Methods and compositions for treating catecholaminergic polymorphic ventricular tachycardia with a viral vector

Abstract

The present disclosure relates to compositions and methods for the treatment of catecholaminergic polymorphic ventricular tachycardia (CPVT). Several embodiments provided for herein relate to virally-mediated transfer of a gene to host cells to induce expression of an encoded polypeptide, protein or other product in the host in order to ameliorate one or more symptom of CPVT. In several embodiments, the methods and compositions relate to recombinant adeno-associated virus (rAAV) vectors encoding human cardiac calsequestrin-2 (CASQ2) in order to treat CPVT.

Description

AAVAN.106WO PATENTMETHODS AND COMPOSITIONS FOR TREATING CATECHOLAMINERGIC POLYMORPHIC VENTRICULAR TACHYCARDIA WITH A VIRAL VECTORRELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Nos.63 / 745790, filed January 15, 2025, 63 / 858705, filed August 6, 2025, and 63 / 894527, filed October 6, 2025, which are hereby incorporated by reference herein in their entirety.INCORPORATION BY REFERENCE OF MATERIAL IN SEQUENCE LISTING

[0002] This application incorporates the material provided in the accompanying XML file entitled SequenceListing_AAVAN106WO.xml, created January 14, 2026, which is 30,742 bytes in size.BACKGROUND

[0003] Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a serious and life-threatening disease that primarily manifests in children in the first and second decades of life (Behere & Weindling, 2016). with a median age of disease onset between 7 and 10 years (Kallas et al., 2021; Perez-Riera et al., 2018). CPVT is an inherited cardiac arrhythmia syndrome characterized by adrenergically induced polymorphic arrhythmias in the presence of a normal resting sinus rhythm and structurally normal heart. In a normal heart, the cardiac cycle is driven by a precisely timed process called excitation-contraction coupling or calcium-induced calcium release (OCR). CPVT is caused by a loss of calcium homeostasis between the cytosolic and intracellular compartments, namely the sarcoplasmic reticulum (SR), resulting in aberrantly timed contraction. Gene therapy is contemplated as a method of treating CPVT.SUMMARY

[0004] CPVT may be caused by autosomal dominant mutations in the RYR2 gene, resulting in aberrantly timed contractions. The compositions and methods described hereinrelate to delivery of compositions that include nucleic acids encoding a functional calsequestrin 2 (CASQ2) protein to a patient suffering from or having symptoms of CPVT related to mutant RYR2.

[0005] The present disclosure relates to compositions and methods of gene therapy vectors for treating CPVT.

[0006] Some embodiments provided herein relate to an expression cassette. In some embodiments, the expression cassette includes a human calsequestrin 2 (CASQ2) coding sequence (SEQ ID NO: 9), a promoter (SEQ ID NO: 7) operably linked to the CASQ coding sequence, and an intron (SEQ ID NO: 8). In some embodiments, the CASQ2 coding sequence is codon optimized, for example, as set forth in SEQ ID NO: 9. In some embodiments, the codon optimization is not sequence specific. In some embodiments, the expression cassette is flanked by inverted terminal repeats (ITRs). In some embodiments, an upstream inverted terminal repeat includes SEQ ID NO: 6. In some embodiments, a downstream inverted terminal repeat includes SEQ ID NO: 11. In some embodiments, the expression cassette does not include intron sequences from a CASQ2 gene. In some embodiments, the expression cassette includes a 3’ untranslated region (3’ UTR), including, for example, sequences from the CASQ2 gene. In some embodiments, the expression cassette includes a polyadenylation signal (SEQ ID NO: 10). In some embodiments, the promoter includes a cardiac specific promoter. In some embodiments, the promoter includes a desmin (SEQ ID NO: 7), TNNT2, TNNI3, creatine kinase, myogenin, alpha myosin heavy chain, or natriuretic peptide promoter. In some embodiments, the promoter includes a human promoter.

[0007] Some embodiments provided herein relate to recombinant adeno-associated virus (rAAV) vectors. In some embodiments, the rAAV vectors include any of the expression cassettes described herein (SEQ ID NO: 5). In some embodiments, the rAAV vector includes a single-stranded AAV (ssAAV). In some embodiments, the rAAV vector includes a self-complementary AAV (scAAV). In some embodiments, the rAAV is serotype 8, serotype 9 or serotype rh74.

[0008] Some embodiments provided herein relate to compositions that include a pharmaceutically acceptable carrier and any of the rAAV vectors (SEQ ID NO: 5) as described herein.

[0009] Some embodiments provided herein relate to methods of treating a cardiac disorder. In some embodiments, the methods include administering to a subject in need of treatment a therapeutically effective amount of a recombinant adeno-associated virus (rAAV). In some embodiments, the rAAV includes an expression cassette including a human CASQ2 coding sequence operably linked to a promoter (SEQ ID NO: 5). In some embodiments, the expression cassette includes an intron (SEQ ID NO: 8). In some embodiments, the expression cassette includes a polyadenylation signal (SEQ ID NO: 10). In some embodiments, the expression cassette includes a 3’ UTR. In some embodiments, the expression cassette is flanked by inverted terminal repeats (ITRs) (SEQ ID NO: 6 and / or SEQ ID NO: 11). In some embodiments, administration of the rAAV results in expression of a therapeutically effective amount of human CASQ2, thereby treating the cardiac disorder. In some embodiments, the intron is not a CASQ2 intron. In some embodiments, the expression cassette does not include a CASQ2 3’ UTR sequence. In some embodiments, the expression cassette comprises an intron, such as a synthetic intron, an endogenous CASQ2 intron, a heterologous intron, e.g., pol II or pol III intron, a beta-globin intron, or the like. In some embodiments, the cardiac disorder is CPVT. hi some embodiments, the CPVT is caused by a mutation in a RYR2 gene. In some embodiments, the RYR2 mutation is R4497C. In some embodiments, the RYR2 mutation is R2474S. In some embodiments, the RYR2 mutation is D3638A. In some embodiments, the subject in need of treatment has arrhythmia. In some embodiments, the subject in need of treatment does not have arrhythmia. In some embodiments, the subject in need of treatment does not have heart failure. In some embodiments, the subject in need of treatment is asymptomatic. In some embodiments, the therapeutically effective amount of expressed human CASQ2 is expressed in cardiac tissue. In some embodiments, the therapeutically effective amount of expressed human CASQ2 improves heart rhythms. In some embodiments, the subject is a human subject. In some embodiments, the method reduces arrhythmia in the subject. In some embodiments, the method restores calcium homeostasis in the subject. In some embodiments, the method reduces delayed after polarization events. In some embodiments, the method reduces diastolic calcium leak through a RYR2 channel into a cytosol. In some embodiments, the therapeutically effective amount of expressed human CASQ2 localizes to sarcoplasmic reticulum.

[0010] Some embodiments provided herein relate to methods of increasing or upregulating expression of CASQ2 in a cell. In some embodiments, the methods include transducing the cell with a recombinant adeno- associated virus (rAAV) vector. In some embodiments, the rAAV vector includes a nucleic acid expression cassette. In some embodiments, the nucleic acid expression cassette includes a functional human CASQ2 coding sequence (SEQ ID NO: 9) operably linked to a promoter (SEQ ID NO: 7). In some embodiments, the expression cassette is flanked by ITRs (SEQ ID NO: 6 and / or SEQ ID NO: 11). In some embodiments, expression of functional human CASQ2 (SEQ ID NO: 4) is increased in the cell.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 shows a non-limiting example of an embodiment of a diagram of a rAAV-CASQ2 expression construct as described herein.

[0012] FIG. 2 shows a non-limiting example of the percentage of animals with positive ventricular tachycardia (VT) 28 and 56 days after treatment with rAAV-CASQ2.

[0013] FIG. 3 shows a non-limiting example of CASQ2 biodistribution (vg / pg of gDNA) in heart, liver, and gastrocnemius tissues from RYR2R4496C / + and WT mice 28 days after treatment with rAAV-CASQ2.

[0014] FIG. 4 shows a non-limiting example of CASQ2 mRNA (single-strand copies / 100 ng total RNA) in heart, liver, and gastrocnemius tissues from RYR2R4496C / + and WT mice 28 days after treatment with rAAV-CASQ2.

[0015] FIG. 5 shows a non-limiting example of rAAV-CASQ2 DNA (vg / pg of gDNA) in C57B1 / 6 wild type mouse hearts 14, 28, 42, 56, 84, 112, and 168 days after treatment with rAAV-CASQ2.

[0016] FIG. 6 shows a non-limiting example of CASQ2 concentration (ng / mg) in heart tissue from wild type (WT) mice 7, 14, 28, 42, 56, 84, 112, and 168 days after treatment with rAAV-CASQ2.

[0017] FIG. 7 shows a non-limiting example of dose-dependent rAAV-CASQ2 biodistribution (vg / pg of gDNA) to the heart in wild type (WT) and RYR2R4496C / +mice 3 months post-rAAV-CASQ2 dosing.

[0018] FIG. 8 shows a non-limiting example of dose-dependent rAAV-CASQ2 mRNA expression (single- strand copies / 100 ng total RNA) in the heart in wild type (WT) and RYR2R4496C / + mice 3 months post-rAAV-CASQ2 dosing.

[0019] FIG. 9 shows a non-limiting example of dose-dependent CASQ2 mRNA expression in heart, gastrocnemius, and liver in wild type and RYR2R4496C / +mice 3 months post-rAAV-CASQ2 dosing.

[0020] FIG. 10 shows a non-limiting example of the percentage of animals with sustained ventricular tachycardia (VT) 3 months post-rAAV-CASQ2 dosing in wild type (WT) and RYR2R4496C / +mice.

[0021] FIG. 11 shows non-limiting examples of echocardiograms (ECGs) from wild type (WT) and RYR2R4496C / +transgenic mice 85 days after dosing with vehicle or rAAV-CASQ2 and following challenge with P-adrenergic agents.

[0022] FIG. 12 shows a non-limiting example of rAAV-CASQ2 dose-dependent prevention of arrhythmia relative to model penetrance in wild type (WT) and RYR2R4496C / +transgenic mice.

[0023] FIG. 13A-13C show non-limiting examples of liver chemistry in non-human primates after rAAV-CASQ2 administration at Day 28: aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) (EIG. 13A); coagulation: activated partial thromboplastic time (APTT), fibrinogen (FIB), and prothrombin time (PT) (FIG. 13B); and creatine kinase (CK) and cardiac troponin I (FIG. 13C). ULN means upper limit normal. Data presented as group mean.

[0024] FIGs. 14A-14F show non-limiting examples of results obtained from an additional study in non-human primates (NHPs). FIG. 14A shows non-limiting examples of liver chemistry: alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in NHPs at Day 84 after rAAV8-CASQ2 administration. FIG. 14B. shows non-limiting examples of liver chemistry: coagulation: activated partial thromboplastic time (APTT), fibrinogen (FIB), and prothrombin time (PT). For FIGs. 14A and 14B, data is presented as group mean. FIG. 14C shows a non-limiting example of the biodistribution of rAAV8-CASQ2 3 months after administration. FIG. 14D shows a non-limiting example of the biodistribution of rAAV8-CASQ26 months after administration. FIG. 14E shows a nonlimiting example of expression of the CASQ2 transgene 6 months after administration ofrAAV8-CASQ2 to NHPs. FIG. 14F shows anon-limiting example of CASQ2 protein in NHP heart tissue at 3 and 6 months post administration of rAAV8-CASQ2.

[0025] FIG. 15 shows a non-limiting example of hCASQ2 expression and normalized against vinculin (hCASQ2 / Vinculin) over time in heart tissue from wild type (WT) mice 28, 56, 84, and 165 days after dosing with rAAV-CASQ2.

[0026] FIG. 16 shows a non-limiting example of hCASQ2 expression (ng / mg) in heart tissue from non-human primates 6 months after treatment with mid (8E13 vg / kg) and high (2E14 vg / kg) doses of rAAV-CASQ2.

[0027] FIG. 17 shows a non-limiting example of an in vitro experimental design used to elucidate the effect of RYR2 mutations on human induced pluripotent stem cell (hiPSC) cardiomyocytes.

[0028] FIG. 18A shows non-limiting examples of impedance traces of spontaneous contractions from isogenic wild type control (left) and RYR2 E2311D mutant (right) hiPSC-cardiomyocytes.

[0029] FIG. 18B shows non-limiting examples of impedance traces of spontaneous contractions from RYR2 E2311D mutant hiPSC-cardiomyocytes transduced with AAV-Luc (left) or AAV-CASQ2 (right).

[0030] FIG. 18C shows a non-limiting example of average spontaneous beat rates of isogenic control and RYR2 E2311D mutant hiPSC-cardiomyocytes across three treatment groups. Two-way ANOVA with Tukey’s multiple comparisons, n=12 wells.

[0031] FIG. 19A shows non-limiting examples of impedance traces of spontaneous contractions with isoproterenol (1 pM) treatment from isogenic wild type control (left) and RYR2 E2311D mutant (right) hiPSC-cardiomyocytes.

[0032] FIG. 19B shows non-limiting examples of impedance traces of spontaneous contractions with isoproterenol (1 pM) treatment from RYR2 E2311D mutant hiPSC-cardiomyocytes transduced with AAV-Luc (left) or AAV-CASQ2 (right).

[0033] FIG. 19C shows a non-limiting example of average spontaneous beat rates of isogenic control and RYR2 E2311D mutant hiPSC-cardiomyocytes after isoproterenol (1 pM) treatment. Two-way ANOVA with Tukey’s multiple comparisons, n=12 wells.

[0034] FIG. 19D shows a non-limiting example of beat rate regularity measured by the coefficient of variation (CoV) for isogenic control and RYR2 E2311D mutant hiPSC-cardiomyocytes after isoproterenol (1 pM) treatment. Two-way ANOVA with Tukey’s multiple comparisons, n=12 wells.

[0035] FIG. 20A shows non-limiting examples of impedance traces of contractions with isoproterenol (1 pM) and 2.5Hz pacing from isogenic wild type control (left) and RYR2 E2311D mutant (right) hiPSC-cardiomyocytes. Two-way ANOVA with Tukey’s multiple comparisons.

[0036] FIG. 20B shows non-limiting examples of impedance traces of contractions with isoproterenol (1 pM) and 2.5Hz pacing from RYR2 E2311D mutant hiPSC-cardiomyocytes transduced with AAV-Luc (left) or AAV-CASQ2 (right). Two-way ANOVA with Tukey’s multiple comparisons.

[0037] FIG. 20C shows non-limiting examples of average beat rates of isogenic control and RYR2 E231 ID mutant hiPSC-cardiomyocytes in response to 2.5Hz pacing before (left) and after (right) isoproterenol (1 pM). Dashed line at 150 bpm indicates pacing frequency. Two-way ANOVA with Tukey’s multiple comparisons, n=12 wells.

[0038] FIG. 20D shows non-limiting examples of the proportion of wells captured with 2.5Hz pacing before (left) and after (right) isoproterenol (1 pM). Wells captured out of total wells treated shown below x-axis. Chi-square statistical analysis.DETAILED DESCRIPTION

[0039] Reference is made to particular features and / or non-limiting embodiments of the disclosure. It is to be understood that the disclosure in this specification includes all possible combinations of such particular features. For example, where a particular feature is disclosed in the context of a particular aspect or embodiment, or a particular claim, that feature may also be used, to the extent possible, in combination with and / or in the context of other particular aspects and embodiments, generally.

[0040] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by those of skill in the art. All patents, applications, published applications and other publications referenced herein are incorporated by reference in their entirety unless stated otherwise. In the event that there is a plurality of definitions for a term herein, those in this section prevail unless stated otherwise.

