Junctophilin-2 (JPH2) gene therapy using AAV vectors

Abstract

Provided herein is a gene therapy for JPH2 (junctophilin-2), for example, using an adeno-associated virus (AAV) vector. The promoter of the vector can be the MHCK7 promoter or the cardiac troponin T (HTNNT2) promoter. The capsid can be the AAV9 or AAVrh74 capsid or a functional variant thereof. Other promoters or capsids may be used. Further provided are methods of treatment, such as by intravenous, intracoronary, intracarotid or intracardiac administration of rAAV vectors, as well as other compositions and methods. TIFF2024546103000093.tif151154

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 287,393, filed December 8, 2021, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0002] SEQUENCE LISTING STATEMENT The sequence listing associated with this application is provided in text form in lieu of a paper copy and is incorporated herein by reference. The text file containing the sequence listing is named ROPA_025_01WO_SeqList_ST26.xml. The text file is approximately 272,918 bytes, was created on December 6, 2022, and has been submitted electronically via EFS-Web. [Background technology]

[0003] background Both hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are severe and fatal diseases. Loss-of-function mutations in the gene junctophilin-2 (JPH2) are associated with HCM and DCM as well as other cardiac diseases, such as atrial fibrillation (AF).

[0004] Junctophilin-2 (JPH2) mediates transverse (T) tubule-associated cardiac L-type Ca2+ transporter located in the sarcoplasmic reticulum within the junctional membrane complex (JMC) of cardiomyocytes. 2+ It is a structural protein that acts as a bridge between the Ca channel and the type 2 ryanodine receptor. Effective signaling between these channels ensures sufficient Ca delivery, which is required for normal cardiac contractility. 2+ Downregulation of JPH2 is detected by disruption of the JMC intracellular domain, a common feature of the failing heart.

[0005] Current treatment methods, including drug therapy and cardiac ablation, are still ineffective for JPH2-deficient cardiomyopathy patients.Therefore, there is still an unmet need in the art for the treatment of JPH2-related disease and disorders, including cardiomyopathy and other heart diseases.The compositions and methods disclosed herein address this need. Summary of the Invention

[0006] overview The present disclosure relates generally to gene therapy for diseases or disorders, such as cardiac diseases or disorders, using vectors expressing JPH2 or functional variants thereof.

[0007] In one aspect, the disclosure provides a polynucleotide comprising an expression cassette and optionally flanking adeno-associated virus (AAV) inverted terminal repeats (ITRs), the polynucleotide comprising a polynucleotide sequence encoding junctophilin-2 (JPH2) or a functional variant thereof, operably linked to a promoter.

[0008] In some embodiments, the promoter is a cardiac specific promoter. In some embodiments, the promoter is a muscle specific promoter. In some embodiments, the promoter is a cardiomyocyte specific promoter.

[0009] In some embodiments, the promoter is a myosin heavy chain creatine kinase 7 (MHCK7) promoter. In some embodiments, the MHCK7 promoter shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:31.

[0010] In some embodiments, the promoter is a cardiac troponin T (hTNNT2) promoter. In some embodiments, the hTNNT2 promoter shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 32. In some embodiments, the expression cassette comprises exon 1 of the cardiac troponin T (hTNNT2) gene, where optionally the hTNNT2 promoter and exon 1 together share at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 32.

[0011] In some embodiments, the promoter is a ubiquitous promoter, optionally a CMV promoter or a CAG promoter.

[0012] In some embodiments, the expression cassette comprises a polyA signal. In some embodiments, the polyA signal is human growth hormone (hGH) polyA.

[0013] In some embodiments, the expression cassette comprises a Woodchuck Hepatitis Virus post-transcriptional regulatory element (WPRE), optionally a WPRE(x).

[0014] In some embodiments, the expression cassette comprises green fluorescent protein (GFP).

[0015] In some embodiments, the junctophilin-2 (JPH2) or functional variant thereof is JPH2. In some embodiments, the JPH2 is human JPH2. In some embodiments, the polynucleotide sequence encoding JPH2 is a human JPH2 polynucleotide.

[0016] In some embodiments, the polynucleotide sequence encoding JPH2 shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:2.

[0017] In some embodiments, the polynucleotide sequence encoding JPH2 shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:4.

[0018] In some embodiments, the polynucleotide sequence encoding JPH2 shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:6.

[0019] In some embodiments, the polynucleotide sequence encoding JPH2 shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:8.

[0020] In some embodiments, the polynucleotide sequence encoding JPH2 shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:10.

[0021] In some embodiments, the polynucleotide comprises at least about 3.0 kb, at least about 3.2 kb, at least about 3.4 kb, at least about 3.5 kb, at least about 3.7 kb, at least about 4.0 kb, at least about 4.1 kb, at least about 4.2 kb, at least about 4.3 kb, at least about 4.4 kb, at least about 4.5 kb, at least about 4.6 kb, at least about 4.7 kb, at least about 4.8 kb or at least about 5.0 kb.

[0022] In some embodiments, the polynucleotide comprises at most about 3.1 kb, at most about 3.3 kb, at most about 3.5 kb, at most about 3.7 kb, at most about 3.9 kb, at most about 4.1 kb, at most about 4.2 kb, at most about 4.3 kb, at most about 4.4 kb, at most about 4.5 kb, at most about 4.6 kb, at most about 4.7 kb, at most about 4.8 kb, at most about 4.9 kb, or at most about 5.0 kb.

[0023] In some embodiments, the polynucleotide comprises between 4.4 kb and 5.0 kb, between 4.4 kb and 4.9 kb, or between 4.4 kb and 4.8 kb, or the polynucleotide comprises between 4.0 kb and 4.6 kb, between 4.0 kb and 4.5 kb, or between 4.0 kb and 4.4 kb, or the polynucleotide comprises between 4.0 kb and 4.3 kb, between 4.0 kb and 4.2 kb, or between 4.0 kb and 4.1 kb, or the polynucleotide comprises between 3.0 kb and 3.9 kb, between 3.0 kb and 3.8 kb, or between 3.0 kb and 3.7 kb.

[0024] In some embodiments, JPH2 or a functional variant thereof comprises at least 600 or at least 630 amino acids.

[0025] In some embodiments, JPH2 or a functional variant thereof comprises at least 600 or at least 696 amino acids.

[0026] In some embodiments, JPH2 or a functional variant thereof comprises at least 100 or at least 129 amino acids.

[0027] In some embodiments, the expression cassette is flanked by 5' and 3' inverted terminal repeats (ITRs). In some embodiments, the ITRs are AAV2 ITRs and / or the ITRs share at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with any one of SEQ ID NOs: 15-21.

[0028] In one aspect, the present disclosure provides a gene therapy vector comprising the polynucleotide described in the present disclosure. In some embodiments, the gene therapy vector is a recombinant adeno-associated virus (rAAV) vector. In some embodiments, the rAAV vector is AAV9 or a functional variant thereof. In some embodiments, the rAAV vector comprises a capsid protein that shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with any one of SEQ ID NO: 97. In some embodiments, the rAAV vector is AAVrhlO or a functional variant thereof. In some embodiments, the rAAV vector comprises a capsid protein that shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with any one of SEQ ID NO: 99. In some embodiments, the rAAV vector is AAV6 or a functional variant thereof. In some embodiments, the rAAV vector comprises a capsid protein that shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NO: 98.

[0029] In some embodiments, the rAAV vector is AAVrh74 or a functional variant thereof. In some embodiments, the rAAV vector comprises a capsid protein that shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NO: 100.

[0030] In one aspect, the present disclosure provides a method of treating and / or preventing a disease or disorder in a subject in need thereof, comprising administering to the subject a vector of the present disclosure.

[0031] In some embodiments, the disease or disorder is a cardiac disorder. In some embodiments, the cardiac disorder is a cardiomyopathy, such as hypertrophic cardiomyopathy (HCM) or dilated cardiomyopathy (DCM). In some embodiments, the disease or disorder is arrhythmia. In some embodiments, the arrhythmia is atrial fibrillation. In some embodiments, the arrhythmia is sinus node disease. In some embodiments, the disease or disorder is familial hypertrophic cardiomyopathy17. In some embodiments, the disease or disorder is heart failure.

[0032] In some embodiments, the subject is a mammal. In some embodiments, the subject is a primate. In some embodiments, the subject is a human.

[0033] In some embodiments, the subject has a mutation in the JPH2 gene. In some embodiments, the subject has a truncated variant of JPH2.

[0034] In some embodiments, the vector is administered intravenously, intracardially, intracoronarily, intracardially, and / or by cardiac catheterization. In certain embodiments, any route of administration may be performed by infusion or injection.

[0035] In some embodiments, administration increases JPH2 expression by at least about 5%. In some embodiments, administration increases JPH2 expression by at least about 30%. In some embodiments, administration increases JPH2 expression by at least about 70%. In some embodiments, administration increases JPH2 expression by about 5% to about 10%. In some embodiments, administration increases JPH2 expression by about 30% to about 50%. In some embodiments, administration increases JPH2 expression by about 50% to about 70%. In some embodiments, administration increases JPH2 expression by about 70% to about 100%.

[0036] In one aspect, the present disclosure provides a method for treating and / or preventing disease or disorder.In some embodiments, the method comprises administering an effective amount of vector.In some embodiments, the disease or disorder is associated with or caused by the cleavage of JPH2 in a subject.In some embodiments, the method comprises administering a pharmaceutical composition comprising an effective amount of vector.

[0037] In some embodiments, the method comprises administering about 1×10 11 Vector genome ~ approx. 1 × 10 13 administering to the subject a vector of the vector genome, 12 Vector genome ~ approx. 1 × 10 14 administering to the subject a vector of the vector genome, 13 Vector genome ~ approx. 1 × 10 15 Administering a vector genome, or about 1×10 15 Vector genome ~ approx. 1 × 10 17 Administering a vector genome, or about 1×10 17 Vector genome ~ approx. 1 × 10 18 The vector may be administered to a subject at any one of a range of 2 to 4 mg / kg / day, or ...

[0038] In one aspect, the present disclosure provides a pharmaceutical composition comprising a vector of the present disclosure.

[0039] In one aspect, the present disclosure provides a kit comprising a vector of the present disclosure or a pharmaceutical composition of the present disclosure and, optionally, instructions for use.

[0040] In one aspect, the disclosure provides for the use of a vector of the disclosure in the treatment of a disease or disorder, optionally by a method of the disclosure.

[0041] In one aspect, the disclosure provides a vector according to the disclosure for use in treating a disease or disorder, optionally by a method of the disclosure.

[0042] In one aspect, the disclosure provides a polynucleotide comprising a polynucleotide sequence sharing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 26-30 or any one of SEQ ID NOs: 76-95.

[0043] In some embodiments, the promoter is an MHCK7 promoter. In some embodiments, the MHCK7 promoter shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:31.

[0044] Various other aspects and embodiments are disclosed in the following detailed description. The present disclosure is limited only by the appended claims. [Brief description of the drawings]

[0045] [Figure 1] A diagram illustrating a non-limiting example of a vector genome is shown. The complete polynucleotide sequence of the vector genome is SEQ ID NO: 26. The MHCK7 promoter described herein is labeled "Enhancer / MHCK7" in the diagram. [Diagram 2] A diagram illustrating a non-limiting example of a vector genome is shown. The complete polynucleotide sequence of the vector genome is SEQ ID NO:27. [Diagram 3] A diagram illustrating a non-limiting example of a vector genome is shown. The complete polynucleotide sequence of the vector genome is SEQ ID NO: 28. The MHCK7 promoter described herein is labeled "Enhancer / MHCK7" in the diagram. [Figure 4] A diagram illustrating a non-limiting example of a vector genome is shown. The complete polynucleotide sequence of the vector genome is SEQ ID NO:29. [Diagram 5]A diagram illustrating a non-limiting example of a vector genome is shown. The complete polynucleotide sequence of the vector genome is SEQ ID NO: 30. [Figure 6] A diagram illustrating a non-limiting example of a vector genome is shown. The complete polynucleotide sequence of the vector genome is SEQ ID NO: 76. The MHCK7 promoter described herein is labeled "Enhancer / MHCK7" in the diagram. [Figure 7] A diagram illustrating a non-limiting example of a vector genome is shown. The complete polynucleotide sequence of the vector genome is SEQ ID NO: 77. [Figure 8] A diagram illustrating a non-limiting example of a vector genome is shown. The complete polynucleotide sequence of the vector genome is SEQ ID NO: 78. The MHCK7 promoter described herein is labeled "Enhancer / MHCK7" in the diagram. [Figure 9] A diagram illustrating a non-limiting example of a vector genome is shown. The complete polynucleotide sequence of the vector genome is SEQ ID NO:79. [Figure 10] A diagram illustrating a non-limiting example of a vector genome is shown. The complete polynucleotide sequence of the vector genome is SEQ ID NO: 80. [Figure 11] A diagram illustrating a non-limiting example of a vector genome is shown. The complete polynucleotide sequence of the vector genome is SEQ ID NO: 81. The MHCK7 promoter described herein is labeled "Enhancer / MHCK7" in the diagram. [Figure 12] A diagram illustrating a non-limiting example of a vector genome is shown. The complete polynucleotide sequence of the vector genome is SEQ ID NO: 82. [Figure 13] A diagram illustrating a non-limiting example of a vector genome is shown. The complete polynucleotide sequence of the vector genome is SEQ ID NO: 83. The MHCK7 promoter described herein is labeled "Enhancer / MHCK7" in the diagram. [Figure 14] A diagram illustrating a non-limiting example of a vector genome is shown. The complete polynucleotide sequence of the vector genome is SEQ ID NO:84. [Figure 15] A diagram illustrating a non-limiting example of a vector genome is shown. The complete polynucleotide sequence of the vector genome is SEQ ID NO: 85. [Figure 16] A diagram illustrating a non-limiting example of a vector genome is shown. The complete polynucleotide sequence of the vector genome is SEQ ID NO: 86. The MHCK7 promoter described herein is labeled "Enhancer / MHCK7" in the diagram. [Figure 17] A diagram illustrating a non-limiting example of a vector genome is shown. The complete polynucleotide sequence of the vector genome is SEQ ID NO: 87. [Figure 18] A diagram illustrating a non-limiting example of a vector genome is shown. The complete polynucleotide sequence of the vector genome is SEQ ID NO: 88. The MHCK7 promoter described herein is labeled "Enhancer / MHCK7" in the diagram. [Figure 19] A diagram illustrating a non-limiting example of a vector genome is shown. The complete polynucleotide sequence of the vector genome is SEQ ID NO:89. [Figure 20] A diagram illustrating a non-limiting example of a vector genome is shown. The complete polynucleotide sequence of the vector genome is SEQ ID NO:90. [Figure 21] A diagram illustrating a non-limiting example of a vector genome is shown. The complete polynucleotide sequence of the vector genome is SEQ ID NO: 91. The MHCK7 promoter described herein is indicated in the diagram as "Enhancer / MHCK7." [Figure 22] A diagram illustrating a non-limiting example of a vector genome is shown. The complete polynucleotide sequence of the vector genome is SEQ ID NO:92. [Diagram 23]A diagram illustrating a non-limiting example of a vector genome is shown. The complete polynucleotide sequence of the vector genome is SEQ ID NO: 93. The MHCK7 promoter described herein is labeled "Enhancer / MHCK7" in the diagram. [Figure 24] A diagram illustrating a non-limiting example of a vector genome is shown. The complete polynucleotide sequence of the vector genome is SEQ ID NO:94. [Diagram 25] A diagram illustrating a non-limiting example of a vector genome is shown. The complete polynucleotide sequence of the vector genome is SEQ ID NO:95. [Figure 26] Figure 2 shows JPH2 protein expression in the hearts of C57BL / 6J mice. The figure shows Western blots (WB) of JPH2 (upper panel) or the loading control GAPDH (lower panel). [Figure 27] 1 illustrates an experimental timeline. [Figure 28] Figures 28A-28B illustrate left ventricular ejection fraction (EF%) over time (Figure 28A) and at 9 weeks post-TAC (6 weeks post-AAV injection; Figure 28B). Statistical analysis (one-way ANOVA) followed by Dunnett's post-hoc comparison revealed a significant benefit of the AAV-treated group compared to the untreated group (*p≦0.05, **p≦0.01, ***p≦0.001, ****p<0.0001). For Figure 28A, at 5 weeks post-TAC, the lines correspond from top to bottom to FB (Neg CON), AAV9-hTnT-JPH2, AAVrh.74-hTnt-JPH2, AAV9-MHCK7-JPH2, FB (POS CON) and AAVrh.74-MHCK7-JPH2. [Figure 29A]Figures 29A-29F show left ventricular ejection fraction (EF%) over time. Figure 29A: shows results for normal mice, and Figure 29B: shows results for untreated TAC mice. Figure 29C: shows results using AAV9 vector and MHCK7 promoter (AAV9-MHCK7). Figure 29D: shows results using AAVrh.74 vector and MHCK7 promoter (AAVrh.74-MHCK7). Figure 29E: shows results using AAV9 vector and hTnT promoter (AAV9-hTnT). Figure 29F: shows results using AAVrh.74 vector and hTnT promoter (AAVrh.74-hTnT). [Figure 29B] See legend to Figure 29A. [Figure 29C] See legend to Figure 29A. [Figure 29D] See legend to Figure 29A. [Figure 29E] See legend to Figure 29A. [Figure 29F] See legend to Figure 29A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] Detailed Description of the Disclosure The present disclosure provides gene therapy vectors for JPH2 that deliver polynucleotides encoding JPH2 polypeptides or functional variants thereof, together with methods of use, as well as other compositions and methods. In certain embodiments, the present disclosure relates to gene therapy vectors comprising a promoter sequence operably linked to a polynucleotide encoding a JPH2 polypeptide or functional variants thereof. In some embodiments, the promoter is a myosin heavy chain creatine kinase 7 (MHCK7) promoter. In some embodiments, the AAV vector is an AAV9 vector. In some embodiments, the promoter is a MHCK7 promoter and the AAV vector is an AAV9 vector. In some embodiments, the promoter is a hTNNT2 promoter. In some embodiments, the promoter is a hTNNT2 promoter and the AAV vector is an AAV9 vector. In some embodiments, the JPH2 is human JPH2. In some embodiments, the JPH2 is human JPH2 isoform 1 (SEQ ID NO:1). In some embodiments, the JPH2 is human JPH2 isoform 2 (SEQ ID NO:108). In some embodiments, the AAV vector is a rh74 vector. In some embodiments, the promoter is a MHCK7 promoter and the AAV vector is a rh74 vector. In some embodiments, the promoter is a hTNNT2 promoter. In some embodiments, the promoter is a hTNNT2 promoter and the AAV vector is a rh74 vector. In some embodiments, the JPH2 is human JPH2.

