B-cell lymphoma 2-associated anthanogene 3 (BAG3) gene therapy using AAV vectors

Abstract

Provided herein is, for example, gene therapy for BAG3 (B-cell lymphoma 2 associated anthanogene 3) using an adeno-associated virus (AAV) vector. The promoter of the vector may be an MHCK7 promoter, a cardiac troponin T (hTNNT2) promoter, a heat shock protein 70 (HSP70) promoter, or an ubiquitin C (UBC) promoter. The capsid may be an AAVrh.74 or AAV9 capsid or a functional variant thereof. In certain embodiments, the capsid is an AAVrh.74 capsid or a functional variant thereof. Other promoters or capsids may also be used. Methods of treatment, such as by intravenous, intracoronary, intracarotid, or intracardiac administration of the AAV vector, as well as other compositions and methods are also provided. TIFF2025512574000073.tif19142

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 333,798, filed April 22, 2022, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0002] Description of sequence listing The sequence listing XML associated with this application is provided in XML file format and is incorporated herein by reference. The name of the XML file containing the sequence listing XML is ROPA_028_01WO_SeqList_ST26.xml. The XML file is 156,193 bytes, was created on April 19, 2023, and was submitted electronically via the USPTO Patent Center. [Background technology]

[0003] background Heart failure and cardiomyopathy are both severe and fatal diseases. Loss-of-function mutations in the gene B-cell lymphoma 2-associated anthanogene 3 (BAG3) have been implicated as causing cardiomyopathies such as dilated cardiomyopathies (DCM), including BAG3-associated dilated cardiomyopathy, BAG3-associated myofibrillar myopathy, familial sporadic dilated cardiomyopathy, and dilated cardiomyopathy 1hh (CMD1HH). Downregulation of BAG3 has also been associated with cardiac dysfunction and heart failure.

[0004] BAG3 mediates chaperone-assisted autophagy by acting as a co-chaperone with Hsp70, blocks apoptosis by coupling with Bcl-2, preserves sarcomere integrity by coupling actin filaments to the Z-disk, and enhances β-agonist-stimulated excitation-contraction by binding β-adrenergic receptors and L-type Ca2+ channels. Downregulation of BAG3 disrupts the chaperone-assisted selective autophagy (CASA) complex that mediates the turnover of cardiac proteins such as filamin C.

[0005] Current treatment methods, including pharmacological therapy and myocardial ablation, are still ineffective for patients with BAG3-deficient cardiomyopathy.Therefore, there is still an unmet need in the art for the treatment of diseases and disorders associated with BAG3, including cardiomyopathy and other cardiac conditions.The compositions and methods disclosed herein address this need. Summary of the Invention

[0006] overview The present invention relates generally to gene therapy vectors and gene therapy directed to a disease or disorder, for example a cardiac disease or disorder, using vectors expressing BAG3 or a functional variant thereof. [Brief description of the drawings]

[0007] [Figure 1] Schematic diagram of a non-limiting example of a vector genome, the complete polynucleotide sequence of which is SEQ ID NO: 107. [Diagram 2] Schematic diagram of a non-limiting example of a vector genome, the complete polynucleotide sequence of which is SEQ ID NO: 108. [Diagram 3] Schematic diagram of a non-limiting example of a vector genome, the complete polynucleotide sequence of which is SEQ ID NO: 109. [Figure 4] Schematic diagram showing a non-limiting example of a vector genome, the complete polynucleotide sequence of which is SEQ ID NO: 110. [Diagram 5] Schematic diagram of a non-limiting example of a vector genome, the complete polynucleotide sequence of which is SEQ ID NO: 111. [Figure 6] Schematic diagram of a non-limiting example of a vector genome, the complete polynucleotide sequence of which is SEQ ID NO: 112. [Figure 7] Schematic diagram showing a non-limiting example of a vector genome, the complete polynucleotide sequence of which is SEQ ID NO: 113. [Figure 8] Schematic diagram of a non-limiting example of a vector genome, the complete polynucleotide sequence of which is SEQ ID NO: 114. [Figure 9] Figure 1 shows expression of BAG3 in CHO-Lec2 cells after transduction with the indicated vectors. Cells were transduced with 3E6 MOI for each vector, and 6 days later, cell lysates were harvested and Western blots were performed using anti-BAG3 polyclonal antibody (abcam ab225561 1:2,500). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] Detailed Description of the Invention The present disclosure provides a gene therapy vector that delivers a polynucleotide that codes for BAG3 polypeptide or its functional fragment or variant, together with methods of use and other compositions and methods.In certain aspects, the present disclosure relates to a gene therapy vector that comprises a promoter sequence that is operably linked to a polynucleotide that codes for BAG3 polypeptide or its functional fragment or variant.In certain aspects, the promoter is a heterologous promoter, i.e., a promoter that is not normal or endogenous to the BAG3 gene in mammalian cells, or a protein that is not normal or endogenous to the gene delivery vector, such as AAV.In some aspects, the BAG3 is human BAG3.

[0009] In some embodiments, the AAV vector is an AAVrh.74 vector. In some embodiments, the promoter is an MHCK7 promoter and the AAV vector is an AAVrh.74 vector. In some embodiments, the promoter is an hTNNT2 promoter. In some embodiments, the promoter is an hTNNT2 promoter and the AAV vector is an AAVrh.74 vector. In some embodiments, the promoter is an HSP70 promoter. In some embodiments, the promoter is an HSP70 promoter and the AAV vector is an AAVrh.74 vector. In some embodiments, the promoter is an ubiquitin C (UBC) promoter. In some embodiments, the promoter is an UBC promoter and the AAV vector is an AAVrh.74 vector. In some embodiments, the promoter is a CAG promoter or a chicken beta actin promoter. In some embodiments, the promoter is a CAG promoter or a chicken beta actin promoter and the AAV vector is an AAVrh.74 vector. In some embodiments, the BAG3 is human BAG3.

[0010] In some embodiments, the AAV vector is an AAV9 vector. In some embodiments, the promoter is a myosin heavy chain creatine kinase 7 (MHCK7) promoter. In some embodiments, the promoter is a MHCK7 promoter and the AAV vector is an AAV9 vector. In some embodiments, the promoter is a hTNNT2 promoter. In some embodiments, the promoter is a hTNNT2 promoter and the AAV vector is an AAV9 vector. In some embodiments, the promoter is a heat shock protein 70 (HSP70) promoter. In some embodiments, the promoter is a HSP70 promoter and the AAV vector is an AAV9 vector. In some embodiments, the promoter is a ubiquitin C (UBC) promoter. In some embodiments, the promoter is a UBC promoter and the AAV vector is an AAV9 vector. In some embodiments, the promoter is a CAG promoter or a chicken beta actin promoter. In some embodiments, the promoter is a CAG promoter or a chicken beta actin promoter and the AAV vector is an AAV9 vector. In some embodiments, the BAG3 is human BAG3.

[0011] The present disclosure further provides a method for treating disease or disorder in a subject by administering the gene therapy vector of the present disclosure to the subject.In certain embodiments, the disease or disorder is heart failure.In certain embodiments, the disease or disorder is cardiomyopathy, such as dilated cardiomyopathy.In certain embodiments, the disease or disorder is BAG3-associated dilated cardiomyopathy, BAG3-associated myofibrillar myopathy, familial sporadic dilated cardiomyopathy, and dilated cardiomyopathy 1hh (CMD1HH).

[0012] In certain embodiments, the subject being treated is a heart failure patient with one or more mutations or truncations in the BAG3 gene. The expression level of BAG3 is decreased as the heart deteriorates due to multiple etiologies, including human heart failure. The gene BAG3 encodes the protein B-cell lymphoma 2-associated anthanogene 3 (BAG3). BAG3 is involved in protein quality control (PQC). PQC includes chaperone and proteolytic systems, including the ubiquitin-proteasome system (UPS) and the autophagy-lysosomal system, which help minimize protein toxicity. Chaperones function as the first line of defense by either refolding misfolded proteins or promoting the degradation of misfolded or damaged proteins that are not suitable for refolding via the UPS or autophagy. BAG3 regulates the ATPase activity of the HSP70 family, a class of chaperones, and is primarily expressed in striated muscle.

[0013] BAG3 is also involved in chaperone-assisted selective autophagy (CASA). CASA is a cargo-selective form of autophagy that is mediated through the HSC70-BAG3-HSPB8 complex and allows ubiquitination of select proteins recognized by HSC70 via the CHIP ubiquitin ligase, and sequestration by autophagosomes via the LC3 adaptor p62 / SQSTM in the presence of synaptopodin2 or DNAJB6. In muscle, BAG3 is particularly important in maintaining sarcomere structure and integrity through its interaction with HSC70 and the actin-capping sarcomere protein (actin-capping protein β1).

[0014] Genome-wide association studies (GWAS) have identified several mutations in BAG3, such as E455K, in patients with DCM. Furthermore, mouse models (e.g., BAG3 cKO or BAG3 E455K KI) have demonstrated that loss of function of BAG3 induces the development of DCM by attenuating PQC across a specific subset of proteins involved in metabolic and contractile function in cardiomyocytes.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.Methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, but suitable methods and materials are described below.In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting.

[0016] All publications and patents mentioned in this specification are incorporated herein by reference in their entirety, as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.In the event of any discrepancy, this application, including any definitions herein, shall prevail.However, the reference to any references, papers, publications, patents, patent publications, and patent applications cited in this specification is not intended to be, and should not be, interpreted as an admission or any form of suggestion that they constitute relevant prior art or form part of the common general knowledge in any country in the world.

[0017] In this description, any concentration range, percentage range, ratio range, or integer range will be understood to include any integer value within the stated range, as well as fractions thereof (such as tenths and hundredths of integers), where appropriate, unless otherwise specified. The term "about", when immediately preceding a number or numeral, means that the number or numeral ranges ±10%. The terms "a" and "an" as used herein, unless otherwise specified, will be understood to refer to "one or more" of the listed components. The use of alternatives (e.g., "or") will be understood to mean any one, both, or any combination thereof of the alternatives. The term "and / or" will be understood to mean any one or both of the alternatives. As used herein, the terms "include" and "comprise" are used interchangeably.

[0018] As used herein, the term "over time in a subject" refers to effects in a subject that occur over about a day, about a month, about a year, about a decade, and / or about several decades.

[0019] As used herein, the terms "identity" and "identical" with respect to a polypeptide or polynucleotide sequence refer to the percentage of exact match residues in the alignment of its "query" sequence to a "subject" sequence, such as the alignment obtained by the BLAST algorithm. Identity is calculated over the entire length of the subject sequence, unless otherwise specified. Thus, a query sequence "shares at least x% identity with" a subject sequence when the query sequence is aligned with the subject sequence, and at least x% (truncated) of the residues in the subject sequence are aligned as exact matches to the corresponding residues in the query sequence. If the subject sequence has variable positions (e.g., residues designated as X), any alignment to any residue in the query sequence is counted as a match. Sequence alignment can be performed using the NCBI Blast service (BLAST+ version 2.12.0).

[0020] As used herein, the term "operably linked" refers to the functional relationship between two or more nucleic acid (e.g., DNA) segments. Typically, the term refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter sequence is functionally linked to a coding sequence if it stimulates or regulates the transcription of the coding sequence in a suitable host cell or other expression system. Generally, a promoter transcriptional regulatory sequence that is functionally linked to a transcribed sequence is physically contiguous with the transcribed sequence, i.e., cis-acting. On the other hand, some transcriptional regulatory sequences, such as enhancers, do not need to be physically contiguous with or located close to the coding sequence that they enhance transcription.

[0021] As used herein, "AAV vector" or "rAAV vector" refers to a recombinant vector that contains one or more polynucleotides of interest (i.e., transgenes) flanked by AAV inverted terminal repeats (ITRs).Such AAV vectors can replicate and be packaged into infectious viral particles when present in a host cell that is transfected with a plasmid that encodes and expresses rep and cap gene products.Alternatively, AAV vectors can be packaged into infectious particles using a host cell that is stably engineered to express rep and cap genes.

[0022] As used herein, "AAV virion" or "AAV virus particle" or "AAV vector particle" refers to a virus particle that is composed of at least one AAV capsid protein and the polynucleotide AAV vector that is contained in the capsid.As used herein, when the particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to mammalian cells), the particle is typically referred to as "AAV vector particle" or simply "AAV vector".Therefore, the production of AAV vector particle necessarily includes the production of AAV vector, since such vector is contained within the AAV vector particle.

[0023] As used herein, "promoter" refers to a polynucleotide sequence capable of promoting the initiation of RNA transcription from a polynucleotide in a eukaryotic cell.

[0024] As used herein, "vector genome" refers to a polynucleotide sequence that is packaged by a vector (e.g., a rAAV virion), including flanking sequences (in AAV, inverted terminal repeat sequences). The terms "expression cassette" and "polynucleotide cassette" refer to the portion of the vector genome between the flanking ITR sequences. "Expression cassette" implies that the vector genome contains at least one gene product-encoding gene operably linked to an element (e.g., a promoter) that directs expression.

[0025] As used herein, the term "patient in need" or "subject in need" refers to a patient or subject at risk of or suffering from a disease, disorder, or condition suitable for treatment or improvement with the recombinant gene therapy vector or gene editing system disclosed herein. A patient or subject in need may be, for example, a patient or subject diagnosed with a heart-related disorder. The subject may have a mutation in the BAG3 gene or a deletion of all or part of the BAG3 gene or gene regulatory sequence, which causes abnormal interaction between BAG3 and HSP70. "Subject" and "patient" are used interchangeably herein. The subject treated by the methods described herein may be an adult or a child. The subject may be of a range of ages.

[0026] As used herein, the term "variant" refers to a protein that has one or more amino acid substitutions, insertions, or deletions compared to a parent protein.As used herein, the term "functional variant" refers to a protein that has one or more amino acid substitutions, insertions, or deletions compared to a parent protein and retains one or more desired activities of the parent protein.

[0027] As used herein, "BAG3", "BAG3 molecule", "BCL2-associated athanogene 3 (BAG3) gene", "BCL2-associated athanogene 3 (BAG3) molecule" includes all family members, variants, cDNA sequences, alleles, fragments, species, coding and non-coding sequences, sense and antisense polynucleotide strands, and the like. Similarly, "BAG3", "BAG3 molecule", "BCL2-associated athanogene 3 (BAG3) molecule" also refers to BAG3 polypeptides or fragments, proteins, variants, derivatives, and the like thereof. The term "molecule" encompasses both nucleic acid and amino acid sequences of BAG3, unless otherwise specified.

[0028] As used herein, the term "fragment" refers to a fragment of a protein. As used herein, the term "functional fragment" refers to a protein that has one or more deletions compared to the parent protein and retains one or more desired activities of the parent protein. In certain embodiments, a fragment may be a portion of a protein that lacks a stretch of amino acids at the N-terminus and / or C-terminus. A fragment may include less than 99%, less than 95%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, or less than 20% of the full-length protein.

[0029] As used herein, "treating" refers to improving one or more symptoms of disease or disorder.The term "preventing" refers to delaying, suppressing, or interrupting the onset of one or more symptoms of disease or disorder, or slowing down the progression of disease or disorder associated with BAG3, such as dilated cardiomyopathy (DCM).

