Optimized Expression Cassettes for Gene Therapy

Abstract

In some aspects, cardiac-specific expression cassettes are provided herein. In some aspects, provided herein are expression cassettes comprising a polynucleotide sequence encoding a gene product for the treatment of cardiac disease, the polynucleotide sequence being operably linked to a promoter (e.g., a cardiac-specific promoter) and, optionally, an enhancer (e.g., a cardiac-specific enhancer). In some aspects, the present disclosure provides recombinant adeno-associated virus (rAAV) virions, comprising a viral genome comprising a capsid protein and an expression cassette comprising a polynucleotide sequence encoding a therapeutic gene product, e.g., a dwarf open reading frame (DWORF) polypeptide, operably linked to a promoter, the expression cassette being flanked by inverted terminal repeats. The present disclosure further provides pharmaceutical compositions and methods for treating or preventing cardiac disease.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 219,651, filed July 8, 2021, which is incorporated by reference in its entirety.

[0002] The present invention relates generally to gene therapy, including optimized gene expression cassettes, recombinant adeno-associated virus (AAV) virions, and methods for using same to treat and prevent heart disease. Reference to Electronic Sequence Listing

[0003] The contents of the electronic sequence listing (TENA_021_02WO_SeqList_ST26.xml; size: 430,204 bytes; and creation date: July 8, 2022) are incorporated herein by reference in their entirety. [Background technology]

[0004] Cardiomyopathy is responsible for approximately half of cardiac-related deaths. It is estimated that approximately 1 in 250 to 1 in 10,000 people suffer from some form of cardiomyopathy (McKenna et al. Circ Res. 121:722-730 (2017)). Despite major efforts in screening, diagnosis, and treatment strategies, the prevalence of cardiomyopathy and the incidence of cardiomyopathy-related deaths remain high (Brieler et al. Am Fam Physician. 96:640-646 (2017)).

[0005] Cardiomyopathy refers to a group of heart conditions that occur when its ability to pump blood is impaired. A decrease in the proper functioning of the myocardium, such as contractile dysfunction, can lead to myocardial infarction, heart failure, blood clots, valve problems, and cardiac arrest. Cardiomyopathy can be divided into primary and secondary categories that result in various phenotypes (McKenna et al. Circ Res. 121:722-730 (2017)). Primary cardiomyopathies can be genetic, acquired, or mixed in etiology. Genetic cardiomyopathies are inherited and include arrhythmogenic right ventricular dysplasia, hypertrophy, ion channelopathy, left ventricular compression, and mitochondrial myopathy. Acquired cardiomyopathies lead to cardiac complications and are primarily due to non-secondary non-genetic causes, including myocarditis, peripartum, tachycardia-induced cardiomyopathies, and stress-induced cardiomyopathies. Cardiomyopathy with mixed etiology is caused by a combination of non-genetic and genetic factors and includes dilated and restrictive cardiomyopathies. Secondary cardiomyopathies refer to heart disease resulting from extra-cardiovascular causes. The underlying causes of secondary cardiomyopathies may be endocrine, infectious, exposure to toxins, autoimmune-related, nutritional, and / or neuromuscular.

[0006] Cardiomyocytes play a central role in cardiomyopathies. Cardiomyocytes, also called cardiac muscle cells, cardiac myocytes, or myocardiocytes, are the cardiac cells that make up the heart muscle and are responsible for the contractile function that allows the heart to act as a pump. There are many mechanisms that reduce the ability of cardiomyocytes to function properly (Dadson et al. Clin Sci (Lond) 131:1375-1392 (2017)). In arrhythmogenic right ventricular cardiomyopathy, progressive replacement of cardiomyocytes with fibrotic tissue leads to electrical isolation of cardiomyocytes and atrophy of the ventricular myocardium, the main structure responsible for contractile function in the heart. In mitochondrial cardiomyopathy, defects in ATP production have a direct effect on contractile function in cardiomyocytes with high metabolic demands. Cardiomyopathy can also be caused by a loss of activity in regulatory enzymes, such as sarco / endoplasmic reticulum calcium ATPase (SERCA), resulting in a loss of normal Ca production. 2+It manifests as a result of abnormal contractile function resulting from loss of ion release, uptake, and sequestration processes (Lennon et al. Int J Mol Med. 7:131-41 (2001)).

[0007] Treatment strategies for cardiomyopathies are needed.

[0008] Gene therapy approaches for the treatment of cardiac disease often use vectors configured to transduce cardiac cells and express a transgene in a cardiac tissue-specific manner. Adeno-associated virus (AAV) vectors, cardiac-specific promoters, or both in combination may be used to deliver a polynucleotide encoding a gene product (e.g., a therapeutic protein) to cardiac tissue, thereby expressing the gene product in that tissue to treat cardiac disease.

[0009] However, achieving high expression of the gene product remains difficult, especially in cardiac cells. Given these difficulties, there remains a need in the art for improved gene therapy vectors for cardiac disease. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] McKenna et al.Circ Res.121:722-730(2017 [Non-Patent Document 2] Brieler et al.Am Fam Physician.96:640-646(2017) [Non-Patent Document 3] Dadson et al.Clin Sci(Lond)131:1375-1392(2017) [Non-Patent Document 4] Lennon et al.Int J Mol Med.7:131-41(2001) Summary of the Invention

[0011] In some aspects, the present invention generally relates to vectors for delivery of polynucleotides encoding dwarf open reading frames (DWORFs) or other transgenes to cardiac cells, e.g., cardiomyocytes. Disclosed herein are recombinant adeno-associated virus virions (rAAV virions) that contain expression cassettes and capsid proteins that effectively deliver DWORF polynucleotides into cardiac cells, along with related compositions and methods. In any aspect described herein in which a DWORF transgene is referenced, DWORF can be replaced by a reference to another transgene expression desired in cardiac cells. In some embodiments, when AAV-based expression vectors and virions are referenced, the present disclosure also contemplates the use of other viral and non-viral vectors for delivery of transgenes. In particular, any viral and non-viral vectors that can be used for delivery of transgenes into cardiac cells are provided herein.

[0012] In some aspects, provided herein is an expression cassette comprising a polynucleotide sequence comprising: i) one or more promoters, optionally wherein the one or more promoters are cardiac-specific promoters; and ii) one or more copies of a transgene, optionally encoding a polypeptide for treating or preventing heart disease or alleviating symptoms associated with heart disease; In addition to elements (i) and (ii), the expression cassette comprises one or more of the following: iii) one or more enhancers, optionally wherein the one or more enhancers are cardiac-specific enhancers; iv) the one or more copies of the transgene is at least two copies of the transgene; v) the polynucleotide sequence contains one or more introns; and / or vi) at least one copy of the transgene(s) is codon-optimized.

[0013] In some embodiments of the expression cassettes described above, in addition to elements (i) and (ii), the expression cassette comprises one, two, three, or all four of elements (iii), (iv), (v), and (vi) (any combination of elements (iii), (iv), (v), and (vi) can be used). In some embodiments of the expression cassettes described above, in addition to elements (i) and (ii), the expression cassette comprises one or more enhancers, the one or more enhancers are cardiac-specific enhancers, and / or the polynucleotide sequence comprises one or more introns. In some embodiments of the expression cassettes described above, in addition to elements (i) and (ii), the expression cassette comprises one or more enhancers, the one or more enhancers are cardiac-specific enhancers, and the polynucleotide sequence comprises one or more introns. In some embodiments, the one or more introns improve or can improve the efficiency of transgene expression. In some embodiments of the expression cassette described above, in addition to elements (i) and (ii), the expression cassette comprises two copies of a transgene, the two copies being non-identical, and optionally, a first copy is codon-optimized and the second copy is not a codon-optimized nucleotide sequence encoding the transgene. In some embodiments of the expression cassette described above, in addition to elements (i) and (ii), the expression cassette comprises two copies of a transgene, the two copies being non-identical, and optionally, a first copy is codon-optimized and the second copy is not a codon-optimized nucleotide sequence encoding the transgene, and further the polynucleotide sequence comprises one or more introns.In some embodiments of the expression cassette described above, in addition to elements (i) and (ii), the expression cassette comprises two copies of a transgene, the two copies being non-identical to each other, and optionally, the first copy is codon-optimized and the second copy is not a codon-optimized nucleotide sequence encoding the transgene, the polynucleotide sequence further comprises one or more introns, and the polynucleotide sequence further comprises one or more enhancers (e.g., the one or more enhancers are cardiac-specific enhancers). In some embodiments, the one or more introns improve or can improve the efficiency of transgene expression. In some embodiments where two copies of a transgene are used, two copies of a promoter are also used.

[0014] In some embodiments of the expression cassette, the polynucleotide sequence comprises one or more promoters, and the one or more promoters are cardiac-specific enhancers. In some embodiments of the expression cassette, at least one promoter is a cardiac-specific promoter, or all of the promoters are cardiac-specific promoters. In some embodiments of the expression cassette, the polynucleotide sequence comprises a single promoter. In some embodiments of the expression cassette, the polynucleotide sequence comprises two promoters. In some embodiments of the expression cassette, at least one promoter of the one or more promoters is a chicken cTnT promoter. In some embodiments, the chicken cTnT promoter shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 11. In some embodiments, the chicken cTnT promoter comprises SEQ ID NO: 11. In some embodiments of the expression cassette, at least one promoter of the one or more promoters is a human cTnT promoter. In some embodiments, the human cTnT promoter shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 12 or SEQ ID NO: 13. In some embodiments, the human cTnT promoter comprises SEQ ID NO: 12 or SEQ ID NO: 13. In some embodiments, the expression cassette comprises a chicken cTnT promoter and a human cTnT promoter.

[0015] In some embodiments of the expression cassette, the polynucleotide sequence comprises one or more copies of a transgene, the transgene encoding a polypeptide for treating or preventing heart disease or alleviating symptoms associated with heart disease.