[0041] CPVT is a serious and life-threatening disease that primarily manifests in the first and second decades of life (Behere & Weindling, 2016). CPVT is an inherited cardiac arrhythmia syndrome characterized by adrenergically induced polymorphic ventricular tachycardia in the presence of a normal resting electrocardiogram and structurally normal heart.

[0042] CPVT manifestations typically involve syncope, cardiac arrest, seizure-like events, and / or sudden cardiac death (SCD). The most common symptoms / signs include syncope (52% to 100%), cardiac arrest (8% to 48%), seizure-like events (40%), and hypoxic-ischemic encephalopathy (20%) (Leenhardt et al., 1995; Priori et al., 2002; Song et al., 2010; Sy et al., 2011). CPVT is a significant cause of sudden death at a young age and mortality is high (historically up to 50%) (Celiker et al., 2009). History of SCD among family members show that approximately 30% experience a cardiac arrest or sudden death as a first manifestation, and that 3 of every 4 children with CPVT present with life-threatening symptoms, which often occur during resting wakeful activities highlighting the unpredictable nature of CPVT (Roston et al., 2015).

[0043] CPVT may be caused by a loss of calcium homeostasis between the cytosolic and intracellular compartments, namely the sarcoplasmic reticulum (SR), resulting in aberrantly timed contraction. In a normal heart, the cardiac cycle is driven by a precisely timed process called excitation-contraction coupling or calcium-induced calcium release (CICR).

[0044] Disruption of the CICR pathway may cause abnormal calcium movement between the SR lumen and the cytosol during diastole. The increased diastolic cytosolic calcium may lead to trigger activity and subsequent membrane depolarization, which may propagate to neighboring cardiomyocytes, leading to unstable ventricular rhythms and even ventricular fibrillation.

[0045] CPVT may result from numerous mutations, including mutations in the ry anodine receptor 2 (RYR2) gene encoding the RYR2 protein (resulting in CPVT type 1) or the calsequestrin 2 (CASQ2) gene. Mutations in different functional domains of the RYR2 protein may result in defects in the calcium pore, tetramerization, or activation via cytosolic or lumen binding sights, for example.

[0046] Mutations in RYR2 may cause CPVT by destabilizing the RYR2 channel resulting in more time spent in the open conformation leading to aberrant calcium movement from the SR lumen into the cytosol during diastole and disruption of the cardiac cycle.

[0047] Patients with CPVT are at risk of sudden cardiac death (SCD). Consequently, there is a continuous psychological burden of illness and a significant impact on the quality of life of affected children, adults, and caregivers. Even in the absence of symptoms, CPVT patients experience restrictive and debilitating effects on their quality of life. Besides athletic activities, which patients are typically counselled to avoid (a significant challenge in the pediatric population), CPVT patients are at risk of syncopal events and SCD following acute emotional stress.

[0048] To date, there are no medicines approved that address the underlying cause of CPVT. Pharmacotherapy requires patients regularly, without interruption, to adhere to their prescribed pharmacological therapy (a particularly difficult challenge in children) as a single missed dose could allow the occurrence of a potentially lethal arrhythmia.

[0049] Current treatment options offer limited cardiac protection, leaving patients vulnerable to breakthrough cardiac events and in need of a curative gene therapy approach. Embodiments provided herein relate to methods for increasing CASQ2 levels. In some embodiments, the methods include stabilizing RYR2 in a closed state. In some embodiments, the methods serve as a dynamic luminal SR calcium storage, thereby preventing Ca+ release during diastole. In some embodiments, the methods relate to CASQ2 overexpression in cardiomyocytes to correct beta-adrenergic stimulated-induction of ventricular tachycardia (VT) in CPVT RYR2 mutations. In some embodiments, efficacy of the methods was assessed using hiPSC-derived cardiomyocytes carrying a disease relevant pathogenic mutation (RYR2E2311D). Without being bound by theory, increased CASQ2 levels enhance buffering of free Ca2+in the sarcoplasmic reticulum (SR) resulting in stabilization of RYR2 in its closed conformation making diastolic Ca2+leak through the RYR2 channel into the cytosol less likely in context of RYR2 variants. Stabilization of RYR2 in its closed conformation supports maintenance of normal cardiac rhythm and protects against triggered activity and arrhythmias.

[0050] The term “isoform” has its ordinary meaning as understood in light of the specification and may include a member of a set of highly similar proteins that originate from a single gene or gene family. Protein isoforms may be formed from alternative splicings orvariable promoter usages. Through RNA splicing, mRNA has the ability to select different protein-coding segments (exons) of a gene, or even different parts of exons from RNA to form different mRNA sequences. Each unique sequence produces a specific form of a protein commonly referred to as an isoform.

[0051] The term “coding sequence” has its ordinary meaning as understood in light of the specification and may include part of a gene’s DNA or RNA that codes for a protein. A coding sequence is made up of triplets of nucleotides also known as codons. Each codon encodes an amino acid in the protein. A coding sequence usually starts with an AUG or ATG initiation codon and ends with a stop codon.

[0052] The term “subject” has its ordinary meaning as understood in light of the specification and may include a mammal that is the object of treatment using a method or composition as provided for herein. “Mammal” includes, without limitation, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cows, horses, primates, such as monkeys, chimpanzees, and apes, and humans. In some embodiments, the subject is human.

[0053] The terms “treating,” “treatment,” “therapeutic,” or “therapy” have their ordinary meaning as understood in light of the specification and do not necessarily mean total cure or abolition of the disease or condition. Any alleviation of any undesired signs or symptoms of a disease or condition, to any extent, may be considered treatment and / or therapy. To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject. Treatment may also include reversing damage done to a tissue or tissues in the body. Treatment may stimulate tissue regeneration or halt continued degeneration of a tissue. Treatment efficacy may be determined by conducting tests and comparing results to baselines measured before treatment was administered. In some cases, treatment may be determined by conducting tests and comparing results to results from individuals who have not been treated and may or may not be in need of treatment. Non-limiting examples of tests that may be administered to determine treatment efficacy include tests that measure molecules in blood or urine and physical tests that measure heart function such as an electrocardiogram, ejection fraction percentages, end-diastolic volume (EDV) and end-systolic volume (ESV). Tests may also measure expression levels and / or localization of the protein being expressed from the rAAV vector. Treatmentefficacy may also be evaluated by measuring levels of an endogenous mutated gene product, including a protein and / or an RNA.

[0054] The term “effective amount.” as used herein, has its ordinary meaning as understood in light of the specification and may include an amount that is capable of treating or improving a disease or condition or otherwise capable of producing an intended therapeutic effect, such as reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0055] The term “nucleic acid” sequence has its ordinary meaning as understood in light of the specification and may include a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequence. The term captures sequences that include any of the known base analogues of DNA and RNA such as, but not limited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxyl-methyl) uracil, 5 -fluorouracil, 5 -bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine. N6-isopentenyladenine, 1-methyladenine, 1 -methylpseudouracil, 1-methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5 -methylaminomethyluracil, 5 -methoxy- aminomethyl -2-thiouracil, beta-D-mannosylqueosine, 5 ’-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-5 -oxy acetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5 -methyluracil, N- uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.

[0056] The term “polynucleotide,” has its ordinary meaning as understood in light of the specification and may include a polymeric form of nucleotides of any length, including DNA, RNA, or analogs thereof. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment described herein, a polynucleotide, encompasses both the double-stranded formand each of two complementary single-stranded forms known or predicted to make up the double-stranded form.

[0057] The term “identity” has its ordinary meaning as understood in light of the specification and may include an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) may be compared by determining their “percent identity.” The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100.

[0058] For the purpose of describing the relative position of nucleotide sequences in a particular nucleic acid molecule throughout the instant application, such as when a particular nucleotide sequence is described as being situated “upstream,” “downstream,” “3’,” or “5”’ relative to another sequence, it is to be understood that it is the position of the sequences in the “sense” or “coding” strand of a DNA molecule that is being referred to as is conventional in the art.

[0059] Sequence identity may be determined by aligning sequences using algorithms, such as BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, (Genetics Computer Group, 575 Science Dr., Madison, Wis.), using default gap parameters, or by inspection, and the best alignment (for example, resulting in the highest percentage of sequence similarity over a comparison window). Percentage of sequence identity is calculated by comparing two optimally aligned sequences over a window of comparison, determining the number of positions at which the identical residues occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of matched and mismatched positions not counting gaps in the window of comparison (for example, the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Unless otherwise indicated the window of comparison between two sequences is defined by the entire length of the shorter of the two sequences.

[0060] The term “recombinant,” has its ordinary meaning as understood in light of the specification and may include a polynucleotide that is the product of various combinations of cloning, restriction or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature and / or a combination of polynucleotides andviral proteins that is not found in nature. A “recombinant virus” is a viral particle comprising a “recombinant polynucleotide.” The terms respectively include replicates of the original polynucleotide construct and progeny of the original virus construct.

[0061] The term “gene,” has its ordinary meaning as understood in light of the specification and may include a polynucleotide containing at least one open reading frame that is capable of encoding a particular gene product. Any of the polynucleotide sequences described herein may be used to identify larger fragments or full-length coding sequences of the genes with which they are associated. Methods of isolating larger fragment sequences are known to those of skill in the art.

[0062] The term “transgene,” has its ordinary meaning as understood in light of the specification and may include a nucleic acid sequence to be positioned within a viral vector and encoding a polypeptide, protein or other product of interest. The transgenes of the present disclosure relate to the improvement of one or more heart conditions, such as cardiomyopathies as provided for herein. In some embodiments, the sequence is codon-optimized.

[0063] As used herein “codon optimized” has its ordinary meaning as understood in light of the specification and may refer to nucleic acid encoding the polypeptide, protein or other product of interest that includes a codon-optimized nucleotide sequence. Codon optimization may be performed to improve expression, stability, translational efficiency, or other properties in a selected host cell or organism. Such codon-optimized sequences may differ in nucleotide sequence from a reference or naturally occurring sequence while encoding the same amino acid sequence, and are not limited to any particular nucleotide sequence. Codon optimization may take into account codon usage bias, GC content, mRNA secondary structure, regulatory motifs, or other sequence features relevant to the intended expression system. In some embodiments, the sequences described herein are not limited to any specific nucleotide sequence and may include sequence variants, including codon-optimized variants, that encode the same or a functionally equivalent amino acid sequence. Codon optimization may be performed using any suitable method or algorithm and may be tailored to a particular host cell, tissue, or organism. Such variants are expressly contemplated as within the scope of the disclosure, provided that the encoded polypeptide, protein or other product of interest retains the desired functional activity. In some embodiments, the sequences described herein include a synthetic or engineered sequence designed for improved expression relative to a wild-type or reference sequence. The disclosure expressly encompasses sequences that are optimized, altered, or modified at the codon level without altering the encoded amino acid sequence or biological function.

[0064] The terms “gene transfer” or “gene delivery” have their ordinary meaning as understood in light of the specification and may include methods or systems for inserting DNA, such as a transgene, into host cells, such as those of a subject afflicted with a cardiomyopathy. In several embodiments, gene transfer yields transient expression of nonintegrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g., episomes). In additional embodiments, gene transfer results in integration of transferred genetic material into the genomic DNA of host cells.

[0065] The terms “regulatory element” or “regulatory sequence,” or variations thereof, have their ordinary meaning as understood in light of the specification and may include a nucleotide sequence that participates in functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. Regulatory elements may be enhancing or inhibitory in nature, depending on the embodiment. Non-limiting examples of regulatory elements include transcriptional regulatory sequences such as promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (“IRES”), enhancers, and the like. These elements collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell, though not all of these sequences need always be present. It shall be appreciated that the structural components of a rAAV vector as provided for herein may be listed in individual paragraphs solely for clarity and may be used together in combination. For example, any regulatory element or other component may be used in combination with any transgene (or transgenes) provided for herein.

[0066] The term “promoter” has its ordinary meaning as understood in light of the specification and may include a polynucleotide that interacts with an RNA polymerase and initiates transcription usually located downstream (in the 3' direction) from the promoter. Promoters may initiate transcription of a coding region (e.g., a transgene) and / or a non-coding region such as a non-coding RNA (e.g. microRNA).

[0067] The term “operably linked” has its ordinary meaning as understood in light of the specification and may include an arrangement of elements wherein the components are configured to perform a function. For example, regulatory sequences operably linked to a coding sequence result in the expression of the coding sequence. Depending on the embodiment, a regulatory sequence need not be contiguous with the coding sequence. Thus, for example, one or more untranslated, yet transcribed, sequences may be present between a promoter sequence and a coding sequence, with those two sequences still being considered “operably linked.”

[0068] The term “intron” has its ordinary meaning as understood in light of the specification and may include the sequence of DNA between exons that is initially copied into RNA, but is cut out of the final mature messenger RNA transcript. Introns do not code for amino acids that make up the protein coded for by the gene. Introns are removed from the transcribed RNA by the process of splicing. A chimeric intron in a plasmid refers to a synthetic intron sequence created by combining different parts of different introns from various genes. In plasmids, chimeric introns may enhance gene expression in eukaryotic cells. Chimeric introns may or may not be in the coding sequence of a transgene. Chimeric introns may be inserted in various positions within the plasmid such as between the promoter and start codon or after the stop codon.

[0069] The term “vector” has its ordinary meaning as understood in light of the specification and may include any molecular vehicle, such as a plasmid, phage, transposon, cosmid, chromosome, virus, viral particle, virion, etc. that may transfer gene sequences (e.g., a transgene) to or between cells of interest.

[0070] The term “contacting” as used herein has its ordinary meaning as understood in light of the specification and may include an interaction between an rAAV or an rAAV vector and a cell that results in the transfer of the genetic material through the cell membrane and into the cell where it is expressed and / or has a biological effect. The mechanisms that result in the rAAV or rAAV vector being transferred through the cell membrane include but are not limited to transduction and transfection.

[0071] The term “expression vector” has its ordinary meaning as understood in light of the specification and may include a vector comprising a region of nucleic acid (e.g., a transgene) that encodes a gene product (e.g., a polypeptide or protein) of interest. In someembodiments, vectors are used for achieving expression, e.g., stable expression, of a protein in an intended target cell. An expression vector may also comprise control elements operatively linked to the transgene to facilitate expression of the encoded protein in the target cell. “Expression construct” and “expression vector” may be used interchangeably.

[0072] The term “expression cassette” as used herein has its ordinary meaning as understood in light of the specification and may include a gene or genes together with the combination of one or more regulatory elements to which the gene(s) are operably linked and which promote and / or regulate expression of the gene(s). A promoter is considered to be “operably linked” to a nucleic acid sequence when the promoter is in a functional location and orientation relative to the sequence such that the promoter is able to initiate transcription of that sequence. Examples of regulatory elements that may be included in an expression cassette include but are not limited to promoters, introns, polyadenylation signals and 3’ untranslated regions (3’ UTRs). Expression cassettes may be components of plasmids. Expression cassettes may also be flanked by AAV inverted terminal repeats (ITRs) and be components of rAAVs.

[0073] The term “AAV” is an abbreviation for adeno-associated virus and has its ordinary meaning as understood in light of the specification and may include the virus itself or derivatives thereof. The term AAV covers all subtypes and both naturally occurring and recombinant forms, unless otherwise indicated. The abbreviation “rAAV” refers to recombinant adeno-associated virus, also referred to as “recombinant AAV vector” or “rAAV vector.” A recombinant AAV comprises a polynucleotide sequence not of AAV origin (e.g., a transgene). The term “AAV” includes AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3). AAV serotype 4 (AAV4), AAV serotype 5 (AAV5). AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), serotype rhlO (AAVrhlO), serotype rh74 (AAVrh74), AAV-SLB101, or a pseudotyped rAAV (e.g., AAV2 / 9. referring an AAV vector with the genome of AAV2 (e.g., the ITRs of AAV2) and the capsid of AAV9) or modifications thereof. In some embodiments, the serotype for delivery to human patients affected by a cardiomyopathy is one of AAV9, serotype rh74, serotype rhlO, AAV-8, or AAV-SLB 101. In some embodiments, the AAV is engineered to be cardiotropic. In some embodiments, the cardiotropic AAV is engineered to comprise an RGD amino acid sequence in its capsid.