[0030] Adeno-associated virus (AAV) is a replication-deficient parvovirus whose single-stranded DNA genome is approximately 4.7 kb long, including two inverted terminal repeats (ITRs) of approximately 145 nucleotides. There are several known variants of AAV, which may be classified according to antigenic epitopes and are also called serotypes. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077; the complete genome of AAV-2 is provided in GenBank Accession No. NC_001401 and Srivastava et al., J. Virol., 45: 555-564 (1983), the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829; the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829; the AAV-5 genome is provided in GenBank Accession No. AF085716; the complete genome of AAV-6 is provided in GenBank Accession No. NC_001862; at least a portion of the AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively; and the AAV-9 genome is provided in Gao et al., J. Virol., 78: The AAV-10 genome is provided in Mol. Ther., 13(1): 67-76 (2006); the AAV-11 genome is provided in Virology, 330(2): 375-383 (2004). The sequence of the AAVrh.74 genome is provided in U.S. Patent No. 9,434,928, which is incorporated herein by reference. Cis-acting sequences that direct viral DNA replication (rep), encapsidation / packaging, and host cell chromosomal integration are contained within the AAV ITRs.Three AAV promoters (designated p5, p19, and p40 from their relative map positions) direct the expression of two AAV internal open reading frames encoding the rep and cap genes. The two rep promoters (p5 and p19), coupled with differential splicing of a single AAV intron (at nucleotides 2107 and 2227), result in the production of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The Rep proteins have multiple enzymatic properties that are ultimately responsible for replicating the viral genome. The cap gene is expressed from the p40 promoter and encodes three capsid proteins, VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites are responsible for the production of the three associated capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158: 97-129 (1992).

[0031] AAV has unique features that make it attractive as a vector for delivering foreign DNA to cells, for example in gene therapy. AAV infection of cells in culture is non-cytopathic, and natural infection of humans and other animals is silent and asymptomatic. Moreover, AAV can infect many mammalian cells, allowing many different tissues to be targeted in vivo. Moreover, AAV can transduce slow-dividing and non-dividing cells and essentially persist for the lifetime of such cells as nuclear episomes (extrachromosomal elements) where active transcription occurs. The AAV viral genome is inserted as cloned DNA into plasmids, making the construction of recombinant genomes feasible. Furthermore, signals directing AAV replication and genome encapsidation are contained within the ITRs of the AAV genome, allowing some or all of the internal 4.7 kb of the genome (encoding replication proteins and structural capsid protein, rep-cap) to be replaced with foreign DNA. To generate AAV vectors, the rep and cap proteins may be supplied in trans. Another significant feature of AAV is that it exhibits high thermostability. AAV readily survives the conditions used to inactivate adenovirus (56°C to 65°C for several hours), making cryopreservation of AAV less important. AAV can even be lyophilized. Finally, AAV-infected cells are no longer resistant to superinfection.

[0032] Gene delivery viral vectors useful in the practice of the present invention can be constructed using methodologies well known in the art of molecular biology.Typically, a viral vector carrying a transgene is assembled from a polynucleotide encoding the transgene, appropriate regulatory elements, and elements required for the production of viral proteins that mediate cell transduction.Such recombinant viruses can be produced by techniques known in the art, for example, by transfecting packaging cells or by transient transfection with helper plasmids or helper viruses.Typical examples of viral packaging cells include, but are not limited to, HeLa cells, SF9 cells (optionally used with baculovirus helper vectors), HEK293 cells, etc.As described in US20170218395A1, AAV vectors can be produced using a herpes virus-based system. Detailed protocols for producing such replication-defective recombinant viruses can be found, for example, in WO95 / 14785, WO96 / 22378, U.S. Pat. No. 5,882,877, U.S. Pat. No. 6,013,516, U.S. Pat. No. 4,861,719, U.S. Pat. No. 5,278,056, and WO94 / 19478, the complete contents of each of which are incorporated herein by reference.

[0033] The present disclosure contemplates compositions and methods of use related to B-cell lymphoma 2-associated anthanogene 3 (BAG3) proteins or polypeptides. In certain embodiments, the subject being treated has one or more mutations in the BAG3 gene or has downregulated expression or levels of BAG3 mRNA and / or protein. Mutations or downregulation of BAG3 that disrupt interaction with HSP70 are known to be associated with cardiomyopathy and heart failure, including diseases such as those described in Fang et al. J Clin Invest. 127(8):3189-320 (2017) and Kieserman et al. J Am Heart Assoc. 8(10) (2019), as well as other sources. Therefore, viral vector-mediated delivery of the BAG3 gene can serve as a viable therapy for BAG3-related human diseases, such as dilated cardiomyopathy and heart failure.

[0034] Mutations in the BAG3 gene have been identified in people with BAG3-associated dilated cardiomyopathy (CMD1HH). (See "CMD1HH" at NCBI MedGen). The condition is an inherited heart disorder characterized by left or biventricular heart dilation. Symptoms include dyspnea, fainting, collapse, fatigue, and chest pain. DCM can lead to death and can affect both children and adults. In certain embodiments, the BAG3 mutation comprises a nucleotide variant in-frame insertion compared to the wild-type BAG3 gene sequence. In certain embodiments, the in-frame insertion encodes an amino acid, e.g., a non-polar amino acid. In certain embodiments, the in-frame insertion comprises a 3-nucleotide insertion that adds an alanine at position 160.

[0035] In some embodiments, BAG3 comprises one or more amino acid substitutions. For example, BAG3 can have the amino acid substitution C151R numbering relative to SEQ ID NO: 1 (as described in Villard, E. et al. Eur Heart J. 2011 May; 32(9):1065-1076).

[0036] BAG3 may contain one or more amino acid substitutions, insertions, or deletions (collectively, mutations) that alter BAG3 protein-protein binding interactions. For example, BAG3 C151R may increase interaction with actin-binding protein filamin A, hippo pathway kinase STK38, and E3 ubiquitin protein ligases DDB1 and TRIM21, while decreasing interaction with small heat shock protein HSPB7 and co-chaperone DNAJB1.

[0037] In some embodiments, the BAG3 protein comprises one or more amino acid substitutions at position C-151 relative to a reference BAG3 protein.

[0038] In some embodiments, BAG3 comprises one or more amino acid modifications, e.g., substitutions, including, but not limited to, any of those disclosed in PCT Patent Application Publication Nos. WO2015117010A or WO2019237002A1, U.S. Patent No. 11,236,389, or U.S. Patent Application Publication Nos. US20180296703 or US20210254159, which are incorporated by reference in their entireties.

[0039] The polypeptide sequence and polynucleotide coding sequence of human BAG3 are shown below: BAG3-wild type (SEQ ID NO: 1) - 575 amino acids TIFF2025512574000002.tif38159 BAG3-wild type (SEQ ID NO: 2) - 1728 nucleotide bases TIFF2025512574000003.tif100158TIFF2025512574000004.tif148151

[0040] In some embodiments, the BAG3 protein comprises a polypeptide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1. In some embodiments, the BAG3 protein comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2. In some embodiments, the BAG3 protein is a wild-type or naturally occurring BAG3 protein, e.g., human BAG3.

[0041] In some aspects, the disclosure provides a recombinant adeno-associated virus (rAAV) virion comprising a capsid and a vector genome, the vector genome comprising a polynucleotide sequence encoding BAG3 or a functional variant thereof, operably linked to a promoter. In some aspects, the disclosure provides a recombinant adeno-associated virus (rAAV) virion comprising a capsid and a vector genome, the vector genome comprising a polynucleotide sequence encoding BAG3, operably linked to a promoter. The polynucleotide encoding BAG3 may comprise a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2.

[0042] The AAV virions of the present disclosure comprise a vector genome, which may comprise an expression cassette (or a polynucleotide cassette for gene editing applications that do not require expression of a polynucleotide sequence).

[0043] Optionally, the polynucleotide sequence encoding the vector genome may include a Kozak sequence, including but not limited to, GCCACCATGG (SEQ ID NO: 11). The Kozak sequence may overlap with the polynucleotide sequence encoding the BAG3 protein or a functional variant thereof.

[0044] In some embodiments, the Kozak sequence is TIFF2025512574000005.tif62128, or an alternative Kozak sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% to any of SEQ ID NOs: 13, 45, 46, 47, 49, or 14.

[0045] In some embodiments, the vector genome does not include a Kozak sequence.

[0046] The vector genome may include 5' and / or 3' inverted terminal repeats (ITRs). Any suitable ITRs may be used. The ITRs may be AAV ITRs from the same serotype as the capsid present in the AAV virion, or from a different serotype than the capsid (e.g., AAV2 ITRs may be used with AAV virions having AAV9 or AAVrh74 capsids). In each case, the serotype of the capsid dictates the name applied to the virion. The ITRs are generally the 5'-most and 3'-most elements of the vector genome. The vector genome also generally includes, in 5' to 3' order, a promoter, a transgene, a 3' untranslated region (UTR) sequence (e.g., a WPRE element), and a polyadenylation sequence. In some forms, the vector genome includes an enhancer element (generally 5' to the promoter) and / or an exon (generally 3' to the promoter). In some forms, vector genome generally comprises green fluorescent protein (GFP) protein at the 3' side of the transgene.In some forms, the vector genome of the present disclosure encodes a partial or complete transgene sequence that is used as a repair template in gene editing system.In such forms, vector genome can comprise an exogenous promoter, or gene editing system can insert transgene into a genomic locus that has an endogenous promoter, such as a cardiac or muscle cell specific promoter.

[0047] In some embodiments, the 5'ITR comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:15. TIFF2025512574000006.tif17154

[0048] In some embodiments, the 5'ITR comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:16. TIFF2025512574000007.tif17156

[0049] In some embodiments, the 5'ITR comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:17. TIFF2025512574000008.tif17154

[0050] In some embodiments, the 5'ITR comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:18. TIFF2025512574000009.tif17156

[0051] In some embodiments, the 3'ITR comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:19. TIFF2025512574000010.tif17154

[0052] In some embodiments, the 3' ITR comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:20. TIFF2025512574000011.tif17154

[0053] In some embodiments, the 3'ITR comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:21. TIFF2025512574000012.tif17144

[0054] In some embodiments, the vector genome comprises one or more filler sequences that are at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, e.g., SEQ ID NO: 22; SEQ ID NO: 23; or SEQ ID NO: 24. TIFF2025512574000013.tif55155

[0055] In some embodiments, the polynucleotide sequence encoding the BAG3 protein or its functional variant is operably linked to a promoter. In some embodiments, the promoter is a MHCK7 promoter. In some embodiments, the promoter is a TNNT2 promoter. In some embodiments, the promoter is a HSP70 promoter. In some embodiments, the promoter is a UBC promoter. In some embodiments, the promoter is a CAG promoter or a chicken β-actin promoter.

[0056] The present disclosure contemplates the use of various promoters. Promoters useful in embodiments of the present disclosure include, but are not limited to, cytomegalovirus (CMV) promoter, phosphoglycerate kinase (PGK) promoter, or CMV enhancer and a promoter sequence (CAG) consisting of a portion of the chicken beta-actin promoter and rabbit beta-globin gene. In some cases, the promoter can be a synthetic promoter. An exemplary synthetic promoter is provided by Schlabach et al. PNAS USA. 107(6):2538-43 (2010). In some embodiments, the promoter comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 25. TIFF2025512574000014.tif80137

[0057] In some embodiments, the polynucleotide sequence encoding the BAG3 protein or its functional variant is operably linked to an inducible promoter. The inducible promoter can be configured to transcriptionally express or not transcriptionally express the polynucleotide sequence in response to the addition or accumulation of an agent, or in response to the removal, degradation, or dilution of the agent. The agent can be a drug. The agent can be tetracycline or one of its derivatives, including but not limited to doxycycline. In some cases, the inducible promoter is a tet-on promoter, a tet-off promoter, a chemically regulated promoter, a physically regulated promoter (i.e., a promoter that responds to the presence or absence of light or low or high temperature). Inducible promoters include heavy metal ion inducible promoters (such as the mouse mammary tumor virus (mMTV) promoter or various growth hormone promoters) and promoters from T7 phage that are active in the presence of T7 RNA polymerase. This list of inducible promoters is non-limiting.

[0058] In some embodiments, the promoter is a tissue-specific promoter, such as a promoter capable of directing expression in cardiac cells to a higher degree than in non-cardiac cells. In some embodiments, the tissue-specific promoter is selected from any of a variety of cardiac tissue-specific or cardiac cell-specific promoters, including but not limited to those promoter / enhancer region combinations, such as desmin (Des), alpha-myosin heavy chain (α-MHC), myosin light chain 2 (MLC-2), cardiac troponin C (cTnC), cardiac troponin T (hTNNT2), muscle creatine kinase (CK), and MHCK7. In some embodiments, the promoter is inducible to an environmental stimulus. In some embodiments, the promoter is a heat shock protein 70 (HSP70) promoter. In some embodiments, the promoter is a ubiquitous promoter. "Ubiquitous promoter" refers to a promoter that is not tissue-specific under experimental or clinical conditions. In some embodiments, the ubiquitous promoter is a cytomegalovirus (CMV) promoter, a cytomegalovirus ( C ytomegalovirus) early enhancer element chicken beta-actin ( A ctin) gene intron and rabbit beta-globin ( G lobin gene splice acceptor together with the (CAG) promoter, the ubiquitin C (UBC) promoter, the phosphoglycerate kinase (PGK) promoter, the eukaryotic translation elongation factor 1 alpha 1 (EF1-alpha) promoter, the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, the simian virus 40 (SV40) promoter, the hepatitis B virus (HBV) promoter, the chicken beta-actin promoter, and the human beta-actin promoter.

[0059] In some embodiments, the promoter sequence is selected from Table 1. In some embodiments, the promoter comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 31-48, 115, or 116. In some embodiments, the promoter comprises a fragment of the polynucleotide sequence of any one of SEQ ID NOs: 31-48, such as a fragment that comprises at least 25%, at least 50%, at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of any one of SEQ ID NOs: 31-51, 115, or 116. For example, in some embodiments, the promoter is a fragment of the UBC promoter consisting of about the first 400 nucleotides of the UBC promoter sequence of SEQ ID NO:116.

[0060] [Table 1] TIFF2025512574000016.tif242166TIFF2025512574000017.tif246166TIFF20255125740 00018.tif246166TIFF2025512574000019.tif246166TIFF2025512574000020.tif242166 TIFF2025512574000021.tif235166TIFF2025512574000022.tif246166TIFF20255125740 00023.tif235166TIFF2025512574000024.tif247166TIFF2025512574000025.tif198166

[0061] In certain embodiments, the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31. In certain embodiments, the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 32. In certain embodiments, the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 33. In certain embodiments, the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 34. In certain embodiments, the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 35. In certain embodiments, the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 36. In certain embodiments, the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 37. In certain embodiments, the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 38. In certain embodiments, the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 39.In certain embodiments, the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 40. In certain embodiments, the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 41. In certain embodiments, the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 42. In certain embodiments, the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 43. In certain embodiments, the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 44. In certain embodiments, the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 48. In certain embodiments, the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 115. In certain embodiments, the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 116.

[0062] Further illustrative examples of promoters are the SV40 late promoter from simian virus 40, the baculovirus polyhedron enhancer / promoter element, herpes simplex virus thymidine kinase (HSV tk), the immediate early promoter from cytomegalovirus (CMV), and various retroviral promoters containing LTR elements. A wide variety of other promoters are known and generally available in the art, and the sequences of many such promoters are available in sequence databases such as the GenBank database.