[0047] The present disclosure further provides a method for treating disease or disorder in a subject by administering the gene therapy vector of the present disclosure to the subject.In some embodiments, the disease or disorder is atrial fibrillation.In some embodiments, the disease or disorder is arrhythmia.In some embodiments, the disease or disorder is sinus node disease.In some embodiments, the disease or disorder is familial hypertrophic cardiomyopathy 17.Mutations in JPH2 are associated with familial hypertrophic cardiomyopathy 17 and atrial fibrillation.

[0048] In certain embodiments, the subject to be treated is a heart failure patient with one or more mutations or truncations in JPH2 gene.The expression level of JPH2 is reduced in the failing heart of multiple etiologies, including human heart failure.Heart failure patients have JPH2 fragments that are generated during cardiac stress.

[0049] The JPH2 gene encodes the junctionophilin-2 (JPH2) protein. JPH2 mediates the expression of type 2 ryanodine receptors in the sarcoplasmic reticulum within the junctional membrane complex (JMC) of cardiomyocytes and the transverse (T) tubule-associated cardiac L-type Ca2+ receptors in the plasma membrane. 2+ Its structure maintains a 12-15 nm gap between the sarcolemma and sarcoplasmic reticulum membranes of the cardiac dyad, allowing the flow of Ca from the cell surface to the intracellular Ca channel. 2+ JPH2 provides a structural basis for functional crosstalk between JPH2 and release channels. JPH2 is required for normal excitation-contraction coupling in cardiomyocytes and contributes to the organization of triple junctions in skeletal muscle.

[0050] After cardiac stress, JPH2 increased Ca 2+ It is cleaved by the dependent protease calpain, thereby releasing the N-terminal fragment (JPH2NT), which translocates to the nucleus, binds to genomic DNA, and regulates the expression of various genes in cardiomyocytes. Stress-induced proteolysis of JPH2 disrupts the ultrastructural organization and promotes the progression of heart failure.

[0051] Calpain cleavage is one of the main mechanisms underlying the loss of JPH2 levels in failing hearts. Calpain-1 and -2 activity are increased in cardiac tissues subjected to stress (i.e., ischemia, oxidative stress, HF), and thus JPH2 Ca upregulation is impaired under pathological conditions. 2+Dependent proteolysis has been observed. Human JPH2 contains three calpain cleavage sites. Calpain-1 and calpain-2 can cleave JPH2 at amino acid positions 572 and 573 of SEQ ID NO: 1. Calpain-1 can also cleave JPH2 at sites found at amino acid positions 155 and 156, and amino acid positions 204 and 205 of human JPH2, isoform 1, as set forth in SEQ ID NO: 1. Additional calpain-2 cleavage sites are disclosed in Weninger et al. Sci Rep 12, 10387 (2022), which is incorporated by reference in its entirety.

[0052] In some embodiments of the present disclosure, a polynucleotide encoding JPH2 for use in making a gene therapy vector may include an alanine substitution for amino acid numbers 155 and 156 of the JPH2 reference sequence set forth in SEQ ID NO: 1. In some embodiments, a polynucleotide encoding JPH2 for use in making a gene therapy vector may include an alanine substitution for amino acid numbers 204 and 205 of the JPH2 reference sequence set forth in SEQ ID NO: 1. In some embodiments, a polynucleotide encoding JPH2 for use in making a gene therapy vector may include an alanine substitution for amino acid numbers 573 and 573 of the JPH2 reference sequence set forth in SEQ ID NO: 1. In some embodiments, a polynucleotide encoding JPH2 for use in making a gene therapy vector may include an alanine substitution for amino acid numbers 155, 156, 204, 205, 572 and 573 of the JPH2 reference sequence set forth in SEQ ID NO: 1, or any combination thereof.

[0053] In some embodiments, at least one calpain cleavage site is removed from the polynucleotide encoding JPH2 by replacing at least one cleavage site amino acid with alanine.In some embodiments, at least one calpain cleavage site is removed from the polynucleotide encoding JPH2 by replacing at least one cleavage site amino acid with an amino acid with similar properties or by conservative amino acid substitution, such as alanine instead of valine, lysine instead of arginine, alanine instead of leucine, and serine instead of threonine.According to the present disclosure, the polynucleotide encoding JPH2 or its functional variant, which comprises at least 129, at least 600, at least 630, or at least 696 amino acids, can be utilized in the creation of gene therapy vectors. The resulting vectors can be utilized in the treatment of diseases or disorders, such as JPH2-associated diseases or disorders, such as atrial fibrillation, arrhythmias, sinus node disease, hypertensive heart disease, heart failure, cardiac hypertrophy, atrial fibrosis, myocardial infarction, symptomatic sick sinus syndrome, atrial disease, myocardial infarction, familial hypertrophic cardiomyopathy, etc.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains.Methods and materials similar or equivalent to those described herein can be used in the practice of this disclosure, and suitable methods and materials are described below.All publications, patent applications, patents and other references mentioned herein are expressly incorporated by reference in their entirety.In case of conflict, the present specification, including definitions, shall prevail.In addition, the materials, methods and examples described herein are illustrative only and are not intended to be limiting.

[0055] All publications and patents mentioned in this specification are incorporated herein by reference in their entirety, as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.In case of conflict, this application, including any definitions herein, shall prevail.However, the mention of any references, articles, publications, patents, patent publications and patent applications cited in this specification is not intended to be, and should not be interpreted as, an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0056] In this description, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value in the recited range, and fractions thereof, where appropriate (e.g., tenths and hundredths of integers, etc.). The term "about", when immediately preceding a number or number, means that the number or number is within a range of plus or minus 10%. As used herein, the terms "a" and "an" should be understood to refer to "one or more" of the recited components, unless otherwise indicated. The use of alternatives (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. The term "and / or" should be understood to mean either one or both of the alternatives. As used herein, the terms "include" and "comprise" are used interchangeably.

[0057] As used herein, the terms "identity" and "identical" refer to the percentage of perfectly matching residues in an alignment of a "query" sequence with a "subject" sequence, such as an alignment generated by the BLAST algorithm, for a polypeptide or polynucleotide sequence. Identity is calculated over the entire length of the subject sequence, unless otherwise specified. Thus, a query sequence "shares at least x% identity" with a subject sequence if, when aligned with the subject sequence, at least x% (truncated) of the residues in the subject sequence are aligned perfectly with the corresponding residues in the query sequence. If the subject sequence has variable positions (e.g., residues designated by X), alignment to any residue in the query sequence is counted as a match. Sequence alignment may be performed using the NCBI Blast service (BLAST+ version 2.12.0).

[0058] As used herein, the term "operably linked" refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter sequence is operably linked to a coding sequence if it stimulates or regulates the transcription of the coding sequence in a suitable host cell or other expression system. Generally, promoter transcriptional regulatory sequences operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, do not need to be physically contiguous or located adjacent to the coding sequence whose transcription they enhance.

[0059] As used herein, "AAV vector" or "rAAV vector" refers to a recombinant vector that contains one or more polynucleotides of interest (or transgenes) flanked by AAV inverted terminal repeats (ITRs). Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell transfected with a plasmid that encodes and expresses the rep and cap gene products. Alternatively, AAV vectors can be packaged into infectious particles using host cells that have been stably engineered to express the rep and cap genes.

[0060] As used herein, "AAV virion" or "AAV virus particle" or "AAV vector particle" refers to a virus particle that is composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector.As used herein, when a particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene that is delivered to mammalian cells), it is usually referred to as "AAV vector particle" or simply "AAV vector".Therefore, the production of AAV vector particle necessarily includes the production of AAV vector, since the vector is contained within the AAV vector particle.

[0061] As used herein, "promoter" refers to a polynucleotide sequence capable of promoting the initiation of RNA transcription from a polynucleotide in a eukaryotic cell.

[0062] As used herein, "vector genome" refers to a polynucleotide sequence packaged by a vector (e.g., a rAAV virion), including flanking sequences (in AAV, inverted terminal repeats). In AAV, the terms "expression cassette" and "polynucleotide cassette" refer to the portion of the vector genome between the flanking ITR sequences. "Expression cassette" means that the vector genome contains at least one gene encoding a gene product operably linked to an element (e.g., a promoter) that drives expression, including any regulatory and / or enhancer elements. "Polynucleotide cassette" refers to the portion of the vector genome that contains at least one gene encoding a gene product operably linked to an element (e.g., a promoter) that drives expression, including any regulatory and / or enhancer elements.

[0063] As used herein, the term "patient in need" or "subject in need" refers to a patient or subject at risk of or suffering from a disease, disorder, or condition that is suitable for treatment or improvement using a recombinant gene therapy vector or gene editing system disclosed herein. A patient or subject in need may be, for example, a patient or subject diagnosed with a heart-related disorder. The subject may have a mutation in the JPH2 gene, or a deletion of all or part of the JPH2 gene, or a deletion of a gene regulatory sequence, causing abnormal expression and / or nuclear translocation of the JPH2 protein. "Subject" and "patient" are used interchangeably herein. The subject treated by the methods described herein may be an adult or a child. The age of the subject may vary.

[0064] As used herein, the term "variant" refers to a protein that has one or more amino acid substitutions, insertions, or deletions compared to a parent protein.As used herein, the term "functional variant" refers to a protein that has one or more amino acid substitutions, insertions, or deletions compared to a parent protein and retains one or more desired activities of the parent protein.

[0065] As used herein, "treat" refers to improve one or more symptoms of disease or disorder.The term "prevent" refers to delay or stop the onset of one or more symptoms of disease or disorder, or slow down the progression of JPH2-related disease or disorder, such as familial hypertrophic cardiomyopathy 17.

[0066] In certain embodiments, "administration" can be performed by injection, catheterization, and / or infusion. In some embodiments, the vector is administered by intravenous infusion, intravenous injection, intracardiac injection, intracardiac injection, intracoronary injection, intracoronary injection, and / or cardiac catheterization.

[0067] Adeno-associated virus (AAV) is a replication-deficient parvovirus whose single-stranded DNA genome is approximately 4.7 kb long and contains two approximately 145 nucleotide inverted terminal repeats (ITRs). There are several known variants of AAV, which are sometimes classified according to antigenic epitopes and are called serotypes. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077, the complete genome of AAV-2 is provided in GenBank Accession No. NC_001401 and in Srivastava et al., J. Virol., 45: 555-564 (1983), the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829, the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829, the AAV-5 genome is provided in GenBank Accession No. AF085716, the complete genome of AAV-6 is provided in GenBank Accession No. NC_00 1862, at least portions of the AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively, and the AAV-9 genome is provided in Gao et al., J. Virol., 78: The AAV-10 genome is provided in Mol. Ther., 13(1): 67-76 (2006), and the AAV-11 genome is provided in Virology, 330(2): 375-383 (2004). The sequence of the AAVrh.74 genome is provided in U.S. Patent No. 9,434,928, which is incorporated herein by reference. Cis-acting sequences directing viral DNA replication (rep), encapsidation / packaging and host cell chromosome integration are contained within the AAV ITR. Three AAV promoters (designated p5, p19 and p40 for their relative map positions) drive the expression of two AAV internal reading frames encoding the rep and cap genes.Two rep promoters (p5 and p19), coupled with differential splicing of a single AAV intron (at nucleotide positions 2107 and 2227), result in the production of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The Rep proteins possess multiple enzymatic properties that are ultimately responsible for the replication of the viral genome. The cap gene is expressed from the p40 promoter and encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites are responsible for the production of the three associated capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158: 97-129 (1992).