[0074] The term “AAV” or “AAV particle” or “rAAV particle” have their ordinary meaning as understood in light of the specification and may include a viral particle composed of at least an AAV capsid protein and an encapsidated polynucleotide.

[0075] The term “heterologous” has its ordinary meaning as understood in light of the specification and may include genotypically distinct origins. For example, a heterologous polynucleotide is one derived from a different species as compared to a reference species (for example a human gene inserted into a viral plasmid is a heterologous gene). A promoter removed from its native coding sequence and operatively linked to a coding sequence with which it is not naturally found linked is a heterologous promoter. In some embodiments, a heterologous promoter may also be referred to as a recombinant promoter. A promoter that drives the expression of its native or natural gene is referred to as a native promoter. Other genetic elements that may also be referred to as native elements because they are used to regulate their endogenous genes, include but are not limited to introns, 5’ UTRS, 3’ UTRS, and poly adenylation signals.

[0076] As used herein, the term “kit” has its ordinary meaning as understood in light of the specification and may include variations of a portable, self-contained enclosure that includes at least one set of components to conduct one or more of the diagnostic or therapeutic methods of the present disclosure.

[0077] The term “earner” has its ordinary meaning as understood in light of the specification and may include a diluent, adjuvant, excipient, or vehicle with which the rAAV particle or preparation, and / or rAAV vectors is administered. Such pharmaceutical carriers may be sterile liquids. For example, saline solutions and aqueous dextrose and glycerol solutions may be employed as liquid carriers.

[0078] Terms and phrases used in this application, and variations thereof, especially in the appended claims, unless otherwise expressly stated, should be construed as open ended as opposed to limiting. As examples of the foregoing, the term ‘including’ should be read to mean ‘including, without limitation,’ ‘including but not limited to,’ or the like; the term ‘comprising’ as used herein is synonymous with ‘including,’ ‘containing,’ or ‘characterized by,’ and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps; the term ‘having’ should be interpreted as ‘having at least;’ the term ‘includes’ should be interpreted as ‘includes but is not limited to;’ the term ‘example’ is usedto provide exemplary instances of the item in discussion, not an exhaustive or limiting list thereof; and use of terms like ‘preferably,’ ‘preferred,’ ‘desired,’ or ‘desirable,’ and words of similar meaning should not be understood as implying that certain features are critical, essential, or even important to the structure or function, but instead as merely intended to highlight alternative or additional features that may or may not be utilized in a particular embodiment. In addition, the term “comprising” is to be interpreted synonymously with the phrases “having at least” or “including at least.” When used in the context of a process, the term “comprising” means that the process includes at least the recited steps, but may include additional steps. When used in the context of a compound, composition or device, the term “comprising” means that the compound, composition, or device includes at least the recited features or components, but may also include additional features or components. Likewise, a group of items linked with the conjunction ‘and’ should not be read as requiring that each and every one of those items be present in the grouping, but rather should be read as ‘and / or’ unless expressly stated otherwise. Similarly, a group of items linked with the conjunction ‘or’ should not be read as requiring mutual exclusivity among that group, but rather should be read as ‘and / or’ unless expressly stated otherwise.

[0079] With respect to the use of substantially any plural and / or singular terms herein, those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for sake of clarity. The indefinite article “a” or “an” does not exclude a plurality. A single processor or other unit may fulfill the functions of several items recited in the claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

[0080] The ranges disclosed herein also encompass any and all overlap, sub-ranges, and combinations thereof. Language such as “up to,” “at least.” “greater than,” “less than,” “between,” and the like includes the number recited. Numbers preceded by a term such as “about” or “approximately” include the recited numbers. For example, “about 90%” includes “90%. ” In some embodiments, at least 95% homologous or identical includes 96%, 97%, 98%, 99%, and 100% homologous or identical to the reference sequence. In addition, when asequence is disclosed as “comprising” a nucleotide or amino acid sequence, such a reference shall also include, unless otherwise indicated, that the sequence “comprises,” “consists of’ or “consists essentially of’ the recited sequence.

[0081] A non-limiting embodiment of the vector genome is provided in FIG. 1. rAAV-CASQ2 (including for example, an embodiment having a sequence as set forth in SEQ ID NO: 5) is a recombinant adeno-associated viral (rAAV) vector designed to deliver a functional CASQ2 coding sequence (SEQ ID NO: 9). In some embodiments, a nucleic acid sequence of the rAAV vector includes a promoter (SEQ ID NO: 7). In some embodiments, a nucleic acid sequence includes a polyadenylation signal (SEQ ID NO: 10). In some embodiments, the polyadenylation signal is a synthetic minimal poly adenylation signal. In some embodiments, rAAV-CASQ2 is used to treat CPVT. In some embodiments, the vector genome is modified with embodiments of regulatory elements (for example, a different promoter) and / or modifications described herein. In some embodiments, modified versions of the vector genome in FIG. 1 are used to treat subjects with CPVT.

[0082] In some embodiments, a single stranded genome includes two AAV2 ITRs (SEQ ID NO: 6 and SEQ ID NO: 11) at each end of the expression cassette. In some embodiments, each ITR is a palindromic sequence. In some embodiments, a tertiary structure and recognition sequences of the ITR direct the viral Rep protein and host cell proteins to replicate and encapsidate vector genomes.

[0083] In some embodiments, an expression cassette includes a human promoter element, a human intron element, a codon optimized human CASQ2 coding sequence (SEQ ID NO: 9) encoded by the human CASQ2 exons (native intron sequences are removed), and a polyadenylation signal (SEQ ID NO: 10). In some embodiments, a DES promoter (SEQ ID NO: 7) governs transgene expression primarily in muscle tissue and has been shown to be active in the myocardium. In some embodiments, codon optimization of the CASQ2 open reading frame is designed to maximize expression of the CASQ2 coding sequence in human cells. In some embodiments, the CASQ2 coding sequence is CpG depleted.

[0084] In some embodiments, the rAAV-CASQ2 comprises a recombinant, nonreplicating, adeno-associated virus vector encoding a codon optimized human calsequestrin 2 (CASQ2) (SEQ ID NO: 9) under control of the human promoter, preceded by a human intron,and followed by a polyadenylation sequence, flanked on each side by adeno-associated virus inverted terminal repeats (ITRs) (SEQ ID NO: 6 and SEQ ID NO: 11) such as AAV2.Treatment of CPVT

[0085] In some embodiments, rAAV-CASQ2 (SEQ ID NO: 5) is administered through systemic IV administration and transduction of cardiomyocytes. In some embodiments, rAAV-CASQ2 is administered by intramuscular injections (e.g., cardiac muscle). In some embodiments, 1AAV-CASQ2 is administered directly to the heart. In some embodiments, the expression cassette is maintained episomally in the nucleus where the CASQ2 exogenous coding sequence is transcribed, the messenger ribonucleic acid (mRNA) is exported from the nucleus and translated at the ribosome, and mature CASQ2 protein is shuttled to the SR lumen following normal cellular pathways.

[0086] In some embodiments, overexpression of CASQ2 in subjects with CPVT caused by mutations in RYR2 may buffer free SR luminal calcium and increase diastolic RYR2 closed state probability, such that diastolic calcium leak through the RYR2 channel into the cytosol is less likely. In some embodiments, this mechanism supports maintenance of normal cardiac rhythm and protects against triggered RYR2 channel activity and arrhythmias.

[0087] In some embodiments, subjects with CPVT caused by mutations in RYR2, are administered rAAV-CASQ2 (SEQ ID NO: 5) to overexpress exogenous CASQ2 protein (SEQ ID NO: 4) which may function in tandem with wild type (WT) CASQ2. In some embodiments, polymerized CASQ2 binds RYR2 and helps maintain closed state probability, while increasing levels of SR luminal CASQ2 protein, which may bind a higher level of calcium and buffer against and / or prevent diastolic calcium leak through the RYR2 channel that would otherwise lead to trigger activity and arrhythmic events.

[0088] In some embodiments, a subject may have one or more mutation that give rise to CPVT. In some embodiments, a mutation in the RYR2 gene may result in an increase in the release of cytosolic calcium in cardiac myocytes that are contacted with a RYR2 agonist relative to a cardiac myocyte that is not contacted with the RYR2 agonist. In some embodiments, mutation in RYR2 includes one or more of the following mutations: R4497C, N4104K, N4895D, R176Q, G178A, G203C, R420W, L433P, P1067S, S2246L, G2273R, F2307L, E2311D, P2328S, L2344P, R2474S, T2504M, D3638A, K3717R, L3778F, G3926D,G3946S, Q4159P, V4319-K4324dup, R4651I, V4653F, V4771I, F4851L, A4860G, I4867M, P4902S, and / or R4959Q. Others are E189D, G230C, G357S, A165D, R2401H, N23861, R2267H, Q4201R, S4153R, R4497C, S4565R, K4750Q, I4855M, R414L, I419F, P164S, A77V, R414C, V186M, P164S, R169Q, L62F, M81L, P164S, E243K, F329L, R332W, V377M, G357S, T415R, R420Q, V507I, A549V, S616L, H240R, D242V, E243K, R169L, R739H, R1013Q, R1051P, A1136V. T1107M, E1724K, E1837K. E2045G, V1810L, V2306I, A2387P, A2403T, L2487I, A2254V, A2394G, V2475F, R2359Q, R2404T, V2113M, Y2156C, H2168Q, E2183V, D2216V, E2296Q, F2307L, V2321M, R2404T, R2420W, M2389L, H2217Y, C2402Y, G2337V, L2527W, E2296K, V2193L, C2277R, G3037D, Q4201R, N4097S, E4146K, T4158P, F4020L, E4076K, N4104I, H4108N, H4108Q, S3938R, T4196A, L4105F, R4144C, L3879P, Q3925E, G3946A, S3959L, M3972I, D3973H. L3974Q, K3997E, S4124G, Y4149s, R4157Q, Q4159P, N4178S, E4187Q, S3799P, G3946D, D3977Y, A4091V, A4091T, F4174L, A4282V, R4307C, G4315E, R4497C, N4895D, E4950K, N4504I, A4608P, V4880A, M4504I, A4607P, F4499C. A4510T, G4671R. I4848V. A4556T, 4657-4658EY insertion, G4671R, G4662S, H4762P, P4902S, F4511LE, 4431K, E4611K, S4565R, E4611K, W4645R, K4650E, N4736, Del, R4790Q, K4805R, R4822H, G4936R, G4671V, and / or D4631V. In some embodiments, the subject may have two or more of the RYR2 mutations as set forth above. In some embodiments, the subject may have one or more CPVT-related mutation.

[0089] In some embodiments, a transgene is employed to correct, reduce, eliminate, or otherwise ameliorate gene deficiencies in which normal genes are expressed at less than normal levels or are expressed at normal or near-normal levels but have a gene product with abnormal activity, or deficiencies in which the functional gene product is not expressed. In several embodiments, the transgene sequence encodes a therapeutic protein or polypeptide which is to be expressed in a host cell.Regulatory Elements

[0090] In some embodiments, the rAAV vector includes one or more regions comprising a sequence that facilitates expression of the heterologous nucleic acid, e.g., regulatory sequences operatively linked to the heterologous nucleic acid. In some embodiments, a promoter drives transcription of the nucleic acid sequence that it regulates andmay be located at or near the transcriptional start site of a gene. Tn some embodiments, a promoter has a length of 100 to 3000 nucleotides. In some embodiments, a promoter has a length of 100 to 3000 nucleotides or length between 100 and 3000 nucleotides, including for example 300, 400, 450, 500, 600, 800, 1000 or 2000 nucleotides or another length in between 100 and 3000 nucleotides. In some embodiments, a promoter is operably linked to a nucleic acid or a sequence of a nucleic acid (nucleotide sequence).

[0091] Promoters that may be used in accordance with the present disclosure may include any promoter that may drive the expression of the transgenes in a tissue of the subject. In some embodiments, the promoter may be a tissue- specific promoter, such as for the heart. In some embodiments, the promoter that may be used in accordance with the present disclosure is a cardiac-specific promoter. Non-limiting examples of tissue-specific promoters and / or regulatory elements that may be used include TNNT2, desmin, TNNI3, creatine kinase, myogenin, alpha myosin heavy chain, natriuretic peptide, cardiac troponin C, cardiac troponin I, and cardiac troponin T (cTnT). Non-limiting examples of sequence elements that regulate gene expression include promoters, insulators, silencers, response elements, introns, enhancers, initiation sites, termination signals, and poly(A) signals and poly(A) tails. Any combination of such regulatory sequences is contemplated herein (e.g., a promoter and an enhancer).

[0092] In some embodiments, the promoter may be, without limitation, a promoter from one of the following genes: TNNT2, desmin (SEQ ID NO: 7), a-myosin heavy chain gene, 6- myosin heavy chain gene, myosin light chain 2v (MLC-2v) gene, myosin light chain 2a gene, CARP gene, cardiac a-actin gene, cardiac m2 muscarinic acetylcholine gene, atrial natriuretic factor gene (ANF), cardiac sarcoplasmic reticulum Ca-ATPase gene, skeletal a-actin gene; or an artificial cardiac promoter derived from MLC-2v gene (for example, as set forth in SEQ ID NO: 19). In some embodiments, the promoter is a promoter from a human form of a gene. In some embodiments, the promoter is less than 3 kb. In some embodiments, the promoter comprises 3 kb or 2 kb or 1 kb or 0.5 kb of the promoter or sizes between these values. In some embodiments, the promoter comprises a TNNT2 promoter, including, for example, TNNT2p-607 (SEQ ID NO: 12). TNNT2p-600 (SEQ ID NO: 13), TNNT2p-500 (SEQ ID NO: 14), TNNT2p-455 (SEQ ID NO: 15), TNNT2p-400 (SEQ ID NO: 16), TNNT2p-300 (SEQ ID NO: 17). In some embodiments, the promoter comprises the C5-C12 promoter(SEQ TD NO: 18). Tn some embodiments, the promoter comprises the MLCv promoter (SEQ ID NO: 19).

[0093] Synthetic promoters are also contemplated herein. In some embodiments, synthetic promoter may comprise, for example, regions of known promoters, regulatory elements, transcription factor binding sites, enhancer elements, repressor elements, and the like.

[0094] In some embodiments, the expression vector includes one or more introns. In some embodiments, the intron is located between the promoter and the start codon. In some embodiments, the intron is a synthetic intron, an endogenous CASQ2 intron, a heterologous intron, e.g., pol II or pol III intron, human beta globin intron (SEQ ID NO: 8), or the like. In some embodiments, the intron is a chimeric intron. In some embodiments, the intron is an intron from a virus. In some embodiments, the intron is flanked by exons of the protein being expressed. In some embodiments, the expression vector includes one or more CASQ2 introns. In some embodiments, the expression vector does not include introns from the CASQ2 gene. In some embodiments, the expression vector includes one or more introns that are not CASQ2 introns.