[0063] In some cases, the vector of the present disclosure further comprises one or more regulatory elements selected from the group consisting of an enhancer, an intron, a polyA signal, a 2A peptide coding sequence, a WPRE (woodchuck hepatitis virus post-transcriptional regulatory element), and a HPRE (hepatitis B post-transcriptional regulatory element). In certain embodiments, the vector comprises a mutant or modified WPRE (collectively referred to as WPRE(x), e.g., a WPRE or a mutant or modified WPRE as disclosed in any of SEQ ID NOs: 62-67 or 70, optionally a modified WPRE as disclosed in SEQ ID NO: 63 (WPRE Mut6; see Zanta-Boussif, M., Charrier, S., Brice-Ouzet, A. et al. Validation of a mutated PRE sequence allowing high and sustained transgene expression while abrogating WHV-X protein synthesis: application to the gene therapy of WAS. Gene Ther 16, 605-619 (2009). https: / / doi.org / 10.1038 / gt.2009.3). In certain embodiments, the modified WPRE comprises a modification of the start codon of the WHV-X protein, thus preventing its expression.

[0064] In some embodiments, the vector comprises a CMV enhancer.

[0065] In certain embodiments, the vector comprises one or more enhancers. In certain embodiments, the enhancer is a CMV enhancer sequence, a GAPDH enhancer sequence, a β-actin enhancer sequence, or an EF1-α enhancer sequence. The sequences of the above are known in the art. For example, the sequence of the CMV immediate early (IE) enhancer is SEQ ID NO: 50. TIFF2025512574000026.tif24154

[0066] In certain embodiments, the vector comprises one or more introns, hi certain embodiments, the intron is a rabbit globin intron sequence, a chicken β-actin intron sequence, a synthetic intron sequence, an SV40 intron, or an EF1-α intron sequence.

[0067] In certain embodiments, the vector comprises a polyA sequence. In certain embodiments, the polyA sequence is rabbit globin polyA sequence, human growth hormone polyA sequence, bovine growth hormone polyA sequence, PGK polyA sequence, SV40 polyA sequence, or TK polyA sequence. In some embodiments, the polyA signal can be bovine growth hormone polyadenylation signal (bGHpA).

[0068] In certain embodiments, the vector comprises one or more transcript stabilizing elements. In certain embodiments, the transcript stabilizing elements are a WPRE sequence, a HPRE sequence, a scaffold attachment region, a 3' UTR, or a 5' UTR. In certain embodiments, the vector comprises both a 5' UTR and a 3' UTR.

[0069] In some embodiments, the vector comprises a 5' untranslated region (UTR) selected from Table 2. In some embodiments, the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 51-61.

[0070] [Table 2] TIFF2025512574000028.tif247166TIFF2025512574000029.tif242166TIFF2025512574000030.tif103166

[0071] In some embodiments, the vector comprises a 3' untranslated region selected from Table 3. In some embodiments, the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 62-70.

[0072] [Table 3] TIFF2025512574000032.tif244166TIFF2025512574000033.tif244166TIFF2025512574000034.tif244166

[0073] In some embodiments, the vector comprises a polyadenylation (polyA) signal selected from Table 4. In some embodiments, the polyA signal comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs 71-75.

[0074] [Table 4] TIFF2025512574000036.tif90166

[0075] Illustrative vector genomes are shown in Figures 1-8 and are provided as SEQ ID NOs: 107-114. In some embodiments, the vector genome comprises, consists essentially of, or consists of a polynucleotide sequence sharing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 26-30 and 76-95, optionally with or without associated ITR sequences.

[0076] In certain embodiments, the vector genome comprises, in 5' to 3' order, 5'ITR; MHCK7 promoter; SV-40 chimeric intron, BAG3 transgene; WPRE(x) element; human GH poly(A) signal (hGH) sequence; and 3'ITR. The vector genome may comprise, in 5' to 3' order, the polynucleotide sequence SEQ ID NO: 107; or a polynucleotide sequence that shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with each of the foregoing. In certain embodiments, the vector genome is packaged in an AAV9 vector or an AAVrh74 vector. In certain embodiments, the vector genome is packaged in an AAVrh74 vector. TIFF2025512574000037.tif47158TIFF2025512574000038.tif245151TIFF2025512574000039.tif245151TIFF2025512574000040.tif16151

[0077] In certain embodiments, the vector genome comprises, in 5' to 3' order, 5'ITR; hTNNT2 promoter; BAG3 transgene; WPRE(x) element; human GH poly(A) signal (hGH) sequence; and 3'ITR. The vector genome may comprise, in 5' to 3' order, the polynucleotide sequence SEQ ID NO: 108; or a polynucleotide sequence that shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 vector or an AAVrh74 vector. In certain embodiments, the vector genome is packaged into an AAVrh74 vector. TIFF2025512574000041.tif161158TIFF2025512574000042.tif245151TIFF2025512574000043.tif95151

[0078] In certain embodiments, the vector genome comprises, in 5' to 3' order, 5'ITR; HSP70 promoter; BAG3 transgene; WPRE(x) element; human GH poly(A) signal (hGH) sequence; and 3'ITR. The vector genome may comprise, in 5' to 3' order, the polynucleotide sequence SEQ ID NO: 109; or a polynucleotide sequence that shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 vector or an AAVrh74 vector. In certain embodiments, the vector genome is packaged into an AAVrh74 vector. TIFF2025512574000044.tif82158TIFF2025512574000045.tif245151TIFF2025512574000046.tif165151

[0079] In certain embodiments, the vector genome comprises, in 5' to 3' order, 5'ITR; MHCK7 promoter; SV-40 chimeric intron; BAG3 transgene; enhanced green fluorescent protein (eGFP); WPRE(x) element; human GH poly(A) signal (hGH) sequence; and 3'ITR. The vector genome may comprise, in 5' to 3' order, the polynucleotide sequence SEQ ID NO: 110; or a polynucleotide sequence that shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 vector or an AAVrh74 vector. In certain embodiments, the vector genome is packaged into an AAVrh74 vector. TIFF2025512574000047.tif206159

[0080] In certain embodiments, the vector genome comprises, in 5' to 3' order, 5'ITR; TNNT2 promoter; BAG3 transgene; enhanced green fluorescent protein (eGFP); WPRE(x) element; human GH poly(A) signal (hGH) sequence; and 3'ITR. The vector genome may comprise, in 5' to 3' order, the polynucleotide sequence SEQ ID NO: 111; or a polynucleotide sequence that shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with each of the foregoing. In certain embodiments, the vector genome is packaged in an AAV9 vector or an AAVrh74 vector. In certain embodiments, the vector genome is packaged in an AAVrh74 vector.

[0081] TIFF2025512574000048.tif221158TIFF2025512574000049.tif245152TIFF2025512574000050.tif148152

[0082] In certain embodiments, the vector genome comprises, in 5' to 3' order, 5'ITR; HSP70 promoter; BAG3 transgene; enhanced green fluorescent protein (eGFP); WPRE(x) element; human GH poly(A) signal (hGH) sequence; and 3'ITR. The vector genome may comprise, in 5' to 3' order, the polynucleotide sequence SEQ ID NO: 112; or a polynucleotide sequence that shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 vector or an AAVrh74 vector. In certain embodiments, the vector genome is packaged into an AAVrh74 vector. TIFF2025512574000051.tif29158TIFF2025512574000052.tif245151TIFF2025512574000053.tif245152TIFF2025512574000054.tif86151

[0083] In certain embodiments, the vector genome comprises, in 5' to 3' order, 5'ITR; CMV enhancer, chicken β-actin promoter; chimeric intron; BAG3 transgene; enhanced green fluorescent protein (eGFP); WPRE(x) element; human GH poly(A) signal (hGH) sequence; and 3'ITR. The vector genome may comprise, in 5' to 3' order, polynucleotide sequence SEQ ID NO: 113; or a polynucleotide sequence that shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 vector or an AAVrh74 vector. In certain embodiments, the vector genome is packaged into an AAVrh74 vector. TIFF2025512574000055.tif82158TIFF2025512574000056.tif245151TIFF2025512574000057.tif245152TIFF2025512574000058.tif201152

[0084] In certain embodiments, the vector genome comprises, in 5' to 3' order, 5'ITR; UBC promoter; BAG3 transgene; WPRE(x) element; human GH poly(A) signal (hGH) sequence; and 3'ITR. The vector genome may comprise, in 5' to 3' order, the polynucleotide sequence SEQ ID NO: 114; or a polynucleotide sequence that shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 vector or an AAVrh74 vector. In certain embodiments, the vector genome is packaged into an AAVrh74 vector. TIFF2025512574000059.tif227159

[0085] In each case, the WPRE element may or may not be present, as is optional.

[0086] Adeno-associated virus vector AAV vectors useful in practicing the present invention can be packaged into AAV virions (virus particles) using a variety of systems, including adenovirus-based systems and helper-free systems. Standard methods in AAV biology include those described in Kwon and Schaffer. Pharm Res. (2008) 25(3):489-99; Wu et al. Mol. Ther. (2006) 14(3):316-27. Burger et al. Mol. Ther. (2004) 10(2):302-17; Grimm et al. Curr Gene Ther. (2003) 3(4):281-304; Deyle DR, Russell DW. Curr Opin Mol Ther. (2009) 11(4):442-447; McCarty et al. Gene Ther. (2001) 8(16):1248-54; and Duan et al. Mol Ther. (2001) 4(4):383-91. Helper-free systems include those described in US 6,004,797; US 7,588,772; and US 7,094,604.

[0087] The AAV DNA in the rAAV genome can be derived from any AAV variant or serotype from which recombinant viruses can be derived, including, but not limited to, AAV variants or serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAVrh.74, and AAVrh10. The production of pseudotyped rAAV is disclosed, for example, in WO 01 / 83692. Other types of rAAV variants are also contemplated, such as rAAV with capsid mutations. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014). The nucleotide sequences of the genomes of various AAV serotypes are known in the art.

[0088] In some cases, rAAV comprises a self-complementary genome. As defined herein, a rAAV comprising a "self-complementary" genome or a "double-stranded" genome refers to a rAAV that has been engineered such that the coding region of the rAAV is configured to form an intramolecular double-stranded DNA template, as described in McCarty et al. Self-complementary recombinant adeno-associated virus (scAAV) vectors facilitate efficient transduction independent of DNA synthesis. Gene Therapy. 8 (16): 1248-54 (2001). The present disclosure contemplates the use of a rAAV comprising a self-complementary genome in some cases, because upon infection (such transduction), the two complementary halves of the scAAV combine to form a single double-stranded DNA (dsDNA) ready for immediate replication and transcription without waiting for cell-mediated synthesis of the second strand of the rAAV genome. It will be appreciated that instead of the entire coding capacity found in rAAV (4.7-6 kb), a rAAV containing a self-complementary genome may retain only about half that amount (about 2.4 kb).

[0089] In other cases, rAAV vectors contain single-stranded genomes. As defined herein, "single-stranded" genome refers to genomes that are not self-complementary. In many cases, non-recombinant AAV has a single-stranded DNA genome. There are some suggestions that rAAV should be scAAV to achieve efficient cell transduction. While the present disclosure contemplates rAAV vectors that may have single-stranded genomes rather than self-complementary genomes, it is considered that other genetic modifications of rAAV vectors may also be beneficial in obtaining optimal gene transcription in target cells.

[0090] In some cases, the rAAV vector is of serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, or AAVrh74. The production of pseudotyped rAAV is disclosed, for example, in WO 01 / 83692. Other types of rAAV variants are also contemplated, such as rAAV with capsid mutations. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014). In certain embodiments, the rAAV vector is of serotype AAVrh74. In some cases, the rAAV vector is of serotype AAV9. In some embodiments, the rAAV vector is of serotype AAV9 and comprises a single-stranded genome. In some embodiments, the rAAV vector is of serotype AAV9 and comprises a self-complementary genome. In some embodiments, the rAAV vector comprises an inverted terminal repeat (ITR) sequence of AAV2. In some embodiments, the rAAV vector comprises an AAV2 genome, such that the rAAV vector is an AAV-2 / 9 vector, an AAV-2 / 6 vector, or an AAV-2 / 8 vector.

[0091] The full length sequence and capsid gene sequence of most of the known AAV are provided in US Pat. No. 8,524,446, which is incorporated herein in its entirety.

[0092] AAV vector can comprise wild-type AAV sequence or comprise one or more modifications to wild-type AAV sequence.In certain embodiments, AAV vector comprises one or more amino acid modifications, which are optionally substitutions, deletions, or insertions, in capsid protein, which are optionally VP1, VP2, and / or VP3.In certain embodiments, the modifications reduce immunogenicity when AAV vector is given to a subject.

[0093] The capsid protein of the rAAV can be modified to target the rAAV to a specific target tissue of interest, such as cardiomyocytes, hi some embodiments, the rAAV is injected directly into the intraventricular space of a subject.

[0094] In some embodiments, the rAAV virion is an AAVrh.74rAAV virion. The capsid can be an AAVrh.74 capsid or a functional variant thereof. In some embodiments, the AAVrh.74 capsid shares at least 98%, 99%, or 100% identity with a reference AAVrh.74 capsid, such as SEQ ID NO: 100. In certain embodiments, the rAAVrh.74rAAV virion is preferred for targeting adults with DCM and has greater safety and / or efficacy than other AAV serotypes.

[0095] In some embodiments, the rAAV virion is an AAV2 rAAV virion. The capsid can be an AAV2 capsid or a functional variant thereof. In some embodiments, the AAV2 capsid shares at least 98%, 99%, or 100% identity with a reference AAV2 capsid, such as SEQ ID NO: 96. TIFF2025512574000060.tif145133TIFF2025512574000061.tif222122

[0096] In some embodiments, the rAAV virion is an AAV9 rAAV virion. The capsid can be an AAV9 capsid or a functional variant thereof. In some embodiments, the AAV9 capsid shares at least 98%, 99%, or 100% identity with a reference AAV9 capsid, such as SEQ ID NO: 97. TIFF2025512574000062.tif214133TIFF2025512574000063.tif144128

[0097] In some embodiments, the rAAV virion is an AAV6 rAAV virion. The capsid can be an AAV6 capsid or a functional variant thereof. In some embodiments, the AAV6 capsid shares at least 98%, 99%, or 100% identity with a reference AAV6 capsid, such as SEQ ID NO: 98. TIFF2025512574000064.tif57133TIFF2025512574000065.tif238121TIFF2025512574000066.tif58128

[0098] In some embodiments, the rAAV virion is an AAVrh.10rAAV virion. The capsid can be an AAVrh.10 capsid or a functional variant thereof. In some embodiments, the AAVrh.10 capsid shares at least 98%, 99%, or 100% identity with a reference AAVrh.10 capsid, such as SEQ ID NO: 99. TIFF2025512574000067.tif143133TIFF2025512574000068.tif222121

[0099] In some embodiments, the capsid protein is encoded by a polynucleotide that is plasmid-supplied in trans to the transfer plasmid. The polynucleotide sequence of wild-type AAVrh74 cap is provided as SEQ ID NO:100. TIFF2025512574000069.tif139154

[0100] The disclosure further provides protein sequences of AAVrh74 VP1, VP2, and VP3, including SEQ ID NOs: 101-103, and homologs or functional variants thereof. TIFF2025512574000070.tif148160

[0101] In certain instances, the AAVrh74 capsid comprises the amino acid sequence set forth in SEQ ID NO: 101. In some embodiments, the rAAV vector comprises a polypeptide comprising, consisting essentially of, or even consisting of a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of AAVrh74 VP1 set forth in, e.g., SEQ ID NO: 101. In some embodiments, the rAAV vector comprises a polypeptide that comprises, consists essentially of, or even consists of a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, and more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of AAVrh74 VP2 as set forth in, e.g., SEQ ID NO:102. In some embodiments, the rAAV vector comprises a polypeptide that comprises, consists essentially of, or even consists of a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, and more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of AAVrh74 VP3 as set forth in, e.g., SEQ ID NO:103.