[0068] AAV possesses unique characteristics that make it attractive as a vector for delivering foreign DNA to cells, for example in gene therapy. AAV infection of cells in culture is noncytopathic, and natural infection of humans and other animals is silent and asymptomatic. Furthermore, AAV can infect many mammalian cells, allowing the possibility of targeting many different tissues in vivo. Furthermore, AAV can transduce slowly dividing and nondividing cells and persist essentially for the life of these cells as transcriptionally active nuclear episomes (extrachromosomal elements). The AAV proviral genome is inserted as cloned DNA within a plasmid, making the construction of recombinant genomes feasible. Furthermore, signals directing AAV replication and genome encapsidation are contained within the ITRs of the AAV genome, so that part or all of the internal ∼4.3 kb of the genome (encoding the replication and structural capsid protein, rep-cap) may be replaced with foreign DNA. To generate AAV vectors, the rep and cap proteins may be provided in trans. Another important feature of AAV is that it is a highly stable and potent virus. It easily survives the conditions used to inactivate adenovirus (56°C to 65°C for several hours), mitigating the importance of cryopreservation of AAV. AAV can also be lyophilized. Finally, AAV-infected cells are not resistant to superinfection.

[0069] Gene delivery viral vectors useful in the practice of the present disclosure can be constructed using methodologies well known in the art of molecular biology. Typically, a viral vector carrying a transgene is constructed from a polynucleotide encoding the transgene, appropriate regulatory elements, and elements required for the production of viral proteins, which mediate cell transduction. Such recombinant viruses can be produced by techniques well known in the art, for example, by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of viral packaging cells include, but are not limited to, HeLa cells, SF9 cells (optionally with baculovirus helper vectors), HEK293 cells, and the like. AAV vectors can be produced using a herpes virus-based system, as described in US2017 / 0218395A1. Detailed protocols for producing such replication-defective recombinant viruses can be found, for example, in WO95 / 14785, WO96 / 22378, U.S. Pat. No. 5,882,877, U.S. Pat. No. 6,013,516, U.S. Pat. No. 4,861,719, U.S. Pat. No. 5,278,056 and WO94 / 19478, the entire contents of each of which are incorporated herein by reference.

[0070] The present disclosure contemplates compositions and methods of use related to junctophilin-2 (JPH2) proteins or polypeptides. Stress-induced truncation of JPH2 is known to be associated with cardiomyopathy and heart failure, including diseases such as those described in Beavers et al. Cardiovascular Research 103:198-205 (2014) and other sources. Details regarding truncated variants of JPH2 protein can be found, for example, in U.S. Patent Application No. 2019 / 0307899, the entire contents of each of which are incorporated herein by reference. Therefore, viral vector-mediated delivery of JPH2 gene can serve as a viable treatment for JPH2-related human diseases, such as cardiomyopathy and heart failure.

[0071] Mutations in the JPH2 gene have been identified in people with familial hypertrophic cardiomyopathy 17 (CMH17). (See "CMH17", NCBI MedGen). The condition is an inherited heart disease characterized by ventricular hypertrophy, usually asymmetric and often involving the interventricular septum. Symptoms include dyspnea, syncope, collapse, palpitations and chest pain, which can be easily provoked by exercise. The disorder has inter- and intrafamilial variability, ranging from a benign form to a malignant form with a high risk of heart failure and sudden cardiac death.

[0072] In some embodiments, JPH2 comprises one or more amino acid substitutions selected from the following: mutation of one or more residues in the predicted calpain 1 cleavage site numbered relative to SEQ ID NO: 1 (V155A, R156K, L204A, L205A, R572K or T573S). That is, the JPH2 protein may comprise one or more, two or more, three or more, or four or more amino acid substitutions selected from the group consisting of R572A or R572K, T573A or T573S, V155A, R156A or R156K, L204A and L205A. Alternative conservative or non-conservative mutations or substitutions at any of these sites may be used, including, but not limited to, one or more, two or more, three or more, or four or more amino acid substitutions selected from the group consisting of R572X, T573X, V155X, R156X, L204X and L205X, where X represents any natural or unnatural amino acid other than an amino acid present in the reference JPH2 protein.

[0073] The term "conservative substitution" as used herein refers to the replacement of one or more amino acids with another biologically similar residue. Examples include the replacement of amino acid residues with similar properties, such as small amino acids, acidic amino acids, polar amino acids, basic amino acids, hydrophobic amino acids and aromatic amino acids. In the following diagram, conservative substitutions of amino acids are grouped according to physicochemical properties: I: neutral, hydrophilic; II: acid and amide; III: basic; IV: hydrophobic; V: aromatic, bulky amino acids. TIFF2024546103000002.tif48128

[0074] In the diagram below, conservative substitutions of amino acids are grouped according to their physicochemical properties: VI: neutral or hydrophobic, VII: acidic, VIII: basic, IX: polar, and X: aromatic. TIFF2024546103000003.tif57128

[0075] Specific mutations contemplated by the present disclosure include R572A or R572K; T573A or T573S; V155A; R156A or R156K; L204A; or L205A.In some embodiments, amino acid substitution disrupts intramolecular or intermolecular interface.In some embodiments, amino acid substitution disrupts intramolecular or intermolecular interface while maintaining one or more characteristics of residue, such as charge, size and / or hydrophobicity.

[0076] Activated JPH2 may contain one or more amino acid substitutions, insertions or deletions (collectively, mutations) that protect JPH2 from calpain 1-mediated cleavage, thereby reducing JPH2 cleavage by calpain. For example, JPH2 may contain a mutation at one calpain 1 site that reduces binding and subsequent cleavage by calpain 1, or a mutation at three calpain 1 sites that reduces binding and subsequent cleavage by calpain 1.

[0077] Various further aspects of JPH2 are shown in Table 1.

[0078] Table 1: Exemplary combinations of amino acid substitutions TIFF2024546103000004.tif10166

[0079] In some embodiments, the JPH2 protein comprises one or more amino acid substitutions at positions Arg-572 and Thr-573 relative to a reference JPH2 protein.

[0080] In some embodiments, the JPH2 protein comprises one or more amino acid substitutions at positions Val-155, Arg-156, Leu204, Leu205, Arg-572, and Thr-573 relative to a reference JPH2 protein.

[0081] In some embodiments, the JPH2 protein comprises one or more amino acid substitutions selected from R572A, R572K, T573A, T573S, V155A, R156A, R156K, L204A and / or L205A compared to a reference JPH2 protein.

[0082] In some embodiments, the JPH2 protein comprises R572A and T573A amino acid substitutions compared to a reference JPH2 protein.

[0083] In some embodiments, the JPH2 protein comprises the following amino acid substitutions compared to a reference JPH2 protein: R572K and T573S.

[0084] In some embodiments, the JPH2 protein comprises the following amino acid substitutions compared to a reference JPH2 protein: V155A, R156A, L204A, L205A, R572A and T573A.

[0085] In some embodiments, the JPH2 protein comprises the following amino acid substitutions compared to a reference JPH2 protein: V155A, R156K, L204A, L205A, R572K and T573S.

[0086] The native and polynucleotide coding sequences for human JPH2 isoform 1 and isoform 2 proteins are shown below: JPH2-wild type, isoform 1 (SEQ ID NO: 1) - 696 amino acids TIFF2024546103000005.tif78128 JPH2-wild type, transcript 1 (SEQ ID NO: 2) - 2091 nucleotide bases TIFF2024546103000006.tif139159 JPH2-wild type, isoform 2 (SEQ ID NO: 108) - 696 amino acids TIFF2024546103000007.tif16128 JPH2-wild type, transcript 2 (SEQ ID NO: 107) - 390 nucleotide bases TIFF2024546103000008.tif27159

[0087] In some embodiments, the JPH2 protein comprises a polypeptide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1. In some embodiments, the JPH2 polynucleotide comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 2. In some embodiments, the JPH2 protein is a wild-type or native JPH2 protein, e.g., human JPH2.

[0088] In some embodiments, the JPH2 protein comprises a polypeptide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 108. In some embodiments, the JPH2 polynucleotide comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 107. In some embodiments, the JPH2 protein is a wild-type or native JPH2 protein, e.g., human JPH2.

[0089] The present disclosure contemplates compositions and methods of use related to JPH2 proteins or polynucleotides having calpain 1 binding site mutations. The 1mutAA mutant of JPH2 comprises a polypeptide sequence that includes the amino acid substitutions R572A and T573A (SEQ ID NO: 3). The 1mutAA mutant of JPH2 comprises a polynucleotide that encodes the amino acid substitutions R572A and T573A (SEQ ID NO: 4).

[0090] JPH2-1mutAA (SEQ ID NO: 3) - 696 amino acids TIFF2024546103000009.tif78128 JPH2-1mutAA (SEQ ID NO: 4) - 2091 nucleotide bases TIFF2024546103000010.tif140159

[0091] In some embodiments, the JPH2 protein comprises a polypeptide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 3. In some embodiments, the JPH2 protein comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 4. In some embodiments, the JPH2 protein is a mutant JPH2 protein.

[0092] The present disclosure contemplates compositions and methods of use related to JPH2 proteins or polynucleotides having calpain 1 binding site mutations. The 1mutKS mutant of JPH2 comprises a polypeptide sequence that includes the amino acid substitutions R572K and T573S (SEQ ID NO: 5). The 1mutKS mutant of JPH2 comprises a polynucleotide that encodes the amino acid substitutions R572K and T573S (SEQ ID NO: 6). JPH2-1mutKS (SEQ ID NO: 5) - 696 amino acids TIFF2024546103000011.tif78128 JPH2-1mutKS (SEQ ID NO: 6) - 2091 nucleotide bases TIFF2024546103000012.tif139159

[0093] In some embodiments, the JPH2 protein comprises a polypeptide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 5. In some embodiments, the JPH2 protein comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 6.

[0094] The present disclosure contemplates compositions and methods of use related to JPH2 proteins or polynucleotides having calpain 1 binding site mutations. The 3mutAA mutant of JPH2 comprises a polypeptide sequence comprising the amino acid substitutions V155A, R156A, L204A, L205A, R572A and T573A (SEQ ID NO: 7). The 3mutAA mutant of JPH2 comprises a polynucleotide encoding the amino acid substitutions V155A, R156A, L204A, L205A, R572A and T573A (SEQ ID NO: 8). JPH2-3mutAA (SEQ ID NO: 7) - 696 amino acids TIFF2024546103000013.tif78128 JPH2-3mutAA (SEQ ID NO: 8) - 2091 nucleotide bases TIFF2024546103000014.tif139159

[0095] In some embodiments, the JPH2 protein comprises a polypeptide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 7. In some embodiments, the JPH2 protein comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 8.

[0096] The present disclosure contemplates compositions and methods of use related to JPH2 proteins or polynucleotides having calpain 1 binding site mutations. The 3mutAKAAKS mutant of JPH2 comprises a polypeptide sequence comprising amino acid substitutions V155A, R156K, L204A, L205A, R572K and T573S (SEQ ID NO: 9). The 3mutAKAAKS mutant of JPH2 comprises a polynucleotide encoding amino acid substitutions V155A, R156K, L204A, L205A, R572K and T573S (SEQ ID NO: 10). JPH2-3mutAKAAKS (SEQ ID NO: 9) - 696 amino acids TIFF2024546103000015.tif78128 JPH2-3mutAKAAKS (SEQ ID NO: 10) - 2091 nucleotide bases TIFF2024546103000016.tif139159

[0097] In some embodiments, the JPH2 protein comprises a polypeptide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 9. In some embodiments, the JPH2 protein comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 10.

[0098] In some embodiments, the disclosure provides a recombinant adeno-associated virus (rAAV) virion comprising a capsid and a vector genome, the vector genome comprising a polynucleotide sequence encoding JPH2 or a functional variant thereof operably linked to a promoter. In some embodiments, the disclosure provides a recombinant adeno-associated virus (rAAV) virion comprising a capsid and a vector genome, the vector genome comprising a polynucleotide sequence encoding JPH2 operably linked to a promoter. In some embodiments, the JPH2 protein comprises a polypeptide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1. A polynucleotide encoding JPH2 can comprise a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:2.

[0099] Optionally, the polynucleotide sequence encoding the vector genome may comprise a Kozak sequence, including but not limited to GCCACCATGG (SEQ ID NO: 11). The Kozak sequence may overlap with the polynucleotide sequence encoding the JPH2 protein or a functional variant thereof. For example, the vector genome may comprise a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 12 (the first 10 nucleotides constitute the Kozak sequence). SEQ ID NO: 12 TIFF2024546103000017.tif95150TIFF2024546103000018.tif182150

[0100] In some embodiments, the Kozak sequence is as follows: TIFF2024546103000019.tif63128 or any one of these alternative Kozak sequences.

[0101] In some embodiments, the vector genome does not comprise a Kozak sequence.

[0102] The AAV virion of the present disclosure comprises a vector genome. The vector genome may comprise an expression cassette (or a polynucleotide cassette for gene editing applications that do not require expression of a polynucleotide sequence). Any suitable inverted terminal repeat (ITR) may be used. The ITR may be an AAV ITR from the same serotype as the capsid present in the AAV virion, or may be an AAV ITR from a different serotype than the capsid (e.g., an AAV2 ITR may be used in an AAV virion having an AAV9 capsid or an AAVrh74 capsid). In either case, the serotype of the capsid determines the name applied to the virion. The ITR is generally the 5'-most and 3'-most element of the vector genome. The vector genome also generally comprises, in the order of 5' to 3' direction, a promoter, a transgene, a 3' untranslated region (UTR) sequence (e.g., a WPRE element), and a polyadenylation sequence. In a variation, the vector genome comprises an enhancer element (generally 5' of the promoter) and / or an exon (generally 3' of the promoter). In a variation, the vector genome comprises a green fluorescent protein (GFP) protein, generally 3' of the transgene. In a variation, the vector genome of the present disclosure encodes a partial or complete transgene sequence that is used as a repair template in a gene editing system. In such a variation, the vector genome may comprise an exogenous promoter, or the gene editing system may insert the transgene into a locus in the genome that has an endogenous promoter, such as a cardiac-specific promoter or a muscle cell-specific promoter.

[0103] In some embodiments, the 5' ITR comprises an AAV2 ITR. In some embodiments, the 5' ITR comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:15.

[0104] In some embodiments, the 5' ITR comprises an AAV2 ITR. In some embodiments, the 5' ITR comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:16.

[0105] In some embodiments, the 5' ITR comprises an AAV2 ITR. In some embodiments, the 5' ITR comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 17).

[0106] In some embodiments, the 5' ITR comprises an AAV2 ITR. In some embodiments, the 5' ITR comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:18.

[0107] In some embodiments, the 3' ITR comprises an AAV2 ITR. In some embodiments, the 5' ITR comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:19.

[0108] In some embodiments, the 3' ITR comprises an AAV2 ITR. In some embodiments, the 5' ITR comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:20.

[0109] In some embodiments, the 3' ITR comprises an AAV2 ITR. In some embodiments, the 5' ITR comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:21.

[0110] In some embodiments, the vector genome comprises one or more filler sequences, e.g., one or more filler sequences that are at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 22; SEQ ID NO: 23; or SEQ ID NO: 24.

[0111] In some embodiments, the polynucleotide sequence encoding the JPH2 protein or its functional variant is operably linked to a promoter. In certain embodiments, the promoter is the MHCK7 promoter. In certain embodiments, the promoter is the TNNT2 promoter.

[0112] The present disclosure contemplates the use of various promoters. Promoters useful in embodiments of the present disclosure include, but are not limited to, cytomegalovirus (CMV) promoter, phosphoglycerate kinase (PGK) promoter, or CMV enhancer and a promoter sequence consisting of a portion of chicken β-actin promoter and rabbit β-globin gene (CAG). In some cases, the promoter may be a synthetic promoter. An exemplary synthetic promoter is provided by Schlabach et al. PNAS USA. 107(6):2538-43 (2010). In some embodiments, the promoter comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 25.