[0016] In some embodiments of the expression cassette, the one or more copies of the transgene are at least two copies of the transgene. In some embodiments of the expression cassette, the one or more copies of the transgene are two copies of the transgene. In some embodiments, the one or more copies of the transgene are at least two copies of the transgene, and the polynucleotide sequence comprises at least two promoters, each operably linked to at least two copies of the transgene. In some embodiments, the one or more copies of the transgene are two copies of the transgene, and the polynucleotide sequence comprises two promoters, each operably linked to two copies of the transgene. In some embodiments of the expression cassette that comprises two copies of the transgene, the two "copies" are not identical. Without being bound by any theory, DNA recombination within the vector may be prevented by using two nucleic acid sequences that encode polypeptides that are not identical. In some embodiments, the expression cassette comprises one copy of a transgene with a native DNA sequence encoding the polypeptide and one copy of a transgene with a codon-optimized DNA sequence encoding the polypeptide. In some embodiments of an expression cassette that includes two copies of a transgene, the first copy of the transgene is sufficiently different from the second copy of the transgene to prevent DNA recombination.

[0017] In some embodiments of the expression cassette, the polynucleotide sequence comprises one or more enhancers, and optionally, the one or more enhancers are cardiac-specific enhancers. In some embodiments of the expression cassette, the polynucleotide sequence comprises two or more enhancers (e.g., two, three, or four enhancers). In some embodiments of the expression cassette, the one or more enhancers are cardiac-specific enhancers (e.g., at least one enhancer is a cardiac-specific enhancer, or two, three, or four, or all of the enhancers are cardiac-specific enhancers). In some embodiments of the expression cassette, the polynucleotide sequence comprises an enhancer. In some embodiments of the expression cassette, the polynucleotide sequence does not comprise an enhancer. In some embodiments, the one or more cardiac-specific enhancers are selected from an ACTC1 enhancer and an αMHC enhancer. In some embodiments, the ACTC1 enhancer shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:78. In some embodiments, the ACTC1 enhancer comprises SEQ ID NO: 78. In some embodiments, the aMHC enhancer shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 79. In some embodiments, the aMHC enhancer comprises SEQ ID NO: 79. In some embodiments, the expression cassette comprises an aMHC enhancer and an ACTC1 enhancer. In some embodiments of the expression cassette, the enhancer sequence comprises an aMHC enhancer followed by an ACTC1 enhancer. In some embodiments of the expression cassette, the enhancer sequence comprises an ACTC1 enhancer followed by an aMHC enhancer.

[0018] In some embodiments of the expression cassette, the polynucleotide sequence comprises one or more introns. In some embodiments of the expression cassette, the polynucleotide sequence comprises one intron. In some embodiments of the expression cassette, the polynucleotide sequence comprises two introns. In some embodiments of the expression cassette, the polynucleotide sequence comprises more than two introns. In some embodiments of the expression cassette, the one or more introns are the same. In some embodiments of the expression cassette, the one or more introns are different from each other. In some embodiments, the expression cassette comprises an intron, the intron being selected from a CMV intron and a chimeric intron. In some embodiments, the CMV intron shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:80. In some embodiments, the CMV intron comprises SEQ ID NO:80. In some embodiments, the chimeric intron shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:81. In some embodiments, the chimeric intron comprises SEQ ID NO: 81. In some embodiments, the expression cassette comprises a CMV intron and a chimeric intron. In some embodiments, the expression cassette does not comprise an intron (e.g., does not comprise a CMV intron or a chimeric intron).

[0019] In some embodiments of the expression cassette, at least one copy of the one or more copies of the transgene is codon optimized (e.g., codon optimized for optimal human expression). In some embodiments of the expression cassette, two copies of the transgene are codon optimized. In some embodiments of the expression cassette, a first copy of the transgene is codon optimized and a second copy of the transgene is not codon optimized (e.g., the native DNA sequence) or is otherwise different from the first copy. In some embodiments of the expression cassette, the first copy of the transgene is sufficiently different from the second copy of the transgene to prevent DNA recombination.

[0020] In some embodiments, the expression cassette further comprises one or more (e.g., two) post-transcriptional regulatory elements ("PTRE"). In some embodiments, the expression cassette further comprises one or more (e.g., two) WPRE sequences. In some embodiments, the expression cassette comprises one WPRE sequence. In some embodiments, the WPRE sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:26. In some embodiments, the WPRE sequence comprises SEQ ID NO:26. In some embodiments, the expression cassette does not comprise a WPRE sequence.

[0021] In some embodiments, the expression cassette further comprises one or more polyadenylation sequences ("p(A)"). In some embodiments, the expression cassette comprises one polyadenylation sequence. In some embodiments, the expression cassette comprises two polyadenylation sequences. In some embodiments, the polyadenylation sequence is selected from a BGH polyadenylation sequence and an SV40 polyadenylation sequence. In some embodiments, the BGH polyadenylation sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:27. In some embodiments, the BGH polyadenylation sequence comprises SEQ ID NO:27. In some embodiments, the SV40 polyadenylation sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:28. In some embodiments, the SV40 polyadenylation sequence comprises SEQ ID NO:28. In some embodiments, the expression cassette comprises a BGH polyadenylation sequence and an SV40 polyadenylation sequence.

[0022] In some embodiments, the expression cassette comprises a 5' to 3' arrangement of elements selected from any one of the following: (i) 5′-promoter-intron-transgene-PTRE-p(A)-3′; (ii) 5′-promoter-transgene-PTRE-p(A)-promoter-transgene-PTRE-p(A); (iii) 5′-enhancer-promoter-transgene-PTRE-p(A)-3′; (iv) 5′-enhancer-promoter-intron-transgene-PTRE-p(A)-3′; (v) 5'-enhancer-enhancer-promoter-transgene-PTRE-p(A)-3'; (vi) 5'-enhancer-enhancer-promoter-intron-transgene-PTRE-p(A)-3'; (vii) 5'-enhancer-promoter-intron-transgene-PTRE-p(A)-p(A)-transgene-intron-promoter-enhancer-3'; (viii) 5'-enhancer-promoter-intron-transgene-PTRE-p(A)-enhancer-promoter-intron-transgene-p(A)-3'; (ix) 5'-p(A)-PTRE-transgene-intron-promoter-enhancer-enhancer-promoter-intron-transgene-p(A)-3'; (x) 5'-promoter-intron-transgene-PTRE-p(A)-p(A)-transgene-intron-promoter-3'; (xi) 5'-promoter-intron-transgene-PTRE-p(A)-promoter-intron-transgene-p(A)-3'; and (xii) 5'-p(A)-PTRE-transgene-intron-promoter-promoter-intron-transgene-p(A)-3'.

[0023] In some embodiments of the expression cassette, the transgene has an increased expression level compared to an expression cassette comprising a polynucleotide having a 5' to 3' arrangement of elements comprising: 5'-promoter-transgene-WPRE-p(A)-3'. In some embodiments, the increased expression level is between about 1.5-fold and about 150-fold. In some embodiments, the increased expression level is at least 2-fold, at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, at least 75-fold, or at least 100-fold.

[0024] In some embodiments, the expression cassette is flanked by ITRs. In some embodiments, the ITRs share at least 90%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 14 and SEQ ID NO: 15. In some embodiments, the ITRs comprise one or more of SEQ ID NO: 14 and SEQ ID NO: 15.

[0025] In some embodiments, the expression cassette comprises from about 1.9 kb to about 3.7 kb, hi some embodiments, the expression cassette comprises from about 2.5 kb to about 3.7 kb, and optionally from about 2.8 kb to about 3.6 kb.

[0026] In some embodiments, the transgene in the expression cassette optionally encodes a polypeptide useful in treating a cardiac disease or disorder when a wild-type copy of the gene is introduced into a subject, hi some embodiments, the transgene in the expression cassette encodes a polypeptide that is associated with a cardiac disease (e.g., a loss-of-function mutation in the gene encoding the polypeptide is associated with a cardiac disease).

[0027] In some embodiments, the transgene in the expression cassette encodes a polypeptide selected from DWORF, JPH2, BAG3, CRYAB, Lamin A isoform of LMNA, Lamin C isoform of LMNA, TNNI3, PLN, LAMP2a, LAMP2b, LAMP2c, DPI isoform of DSP, DPII isoform of DSP, DSG2, and JUP. In some embodiments, the expression cassette comprises a transgene that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQ ID NO:227, or SEQ ID NO:229. In some embodiments, the polypeptide shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:208, SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, or SEQ ID NO:230.

[0028] In some embodiments, the transgene in the expression cassette encodes a DWORF polypeptide. In some embodiments, the transgene shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:33, SEQ ID NO:44, SEQ ID NO:76, or SEQ ID NO:77. In some embodiments, the polypeptide shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:32, or SEQ ID NO:43.