[0102] In some embodiments, the rAAV virion is an AAV-PHP.B rAAV virion or a neurotrophic variant, such as, but not limited to, those disclosed in International Patent Publication Nos. WO 2015 / 038958 A1 and WO 2017 / 100671 A1. For example, the AAV capsid can include at least four consecutive amino acids from the sequence TLAVPFK (SEQ ID NO: 105) or KFPVALT (SEQ ID NO: 106), e.g., inserted between the sequence encoding amino acids 588 and 589 of AAV9.

[0103] The capsid may be an AAV-PHP.B capsid or a functional variant thereof. In some embodiments, the AAV-PHP.B capsid shares at least 98%, 99%, or 100% identity with a reference AAV-PHP.B capsid, such as SEQ ID NO: 104. TIFF2025512574000071.tif200133TIFF2025512574000072.tif175128

[0104] Additional AAV capsids for use in the rAAV virions of the present disclosure include those disclosed in Patent Publication Nos. WO 2009 / 012176 A2 and WO 2015 / 168666 A2.

[0105] Without being bound by theory, the inventors conclude that AAVrh.74, AAV9 vector, or AAVrh.10 vector confers desired cardiac tropism to the vector. Without being bound by theory, the inventors further conclude that AAVrh.74, AAV9 vector, or AAVrh.10 vector can confer desired cardiac cell specificity.

[0106] In one aspect, the disclosure provides a pharmaceutical composition comprising a rAAV virion of the disclosure and one or more pharma- ceutically acceptable carriers, diluents, or excipients.

[0107] For the purpose of administration, optionally by injection, various solutions can be used, such as sterile aqueous solutions. Such aqueous solutions can be buffered, if desired, and the liquid diluent is first made isotonic with physiological saline or glucose. Solutions of rAAV as a free acid (DNA contains an acidic phosphate group) or pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant, such as poloxamer 188, for example, at 0.001% or 0.01%. Dispersions of rAAV can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and oils. Under ordinary conditions of storage and use, such preparations contain a preservative to prevent the growth of microorganisms. In this regard, all sterile aqueous media employed are readily available by standard techniques well known to those skilled in the art.

[0108] Pharmaceutical forms suitable for injectable use include, but are not limited to, sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid to the extent that easy syringability exists. The form must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, such as, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include an isotonic agent, for example, sugar or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0109] Sterile injectable solution can be prepared by incorporating rAAV in the required amount in a suitable solvent together with various other components listed above, and then optionally performing filtration sterilization.Generally, dispersion is prepared by incorporating sterilized active ingredient in a sterile medium that contains basic dispersion medium and other necessary components listed above.In the case of sterile powder for preparing sterile injectable solution, one specific preparation method is vacuum drying and freeze-drying technique, which gives powder containing active ingredient plus any additional desired ingredient from its previously sterilized filtered solution.

[0110] In another aspect, the disclosure includes a kit comprising a rAAV virion of the disclosure and instructions for use.

[0111] In one aspect, the present disclosure provides a method for increasing BAG3 activity in a cell, comprising contacting the cell with the rAAV of the present disclosure. In another aspect, the present disclosure provides a method for increasing BAG3 activity in a subject, comprising administering the rAAV of the present disclosure to the subject. In some embodiments, the cell and / or the subject lacks expression levels and / or activity of BAG3 messenger RNA or BAG3 protein, and / or comprises a loss-of-function mutation in BAG3. In some embodiments, the cell and / or the subject lacks expression levels and / or activity of BAG3 messenger RNA or BAG3 protein. The cell can be a cardiac cell, e.g., a cardiomyocyte. In certain embodiments, the subject is a mammal, e.g., a human.

[0112] In some embodiments, the methods promote survival of cardiac cells, e.g., cardiomyocytes, in cell culture and / or in vivo. In some embodiments, the methods promote and / or restore cardiac function.

[0113] In another aspect, the present disclosure provides a method for treating a disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of the rAAV virion of the present disclosure.In some embodiments, the disease or disorder is a cardiac disease or disorder.Illustrative cardiac disorders include heart failure, dilated cardiomyopathy (DCM), such as BAG3-associated dilated cardiomyopathy, BAG3-associated myofibrillar myopathy, familial isolated dilated cardiomyopathy, or dilated cardiomyopathy 1hh (CMD1HH), hypertrophic cardiomyopathy, atrial fibrillation, arrhythmia, sinus node disease, hypertensive heart disease, cardiac hypertrophy, atrial fibrosis, myocardial infarction, sick sinus syndrome, atrial disease, and myocardial infarction.In certain embodiments, the subject is suffering from or at risk of DCM. In certain embodiments, the subject has a cardiomyopathy, for example, dilated cardiomyopathy (DCM), such as BAG3-associated dilated cardiomyopathy, BAG3-associated myofibrillary myopathy, familial sporadic dilated cardiomyopathy, or dilated cardiomyopathy 1hh (CMD1HH). In certain embodiments, the subject is a mammal, for example, a human, with a loss-of-function mutation in the BAG3 gene. In certain embodiments, the subject is a mammal, for example, a human, with a mutation in BAG3, for example, E455K. In certain methods, treatment with rAAV virions results in expression of the BAG3 protein encoded by the rAAV virion in the subject, for example, in the subject's heart or heart tissue. In certain embodiments, treatment with rAAV virions results in at least 2-fold, at least 5-fold, at least 10-fold, or more detectable BAG3 protein levels in the subject's heart.

[0114] AAV-mediated delivery of BAG3 protein to the heart can prolong life, prevent or attenuate cardiac cell degeneration, heart failure, scarring, reduced ejection fraction, arrhythmia, angina, exercise intolerance, angina (chest pain), sudden cardiac death, exertional muscle pain, and spasms.AAV-mediated delivery of BAG3 protein to the heart can show improvement or prevent the usual disease course detected by the use of echocardiography, pathological electrocardiogram, cardiac MRI, cardiac biopsy, reduce ventricular arrhythmia, and / or reduce sudden cardiac death.

[0115] The method disclosed herein can provide effective biodistribution in heart.The method can provide sustained expression in all or a substantial part of cardiac cells, such as cardiomyocytes.Notably, the method disclosed herein can provide long-term expression of BAG3 protein throughout the life of subject after administration of AAV vector.In some embodiments, BAG3 protein expression in response to treatment lasts for at least 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 15 years, 20 years, 25 years, 30 years, 35 years, or 40 years.

[0116] Combination therapy is also contemplated by the present invention.The combination of the method of the present invention with standard medical treatment (e.g., corticosteroids or local pressure reducing drugs) is particularly contemplated, as well as combination with new treatments.In some cases, subjects can be treated with a combination of steroids and / or immunosuppressants to prevent or reduce immune response to administration of rAAV as described herein.

[0117] In some embodiments, the AAV vector (e.g., an AAV9, AAVrh.74, or AAVrh.10 vector) is administered at a dose of about 1×10 AAV vectors (vg) per kilogram of total subject body weight (vg / kg). 12 ~5×10 14 Vector genome (vg) or approximately 1 × 10 12 and 6×10 14 In some embodiments, the AAV vector is administered at a dose of about 1×10 13 ~5×10 14 In some embodiments, the AAV vector is administered at a dose of about 5×10 13 ~3×10 14 In some embodiments, the AAV vector is administered at a dose of about 5×10 13 ~1×10 14 In some embodiments, the AAV vector is administered at a dose of about 1×10 12 Less than 3×10 vg / kg 12 Less than 5 × 10 vg / kg12 Less than 7×10 vg / kg 12 Less than 1×10 vg / kg 13 Less than 3×10 vg / kg 13 Less than 5 × 10 vg / kg 13 Less than 7×10 vg / kg 13 Less than 1×10 vg / kg 14 Less than 3×10 vg / kg 14 Less than 5 × 10 vg / kg 14 Less than 7×10 vg / kg 14 Less than 1×10 vg / kg 15 Less than 3×10 vg / kg 15 Less than 5×10 vg / kg 15 vg / kg or less than about 7 × 10 15 In certain embodiments, the AAV vector delivered at any of these doses is an AAV9 vector or an AAV rh74 vector. In some cases, it may be advantageous to use an AAV rh74 vector at a higher dose than an AAV9 vector. In some embodiments, the AAV vector, such as an AVrh.74 vector, is administered at a dose of at least about 5×10 13 vg / kg, at least about 6 × 10 13 vg / kg, at least about 7 × 10 13 vg / kg, at least about 8 × 10 13 vg / kg, at least about 9 × 10 13 vg / kg, at least about 1 × 10 14 vg / kg, at least about 2 × 10 14 vg / kg, at least about 3 × 10 14 vg / kg, at least about 4 × 10 14 vg / kg, at least about 5 × 10 14 vg / kg, at least about 6 × 10 14 vg / kg, or at least about 7 × 10 14 It is administered at a dose of vg / kg.

[0118] In some embodiments, the AAV vector (e.g., an AAV9, AAVrh.74, or AAVrh.10 vector) is administered at a concentration of about 1×10 12vg / kg, approx. 3×10 12 vg / kg, approx. 5×10 12 vg / kg, approximately 7×10 12 vg / kg, approximately 1×10 13 vg / kg, approx. 3×10 13 vg / kg, approx. 5×10 13 vg / kg, approximately 7×10 13 vg / kg, approximately 1×10 14 vg / kg, approx. 3×10 14 vg / kg, approx. 5×10 14 vg / kg, approximately 7×10 14 vg / kg, approximately 1×10 15 vg / kg, approx. 3×10 15 vg / kg, approx. 5×10 15 vg / kg, or approximately 7 × 10 15 vg / kg. In certain embodiments, the AAV vector delivered in any of these doses is an AAV9 vector or an AAV rh74 vector.

[0119] In some embodiments, the AAV vector (e.g., an AAV9, AAVrh.74, or AAVrh.10 vector) is administered at a concentration of 1×10 12 vg / kg, 3 × 10 12 vg / kg, 5×10 12 vg / kg, 7×10 12 vg / kg, 1×10 13 vg / kg, 3 × 10 13 vg / kg, 5×10 13 vg / kg, 7×10 13 vg / kg, 1×10 14 vg / kg, 3 × 10 14 vg / kg, 5×10 14 vg / kg, 7×10 14 vg / kg, 1×10 15 vg / kg, 3 × 10 15 vg / kg, 5×10 15 vg / kg, or 7 × 10 15 vg / kg. In certain embodiments, the AAV vector delivered in any of these doses is an AAV9 vector or an AAV rh74 vector.

[0120] In some embodiments, the AAV vector (e.g., an AAV9, AAVrh.74, or AAVrh.10 vector) is administered at a dose of about 1×10 AAV vectors (vg) per kilogram of total subject body weight (vg / kg). 12 ~5×10 14 In some embodiments, the AAV vector is administered systemically at a dose of about 1×10 13 ~5×10 14 In some embodiments, the AAV vector is administered systemically at a dose of about 5×10 13 ~3×10 14 In some embodiments, the AAV vector is administered systemically at a dose of about 5×10 13 ~1×10 14 In some embodiments, the AAV vector is administered systemically at a dose of about 1×10 12 Less than 3×10 vg / kg 12 Less than 5 × 10 vg / kg 12 Less than 7×10 vg / kg 12 Less than 1×10 vg / kg 13 Less than 3×10 vg / kg 13 Less than 5 × 10 vg / kg 13 Less than 7×10 vg / kg 13 Less than 1×10 vg / kg 14 Less than 3×10 vg / kg 14 Less than 5 × 10 vg / kg 14 Less than 7×10 vg / kg 14 Less than 1×10 vg / kg 15 Less than 3×10 vg / kg 15 Less than 5 × 10 vg / kg 15 vg / kg or less than about 7 × 10 15 In some embodiments, the AAV vector, e.g., the AVrh.74 vector, is administered systemically at a dose of at least about 5×10 13 vg / kg, at least about 6 × 10 13 vg / kg, at least about 7 × 10 13 vg / kg, at least about 8 × 10 13 vg / kg, at least about 9 × 1013 vg / kg, at least about 1 × 10 14 vg / kg, at least about 2 × 10 14 vg / kg, at least about 3 × 10 14 vg / kg, at least about 4 × 10 14 vg / kg, at least about 5 × 10 14 vg / kg, at least about 6 × 10 14 vg / kg, or at least about 7 × 10 14 vg / kg. In certain embodiments, the AAV vector delivered in any of these doses is an AAV9 vector or an AAV rh74 vector.

[0121] In some embodiments, the AAV vector (e.g., an AAV9, AAVrh.74, or AAVrh.10 vector) is administered at a concentration of about 1×10 12 vg / kg, approx. 3×10 12 vg / kg, approx. 5×10 12 vg / kg, approximately 7×10 12 vg / kg, approximately 1×10 13 vg / kg, approx. 3×10 13 vg / kg, approx. 5×10 13 vg / kg, approximately 7×10 13 vg / kg, approximately 1×10 14 vg / kg, approx. 3×10 14 vg / kg, approx. 5×10 14 vg / kg, approximately 7×10 14 vg / kg, approximately 1×10 15 vg / kg, approx. 3×10 15 vg / kg, approx. 5×10 15 vg / kg, or approximately 7 × 10 15 vg / kg. In certain embodiments, the AAV vector delivered at any of these doses is an AAV9 vector or an AAV rh74 vector.

[0122] In some embodiments, the AAV vector (e.g., an AAV9, AAVrh.74, or AAVrh.10 vector) is administered at a concentration of 1×10 12 vg / kg, 3 × 10 12 vg / kg, 5×1012 vg / kg, 7×10 12 vg / kg, 1×10 13 vg / kg, 3 × 10 13 vg / kg, 5×10 13 vg / kg, 7×10 13 vg / kg, 1×10 14 vg / kg, 3 × 10 14 vg / kg, 5×10 14 vg / kg, 7×10 14 vg / kg, 1×10 15 vg / kg, 3 × 10 15 vg / kg, 5×10 15 vg / kg, or 7 × 10 15 vg / kg. In certain embodiments, the AAV vector delivered at any of these doses is an AAV9 vector or an AAV rh74 vector.