[0113] In some embodiments, the polynucleotide sequence encoding the JPH2 protein or its functional variant is operably linked to an inducible promoter. The inducible promoter may be configured to transcriptionally express or not transcriptionally express the polynucleotide sequence in response to the addition or accumulation of an agent, or in response to the removal, degradation or dilution of the agent. The agent may be a drug. The agent may be tetracycline or one of its derivatives, such as, but not limited to, doxycycline. In some cases, the inducible promoter is a tet-on promoter, a tet-off promoter, a chemically regulated promoter, a physically regulated promoter (i.e., a promoter that responds to the presence or absence of light or to low or high temperatures). Inducible promoters include heavy metal ion inducible promoters (such as mouse mammary tumor virus (mMTV) promoter or various growth hormone promoters), and promoters from T7 phage that are active in the presence of T7 RNA polymerase. This list of inducible promoters is non-limiting.

[0114] In some cases, the promoter is a tissue-specific promoter, for example, a promoter that can drive expression in cardiac cells to a greater extent than in non-cardiac cells. In some embodiments, the tissue-specific promoter is selected from any of a variety of cardiac cell-specific promoters, including, but not limited to, desmin (Des), α-myosin heavy chain (α-MHC), myosin light chain 2 (MLC-2), cardiac troponin C (cTnC), cardiac troponin T (hTNNT2), muscle creatine kinase (CK), and their promoter / enhancer region combinations, such as MHCK7. In some cases, the promoter is a ubiquitous promoter. "Ubiquitous promoter" refers to a promoter that is not tissue-specific under experimental or clinical conditions. In some cases, the ubiquitous promoter is a promoter that is not tissue-specific under experimental or clinical conditions, such as cytomegalovirus (CMV), cytomegalovirus early enhancer element with splice acceptor of rabbit β-globin gene, chicken β-actin gene intron ( C ytomegalovirus early enhancer element chicken beta- A ctin gene intron with the splice acceptor of the rabbit beta- G lobin gene (CAG), ubiquitin C (UBC), phosphoglycerate kinase (PGK), eukaryotic translation elongation factor 1α1 (EF1-α), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), simian virus 40 (SV40), hepatitis B virus (HBV), chicken β-actin, and human β-actin promoter.

[0115] In some embodiments, the promoter sequence is selected from Table 3. In some embodiments, the promoter comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 31-51. In some embodiments, the promoter comprises a fragment of the polynucleotide sequence of any one of SEQ ID NOs: 31-51, such as a fragment comprising at least 25%, at least 50%, at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of any one of SEQ ID NOs: 31-51.

[0116] (Table 3) TIFF2024546103000020.tif161166TIFF2024546103000021.tif228166TIFF2024546103000022.tif228166TIFF20245461030 00023.tif228166TIFF2024546103000024.tif228166TIFF2024546103000025.tif219166TIFF2024546103000026.tif238166

[0117] In certain embodiments, the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 31. In certain embodiments, the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 32. In certain embodiments, the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 33.

[0118] Further illustrative examples of promoters are the SV40 late promoter from Simian Virus 40, the baculovirus polyhedron enhancer / promoter element, the herpes simplex virus thymidine kinase (HSV tk), the immediate early promoter from cytomegalovirus (CMV) and various retroviral promoters including the LTR element. A wide variety of other promoters are known in the art and are publicly available, and the sequences of many such promoters are available in sequence databases such as the GenBank database.

[0119] In some cases, the vectors of the present disclosure further comprise one or more regulatory elements selected from the group consisting of an enhancer, an intron, a polyA signal, a 2A peptide coding sequence, a WPRE (woodchuck hepatitis virus post-transcriptional regulatory element), and a HPRE (hepatitis B virus post-transcriptional regulatory element).

[0120] In some embodiments, the vector comprises a CMV enhancer.

[0121] In certain embodiments, the vector comprises one or more enhancers. In certain embodiments, the enhancer is a CMV enhancer sequence, a GAPDH enhancer sequence, a β-actin enhancer sequence or an EF1-α enhancer sequence. The above sequences are known in the art. For example, the sequence of the CMV immediate early (IE) enhancer is SEQ ID NO: 50.

[0122] In certain embodiments, the vector comprises one or more introns. In particular embodiments, the intron is a rabbit globin intron sequence, a chicken β-actin intron sequence, a synthetic intron sequence, an SV40 intron, or an EF1-α intron sequence.

[0123] In certain embodiments, the vector comprises a polyA sequence. In particular embodiments, the polyA sequence is a rabbit globin polyA sequence, a human growth hormone polyA sequence, a bovine growth hormone polyA sequence, a PGK polyA sequence, an SV40 polyA sequence, or a TK polyA sequence. In some embodiments, the polyA signal can be a bovine growth hormone polyadenylation signal (bGHpA).

[0124] In certain embodiments, the vector comprises one or more transcript stabilizing elements. In particular embodiments, the transcript stabilizing element is a WPRE sequence, a HPRE sequence, a scaffold attachment region, a 3' UTR or a 5' UTR. In particular embodiments, the vector comprises both a 5' UTR and a 3' UTR.

[0125] In some embodiments, the vector comprises a 5' untranslated region (UTR) selected from Table 4. In some embodiments, the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 51-61.

[0126] (Table 4) TIFF2024546103000027.tif85166TIFF2024546103000028.tif229166TIFF2024546103000029.tif228166TIFF2024546103000030.tif170166

[0127] In some embodiments, the vector comprises a 3' untranslated region selected from Table 5. In some embodiments, the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 62-70.

[0128] (Table 5) TIFF2024546103000031.tif219166TIFF2024546103000032.tif229166TIFF2024546103000033.tif166166

[0129] In some embodiments, the vector comprises a polyadenylation (polyA) signal selected from Table 6. In some embodiments, the polyA signal comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs 71-75.

[0130] (Table 6) TIFF2024546103000034.tif201166

[0131] Exemplary vector genomes are depicted in Figures 1-25 and provided as SEQ ID NOs: 26-30 and 76-95. In some embodiments, the vector genome comprises, consists essentially of, or comprises a polynucleotide sequence that shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with any one of SEQ ID NOs: 26-30 and 76-95, optionally with or without ITR sequences, or comprises a polynucleotide sequence that shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with any one of SEQ ID NOs: 26-30 and 76-95, optionally with or without ITR sequences. 26-30 and 76-95. The present disclosure also contemplates the expression cassettes of the exemplary vector genomes depicted in Figures 1-25 and sequences comprising same, such as sequences set forth in SEQ ID NOs: 26-30 and 76-95, but lacking the 5' and 3' ITRs, and variants thereof sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with any of the foregoing.

[0132] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; an MHCK7 promoter; a JPH2 transgene; a WPRE(x) element; a human GH poly(A) signal (hGH) sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, a polynucleotide sequence SEQ ID NO: 26; or a polynucleotide sequence sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with any of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 or AAVrh74 vector. The JPH2 transgene of this embodiment is a full-length wild-type transgene, i.e., a transgene encoding at least 600 or at least 630 amino acids of JPH2. SEQ ID NO: 26 TIFF2024546103000035.tif103159TIFF2024546103000036.tif171159

[0133] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; an hTnnT2 promoter; a JPH2 transgene; a WPRE(x) element; an hGH sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, any one of the polynucleotide sequences SEQ ID NO: 27; or a polynucleotide sequence sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 or AAVrh74 vector. The JPH2 transgene of this embodiment is a full-length wild-type transgene, i.e., a transgene encoding at least 600 or at least 630 amino acids of JPH2. SEQ ID NO: 27 TIFF2024546103000037.tif203159TIFF2024546103000038.tif47159

[0134] In certain embodiments, the vector genome comprises, in 5' to 3' order, 5' ITR; hTnnT2 promoter; JPH2 transgene; GFP tag; WPRE(x) element; hGH sequence; and 3' ITR. The vector genome may comprise, in 5' to 3' order, any one of the polynucleotide sequences SEQ ID NO: 28; or a polynucleotide sequence sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 or AAVrh74 vector. The JPH2 transgene of this embodiment is a full-length wild-type transgene, i.e., a transgene encoding at least 600 or at least 630 amino acids of JPH2. SEQ ID NO: 28 TIFF2024546103000039.tif103159TIFF2024546103000040.tif215159

[0135] In certain embodiments, the vector genome comprises, in 5' to 3' order, 5' ITR; hTnnT2 promoter; JPH2 transgene; GFP tag; WPRE(x) element; hGH sequence; and 3' ITR. The vector genome may comprise, in 5' to 3' order, any one of the polynucleotide sequences SEQ ID NO: 29; or a polynucleotide sequence sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 or AAVrh74 vector. The JPH2 transgene of this embodiment is a full-length wild-type transgene, i.e., a transgene encoding at least 600 or at least 630 amino acids of JPH2. SEQ ID NO: 29 TIFF2024546103000041.tif159159TIFF2024546103000042.tif143159

[0136] In certain embodiments, the vector genome comprises, in 5' to 3' order, 5' ITR; CMV enhancer element; CMV promoter; JPH2 transgene; GFP tag; WPRE(x) element; hGH sequence; and 3' ITR. The vector genome may comprise, in 5' to 3' order, any one of the polynucleotide sequences SEQ ID NO: 30; or a polynucleotide sequence sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 or AAVrh74 vector. The JPH2 transgene of this embodiment is a full-length wild-type transgene, i.e., a transgene encoding at least 600 or at least 630 amino acids of JPH2. SEQ ID NO: 30 TIFF2024546103000043.tif7159TIFF2024546103000044.tif227159TIFF2024546103000045.tif71159

[0137] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; an MHCK7 promoter; a JPH2 1mutAA (R572A and T573A) transgene; a WPRE(x) element; an hGH sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, the polynucleotide sequence SEQ ID NO: 76; or a polynucleotide sequence sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 or AAVrh74 vector. The JPH2 transgene of this embodiment is a full-length 1mutAA (R572A and T573A) transgene, i.e., a transgene encoding at least 600 or at least 630 amino acids of JPH2. SEQ ID NO: 76 TIFF2024546103000046.tif79159TIFF2024546103000047.tif195159

[0138] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; an hTnnT2 promoter; a JPH2 1mutAA (R572A and T573A) transgene; a WPRE(x) element; an hGH sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, any one of the polynucleotide sequences SEQ ID NO: 77; or a polynucleotide sequence sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 or AAVrh74 vector. The JPH2 transgene of this embodiment is a full-length JPH2 1mutAA (R572A and T573A) transgene, i.e., a transgene encoding at least 600 or at least 630 amino acids of JPH2. SEQ ID NO: 77 TIFF2024546103000048.tif179159TIFF2024546103000049.tif71159

[0139] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; an MHCK7 promoter; a JPH2 1mutAA (R572A and T573A) transgene; a GFP tag; a WPRE(x) element; an hGH sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, the polynucleotide sequence SEQ ID NO: 78; or a polynucleotide sequence sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 or AAVrh74 vector. The JPH2 transgene of this embodiment is a full-length 1mutAA (R572A and T573A) transgene, i.e., a transgene encoding at least 600 or at least 630 amino acids of JPH2. SEQ ID NO: 78 TIFF2024546103000050.tif79159TIFF2024546103000051.tif227159TIFF2024546103000052.tif11159

[0140] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; an hTnnT2 promoter; a JPH2 1mutAA (R572A and T573A) transgene; a GFP tag; a WPRE(x) element; an hGH sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, any one of the polynucleotide sequences SEQ ID NO: 79; or a polynucleotide sequence sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 or AAVrh74 vector. The JPH2 transgene of this embodiment is a full-length JPH2 1mutAA (R572A and T573A) transgene, i.e., a transgene encoding at least 600 or at least 630 amino acids of JPH2. SEQ ID NO: 79 TIFF2024546103000053.tif139159TIFF2024546103000054.tif163159

[0141] In certain embodiments, the vector genome comprises, in 5' to 3' order, the 5' ITR; a CMV enhancer element; a CMV promoter; a JPH2 1mutAA (R572A and T573A) transgene; a GFP tag; a WPRE(x) element; an hGH sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, any one of the polynucleotide sequences SEQ ID NO: 80; or a polynucleotide sequence sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 or AAVrh74 vector. The JPH2 transgene of this embodiment is a full-length 1mutAA (R572A and T573A) transgene, i.e., a transgene encoding at least 600 or at least 630 amino acids of JPH2. SEQ ID NO: 80 TIFF2024546103000055.tif203159TIFF2024546103000056.tif103159

[0142] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; an MHCK7 promoter; a JPH2 1mutKS (R572K and T573S) transgene; a WPRE(x) element; an hGH sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, the polynucleotide sequence SEQ ID NO: 81; or a polynucleotide sequence sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 or AAVrh74 vector. The JPH2 transgene of this embodiment is a full-length 1mutKS (R572K and T573S) transgene, i.e., a transgene encoding at least 600 or at least 630 amino acids of JPH2. SEQ ID NO: 81 TIFF2024546103000057.tif47159TIFF2024546103000058.tif227159

[0143] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; an hTnnT2 promoter; a JPH2 1mutKS (R572K and T573S) transgene; a WPRE(x) element; an hGH sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, any one of the polynucleotide sequences SEQ ID NO: 82; or a polynucleotide sequence sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 or AAVrh74 vector. The JPH2 transgene of this embodiment is a full-length JPH2 1mutKS (R572K and T573S) transgene, i.e., a transgene encoding at least 600 or at least 630 amino acids of JPH2. SEQ ID NO: 82 TIFF2024546103000059.tif151159TIFF2024546103000060.tif99159

[0144] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; an MHCK7 promoter; a JPH2 1mutKS (R572K and T573S) transgene; a GFP tag; a WPRE(x) element; an hGH sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, the polynucleotide sequence SEQ ID NO: 83; or a polynucleotide sequence sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 or AAVrh74 vector. The JPH2 transgene of this embodiment is a full-length 1mutKS (R572K and T573S) transgene, i.e., a transgene encoding at least 600 or at least 630 amino acids of JPH2. SEQ ID NO: 83 TIFF2024546103000061.tif51159TIFF2024546103000062.tif227159TIFF2024546103000063.tif39159

[0145] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; an hTnnT2 promoter; a JPH2 1mutKS (R572K and T573S) transgene; a GFP tag; a WPRE(x) element; an hGH sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, any one of the polynucleotide sequences SEQ ID NO: 84; or a polynucleotide sequence sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 or AAVrh74 vector. The JPH2 transgene of this embodiment is a full-length JPH2 1mutKS (R572K and T573S) transgene, i.e., a transgene encoding at least 600 or at least 630 amino acids of JPH2. SEQ ID NO: 84 TIFF2024546103000064.tif111159TIFF2024546103000065.tif191159

[0146] In certain embodiments, the vector genome comprises, in 5' to 3' order, the 5' ITR; a CMV enhancer element; a CMV promoter; a JPH2 1mutKS (R572K and T573S) transgene; a GFP tag; a WPRE(x) element; an hGH sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, any one of the polynucleotide sequences SEQ ID NO: 85; or a polynucleotide sequence sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 or AAVrh74 vector. The JPH2 transgene of this embodiment is a full-length 1mutKS (R572K and T573S) transgene, i.e., a transgene encoding at least 600 or at least 630 amino acids of JPH2. SEQ ID NO: 85 TIFF2024546103000066.tif179159TIFF2024546103000067.tif127159