[0029] In some embodiments, the expression cassette comprises a 5' to 3' arrangement of elements selected from any one of the following: (i) 5'-human or chicken TnT promoter-chimeric intron-transgene-WPRE-p(A)-3'; (ii) a first copy of 5'-first human or chicken TnT promoter-transgene-WPRE-p(A)-second copy of second human or chicken TnT promoter-transgene-WPRE-p(A), optionally wherein the first and second promoter sequences and the first and second copies of the transgene are in the same forward orientation; (iii) 5′-human TnT promoter-CMV intron-transgene-WPRE-bGHpA-3′; (iv) 5′-ACTC1e enhancer-heart-human TnT promoter-transgene-WPRE-bGHpA-3′; (v) 5′-αMHCe enhancer-human TnT promoter-transgene-WPRE-bGHpA-3′; (vi) 5′-ACTC1e enhancer-human TnT promoter-CMV intron-transgene-WPRE-bGHpA-3′; (vii) 5'-αMHCe enhancer-human TnT promoter-CMV intron-transgene-WPRE-bGHpA-3'; (viii) 5′-ACTC1e enhancer-αMHCe enhancer-human TnT promoter-transgene-WPRE-bGHpA-3′; (ix) 5'-αMHCe enhancer-ACTC1e enhancer-human TnT promoter-transgene-WPRE-bGHpA-3'; (x) 5'-ACTC1e enhancer-αMHCe enhancer-human TnT promoter-CMV intron-transgene-WPRE-bGHpA-3'; (xi) 5'-αMHCe enhancer-ACTC1e enhancer-human TnT promoter-CMV intron-transgene-WPRE-bGHpA-3'; (xii) a 5'-human TnT promoter transgene with a codon-optimized polynucleotide sequence -WPRE-bGHpA-3'; (xiii) 5'-αMHCe enhancer-human TnT promoter-CMV intron-first transgene-WPRE-bGHpA-SV40pA-second transgene (e.g., with a codon-optimized polynucleotide sequence)-chimeric intron-chicken TnT promoter-ACTC1e enhancer-3', optionally wherein the first transgene and human TnT promoter are in a forward orientation and the second transgene and chicken TnT promoter are in a reverse orientation; (xiv) 5'-αMHCe enhancer-human TnT promoter-CMV intron-first transgene-WPRE-bGHpA-ACTC1e enhancer-chicken TnT promoter-chimeric intron-second transgene (e.g., with a codon-optimized polynucleotide sequence)-SV40pA-3', optionally wherein the first transgene, human TnT promoter, second transgene, and chicken TnT promoter are in a forward orientation; (xv) 5'-bGHpA-WPRE-first transgene-CMV intron-human TnT promoter-αMHCe enhancer-ACTC1e enhancer-chicken TnT promoter-chimeric intron-second transgene (e.g., with a codon-optimized polynucleotide sequence)-SV40pA-3', optionally wherein the first transgene and human TnT promoter are in a reverse orientation and the second transgene and chicken TnT promoter are in a forward orientation; (xvi) 5'-human TnT promoter-CMV intron-first transgene-WPRE-bGHpA-pSV40pA-second transgene (e.g., with a codon-optimized polynucleotide sequence)-chimeric intron-chicken TnT promoter-3', optionally with the first transgene and human TnT promoter in a forward orientation and the second transgene and chicken TnT promoter in a reverse orientation; and (xvii) 5'-human TnT promoter-CMV intron-first transgene-WPRE-bGHpA-chicken TnT promoter-chimeric intron-second transgene (e.g., with a codon optimized polynucleotide sequence)-SV40pA-3', optionally with the first transgene, human TnT promoter, second transgene, and chicken TnT promoter in a forward orientation; and (xix) 5'-bGHpA-WPRE-first transgene-CMV intron-human TnT promoter-chicken TnT promoter-chimeric intron-second transgene (e.g., with a codon optimized polynucleotide sequence)-SV40pA-3', optionally with the first transgene and human TnT promoter in a reverse orientation and the second transgene and chicken TnT promoter in a forward orientation.

[0030] In some embodiments, the expression cassette comprises a 5' to 3' arrangement of elements selected from any one of the following: (i) 5′-ACTC1e enhancer-αMHCe enhancer-human TnT promoter-CMV intron-transgene-WPRE-bGHpA-3′; (ii) 5′-αMHCe enhancer-ACTC1e enhancer-human TnT promoter-CMV intron-transgene-WPRE-bGHpA-3′; (iii) 5'-αMHCe enhancer-human TnT promoter-CMV intron-first transgene-WPRE-bGHpA-SV40pA-second transgene (e.g., with a codon-optimized polynucleotide sequence)-chimeric intron-chicken TnT promoter-ACTC1e enhancer-3', optionally with the first transgene and human TnT promoter in a forward orientation and the second transgene and chicken TnT promoter in a reverse orientation; (iv) 5'-αMHCe enhancer-human TnT promoter-CMV intron-first transgene-WPRE-bGHpA-ACTC1e enhancer-chicken TnT promoter-chimeric intron-second transgene (e.g., with a codon-optimized polynucleotide sequence)-SV40pA-3', optionally with the first transgene, human TnT promoter, second transgene, and chicken TnT promoter in a forward orientation; (v) 5'-bGHpA-WPRE-first transgene-CMV intron-human TnT promoter-αMHCe enhancer-ACTC1e enhancer-chicken TnT promoter-chimeric intron-second transgene (e.g., with a codon-optimized polynucleotide sequence)-SV40pA-3', optionally wherein the first transgene and human TnT promoter are in a reverse orientation and the second transgene and chicken TnT promoter are in a forward orientation; (vi) 5'-human TnT promoter-CMV intron-first transgene-WPRE-bGHpA-pSV40pA-second transgene (e.g., with a codon-optimized polynucleotide sequence)-chimeric intron-chicken TnT promoter-3', optionally with the first transgene and human TnT promoter in a forward orientation and the second transgene and chicken TnT promoter in a reverse orientation; (vii) 5'-human TnT promoter-CMV intron-first transgene-WPRE-bGHpA-chicken TnT promoter-chimeric intron-second transgene (e.g., with a codon-optimized polynucleotide sequence)-SV40pA-3', optionally with the first transgene, human TnT promoter, second transgene, and chicken TnT promoter in a forward orientation; and (viii) 5'-bGHpA-WPRE-first transgene-CMV intron-human TnT promoter-chicken TnT promoter-chimeric intron-second transgene (e.g., with a codon-optimized polynucleotide sequence)-SV40pA-3', optionally wherein the first transgene and the human TnT promoter are in a reverse orientation and the second transgene and the chicken TnT promoter are in a forward orientation.

[0031] In some embodiments, the expression cassette is a recombinant expression cassette.

[0032] In some aspects, provided herein is a recombinant vector comprising any of the expression cassettes described herein. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a non-viral vector.

[0033] In some aspects, provided herein are recombinant adeno-associated virus (rAAV) virions comprising a viral genome comprising a capsid protein and any of the expression cassettes described herein, wherein the expression cassette is flanked by inverted terminal repeats (ITRs). In some embodiments, the ITRs share at least 90%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 14 and SEQ ID NO: 15. In some embodiments, the ITRs share at least 90%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 14 and SEQ ID NO: 15. In some embodiments, the capsid protein shares at least 98%, at least 99%, or 100% identity with the AAV9 capsid protein (SEQ ID NO: 143). In some embodiments, the capsid protein shares at least 98%, at least 99%, or 100% identity with the AAV5 capsid protein (SEQ ID NO: 144). In some embodiments, the capsid protein is a chimeric capsid protein. In some embodiments, the capsid protein is an AAV5 / AAV9 chimeric capsid protein. In some embodiments, the capsid protein is selected from any one of SEQ ID NOs: 145-200.

[0034] In some aspects, provided herein is a pharmaceutical composition comprising any of the vectors described herein or any of the rAAV virions described herein and a pharma- ceutical acceptable carrier.

[0035] In some aspects, provided herein are kits that include any of the pharmaceutical compositions described herein, or any component of such a pharmaceutical composition (e.g., a vector or a rAAV virion).

[0036] In some aspects, provided herein are methods of increasing expression of a polypeptide in a cardiac cell or cardiac tissue, comprising contacting a cell with any vector described herein, any rAAV virion described herein, or any pharmaceutical composition described herein. In some embodiments, the cardiac cell is a cardiomyocyte. In some embodiments, the cardiac tissue is cardiac tissue. In some embodiments, the polypeptide expression is increased between about 1.5-fold and 150-fold. In some embodiments, the polypeptide expression is increased at least 2-fold, at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, at least 75-fold, or at least 100-fold. In some embodiments, the contacting is in vitro. In some embodiments, the contacting is in vivo.

[0037] In some aspects, provided herein are methods of increasing polypeptide expression in a subject, comprising administering to the subject any vector described herein, any rAAV virion described herein, or any pharmaceutical composition described herein. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, after administration, polypeptide expression is increased in the subject's heart. In some embodiments, the subject being treated has or is at risk for heart disease. In some embodiments, the subject being treated has a borderline or reduced ejection fraction. In some embodiments, the subject being treated has a normal ejection fraction. In some embodiments, the subject being treated has a genetic mutation associated with heart disease (e.g., a mutation in the PLN gene). In some embodiments, the subject has low or undetectable expression levels of the polypeptide encoded by the transgene compared to healthy subjects.

[0038] In some aspects, provided herein is a method of treating or preventing a cardiac disease or disorder in a subject in need thereof, comprising administering to the subject any of the vectors described herein, any of the rAAV virions described herein, or any of the pharmaceutical compositions described herein. In some embodiments, the subject being treated has a cardiac disease or disorder. In some embodiments, the subject being treated is at risk of developing a cardiac disease or disorder. In some embodiments, the cardiac disease or disorder is a cardiomyopathy. In some embodiments, the cardiomyopathy is a dilated cardiomyopathy. In some embodiments, the cardiac disease or disorder is a myocardial infarction. In some embodiments, the myocardial infarction is a chronic myocardial infarction. In some embodiments, the subject has a genetic risk allele for a cardiac disease or disorder. In some embodiments, the subject has a genetic risk allele for a cardiac disease or disorder due to a genetic mutation. In some embodiments, the subject has a genetic risk allele for a cardiac disease or disorder due to a genetic mutation in the PLN gene (e.g., one or more mutations in the PLN gene described herein or known in the art). In some embodiments, the cardiac disease or disorder is associated with reduced ejection fraction (HFrEF). In some embodiments, the impaired cardiac disease is associated with preserved ejection fraction (HFpEF). In some embodiments, the method leads to expression of a polypeptide encoded by the transgene in the heart of the subject. In some embodiments, the method leads to expression of a polypeptide encoded by the transgene in cardiomyocytes of the subject. In some embodiments, the method does not result in detectable expression of a polypeptide encoded by the transgene in muscles of the subject other than the heart, in the liver of the subject, and / or in cardiac fibroblasts of the subject. In some embodiments, the method improves one or more measures of cardiac function, optionally fractional shortening and / or left ventricular internal diameter (LVID). In some embodiments, the improvement in cardiac function is observed at 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, and / or 24 weeks or more after administration. In some embodiments, the administration is systemic.In some embodiments, the systemic administration is selected from intravenous injection or intracoronary injection. In some embodiments, when the rAAV virion is administered, it is administered as a unit dose. In some embodiments, the unit dose is about 3×10 14 vg / kg or less, approximately 2×10 14 vg / kg or less, approximately 1×10 14 vg / kg or less, approximately 9 × 10 13 vg / kg or less, approximately 8 × 10 13 vg / kg or less, approximately 7 × 10 13 vg / kg or less, approximately 6×10 13 vg / kg or less, approximately 5 × 10 13 vg / kg or less, approximately 4 × 10 13 vg / kg or less, approximately 3 × 10 13 vg / kg or less, approximately 2×10 13 vg / kg or less, or approximately 1 × 10 13 vg / kg or less. In some embodiments, the subject being treated is a mammal. In some embodiments, the subject being treated is a human.