[0123] In some embodiments, the AAV vector (e.g., an AAV9, AAVrh.74, or AAVrh.10 vector) is administered at a dose of about 1×10 AAV vectors (vg) per kilogram of total subject body weight (vg / kg). 12 ~5×10 14 In some embodiments, the AAV vector is administered intravenously at a dose of about 1×10 13 ~5×10 14 In some embodiments, the AAV vector is administered intravenously at a dose of about 5×10 13 ~3×10 14 In some embodiments, the AAV vector is administered intravenously at a dose of about 5×10 13 ~1×10 14 In some embodiments, the AAV vector is administered intravenously at a dose of about 1×10 12 Less than 3×10 vg / kg 12 Less than 5×10 vg / kg 12 Less than 7×10 vg / kg 12 Less than 1×10 vg / kg 13 Less than 3×10 vg / kg 13 Less than 5×10 vg / kg 13Less than 7×10 vg / kg 13 Less than 1×10 vg / kg 14 Less than 3×10 vg / kg 14 Less than 5×10 vg / kg 14 Less than 7×10 vg / kg 14 Less than 1×10 vg / kg 15 Less than 3×10 vg / kg 15 Less than 5×10 vg / kg 15 vg / kg or less than about 7 × 10 15 In some embodiments, the AAV vector, e.g., the AVrh.74 vector, is administered intravenously at a dose of at least about 5×10 13 vg / kg, at least about 6 × 10 13 vg / kg, at least about 7 × 10 13 vg / kg, at least about 8 × 10 13 vg / kg, at least about 9 × 10 13 vg / kg, at least about 1 × 10 14 vg / kg, at least about 2 × 10 14 vg / kg, at least about 3 × 10 14 vg / kg, at least about 4 × 10 14 vg / kg, at least about 5 × 10 14 vg / kg, at least about 6 × 10 14 vg / kg, or at least about 7 × 10 14 vg / kg. In certain embodiments, the AAV vector delivered in any of these doses is an AAV9 vector or an AAV rh74 vector.

[0124] In some embodiments, the AAV vector (e.g., an AAV9, AAVrh.74, or AAVrh.10 vector) is administered at a concentration of about 1×10 12 vg / kg, approx. 3×10 12 vg / kg, approx. 5×10 12 vg / kg, approximately 7×10 12 vg / kg, approximately 1×10 13 vg / kg, approx. 3×10 13 vg / kg, approx. 5×10 13 vg / kg, approximately 7×10 13vg / kg, approximately 1×10 14 vg / kg, approx. 3×10 14 vg / kg, approx. 5×10 14 vg / kg, approximately 7×10 14 vg / kg, approximately 1×10 15 vg / kg, approx. 3×10 15 vg / kg, approx. 5×10 15 vg / kg, or approximately 7 × 10 15 It is given intravenously at a dose of 1000 mg / kg.

[0125] In some embodiments, the AAV vector (e.g., an AAV9, AAVrh.74, or AAVrh.10 vector) is administered at a concentration of 1×10 12 vg / kg, 3 × 10 12 vg / kg, 5×10 12 vg / kg, 7×10 12 vg / kg, 1×10 13 vg / kg, 3 × 10 13 vg / kg, 5×10 13 vg / kg, 7×10 13 vg / kg, 1×10 14 vg / kg, 3 × 10 14 vg / kg, 5×10 14 vg / kg, 7×10 14 vg / kg, 1×10 15 vg / kg, 3 × 10 15 vg / kg, 5×10 15 vg / kg, or 7 × 10 15 vg / kg intravenously. In certain embodiments, the AAV vector delivered in any of these doses is an AAV9 vector or an AAV rh74 vector.

[0126] Patient functional improvement, clinical benefit, or evidence of efficacy may be evident by improvement in New York Heart Association functional class (NYHA class), echocardiography (stabilization or improvement of cardiac output, left ventricular ejection fraction, fractional shortening, left ventricular outflow tract obstruction, left ventricular wall thickness, left or right ventricular volumes, velocity time integral, time constant of ventricular relaxation, regurgitant volume, and reduction in left ventricular end diastolic diameter, LVEDD, and reduction in right ventricular area), electrocardiography (stabilization or improvement of ST segment changes, T wave inversions, Q waves, atrial fibrillation, and / or supraventricular tachycardia), cardiac MRI, cardiac biopsy, reduction in ventricular arrhythmias, reduction in sudden cardiac death, and / or reduction or absence of further episodes of myocardial derangements.

[0127] The effective dose of the composition can be administered by any standard route in the art, including, but not limited to, systemic, local, direct injection, intravenous injection, and intracardiac administration. In some cases, administration includes systemic, local, direct injection, intravenous injection, and intracardiac injection. Administration can be performed by cardiac catheterization.

[0128] In some embodiments, the present disclosure provides for local and systemic administration of effective doses of the rAAV and compositions of the present invention. For example, systemic administration can be administration into the circulatory system to affect the entire body. Systemic administration includes parenteral administration via injection, infusion, or implantation. Routes of administration of the compositions disclosed herein include intravenous ("IV"), intraperitoneal ("IP"), intramuscular ("IM"), intralesional, or subcutaneous ("SC") administration, or implantation of sustained release devices, such as mini-osmotic pumps, depot formulations, and the like. In some embodiments, the method of the present disclosure includes administering the AAV vector or pharmaceutical composition of the present disclosure by intravenous, intramuscular, intraarterial, intrarenal, intraurethral, ​​intracardiac, intracoronary, intramyocardial, intradermal, epidural, subcutaneous, intraperitoneal, intraventricular, or iontophoretic administration.

[0129] Specifically, the administration of the rAAV of the present invention can be achieved by using any physical method that delivers the rAAV recombinant vector to the target tissue of an animal. Administration includes, but is not limited to, injection into the heart. In some embodiments, the method includes administering the rAAV to the blood vessels of the coronary circulation in vivo, such as retrograde coronary sinus injection.

[0130] In some aspects, the disclosed method includes intracardiac delivery. Infusion can be performed using a special cannula, catheter, syringe / needle using an infusion pump. Administration can include delivery of an effective amount of rAAV virion or a pharmaceutical composition comprising rAAV virion to the heart. This can be accomplished, for example, via intravenous, intramuscular, intraarterial, intrarenal, intraurethral, ​​intracardiac, intracoronary, intramyocardial, intradermal, epidural, subcutaneous, intraperitoneal, intraventricular, or iontophoretic administration. The disclosed composition can also be administered intravenously.

[0131] Effect of rAAV administration In some aspects, administration of the rAAV of the present disclosure has a beneficial effect on the subject.

[0132] lifespan In some embodiments, administration of a rAAV of the present disclosure can extend the lifespan of a subject compared to a subject not administered a rAAV of the present disclosure or compared to a baseline.

[0133] In some embodiments, administration of a rAAV of the present disclosure extends lifespan by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, or at least about 500% compared to a subject not administered a rAAV of the present disclosure or a baseline.

[0134] In some embodiments, administration of a rAAV of the present disclosure extends lifespan by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 35%, or about 35% to about 40% compared to a subject not administered a rAAV of the present disclosure or to a baseline. %, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 95% to about 100%, about 100% to about 200%, about 200% to about 300%, about 300% to about 400%, or about 400% to about 500%.

[0135] ejection fraction In some embodiments, administration of a rAAV of the present disclosure limits the decrease in, restores, and / or increases the ejection fraction in a subject compared to a subject not administered a rAAV of the present disclosure or to a baseline. In some embodiments, administration of a rAAV of the present disclosure limits the decrease in, restores, and / or increases the ejection fraction in a subject over time.

[0136] In some embodiments, administration of a rAAV of the present disclosure restores and / or increases the ejection fraction by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to a subject not administered a rAAV of the present disclosure or to a baseline.

[0137] In some embodiments, administration of a rAAV of the disclosure limits the decrease in ejection fraction to less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 45%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, at least about 75%, less than about 80%, less than about 85%, less than about 90%, less than about 95%, or less than about 100% compared to subjects not administered a rAAV of the disclosure or to baseline.

[0138] In some embodiments, administration of a rAAV of the disclosure restores and / or increases ejection fraction by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% over time in a subject.

[0139] In some embodiments, administration of a rAAV of the disclosure limits the decrease in ejection fraction over time in a subject to less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 45%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, at least about 75%, less than about 80%, less than about 85%, less than about 90%, less than about 95%, or less than about 100%.

[0140] In some embodiments, administration of a rAAV of the disclosure increases or decreases the ejection fraction by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, or about 16% to about 20%, or about 22% to about 24%, or about 26% to about 28%, or about 28% to about 30%, or about 29% to about 31%, or about 32% to about 35%, or about 33% to about 36%, or about 37% to about 38%, or about 39% to about 40%, or about 41% to about 42%, or about 42% to about 45%, or about 43% to about 46%, or about 44% to about 47%, or about 45% to about 48%, or about 45% to about 49%, or about 46% to about 50%, or about 47% to about 50%, or about 48% to about 50%, or about 49% to about 50%, or about 50% to about 50%, or about 50% to about 50%, or about 50% to about 50%, or about 60% to about 60%, or about 60% to about 60%, or about 60% to about 70%, or about 60% to about 70%, or about 70% to about 80%, or about 70% to about 80%, or about 80% to Restore and / or increase by about 15% to about 20%, about 20% to about 35%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%.

[0141] In some embodiments, administration of an rAAV of the disclosure increases or decreases the ejection fraction over time in a subject by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, Restore and / or increase by about 20% to about 35%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%.

[0142] In some embodiments, administration of an rAAV of the disclosure reduces ejection fraction by less than about 1% to less than about 90%, less than about 20% to less than about 80%, less than about 30% to less than about 80%, less than about 40% to less than about 80%, less than about 50% to less than about 80%, less than about 1% to less than about 2%, less than about 2% to less than about 3%, less than about 3% to less than about 4%, less than about 4% to less than about 5%, less than about 5% to less than about 6%, less than about 6% to less than about 7%, less than about 7% to less than about 8%, less than about 8% to less than about 9%, about 9% to less than 10%, or less than about 10% to about 15% compared to a subject not administered an rAAV of the disclosure or to a baseline. less than about 15% to less than about 20%, less than about 20% to less than about 35%, less than about 25% to less than about 30%, less than about 30% to less than about 35%, less than about 35% to less than about 40%, less than about 40% to less than about 45%, less than about 45% to less than about 50%, less than about 50% to less than about 55%, less than about 55% to less than about 60%, less than about 60% to less than about 65%, less than about 65% to less than about 70%, less than about 70% to less than about 75%, less than about 75% to less than about 80%, less than about 80% to less than about 85%, less than about 85% to less than about 90%, less than about 90% to less than about 95%, or about 95% to less than about 100%.

[0143] In some embodiments, administration of an rAAV of the disclosure results in a decrease in ejection fraction over time in a subject of between about 1% and about 90%, between about 20% and about 80%, between about 30% and about 80%, between about 40% and about 80%, between about 50% and about 80%, between about 1% and about 2%, between about 2% and about 3%, between about 3% and about 4%, between about 4% and about 5%, between about 5% and about 6%, between about 6% and about 7%, between about 7% and about 8%, between about 8% and about 9%, between about 9% and about 10%, between about 10% and about 15%, between about 15% and about 20%, between about 20% and about 25%, between about 25% and about 30%, between about 30% and about 25%, between about 25% and about 35%, between about 30 ... The range is limited to less than about 20%, less than about 20% to less than about 35%, less than about 25% to less than about 30%, less than about 30% to less than about 35%, less than about 35% to less than about 40%, less than about 40% to less than about 45%, less than about 45% to less than about 50%, less than about 50% to less than about 55%, less than about 55% to less than about 60%, less than about 60% to less than about 65%, less than about 65% to less than about 70%, less than about 70% to less than about 75%, less than about 75% to less than about 80%, less than about 80% to less than about 85%, less than about 85% to less than about 90%, less than about 90% to less than about 95%, or about 95% to less than about 100%.

[0144] Left ventricular ejection fraction (LVEF) In some embodiments, administration of a rAAV of the present disclosure prevents a decrease in, restores, and / or increases the LVEF in a subject compared to a subject not administered a rAAV of the present disclosure or to a baseline. In some embodiments, administration of a rAAV of the present disclosure limits a decrease in, restores, and / or increases the LVEF in a subject over time.

[0145] In some embodiments, administration of a rAAV of the present disclosure restores and / or increases LVEF by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to a subject not administered a rAAV of the present disclosure or to a baseline.

[0146] In some embodiments, administration of a rAAV of the disclosure limits the decrease in LVEF to less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 45%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, at least about 75%, less than about 80%, less than about 85%, less than about 90%, less than about 95%, or less than about 100% compared to subjects not administered a rAAV of the disclosure or to baseline.

[0147] In some embodiments, administration of a rAAV of the disclosure restores and / or increases LVEF by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% in a subject over time.

[0148] In some embodiments, administration of a rAAV of the disclosure limits the decrease in LVEF over time in a subject to less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 45%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, at least about 75%, less than about 80%, less than about 85%, less than about 90%, less than about 95%, or less than about 100%.

[0149] In some embodiments, administration of a rAAV of the disclosure increases LVEF by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, or about 16% to about 20% compared to a subject not administered a rAAV of the disclosure or to baseline. Restore and / or increase by about 15% to about 20%, about 20% to about 35%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%.

[0150] In some embodiments, administration of an rAAV of the disclosure may improve LVEF over time in a subject by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, Restore and / or increase by about 20% to about 35%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%.

[0151] In some embodiments, administration of an rAAV of the present disclosure reduces LVEF by less than about 1% to less than about 90%, less than about 20% to less than about 80%, less than about 30% to less than about 80%, less than about 40% to less than about 80%, less than about 50% to less than about 80%, less than about 1% to less than about 2%, less than about 2% to less than about 3%, less than about 3% to less than about 4%, less than about 4% to less than about 5%, less than about 5% to less than about 6%, less than about 6% to less than about 7%, less than about 7% to less than about 8%, less than about 8% to less than about 9%, about 9% to less than 10%, or less than about 10% to about 15% compared to a subject not administered an rAAV of the present disclosure or to baseline. %, less than about 15% to less than about 20%, less than about 20% to less than about 35%, less than about 25% to less than about 30%, less than about 30% to less than about 35%, less than about 35% to less than about 40%, less than about 40% to less than about 45%, less than about 45% to less than about 50%, less than about 50% to less than about 55%, less than about 55% to less than about 60%, less than about 60% to less than about 65%, less than about 65% to less than about 70%, less than about 70% to less than about 75%, less than about 75% to less than about 80%, less than about 80% to less than about 85%, less than about 85% to less than about 90%, less than about 90% to less than about 95%, or about 95% to less than about 100%.

[0152] In some embodiments, administration of an rAAV of the disclosure reduces LVEF over time in a subject by about 1% to less than about 90%, about 20% to less than about 80%, about 30% to less than about 80%, about 40% to less than about 80%, about 50% to less than about 80%, about 1% to less than about 2%, about 2% to less than about 3%, about 3% to less than about 4%, about 4% to less than about 5%, about 5% to less than about 6%, about 6% to less than about 7%, about 7% to less than about 8%, about 8% to less than about 9%, about 9% to less than about 10%, about 10% to less than about 15%, or about 15%. to less than about 20%, less than about 20% to less than about 35%, less than about 25% to less than about 30%, less than about 30% to less than about 35%, less than about 35% to less than about 40%, less than about 40% to less than about 45%, less than about 45% to less than about 50%, less than about 50% to less than about 55%, less than about 55% to less than about 60%, less than about 60% to less than about 65%, less than about 65% to less than about 70%, less than about 70% to less than about 75%, less than about 75% to less than about 80%, less than about 80% to less than about 85%, less than about 85% to less than about 90%, less than about 90% to less than about 95%, or about 95% to less than about 100%.