[0147] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; an MHCK7 promoter; a JPH2 3mutAA (V155A, R156A, L204A, L205A, R572A and T573A) transgene; a WPRE(x) element; an hGH sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, the polynucleotide sequence SEQ ID NO: 86; or a polynucleotide sequence sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 or AAVrh74 vector. The JPH2 transgene in this embodiment is a full-length 3mutAA (V155A, R156A, L204A, L205A, R572A and T573A) transgene, i.e. a transgene encoding at least 600 or at least 630 amino acids of JPH2. SEQ ID NO: 86 TIFF2024546103000068.tif19159TIFF2024546103000069.tif227159TIFF2024546103000070.tif27159

[0148] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; an hTnnT2 promoter; a JPH2 3mutAA (V155A, R156A, L204A, L205A, R572A and T573A) transgene; a WPRE(x) element; an hGH sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, any one of the polynucleotide sequences SEQ ID NO: 87; or a polynucleotide sequence sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 or AAVrh74 vector. The JPH2 transgene in this embodiment is a full-length JPH2 3mutAA (V155A, R156A, L204A, L205A, R572A and T573A) transgene, i.e., a transgene encoding at least 600 or at least 630 amino acids of JPH2. SEQ ID NO: 87 TIFF2024546103000071.tif115159TIFF2024546103000072.tif135159

[0149] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; an MHCK7 promoter; a JPH2 3mutAA (V155A, R156A, L204A, L205A, R572A and T573A) transgene; a GFP tag; a WPRE(x) element; an hGH sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, the polynucleotide sequence SEQ ID NO: 88; or a polynucleotide sequence sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 or AAVrh74 vector. The JPH2 transgene in this embodiment is a full-length 3mutAA (V155A, R156A, L204A, L205A, R572A and T573A) transgene, i.e. a transgene encoding at least 600 or at least 630 amino acids of JPH2. SEQ ID NO: 88 TIFF2024546103000073.tif7159TIFF2024546103000074.tif227159TIFF2024546103000075.tif83159

[0150] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; an hTnnT2 promoter; a JPH2 3mutAA (V155A, R156A, L204A, L205A, R572A and T573A) transgene; a GFP tag; a WPRE(x) element; an hGH sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, any one of the polynucleotide sequences SEQ ID NO: 89; or a polynucleotide sequence sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 or AAVrh74 vector. The JPH2 transgene in this embodiment is a full-length JPH2 3mutAA (V155A, R156A, L204A, L205A, R572A and T573A) transgene, i.e., a transgene encoding at least 600 or at least 630 amino acids of JPH2. SEQ ID NO: 89 TIFF2024546103000076.tif59159TIFF2024546103000077.tif227159TIFF2024546103000078.tif15159

[0151] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; a CMV enhancer element; a CMV promoter; a JPH2 3mutAA (V155A, R156A, L204A, L205A, R572A and T573A) transgene; a GFP tag; a WPRE(x) element; an hGH sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, any one of the polynucleotide sequences SEQ ID NO: 90; or a polynucleotide sequence sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 or AAVrh74 vector. The JPH2 transgene in this embodiment is a full-length JPH2 3mutAA (V155A, R156A, L204A, L205A, R572A and T573A) transgene, i.e., a transgene encoding at least 600 or at least 630 amino acids of JPH2. SEQ ID NO: 90 TIFF2024546103000079.tif127159TIFF2024546103000080.tif179159

[0152] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; an MHCK7 promoter; a JPH2 3mutAKAAKS (V155A, R156K, L204A, L205A, R572K and T573S) transgene; a WPRE(x) element; an hGH sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, any one of the polynucleotide sequences SEQ ID NO: 91; or a polynucleotide sequence sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 or AAVrh74 vector. The JPH2 transgene in this embodiment is a full-length JPH2 3mutAKAAKS (V155A, R156K, L204A, L205A, R572K and T573S) transgene, i.e., a transgene encoding at least 600 or at least 630 amino acids of JPH2. SEQ ID NO: 91 TIFF2024546103000081.tif195159TIFF2024546103000082.tif79159

[0153] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; an hTnnT2 promoter; a JPH2 3mutAKAAKS (V155A, R156K, L204A, L205A, R572K and T573S) transgene; a WPRE(x) element; an hGH sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, any one of the polynucleotide sequences SEQ ID NO: 92; or a polynucleotide sequence sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 or AAVrh74 vector. The JPH2 transgene in this embodiment is a full-length JPH2 3mutAKAAKS (V155A, R156K, L204A, L205A, R572K and T573S) transgene, i.e., a transgene encoding at least 600 or at least 630 amino acids of JPH2. SEQ ID NO: 92 TIFF2024546103000083.tif63159TIFF2024546103000084.tif187159

[0154] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; an MHCK7 promoter; a JPH2 3mutAKAAKS (V155A, R156K, L204A, L205A, R572K and T573S) transgene; a GFP tag; a WPRE(x) element; an hGH sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, any one of the polynucleotide sequences SEQ ID NO: 93; or a polynucleotide sequence sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 or AAVrh74 vector. The JPH2 transgene in this embodiment is a full-length JPH2 3mutAKAAKS (V155A, R156K, L204A, L205A, R572K and T573S) transgene, i.e., a transgene encoding at least 600 or at least 630 amino acids of JPH2. SEQ ID NO: 93 TIFF2024546103000085.tif179159TIFF2024546103000086.tif139159

[0155] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; an hTnnT2 promoter; a JPH2 3mutAKAAKS (V155A, R156K, L204A, L205A, R572K and T573S) transgene; a GFP tag; a WPRE(x) element; an hGH sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, any one of the polynucleotide sequences SEQ ID NO: 94; or a polynucleotide sequence sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 or AAVrh74 vector. The JPH2 transgene in this embodiment is a full-length JPH2 3mutAKAAKS (V155A, R156K, L204A, L205A, R572K and T573S) transgene, i.e., a transgene encoding at least 600 or at least 630 amino acids of JPH2. SEQ ID NO: 94 TIFF2024546103000087.tif3159TIFF2024546103000088.tif227159TIFF2024546103000089.tif71159

[0156] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; a CMV enhancer element; a CMV promoter; a JPH2 3mutAKAAKS (V155A, R156K, L204A, L205A, R572K and T573S) transgene; a GFP tag; a WPRE(x) element; an hGH sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, any one of the polynucleotide sequences SEQ ID NO: 95; or a polynucleotide sequence sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 or AAVrh74 vector. The JPH2 transgene in this embodiment is a full-length 3mutAKAAKS (V155A, R156K, L204A, L205A, R572K and T573S) transgene, i.e., a transgene encoding at least 600 or at least 630 amino acids of JPH2. SEQ ID NO: 95 TIFF2024546103000090.tif71159TIFF2024546103000091.tif227159TIFF2024546103000092.tif7159

[0157] In either case, the optional WPRE element may or may not be present.

[0158] Adeno-associated viral vectors and uses thereof AAV vectors useful in practicing the present disclosure can be packaged into AAV virions (virus particles) using a variety of systems, including adenovirus-based and helper-free systems. Standard methods in AAV biology include those described in Kwon and Schaffer. Pharm Res. (2008) 25(3):489-99; Wu et al. Mol. Ther. (2006) 14(3):316-27. Burger et al. Mol. Ther. (2004) 10(2):302-17; Grimm et al. Curr Gene Ther. (2003) 3(4):281-304; Deyle DR, Russell DW. Curr Opin Mol Ther. (2009) 11(4):442-447; McCarty et al. Gene Ther. (2001) 8(16):1248-54; and Duan et al. Mol Ther. (2001) 4(4):383-91. Helper-free systems include those described in U.S. Patent Nos. 6,004,797; 7,588,772; and 7,094,604;

[0159] The AAV DNA in the rAAV genome can be from any AAV variant or serotype from which a recombinant virus can be derived, including, but not limited to, AAV variants or serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAVrh.74 and AAVrh10. The production of pseudotyped rAAV is disclosed, for example, in WO 01 / 83692. Other types of rAAV variants are also contemplated, such as rAAVs with capsid mutations. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014). The nucleotide sequences of the genomes of various AAV serotypes are known in the art.

[0160] In some cases, rAAVs contain self-complementary genomes. As defined herein, rAAVs containing "self-complementary" or "double-stranded" genomes refer to rAAVs engineered such that the coding region of the rAAV constitutes an intramolecular double-stranded DNA template, as described in McCarty et al. Self-complementary recombinant adeno-associated virus (scAAV) vectors promoter efficient transduction independently of DNA synthesis. Gene Therapy. 8 (16): 1248-54 (2001). The present disclosure contemplates the use of rAAVs containing self-complementary genomes in some cases, because upon infection (such as transduction), rather than waiting for cell-mediated synthesis of the second strand of the rAAV genome, the two complementary halves of the scAAV associate to form one double-stranded DNA (dsDNA) unit, ready for immediate replication and transcription. It will be appreciated that instead of the full coding capacity found in rAAV (4.7-6 kb), rAAV containing self-complementary genomes may only retain about half that amount (approximately 2.4 kb).

[0161] In other cases, rAAV vectors contain single-stranded genomes. As defined herein, "single-standard" genome refers to genomes that are not self-complementary. In most cases, non-recombinant AAV has a single-stranded DNA genome. Some have indicated that rAAV should be scAAV to achieve efficient cell transduction. However, the present disclosure contemplates rAAV vectors that can have single-stranded genomes rather than self-complementary genomes, with the understanding that other genetic modifications of rAAV vectors may be beneficial to obtain optimal gene transcription in target cells.

[0162] In some cases, the rAAV vector is of serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10 or AAVrh74. The production of pseudotyped rAAV is disclosed, for example, in WO 01 / 83692. Other types of rAAV variants are also contemplated, such as rAAV with capsid mutations. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014). In some cases, the rAAV vector is of serotype AAV9. In some embodiments, the rAAV vector is of serotype AAV9 and comprises a single-stranded genome. In some embodiments, the rAAV vector is of serotype AAV9 and comprises a self-complementary genome. In some embodiments, the rAAV vector comprises an inverted terminal repeat (ITR) sequence of AAV2. In some embodiments, the rAAV vector comprises an AAV2 genome, such that the rAAV vector is an AAV-2 / 9 vector, an AAV-2 / 6 vector, or an AAV-2 / 8 vector.

[0163] The full-length and capsid gene sequences of most of the known AAVs are provided in US Pat. No. 8,524,446, which is incorporated herein in its entirety.

[0164] AAV vector may comprise wild-type AAV sequence or may comprise one or more modifications to wild-type AAV sequence.In certain embodiments, AAV vector comprises one or more amino acid modifications, optionally substitutions, deletions or insertions, in capsid protein, optionally VP1, VP2 and / or VP3.In certain embodiments, modifications provide reduced immunogenicity when AAV vector is provided to subject.

[0165] The capsid protein of the rAAV may be modified to target the rAAV to a specific target tissue of interest, such as cardiomyocytes. In some embodiments, the rAAV is injected directly into the intraventricular space of a subject.

[0166] In some embodiments, the rAAV virion is an AAV2 rAAV virion. The capsid can be an AAV2 capsid or a functional variant thereof. In some embodiments, the AAV2 capsid shares at least 98%, 99% or 100% identity with a reference AAV2 capsid, e.g., SEQ ID NO: 96.

[0167] In some embodiments, the rAAV virion is an AAV9 rAAV virion. The capsid can be an AAV9 capsid or a functional variant thereof. In some embodiments, the AAV9 capsid shares at least 98%, 99% or 100% identity with a reference AAV9 capsid, e.g., SEQ ID NO: 97.

[0168] In some embodiments, the rAAV virion is an AAV6 rAAV virion. The capsid can be an AAV9 capsid or a functional variant thereof. In some embodiments, the AAV6 capsid shares at least 98%, 99% or 100% identity with a reference AAV6 capsid, e.g., SEQ ID NO: 98.

[0169] In some embodiments, the rAAV virion is an AAVrh.10 rAAV virion. The capsid can be an AAV9 capsid or a functional variant thereof. In some embodiments, the AAVrh.10 capsid shares at least 98%, 99% or 100% identity with a reference AAVrh.10 capsid, e.g., SEQ ID NO: 99.

[0170] In some embodiments, the capsid protein is encoded by a polynucleotide provided on a plasmid in trans to a transfer plasmid. The polynucleotide sequence of the wild-type AAVrh74 cap is provided as SEQ ID NO: 100.

[0171] The disclosure further provides protein sequences of AAVrh74 VP1, VP2 and VP3, including SEQ ID NOs: 101-103, and homologs or functional variants thereof.

[0172] In certain cases, the AAVrh74 capsid comprises the amino acid sequence set forth in SEQ ID NO: 101. In some embodiments, the rAAV vector comprises a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of AAVrh74 VP1, e.g., as set forth in SEQ ID NO: 101, or a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of AAVrh74 VP1, e.g., as set forth in SEQ ID NO: 101. VP1, or further comprising a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of AAVrh74 VP1, e.g., as set forth in SEQ ID NO: 101.In some embodiments, the rAAV vector comprises a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of AAVrh74 VP2, e.g., as set forth in SEQ ID NO: 102, or a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of AAVrh74 VP2, e.g., as set forth in SEQ ID NO: 102. VP2、or further comprising a polypeptide that is at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of AAVrh74 VP2, as set forth in, for example, SEQ ID NO: 102.In some embodiments, the rAAV vector comprises a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of AAVrh74 VP3, e.g., as set forth in SEQ ID NO: 103, or a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of AAVrh74 VP3, e.g., as set forth in SEQ ID NO: 103. VP3、55%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of AAVrh74 VP3, or further comprising a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of AAVrh74 VP3, e.g., as set forth in SEQ ID NO: 103.

[0173] In some embodiments, the rAAV virion is an AAV-PHP.B rAAV virion or a neutrotrophic variant thereof, such as, but not limited to, those disclosed in International Patent Publication Nos. WO 2015 / 038958 A1 and WO 2017 / 100671 A1. For example, the AAV capsid can include at least four consecutive amino acids from the sequence TLAVPFK (SEQ ID NO: 105) or KFPVALT (SEQ ID NO: 106), e.g., inserted between the sequence encoding amino acid numbers 588 and 589 of AAV9.

[0174] The capsid can be an AAV-PHP.B capsid or a functional variant thereof. In some embodiments, the AAV-PHP.B capsid shares at least 98%, 99% or 100% identity with a reference AAV-PHP.B capsid, e.g., SEQ ID NO: 104.

[0175] Additional AAV capsids for use in the rAAV virions of the present disclosure include those disclosed in Patent Publication Nos. WO 2009 / 012176 A2 and WO 2015 / 168666 A2.

[0176] Without being bound by theory, the inventors have determined that the AAV9 vector, the AAVrh.74 or the AAVrh.10 vector confers the desired cardiac tropism to the vector. Without being bound by theory, the inventors have further determined that the AAV9 vector, the AAVrh.74 or the AAVrh.10 vector may provide the desired specificity for cardiac cells.

[0177] In one aspect, the disclosure provides a pharmaceutical composition comprising a rAAV virion of the disclosure and one or more pharma- ceutically acceptable carriers, diluents, or excipients.