[0095] In some embodiments, the rAAV vectors of the present disclosure comprise a polyadenylation (pA) signal. Non-limiting poly adenylation signals include nucleotide sequences comprising, for example, a polyadenylation signal from human growth hormone (hGH), a polyadenylation signal from simian virus 40 (sV40 late), a synthetic polyadenylation signal from rabbit beta-globin (SPA), a polyadenylation signal from bovine growth hormone (bGH), a polyadenylation signal from the human beta globin gene (SEQ ID NO: 8), truncations of any of the foregoing, and combinations of the foregoing. In some embodiments, the expression vector includes a 3’ untranslated region (3’ UTR). In some embodiments, the 3’ UTR is from the human CASQ2 gene. In some embodiments, the 3’ UTR is not from the human CASQ2 gene. In some embodiments, the 3’ UTR is from any suitable gene known to those of skill in the art, including, for example, the globin gene (including, for example, a beta globin gene, such as a human beta globin gene, an alpha globin gene, or a zeta globin gene), an actin gene, a tubulin gene, a microtubule gene, a synapsin gene, or variants thereof, or from other suitable genes.Expression Cassette

[0096] In some embodiments, an expression cassette includes a transgene and its regulatory sequences. In some embodiments, the cassette is designed to be expressed from a rAAV. In some embodiments, the expression cassette is flanked by 5' (SEQ ID NO: 6) and 3' (SEQ ID NO: 11) AAV ITRs. In some embodiments, the ITRs are full-length, or one or both of the ITRs may be truncated. In some embodiments, the AAV capsid is from a different AAV serotype than the AAV serotype from which the ITRs are derived. In some embodiments, the ITRs of AAV serotype 2 are used. In some embodiments, the ITRs of AAV serotype 1 are used. However, ITRs from other suitable sources may be selected. Some embodiments include at least one or a plurality of spacer sequences inserted within the construct. Additionally, a construct may comprise any number of promoter or regulatory sequences to alter or change the expression of a CASQ2 coding sequence. In some embodiments, from the 5’ to 3’ end, the AAV genome comprises an AAV ITR (SEQ ID NO: 6), promoter (SEQ ID NO: 7), intron (SEQ ID NO: 8), transgene (SEQ ID NO: 9), a poly A signal (SEQ ID NOTO), and a second ITR (SEQ ID NO: 11). In some embodiments capsid protein VP1 is set forth in SEQ ID NO: 1. In some embodiments, capsid protein VP2 is set forth in SEQ ID NO: 2. In some embodiments, capsid protein VP3 is set forth in SEQ ID NO: 3.

[0097] FIG. 1 depicts an embodiment of a construct described herein (including for example, a construct having a sequence as set forth in SEQ ID NO: 5). At the 5’ end, an AAV ITR (IRL-L) (SEQ ID NO: 6) and promoter (SEQ ID NO: 7) are present. An intron (SEQ ID NO: 8) is disposed between the promoter and the CASQ2 transgene (SEQ ID NO: 9). The construct further comprises a polyadenylation site (SEQ ID NO: 10) following the CASQ2 transgene (SEQ ID NO: 9) and a 3’ ITR (ITR-R) (SEQ ID NO: 11). The constructs provided herein can further comprise one or a plurality of spacer (stuffer) sequences inserted at any point within the construct, such as between one or more of the sequences depicted in FIG. 1.

[0098] In some embodiments, the expression cassette comprises a CASQ2 transgene (SEQ ID NO: 9) and associated regulatory sequences but does not include a region modulating endogenous CASQ2 gene expression. In some embodiments, a construct comprising the expression cassette with the functional CASQ2 transgene is administered. In some embodiments, the expression of the functional CASQ2 transgene is sufficient to providetherapeutic benefits to a subject. In some embodiments, the amino acid sequence of CASQ2 is set forth in SEQ ID NO: 4.Vector

[0099] Further provided herein are rAAV particles or rAAV preparations containing such particles. In several embodiments, rAAV particles comprise a viral capsid (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3) and one or more transgene as described herein, which is encapsidated by the viral capsid. Methods of producing rAAV particles are known in the art and are commercially available (see, e.g., Zolotukhin et al. Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors. Methods 28 (2002) 158-167; and U.S. Patent Application Publication Numbers US 2007 / 0015238 and US 2012 / 0322861, which are incorporated herein by reference; and plasmids and kits available from ATCC and Cell Biolabs, Inc.). For example, a plasmid containing the rAAV vector may be combined with one or more helper plasmids, e.g., that contain a rep gene (e.g., encoding Rep78, Rep68, Rep52 and Rep40) and a cap gene (encoding VP1, SEQ ID NO: 1, VP2, SEQ ID NO: 2, and VP3 SEQ ID NO: 3, including a modified VP3 region as described herein), and transfected into a producer cell line such that the rAAV particle may be packaged and subsequently purified.

[0100] The rAAV particles or particles within an rAAV preparation disclosed herein may be of any AAV serotype, including any derivative or pseudotype (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 2 / 1, 2 / 5, 2 / 8, 2 / 9, 3 / 1, 3 / 5, 3 / 8, or 3 / 9). As used herein, the serotype of an rAAV or an rAAV particle refers to the serotype of the capsid proteins of the recombinant virus. In some embodiments, the rAAV particle is rAAV6, rAAV8 or rAAV9. In some embodiments, the rAAV particle is rAAV8. In some embodiments, the rAAV particle is rAAV6. In some embodiments, the rAAV particle is rAAVrh74. Non-limiting examples of derivatives, pseudotypes, and / or other vector types include, but are not limited to, AAVrh.10, AAVrh74, AAV2 / 1, AAV2 / 5, AAV2 / 6, AAV2 / 8, AAV2 / 9, AAV2-AAV3 hybrid, AAVhu.14, AAV3a / 3b, AAVrh32.33, AAV-HSC15, AAV- HSC17, AAVhu.37, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC15 / 17, AAVM41, AAV9.45, AAV6(Y445F / Y731F), AAV2.5T, AAV-HAE1 / 2, AAV clone 32 / 83, AAVShHIO, AAV2 (Y->F), AAV8 (Y733F), AAV2.15, AAV2.4, AAVM41, and AAVr3.45.

[0101] In some embodiments, the AAV is an AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8). AAV serotype 9 (AAV9), serotype rhlO AAV, serotype rh74 AAV, or a pseudotyped rAAV (e.g., AAV2 / 9, referring an AAV vector with the genome of AAV2 (e.g., the ITRs of AAV2) and the capsid of AAV9

[0102] In some embodiments, the AAV is an AAV serotype 8 vector. In some embodiments, the AAV has about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity to an AAV serotype 8 vector, or a percent identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the AAV has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 70%-75%, 75%-100%, 75%-95%, 75%-90%, 75%-75%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to an AAV serotype 8 vector. In some embodiments, the AAV is an AAV serotype rh74 vector. In some embodiments, the AAV has about 70%. 75%, 80%, 85%, 90%, 95%, or 100% identity to an AAV serotype rh74 vector, or a percent identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the AAV has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 70%-75%, 75%-100%, 75%-95%, 75%-90%, 75%-75%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to an AAV serotype rh74 vector .In some embodiments, the AAV is an AAV serotype 9 vector. In some embodiments, the AAV has about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity to an AAV serotype 9 vector, or a percent identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the AAV has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 70%-75%, 75%-100%, 75%-95%, 75%-90%, 75%-75%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to an AAV serotype 9 vector.

[0103] Certain AAV serotypes and derivatives / pseudotypes, and methods of producing such derivatives / pseudotypes are known in the art (see, e.g., Mol Ther. 2012 Apr;20(4):699- 708. doi: 10.1038 / mt.2011.287. Epub 2012 Jan 24. The AAV vector toolkit: poised at the clinical crossroads. Asokan Al, Schaffer DV, Samulski RJ.). Methods for producing and using pseudotyped rAAV vectors are known in the art (see, e.g., Duan et al. J.Virok, 75:7662-7671, 2001; Halbert et al, J. Virol., 74:1524-1532, 2000; Zolotukhin et al, Methods, 28:158-167, 2002; and Auricchio et al., Hum. Molec. Genet., 10:3075-3081, 2001).

[0104] In some embodiments, the rAAV vectors of the present disclosure further comprise a polyadenylation (pA) signal. In some embodiments, the pA signal is from a globin gene (including, for example, a beta globin gene, such as a human beta globin gene (SEQ ID NO: 8). an alpha globin gene, or a zeta globin gene), an actin gene, a microtubule gene, SV40, a growth hormone gene, a polyoma virus, or a thymidine kinase gene, or variants thereof, or from other suitable genes. In some embodiments, the pA signal is from the human CASQ2 gene.

[0105] In some embodiments, the rAAV vectors of the present disclosure comprise at least, in order from 5’ to 3’, a first adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence (SEQ ID NO: 6), a promoter (SEQ ID NO: 7) operably linked to a transgene (SEQ ID NO: 9), an intron (SEQ ID NO: 8) disposed between the promoter and the transgene; a polyadenylation signal (SEQ ID NO: 10), and a second AAV inverted terminal repeat (ITR) sequence (SEQ ID NO: 11). In some embodiments, the inverted terminal repeat (ITR) sequences have sequences as set forth in SEQ ID NO: 6 and / or SEQ ID NO: 11 or have at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 6 and / or SEQ ID NO: 11. In some embodiments, the promoter includes a cardiac specific promoter. In some embodiments, the promoter includes a human Desmin promoter. In some embodiments, the human Desmin promoter has a sequence as set forth in SEQ ID NO: 7 or has at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 7. In some embodiments, the expression cassette includes a naturally occurring intron. In some embodiments, the intron is disposed between the promoter and the CASQ2 coding sequence. In some embodiments, the intron is 3’ of the coding sequence. In some embodiments, the intron has a sequence as set forth in SEQ ID NO: 8 or has at least about 85%, 90%, 95%. 96%. 97%, 98%, or 99% sequence identity to SEQ ID NO: 8. In some embodiments, the expression cassette further includes a transgene encoding CASQ2. In some embodiments, the CASQ2 transgene is codon optimized and has a sequence as set forth in SEQ ID NO: 9 or has at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 9. In some embodiments, the CASQ2 sequence is wild type and not codon optimized. In some embodiments, the expression cassette further includes a polyadenylation signal. In someembodiments, the polyadenylation signal has a sequence as set forth in SEQ ID NO: 10 or has at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 10.

[0106] Some embodiments provided herein relate to recombinant adeno-associated virus (rAAV) vectors. In some embodiments, the rAAV vectors include any of the polynucleotides as described herein. In some embodiments, the rAAV vector is rAAV-CASQ2 and has a sequence as set forth in SEQ ID NO: 5 or has at least about 85%, 90%. 95%. 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 5. In some embodiments, the rAAV vector genome is circular, hi some embodiments, the rAAV vector genome is linear. In some embodiments, the rAAV vector genome is single- stranded. In some embodiments, the rAAV vector genome is double- stranded. In some embodiments, the rAAV genome vector is a self-complementary rAAV vector.Recombinant Adeno-Associated Virus Vectors and Therapeutic Use Thereof

[0107] Many serotypes of AAV have been cloned and sequenced. Of the first six AAV serotypes, serotype 2 is widely characterized and therefore often used in gene transfer studies. Embodiments provided herein include various AAV serotypes, including, for example AAV8, AAV9, AAV20, AAVrh74, AAVrhlO, and the like. In some embodiments, administration of a given serotype may be performed in connection with an immune management regimen. In some embodiments, an immune management regimen includes administration of one or more agents that function, for example, as B cell depletors, alone, or in conjunction with one or more agents that, for example, inhibit one or more aspects of the mTOR pathway. In some embodiments, an anti-CD20 antibody is administered. In some embodiments, rapamycin is administered. In some embodiments, a steroid, such as prednisone, methylprednisolone, or the like is administered alone or in combination with the above agents. In several embodiments, this allows for the administration of a given serotype rAAV with reduced, limited or no immune response to a subsequent dosing of the rAAV.

[0108] The therapeutic rAAV vectors, therapeutic rAAV particles, or the composition comprising the therapeutic rAAV particles of the present disclosure, may be used in a gene therapy for heart diseases in a human subject in need thereof, such as CPVT as provided for herein. The therapeutic rAAV vectors, particles, and compositions comprising the therapeutic rAAV particles may be used for treatment of such catecholaminergic polymorphicventricular tachycardia (CPVT) when administered to a subject in need thereof, e.g., via intravenous delivery, vascular delivery into the coronary arteries and / or direct injection to the heart. The therapeutic rAAV vectors, particles, and compositions comprising the rAAV particles drive the concurrent expression of CASQ2 in the cardiomyocytes of the subject.

[0109] In some embodiments, there are provided amino acid sequences that correspond to any of the nucleic acids disclosed herein, while accounting for degeneracy of the nucleic acid code. Furthermore, those sequences (whether nucleic acid or amino acid) that vary from those expressly disclosed herein (and / or included in the accompanying sequence listing), but have functional similarity or equivalency are also contemplated within the scope of the present disclosure. The foregoing includes mutants, truncations, substitutions, or other types of modifications.

[0110] In accordance with some embodiments described herein, any of the sequences may be used, or a truncated or mutated form of any of the sequences disclosed herein (and / or included in the accompanying sequence listing) may be used and in any combination.

[0111] The promoter driving expression of the therapeutic nucleic acid may be, but is not limited to, a constitutive promoter, an inducible promoter, a tissue-specific promoter, a muscle-specific promoter, or a synthetic promoter. In some embodiments, the promoter is a muscle-specific promoter. A constitutive promoter may be, but is not limited to, a Herpes Simplex virus (HSV) promoter, a thymidine kinase (TK) promoter, a Rous Sarcoma Virus (RSV) promoter, a Simian Virus 40 (SV40) promoter, a Mouse Mammary Tumor Virus (MMTV) promoter, an Adenovirus E1A promoter, a cytomegalovirus (CMV) promoter, a mammalian housekeeping gene promoter, or a [3-actin promoter. An inducible promoter may be, but is not limited to, a cytochrome P450 gene promoter, a heat shock protein gene promoter, a metallothionein gene promoter, a hormone-inducible gene promoter, an estrogen gene promoter, or a tetVP16 promoter that is responsive to tetracycline. A muscle- specific promoter may be, but is not limited to, desmin promoter, a creatine kinase promoter, a myogenin promoter, an alpha myosin heavy chain promoter, or a natriuretic peptide promoter.

[0112] In some embodiments, the therapeutic rAAV promoter comprises a muscle-or cardiomuscle-specific promoter.

[0113] In some embodiments, the therapeutic rAAV includes serotype 1, serotype 2, serotype 3, serotype 4, serotype 5, serotype 6, serotype 7, serotype 8, serotype 9, serotype10, serotype 11, serotype 12, serotype rhlO, or serotype rh74. The therapeutic rAAV can also be a pseudo-type rAAV, including pseudo-type rAAV of any of the above rAAVs.Pharmaceutical Formulations and Administration

[0114] Compositions described herein may further include a pharmaceutically acceptable excipient, buffer, carrier, diluent or other medicinal agents or combinations thereof, and may be formulated for administration to a host cell ex vivo or in situ or in vivo in an animal, and particularly a human being. In some embodiments, compositions may further comprise a liposome, a lipid, a lipid complex, a microsphere, a microparticle, a nanosphere, and / or a nanoparticle, or may be otherwise formulated for administration to the cells, tissues, organs, or body of a subject in need thereof. Such compositions may be formulated for use in a variety of therapies, such as for example, in the amelioration, prevention, and / or treatment of conditions such as peptide deficiency, polypeptide deficiency, peptide overexpression, polypeptide overexpression, including for example, conditions which result in diseases or disorders as described herein.

[0115] Formulations including pharmaceutically-acceptable excipients and / or carrier solutions are well known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., oral, intravenous, parenteral, intra-arterial, intramuscular (e.g., cardiac muscle), intravitreal, intranasal, intra-articular, subcutaneous and intraperitoneal administration and formulation.

[0116] In some embodiments, formulations may contain at least about 0.1% of the therapeutic agent (e.g., therapeutic rAAV particle or preparation) or more, although the percentage of the active ingredient(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 70% or 90% or more of the weight or volume of the total formulation. Naturally, the amount of therapeutic agent(s) in each therapeutically useful composition may be prepared in such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art when preparing such pharmaceutical formulations. Additionally, a variety of dosages and treatment regimens may be desirable.