[0039] In one aspect, the disclosure provides a recombinant adeno-associated virus (rAAV) virion comprising a viral genome comprising an expression cassette comprising a polynucleotide sequence encoding a capsid protein and a dwarf open reading frame (DWORF) polypeptide operably linked to a promoter, wherein the expression cassette is flanked by inverted terminal repeats.

[0040] In some embodiments, the DWORF polypeptide shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with a sequence selected from SEQ ID NOs: 1, 3, 4, 7, 9, 23, and 43. In some embodiments, the DWORF polypeptide is selected from SEQ ID NOs: 1, 3, 4, 7, 9, 23, and 43.

[0041] In some embodiments, the promoter is a chicken cTnT promoter. In some embodiments, the chicken cTnT promoter shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 11. In some embodiments, the chicken cTnT promoter comprises SEQ ID NO: 11. In some embodiments, the promoter is a human cTnT promoter. In some embodiments, the human cTnT promoter shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 12 or SEQ ID NO: 13. In some embodiments, the human cTnT promoter comprises SEQ ID NO: 12 or SEQ ID NO: 13.

[0042] In some embodiments, the expression cassette further comprises one or more enhancers. In some embodiments, the enhancer, the one or more enhancers are selected from an ACTC1 cardiac enhancer and an αMHC enhancer. In some embodiments, the ACTC1 cardiac enhancer shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 78. In some embodiments, the ACTC1 cardiac enhancer comprises SEQ ID NO: 78. In some embodiments, the αMHC enhancer shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 79. In some embodiments, the αMHC enhancer comprises SEQ ID NO: 79.

[0043] In some embodiments, the expression cassette further comprises an intron. In some embodiments, the intron is selected from a CMV intron and a chimeric intron. In some embodiments, the CMV intron shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 80. In some embodiments, the CMV intron comprises SEQ ID NO: 80. In some embodiments, the chimeric intron shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 81. In some embodiments, the chimeric intron comprises SEQ ID NO: 81.

[0044] In some embodiments, the expression cassette further comprises a WPRE sequence. In some embodiments, the WPRE sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 26. In some embodiments, the WPRE sequence comprises SEQ ID NO: 26.

[0045] In some embodiments, the expression cassette further comprises a polyadenylation sequence. In some embodiments, the polyadenylation sequence is selected from a BGH polyadenylation sequence and an SV40 polyadenylation sequence. In some embodiments, the BGH polyadenylation sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:27. In some embodiments, the BGH polyadenylation sequence comprises SEQ ID NO:27. In some embodiments, the SV40 polyadenylation sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:28. In some embodiments, the SV40 polyadenylation sequence comprises SEQ ID NO:28.

[0046] In some embodiments, the expression cassette is flanked by ITRs. In some embodiments, the ITRs share at least 90%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 14 and SEQ ID NO: 15. In some embodiments, the ITRs comprise one or more of SEQ ID NO: 14 and SEQ ID NO: 15.

[0047] In some embodiments, the expression cassette comprises a single promoter. In some embodiments, the expression cassette comprises two promoters. In some embodiments, the expression cassette comprises a single copy of a sequence encoding a DWORF polypeptide. In some embodiments, the expression cassette comprises two copies of a sequence encoding a DWORF polypeptide. In some embodiments, when the expression cassette comprises two copies of a sequence encoding a DWORF polypeptide, the two "copies" are not identical. Without being bound by any theory, by using two nucleic acid sequences encoding non-identical polypeptides, DNA recombination within the vector may be prevented. In some embodiments, the expression cassette comprises one copy having a native DNA sequence encoding a DWORF polypeptide and one copy having a codon-optimized DNA sequence encoding a DWORF polypeptide. In some embodiments, the expression cassette comprises two copies of a sequence encoding a DWORF polypeptide, one copy being codon-optimized and one copy not being codon-optimized. In some embodiments, the expression cassette comprises one, two, three, or four enhancers. In some embodiments, the expression cassette comprises one or two introns. In some embodiments, the expression cassette comprises one or two WPRE sequences. In some embodiments, the expression cassette comprises one or two polyadenylation sequences.

[0048] In some embodiments, the expression cassette comprises about 3.2 kb, about, about 3.3 kb, about 3.4 kb, about 3.5 kb, about 3.6 kb, about 3.7 kb or less. In some embodiments, the expression cassette comprises about 1.9 kb, about 2.1 kb, about 2.2 kb, about 2.3 kb, about 2.4 kb, about 2.5 kb, about 2.6 kb, about 2.7 kb, about 2.8 kb, about 2.9 kb, about 3.0 kb, about 3.1 kb, about 3.2 kb or more.

[0049] In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NOs: 20-24 or 45-75. In some embodiments, the expression cassette comprises any one of SEQ ID NOs: 20-24 or 45-75. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 61. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 62. In some embodiments, the expression cassette comprises SEQ ID NO: 62. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 63. In some embodiments, the expression cassette comprises SEQ ID NO:63.

[0050] In some embodiments, the capsid protein shares at least 98%, at least 99%, or 100% identity with the AAV9 capsid protein (SEQ ID NO: 143). In some embodiments, the capsid protein shares at least 98%, at least 99%, or 100% identity with the AAV5 capsid protein (SEQ ID NO: 144). In some embodiments, the capsid protein is a chimeric capsid protein. In some embodiments, the capsid protein is an AAV5 / AAV9 chimeric capsid protein. In some embodiments, the capsid protein is selected from any one of SEQ ID NOs: 145-200.

[0051] In one aspect, the disclosure provides an expression cassette comprising a polynucleotide sequence encoding a dwarf open reading frame (DWORF) polypeptide operably linked to a promoter. In some embodiments, the DWORF polypeptide shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NOs: 1, 3, 4, 7, 9, 23, and 43. In some embodiments, the DWORF polypeptide is selected from SEQ ID NOs: 1, 3, 4, 7, 9, 23, and 43.

[0052] In some embodiments, the promoter is a chicken cTnT promoter. In some embodiments, the chicken cTnT promoter shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 11. In some embodiments, the chicken cTnT promoter comprises SEQ ID NO: 11. In some embodiments, the promoter is a human cTnT promoter. In some embodiments, the human cTnT promoter shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 12 or SEQ ID NO: 13. In some embodiments, the human cTnT promoter comprises SEQ ID NO: 12 or SEQ ID NO: 13.

[0053] In some embodiments, the expression cassette further comprises one or more enhancers. In some embodiments, the enhancer, the one or more enhancers are selected from an ACTC1 cardiac enhancer and an αMHC enhancer. In some embodiments, the ACTC1 cardiac enhancer shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 78. In some embodiments, the ACTC1 cardiac enhancer comprises SEQ ID NO: 78. In some embodiments, the αMHC enhancer shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 79. In some embodiments, the αMHC enhancer comprises SEQ ID NO: 79.

[0054] In some embodiments, the expression cassette further comprises an intron. In some embodiments, the intron is selected from a CMV intron and a chimeric intron. In some embodiments, the CMV intron shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 80. In some embodiments, the CMV intron comprises SEQ ID NO: 80. In some embodiments, the chimeric intron shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 81. In some embodiments, the chimeric intron comprises SEQ ID NO: 81.

[0055] In some embodiments, the expression cassette further comprises a WPRE sequence. In some embodiments, the WPRE sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 26. In some embodiments, the WPRE sequence comprises SEQ ID NO: 26.

[0056] In some embodiments, the expression cassette further comprises a polyadenylation sequence. In some embodiments, the polyadenylation sequence is selected from a BGH polyadenylation sequence and an SV40 polyadenylation sequence. In some embodiments, the BGH polyadenylation sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:27. In some embodiments, the BGH polyadenylation sequence comprises SEQ ID NO:27. In some embodiments, the SV40 polyadenylation sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:28. In some embodiments, the SV40 polyadenylation sequence comprises SEQ ID NO:28.

[0057] In some embodiments, the expression cassette is flanked by ITRs. In some embodiments, the ITRs share at least 90%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 14 and SEQ ID NO: 15. In some embodiments, the ITRs comprise one or more of SEQ ID NO: 14 and SEQ ID NO: 15.

[0058] In some embodiments, the expression cassette comprises a single promoter. In some embodiments, the expression cassette comprises two promoters. In some embodiments, the expression cassette comprises a single copy of a sequence encoding a DWORF polypeptide. In some embodiments, the expression cassette comprises two copies of a sequence encoding a DWORF polypeptide. In some embodiments, the expression cassette comprises one, two, three, or four enhancers. In some embodiments, the expression cassette comprises one or two introns. In some embodiments, the expression cassette comprises one or two WPRE sequences. In some embodiments, the expression cassette comprises one or two polyadenylation sequences.

[0059] In some embodiments, the expression cassette comprises about 3.2 kb, about, about 3.3 kb, about 3.4 kb, about 3.5 kb, about 3.6 kb, about 3.7 kb or less. In some embodiments, the expression cassette comprises about 1.9 kb, about 2.1 kb, about 2.2 kb, about 2.3 kb, about 2.4 kb, about 2.5 kb, about 2.6 kb, about 2.7 kb, about 2.8 kb, about 2.9 kb, about 3.0 kb, about 3.1 kb, about 3.2 kb or more.

[0060] In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NOs: 20-24 or 45-75. In some embodiments, the expression cassette comprises any one of SEQ ID NOs: 20-24 or 45-75. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 61. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 62. In some embodiments, the expression cassette comprises SEQ ID NO: 62. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 63. In some embodiments, the expression cassette comprises SEQ ID NO:63.

[0061] In some embodiments, the expression cassette comprises a 5' inverted terminal repeat and a 3' inverted terminal repeat.