[0153] Right ventricular ejection fraction (RVEF) In some embodiments, administration of a rAAV of the present disclosure prevents a decrease in RVEF, restores and / or increases the RVEF in a subject compared to a subject not administered a rAAV of the present disclosure or to a baseline. In some embodiments, administration of a rAAV of the present disclosure limits a decrease in LVEF, restores and / or increases the RVEF in a subject over time.

[0154] In some embodiments, administration of a rAAV of the disclosure restores and / or increases RVEF by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to a subject not administered a rAAV of the disclosure or to a baseline.

[0155] In some embodiments, administration of a rAAV of the disclosure limits the decrease in RVEF to less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 45%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, at least about 75%, less than about 80%, less than about 85%, less than about 90%, less than about 95%, or less than about 100% compared to subjects not administered a rAAV of the disclosure or to baseline.

[0156] In some embodiments, administration of a rAAV of the disclosure restores and / or increases RVEF by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% in a subject over time.

[0157] In some embodiments, administration of a rAAV of the disclosure limits the decrease in RVEF over time in a subject to less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 45%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, at least about 75%, less than about 80%, less than about 85%, less than about 90%, less than about 95%, or less than about 100%.

[0158] In some embodiments, administration of a rAAV of the disclosure increases RVEF by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, or about 16% to about 20% compared to a subject not administered a rAAV of the disclosure or to baseline. Restore and / or increase by about 15% to about 20%, about 20% to about 35%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%.

[0159] In some embodiments, administration of an rAAV of the disclosure increases or decreases RVEF over time in a subject by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, Restore and / or increase by about 20% to about 35%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%.

[0160] In some embodiments, administration of an rAAV of the disclosure results in a decrease in RVEF of less than about 1% to less than about 90%, less than about 20% to less than about 80%, less than about 30% to less than about 80%, less than about 40% to less than about 80%, less than about 50% to less than about 80%, less than about 1% to less than about 2%, less than about 2% to less than about 3%, less than about 3% to less than about 4%, less than about 4% to less than about 5%, less than about 5% to less than about 6%, less than about 6% to less than about 7%, less than about 7% to less than about 8%, less than about 8% to less than about 9%, about 9% to less than 10%, or less than about 10% to about 15% compared to a subject not administered an rAAV of the disclosure or to baseline. %, less than about 15% to less than about 20%, less than about 20% to less than about 35%, less than about 25% to less than about 30%, less than about 30% to less than about 35%, less than about 35% to less than about 40%, less than about 40% to less than about 45%, less than about 45% to less than about 50%, less than about 50% to less than about 55%, less than about 55% to less than about 60%, less than about 60% to less than about 65%, less than about 65% to less than about 70%, less than about 70% to less than about 75%, less than about 75% to less than about 80%, less than about 80% to less than about 85%, less than about 85% to less than about 90%, less than about 90% to less than about 95%, or about 95% to less than about 100%.

[0161] In some embodiments, administration of an rAAV of the disclosure results in a decrease in RVEF over time in a subject of between about 1% and about 90%, between about 20% and about 80%, between about 30% and about 80%, between about 40% and about 80%, between about 50% and about 80%, between about 1% and about 2%, between about 2% and about 3%, between about 3% and about 4%, between about 4% and about 5%, between about 5% and about 6%, between about 6% and about 7%, between about 7% and about 8%, between about 8% and about 9%, between about 9% and about 10%, between about 10% and about 15%, between about 15% and about 20%. to less than about 20%, less than about 20% to less than about 35%, less than about 25% to less than about 30%, less than about 30% to less than about 35%, less than about 35% to less than about 40%, less than about 40% to less than about 45%, less than about 45% to less than about 50%, less than about 50% to less than about 55%, less than about 55% to less than about 60%, less than about 60% to less than about 65%, less than about 65% to less than about 70%, less than about 70% to less than about 75%, less than about 75% to less than about 80%, less than about 80% to less than about 85%, less than about 85% to less than about 90%, less than about 90% to less than about 95%, or about 95% to less than about 100%.

[0162] Right ventricular (RV) area In some embodiments, administration of a rAAV of the present disclosure prevents, reverses, and / or reduces an increase in RV area in a subject compared to a subject not administered a rAAV of the present disclosure or to a baseline. In some embodiments, administration of a rAAV of the present disclosure prevents, reverses, and / or reduces an increase in RV area in a subject over time.

[0163] In some embodiments, administration of a rAAV of the disclosure prevents an increase in RV area, restores and / or reduces the RV area by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to a subject not administered a rAAV of the disclosure or to a baseline.

[0164] In some embodiments, administration of a rAAV of the present disclosure provides an improvement in pulmonary function by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, about 2% to about 30%, about 3% to about 35%, about 3% to about 36%, about 3% to about 37%, about 3% to about 38%, about 3% to about 39%, about 4% to about 40 ... 0% to about 35%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%, prevents an increase in the RV area, restores and / or reduces the RV area.

[0165] In some embodiments, administration of a rAAV of the disclosure prevents an increase in RV area, restores and / or reduces RV area by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% in a subject over time.

[0166] In some embodiments, administration of an rAAV of the disclosure reduces or decreases the efficacy of an rAAV over time in a subject by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 35%, about 30% to about 4 ...50%, about 40% to about 50%, about 40% to about 50%, about 40% to about 50%, about 40% to about 50%, about 40% to about 50%, about 40% to about 50%, about 40% to about 50%, about 40% to about 50%, about 40% to about 50%, about 40% to about 50%, about 40% to about 50%, about 40% to about 50%, about 40% to about 50%, 5% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%, inhibiting an increase in RV area and restoring and / or reducing said RV area.

[0167] Left ventricular (LV) area In some embodiments, administration of a rAAV of the present disclosure prevents an increase in, restores, and / or reduces LV area in a subject compared to a subject not administered a rAAV of the present disclosure or to a baseline. In some embodiments, administration of a rAAV of the present disclosure prevents an increase in, restores, and / or reduces RV area in a subject over time.

[0168] In some embodiments, administration of a rAAV of the disclosure prevents an increase in LV area, restores and / or reduces the LV area by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to a subject not administered a rAAV of the disclosure or to a baseline.

[0169] In some embodiments, administration of a rAAV of the present disclosure provides an improvement in pulmonary function by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, about 2% to about 30%, about 3% to about 35%, about 3% to about 36%, about 3% to about 37%, about 3% to about 38%, about 3% to about 39%, about 4% to about 40 ... 0% to about 35%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%, prevents an increase in LV area, restores and / or reduces the LV area.

[0170] In some embodiments, administration of a rAAV of the disclosure prevents an increase in LV area, restores and / or reduces the LV area by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% in a subject over time.

[0171] In some embodiments, administration of an rAAV of the disclosure reduces or decreases the efficacy of an rAAV over time in a subject by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 35%, about 30% to about 4 ...50%, about 40% to about 50%, about 40% to about 50%, about 40% to about 50%, about 40% to about 50%, about 40% to about 50%, about 40% to about 50%, about 40% to about 50%, about 40% to about 50%, about 40% to about 50%, about 40% to about 50%, about 40% to about 50%, about 40% to about 50%, about 40% to about 50%, 5% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%, preventing an increase in LV area and restoring and / or reducing the LV area.

[0172] Right ventricular (RV) velocity time integral (VTI) In some embodiments, administration of a rAAV of the present disclosure limits the decrease in, restores, and / or increases RV VTI in a subject compared to a subject not administered a rAAV of the present disclosure or to a baseline. In some embodiments, administration of a rAAV of the present disclosure limits the decrease in, restores, and / or increases RV VTI in a subject over time.

[0173] In some embodiments, administration of a rAAV of the present disclosure restores and / or increases RV VTI by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to subjects not administered a rAAV of the present disclosure or to baseline.

[0174] In some embodiments, administration of a rAAV of the present disclosure limits the reduction in RV VTI to less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 45%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, at least about 75%, less than about 80%, less than about 85%, less than about 90%, less than about 95%, or less than about 100% compared to subjects not administered a rAAV of the present disclosure or to baseline.

[0175] In some embodiments, administration of a rAAV of the disclosure restores and / or increases RV VTI by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% in a subject over time.

[0176] In some embodiments, administration of a rAAV of the disclosure limits the decrease in RV VTI over time in a subject to less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 45%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, at least about 75%, less than about 80%, less than about 85%, less than about 90%, less than about 95%, or less than about 100%.

[0177] In some embodiments, administration of a rAAV of the present disclosure increases RV VTI by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 35%, about 25% to about 30%, or about 35% to about 40% compared to a subject not administered a rAAV of the present disclosure or to a baseline. %, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100% recovery and / or increase.

[0178] In some embodiments, administration of an rAAV of the disclosure reduces RV VTI over time in a subject by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 35%, about 25% to about 35%, about 30% to about 35%, about 35 ... Restore and / or increase by 0%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%.

[0179] In some embodiments, administration of a rAAV of the disclosure results in a reduction in RV VTI of less than about 1% to less than about 90%, less than about 20% to less than about 80%, less than about 30% to less than about 80%, less than about 40% to less than about 80%, less than about 50% to less than about 80%, less than about 1% to less than about 2%, less than about 2% to less than about 3%, less than about 3% to less than about 4%, less than about 4% to less than about 5%, less than about 5% to less than about 6%, less than about 6% to less than about 7%, less than about 7% to less than about 8%, less than about 8% to less than about 9%, less than about 9% to less than about 10%, less than about 10% to less than about 15%, less than about 15% to less than about 20%, less than about 20% to less than about 35%. %, less than about 25% to less than about 30%, less than about 30% to less than about 35%, less than about 35% to less than about 40%, less than about 40% to less than about 45%, less than about 45% to less than about 50%, less than about 50% to less than about 55%, less than about 55% to less than about 60%, less than about 60% to less than about 65%, less than about 65% to less than about 70%, less than about 70% to less than about 75%, less than about 75% to less than about 80%, less than about 80% to less than about 85%, less than about 85% to less than about 90%, less than about 90% to less than about 95%, or about 95% to less than about 100%.

[0180] In some embodiments, administration of an rAAV of the disclosure results in a reduction in RV VTI over time in a subject by less than about 1% to less than about 90%, less than about 20% to less than about 80%, less than about 30% to less than about 80%, less than about 40% to less than about 80%, less than about 50% to less than about 80%, less than about 1% to less than about 2%, less than about 2% to less than about 3%, less than about 3% to less than about 4%, less than about 4% to less than about 5%, less than about 5% to less than about 6%, less than about 6% to less than about 7%, less than about 7% to less than about 8%, less than about 8% to less than about 9%, about 9% to less than 10%, less than about 10% to less than about 15%, less than about 15% to less than about 20%, less than about 20% to less than about 35%. %, less than about 25% to less than about 30%, less than about 30% to less than about 35%, less than about 35% to less than about 40%, less than about 40% to less than about 45%, less than about 45% to less than about 50%, less than about 50% to less than about 55%, less than about 55% to less than about 60%, less than about 60% to less than about 65%, less than about 65% to less than about 70%, less than about 70% to less than about 75%, less than about 75% to less than about 80%, less than about 80% to less than about 85%, less than about 85% to less than about 90%, less than about 90% to less than about 95%, or about 95% to less than about 100%.

[0181] Left ventricular (LV) velocity time integral (VTI) In some embodiments, administration of a rAAV of the present disclosure limits the decrease in, restores, and / or increases LV VTI in a subject compared to a subject not administered a rAAV of the present disclosure or to a baseline. In some embodiments, administration of a rAAV of the present disclosure limits the decrease in, restores, and / or increases LV VTI in a subject over time.

[0182] In some embodiments, administration of a rAAV of the disclosure restores and / or increases LV VTI by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to subjects not administered a rAAV of the disclosure or to baseline.

[0183] In some embodiments, administration of a rAAV of the disclosure limits the decrease in LV VTI to less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 45%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, at least about 75%, less than about 80%, less than about 85%, less than about 90%, less than about 95%, or less than about 100% compared to subjects not administered a rAAV of the disclosure or to baseline.

[0184] In some embodiments, administration of a rAAV of the disclosure restores and / or increases LV VTI by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% in a subject over time.

[0185] In some embodiments, administration of a rAAV of the disclosure limits the decrease in LV VTI over time in a subject to less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 45%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, at least about 75%, less than about 80%, less than about 85%, less than about 90%, less than about 95%, or less than about 100%.

[0186] In some embodiments, administration of a rAAV of the disclosure increases LV VTI by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 35%, about 25% to about 30%, or about 35% to about 40% compared to a subject not administered a rAAV of the disclosure or to baseline. %, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100% recovery and / or increase.

[0187] In some embodiments, administration of an rAAV of the disclosure increases LV VTI over time in a subject by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 35%, about 25% to about 35%, about 30% to about 35%, about 35 ... Restore and / or increase by 0%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%.

[0188] In some embodiments, administration of a rAAV of the disclosure reduces LV VTI by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 35%, or about 35% to about 40%. %, less than about 25% to less than about 30%, less than about 30% to less than about 35%, less than about 35% to less than about 40%, less than about 40% to less than about 45%, less than about 45% to less than about 50%, less than about 50% to less than about 55%, less than about 55% to less than about 60%, less than about 60% to less than about 65%, less than about 65% to less than about 70%, less than about 70% to less than about 75%, less than about 75% to less than about 80%, less than about 80% to less than about 85%, less than about 85% to less than about 90%, less than about 90% to less than about 95%, or about 95% to less than about 100%.

[0189] In some embodiments, administration of an rAAV of the disclosure results in a decrease in LV VTI over time in a subject of between about 1% and about 90%, between about 20% and about 80%, between about 30% and about 80%, between about 40% and about 80%, between about 50% and about 80%, between about 1% and about 2%, between about 2% and about 3%, between about 3% and about 4%, between about 4% and about 5%, between about 5% and about 6%, between about 6% and about 7%, between about 7% and about 8%, between about 8% and about 9%, between about 9% and about 10%, between about 10% and about 15%, between about 15% and about 20%, between about 20% and about 35%, %, less than about 25% to less than about 30%, less than about 30% to less than about 35%, less than about 35% to less than about 40%, less than about 40% to less than about 45%, less than about 45% to less than about 50%, less than about 50% to less than about 55%, less than about 55% to less than about 60%, less than about 60% to less than about 65%, less than about 65% to less than about 70%, less than about 70% to less than about 75%, less than about 75% to less than about 80%, less than about 80% to less than about 85%, less than about 85% to less than about 90%, less than about 90% to less than about 95%, or about 95% to less than about 100%.

[0190] Left ventricular (LV) fibrosis In some embodiments, administration of a rAAV of the present disclosure prevents an increase in LV fibrosis and / or reduces LV fibrosis in a subject compared to a subject not administered a rAAV of the present disclosure or to a baseline. In some embodiments, administration of a rAAV of the present disclosure prevents an increase in LV fibrosis and / or reduces LV fibrosis over time in a subject.

[0191] In some embodiments, administration of a rAAV of the present disclosure prevents an increase in LV fibrosis and / or reduces LV fibrosis by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to subjects not administered a rAAV of the present disclosure or to a baseline.