[0117] In some embodiments, the therapeutic rAAV particles or preparations may be delivered in suitably formulated pharmaceutical compositions disclosed herein; either subcutaneously, intracardially, intraocularly, intravitreally, parenterally, subcutaneously, intravenously, intracerebro-ventricularly, intramuscularly, intrathecally, orally, intraperitoneally, by oral or nasal inhalation, or by direct injection to one or more cells (e.g„ cardiomyocytes and / or other heart cells), tissues, or organs. In some embodiments, the therapeutic rAAV particles or the composition comprising the therapeutic rAAV particles described herein are delivered systemically via intravenous injection, particularly in those for treating a human. In some embodiments, the therapeutic rAAV particles or the composition comprising the therapeutic rAAV particles described herein are injected directly into the heart of the subject. Direct injection to the heart may comprise injection into one or more of the myocardial tissues, the cardiac lining, or the skeletal muscle surrounding the heart, for example, using a needle catheter. In some embodiments, direct injection is into the heart, for example, if delivery is performed concurrently with a surgical procedure or interventional procedure whereby access to the heart is improved. In some embodiments, the interventional procedure includes any procedure wherein coronary or pulmonary perfusion is altered. In some embodiments, the interventional procedure includes one or more of percutaneous administration, catheterization, or coronary retroperfusion.

[0118] Other aspects of the present disclosure relate to methods and preparations for use with a subject, such as human or non-human subjects, a host cell in situ in a subject, or a host cell derived from a subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is a companion animal. “A companion animal.” as used herein, refers to pets and other domestic animals. Non-limiting examples of companion animals include dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and other animals such as mice, rats, guinea pigs, and hamsters. In some embodiments, the subject is a human subject.

[0119] In some embodiments, one or more pharmaceutically acceptable excipients (including vehicles, carriers, diluents, and / or delivery polymers) are added to the pharmaceutical compositions including a therapeutic, thereby forming a pharmaceutical formulation suitable for in vivo delivery to a subject, such as a human.

[0120] Some embodiments herein relate to the use of one or more excipients, for example, but not limited to, amino acids, salts, base buffers, detergents, and, optionally, stabilizers, in a formulation to promote recovery of a sample from the formulation following one or more stressors, for example, but not limited to, agitation stress, shear stress, freeze-thaw stress, thermal stress, or any combination therein, as compared to the same formulation without the one or more excipients. In some embodiments, the one or more excipients in the formulation promote recovery of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%. 94%, 95%, 96%, 97%, 98%, 99%, or 100% more of a sample from the formulation buffer following from agitation stress, shear stress, freeze-thaw stress, thermal stress, or any combination therein, as compared to the same formulation without the one or more excipients.

[0121] As described herein, the inclusion of one or more amino acids may make a sample resistant to one or more stressors including, but not limited to, agitation stress, shear stress, freeze-thaw stress, thermal stress, or any combination therein, as compared to the same formulation without the one or more amino acid. Some embodiments herein relate to the use of one or more amino acid in a formulation to promote recovery from and / or resistance to one or more stressor, for example, but not limited to, agitation stress, shear stress, freeze-thaw stress, thermal stress, or any combination therein, as compared to the same formulation buffer without the one or more amino acid. In some embodiments, the one or more amino acid in the formulation promotes recovery of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% more of a sample from the formulation buffer following agitation stress, shear stress, freeze-thaw stress, thermal stress, or any combination therein, or an amount of sample that is in a range defined by any two of the preceding values.

[0122] In some embodiments, the one or more amino acid in the formulation promotes increased sample resistance to freeze-thaw (FT) cycling, as compared to the same formulation buffer without the one or more amino acids. In some embodiments, the one or more amino acids in the formulation promotes increased resistance of a sample in the formulation to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 freeze-thaw cycles, or increased resistance to a number of FT cycles that are in a range defined by any two of the preceding values, as compared to the same formulation without the one or more amino acids. In some embodiments,the formulation promotes recovery of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% more of a sample from the formulation buffer following 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 FT cycles, or an amount of sample that is in a range defined by any two of the preceding values.

[0123] In some embodiments, the one or more amino acids in the formulation promote increased sample resistance to agitation, as compared to the same formulation without the one or more amino acids. In some embodiments, the one or more amino acids in the formulation promote recovery of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% more of a sample from the formulation buffer following agitation at about 1 rpm, 2 rpm, 3 rpm, 4 rpm, 5 rpm, 6 rpm, 7 rpm, 8 rpm, 9 rpm, 10 rpm, 20 rpm, 25 rpm, 30 rpm, 40 rpm, 50 rpm, 60 rpm, 70 rpm, 75 rpm, 80 rpm, 90 rpm, 100 rpm, 125 rpm, 150 rpm, 175 rpm, 200 rpm, 250 rpm, 300 rpm, 400 rpm, or 500 rpm, as compared to the same formulation buffer without the one or more amino acids.

[0124] In some embodiments, the one or more amino acid in the formulation buffer promote increased sample resistance to thermal stress as compared to the same formulation without the one or more amino acids. In some embodiments, the one or more amino acids in the formulation promote recovery of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% more of a sample from the formulation following exposure to temperatures of about 1°C, 2°C, 3°C. 4°C, 5°C, 6°C, 7°C. 8°C, 9°C, 10°C, 20°C, 25°C, 30°C. 40°C, 45°C, 50°C, 55°C, or 60°C, as compared to the same formulation without the one or more amino acids.

[0125] In some embodiments, the formulations disclosed herein comprise one or more amino acid. In some embodiments, the one or more amino acid increase the stress resistance of the formulation as compared to the stress resistance of the same formulation without the one or more amino acid. For example, in some embodiments, the one or more amino acid increases resistance to agitation stress, shear stress, freeze-thaw stress, thermal stress, or any combination therein. In some embodiments, the one or more amino acid comprises alanine, arginine, glutamic acid, glycine, methionine, proline, and / or serine. In some embodiments, the formulation comprises about 0.1 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM,5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 75 mM, 80 mM, 90 mM, 100 mM, 125 mM, 250 mM, 175 mM, or 200 mM alanine, arginine, glutamic acid, glycine, methionine, proline, and / or serine, or an amount of alanine, arginine, glutamic acid, glycine, methionine, proline, and / or serine that is in a range defined by any two of the preceding values. For example, in some embodiments, the formulation comprises between about 0.1-200 mM, 0.1-150 mM, 0.1-100 mM, 0.1-75 mM, 0.1-50 mM, 0.1-25 mM, 0.1-10 mM, 0.1-1 mM, 1-200 mM, 1-250 mM, 1-100 mM, 1-75 mM, 1-50 mM, 1-25 mM. 1-10 mM, 10-200 mM, 10-150 mM. 10-100 mM, 10-75 mM, 10-50 mM, 10-25 mM, 25-200 mM, 25-150 mM, 25-100 mM, 25-75 mM, 25-50 mM, 50-200 mM, 50-150 mM, 50-100 mM, 100-200 mM, 100-150 mM, or 150-200 mM, alanine, arginine, glutamic acid, glycine, methionine, proline, and / or serine. In some embodiments, the one or more amino acid comprises arginine. In some embodiments, the formulation comprises about 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 60 mM, or 70 mM, arginine, or an amount of arginine in a range defined by any two of the preceding values. For example, in some embodiments, the formulation comprises between about 20-70 mM, 20-60 mM, 20-50 mM, 20-40 mM, 20-30 mM, 30-70 mM, 30-60 mM, 30-50 mM, 30-40 mM, 40-70 mM, 40-60 mM, 40-50 mM, 50-70 mM, 50-60 mM, or 60-70 mM arginine. In some embodiments, the one or more amino acid comprises proline. In some embodiments, the formulation comprises about 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 60 mM, or 70 mM proline, or an amount of proline that is in a range defined by any two of the preceding values. For example, in some embodiments, the formulation comprises between about 20-70 mM, 20-60 mM, 20-50 mM, 20-40 mM, 20-30 mM, 30-70 mM. 30-60 mM, 30-50 mM, 30-40 mM, 40-70 mM, 40-60 mM, 40-50 mM, 50-70 mM, 50-60 mM, or 60-70 mM proline. In some embodiments, the one or more amino acid comprises arginine and proline. In some embodiments, the formulation comprises about 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 60 mM, or 70 mM, arginine, or an amount of arginine that is in a range defined by any two of the preceding values and about 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 60 mM, or 70 mM, proline, or an amount of proline that is in a range defined by any two of the preceding values. For example, in some embodiments, the formulation comprises between about 20-70 mM, 20-60 mM, 20-50 mM, 20-40 mM, 20-30 mM, 30-70 mM, 30-60 mM, 30-50 mM, 30-40 mM, 40-70 mM, 40-60 mM, 40-50 mM, 50-70 mM, 50-60 mM, or 60-70 mM arginine and about 20-70 mM, 20-60 mM, 20-50 mM, 20-40mM, 20-30 mM, 30-70 mM, 30-60 mM, 30-50 M, 30-40 mM, 40-70 mM, 40-60 mM, 40-50 mM, 50-70 mM, 50-60 mM, or 60-70 mM proline.

[0126] In some embodiments, the formulation comprises one or more base buffer. In some embodiments, the one or more base buffer comprise acetate, citrate, histidine, phosphate, succinate, Tris, and / or Tris HC1. In some embodiments, the formulation comprises about 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, or 25 mM, of one or more of acetate, citrate, histidine, phosphate, succinate, and / or Tris, or an amount of acetate, citrate, histidine, phosphate, succinate, Tris, and / or Tris HC1, that is in a range defined by any of the preceding values. In some embodiments, the one or more base buffer comprises Tris. In some embodiments, the formulation comprises about 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, or 25 mM, Tris and / or Tris HC1 or an amount of Tris and / or Tris HC1 that is in a range defined by any of the preceding values. In some embodiments, the formulation comprises about 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, or 25 mM, or an amount of Tris or Tris HC1, that is in a range defined by any of the preceding values. In some embodiments, the formulation comprises about 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, or 25 mM, tris and Tris-HCl in combination or in an amount of Tris and Tris HCL, that is in a range defined by any of the preceding values. In several embodiments, the buffer comprises about 20 mM Tris. In some embodiments, the buffer comprises 20 mM Tris. In some embodiments, the buffer comprises about 20 mM of Tris and Tris-HCl in combination. In some embodiments, the buffer comprises 20 mM of Tris and Tris-HCl in combination.

[0127] In some embodiments, the formulation comprises one or more salts. In some embodiments, the one or more salt comprises MgC12 and / or NaCl. In some embodiments, the formulation comprises about 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 57 mM, 60 mM, 70 mM, 75 mM, 80 mM, 90 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, or 250mM MgC12 and / or NaCl, or an amount of MgC12 and / or NaCl that is in a range defined by any two of the preceding values. In someembodiments, the one or more salt comprises MgC12. In some embodiments, the formulation comprises about 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 2 mM, 3 mM, 4 mM, or 5 mM MgC12, or an amount of MgC12 that is in a range defined by any two of the preceding values. For example, in some embodiments, the formulation comprises between about 0.1-5 mM, 0.1-3 mM, 0.1-1 mM, 0.1-0.5 mM, 0.5-5 mM, 0.5-3 mM, 0.5-1 mM, 1-5 mM, 1-3 mM, or 3-5 mM MgC12. In some embodiments, the formulation comprises about 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 57 mM, 60 mM, 70 mM, 75 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 125 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 175 mM, 180mM, 190 mM, 200 mM, or 250 mM NaCl, or an amount of NaCl that is in a range defined by any two of the preceding values. In some embodiments, the one or more salt in the formulation promotes recovery of a sample from the formulation following exposure to one or more stressor, for example, but not limited to, agitation stress, shear stress, freeze-thaw stress, thermal stress, or any combination therein. In some embodiments, the stability of the formulation under forced degradation conditions due to storage at rapid temperature shits and multiple freeze-thaw cycles is improved by the addition of sodium chloride.

[0128] In some embodiments, the formulation comprises one or more detergent. In some embodiments, the formulation comprises one or more non-ionic detergent. In some embodiments, the formulation comprises one or more ionic, anionic, and / or cationic detergent. In some embodiments, the formulation comprises one or more polysorbate, for example, but not limited to, polysorbate 20, 40, 60, 65, and / or 80. In some embodiments, the formulation comprises one or more sorbitan, for example, but not limited to sorbitan stearate, sorbitan laurate, sorbitan sesquioleate, sorbitan oleate, sorbitan tristearate, sorbitan palmitate and / or sorbitan trioleate. In some embodiments, the formulation comprises one or more poloxamer, for example, but not limited to, poloxamer 68. 88. 98, 108. 124. 188, 237, 338, and 407. In some embodiments, the detergent comprises sodium lauryl sulfate. In some embodiments, the formulation comprises benzalkonium chloride. In some embodiments, the formulation comprises centrimonium bromide. In some embodiments, the formulation comprises cocamidopropyl betaine. In some embodiments, the formulation comprises sodium cocoamphoacetate. In some embodiments, the one or more detergent comprises P188 and / or PS80. In some embodiments, the formulation comprises about 0.001%, 0.002%, 0.003%,0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1 %, P188 and / or polysorbate 80 (PS80), or an amount of P188 and / or PS80 that is in a range defined by any two of the preceding values.

[0129] In some embodiments, the formulation comprises one or more stabilizer. In some embodiments, the one or more stabilizer comprise HSA, cyclodextrin, sucrose, and / or sorbitol. In some embodiments, the formulation comprises about 0.00%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, HSA, cyclodextrin, sucrose, and / or sorbitol, or an amount of HSA, cyclodextrin, sucrose, and / or sorbitol in a range defined by any two of the preceding values. In some embodiments, the formulation comprises about 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, or 300 mM, HSA, cyclodextrin, sucrose, and / or sorbitol, or an amount of HSA, cyclodextrin, sucrose, and / or sorbitol that is in a range defined by any two of the preceding values.

[0130] In some embodiments, the pH of the formulation is maintained. In some embodiments, the formulation comprises a pH between about 7 and 9. In some embodiments, the formulation comprises a pH of about 7.0, 7.1. 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, or a pH that is in a range defined by any two of the preceding values. In several embodiments, the formulation has a pH between 7.5 and 8.5. In several embodiments, the formulation has a pH between 7.7 and 8.3. In several embodiments, the formulation has a pH of about 8. In several embodiments, the formulation has a pH of 8.

[0131] The rAAVs or pharmaceutical compositions as described herein may be formulated for administration to host cell ex vivo or in situ in an animal, including a human being. In some embodiments, a plurality of injections, or other means of administration, are provided, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more injections. Means of administration may be combined, if desired. In some embodiments, administration of the dosing is intramuscular.

[0132] In some embodiments, the number of rAAV particles administered to a subject may range from about 103to about 1016particles / mL, such as. for example, about 103, 104, 105, 106, 107, 108, 109, 1010, 1011, 1012, 1013, 1014, 1015, or 1016particles / mL. In someembodiments, the number of rAAV particles administered to a subject may range from about 1012to about 1016particles / mL. In some embodiments, the number of rAAV particles administered to a subject may range from about 1013to about 1015particles / mL. In some embodiments, the number of rAAV particles administered to a subject may range from about 103to about 1016vector genomes (vg) / kg, such as for example, about 103, 104, 105, 106, 107, 108. 109, IO10, 1011, 1012, 1013, 1014, 1015, or 1016vg / kg. In some embodiments, the number of rAAV particles administered to a subject may range from about 1012to about 1016vg / kg. In some embodiments, the number of rAAV particles administered to a subject may range from about 1013to about 1015vg / kg. The rAAV particles may be administered as a single dose or divided into two or more doses as required to ameliorate the particular disease or disorder being treated.