[0062] In one aspect, the disclosure provides a pharmaceutical composition comprising the rAAV virion disclosed herein and a pharma- ceutical acceptable diluent. In another aspect, the disclosure provides a kit comprising the pharmaceutical composition provided herein.

[0063] In one aspect, the disclosure provides a method of increasing DWORF expression in a cell, comprising contacting the cell with a rAAV virion disclosed herein or a pharmaceutical composition disclosed herein. In some embodiments, the cell is a cardiac cell. In some embodiments, the cardiac cell is a cardiomyocyte. In some embodiments, DWORF expression is increased between about 1.5-fold and 150-fold. In some embodiments, the contacting is in vitro. In some embodiments, the contacting is in vivo.

[0064] In one aspect, the disclosure provides a method of increasing DWORF expression in a tissue, comprising contacting the tissue with a rAAV virion disclosed herein or a pharmaceutical composition disclosed herein. In some embodiments, the tissue is cardiac tissue. In some embodiments, DWORF expression is increased between about 1.5-fold and 150-fold. In some embodiments, the contacting is in vitro. In some embodiments, the contacting is in vivo.

[0065] In one aspect, the disclosure provides a method of increasing DWORF expression in an organ comprising contacting the organ with a rAAV virion disclosed herein or a pharmaceutical composition disclosed herein, hi some embodiments, DWORF expression is increased between about 1.5-fold and 150-fold.

[0066] In some embodiments, the organ is the heart. In some embodiments, the heart is diseased or at risk for heart disease. In some embodiments, the heart has a reduced or borderline ejection fraction. In some embodiments, the heart has a normal ejection fraction.

[0067] In some embodiments, the heart comprises a genetic mutation associated with heart disease. In some embodiments, the genetic mutation is a PLN mutation. In some embodiments, the heart has low or undetectable DWORF expression compared to a healthy heart. In some embodiments, the contacting is in vitro. In some embodiments, the contacting is in vivo.

[0068] In one aspect, the disclosure provides a method of increasing DWORF expression in a subject, the method comprising administering to the subject a rAAV virion disclosed herein or a pharmaceutical composition disclosed herein. In some embodiments, the subject is an animal. In some embodiments, the subject is a human. In some embodiments, DWORF expression is increased in the heart of the subject. In some embodiments, the subject has or is at risk for heart disease. In some embodiments, the subject has a borderline or reduced ejection fraction. In some embodiments, the subject has a normal ejection fraction. In some embodiments, the subject has a genetic mutation associated with heart disease. In some embodiments, the genetic mutation is a PLN mutation. In some embodiments, the subject has a low or undetectable level of DWORF expression compared to a healthy subject.

[0069] In one aspect, the disclosure provides a method of treating a cardiac disease or disorder in a subject in need thereof comprising administering to the subject a rAAV virion disclosed herein or a pharmaceutical composition disclosed herein.

[0070] In some embodiments, the subject has a cardiac disease or disorder. In some embodiments, the subject is at risk for developing a cardiac disease or disorder. In some embodiments, the cardiac disease or disorder is cardiomyopathy. In some embodiments, the cardiac disease or disorder is dilated cardiomyopathy. In some embodiments, the cardiac disease or disorder is myocardial infarction. In some embodiments, the cardiac disease or disorder is chronic myocardial infarction. In some embodiments, the cardiac disease or disorder is acute myocardial infarction.

[0071] In some embodiments, the subject has an inherited risk allele for a cardiac disease or disorder. In some embodiments, the inherited risk allele comprises a mutation in the PLN gene. In some embodiments, the mutation in the PLN gene is a PLN promoter mutation. In some embodiments, the mutation in the PLN gene is a PLNL39stop mutation. In some embodiments, the mutation in the PLN gene is an RC9 mutation. In some embodiments, the mutation in the PLN gene is an R9L mutation. In some embodiments, the mutation in the PLN gene is a PLN gene duplication. In some embodiments, the mutation in the PLN gene is an R14del mutation.

[0072] In some embodiments, the cardiac disease or disorder is associated with reduced ejection fraction (HFrEF). In some embodiments, the cardiac disease or disorder is associated with preserved ejection fraction (HFpEF).

[0073] In some embodiments, the method results in expression of a DWORF polypeptide in the heart of the subject.In some embodiments, the method results in expression of a DWORF polypeptide in cardiomyocytes.

[0074] In some embodiments, the method does not cause detectable expression of DWORF polypeptide in the muscle of the subject, except the heart.In some embodiments, the method does not cause detectable expression of DWORF polypeptide in the liver of the subject.In some embodiments, the method does not cause detectable expression of DWORF polypeptide in cardiac fibroblasts.

[0075] In some embodiments, the method improves one or more measures of cardiac function, optionally fractional shortening and / or left ventricular internal diameter (LVID). In some embodiments, the improvement in cardiac function is observed at weeks 2 to 16. In some embodiments, the method reduces cardiac remodeling. In some embodiments, the method counteracts a decrease in DWORF expression in a subject suffering from or at risk for cardiac disease.

[0076] In some embodiments, the rAAV virions are administered by systemic administration, hi some embodiments, the systemic administration is selected from intravenous injection or intracoronary injection.

[0077] In some embodiments, the rAAV is administered as a unit dose. In some embodiments, the unit dose is about 3×10 14 vg / kg or less, approximately 2×10 14 vg / kg or less, approximately 1×10 14 vg / kg or less, approximately 9 × 10 13 vg / kg or less, approximately 8 × 10 13 vg / kg or less, approximately 7 × 10 13 vg / kg or less, approximately 6×10 13 vg / kg or less, approximately 5 × 10 13 vg / kg or less, approximately 4 × 10 13 vg / kg or less, approximately 3 × 10 13 vg / kg or less, approximately 2×10 13 vg / kg or less, or approximately 1 × 10 13 vg / kg or less.

[0078] In one aspect, the disclosure provides a method of alleviating one or more symptoms of a cardiac disease or disorder in a subject in need thereof comprising administering a rAAV virion disclosed herein or a pharmaceutical composition disclosed herein.

[0079] In one aspect, the disclosure provides a method of ameliorating one or more symptoms of a cardiac disease or disorder in a subject in need thereof comprising administering a rAAV virion disclosed herein or a pharmaceutical composition disclosed herein.

[0080] In one aspect, the disclosure provides a method of preventing one or more symptoms of a cardiac disease or disorder in a subject in need thereof comprising administering a rAAV virion disclosed herein or a pharmaceutical composition disclosed herein.

[0081] In one aspect, the disclosure provides an expression cassette comprising a polynucleotide comprising a 5' to 3' arrangement of elements, the elements comprising: i) one or more promoters; ii) optionally, one or more enhancers; iii) optionally, one or more introns; iv) one or more transgenes; v) optionally, one or more WPRE sequences; and vi) optionally, one or more polyadenylation sequences, p(A). In some embodiments, the 5' to 3' arrangement of the elements is selected from: i) 5'-promoter-intron-transgene-WPRE-p(A)-3'; ii) 5'-enhancer-promoter-transgene-WPRE-p(A)-3'; iii) 5'-enhancer-enhancer-promoter-transgene-WPRE-p(A)-3'; iv) 5'-enhancer-enhancer-promoter-intron-transgene-WPRE-p(A)-3'; v) 5'-enhancer-enhancer-promoter-intron-transgene-WPRE-p(A)-3'; vi) 5'-enhancer-promoter-intron-transgene-WPRE-p(A)-p(A)-transgene-intron-promoter-enhancer-3'; vi i) 5'-enhancer-promoter-intron-transgene-WPRE-p(A)-enhancer-promoter-intron-transgene-p(A)-3'; viii) 5'-p(A)-WPRE-transgene-intron-promoter-enhancer-enhancer-promoter-intron-transgene-p(A)-3'; ix) 5'-promoter-intron-transgene-WPRE-p(A)-p(A)-transgene-intron-promoter-3'; x) 5'-promoter-intron-transgene-WPRE-p(A)-promoter-intron-transgene-p(A)-3'; and xi) 5'-p(A)-WPRE-transgene-intron-promoter-promoter-intron-transgene-p(A)-3'. In some embodiments, the transgene has an increased expression level compared to a second expression cassette comprising a polynucleotide having a 5' to 3' arrangement of elements comprising: 5'-promoter-transgene-WPRE-p(A)-3'.In some embodiments, the increased expression level is between about 1.5-fold and about 150-fold compared to the second expression cassette.

[0082] In one aspect, the disclosure provides a recombinant adeno-associated virus (rAAV) virion comprising a viral genome comprising a capsid protein and an expression cassette of any one of those disclosed herein, wherein the expression cassette is flanked by inverted terminal repeats. In some embodiments, the expression cassette comprises a transgene, the transgene encoding a polypeptide for use in treating or preventing cardiac disease or alleviating symptoms associated with cardiac disease. In some embodiments, the capsid protein is selected from any one of SEQ ID NOs: 145-200. [Brief description of the drawings]

[0083] [Figure 1] FIG. 1 shows a diagram of an illustrative embodiment of an expression cassette comprising a polynucleotide encoding a promoter, a DWORF polypeptide, a WPRE sequence, and a poly(A) signal sequence flanked by AAV inverted terminal repeats.

[0084] [Diagram 2] FIG. 2 is a graph showing expression of GFP delivered to human induced pluripotent stem cell-derived cardiomyocytes in vitro using an embodiment of an expression cassette packaged into rAAV virions.

[0085] [Diagram 3] FIG. 3 is a graph showing expression of human DWORF polypeptide delivered in vivo in a mouse model using an embodiment of an expression cassette packaged into rAAV virions using AAV9 capsid proteins.

[0086] [Figure 4-1]FIG. 4A is a graph showing expression of human DWORF in induced pluripotent stem cell-derived cardiomyocytes using an embodiment of an expression cassette in which one of several AAV capsid proteins described herein is packaged into a rAAV virion.

[0087] [Figure 4-2] FIG. 4B is a series of images showing expression of GFP using an embodiment of an expression cassette described herein in which one of several AAV capsid proteins described herein was packaged into rAAV virions.