[0192] In some embodiments, administration of an rAAV of the disclosure improves or reduces the pulmonary function by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, or about 20% to about 30%, compared to a subject not administered an rAAV of the disclosure or to a baseline. about 20% to about 35%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100% to prevent an increase in LV fibrosis and / or reduce said LV fibrosis.

[0193] In some embodiments, administration of a rAAV of the present disclosure prevents an increase in LV fibrosis and / or reduces LV fibrosis by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% in a subject over time.

[0194] In some embodiments, administration of an rAAV of the disclosure reduces or decreases the efficacy of an rAAV over time in a subject by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 35%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100% to prevent an increase in LV fibrosis and / or reduce said LV fibrosis.

[0195] Right ventricular (RV) fibrosis In some embodiments, administration of a rAAV of the present disclosure prevents an increase in RV fibrosis and / or reduces RV fibrosis in a subject compared to a subject not administered a rAAV of the present disclosure or to a baseline. In some embodiments, administration of a rAAV of the present disclosure prevents an increase in RV fibrosis and / or reduces RV fibrosis over time in a subject.

[0196] In some embodiments, administration of a rAAV of the present disclosure prevents an increase in RV fibrosis and / or reduces RV fibrosis by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to subjects not administered a rAAV of the present disclosure or to a baseline.

[0197] In some embodiments, administration of an rAAV of the disclosure improves or reduces the pulmonary function by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, or about 20% to about 30%, compared to a subject not administered an rAAV of the disclosure or to a baseline. about 20% to about 35%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100% to prevent an increase in RV fibrosis and / or reduce said RV fibrosis.

[0198] In some embodiments, administration of a rAAV of the present disclosure prevents an increase in RV fibrosis and / or reduces RV fibrosis by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% in a subject over time.

[0199] In some embodiments, administration of an rAAV of the disclosure reduces or decreases the efficacy of an rAAV over time in a subject by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 35%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100% to prevent an increase in RV fibrosis and / or reduce said RV fibrosis.

[0200] Premature ventricular contractions (PVCs) In some embodiments, administration of a rAAV of the present disclosure prevents an increase in and / or reduces PVC in a subject compared to a subject not administered a rAAV of the present disclosure or to a baseline. In some embodiments, administration of a rAAV of the present disclosure prevents an increase in and / or reduces PVC in a subject over time.

[0201] In some embodiments, administration of a rAAV of the present disclosure prevents an increase in PVC and / or reduces PVC by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to subjects not administered a rAAV of the present disclosure or to a baseline.

[0202] In some embodiments, administration of a rAAV of the present disclosure provides an improvement in pulmonary function by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, or a similar improvement in pulmonary function compared to a subject not administered a rAAV of the present disclosure or to a baseline. %, about 20% to about 35%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%, preventing an increase in PVC and / or reducing the PVC.

[0203] In some embodiments, administration of a rAAV of the present disclosure prevents an increase in and / or reduces PVC by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% in a subject over time.

[0204] In some embodiments, administration of an rAAV of the present disclosure reduces or decreases the efficacy of rAAV over time in a subject by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 35% , about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%, preventing an increase in PVC and / or reducing the PVC.

[0205] Nonsustained Ventricular Tachycardia (NSVT) In some embodiments, administration of the rAAV of the present disclosure prevents an increase in NSVT and / or reduces NSVT in a subject compared to a subject not administered the rAAV of the present disclosure or to a baseline. In some embodiments, administration of the rAAV of the present disclosure prevents an increase in NSVT and / or reduces NSVT in a subject over time.

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

[0207] In some embodiments, administration of a rAAV of the present disclosure provides an improvement in pulmonary function by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20% compared to a subject not administered a rAAV of the present disclosure or to a baseline. , about 20% to about 35%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%, preventing an increase in NSVT and / or reducing the NSVT.

[0208] In some embodiments, administration of a rAAV of the present disclosure prevents an increase in NSVT and / or reduces NSVT by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% in a subject over time.

[0209] In some embodiments, administration of an rAAV of the disclosure reduces or eliminates about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 35%, Prevents an increase in NSVT and / or reduces NSVT by about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%.

[0210] Ectopic beats In some embodiments, administration of a rAAV of the present disclosure prevents an increase in ectopic beats and / or reduces ectopic beats in a subject compared to a subject not administered a rAAV of the present disclosure or to a baseline. In some embodiments, administration of a rAAV of the present disclosure prevents an increase in ectopic beats and / or reduces ectopic beats in a subject over time.

[0211] In some embodiments, administration of a rAAV of the present disclosure prevents an increase in ectopic beats and / or reduces ectopic beats by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to subjects not administered a rAAV of the present disclosure or to a baseline.

[0212] In some embodiments, administration of an rAAV of the disclosure improves or reduces the pulmonary function by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, or about 20% to about 30%, compared to a subject not administered an rAAV of the disclosure or to a baseline. about 20% to about 35%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%, preventing an increase in ectopic beats and / or reducing the ectopic beats.

[0213] In some embodiments, administration of a rAAV of the present disclosure prevents an increase in ectopic beats and / or reduces ectopic beats by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% in a subject over time.

[0214] In some embodiments, administration of an rAAV of the disclosure reduces or decreases the efficacy of an rAAV over time in a subject by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 35%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%, preventing an increase in ectopic beats and / or reducing said ectopic beats.

[0215] Numbered aspects Aspect 1. A polynucleotide comprising an expression cassette and optionally flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs), the polynucleotide comprises a polynucleotide sequence encoding B-cell lymphoma 2-associated anthanogene 3 (BAG3) or a functional variant thereof operably linked to a promoter, and optionally, the promoter is a heterologous promoter; The polynucleotide. Aspect 2. The polynucleotide of embodiment 1, wherein the promoter is a cardiac-specific promoter. Aspect 3. The polynucleotide of embodiment 1 or embodiment 2, wherein the promoter is a muscle-specific promoter. Aspect 4. The polynucleotide of any one of aspects 1 to 3, wherein the promoter is a cardiomyocyte-specific promoter. Aspect 5. The polynucleotide of any one of aspects 1 to 4, wherein the promoter is a myosin heavy chain creatine kinase 7 (MHCK7) promoter. Aspect 6. The polynucleotide of embodiment 5, wherein the MHCK7 promoter shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:31. Aspect 7. The polynucleotide of any one of aspects 1 to 4, wherein the promoter is a cardiac troponin T (hTNNT2) promoter. Aspect 8. The polynucleotide of embodiment 7, wherein the hTNNT2 promoter shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 32. Aspect 9. The polynucleotide of any one of aspects 1 to 4, wherein the promoter is a human heat shock factor 70 (hHSP70) promoter. Aspect 10. The polynucleotide of embodiment 9, wherein the hHSP70 promoter shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:115. Aspect 11. 11. The polynucleotide of any one of embodiments 1-10, wherein the expression cassette comprises exon 1 of the cardiac troponin T (hTNNT2) gene, and optionally, the hTNNT2 promoter and exon 1 together share at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 32. Aspect 12. The polynucleotide of any one of embodiments 1-4, wherein said promoter is a ubiquitous promoter, optionally a CMV promoter or a CAG promoter or a chicken b-globin promoter or a UBC promoter (SEQ ID NO: 116). Aspect 13. 13. The polynucleotide of any one of embodiments 1 to 12, wherein the expression cassette comprises a polyA signal. Aspect 14. The polynucleotide of embodiment 13, wherein the poly A signal is human growth hormone (hGH) poly A. Aspect 15. The polynucleotide of any one of embodiments 1-14, wherein the expression cassette comprises a Woodchuck Hepatitis Virus post-transcriptional regulatory element (WPRE), and optionally comprises a mutant or modified WPRE (WPRE(x)), and optionally comprises WPRE Mut6 (SEQ ID NO: 63). Aspect 16. 16. The polynucleotide of any one of embodiments 1-15, wherein the expression cassette comprises a polynucleotide sequence encoding green fluorescent protein (GFP). Aspect 17. 17. The polynucleotide of any one of embodiments 1-16, wherein the B-cell Lymphoma 2-associated Anthanogene 3 (BAG3) or a functional fragment or variant thereof is full-length BAG3 or wild-type BAG3. Aspect 18. The polynucleotide of any one of embodiments 1 to 17, wherein the BAG3 is human BAG3. Aspect 19. 19. The polynucleotide of any one of embodiments 1-18, wherein the polynucleotide sequence encoding BAG3 shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:2. Aspect 20. The polynucleotide of any one of embodiments 1 to 19, wherein the polynucleotide sequence encoding BAG3 is a human BAG3 polynucleotide. Aspect 21. 21. The polynucleotide of any one of embodiments 1-20, comprising at least about 3.0 kb, at least about 3.2 kb, at least about 3.4 kb, at least about 3.5 kb, at least about 3.7 kb, at least about 4.0 kb, at least about 4.1 kb, at least about 4.2 kb, at least about 4.3 kb, at least about 4.4 kb, at least about 4.5 kb, at least about 4.6 kb, at least about 4.7 kb, at least about 4.8 kb, or at least about 5.0 kb. Aspect 22. 22. The polynucleotide of any one of embodiments 1-21, comprising up to about 3.1 kb, up to about 3.3 kb, up to about 3.5 kb, up to about 3.7 kb, up to about 3.9 kb, up to about 4.1 kb, up to about 4.2 kb, up to about 4.3 kb, up to about 4.4 kb, up to about 4.5 kb, up to about 4.6 kb, up to about 4.7 kb, up to about 4.8 kb, up to about 4.9 kb, or up to about 5.0 kb. Aspect 23. 23. The polynucleotide of any one of embodiments 1 to 22, comprising from 4.4 kb to 5.0 kb, from 4.4 kb to 4.9 kb, or from 4.4 kb to 4.8 kb, or from 4.0 kb to 4.6 kb, from 4.0 kb to 4.5 kb, or from 4.0 kb to 4.4 kb, or from 4.0 kb to 4.3 kb, from 4.0 kb to 4.2 kb, or from 4.0 kb to 4.1 kb, or from 3.0 kb to 3.9 kb, from 3.0 kb to 3.8 kb, or from 3.0 kb to 3.7 kb. Aspect 24. 24. The polynucleotide of any one of embodiments 1-23, wherein the expression cassette is flanked by 5' and 3' inverted terminal repeats (ITRs). Aspect 25. 25. The polynucleotide of embodiment 24, wherein the ITR is an AAV2 ITR and / or the ITR shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NOs: 15-21. Aspect 26. A gene therapy vector comprising the polynucleotide of any one of embodiments 1 to 25. Aspect 27. The vector of embodiment 26, wherein the gene therapy vector is a recombinant adeno-associated virus (rAAV) vector. Aspect 28. The vector of embodiment 27, wherein the rAAV vector is AAVrh74 or a functional variant thereof. Aspect 29. The vector of embodiment 28, wherein the rAAV vector comprises a capsid protein that shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 100. Aspect 30. The vector of embodiment 27, wherein the rAAV vector is AAV9 or a functional variant thereof. Aspect 31. The vector of embodiment 30, wherein the rAAV vector comprises a capsid protein that shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NO: 97. Aspect 32. The vector of embodiment 27, wherein the rAAV vector is AAV6 or a functional variant thereof. Aspect 33. The vector of embodiment 32, wherein the rAAV vector comprises a capsid protein that shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 98. Aspect 34. The vector of embodiment 27, wherein the rAAV vector is AAVrhlO or a functional variant thereof. Aspect 35. The vector of embodiment 34, wherein the rAAV vector comprises a capsid protein that shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 99. Aspect 36. A method of treating and / or preventing a disease or disorder in a subject in need thereof, comprising administering to said subject a vector according to any one of aspects 26 to 35. Aspect 37. The method of embodiment 36, wherein said disease or disorder is a cardiac disorder. Aspect 38. The method of embodiment 37, wherein the cardiac disorder is a cardiomyopathy, optionally BAG3-associated dilated cardiomyopathy. Aspect 39. The method of embodiment 38, wherein the cardiomyopathy is hypertrophic cardiomyopathy (HCM) (hypertrophy). Aspect 40. The method of embodiment 38, wherein the cardiomyopathy is dilated cardiomyopathy (DCM). Aspect 41. The method of embodiment 37, wherein said disease or disorder is arrhythmia, optionally atrial fibrillation or sinus node disease. Aspect 42. The method of embodiment 37, wherein said disease or disorder is heart failure. Aspect 43. The method of any one of aspects 36 to 42, wherein the subject is a mammal. Aspect 44. The method of embodiment 43, wherein the subject is a primate. Aspect 45. The method of embodiment 44, wherein the subject is a human. Aspect 46. The method of any one of aspects 36 to 45, wherein the subject has a mutation in the BAG3 gene and / or reduced expression of BAG3 when compared to a healthy subject. Aspect 47. The method of any one of aspects 36-46, wherein said vector is administered by intravenous injection, intracardiac injection, intracardiac infusion, and / or cardiac catheterization. Aspect 48. The method of any one of aspects 36-47, wherein said administering increases BAG3 expression by at least about 5%. Aspect 49. The method of any one of aspects 36-47, wherein said administering increases BAG3 expression by at least about 30%. Aspect 50. The method of any one of aspects 36-47, wherein said administering increases BAG3 expression by at least about 70%. Aspect 51. The method of any one of aspects 36-47, wherein the administration increases BAG3 expression by about 5% to about 10%. Aspect 52. The method of any one of aspects 36 to 47, wherein the administration increases BAG3 expression by about 30% to about 50%. Aspect 53. The method of any one of aspects 36 to 47, wherein the administration increases BAG3 expression by about 50% to about 70%. Aspect 54. The method of any one of aspects 36 to 47, wherein the administration increases BAG3 expression by about 70% to about 100%. Aspect 55. The method according to any one of embodiments 36 to 54, for treating and / or preventing said disease or disorder. Aspect 56. The method of any one of aspects 36 to 55, comprising the step of administering an effective amount of said vector. Aspect 57. The method of any one of embodiments 36 to 56, wherein said disease or disorder is associated with or caused by truncation of BAG3 in said subject. Aspect 58. The method of any one of aspects 36 to 57, comprising administering an effective amount of a pharmaceutical composition comprising said vector. Aspect 59. Approximately 1×10 11 Vector genomes ~ approx. 1 × 10 13 administering to the subject about 1×10 vector genomes of the vector; 12 Vector genomes ~ approx. 1 × 10 14 or administering to the subject about 1×10 vector genomes of the vector. 13 Vector genomes ~ approx. 1 × 10 15 59. The method of any one of aspects 36 to 58, comprising administering to said subject said vector a vector genome. Aspect 60. A pharmaceutical composition comprising the vector of any one of embodiments 26 to 35. Aspect 61. A kit comprising the vector of any one of embodiments 26 to 35 or the pharmaceutical composition of embodiment 60, and optionally, instructions for use. Aspect 62. Use of the vector of any one of embodiments 26 to 35, optionally in accordance with the method of any one of embodiments 39 to 63, in treating said disease or disorder. Aspect 63. The vector of any one of embodiments 29 to 38, optionally for use in treating said disease or disorder according to the method of any one of embodiments 36 to 59. Aspect 64. A polynucleotide comprising a polynucleotide sequence sharing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NOs: 107-116. Aspect 65. The polynucleotide of embodiment 64, wherein the promoter is an MHCK7 promoter. Aspect 66. 66. The polynucleotide of embodiment 65, wherein the MHCK7 promoter shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 31. Aspect 67. The polynucleotide of embodiment 64, wherein BAG3 is human BAG3. Aspect 68. A gene therapy vector comprising the polynucleotide of any one of embodiments 64 to 67. Aspect 69. The vector of embodiment 68, wherein the gene therapy vector is a recombinant adeno-associated virus (rAAV) vector. Aspect 70. The vector of embodiment 69, wherein the rAAV vector is an AArh74 vector. Aspect 71. The vector of embodiment 69, wherein the rAAV vector is an AAV9 vector. Aspect 72. A method for treating and / or preventing cardiac damage in a subject identified as having a mutation or truncation in BAG3, the method comprising administering to the subject a vector according to any one of aspects 68 to 71. Aspect 73. The method of embodiment 72, wherein the disease or disorder is a cardiomyopathy, optionally dilated cardiomyopathy (DCM). Aspect 74. The method of embodiment 72, wherein the cardiac disorder is arrhythmia, optionally atrial fibrillation or sinus node disease, or BAG3-associated dilated cardiomyopathy. Aspect 75. The method of embodiment 72, wherein the cardiac disorder is heart failure. Aspect 76. The method of any one of aspects 72 to 75, wherein the subject is a mammal. Aspect 77. The method of any one of embodiments 72-76, wherein said vector is administered by intravenous injection, intracardiac injection, intracardiac infusion, and / or cardiac catheterization.