[0133] The pharmaceutical formulations of the compositions suitable for injectable or intravenous use include sterile aqueous solutions or dispersions. In some embodiments, the formulation is sterile and fluid to the extent that easy syringability exists. In some embodiments, the form is stable under the conditions of manufacture and storage, and is preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, saline, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, vegetable oils or other pharmaceutically acceptable carriers such as those that are Generally Recognized as Safe (GRAS) by the United States Food and Drug Administration. Proper fluidity may 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 dispersion and by the use of surfactants. In fact, there is virtually no limit to other components that may also be included, as long as the additional agents do not cause a significant adverse effect upon contact with the target cells or host tissues. The therapeutic rAAV particles or preparations may thus be delivered along with various other pharmaceutically acceptable agents as required in the particular instance. Such compositions may be purified from host cells or other biological sources, or alternatively may be chemically synthesized as described herein.

[0134] The amount of a therapeutic rAAV particle or preparation and / or a therapeutic rAAV vector composition included in a pharmaceutical composition and time of administration of such compositions will be within the purview of the skilled artisan havingbenefit of the present teachings. Tt is likely, however, that the administration of therapeutically-effective amounts of the compositions of the present disclosure may be achieved by a single administration, such as for example, a single injection of sufficient numbers of infectious particles to provide therapeutic benefit to the patient undergoing such treatment. In some circumstances, it may be desirable to provide multiple or successive administrations of the rAAV particle or preparation, and / or rAAV vector compositions, either over a relatively short, or a relatively prolonged period of time, as may be determined by the medical practitioner overseeing the administration of such compositions.

[0135] Toxicity and efficacy of the compositions utilized in methods described herein may be determined by standard pharmaceutical procedures, using either cells in culture or experimental animals to determine the LD50 (the dose lethal to 50% of the treated population) and the ED50 (the dose that produces a beneficial therapeutic response in 50% of the treated population). The dose ratio between toxicity and efficacy is the therapeutic index and it may be expressed as the ratio LD50 / ED50. Those compositions that exhibit large therapeutic indices are preferred. While compositions that exhibit toxic side effects may be used, care should be taken to design a delivery system that minimizes the potential damage of such side effects. The dosage of compositions as described herein lies generally within a range that includes an ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.

[0136] In some embodiments, one or more pharmaceutically acceptable excipients (including vehicles, carriers, diluents, and / or delivery polymers) are added to the pharmaceutical compositions including a therapeutic, thereby forming a pharmaceutical formulation suitable for in vivo delivery to a subject, such as a human.

[0137] A pharmaceutical composition or medicament includes a pharmacologically effective amount of at least one of the therapeutic and optionally one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients (excipients) are substances other than the Active Pharmaceutical Ingredient (API, therapeutic product) that are intentionally included in the drug delivery system. Excipients do not exert or are not intended to exert a therapeutic effect at the intended dosage. Excipients may act to a) aid in processing of the drag delivery system during manufacture, b) protect, support or enhance stability, bioavailability or patient acceptability of the API, c) assist in productidentification, and / or d) enhance any other attribute of the overall safety, effectiveness, of delivery of the API during storage or use. A pharmaceutically acceptable excipient may or may not be an inert substance.

[0138] Excipients include, but are not limited to absorption enhancers, antiadherents, anti-foaming agents, anti-oxidants, binders, buffering agents, carriers, coating agents, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, and suspending agents.

[0139] The carrier may be, but is not limited to, a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

[0140] The rAAVs or pharmaceutical compositions as described herein, may be formulated for administration to a host cell ex vivo or in situ or in vivo in an animal, and particularly a human being. The rAAVs or pharmaceutical compositions may be administered by a variety of routes. Administration routes include, but are not limited to, oral, intravenous, parenteral, intra-arterial, intramuscular (e.g., cardiac muscle), intravitreal, intranasal, intraarticular, subcutaneous and intraperitoneal administration and / or local delivery to a target tissue. In some embodiments, a plurality of injections, or other administration types, are provided, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more injections. Routes of administration may be combined, if desired. In some embodiments, the dosing is intramuscular administration.

[0141] For administration of an injectable aqueous solution, for example, the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intra-arterial, intramuscular, intravitreal, subcutaneous and intraperitoneal administration. In this connection, a sterile aqueous medium that may be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 mL of isotonic NaCl solution and either added to 1000 mL of hypodermoclysis fluid or injected at the proposed site of infusion, (see, for example, “Remington’s Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). In some embodiments, the rAAV formulations comprise, consist of, or consist essentially of active rAAV ingredient, a mono-basic buffer (e.g., sodium phosphate mono-basic buffer, a dibasic salt (e.g., sodium phosphate di-basic), a sodium-based tonicifier (e.g., sodium chloridetonicifier), a non-sodium tonicifier (e.g., magnesium chloride hexahydrate tonicifier), a surfactant (e.g., poloxamer 188 surfactant), and water. In some embodiments, the rAAV formulations comprise, consist of. or consist essentially of active rAAV ingredient, sodium phosphate mono-basic buffer, sodium phosphate di-based, sodium chloride tonicifier, magnesium chloride hexahydrate tonicifier, poloxamer 188 surfactant, and water. In some embodiments, the active rAAV ingredient is present in the formulation according to the vector genome amounts provided for herein. In some embodiments, the mono-basic buffer (e.g., sodium phosphate mono-basic buffer) is present in the formulation at a concentration between about 0.2 mg / mL and about 0.5 mg / mL. In some embodiments, the di-basic salt (e.g., sodium phosphate di-basic) is present in the formulation at a concentration between about 1.5 mg / mL and about 4 mg / mL. In some embodiments, the sodium-based tonicifier (e.g.. sodium chloride tonicifier) is present in the formulation at a concentration between about 8 mg / mL and about 12 mg / mL. In some embodiments, the non-sodium tonicifier (e.g., magnesium chloride hexahydrate tonicifier) is present in the formulation at a concentration between about 0.1 mg / mL and about 0.35 mg / mL. In some embodiments, the surfactant (e.g., poloxamer 188 surfactant) is present in the formulation at a concentration between about 0.05 mg / mL and about 0.8 mg / mL. In some embodiments, water is present to bring the volume of the formulation (e.g. a dosage unit) to 1 mL.

[0142] Some variation in dosage may occur depending on the condition of the subject being treated. The person responsible for administration may determine the appropriate dose for the individual subject. Moreover, for human administration, preparations may be prepared that meet sterility, pyrogenicity, and the general safety and purity standards as required by, e.g., FDA Office of Biologies standards.

[0143] Sterile injectable solutions may be prepared by incorporating the rAAV particles or preparations in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle that contains the basic dispersion medium and the other ingredients from those enumerated above. In some embodiments, sterile powders for the preparation of sterile injectable solutions are prepared by vacuum-drying and freeze-drying techniques, which yielda powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0144] The administration of therapeutically-effective amounts of the rAAV particles or preparations of the present disclosure may be achieved by a single administration, such as for example, a single injection of sufficient numbers of infectious particles to provide therapeutic benefit to the patient undergoing such treatment. Alternatively, in some circumstances, it may be desirable to provide multiple or successive administrations of the rAAV particle or preparation, either over a relatively short or a relatively prolonged period of time, as may be determined by the medical practitioner overseeing the administration of such compositions.

[0145] If desired, rAAV particles may be administered in combination with other agents as well, such as, e.g., proteins or polypeptides or various pharmaceutically-active agents, including one or more administrations of therapeutic polypeptides, biologically active fragments, or variants thereof. In fact, there is virtually no limit to other components that may also be included, as long as the additional agents do not cause a significant adverse effect upon contact with the target cells or host tissues. The rAAV particles or preparations may thus be delivered along with various other pharmaceutically acceptable agents as required in the particular instance. Such compositions may be purified from host cells or other biological sources or chemically synthesized as described herein.

[0146] In some embodiments, treatment of a subject with a rAAV particles as described herein achieves one, two, three, four, or more of the following effects, including, for example: (i) reduction or amelioration the severity of disease or symptom associated therewith; (ii) reduction in the duration of a symptom associated with a disease; (iii) protection against the progression of a disease or symptom associated therewith; (iv) regression of a disease or symptom associated therewith; (v) protection against the development or onset of a symptom associated with a disease; (vi) protection against the recurrence of a symptom associated with a disease; (vii) reduction in the hospitalization of a subject; (viii) reduction in the hospitalization length; (ix) an increase in the survival of a subject with a disease; (x) a reduction in the number of symptoms associated with a disease; (xi) an enhancement, improvement, supplementation, complementation, or augmentation of the prophylactic or therapeutic effect(s) of another therapy.

[0147] As is apparent to those skilled in the art in view of the teachings of this specification, an effective amount of viral vector to be added may be empirically determined. Administration may be administered in a single dose, a plurality of doses, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosages of administration are well known to those of skill in the art and will vary with the viral vector, the composition of the therapy, the target cells, and the subject being treated. Single and multiple administrations may be carried out with the dose level and pattern being selected by the treating physician.Kits

[0148] Herein are described compositions including one or more of the disclosed rAAV vectors comprised within a kit for diagnosing, preventing, treating or ameliorating one or more symptoms of a heart disease or condition, such as catecholaminergic polymorphic ventricular tachycardia (CPVT) . Such kits may be useful in the diagnosis, prophylaxis, and / or therapy of a human disease, and may be particularly useful in the treatment, prevention, and / or amelioration of one or more symptoms of heart disease, such as CPVT. In some embodiments, the CPVT is caused by or associated with a mutation in an RYR2 allele in the subject’ s genome. In some embodiments, the kit comprises reagents for an assay to detect a mutation in an RYR2 coding sequence in a subject.

[0149] Kits comprising one or more of the disclosed rAAV vectors (as well as one or more virions, viral particles, transformed host cells or pharmaceutical compositions comprising such vectors); and instructions for using such kits in one or more therapeutic, diagnostic, and / or prophylactic clinical embodiments are also provided according to several embodiments. Such kits may comprise one or more reagents, restriction enzymes, peptides, therapeutics, pharmaceutical compounds, or means for delivery of the composition(s) to host cells, or to an animal (e.g., syringes, injectables, and the like). Depending on the embodiment, kits include those for treating, preventing, or ameliorating the symptoms of a disease, deficiency, dysfunction, and / or injury, or may include components for the large-scale production of the viral vectors themselves.

[0150] In some embodiments, a kit includes one or more containers or receptacles comprising one or more doses of any of the therapeutic compositions provided herein. Suchkits may be therapeutic in nature. Tn some embodiments, the kit comprises a unit dosage, meaning a predetermined amount of a composition comprising, for example, a therapeutic composition described herein.

[0151] In some embodiments, one or more of the components of a kit are provided in one or more liquid or frozen solvents. In some embodiments, the solvent is aqueous or nonaqueous. In some embodiments, formulations in the kit are provided as dried powder(s) or in lyophilized form that may be reconstituted upon addition of an appropriate solvent.

[0152] In some embodiments, a kit comprises a label, marker, package insert, bar code and / or reader indicating directions of suitable usage of the kit contents. In some embodiments, the kit may comprise a label, marker, package insert, bar code and / or reader indicating that the kit contents may be administered in accordance with a certain dosage or dosing regimen to treat a subject.

[0153] In addition, a kit may also contain various reagents, including, but not limited to, wash reagents, elution reagents, and concentration reagents. Such reagents may be readily selected from among the reagents described herein, and from among conventional concentration reagents.

[0154] As used herein, the term “kit” may be used to describe variations of the portable, self-contained enclosure that includes at least one set of components to conduct one or more of the diagnostic or therapeutic methods described herein.Combination Therapies

[0155] Some embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

[0156] The compositions of the present disclosure may include rAAV particles or preparations, and / or rAAV vectors, either alone or in combination with one or more additional active ingredients, which may be obtained from natural or recombinant sources or chemically synthesized. In some embodiments, rAAV particles or preparations are administered incombination, either in the same composition or administered as part of the same treatment regimen.

[0157] If desired, rAAV particles may be administered in combination with other agents used for the treatment of CPVT, such as, e.g., proteins or polypeptides or various pharmaceutically-active agents. This may, in some embodiments, reflect for example one or more administrations of therapeutic polypeptides, (e.g.. a recombinant form of a functional peptide or protein that aids to replace or supplement the rAAV-based production of protein encoded by the transgene) biologically active fragments, or variants thereof. The rAAV particles or preparations may thus be delivered along with various other pharmaceutically acceptable agents as required in the particular instance. Such compositions may be purified from host cells or other biological sources, or alternatively may be chemically synthesized as described herein.

[0158] In some embodiments, the additional therapeutic agent comprises an antiinflammatory agent. In some embodiments, an anti-inflammatory agent includes a corticosteroid, cortisone hydrocortisone, hydrocortisone-21 -monoesters (e.g., hydrocortisone-21-acetate, hydrocortisone-21 -butyrate, hydrocortisone-21-propionate, hydrocortisone-21 -valerate, etc.), hydrocortisone-17,21-diesters (e.g., hydrocortisone- 17, 21 -diacetate, hydrocortisone- 17 -acetate- 21-butyrate, hydrocortisone-17,21-dibutyrate, etc.), alclometasone, dexamethasone, flumethasone, prednisolone, methylprednisolone, betamethasone, typically as betamethasone benzoate or betamethasone diproprionate; fluocinonide; prednisone; and triamcinolone, typically as triamcinolone acetonide. In some embodiments, the anti-inflammatory agent is a mast cell degranulation inhibitor, such as, without limitation, cromolyn (5,5'-(2-hydroxypropane-l,3- diyl)bis(oxy)bis(4-oxo-4H-chromene-2-carboxylic acid) (also known as cromoglycate), and 2- carboxylatochromon-5'-yl-2-hydroxypropane derivatives such as bis (acetoxymethyl), disodium cromoglycate, nedocromil (9-ethyl-4,6-dioxo-10-propyl-6,9-dihydro-4H-pyrano[3,2-g]quinoline- 2,8-dicarboxylic acid) and tranilast (2-{[(2E)-3-(3,4-dimethoxyphenyl)prop-2-enoyl]amino}), and lodoxamide (2-[2-chloro-5-cyano-3-(oxaloamino)anilino]-2-oxoacetic acid). In some embodiments, the anti-inflammatory agent is a nonsteroidal anti-inflammatory drugs (NS AIDs), such as. without limitation, aspirin compounds (acetylsalicylates), non-aspirin salicylates, diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin,ketoprofen, meclofenamate, naproxen, naproxen sodium, phenylbutazone, sulindac, and tometin.

[0159] In some embodiments, the anti-inflammatory agent comprises an antihistamine. In some embodiments, the antihistamine includes clemastine, clemastine fumarate (2(R)-[2-[l-(4-Chlorophenyl)-l-phenyl-ethoxy]ethyl-l-methylpyrrolidine), dexmedetomidine, doxylamine, loratidine, desloratidine and promethazine, and diphenhydramine, or pharmaceutically acceptable salts, solvates or esters thereof. In some embodiments, the antihistamine includes, without limitation, azatadine, azelastine, burfroline, cetirizine, cyproheptadine, doxantrozole, etodroxizine, forskolin, hydroxyzine, ketotifen, oxatomide, pizotifen, proxicromil, N,N'- substituted piperazines or terfenadine. In some embodiments, the antihistamine is an Hl antagonist, such as, but not limited to, cetirizine, chlorpheniramine, dimenhydrinate, diphenhydramine, fexofenadine, hydroxyzine, orphenadrine, pheniramine, and doxylamine. In some embodiments, the antihistamine is an H2 antagonist, such as, but not limited to, cimetidine, famotidine, lafutidine, nizatidine, ranitidine, and roxatidine.

