease or condition is a heart disease.

76. The method of claim 75, wherein the heart disease is cardiomyopathy.

77. The method of claim 76, wherein the cardiomyopathy is hypertrophic cardiomyopathy (HCM).

78. The method of any one of claims 74-77, wherein the method improves one or more measures of cardiac function.

79. The method of claim 78, wherein the method increases ejection fraction, reduces left ventricular internal dimension and / or left ventricular mass.

80. The method of any one of claims 74-79, wherein the administering is systemic administration or local administration to the heart.

81. The method of claim 80, wherein the systemic administration is intravenous administration.

82. The method of claim 80, wherein the local administration is by direct injection into the heart or cardiac tissue, intracoronary administration, or retrograde coronary sinus infusion.

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