[0088] [Figure 5-1] FIG. 5A is a graph showing DWORF RNA expression in heart tissue from animals treated with rAAV virions containing embodiments of the expression cassette packaged in one of the five chimeric capsid proteins or the AAV9 capsid protein.

[0089] [Figure 5-2] FIG. 5B is an immunoblot showing DWORF protein levels in heart tissue from animals treated with rAAV virions containing embodiments of the expression cassette packaged in one of the four chimeric capsid proteins or the AAV9 capsid protein.

[0090] [Figure 6-1] FIG. 6A is a graph showing improved ejection fraction in a PLN-R14Δ / Δ mouse model following treatment with an embodiment of an expression cassette packaged into rAAV virions using the AAV9 protein capsid.

[0091] [Figure 6-2] FIG. 6B is a graph showing improved fractional shortening in a PLN-R14Δ / Δ mouse model following treatment with one embodiment of an expression cassette packaged into rAAV virions using the AAV9 protein capsid.

[0092] [Figure 7-1] FIG. 7A shows a diagram of an exemplary expression cassette orientation, including a polynucleotide encoding a promoter, a DWORF polypeptide, a WPRE sequence, and a poly(A) signal sequence flanked by AAV inverted terminal repeats.

[0093] [Figure 7-2] FIG. 7B shows a diagram of an exemplary expression cassette orientation, including a polynucleotide encoding a promoter, one or more enhancers, an intron, a DWORF polypeptide, a WPRE sequence, and a poly(A) signal sequence flanked by AAV inverted terminal repeats.

[0094] [Figure 7-3] FIG. 7C shows a diagram of an exemplary expression cassette orientation, including a polynucleotide encoding a promoter, one or more enhancers, an intron, a DWORF polypeptide, a WPRE sequence, and a poly(A) signal sequence flanked by AAV inverted terminal repeats.

[0095] [Figure 8-1] FIG. 8A is a schematic outlining the strategy for assessing DWORF expression in cardiomyocyte mice injected retro-orbitally with AAV9:DWORF constructs containing various regulatory elements and in vivo configurations.

[0096] [Figure 8-2] FIG. 8B is a Western blot demonstrating expression of DWORF and GAPDH in cardiomyocyte mice injected retro-orbitally with AAV9:DWORF constructs containing various regulatory elements and in vivo configurations.

[0097] [Figure 8-3] FIG. 8C is a chart showing DWORF expression levels in animal models achieved using a panel of rAAV virions containing an expression cassette encoding a DWORF polypeptide.

[0098] [Figure 9] FIG. 9 is a plot showing improved ejection fraction in an animal model of cardiomyopathy treated with a panel of rAAV virions containing an expression cassette encoding a DWORF polypeptide.

[0099] [Figure 10] FIG. 10 is a plot showing preserved ejection fraction in an animal model of cardiomyopathy treated with a panel of rAAV virions containing an expression cassette encoding a DWORF polypeptide.

[0100] [Figure 11-1] FIG. 11A is a schematic diagram of the efficacy study of DWORF gene therapy in the MLP-KO DCM mouse model.

[0101] [Figure 11-2] Figures 11B and 11C demonstrate that the AAV9:DWORF construct containing the novel promoter improves ejection fraction compared to saline controls in the MLP-KO DCM mouse model. [Figure 11-3] Same as above.

[0102] [Figure 11-4] Figures 11D and 11E demonstrate that the AAV9:DWORF construct improved exercise performance, including distance traveled and time to exhaustion, in the MLP-KO DCM mouse model 26 weeks after treatment. [Figure 11-5] Same as above.

[0103] [Figure 12-1] FIG. 12A is a schematic detailing a tolerability study of DWORF gene therapy in naive mice.

[0104] [Figure 12-2]FIG. 12B demonstrates that AAV9:pHZ21 was well tolerated in naive mice at doses up to 2×10 vg / kg, with no differences in body weight, ejection fraction, heart rate, and left ventricular mass (LV mass). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0105] In some aspects, described herein are optimized gene therapy expression cassettes and their use in treating cardiac disease. In some aspects, described herein are gene therapy expression cassettes that can mediate high expression of a transgene. In some embodiments, described herein are cardiac-specific gene therapy expression cassettes that can mediate significantly higher expression of a transgene than can be achieved using a cTnT promoter alone (e.g., chicken cTnT promoter alone and / or human cTnT promoter alone) or using the expression cassette depicted in FIG. 7A. In some aspects, described herein are gene therapy expression cassettes that allow for lowering viral load while achieving desired expression of a transgene. In some aspects, described herein are gene therapy expression cassettes that allow for achieving sustained expression of a transgene. In some embodiments, described herein are gene therapy expression cassettes that allow expression of a transgene to be achieved for at least 12 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks, or 26 weeks or more after administration of the gene therapy expression cassette containing the transgene to a subject. In some embodiments, described herein are gene therapy expression cassettes that allow expression of a transgene to be achieved for at least 24 weeks or at least 6 months or more after administration of the gene therapy expression cassette containing the transgene to a subject. In some aspects, administration of the gene therapy expression cassette described herein to a subject results in one or more improvements in cardiac function (e.g., an improvement in ejection fraction or an improvement in exercise capacity). In some embodiments, administration of the gene therapy expression cassette described herein to a subject results in a sustained improvement in cardiac function (e.g., a sustained improvement in ejection fraction or a sustained improvement in exercise capacity). In some embodiments, administration of a gene therapy expression cassette described herein to a subject results in one or more improvements in cardiac function for at least 12 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks, or 26 weeks or more following administration.In some embodiments, administration of a gene therapy expression cassette described herein to a subject results in one or more improvements in cardiac function for at least 24 weeks or at least 6 months, or more, following administration. In some embodiments, the gene therapy expression cassette described herein allows for cardiac cell-specific expression of a transgene (e.g., cardiomyocyte-specific expression of a transgene).

[0106] In some aspects, the disclosure provides viral or non-viral vectors comprising expression cassettes encoding gene products, and methods of use thereof. In some embodiments, the expression cassettes described herein comprise a polynucleotide encoding a gene product operably linked to a cardiac cell-specific promoter and / or enhancer (e.g., any combination of cardiac cell-specific promoters and enhancers described herein, in any orientation described herein, etc.). In some embodiments, the expression cassettes described herein comprise a polynucleotide encoding a gene product and two copies of a cardiac cell-specific promoter (e.g., any combination of such sequences, in any orientation described herein, etc.). In some embodiments, the expression cassettes described herein comprise a polynucleotide encoding a gene product operably linked to a cardiac cell-specific promoter and / or enhancer (e.g., one promoter or any combination of cardiac cell-specific promoters and enhancers described herein, in any orientation described herein, etc.), a WPRE sequence, and / or one or two copies of a polyA sequence (e.g., any combination of such sequences, in any orientation described herein, etc.). In some embodiments, the expression cassettes described herein comprise a polynucleotide encoding a gene product operably linked to a cardiac cell-specific promoter and / or an intron. In some embodiments, the expression cassettes described herein comprise one or two copies of a polynucleotide encoding a gene product, one or two copies of a cardiac cell-specific promoter, one, two or more copies of a cardiac-specific enhancer, and / or one or more intron sequences (e.g., any combination of such sequences in any orientation described herein).In some embodiments, the expression cassettes described herein include one or two copies of a polynucleotide encoding a gene product, one or two copies of a cardiac cell-specific promoter, one, two or more copies of a cardiac specific enhancer, one or more intronic sequences (e.g., any combination of such sequences in any orientation described herein), a WPRE sequence, and one or two copies of a polyA sequence (e.g., any combination of such sequences in any orientation described herein). In some embodiments, a vector comprising an expression cassette described herein can, for example, transduce a cardiac cell. In some embodiments, the targeted cardiac cell expresses the gene product, e.g., providing high levels of expression of the gene product. In some aspects, the disclosure provides pharmaceutical compositions comprising the vectors described herein. In some aspects, the disclosure provides methods for treating a subject diagnosed with or at risk for cardiac disease (e.g., cardiomyopathy) using the vectors and pharmaceutical compositions of the disclosure.

[0107] In some aspects, the present disclosure provides recombinant adeno-associated virus (rAAV) virions as vectors for the expression cassettes described herein.

[0108] Abnormal calcium regulation is a common feature of cardiomyopathies, and reduced sarcoplasmic reticulum calcium ATPase (SERCA) activity plays a central role in both the initiation and progression of the disease. SERCA regulates Ca ATPase activity in cardiac cells, such as cardiomyocytes. 2+ SERCA is a calcium pump that promotes ion uptake, maintenance, and cycling. SERCA activity is regulated by the inhibitory peptide phospholamban. There is significant interest in increasing the activity of SERCA by increasing the abundance of a polypeptide called Dwarf open reading frame (DWORF), which enhances SERCA activity through its direct replacement of the SERCA inhibitory peptide phospholamban. Contacting SERCA with DWORF is a strategy for increasing SERCA activity in cells.

[0109] In some aspects, the present disclosure provides recombinant adeno-associated virus (rAAV) virions comprising a polynucleotide encoding a DWORF polypeptide, or a functional variant thereof, and methods of use thereof. In some embodiments, the rAAV virions described herein comprise a polynucleotide encoding a DWORF polypeptide, or a functional variant thereof, operably linked to a cardiac cell-specific promoter and / or enhancer (e.g., any combination of cardiac cell-specific promoters and enhancers described herein, in any orientation described herein, etc.). In some embodiments, the rAAV virions described herein comprise one or two copies of a polynucleotide encoding a DWORF polypeptide, or a functional variant thereof, a WPRE sequence, and one or two copies of a polyA sequence (e.g., any combination of such sequences, in any orientation described herein, etc.). In some embodiments, the rAAV virions described herein comprise a polynucleotide encoding a DWORF polypeptide, operably linked to a cardiac cell-specific promoter and / or intron. In some embodiments, the rAAV virions described herein comprise one or two copies of a polynucleotide encoding DWORF, one or two copies of a cardiac cell-specific promoter, one, two or more copies of a cardiac-specific enhancer, and / or one or more intron sequences (e.g., any combination of such sequences in any orientation described herein). In some embodiments, the rAAV virions described herein comprise one or two copies of a polynucleotide encoding DWORF, one or two copies of a cardiac cell-specific promoter, one, two or more copies of a cardiac-specific enhancer, one or more intron sequences (e.g., any combination of such sequences in any orientation described herein), a WPRE sequence, and one or two copies of a polyA sequence (e.g., any combination of such sequences in any orientation described herein).In some embodiments, the rAAV virions described herein can be transduced into a host cell genome, for example, into cardiac cells using a polynucleotide with a sequence encoding a DWORF polypeptide operably linked to a cardiac cell-specific promoter region. In some embodiments, the targeted cardiac cells can express the DWORF polypeptide and have increased SERCA activity. Also provided in the present disclosure is a pharmaceutical composition comprising the rAAV virions described herein. In one aspect, the present disclosure provides a method for treating a subject diagnosed with or at risk of cardiomyopathy using the rAAV virions and pharmaceutical compositions of the present disclosure.