[0216] All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to herein and / or listed in the Application Data Sheet are hereby incorporated by reference in their entirety.

[0217] It will be appreciated from the foregoing that specific embodiments of the disclosure have been described herein for purposes of illustration, but that various modifications can be made without deviating from the spirit and scope of the disclosure. EXAMPLES

[0218] Example 1: Preclinical Transgene Expression Test the vectors shown in Figures 1-8. Test the AAV vectors or their respective expression cassettes in vitro using cultured cardiomyocytes (e.g., induced pluripotent stem cell cardiomyocytes, iPSC-CM, AC16, HL-1, C2C12, H9C2) or other cells suitable for transfection or transduction with these constructs. Evaluate the expression of the BAG3 transgene protein by ELISA, immunofluorescence, immunohistochemistry, and Western blot. Detect vector DNA by PCR and BAG3 transgene mRNA by qRT-PCR.

[0219] Considering the difficulty in efficiently maintaining BAG3 KO cardiomyocytes in culture due to the expected high levels of spontaneous apoptosis, cell-based studies using patient iPSC-derived cardiomyocytes may be very challenging.To clarify the benefits of BAG3 transgene expression (following either AAV vector transduction and / or transfection with vector plasmids), we aimed to alleviate / reduce the apoptosis levels of these cells (measured by conventional methods, e.g., TUNEL) after differentiation into cardiomyocytes using C2C12, AC16, and H9C2 cells, as well as cellular models using shRNAs targeting BAG3.

[0220] The expression cassettes shown in Figures 1-3 and 8 were tested after packaging into the AAV.rh74 vector. The resulting AAV vectors were tested in vitro using cultured CHO-Lec2, a standard mutant cell line lacking 70-90% of sialic acid in glycoproteins and gangliosides, which renders the cells susceptible to AAV transduction. Subsequent BAG3 protein expression in transduced CHO-Lec2 cells was assessed by Western blot. Data showing AAVrh.74-BAG3-mediated expression of the transgene protein (BAG3) are presented (Figure 9).

[0221] The UBC promoter induced the highest expression level of BAG3 by WB in CHO-Lec2 cells, followed by hHSP70 and MHCK7. Under these conditions, the hTnnT2 promoter ("hTnT") was found to induce low levels of BAG3 protein expression. Based on these results, it can be concluded that the AAVrh.74 vector can efficiently express BAG3 protein.

[0222] Example 2: Preclinical Efficacy In Vivo in BAG3 KO and BAG3 CKO Mice Selected vectors are tested in vivo using mutant mouse models of cardiomyopathy. The BAG3- / - knockout mouse model displays a severe DCM phenotype and exhibits one or more DCM components of the human disease. The BAG3- / - knockout mouse (e.g., as described in Homma et al., Am J Pathol, 2006) has a single retroviral insertion that selectively disrupts the mouse bag3 gene. Histological analysis of tissues from BAG3- / - mice revealed abnormalities in skeletal and cardiac tissues, but not elsewhere, suggesting a possible myopathy. BAG3- / - knockout mice were indistinguishable from their wild-type (+ / +) and heterozygous (+ / -) littermates during the first week of life. Myocardium from BAG3 null mice displayed degenerative changes, with the atria more affected than the ventricles. KO mice stop gaining weight after 12 days and appear smaller than littermates, with all BAG3- / - animals dying by day 25. After 20 days of age, myofiber degeneration was evident throughout all muscles harvested from BAG3- / - mice. H&E-stained fresh frozen sections of muscles from BAG3- / - animals showed marked variation in myofiber size, with evidence of atrophic fibers and intracellular accumulation of basophilic material. No inflammation, myonecrosis, or other dystrophic pathological abnormalities were observed in BAG3- / - muscles.

[0223] A Cre-inducible cardiac-specific BAG3- / - knockout (also called bis-iCKO) is also tested as a mouse model of a moderate DCM phenotype. This mouse model displays one or more DCM elements of the human disease and is characterized by a cardiomyocyte-specific, tamoxifen-activated BAG3 knockout, referred to as "bis-iCKO" (e.g., as described in Yun et al, Int J Mol Sci 2021). This mouse model allows for developmental control of the loss of expression of BAG3, restricting it to adult myocytes and initiating the progression of molecular and functional events that lead to dilated cardiomyopathy. Bis-iCKO mice were viable at birth but had a marked susceptibility to premature death as a result of DCM and heart failure. While 60% of bis-iCKO mice survived to 28 days of age, morphological and histological analysis of the bis-iCKO hearts revealed significant cardiac hypertrophy in these mice. Echocardiography revealed an age-dependent decline in left ventricular (LV) systolic function (percentage of fractional shortening [FS]) in BAG3-deficient mice.

[0224] The benefit of AAV-mediated BAG3 expression in any of the above or similar models would be evidenced by increased survival, increased weight gain, mitigation of the usual progression of echocardiographic cardiomyopathy (e.g., LVESD, LVEDD), mitigation of right and / or left ventricular size enlargement, and / or mitigation of the typical decline in left ventricular ejection fraction and / or fractional shortening. Histological analysis would reveal benefit by a reduced incidence of disease-associated myofiber disarray, reduced ventricular dilation, reduced ventricular wall thinning, reduced number of apoptotic cells, reduced DNA damage, and reduced disease-associated changes in absolute cardiac size.

[0225] Example 3: Rescue of heart failure in vivo after transverse aortic constriction (TAC) AAV-BAG3 gene therapy with the selected AAV vectors described above is performed essentially as described in Knezevic et al. (JACC, 2016). Different capsid serotypes, such as AAVrh.74 and / or AAV9, are used to package the AAV expression cassette and deliver it in vivo.

[0226] Mouse TAC model: Transverse aortic constriction (TAC) in mice is an experimentally induced cardiac hypertrophy due to pressure overload, followed by heart failure. Compared to other experimental mouse heart failure models, the TAC model results in more reproducible cardiac hypertrophy and a slower course of heart failure development. After TAC in mice, a gradual decrease in ejection fraction and other cardiac performance measures occurs in parallel with a gradual decrease in cardiac BAG3 levels. Male C57BI / 6J mice (approximately 4 months old) are placed under anesthesia and aortic arch is visualized by performing a precordial thoracotomy at the level of the third intercostal space. Constriction is performed by tying a silk suture between the first and second trunks of the aortic arch along a 28-gauge needle. For consistency, stenosis levels are quantified by measuring the change in Doppler velocity of the right and left carotid arteries on the 7th day after surgery. The peak velocity ratio of the right carotid artery to the left carotid artery can range from 5.0 to 6.5, and 2 weeks after TAC, the ejection fraction can range from 40% to 50%.

[0227] Functional support of efficacy by echocardiography: Bioactivity and support of efficacy for cardiac effects in the TAC model are assessed using transthoracic echocardiography at predefined time points including baseline and various post-TAC intervals. To screen for animals with sufficient cardiac dysfunction suitable for this mouse model, the Doppler ratio of peak velocity of the right carotid artery to the left carotid artery (RC / LC) is determined 1 week after TAC, and those that do not meet the criteria (RC / LC of 5.0-6.5) are excluded from the study. Additionally, echocardiography is performed at 2 weeks after TAC, and animals that fall outside the range of ejection fraction (EF) of 40-50% are also excluded. Mice with adequate Doppler RC / EV and EF by echocardiogram are then injected (either intravenously or retroorbitally) with AAV constructs overexpressing BAG3 protein or formulation buffer (FB; vehicle control) at 8 weeks after TAC. Over time, efficacy will be evident in AAV-BAG3-treated animals by significant increases in EF and FS when compared to the FB control group. Echocardiography will reveal a gradual decline in EF and FS over time in FB-injected mice. In contrast, EF and FS will return to control levels in AAV-BAG3-injected animals within weeks of treatment. Histological analysis will reveal a reduction in myocardial fiber disarray.

Claims

1. A polynucleotide containing an expression cassette, The expression cassette comprises a polynucleotide sequence that is functionally linked to the promoter and encodes B-cell lymphoma 2-associated athanogene 3 (BAG3) or a functional variant thereof. The aforementioned polynucleotide.

2. The polynucleotide according to claim 1, wherein the promoter is a heterogeneous promoter.

3. The promoters include the cardiac-specific promoter, muscle-specific promoter, cardiomyocyte-specific promoter, myosin heavy chain creatine kinase 7 (MHCK7) promoter, SEQ ID NO: 31, sharing at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the MHCK7 promoter, SEQ ID NO: 31, the hTNNT2 promoter, sharing at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the cardiac troponin T (hTNNT2) promoter, SEQ ID NO: 32, the human heat shock factor 70 (hHSP70) promoter, SEQ ID NO: The polynucleotide according to claim 1, selected from the group consisting of an hHSP70 promoter, a ubiquitous promoter, a CMV promoter, a CAG promoter, a chicken β-globin promoter, and a UBC promoter having the sequence SEQ ID NO: 116, sharing at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with 115.

4. The polynucleotide according to claim 1, wherein the expression cassette comprises exon 1 of the cardiac troponin T (hTNNT2) gene.

5. The polynucleotide according to claim 1, wherein the promoter is the hTNNT2 promoter, the expression cassette comprises exon 1 of the cardiac troponin T (hTNNT2) gene, and both the hTNNT2 promoter and exon 1 share at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:

32.

6. The polynucleotide according to claim 1, wherein the expression cassette includes a polyA signal.

7. The polynucleotide according to claim 6, wherein the polyA signal is human growth hormone (hGH) polyA.

8. The polynucleotide according to claim 1, wherein the expression cassette comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

9. The polynucleotide according to claim 8, wherein the WPRE is a mutant or modified WPRE.

10. The polynucleotide according to claim 9, wherein the WPRE is WPRE Mut6 (SEQ ID NO: 63).

11. The polynucleotide according to claim 1, wherein the BAG3 is full-length BAG3 or wild-type BAG3.

12. The polynucleotide according to claim 1, wherein the BAG3 is human BAG3.

13. The polynucleotide according to claim 1, wherein the polynucleotide sequence encoding BAG3 shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 2, or is a human BAG3 polynucleotide.

14. The polynucleotide according to claim 1, wherein the expression cassette is adjacent to 5' and 3' inverted terminal repeat sequences (ITRs).

15. The polynucleotide according to claim 14, wherein the ITR is an AAV2 ITR, or shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NO: 15 to 21.

16. A gene therapy vector comprising a polynucleotide according to any one of claims 1 to 15.

17. The vector according to claim 16, wherein the gene therapy vector is a recombinant adeno-associated virus (rAAV) vector.

18. The vector according to claim 17, wherein the rAAV vector is selected from the group consisting of AAVrh74 vector or a functional variant thereof, AAV9 vector or a functional variant thereof, AAV6 vector or a functional variant thereof, and AAVrh10 vector or a functional variant thereof.

19. The vector according to claim 17, wherein the rAAV vector comprises a capsid protein that shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a sequence selected from the group consisting of SEQ ID NO: 100, SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO:

99.

20. A pharmaceutical composition for treating and / or inhibiting a disease or disorder, comprising the vector according to claim 16.

21. The pharmaceutical composition according to claim 20, wherein the disease or disorder is a cardiac disorder.

22. The pharmaceutical composition according to claim 21, wherein the cardiac disorder is cardiomyopathy, arrhythmia, or heart failure.

23. The pharmaceutical composition according to claim 21, wherein the cardiac disorder is a cardiomyopathy selected from the group consisting of BAG3-associated dilated cardiomyopathy, hypertrophic cardiomyopathy (HCM), and dilated cardiomyopathy (DCM).

24. The pharmaceutical composition according to claim 21, wherein the cardiac disorder is an arrhythmia selected from the group consisting of atrial fibrillation and sinoatrial node disease.

25. The pharmaceutical composition according to claim 20 for administration to a mammal.

26. The pharmaceutical composition according to claim 25, wherein the mammal is a primate.

27. The pharmaceutical composition according to claim 26, wherein the primate is a human.

28. The pharmaceutical composition according to claim 20 for administration to subjects having a mutation in the BAG3 gene, decreased BAG3 expression, or both, compared to healthy subjects.

29. The pharmaceutical composition according to claim 20, formulated for administration by a route selected from the group consisting of intravenous injection, intracardiac injection, intracardiac infusion, and cardiac catheterization.

30. The pharmaceutically active composition according to claim 20, wherein administration of the composition increases BAG3 expression to an extent selected from the group consisting of at least about 5%, at least about 30%, at least about 70%, about 5% to about 10%, about 30% to about 50%, about 50% to about 70%, and about 70% to about 100%.

31. The pharmaceutical composition according to claim 20, wherein the disease or disorder is associated with or caused by shortening of BAG3 in the subject.

32. Approximately 1 × 10 per dose 11 A vector genome of approximately 1 × 10⁶ 13 Each vector genome contains approximately 1 × 10⁶ of genomes per dose. 12 A vector genome of approximately 1 × 10⁶ 14 One vector genome, or approximately 1 × 10⁶ per dose. 13 A vector genome of approximately 1 × 10⁶ 15 The pharmaceutical composition according to claim 20, comprising a vector genome.

33. A pharmaceutical composition comprising the vector according to claim 16 for treating and / or inhibiting cardiac impairment in subjects having mutations or shortenings in BAG3.

34. The pharmaceutical composition according to claim 33, wherein the cardiac disorder is selected from the group consisting of cardiomyopathy, dilated cardiomyopathy (DCM), arrhythmia, atrial fibrillation, sinoatrial node disease, BAG3-associated dilated cardiomyopathy, and heart failure.

35. The pharmaceutical composition according to claim 33, formulated for administration by a route selected from the group consisting of intravenous injection, intracardiac injection, intracardiac infusion, and cardiac catheterization.

36. A polynucleotide containing a polynucleotide sequence that shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with one of SEQ ID NO: 107–116.

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