[0178] For purposes of administration, optionally by injection, a variety of solutions can be utilized, including sterile aqueous solutions. Such aqueous solutions can be buffered, if desired, and the liquid diluent can be first rendered isotonic with saline or glucose. Solutions of rAAV as a free acid (DNA contains acidic phosphate groups) or pharmacologically acceptable salts can be prepared, for example, at 0.001% or 0.01%, in water suitably mixed with a surfactant, such as poloxamer 188. Dispersions of rAAV can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In this connection, all sterile aqueous media utilized are readily available by standard techniques well known to those skilled in the art.

[0179] Pharmaceutical forms suitable for injectable use include, but are not limited to, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In some embodiments, isotonic agents, such as sugars or sodium chloride, may be included. Prolonged absorption of the injectable compositions can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0180] Sterile injectable solution can be prepared by incorporating the required amount of rAAV in a suitable solvent with various other components as listed above as necessary, and then sterilizing by filtration.Generally, dispersion is prepared by incorporating sterilized active ingredient into a sterile vehicle that contains basic dispersion medium and other components as listed above.In the case of sterile powder for preparing sterile injectable solution, some preparation methods are vacuum drying and freeze-drying techniques, which produce powder of active ingredient plus any additional desired components from its solution that has been previously sterilized and filtered.

[0181] In another aspect, the disclosure includes a kit comprising a rAAV virion of the disclosure and instructions for use.

[0182] In one aspect, the present disclosure provides a method for increasing JPH2 expression and / or activity in a cell, comprising contacting the cell with the rAAV of the present disclosure. In another aspect, the present disclosure provides a method for increasing JPH2 expression and / or activity in a subject, comprising administering the rAAV of the present disclosure to the subject. In some embodiments, the cell and / or subject is deficient in JPH2 messenger RNA or JPH2 protein expression level and / or activity, and / or comprises a loss-of-function mutation of JPH2. In some embodiments, the cell and / or subject is deficient in JPH2 messenger RNA or JPH2 protein expression level and / or activity, and / or comprises a truncated variant of JPH2 having at most 150 or at most 200 amino acids. The cell may be a cardiac cell, e.g., a cardiomyocyte. In certain embodiments, the subject is a mammal, e.g., a human.

[0183] In some embodiments, the method promotes survival of cardiac cells, e.g., cardiomyocyte cells, in cell culture and / or in vivo. In some embodiments, the method promotes and / or restores cardiac function.

[0184] In another aspect, the present disclosure provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of a rAAV virion of the present disclosure. In some embodiments, the disease or disorder is a cardiac disease or disorder. Exemplary cardiac disorders include heart failure, dilated cardiomyopathy, hypertrophic cardiomyopathy, atrial fibrillation, arrhythmia, sinus node disease, hypertensive heart disease, cardiac hypertrophy, atrial fibrosis, myocardial infarction, symptomatic sick sinus syndrome, atrial disease, myocardial infarction, and familial hypertrophic cardiomyopathy 17 (CMH17). In certain embodiments, the subject is afflicted with or at risk for CMH17. In certain embodiments, the subject is a mammal, e.g., a human, having a loss-of-function mutation in the JPH2 gene. In certain embodiments, the subject is a mammal, e.g., a human, having a stress-induced truncated variant of JPH2. In certain methods, treatment with the rAAV virion results in expression of the JPH2 protein encoded by the rAAV virion in the subject, e.g., in the subject's heart or cardiac tissue. In certain embodiments, treatment with rAAV virions results in at least 2-fold, at least 5-fold, at least 10-fold or more detectable JPH2 protein levels in the subject's heart. In certain embodiments, treatment with rAAV virions results in at least 2-fold, at least 5-fold, at least 10-fold or more detectable JPH2 protein levels in cardiac fibroblasts (CFs) of the subject's heart. In certain embodiments, treatment with rAAV virions results in at least 2-fold, at least 5-fold, at least 10-fold or more detectable JPH2 protein levels in cardiomyocytes of the subject's heart. In certain embodiments, treatment with rAAV virions results in at least 2-fold, at least 5-fold, at least 10-fold or more detectable JPH2 protein levels in smooth muscle cells (SMCs) of the subject's heart. In certain embodiments, treatment with rAAV virions results in at least 2-fold, at least 5-fold, at least 10-fold or more detectable JPH2 protein levels in endothelial cells (ECs) of the subject's heart.In certain embodiments, treatment with rAAV virions results in at least 2-fold, at least 5-fold, at least 10-fold or more JPH2 protein levels detectable in the epicardium of the subject's heart. In certain embodiments, treatment with rAAV virions results in at least 2-fold, at least 5-fold, at least 10-fold or more JPH2 protein levels detectable in the myocardium of the subject's heart. In certain embodiments, treatment with rAAV virions results in at least 2-fold, at least 5-fold, at least 10-fold or more JPH2 protein levels detectable in the endocardium of the subject's heart.

[0185] AAV-mediated delivery of JPH2 protein to the heart may prolong life and prevent or attenuate cardiac cell degeneration, heart failure, scarring, reduced ejection fraction, arrhythmias, angina, exercise intolerance, angina (chest pain), sudden cardiac death, exertional muscle pain, and cramps. AAV-mediated delivery of JPH2 protein to the heart may show improvement from the usual disease course as detected by the use of echocardiography, pathological electrocardiogram, cardiac MRI, cardiac biopsy, reduction in paroxysmal ventricular arrhythmias, and / or reduction in sudden cardiac death, or prevent the usual disease course as detected by the use of echocardiography, pathological electrocardiogram, cardiac MRI, cardiac biopsy, reduction in paroxysmal ventricular arrhythmias, and / or reduction in sudden cardiac death.

[0186] The methods disclosed herein can provide efficient biodistribution of JPH2 in heart. They can provide sustained expression in all or a significant portion of cardiac cells, such as cardiomyocytes. Notably, the methods disclosed herein can provide long-term expression of JPH2 protein throughout the life of a subject after AAV vector administration. In some embodiments, the expression of JPH2 protein in response to treatment lasts for at least 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 15 years, 20 years, 25 years, 30 years, 35 years or 40 years.

[0187] Combination therapy is also contemplated by the present disclosure.The combination of the method of the present disclosure with standard medical treatment (e.g., corticosteroids or local decompression agents) is particularly contemplated, as well as the combination with novel treatments.In some cases, subjects may be treated with a combination of steroids and / or immunosuppressants to suppress or reduce immune response to administration of rAAV as described herein.

[0188] In some embodiments, the AAV vector is about 1 x 10 AAV vector (vg) per kilogram of total subject body weight. 12 ~5×10 14 Vector genome (vg) or approximately 1 × 10 12 ~6×10 14 In some embodiments, the AAV vector is administered at a dose of about 1×10 13 ~5×10 14 In some embodiments, the AAV vector is administered at a dose of about 5×10 13 ~3×10 14 In some embodiments, the AAV vector is administered at a dose of about 5×10 13 ~1×10 14 In certain embodiments, the AAV vector is administered at a dose of about 5×10 13 ~5×10 14 In certain embodiments, the AAV vector is administered at a dose of about 1×10 13 ~1×10 15 In certain embodiments, the AAV vector is administered at a dose of about 5×10 13 ~1×10 14 In certain embodiments, the AAV vector is administered at a dose of about 8×10 13 ~1×10 14 The drug is administered at a dose of 100 mg / kg.

[0189] In some embodiments, the AAV vector is about 1 x 10 12 Less than 3×10 vg / kg 12 Less than 5×10 vg / kg 12Less than 7×10 vg / kg 12 Less than 1×10 vg / kg 13 Less than 3×10 vg / kg 13 Less than 5×10 vg / kg 13 Less than 7×10 vg / kg 13 Less than 1×10 vg / kg 14 Less than 3×10 vg / kg 14 Less than 5×10 vg / kg 14 Less than 7×10 vg / kg 14 Less than 1×10 vg / kg 15 Less than 3×10 vg / kg 15 Less than 5×10 vg / kg 15 Less than 7×10 vg / kg 15 Less than 1×10 vg / kg 16 Less than 3×10 vg / kg 16 Less than 5×10 vg / kg 16 Less than 7×10 vg / kg 16 Less than 1×10 vg / kg 17 Less than 3×10 vg / kg 17 Less than 5×10 vg / kg 17 Less than 7×10 vg / kg 17 Less than 1×10 vg / kg 18 Less than 3×10 vg / kg 18 Less than 5×10 vg / kg 18 vg / kg or less than about 7 × 10 18 In some embodiments, the AAV vector delivered at any of these doses is an AAV9 vector or an AAV rh74 vector. In some cases, it may be advantageous to use a higher dose for the AAV rh74 vector than for the AAV9 vector.

[0190] In some embodiments, the AAV vector is about 1 x 10 12 vg / kg, approx. 3×10 12 vg / kg, approx. 5×10 12 vg / kg, approx. 7×10 12 vg / kg, approximately 1×10 13 vg / kg, approx. 3×10 13vg / kg, approx. 5×10 13 vg / kg, approximately 6×10 13 vg / kg, approximately 7×10 13 vg / kg, approx. 8×10 13 vg / kg, approx. 9×10 13 vg / kg, approximately 1×10 14 vg / kg, approx. 3×10 14 vg / kg, approx. 5×10 14 vg / kg, approximately 7×10 14 vg / kg, approximately 1×10 15 vg / kg, approx. 3×10 15 vg / kg, approx. 5×10 15 vg / kg, approximately 7×10 15 vg / kg, approximately 1×10 16 vg / kg, approx. 3×10 16 vg / kg, approx. 5×10 16 vg / kg, approximately 7×10 16 vg / kg, approximately 1×10 17 vg / kg, approx. 3×10 17 vg / kg, approx. 5×10 17 vg / kg, approximately 7×10 17 vg / kg, approximately 1×10 18 vg / kg, approx. 3×10 18 vg / kg, approx. 5×10 18 vg / kg or approximately 7 × 10 18 vg / kg. In certain embodiments, the AAV vector delivered in any of these doses is an AAV9 vector or an AAV rh74 vector.

[0191] In some embodiments, the AAV vector is 1 x 10 12 vg / kg, 3 × 10 12 vg / kg, 5×10 12 vg / kg, 7×10 12 vg / kg, 1×10 13 vg / kg, 3 × 10 13 vg / kg, 5×10 13 vg / kg, 6×10 13 vg / kg, 7×10 13 vg / kg, 8×10 13 vg / kg, 9×10 13 vg / kg, 1×1014 vg / kg, 3 × 10 14 vg / kg, 5×10 14 vg / kg, 7×10 14 vg / kg, 1×10 15 vg / kg, 3 × 10 15 vg / kg, 5×10 15 vg / kg or 7 × 10 15 vg / kg, 1×10 16 vg / kg, 3 × 10 16 vg / kg, 5×10 16 vg / kg, 7×10 16 vg / kg, 1×10 17 vg / kg, 3 × 10 17 vg / kg, 5×10 17 vg / kg, 7×10 17 vg / kg, 1×10 18 vg / kg, 3 × 10 18 vg / kg, 5×10 18 vg / kg, 7×10 18 vg / kg, or a range between any of these values. In certain embodiments, the AAV vector delivered at any of these doses is an AAV9 vector or an AAV rh74 vector.

[0192] In some embodiments, the AAV vector is about 1 x 10 AAV vector (vg) per kilogram of total subject body weight. 12 ~5×10 14 The AAV vector is administered systemically at a dose (vg / kg) of vector genome (vg). In some embodiments, the AAV vector is administered in an amount of about 1×10 13 ~5×10 14 In some embodiments, the AAV vector is administered systemically at a dose of about 5×10 13 ~3×10 14 In some embodiments, the AAV vector is administered systemically at a dose of about 5×10 13 ~1×10 14 In some embodiments, the AAV vector is administered systemically at a dose of about 1×10 12 Less than 3×10 vg / kg 12 Less than 5×10 vg / kg12 Less than 7×10 vg / kg 12 Less than 1×10 vg / kg 13 Less than 3×10 vg / kg 13 Less than 5×10 vg / kg 13 Less than 7×10 vg / kg 13 Less than 1×10 vg / kg 14 Less than 3×10 vg / kg 14 Less than 5×10 vg / kg 14 Less than 7×10 vg / kg 14 Less than 1×10 vg / kg 15 Less than 3×10 vg / kg 15 Less than 5×10 vg / kg 15 Less than 7×10 vg / kg 15 Less than 1×10 vg / kg 16 Less than 3×10 vg / kg 16 Less than 5×10 vg / kg 16 Less than 7×10 vg / kg 16 Less than 1×10 vg / kg 17 Less than 3×10 vg / kg 17 Less than 5×10 vg / kg 17 Less than 7×10 vg / kg 17 Less than 1×10 vg / kg 18 Less than 3×10 vg / kg 18 Less than 5×10 vg / kg 18 vg / kg or less than about 7 × 10 18 In certain embodiments, the AAV vector delivered at any of these doses is an AAV9 vector or an AAV rh74 vector.

[0193] In some embodiments, the AAV vector is about 1 x 10 12 vg / kg, approx. 3×10 12 vg / kg, approx. 5×10 12 vg / kg, approx. 7×10 12 vg / kg, approximately 1×10 13 vg / kg, approx. 3×10 13 vg / kg, approx. 5×10 13 vg / kg, approximately 6×10 13vg / kg, approximately 7×10 13 vg / kg, approx. 8×10 13 vg / kg, approx. 9×10 13 vg / kg, approximately 1×10 14 vg / kg, approx. 3×10 14 vg / kg, approx. 5×10 14 vg / kg, approximately 7×10 14 vg / kg, approximately 1×10 15 vg / kg, approx. 3×10 15 vg / kg, approx. 5×10 15 vg / kg, approximately 7×10 15 vg / kg, approximately 1×10 16 vg / kg, approx. 3×10 16 vg / kg, approx. 5×10 16 vg / kg, approximately 7×10 16 vg / kg, approximately 1×10 17 vg / kg, approx. 3×10 17 vg / kg, approx. 5×10 17 vg / kg, approximately 7×10 17 vg / kg, approximately 1×10 18 vg / kg, approx. 3×10 18 vg / kg, approx. 5×10 18 vg / kg or approximately 7 × 10 18 vg / kg. In certain embodiments, the AAV vector delivered at any of these doses is an AAV9 vector or an AAV rh74 vector.

[0194] In some embodiments, the AAV vector is 1 x 10 12 vg / kg, 3 × 10 12 vg / kg, 5×10 12 vg / kg, 7×10 12 vg / kg, 1×10 13 vg / kg, 3 × 10 13 vg / kg, 5×10 13 vg / kg, 6×10 13 vg / kg, 7×10 13 vg / kg, 8×10 13 vg / kg, 9×10 13 vg / kg, 1×10 14 vg / kg, 3 × 10 14 vg / kg, 5×1014 vg / kg, 7×10 14 vg / kg, 1×10 15 vg / kg, 3 × 10 15 vg / kg, 5×10 15 vg / kg, 7×10 15 vg / kg, 1×10 16 vg / kg, 3 × 10 16 vg / kg, 5×10 16 vg / kg, 7×10 16 vg / kg, 1×10 17 vg / kg, 3 × 10 17 vg / kg, 5×10 17 vg / kg, 7×10 17 vg / kg, 1×10 18 vg / kg, 3 × 10 18 vg / kg, 5×10 18 vg / kg, 7×10 18 vg / kg. In certain embodiments, the AAV vector delivered at any of these doses is an AAV9 vector or an AAV rh74 vector.