[0160] In some embodiments, the additional therapeutic agent comprises an antibiotic. Non-limiting examples of suitable antibiotics include beta-lactams such as penicillins, aminopenicillins (e.g., amoxicillin, ampicillin, hetacillin, etc.), penicillinase resistant antibiotics (e.g., cioxacillin, dicloxacillin, methicillin, nafcillin, oxacillin, etc.), extended spectrum antibiotics (e.g., axlocillin, carbenicillin, mezlocillin, piperacillin, ticarcillin, etc.); cephalosporins (e.g., cefadroxil, cefazolin, cephalixin, cephalothin, cephapirin, cephradine, cefaclor, cefacmandole. cefmetazole, cefonicid, ceforanide, cefotetan, cefoxitin, cefprozil, cefuroxime, loracarbef, cefixime, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftiofur, ceftizoxime, ceftriaxone, moxalactam, etc.); monobactams such as aztreonam; Carbapenems such as imipenem and eropenem; quinolones (e.g., ciprofloxacin, enrofloxacin, difloxacin, orbifloxacin, marbofloxacin, etc.); chloramphenicols (e.g., chloramphenicol, thiamphenicol, florfenicol, etc.); tetracyclines (e.g., chlortetracycline, tetracycline, oxytetracycline, doxycycline, minocycline, etc.); macrolides (e.g., erythromycin, tylosin, tlimicosin, clarithromycin, azithromycin, etc.); lincosamides (e.g., lincomycin, clindamycin, etc.); aminoglycosides (e.g.. gentamicin, amikacin, kanamycin, apramycin, tobramycin, neomycin, dihydrostreptomycin, paromomycin, etc.); sulfonamides(e.g., sulfadmethoxine, sfulfamethazine, sulfaquinoxaline, sulfamerazine, sulfathiazole, sulfasalazine, sulfadiazine, sulfabromomethazine, suflaethoxypyridazine, etc.); glycopeptides (e.g„ vancomycin, teicoplanin, ramoplanin, and decaplanin; and other antibiotics (e.g., rifampin, nitrofuran, virginiamycin, polymyxins, tobramycin, etc.)).Methods of Treatment and Expressing CASQ2 in cells

[0161] Provided herein are methods of treating a subject with CPVT through delivery of CASQ2 using recombinant adeno-associated viruses (rAAVs). Provided herein are also methods for increasing CASQ2 levels in cells using rAAVs.

[0162] Some embodiments provide for a method of administering to a subject in need of treatment a therapeutically effective amount of a recombinant adeno-associated virus (rAAV), wherein the rAAV comprises a nucleic acid expression construct (SEQ ID NO: 5) comprising a human CASQ2 coding sequence (SEQ ID NO: 9) operably linked to a desmin promoter (SEQ ID NO: 7). wherein the expression construct is flanked by inverted terminal repeats (ITRs) (SEQ ID NO: 6 and / or SEQ ID NO: 11), and wherein said administration results in expression of a therapeutically effective amount of human CASQ2 (SEQ ID NO: 4), thereby treating the CPVT. In some embodiments, the therapeutically effective amount of human CASQ2 ameliorates the symptoms of a subject with CPVT. In some embodiments, the subject with CPVT in need of treatment has a mutation in the RYR2 gene. In some embodiments, the subject with CPVT in need of treatment does not have a mutation in the CASQ2. In some embodiments, the subject in need of treatment has heart failure. In some embodiments, the subject in need of treatment has symptoms associated with dyspnea, fatigue and chest pain, and / or arrhythmia. In some embodiments, the subject in need of treatment does not have heart failure. In some embodiments, the subject in need of treatment is asymptomatic. In some embodiments, the therapeutically effective amount of expressed human CASQ2 is expressed in cardiac tissue. In some embodiments, the therapeutically effective amount of human CASQ2 is localized in the sarcoplasmic reticulum of cardiomyocytes. In some embodiments, the therapeutically effective amount of human CASQ2 eliminates or improves symptoms of arrythmia.

[0163] In some embodiments, the therapeutically effective amount is between about 1 x 1012to about 1 x 1016viral genomes (vg) / kg. In some embodiments, the-M-therapeutically effective amount is about 5 x 1013to about 1 x 1014vg / kg or in a range defined by any two of the preceding values. In several embodiments, the effective amount comprises vg / kg of about 1.0 x 1013. 2.0 x 1013, 3.0 x 1013, 4.0 x 1013, 5.0 x 1013, 6.0 x 1013. 7.0 x 1013, 8.0 x 1013, 9.0 x 1013, 1.0 x 1014, or 2.0 x 1014or an amount of vg / kg that is within a range defined by any two of the preceding values. In some embodiments, the effective amount is about 2.0 x 1013vg / kg. In some embodiments, the effective amount is about 3.0 x 1013vg / kg. In some embodiments, the effective amount is about 4.0 x 1013vg / kg. In some embodiments, the effective amount is about 5.0 x 1013vg / kg. In some embodiments, the effective amount is about 6.0 x 1013vg / kg. In some embodiments, the effective amount is about 7.0 x 1013vg / kg. In some embodiments, the effective amount is about 8.0 x 1013vg / kg. In some embodiments, the effective amount is about 9.0 x 1013vg / kg. In some embodiments, the effective amount is about 1.0 x 1014vg / kg.

[0164] Some embodiments provide for methods of increasing or upregulating expression of human CASQ2 (SEQ ID NO: 4) in a cell, comprising contacting the cell with an adeno-associated virus (AAV) vector (SEQ ID NO: 5) comprising a nucleic acid expression cassette comprising a functional human CASQ2 coding sequence (SEQ ID NO: 9) operably linked to a promoter (SEQ ID NO: 7), wherein the expression construct is flanked by inverted terminal repeats (ITRs) (SEQ ID NO: 6 and / or SEQ ID NO: 11), and wherein expression of functional human CASQ2 (SEQ ID NO: 4)is increased in the cell. In some embodiments, the AAV serotype is AAV2. In some embodiments, the AAV serotype is AAV8. In some embodiments, the AAV serotype is AAV8 / 2.

[0165] Any titles or subheadings used herein are for organization purposes and should not be used to limit the scope of embodiments disclosed herein.EXAMPLES

[0166] The following examples are illustrative only and are not intended to be a limitation on the scope of the disclosure.

[0167] For the examples provided herein, reference is made to Dose Level 1 (8E12 vg / kg), Dose Level 2 (3E13 vg / kg), Dose Level 3 (8E13 vg / kg), and Dose Level 4 (1.6E14 vg / kg) (mice), which represent ascending dosage levels, respectively, all dosed vector genomes (vg) / kg.Example 1

[0168] A RYR2 transgenic mouse model containing the CPVT mutation RYR2R4496C / +as described in Cerrone et al., 2005 and Priori et al., 2002, was used.

[0169] To record the arrhythmogenic response in the mice, an implanted telemetry device was used to detect sinus rhythm or arrhythmias. (Data Sci (DSI) Fll transponder). Animals were greater than 20 g and approximately 9 to 11 weeks old for telemetry surgery. Phenotype penetrance was determined by an ECG following challenge. Mice underwent the challenge once. An untreated control group was used to assess the phenotype penetrance which was accounted for in tabulating outcomes.

[0170] WT mice were dosed with vehicle, and RYR2R4496C / +mice were dosed with either vehicle or 3.0E13 vg / kg (Dose Level 2) or 8.0E13 vg / kg (Dose Level 3) of AAV8-CASQ2 and subjected to a caffeine / epinephrine challenge with telemetry (ECG) at either 28 or 56 days followed by necropsy. Animals were randomized into groups based on body weight stratification using a random number generator. Prior to dosing, mice were surgically implanted with telemetry transponders at 8 to 11 weeks of age, with dosing at 10 to 13 weeks of age.

[0171] 38% of animals dosed with rAAV-CASQ2 (SEQ ID NO: 5) at Dose Level 2 and 42% of animals dosed at Dose Level 3 experienced positive ventricular tachycardia (VT) events after challenge at 28 days post dosing. Approximately 50% of rAAV8-CASQ2 dosed animals (Dose Level 3) experienced positive VT events 56 days post dosing. (FIG. 2), which is consistent with the approximate 51% background penetrance of the disease model.

[0172] At necropsy, there were no macroscopic observations related to the administration of rAAV-CASQ2 (SEQ ID NO: 5) and no adverse events attributed to rAAV-CASQ2. Tissues (heart, gastrocnemius (gastroc), liver) were collected for biodistribution (FIG.3) and mRNA expression (FIG. 4).Example 2

[0173] Adult WT C57B1 / 6 mice were dosed with either vehicle (n=3) or rAAV-CASQ2 (n=8). The rAAV-CASQ2 at Dose Level 2 (3E13 vg / kg) for each terminal interval (Days 7, 14, 28, 42, 56, 84+1, 112+1, and 168+2 post dose). Tissues were collected with time-matched vehicle controls. rAAV-CASQ2 vector and CASQ2 protein levels were analyzed in heart, gastrocnemius, and liver.

[0174] At necropsy for time-matched controls, there were no macroscopic observations related to the administration of rAAV-CASQ2 and no adverse events attributed to rAAV-CASQ2. Biodistribution results from heart tissue are shown in FIG. 5. Protein expression levels over time are shown in FIG. 6. Histology and microscopic evaluation indicated that overexpressed CASQ2 did not cause structural or morphological changes in the heart.Example 3

[0175] RYR2R4496C / +mice and WT mice, each with implanted telemetry (see Example 1, above) were dosed intravenously with rAAV8-CASQ2 (SEQ ID NO: 5) between 10 to 13 weeks of age. WT mice (n=12) and a cohort of RYR2R4496C / +mice (n=24) received vehicle, while remaining RYR2R4496C / +mice in 3 cohorts of n=24 each were dosed with AAV-CASQ2 at 3.0E13 vg / kg (Dose Level 2), 8.0E13 vg / kg (Dose Level 3), or 1.6E14 vg / kg (Dose Level 4), respectively. Assessments were performed at 3 months post dose based on peak transgene expression results (Example 2, FIG. 6, dotted line represents assay limit of quantitation). An expert-blinded read of the ECGs was also performed.

[0176] The bioanalytical results show a dose responsive biodistribution to the heart (FIG. 7) and dose responsive and cardiac selective mRNA expression (FIGs. 8 and 9) that was liver and skeletal muscle detargeted (FIG. 9).

[0177] Results from this example demonstrate rAAV-CASQ2 treatment resulted in a dose related response at the dose level 3 (8.0E13 vg / kg) and dose level 4 (1.6E14 vg / kg) as compared to the vehicle-treated transgenic animals (FIG. 10). The vehicle- treated transgenic RYR2R4496C / +animals showed a similar disease penetrance (52%) as previous studies (Example 1 and Cerrone et al. 2005).

[0178] Electrocardiograms (ECGs) were performed for wild type and RYR2R4496C / +transgenic mice 85 days after dosing with vehicle or rAA-CASQ2 and following challenge (intraperitoneal (IP) dose) with P-adrenergic agents (for example, epinephrine and caffeine). Wild type mice exhibited a normal heart rhythm following challenge. Transgenic mice dosed with vehicle exhibited polymorphic and / or bidirectional arrhythmic morphology. RYR2R4496C / +transgenic mice treated with rAAV-CASQ2 exhibited normal heart rhythm after challenge (FIG. 11).

[0179] FIG. 12 shows rAAV8-CASQ2 treatment in RYR2R4496C / +transgenic mice with a dose responsive prevention of arrhythmia rates upon P-adrenergic challenge. Prevention of arrhythmia was normalized to background model penetrance of 52%.Example 4

[0180] Juvenile male cynomolgus macaques were administered via a single, intravenous infusion either vehicle or rAAV-CASQ2 at, 8.0E12 vg / kg (Low Dose), 3.0E13 vg / kg (Medium Dose), and 8.0E13 vg / kg (High Dose), with n=2 / group. All animals were administered 5 mg / kg methylprednisolone daily by oral gavage starting on Day -7 through the end of testing

[0181] All animals survived to scheduled euthanasia on Day 28. There were no toxicologically relevant observations, changes in body weight, or organ weights that were attributable to rAAV-CASQ2. Pathology results showed no definitive changes in hematology parameters and no changes in coagulation, chemistry, or complement associated with administration of rAAV-CASQ2 (FIGs. 13A-13C).

[0182] Upon necropsy, there were no macroscopic observations and no microscopic changes related to the administration of rAAV-CASQ2. The histopathology evaluation showed no rAAV-CASQ2-related microscopic effects in any tissue at any dose level.Example 5

[0183] Male and female macaques (Macaca fascicularis) were administered by infusion either vehicle or rAAV-CASQ2 at low dose (8E12 vg / kg), medium / mid dose (3E13 vg / kg), or high dose (8E13 vg / kg). The study design is outlined in Table 1. Animals were prescreened and selected negative for neutralizing antibodies to AAV8 and were randomly assigned to groups based on serostatus and stratified by body weights, where possible. An immunosuppression regimen was employed, along with an additional triple combination regimen (TIS) at the high dose level.Table 1: Toxicology in Nonhuman PrimatesAbbreviations: F = female; M = male; PO = per ora / by mouth; QD = quaque die / each day;Q wk = quaque / each weekaDaily PO, methylprednisolone 5 mg / kg; PO QD (oral gavage), Day -7 to Day 28, then taper by Vi dose Q wk.bTriple immunosuppression:• Methylprednisolone 5 mg / kg, PO QD (oral gavage), Day -7 to Day 28, then taper by Yi dose Q wk. • Rapamycin, 0.5 mg / kg, PO QD (oral gavage), Day -7 to Day 28, then taper by Yi dose Q wk.• Rituximab, 20 mg / kg, slow bolus IV, Days -15, -7, 1, 7, 14.

[0184] Liver chemistry results (FIGs. 14A and 14B; 84 days) show safe response with minimal enzyme elevations at Day 4 that resolved by Day 8-15 without intervention, while animals maintained normal signs. Biodistribution of rAAV-CASQ2 was persistent in the heart at 3 months (FIG. 14C) and at 6 months (FIG. 14D), with transgene expression present 6-months post-dosing (FIG. 14E). Dose-dependent expression was observed at both 3 months and 6 months (FIG. 14F).Example 6

[0185] The kinetics of CASQ2 protein expression in mice were evaluated using Western blotting (FIG. 15). Western blotting separated (based on molecular weight differences) endogenous mouse CASQ2 from human CASQ2 protein (i.e., transgene derived protein). Human CASQ2 protein expression in mouse heart lysates was assessed on Day 28, 56, 84, and 168 post-rAAV-CASQ2 dosing and normalized against vinculin (loading control).

[0186] hCASQ2 protein levels are represented as relative quantity (RQ) normalized to the mean of the 28-day cohort samples within each blot. For comparisons of Day 28 to the other time points within a single blot, an unpaired t-test was performed. A one-way ANOVA using Tukey’s multiple comparison post-test was applied to compare Day 56, Day 84 and Day 168 samples.

[0187] Based on the blots, compared to Day 28, an approximately 2-fold increase was observed in human CASQ2 by Day 56, which was significant. Protein levels plateaued from Day 56 through Day 168, with no significant differences in human CASQ2 protein levels between Day 56 and 168.Example 7

[0188] The kinetics of hCASQ2 protein expression was evaluated in non-human primates (NHPs) dosed with rAAV-CASQ2 (FIG. 16). hCASQ2 expression levels were similar between 3 and 6-months post-rAAV-CASQ2-dosing. hCASQ2 protein was increased 1.7-fold and 2.3-fold in the Mid (8E13 vg / kg) and High (2E14 vg / kg) Dose groups compared to endogenous NHP CASQ2 levels, respectively. Human CASQ2 expression levels achieved in the high-Dose group were similar to CASQ2 levels measured in human heart samples (1,147 ± 486 ng / mg tissue protein). Data represented as mean ± SEMExample 8

[0189] The potential of CASQ2 overexpression in cardiomyocytes to correct beta-adrenergic stimulated induction of ventricular tachycardia (VT) was investigated in an in vitro model system harboring a CPVT RYR2 mutation (FIG. 17). Efficacy of CASQ2 overexpression to correct beta-adrenergic stimulated-induction of VT was assessed in human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes carrying a disease-relevant pathogenic mutation (RYR2E2311D). Isogenic controls and RYR2 E2311D mutant (Fujifilm iCell® 01434) hiPSC-cardiomyocytes were divided into an untreated group (UNT), a group transduced with an AAV comprising a luciferase transgene (CTRL), and a treatment group transduced with an AAV comprising a CASQ2 transgene seeded on Nanion CardioExcyte plates at a density of 50,000 cells / well. Cells were cultured for two days prior to transduction. Cells were then treated with formulation buffer or transduced with either AAV-luciferase (Luc) or AAV-CASQ2 (SEQ ID NO: 5) at an MOI of 1E5 vg / cell. Cells were cultured for an additional 4 days prior to recording beat rate: at baseline, with pacing, with isoproterenol stimulation, or with pacing and isoproterenol stimulation using the Nanion CardioExcyte 96.