[0110] term Unless the context indicates otherwise, features of the invention can be used in any combination. Any feature or combination of features described can be excluded or omitted. Certain features of the invention are described in separate embodiments and may also be provided in combination in a single embodiment. Features of the invention are described in a single embodiment and may also be provided separately or in any suitable subcombination.

[0111] Generally, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The detailed description is divided into sections for the convenience of the reader only, and disclosure found in any section may be combined with disclosure in another section.

[0112] The practice of the present disclosure employs, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, cell biology, and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3 rdedition;Ausubel et al.eds.(2007)Current Protocols in Molecular Biology;Methods in Enzymology(Academic Press,Inc.,N.Y.);MacPherson et al.(1991)PCR 1: A Practical Approach(IRL Press at Oxford University Press);MacPherson et al.(1995)PCR 2:A Practical Approach;Harlow and Lane eds.(1999)Antibodies,A Laboratory Manual、freshney(2005)Culture of Animal Cells: A Manual of Basic Technique,5 thedition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; IRL Press (1986) Immobilized Cells and Enzymes; Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology; Manipulating the Mouse Embryo: A Laboratory Manual, 3 rd edition (2002) Cold Spring Harbor Laboratory Press; Sohail (2004) Gene Silencing by RNA Interference: Technology and Application (CRC Press); and Sell (2013) Stem Cells Handbook. See also

[0113] The conjunction "and / or" means both "and" and "or," and a list connected by "and / or" includes all possible combinations of one or more of the listed items.

[0114] As used herein, the term "about," when used to modify a numerical value, indicates that a deviation of up to 10% above and below the numerical value remains within the intended meaning of the recited value.

[0115] "AAV" is an abbreviation for adeno-associated virus. The term encompasses all subtypes of AAV, both natural and recombinant, except where a subtype is indicated. The abbreviation "rAAV" refers to recombinant adeno-associated virus. "AAV" includes AAV or any subtype. "AAV5" refers to AAV subtype 5. "AAV9" means AAV subtype 9. The genomic sequences of the various serotypes of AAV, as well as the sequences of the natural inverted terminal repeats (ITRs), Rep proteins, and capsid subunits, can be found in the literature or in public databases such as GenBank. See, e.g., GenBank Accession Nos. NC_002077 (AAV1), AF063497 (AAV1), NC_001401 (AAV2), AF043303 (AAV2), NC_001729 (AAV3), NC_001829 (AAV4), U89790 (AAV4), NC_006152 (AAV5), AF513851 (AAV7), AF513852 (AAV8), NC_006261 (AAV8), and AY530579 (AAV9).Publications describing AAV include Srivistava et al. (1983) J. Virol. 45:555; Chiorini et al. (1998) J. Virol. 71:6823; Chiorini et al. (1999) J. Virol. 73:1309; Bantel-Schaal et al. al.(1999)J.Virol.73:939;Xiao et al.(1999)J.Virol.73:3994;Muramatsu et al.(1996)Virol.221:208;Shade et al.(1986)J.Virol.58:921;Gao et al. al.(2002)Proc.Nat.Acad.Sci.USA 99:11854;Moris et al.(2004)Virology 33:375-383; International Patent Publication Nos. WO2018 / 222503A1, WO2012 / 145601A2, WO2000 / 028061A2, WO1999 / 61601A2, and WO1998 / 11244A2; U.S. Patent Application Nos. 15 / 782,980 and 15 / 433,322; and U.S. Patent Nos. 10,036,016, 9,790,472 Nos. 9,737,618, 9,434,928, 9,233,131, 8,906,675, 7,790,449, 7,906,111, 7,718,424, 7,259,151, 7,198,951, 7,105,345, 6,962,815, 6,984,517, and 6,156,303.

[0116] "rAAV virion" refers to a viral particle that contains at least one viral capsid protein (e.g., VP1) and an encapsidated rAAV vector (or fragment thereof).

[0117] An "infectious" virion or virus particle is one that contains a competently assembled viral capsid and is capable of delivering polynucleotide components into a cell for which the virion is a tropic.

[0118] "Packaging" refers to a series of intracellular events that result in the assembly of rAAV virions, including encapsidation of the rAAV vector. AAV "rep" and "cap" genes refer to polynucleotide sequences that encode the replication and encapsidation proteins of the adeno-associated virus. AAV rep and cap are referred to herein as AAV "packaging genes." Packaging requires either the helper virus itself, or, more commonly in recombinant systems, helper virus functions (i.e., one or more helper plasmids) provided by a helper-free system. "Helper virus" in reference to AAV refers to a virus capable of replicating and packaging AAV (e.g., wild-type AAV) by mammalian cells. The helper virus can be an adenovirus, a herpesvirus, or a poxvirus, such as vaccinia.

[0119] The term "inverted terminal repeat" or "ITR" as used herein refers to AAV viral cis elements so named because of their symmetry. These elements are essential for efficient propagation of the AAV genome. In some embodiments, the minimum elements essential for ITR function are the Rep binding site and the terminal resolution site plus a variable palindrome sequence that allows hairpin formation.

[0120] The term "parent capsid" or "parent sequence" refers to a reference sequence from which a particle capsid or sequence is derived. Unless otherwise specified, the parent sequence refers to the sequence of a wild-type capsid protein of the same serotype as the engineered capsid protein.

[0121] "Recombinant," when applied to a polynucleotide, means that the polynucleotide is different from a polynucleotide found in nature (e.g., the polynucleotide is the product of various combinations of cloning, restriction or ligation steps as well as other procedures, or the polynucleotide is assembled from synthetic oligonucleotides). A "recombinant" protein is a protein produced from a recombinant polypeptide. A recombinant virion is a virion that includes a recombinant polynucleotide and / or a recombinant protein, e.g., a recombinant capsid protein.

[0122] As used herein, the term "percent sequence identity" and the term "identity", when used to refer to % sequence identity with respect to a reference nucleic acid or amino acid sequence, are the percentage of nucleic acid bases or amino acid residues in a candidate sequence that are identical to the nucleic acid bases or amino acid residues in the reference sequence, respectively, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Methods of sequence alignment are well known in the art. Sequences can be aligned using various computer programs, such as BLAST, available at ncbi.nlm.nih.gov. Alignment can be made using publicly available computer software, such as BLASTp, BLASTn, BLAST-2, ALIGN, or MegAlign Pro (DNASTAR) software. Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996); and Meth. Mol. Biol. 70: 173-187 (1997); J. Mol. Biol. 48: 44. Those skilled in the art can choose an appropriate alignment method depending on various factors, including sequence length, differences, and the presence or absence of insertions or deletions relative to the reference sequence.

[0123] The terms "operably linked" and "operably linked" refer to a nucleic acid sequence placed in a functional relationship with another nucleic acid sequence. These terms, as used herein, have their commonly known meanings in the art. For example, a promoter is operably linked to a gene if it is positioned in a location that allows the promoter to initiate transcription of that gene. An enhancer is operably linked to a gene if it is capable of modulating (e.g., enhancing) expression of that gene when bound by the appropriate transcription factors.

[0124] "Treatment," "treating," and "treat" are defined as acting on a disease, disorder, or condition with an agent to reduce or ameliorate the harmful or any other undesirable effects of the disease, disorder, or condition and / or its symptoms.

[0125] As used herein, the term "effective amount" and the like with respect to the amount of a composition refers to an amount that is sufficient to induce a desired physiological outcome (e.g., treatment of a disease). An effective amount can be administered in one or more administrations, applications, or dosages. Such delivery depends on many variables, including the period for which the individual dosage units are used, the bioavailability of the composition, the route of administration, and the like. However, it is understood that the specific amount of a composition (e.g., rAAV virions) for any particular subject will depend on a variety of factors, including the activity of the particular agent used, the age, weight, general health, sex, and diet of the subject, the time of administration, the rate of excretion, the combination of compositions, the severity of the particular disease being treated, and the form of administration.

[0126] The phrase "pharmaceutical acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0127] The terms "individual," "subject," and "patient" are used interchangeably herein and refer to mammals, including, but not limited to, humans and non-human primates (e.g., monkeys); mammalian sports animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pets (e.g., dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

[0128] As used herein, the term "cardiomyopathy" refers to any disease or dysfunction that directly affects the myocardium. The etiology of the disease or disorder may be, for example, inflammatory, metabolic, toxic, infiltrative, fibrogenic, hematological, genetic, or of unknown origin. Two basic forms are recognized as (1) primary type, consisting of myocardial disease of unknown etiology; and (2) secondary type, consisting of myocardial disease of known etiology or associated with diseases involving other organ systems. "Specific cardiomyopathies" refers to cardiac disease associated with a specific systemic or cardiac disorder; examples include hypertensive and metabolic cardiomyopathies. Cardiomyopathy includes dilated cardiomyopathy (DCM), a disorder in which left and / or right ventricular systolic pump function is impaired, leading to progressive cardiac enlargement; hypertrophic cardiomyopathy, characterized by left ventricular hypertrophy without obvious causes, such as hypertension or aortic stenosis; and restrictive cardiomyopathies, characterized by abnormal diastolic function and excessively stiff ventricular walls that impede ventricular filling. Cardiomyopathy also includes left ventricular noncompaction, arrhythmogenic right ventricular cardiomyopathy, and arrhythmogenic right ventricular dysplasia.