[0195] In some embodiments, the AAV vector is about 1 x 10 AAV vector (vg) per kilogram of total subject body weight. 12 ~5×10 14 The vector genome (vg) is administered intravenously at a dose (vg / kg). In some embodiments, the AAV vector is administered at about 1×10 13 ~5×10 14 In some embodiments, the AAV vector is administered intravenously at a dose of about 5×10 13 ~3×10 14 In some embodiments, the AAV vector is administered intravenously at a dose of about 5×10 13 ~1×10 14 In some embodiments, the AAV vector is administered intravenously at a dose of about 1×10 12 Less than 3×10 vg / kg 12 Less than 5×10 vg / kg 12 Less than 7×10 vg / kg 12 Less than 1×10 vg / kg 13Less than 3×10 vg / kg 13 Less than 5×10 vg / kg 13 Less than 7×10 vg / kg 13 Less than 1×10 vg / kg 14 Less than 3×10 vg / kg 14 Less than 5×10 vg / kg 14 Less than 7×10 vg / kg 14 Less than 1×10 vg / kg 15 Less than 3×10 vg / kg 15 Less than 5×10 vg / kg 15 Less than 7×10 vg / kg 15 Less than 1×10 vg / kg 16 Less than 3×10 vg / kg 16 Less than 5×10 vg / kg 16 Less than 7×10 vg / kg 16 Less than 1×10 vg / kg 17 Less than 3×10 vg / kg 17 Less than 5×10 vg / kg 17 Less than 7×10 vg / kg 17 Less than 1×10 vg / kg 18 Less than 3×10 vg / kg 18 Less than 5×10 vg / kg 18 vg / kg or less than about 7 × 10 18 In certain embodiments, the AAV vector delivered at any of these doses is an AAV9 vector or an AAV rh74 vector.

[0196] In some embodiments, the AAV vector is about 1 x 10 12 vg / kg, approx. 3×10 12 vg / kg, approx. 5×10 12 vg / kg, approx. 7×10 12 vg / kg, approximately 1×10 13 vg / kg, approx. 3×10 13 vg / kg, approx. 5×10 13 vg / kg, approximately 6×10 13 vg / kg, approx. 7×10 13 vg / kg, approximately 8×10 13 vg / kg, approx. 9×10 13vg / kg, approximately 1×10 14 vg / kg, approx. 3×10 14 vg / kg, approx. 5×10 14 vg / kg, approximately 7×10 14 vg / kg, approximately 1×10 15 vg / kg, approx. 3×10 15 vg / kg, approx. 5×10 15 vg / kg, approximately 7×10 15 vg / kg, approximately 1×10 16 vg / kg, approx. 3×10 16 vg / kg, approx. 5×10 16 vg / kg, approximately 7×10 16 vg / kg, approximately 1×10 17 vg / kg, approx. 3×10 17 vg / kg, approx. 5×10 17 vg / kg, approximately 7×10 17 vg / kg, approximately 1×10 18 vg / kg, approx. 3×10 18 vg / kg, approx. 5×10 18 vg / kg or approximately 7 × 10 18 It is given intravenously at a dose of 1000 mg / kg.

[0197] In some embodiments, the AAV vector is 1 x 10 12 vg / kg, 3 × 10 12 vg / kg, 5×10 12 vg / kg, 7×10 12 vg / kg, 1×10 13 vg / kg, 3 × 10 13 vg / kg, 5×10 13 vg / kg, 6×10 13 vg / kg, 7×10 13 vg / kg, 8×10 13 vg / kg, 9×10 13 vg / kg, 1×10 14 vg / kg, 3 × 10 14 vg / kg, 5×10 14 vg / kg, 7×10 14 vg / kg, 1×10 15 vg / kg, 3 × 10 15 vg / kg, 5×10 15 vg / kg, 7×10 15 vg / kg, 1×1016 vg / kg, 3 × 10 16 vg / kg, 5×10 16 vg / kg, 7×10 16 vg / kg, 1×10 17 vg / kg, 3 × 10 17 vg / kg, 5×10 17 vg / kg, 7×10 17 vg / kg, 1×10 18 vg / kg, 3 × 10 18 vg / kg, 5×10 18 vg / kg, 7×10 18 vg / kg intravenously. In certain embodiments, the AAV vector delivered in any of these doses is an AAV9 vector or an AAV rh74 vector.

[0198] Evidence of functional improvement, clinical benefit or efficacy in patients may be evidenced by improvement in New York Heart Association functional class (NYHA class), echocardiography (stabilization or improvement in left ventricular ejection fraction, fractional shortening, left ventricular outflow tract obstruction, left ventricular wall thickness, left or right ventricular volumes, right ventricular area and / or velocity time integral), electrocardiography (stabilization or improvement in ST segment changes, T wave inversions, Q waves, atrial fibrillation and / or supraventricular tachycardia), cardiac MRI, cardiac biopsy, reduction in paroxysmal ventricular arrhythmias, reduction in sudden cardiac death, and / or reduction or lack of further development of fibrofatty deposits.

[0199] Administration of an effective dose of the composition may be by any route standard in the art, including, but not limited to, systemic administration, local administration, direct injection, intravenous administration, or intracardiac administration. In some cases, administration includes systemic injection, local injection, direct injection, intravenous injection, or intracardiac injection. Administration may be performed by cardiac catheterization.

[0200] In some embodiments, the present disclosure provides for local and systemic administration of an effective dose of rAAV and compositions of the present disclosure. For example, systemic administration can be administration into the circulatory system so that the entire body is affected. Systemic administration includes parenteral administration by injection, infusion or implantation. The routes of administration of the compositions disclosed herein include intravenous ("IV") administration, intraperitoneal ("IP") administration, intramuscular ("IM") administration, intralesional administration or subcutaneous ("SC") administration, or implantation of a sustained release device, such as a mini-osmotic pump, a depot formulation, and the like. In some embodiments, the method of the present disclosure includes administering the AAV vector of the present disclosure or a pharmaceutical composition thereof by intravenous, intramuscular, intraarterial, intrarenal, intraurethral, ​​intracardiac, intracoronary, intramyocardial, intradermal, epidural, subcutaneous, intraperitoneal, intraventricular, or iontophoretic administration.

[0201] In particular, administration of the rAAV of the present disclosure can be accomplished using any physical method that delivers the rAAV recombinant vector to the target tissue of an animal, including, but not limited to, injection into the heart.

[0202] In some embodiments, the methods of the disclosure include intracardiac delivery. Infusion can be performed using specialized cannulas, catheters, syringes / needles with an infusion pump. Administration can include delivery of an effective amount of rAAV virions, or pharmaceutical compositions containing rAAV virions, to the heart. These can be accomplished, for example, by intravenous, intramuscular, intraarterial, intrarenal, intraurethral, ​​intracardiac, intracoronary, intramyocardial, intradermal, epidural, subcutaneous, intraperitoneal, intraventricular, or iontophoretic administration. The compositions of the disclosure can also be administered intravenously.

[0203] Effect of rAAV administration In some embodiments, administration of a rAAV of the present disclosure can have a beneficial effect on a subject. For example, administration of a rAAV of the present disclosure can increase the survival of a subject compared to a subject not administered a rAAV of the present disclosure.

[0204] In some embodiments, administration of a rAAV of the disclosure increases viability by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, or at least about 500% compared to a subject not administered a rAAV of the disclosure.

[0205] In some embodiments, administration of a rAAV of the disclosure improves survival by 1% to 90%, 20% to 80%, 30% to 80%, 40% to 80%, 50% to 80%, 1% to 2%, 2% to 3%, 3% to 4%, 4% to 5%, 5% to 6%, 6% to 7%, 7% to 8%, 8% to 9%, 9% to 10%, 10% to 15%, 15% to 20%, 20% to 35%, or 40% to 50% compared to a subject not administered a rAAV of the disclosure. %, 25%-30%, 30%-35%, 35%-40%, 40%-45%, 45%-50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, 95%-100%, 100%-200%, 200%-300%, 300%-400% or 400%-500% increase.

[0206] In some embodiments, administration of a rAAV of the disclosure abrogates the decline in ejection fraction in a subject compared to a subject not administered a rAAV of the disclosure. In some embodiments, administration of a rAAV of the disclosure abrogates the decline in ejection fraction by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to a subject not administered a rAAV of the disclosure.

[0207] In some embodiments, administration of a rAAV of the disclosure reduces or prevents a decrease in ejection fraction by 1% to 90%, 20% to 80%, 30% to 80%, 40% to 80%, 50% to 80%, 1% to 2%, 2% to 3%, 3% to 4%, 4% to 5%, 5% to 6%, 6% to 7%, 7% to 8%, 8% to 9%, 9% to 10%, or 12% compared to a subject not administered a rAAV of the disclosure. Suppresses by 10%-15%, 15%-20%, 20%-35%, 25%-30%, 30%-35%, 35%-40%, 40%-45%, 45%-50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95% or 95%-100%.

[0208] In some embodiments, administration of a rAAV of the present disclosure inhibits an increase in end diastolic diameter (EDD) in a subject compared to a subject not administered a rAAV of the present disclosure. In some embodiments, administration of a rAAV of the disclosure inhibits an increase in end diastolic diameter (EDD) in a subject by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, or at least about 500% compared to a subject not administered a rAAV of the disclosure.

[0209] In some embodiments, administration of a rAAV of the disclosure results in an increase in EDD in a subject of between 1% and 90%, between 20% and 80%, between 30% and 80%, between 40% and 80%, between 50% and 80%, between 1% and 2%, between 2% and 3%, between 3% and 4%, between 4% and 5%, between 5% and 6%, between 6% and 7%, between 7% and 8%, between 8% and 9%, between 9% and 10%, between 10% and 15%, between 15% and 20%, between 20% and 35%, between 25% and 40%, between 35% and 45%, between 3 ... Suppression will be 0%~35%, 25%~30%, 30%~35%, 35%~40%, 40%~45%, 45%~50%, 50%~55%, 55%~60%, 60%~65%, 65%~70%, 70%~75%, 75%~80%, 80%~85%, 85%~90%, 90%~95%, 95%~100%, 100%~200%, 200%~300%, 300%~400% or 400%~500%.

[0210] In some embodiments, administration of a rAAV of the present disclosure inhibits an increase in systolic left ventricular posterior wall thickness (LVPW) in a subject compared to a subject not administered a rAAV of the present disclosure. In some embodiments, administration of a rAAV of the disclosure inhibits the increase in LVPW in a subject by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 300%, at least about 400% or at least about 500% compared to a subject not administered a rAAV of the disclosure.

[0211] In some embodiments, administration of a rAAV of the disclosure results in an increase in LVPW in a subject by 1% to 90%, 20% to 80%, 30% to 80%, 40% to 80%, 50% to 80%, 1% to 2%, 2% to 3%, 3% to 4%, 4% to 5%, 5% to 6%, 6% to 7%, 7% to 8%, 8% to 9%, 9% to 10%, 10% to 15%, 15% to 20%, Suppression by 20%-35%, 25%-30%, 30%-35%, 35%-40%, 40%-45%, 45%-50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, 95%-100%, 100%-200%, 200%-300%, 300%-400% or 400%-500%. EXAMPLES

[0212] Example 1: In vitro preclinical bioactivity and efficacy The vectors exemplified in Figures 1-25 will be tested. The AAV vectors or respective expression cassettes will be tested in vitro using cultured cardiomyocytes (e.g., patient-derived induced pluripotent stem cell cardiomyocytes (iPSC-CMs) or primary cardiomyocytes harvested from animal models) or other cells suitable for transfection or transduction with these constructs. Expression of JPH2 will be assessed by immunofluorescence and Western blotting. Cell-based studies utilizing mutant cardiomyocytes will reveal the benefit of overexpressing the JPH2 transgene (either after transduction with AAV vectors and / or transfection with vector plasmids) by a) normalization of calcium handling and therefore normalization of contractile pace, and / or b) restoration of normal T-tubule structure and / or attenuation of remodeling.

[0213] Example 2: Rescue of heart failure in vivo after aortic coarctation (TAC) AAV-JHP2 gene therapy with the AAV vector of choice described above is performed essentially as described in Reynolds et al. (Int J Cardiol. 2016 Dec 15; 225: 371-380). AAV expression cassettes are packaged and delivered in vivo using different capsid serotypes, such as AAV9 and / or AAV rh.74.

[0214] Mouse TAC model: Aortic coarctation (TAC) in mice is an experimental induction of cardiac hypertrophy and subsequent heart failure due to pressure overload. Compared with other experimental mouse models of heart failure, the TAC model results in more reproducible cardiac hypertrophy and a gradual time course of heart failure development. After TAC in mice, a gradual decline in ejection fraction and other cardiac function indices parallels a gradual decline in cardiac JPH2 levels. Male C57BI / 6J mice (approximately 4 months old) are anesthetized and the aortic arch is visualized by performing an anterior thoracotomy to the level of the third intercostal space. Coarctation is performed by tying a silk suture between the first and second trunks of the aortic arch with a 28-gauge needle. For consistency, the coarctation level is quantified by measuring the change in Doppler flow velocity in the left and right carotid arteries on the 7th day after surgery. The peak velocity ratio of the right carotid artery to the left carotid artery ranges from 5.0 to 6.5, and the ejection fraction 2 weeks after TAC can range from 40% to 50%.

[0215] Functional evidence of efficacy by echocardiography: Evidence of efficacy and bioactivity for cardiac benefit in the TAC model is assessed using transthoracic echocardiography at predefined time points including baseline and various intervals after TAC. To screen for animals with sufficient heart failure to be suitable for this mouse model, the Doppler ratio of peak velocity in the right and left carotid arteries (RC / LC) is determined at 1 week after TAC, and those that do not meet the criteria (RC / LC between 5.0 and 6.5) are excluded from the study. In addition, an echocardiogram is performed at 2 weeks after TAC, and animals with ejection fractions (EF) outside the range of 40-50% are also excluded. Mice with adequate Doppler RC / EV and EF by echocardiography are then injected (either intravenously or retroorbitally) with an AAV construct overexpressing JPH2 protein or formulation buffer (FB; vehicle control) at 3 weeks after Tac. Efficacy is evidenced by a significant increase in EF over time in AAV-JPH2-treated animals compared to the FB control group. Echocardiography will reveal that FB-injected mice will see their EF gradually decline over time, their end diastolic diameter (EDD) increase over time, and their systolic left ventricular posterior wall thickness (LVPW) also increase over time. In contrast, AAV-JPH2-injected animals will see their EF and EDD remain stable or improve slightly over time and their LVPW become greater than FB controls over time after AAV-JPH2 treatment.

[0216] Morphological evidence of efficacy by attenuating transverse tubule remodeling: To test the effect of AAV9-JPH2 on T-tubule structure, isolated cardiomyocytes are evaluated for potential remodeling as a result of TAC. As a result of TAC, FB control-injected mice exhibit typical cardiac remodeling evident by a significant reduction in T-tubule area and T-tubule power, which are measures of the integrity of T-tubule structures in myocytes. Evidence of efficacy of JPH2 overexpression will be observed by mitigation of cardiac remodeling and attenuation of changes in T-tubule area and T-tubule power in AAV-JPH2-injected animals compared to FB-injected controls.

[0217] Functional evidence of efficacy through improved calcium handling: As a result of TAC, sarcoplasmic reticulum (SR) Ca in isolated ventricular myocytes 2+ The treatment was Ca 2+ Significantly impaired as measured by reduced transient amplitude and normal Na 2+ / Ca 2+ Ca using a caffeine dump protocol with exchanger changes 2+ A significant reduction in SR load is observed. Evidence for the benefit or efficacy of AAV-mediated JPH2 overexpression in the TAC model is the Ca upregulation in cardiomyocytes. 2+ Normalization of transient amplitude, SR Ca 2+ Load improvement and Na 2+ / Ca 2+ This will be evident by normalization of the exchanger.