[0190] Four days following transduction, impedance and electrode recordings showed RYR2E2311Dmutant cardiomyocytes have significantly lower spontaneous beatingrates compared to isogenic control (15.4 + / - 2.2 vs. 32.5 + / - 1.8 bpm; FTGs. 18A and 18C). Treatment of RYR2E2311Dcardiomyocytes with rAAV-CASQ2 resulted in an improvement in the spontaneous beating rate compared to CTRL (20.1 + / - 3.3 vs. 16.0 + / - 1.6 bpm; FIGs. 18B and 18C). This pattern was repeated when cells were incubated in the presence of 1 pM isoproterenol, a beta-adrenergic agonist (FIGs. 19A-19C).

[0191] CASQ2 overexpression improves RYR2 E2311D cardiomyocyte's ability to adopt rapid pacing rates and protects from tachycardia induction with isoproterenol (FIGs.20A-20C).

[0192] Addition of isoproterenol (1 pM) exemplified RYR2E2311Dcardiomyocytes’ arrhythmogenicity with higher beating irregularity (0.242 + / - 0.218 vs. 0.015 + / - 0.004, CoV seconds; FIG. 19D) and incidence of VT with 2.5 Hz pacing (7 / 12 vs. 0 / 6 wells) compared to isogenic control (FIG. 20D). Importantly, treatment of RYR2E2311Dcardiomyocytes with CASQ2 rescued beat regularity compared to CTRL (FIG. 19D, 0.012 + / - 0.005 vs. 0.159 + / -0.138, CoV seconds) and incidence of VT (0 / 12 wells) with pacing back to isogenic control levels (FIG. 20D).

[0193] This example demonstrates that AAV-CASQ2 treatment of RYR2 E231 ID cells resulted in improvements in both beat rate and beat rate regularity under beta-adrenergic induced stress conditions. Additionally, treatment improved RYR2 mutant cardiomyocyte’s ability to adapt to rapid pacing rates combined with isoproterenol treatment, conveying protection from stressed induced tachycardia. AAV-CASQ2 treatment led to a dose-responsive increase in CASQ2 mRNA and protein levels. Furthermore, this dose-responsive increase in CASQ2 expression significantly reduced the number of animals experiencing beta-adrenergic stress induced VT.

[0194] It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein. It should also be appreciated that terminology explicitly employed herein that also may appear in any disclosure incorporated by reference should be accorded a meaning most consistent with the particular concepts disclosed herein.

[0195] Reference throughout the specification to “one example”, “another example”, “an example”, and so forth, means that a particular element (e.g., feature, structure, and / or characteristic) described in connection with the example is included in at least one example described herein, and may or may not be present in other examples. In addition, it is to be understood that the described elements for any example may be combined in any suitable manner in the various examples unless the context clearly dictates otherwise. While several examples have been described in detail, it is to be understood that the disclosed examples may be modified. Therefore, the foregoing description is to be considered non-limiting.

[0196] The use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting. The use of the term “having” as well as other forms, such as “have,” “has,” and “had.” is not limiting. As used in this specification, whether in a transitional phrase or in the body of the claim, the terms “comprise(s)” and “comprising” are to be interpreted as having an open-ended meaning. That is, the above terms are to be interpreted synonymously with the phrases “having at least” or “including at least.” For example, when used in the context of a process, the term “comprising” means that the process includes at least the recited steps, but may include additional steps. When used in the context of a compound, composition, or device, the term “comprising” means that the compound, composition, or device includes at least the recited features or components, but may also include additional features or components.

[0197] Features, materials, characteristics, or groups described in conjunction with a particular aspect, or example are to be understood to be applicable to any other aspect or example described in this section or elsewhere in this specification unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The protection is not restricted to the details of any foregoing examples. The protection extends to any one, or any combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any one, or any combination, of the steps of any method or process so disclosed.

[0198] Furthermore, certain features that are described in this disclosure in the context of separate implementations may also be combined in a single implementation.Additionally, various features described in the context of a single implementation may be implemented in multiple implementations separately or in a suitable sub-combination. Moreover, although features may be described above as acting in certain combinations, one or more feature from a claimed combination may be excised from the combination, and the combination may be claimed as a sub-combination or variation of a sub-combination.

[0199] Moreover, while operations may be depicted in the drawings or described in the specification in a particular order, such operations need not be performed in the particular order or in the sequential order shown, but all operations may be performed in the order needed to achieve desirable results. Other operations that are not depicted or described in the example methods and processes may also be incorporated. For example, one or more additional operation may be performed before, after, simultaneously, or between any of the described operations. Further, the operations may be rearranged or reordered in other implementations. Those skilled in the art will appreciate that in some examples, the actual steps taken in the processes illustrated and / or disclosed may differ from those shown in the figures. Depending on the example, certain of the steps described above may be removed or others may be added. Furthermore, the features and attributes of the specific examples disclosed above may be combined in different ways to form additional examples, all of which fall within the scope of the present disclosure.

[0200] For purposes of this disclosure, certain aspects, advantages, and features are described herein. Not necessarily all such advantages may be achieved in accordance with any particular example. Thus, for example, those skilled in the art will recognize that the disclosure may be embodied or carried out in a manner that achieves one advantage or a group of advantages as taught herein without necessarily achieving other advantages as may be taught or suggested herein.

[0201] Conditional language, such as “can,” “could,” “might,” or “may,” unless specifically stated otherwise, or otherwise understood within the context as used, is generally intended to convey that certain examples include, while other examples do not include, certain features, elements, and / or steps. Thus, such conditional language is not generally intended to imply that features, elements, and / or steps are in any way required for one or more examples or that one or more examples necessarily include logic for deciding, with or without user inputor prompting, whether these features, elements, and / or steps are included or are to be performed in any particular example.

[0202] Conjunctive language such as the phrase “at least one of X, Y, and Z,” unless specifically stated otherwise, is otherwise understood with the context as used in general to convey that an item, term, etc. may be either X, Y, or Z. Thus, such conjunctive language is not generally intended to imply that certain examples require the presence of at least one of X, at least one of Y, and at least one of Z.

[0203] Language of degree used herein, such as the terms “approximately,” “about,” “generally,” and “substantially” represent a value, amount, or characteristic close to the stated value, amount, or characteristic that still performs a desired function or achieves a desired result.

[0204] The scope of the present disclosure is not intended to be limited by the specific disclosures of preferred examples in this section or elsewhere in this specification and may be defined by claims as presented in this section or elsewhere in this specification or as presented in the future. The language of the claims is to be interpreted broadly based on the language employed in the claims and not limited to the examples described in the present specification or during the prosecution of the application, which examples are to be construed as non-exclusive. Although certain dependent claims may be described as depending from a single preceding claim, it is to be understood that each such dependent claim may alternatively be construed as depending from any one of the preceding claims, whether independent or dependent, unless expressly stated otherwise. All such alternative dependencies and combinations are expressly contemplated as part of the disclosure.

[0205] The described embodiments and examples of the present disclosure are intended to be illustrative rather than restrictive and are not intended to represent every embodiment or example of the present disclosure, and thus, are not to be limited in scope by the specific embodiments and examples described herein. While the fundamental features of the disclosure as applied to various specific embodiments thereof have been shown, described, and pointed out, it will also be understood that various omissions, substitutions, and changes in the details of the compositions and methods that are disclosed, may become apparent and may be made by those skilled in the art without departing from the spirit of the disclosure. For example, it is expressly intended that all combinations of those method steps that performsubstantially the same function in substantially the same way to achieve the same results are within the scope of the disclosure. Moreover, it should be recognized that method steps shown and / or described in connection with any disclosed form or embodiment of the disclosure may be incorporated in any other disclosed or described or suggested form or embodiment as a general matter of design choice. Further, various modifications and variations may be made without departing from the spirit or scope of the disclosure as set forth in the following claims both literally and in equivalents recognized in law.

Claims

WHAT TS CLAIMED IS:

1. An expression cassette comprising:a codon optimized human cardiac calsequestrin-2 (CASQ2) coding sequence; a promoter operably linked to the CASQ2 coding sequence; and an intron,wherein the expression cassette is flanked by inverted terminal repeats (ITRs).

2. The expression cassette of claim 1, wherein the CASQ2 coding sequence is a codon optimized sequence set forth in SEQ ID NO: 9 or a sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 9.

3. The expression cassette of claim 2, wherein the CASQ2 coding sequence encodes the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4.

4. The expression cassette of claim 1, wherein the intron is not a CASQ2 intron.

5. The expression cassette of claim 1, wherein the intron comprises a human beta globin intron.

6. The expression cassette of claim 5, wherein the human beta globin intron comprises the sequence as set forth in SEQ ID NO: 8.

7. The expression cassette of claim 1, wherein the expression cassette does not comprise a CASQ23’ UTR sequence.

8. The expression cassette of claim 1, wherein the ITR sequence comprises an AAV serotype 2 ITR.

9. The expression cassette of claim 8. wherein the AAV serotype 2 ITR comprises the sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 11.

10. The expression cassette of claim 1, further comprising a polyadenylation signal.

11. The expression cassette of claim 10. wherein the polyadenylation signal comprises a human beta globin polyadenylation signal.

12. The expression cassette of claim 11, wherein the human beta globin polyadenylation signal comprises the sequence as set forth in SEQ ID NO: 10.

13. The expression cassette of claim 1, wherein the promoter comprises a cardiac specific promotor.

14. The expression cassette of claim 1, wherein the promoter is selected from the group consisting of desmin, TNNT2, TNNI3, creatine kinase, myogenin, alpha myosin heavy chain, and natriuretic peptide.

15. The expression cassette of claim 14, wherein the promoter comprises a desmin promoter.

16. The expression cassette of claim 15, wherein the desmin promoter comprises the sequence as set forth in SEQ ID NO: 7.

17. The expression cassette of claim 1, comprising the sequence set forth in SEQ ID NO: 5 or a nucleic acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 5.

18. A recombinant adeno-associated virus (rAAV) vector comprising the expression cassette of claim 1.

19. The rAAV vector of claim 18, wherein the vector is self-complementary.

20. The rAAV vector of claim 18, wherein the rAAV is serotype 8.

21. A composition comprising a pharmaceutically acceptable carrier and the rAAV of claim 18.

22. A method of treating a cardiac disorder in a subject, the method comprising:administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus (rAAV), wherein the rAAV comprises:an expression cassette comprising a codon optimized human cardiac calsequestrin-2 (CASQ2) coding sequence;a promoter operably linked to the CASQ2 coding sequence; and an intron,wherein the expression cassette is flanked by inverted terminal repeats (ITRs), andwherein said administration results in expression of a therapeutically effective amount of human CASQ2, thereby treating the cardiac disorder.

23. The method of claim 22, wherein the CASQ2 coding sequence has a sequence as set forth in SEQ ID NO: 9, or a sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 9.

24. The method of claim 22, wherein the ITRs have a sequence as set forth in SEQ ID NO: 6 and / or SEQ ID NO: 11, or a sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 6 and / or SEQ ID NO: 11.

25. The method of claim 22, wherein the intron is not a CASQ2 intron.

26. The method of claim 22, wherein the expression cassette does not comprise a CASQ23’ UTR sequence.

27. The method of claim 22, wherein the intron comprises a human beta globin intron.

28. The method of claim 27, wherein the human beta globin intron has a sequence as set forth in SEQ ID NO: 8.

29. The method of claim 22, wherein the cardiac disorder is catecholaminergic polymorphic ventricular tachycardia (CPVT).

30. The method of claim 29, wherein the CPVT is caused by a mutation in a RYR2 gene.

31. The method of claim 30, wherein the RYR2 mutation is R4497C.

32. The method of claim 30, wherein the RYR2 mutation is R2474S.

33. The method of claim 30, wherein the RYR2 mutation is D3638A.

34. The method of claim 29, wherein the CPVT is caused by a mutation in a CASQ2 gene.

35. The method of claim 34, wherein the CPVT is not caused by a mutation in a RYR2 gene.

36. The method of claim 22, wherein the subject in need of treatment has arrhythmia.

37. The method of claim 22, wherein the subject in need of treatment does not have heart failure.

38. The method of claim 22, wherein the subject in need of treatment is asymptomatic.

39. The method of claim 22, wherein the therapeutically effective amount of expressed human CASQ2 is expressed in cardiac tissue.

40. The method of claim 22, wherein the therapeutically effective amount of expressed human CASQ2 improves heart rhythms.

41. The method of claim 22, wherein the subject is a human subject.

42. The method of claim 22, wherein the method reduces arrhythmia in the subject.

43. The method of claim 22, wherein the method restores calcium homeostasis in the subject’s heart.

44. The method of claim 22, wherein the method reduces delayed after polarization events.

45. The method of claim 22, wherein the method reduces diastolic calcium leak through a RYR2 channel into a cytosol.

46. The method of claim 22, wherein the subject in need of treatment receives an immunosuppressant prior to administration of said rAAV.

47. The method of claim 22, wherein said immunosuppressant comprises methylprednisolone, rapamycin, rituximab, or combinations thereof.

48. A method of increasing or upregulating expression of human CAS Q2comp rising:contacting the cell with an adeno-associated virus (AAV) vector comprising a nucleic acid expression cassette comprising a functional codon-optimized human CASQ2 coding sequence operably linked to a promoter, and an intron, wherein the expression construct is flanked by inverted terminal repeats (ITRs), andwherein expression of functional human CASQ2 is increased in the cell.

49. The method of claim 48, wherein the CASQ2 coding sequence comprises a sequence as set forth in SEQ ID NO: 9 or a sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%. or 99% sequence identity to SEQ ID NO: 9.

50. The method of claim 49, wherein the CASQ2 coding sequence encodes an amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having at least about 85%, 90%, 95%. 96%. 97%, 98%, or 99% sequence identity to SEQ ID NO: 4.

51. The method of claim 48, wherein the intron comprises a human beta globin intron.

52. The method of claim 51, wherein the human beta globin intron comprises a sequence as set forth in SEQ ID NO: 8.

53. The method of claim 48, wherein the ITR sequence comprises an AAV serotype 2 ITR.

54. The method of claim 53, wherein the AAV serotype 2 TTR comprises a sequence as set forth in SEQ ID NO: 6 or SEQ ID NO: 11.

55. The method of claim 48, further comprising a polyadenylation signal.

56. The method of claim 55, wherein the polyadenylation signal comprises a human beta globin polyadenylation signal.

57. The method of claim 56, wherein the human beta globin polyadenylation signal comprises a sequence as set forth in SEQ ID NO: 10.

58. The method of claim 48, wherein the promoter comprises a desmin promoter.

59. The method of claim 58, wherein the desmin promoter comprises the sequence as set forth in SEQ ID NO: 7.

60. The method of claim 48, comprising the sequence as set forth in SEQ ID NO: 5 or a nucleic acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 5.

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