[0218] Evidence of efficacy through improved expression of transgenic protein and downstream hypertrophic responses: Expression levels of JPH2 protein as a result of AAV administration will be assessed in cardiac lysates by Western blot. Although JPH2 protein levels will be reduced as a result of TAC in FB-injected animals, AAV-mediated overexpression of JPH2 is expected to result in sustained protein levels up to 9 weeks after TAC. Furthermore, quantitative polymerase chain reaction (qPCR) will reveal increased mRNA levels of several pro-hypertrophy markers in FB-injected TAC mice compared to normal sham-operated controls. Increased pro-hypertrophy markers include, but are not limited to, "regulator of calcineurin 1 isoform 4" (Rcan1.4), a marker of "nuclear factor of activated T cells" (NFAT), myosin heavy chain 7 (Myh7), natriuretic peptide type A (Nppa), and natriuretic peptide type B (Nppb). The beneficial effect of JHP2 delivery by AAV gene therapy would be manifested by attenuating or significantly reducing the mRNA levels of one or several of these prohypertrophic markers in cardiac lysates of AAV-JPH2-injected animals compared with FB-injected TAC controls.

[0219] Example 3: Preclinical in vivo efficacy in JPH2-A399S knock-in mice The JPH2-A399S knock-in mouse is a genetic model that captures elements of human disease and represents a variant of hypertrophic cardiomyopathy (HCM), which causes left ventricular hypertrophy and fibrosis by 6 months of age in mutant mice. As a mouse model of human disease, we can further evaluate the potential biological activity and efficacy of AAV overexpression of JPH2.

[0220] Morphological evidence of efficacy by attenuating transverse tubule remodeling: To test the effect of AAV9-JPH2 on T-tubule structure in the JPH2-A399S mouse model, isolated cardiomyocytes will be evaluated for potential remodeling as a result of the A399S mutation. As a result of the A399S mutation, A399S mice injected with FB controls may exhibit typical cardiac remodeling as evidenced by a significant reduction in T-tubule area and T-tubule force, which are measures of the integrity of T-tubule structure in cardiomyocytes. Evidence of efficacy of JPH2 overexpression will be observed by mitigation of cardiac remodeling and attenuation of changes in T-tubule area and T-tubule force in animals injected with AAV-JPH2 compared to A399S knock-in controls injected with FB.

[0221] Functional evidence of efficacy of A399S mutant mice with improved calcium handling: In the JPH2 HCM genetic mouse model, A399S, mice express a mutation similar to that found in humans (A405S), leading to cardiomyocyte hypertrophy and significant fibrosis over the course of weeks to months. A399S mice have been shown to display a variety of features associated with HC, including hypertrophied ventricular septum, increased LV mass, asymmetric LV hypertrophy, reduced diastolic filling and disorganized myofibers. Evidence of therapeutic utility as a result of JPH2 overexpression in the A399S mouse model will be evident by mitigating the above-mentioned abnormal consequences on cardiac morphology and function. Furthermore, sarcoplasmic reticulum (SR) Ca in isolated ventricular myocytes was significantly increased as a result of the A399S knock-in mutation.2+ Processing is normal Na 2+ / Ca 2+ With the change in exchanger, Ca 2+ The decrease in transient amplitude, and Ca 2+ Evidence for the benefit or efficacy of AAV-mediated JPH2 overexpression in the A399S model could be provided by the Ca upregulation in cardiomyocytes. 2+ Normalization of transient amplitude, SR Ca 2+ Improved loading and / or Na 2+ / Ca 2+ This may be evident by normalization of the exchanger.

[0222] Example 4: Preclinical Transgene Expression The expression cassettes illustrated in Figures 1 and 2 were tested after packaging into AAV.rh74 or AAV9 vectors. The resulting AAV vectors (both AAVrh.74 and AAV9) were tested in vivo using C57BL / 6J mice, and the expression levels of JPH2 were evaluated by Western blot (WB) of heart tissue proteins (Figure 26). The MHCK7 promoter produced the highest expression levels of JPH2 by WB in mouse hearts after delivery of AAV9-MHCK7-JPH2 and AAVrh.74-MHCK7-JPH2, respectively. The hTnnT2 promoter ("hTnT") was found to reduce the expression levels of JPH2 protein, and AAVrh.74 produced higher levels of expression than AAV9. Based on these results, it can be concluded that AAVrh.74 and AAV9 vectors can be effectively used to express JPH2 in the heart.

[0223] Example 5: Rescue of heart failure in vivo after aortic coarctation (TAC) AAV-JHP2 gene therapy with the selected AAV vectors described above was performed essentially as described by Reynolds et al. (Int J Cardiol. 2016 Dec 15; 225: 371-380). AAV expression cassettes were packaged and delivered in vivo using the different capsid serotypes AAVrh.74 and AAV9.

[0224] Mouse TAC model: Aortic coarctation (TAC) in mice is an experimental induction of cardiac hypertrophy and subsequent heart failure due to pressure overload. Compared with other experimental mouse models of heart failure, the TAC model results in more reproducible cardiac hypertrophy and a gradual time course of heart failure development. After TAC in mice, a gradual decline in ejection fraction (EF) and other cardiac function indices parallels a gradual decline in cardiac JPH2 levels. To investigate the extent to which AAV-mediated overexpression of JPH2 could be beneficial in this mouse model of cardiac hypertrophy, male C57BL / 6J mice (approximately 4 months old) were anesthetized and the aortic arch was visualized by performing an anterior thoracotomy to the level of the third intercostal space. Coarctation was performed by tying a silk suture between the first and second trunks of the aortic arch with a 28-gauge needle. For consistency, the coarctation level was quantified by measuring changes by echocardiography, and mice whose ejection fraction was in the range of 40%–50% 2 weeks after TAC were selected for this study.

[0225] Functional evidence of efficacy by echocardiography: Evidence of efficacy and bioactivity for cardiac benefit in the TAC model was assessed using transthoracic echocardiography at predefined time points including baseline and various intervals after TAC (Figure 27). To screen for animals with sufficient heart failure to be suitable for this mouse model, echocardiography was performed 2 weeks after TAC and animals with ejection fraction (EF) outside the range of 40-50% were excluded. Mice with adequate EF by echocardiography were then injected with 3x10 AAV constructs overexpressing JPH2 protein at 3 weeks after TAC. 13AAV-JPH2-treated animals were injected retro-orbitally with a dose of 1000 mg / kg or formulation buffer (FB; vehicle control). Results are compared to either sham-operated, FB (POS CON) or TAC-operated, FB (Neg CON) mice. Efficacy was evident in AAV-JPH2-treated animals by a significantly increased EF over time compared to the FB control group (Figures 28A-28B and 29A-29F).

[0226] Echocardiography revealed that mice injected with FB (POS CON) had a gradually reduced EF over time (Figure 28A). In contrast, animals injected with AAV-JPH2 showed a clear halt in the progression of EF loss after TAC surgery, as evidenced by EF data at 9 weeks after AAV-JPH2 treatment (Figure 28B).

[0227] Evidence of alleviation of disease phenotype, albeit to a greater or lesser extent, was observed following both AAVrh.74- and AAV9-mediated JPH2 expression (Figures 29A-29F). These results indicate that both AAVrh.74 and AAV9 may benefit cardiac indications with JPH2 deficiency for which TAC mice are considered an appropriate model. Furthermore, vectors bearing either the hTnT promoter or the MHCK7 promoter have been demonstrated to be effective in treating JPH2-associated deficiency in this mouse model. Nevertheless, considering the time course data (Figure 28A and Figures 29A-29F), more robust and consistent preservation of EF was observed with vector constructs utilizing the hTnT promoter. Furthermore, no animals injected with AAV constructs bearing the hTnT promoter died prior to the planned 9-week sacrifice time point. In contrast, 4 of 8 FB (POS CON) animals died early, 2 of 8 animals in the AAV9-MHCK7 group died early, and 2 of 3 animals in the AAVrh.74-MHCK7 group died early.

[0228] All of the above U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned herein and / or listed in the Application Data Sheets are incorporated herein by reference in their entireties.

[0229] From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims

1. A polynucleotide comprising an expression cassette and optionally flanking adeno-associated virus (AAV) inverted terminal repeats (ITRs), the polynucleotide comprising a polynucleotide sequence encoding junctophilin-2 (JPH2) or a functional variant thereof, operably linked to a promoter.

2. 2. The polynucleotide of claim 1, wherein the promoter is a cardiac troponin T (hTNNT2) promoter, a myosin heavy chain creatine kinase 7 (MHCK7) promoter, a cardiac-specific promoter, a muscle-specific promoter, a cardiomyocyte-specific promoter, or a ubiquitous promoter, optionally a CMV promoter or a CAG promoter. (i) the promoter is the MHCK7 promoter, which shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 31; or (ii) the promoter is an hTNNT2 promoter that shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 33; The polynucleotide of claim 2.

4. The expression cassette (i) comprises exon 1 of the cardiac troponin T (hTNNT2) gene, wherein optionally the promoter and exon 1 of the hTNNT2 together share at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 32; and / or (ii) a polyA signal, optionally including a human growth hormone (hGH) polyA; and / or (iii) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), optionally including a WPRE(x), or a green fluorescent protein (GFP); The polynucleotide of claim 1.

5. The polynucleotide of claim 1, wherein the junctophilin-2 (JPH2) or a functional variant thereof is JPH2 or human JPH2.

6. (i) the polynucleotide sequence encoding JPH2 shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 2, 4, 6, 8, or 10; and / or (ii) the polynucleotide sequence encoding JPH2 is a human JPH2 polynucleotide; The polynucleotide of claim 1.

7. (i) comprising at least about 3.0 kb, at least about 3.2 kb, at least about 3.4 kb, at least about 3.5 kb, at least about 3.7 kb, at least about 4.0 kb, at least about 4.1 kb, at least about 4.2 kb, at least about 4.3 kb, at least about 4.4 kb, at least about 4.5 kb, at least about 4.6 kb, at least about 4.7 kb, at least about 4.8 kb, or at least about 5.0 kb; (ii) containing at most about 3.1 kb, at most about 3.3 kb, at most about 3.5 kb, at most about 3.7 kb, at most about 3.9 kb, at most about 4.1 kb, at most about 4.2 kb, at most about 4.3 kb, at most about 4.4 kb, at most about 4.5 kb, at most about 4.6 kb, at most about 4.7 kb, at most about 4.8 kb, at most about 4.9 kb, or at most about 5.0 kb; (iii) containing 4.4 kb to 5.0 kb, 4.4 kb to 4.9 kb, or 4.4 kb to 4.8 kb, or containing 4.0 kb to 4.6 kb, 4.0 kb to 4.5 kb, or 4.0 kb to 4.4 kb, or containing 4.0 kb to 4.3 kb, 4.0 kb to 4.2 kb, or 4.0 kb to 4.1 kb, or containing 3.0 kb to 3.9 kb, 3.0 kb to 3.8 kb, or 3.0 kb to 3.7 kb; or (iv) JPH2 or a functional variant thereof comprises at least 600 or at least 630 amino acids; The polynucleotide of claim 1.

8. (i) the expression cassette is flanked by 5' and 3' inverted terminal repeats (ITRs), or (ii) the expression cassette is flanked by 5' and 3' inverted terminal repeats (ITRs), and the ITRs are AAV2 ITRs and / or the ITRs share at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with any one of SEQ ID NOs: 15-21; The polynucleotide of claim 1.

9. A gene therapy vector comprising the polynucleotide of claim 1.

10. (i) The gene therapy vector is a recombinant adeno-associated virus (rAAV) vector. (ii) the gene therapy vector is a recombinant adeno-associated virus (rAAV) vector, and the rAAV vector is AAV9, AAVrh10, AAV6, or AAVrh74 or a functional variant thereof; or (iii) the gene therapy vector is a recombinant adeno-associated virus (rAAV) vector, and the rAAV vector is AAV9, AAVrhlO, AAV6, or AAVrh74 or a functional variant thereof, and the AAV vector comprises a capsid protein that shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with any one of SEQ ID NOs: 97-100; The vector of claim 9.

11. A pharmaceutical composition for treating and / or preventing a disease or disorder in a subject in need thereof, comprising the vector of claim 9.

12. (i) the disease or disorder is a cardiac disorder, optionally a cardiomyopathy selected from familial hypertrophic cardiomyopathy17, hypertrophic cardiomyopathy (HCM) (abnormal hypertrophy), and dilated cardiomyopathy (DCM); arrhythmia, optionally atrial fibrillation or sinus node disease; or heart failure; and / or (ii) the disease or disorder is associated with or caused by cleavage of JPH2 in the subject; 12. The pharmaceutical composition of claim 11.

13. (i) the subject is a mammal, a primate, or a human; and / or (ii) the subject has a mutation in the JPH2 gene or a truncating variant of JPH2; 12. The pharmaceutical composition of claim 11.

14. 12. The pharmaceutical composition of claim 11, wherein administration of the pharmaceutical composition increases JPH2 expression by (i) at least about 5%, about 30%, or about 70%, or (ii) about 5% to about 10%, about 30% to about 50%, about 50% to about 70%, or about 70% to about 100%. (i) an effective amount of the vector, or (ii) the vector having about 1×10 11 to about 1×10 13 vector genomes, the vector having about 1×10 12 to about 1×10 14 vector genomes, or the vector having about 1×10 13 to about 1×10 15 vector genomes; 12. The pharmaceutical composition of claim 11, comprising:

16. A pharmaceutical composition comprising the vector of claim 9.

17. 17. A kit comprising the vector of claim 9 or the pharmaceutical composition of claim 16 and optionally instructions for use.

18. A polynucleotide comprising a polynucleotide sequence that shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with any one of SEQ ID NOs: 26-30 or any one of SEQ ID NOs: 76-95. (i) the polynucleotide comprises a sequence encoding the MHCK7 promoter or human JPH2; or (ii) the polynucleotide comprises an MHCK7 promoter, and the MHCK7 promoter shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 31; 19. The polynucleotide of claim 18.

20. A gene therapy vector comprising the polynucleotide of claim 18. (i) the gene therapy vector is a recombinant adeno-associated virus (rAAV) vector, or (ii) The gene therapy vector is a recombinant adeno-associated virus (rAAV) vector, and the rAAV vector is an AAV9 or AAVrh74 vector; The vector of claim 20.

22. 21. A pharmaceutical composition for treating and / or preventing cardiac disorders in a subject identified as having a truncation in JPH2, comprising the vector of claim 20.

23. Heart problems, (i) cardiomyopathy, optionally familial hypertrophic cardiomyopathy, hypertrophic cardiomyopathy (HCM) (abnormal hypertrophy), or dilated cardiomyopathy (DCM); or (ii) arrhythmia, optionally atrial fibrillation or sinus node disease, familial hypertrophic cardiomyopathy, or heart failure; 23. The pharmaceutical composition of claim 22.

24. 23. The pharmaceutical composition of claim 22, wherein the subject is a mammal.

25. 23. The pharmaceutical composition of claim 11 or 22, wherein the vector is administered by intravenous injection, intracardiac injection, intracardiac infusion, and / or cardiac catheterization.

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