[0129] "Heart failure" refers to a pathological condition in which abnormalities in cardiac function involve the failure of the heart to pump blood at a rate commensurate with the demands of metabolic tissues and / or the heart is only able to do so from an abnormally elevated diastolic volume. Heart failure includes systolic and diastolic failure. Patients with heart failure are classified into those with low cardiac output (typically secondary to ischemic heart disease, hypertension, dilated cardiomyopathy, and / or valvular or pericardial disease) and those with elevated cardiac output (typically due to hyperthyroidism, anemia, pregnancy, arteriovenous fistula, beriberi, and Paget's disease). Heart failure includes heart failure with reduced ejection fraction (HFrEF) and heart failure with preserved ejection fraction (HFpEF).

[0130] As used herein, the term "therapeutic gene" refers to a gene that, when expressed, confers a beneficial effect on the cell or tissue in which it is present, or on the mammal in which it is expressed. Examples of beneficial effects include ameliorating a sign or symptom of a condition or disease, preventing or inhibiting a condition or disease, or imparting a desired characteristic. Therapeutic genes include genes that partially or completely correct a genetic defect in a cell or mammal.

[0131] As used herein, the term "cardiac cells" refers to any cell present in the heart that provides cardiac functions, such as cardiac contraction or blood supply, or otherwise helps maintain the structure of the heart. As used herein, cardiac cells include cells present in the epicardium, myocardium, or endocardium of the heart. Cardiac cells also include cells of the cardiovascular system, such as, for example, cardiac myocytes or cardiomyocytes, and cells of the coronary arteries or veins. Other non-limiting examples of cardiac cells include epithelial cells, endothelial cells, fibroblasts, cardiac stem or progenitor cells, cardiac conduction cells, and cardiac pacemaker cells that make up the myocardium, blood vessels, and cardiac cell support structures. Cardiac cells can be derived from stem cells, including, for example, embryonic stem cells or induced pluripotent stem cells.

[0132] Expression cassette Overview of expression cassettes The vectors of the present disclosure may include any of the expression cassettes described herein. In some aspects, the rAAV virions of the present disclosure include a viral genome that includes an expression cassette as shown in FIG. 1, FIG. 7A-7C, or a variation thereof. The expression cassette may include a polynucleotide encoding any of the gene products described herein, or a functional variant thereof, optionally operably linked to a promoter, optionally an intron, optionally a polyadenylation (poly(A)) signal, optionally a woodchuck hepatitis virus posttranscriptional element (WPRE), and optionally a transcription termination signal. The expression cassette may be flanked by inverted terminal repeats (ITRs). These components provide the ability to express the transgene after the host cell is targeted, for example, by the rAAV virion. The promoter sequence, when present, controls the expression of the polynucleotide encoding the gene product.

[0133] The expression cassette may include a polynucleotide encoding a DWORF polypeptide, or a functional variant thereof, optionally operably linked to a promoter, optionally an intron, optionally a polyadenylation (poly(A)) signal, optionally a woodchuck hepatitis virus posttranscriptional element (WPRE), and optionally a transcription termination signal. The promoter sequence, when present, controls the expression of the polynucleotide encoding the DWORF polypeptide, or a functional variant thereof. The promoter sequence may be a cardiac cell-specific promoter. The promoter sequence may further be operably linked to an enhancer, such as any cardiac cell-specific enhancer described herein.

[0134] In any of the constructs shown in Figure 1 and Figures 7A-7C, the DWORF nucleotide sequence can be replaced by a nucleotide sequence encoding another gene product or polypeptide, such as any of the gene products or polypeptides described herein (see, e.g., the description of transgenes and gene products encoded by such transgenes below). Thus, in some embodiments, provided herein are expression cassettes as shown in Figure 1 and Figures 7A-7C, in which the DWORF nucleotide sequence is replaced by a nucleotide sequence encoding another gene product or polypeptide.

[0135] Also, in any of the expression cassettes shown in Table 1, the DWORF nucleotide sequence can be replaced by a nucleotide sequence encoding another gene product or polypeptide, such as any of the gene products or polypeptides described herein (see, e.g., the description of transgenes and gene products encoded by such transgenes below). Additionally, in any of the expression cassettes shown in Table 1, the ITR sequences can be omitted. Thus, in some embodiments, provided herein is any expression cassette shown in Table 1, where the DWORF nucleotide sequence is replaced by a nucleotide sequence encoding another gene product or polypeptide and / or the specified ITR sequences are not present.

[0136] Transgene In some embodiments, the expression cassette of the present disclosure comprises a transgene. The transgene may comprise a nucleotide sequence encoding any polypeptide for use in treating or preventing or alleviating symptoms from a cardiac disease or disorder. The promoters, enhancers, and combinations thereof described herein are operably linked to a transgene that encodes a product. The transgene may be a gene or nucleotide sequence that encodes a product, or a functional fragment thereof. The product may be, for example, a polypeptide or a non-coding nucleotide. By non-coding nucleotide, it is meant that the sequence transcribed from the transgene or nucleotide sequence is not translated into a polypeptide. In some embodiments, the product encoded by the transgene or nucleotide operably linked to the enhancer described herein is a non-coding polynucleotide. The non-coding polynucleotide may be an RNA, such as a microRNA (miRNA or mIR), a short hairpin RNA (shRNA), a long non-coding RNA (lnRNA), and / or a short interfering RNA (siRNA). In some embodiments, the transgene encodes a product that is naturally expressed by cardiac cells, e.g., cardiomyocytes. In some embodiments, the transgene encodes a product that is naturally expressed in a cell type other than a cardiac cell. Without limitation, the cell type other than a cardiac fibroblast can be from any multicellular organism, unicellular organism, or microorganism.

[0137] In some embodiments, the transgene encodes a polypeptide. In some embodiments, the transgene encodes a non-coding polynucleotide, such as, for example, a microRNA (miRNA or mIR).

[0138] In some embodiments, the transgene comprises a sequence encoding a product selected from cadherin, connexin, Cx43, growth factors such as fibroblast growth factor (FGF)-2 and transforming growth factor-β, cytokines such as interleukin (IL)-1β and the IL-6 family, leukemia inhibitory factor, cardiotrophin-1, cardiogenic transcription factors, insulin-like growth factor, GATA4, MEF2C, TBX5, ESRRG, MESP1, MYOCD, ZFPM2, HAND2, miR-1, miR-133, Oct4, Sox2, Klf4, c-Myc, SRF, SMARCD3, Nkx2-5, Akt, PKB, Baf60c, BMP4, miR-208, and miR-499.

[0139] In some embodiments, the transgene is a functional cardiac protein, hi some embodiments, the gene product is a genome editing endonuclease (optionally with a guide RNA, single guide RNA, and / or repair template) that replaces or repairs a non-functional cardiac protein with a functional cardiac protein. Functional cardiac proteins include, but are not limited to, cardiac troponin T; cardiac sarcomeric protein; beta myosin heavy chain; myosin ventricular essential light chain 1; myosin ventricular regulatory light chain 2; cardiac a-actin; a-tropomyosin; cardiac troponin I; cardiac myosin-binding protein C; 4.5 LIM protein 1; titin; 5'-AMP-activated protein kinase subunit gamma-2; troponin I type 3, myosin light chain 2, actin alpha cardiac 1; cardiac LIM protein; caveolin 3 (CAV3); galactosidase alpha (GLA); lysosome-associated membrane protein 2 (LAMP2); mitochondrial transfer RNA glycine (MTTG); mitochondrial transfer RNA isoleucine (MTTI); mitochondrial transfer RNA lysine (MTTK); mitochondrial transfer RNA glutamine (MTTQ); myosin light chain 3 (MYL3); troponin C (TNNC1); transthyretin (TTR); sarcoplasmic reticulum calcium-ATPase 2a (SERCA2a); stromal-derived factor-1 (SDF-1); adenylate cyclase-6 (AC6); beta-ARKct (β-adrenergic receptor kinase C-terminal); fibroblast growth factor (FGF); platelet-derived growth factor (PDGF); vascular endothelial growth factor (VEGF); hepatocyte growth factor; hypoxia-inducible growth factor; thymosin beta 4 (TMSB4X); nitric oxide synthase-3 (NOS3); unocalcin 3 (UCN3); merusin; apolipoprotein E (ApoE); superoxide dismutase (SOD); and S100A1 (small calcium-binding protein; see e.g., Ritterhoff and Most (2012) Gene Ther. 19:613; Kraus et al. (2009) Mol. Cell. Cardiol. 47:445).

[0140] In some embodiments, the transgene is capable of treating or preventing coronary heart disease. In some embodiments, the transgene is capable of encoding vascular endothelial growth factor (VEGF), VEGF isoforms, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-D dNdC , VEGF-A 116A , VEGF-A 165 , VEGF-A 121 , VEGF-2, placenta growth factor (PIGF), fibroblast growth factor 4 (FGF-4), human growth factor (HGF), human granulocyte colony stimulating factor (hGCSF), and hypoxia inducible factor 1α (HIF-1α).

[0141] In some embodiments, the transgene is capable of treating or preventing heart failure. In some embodiments, the transgene is capable of treating or preventing chronic heart failure. In some embodiments, the transgene is capable of treating or preventing chronic heart failure. In some embodiments, the transgene is capable of treating or preventing chronic heart failure. In some embodiments, the transgene is capable of treating or preventing chronic heart failure. In some embodiments, the transgene is capable of treating or preventing chronic heart failure. In some embodiments, the transgene is capable of treating or preventing chronic heart failure. S447X , an antisense oligonucleotide against apolipoprotein B, an antisense oligonucleotide against c-myc, and an E2F oligonucleotide decoy.

[0142] In some embodiments, the transgene encodes a gene product whose expression compensates for a defect in a gene involved in a genetic disorder. The present disclosure includes, for example and without limitation, polynucleotides encoding one or more of the following for use in the disorders indicated in parentheses, or for other disorders caused by each: TAZ (Barth syndrome); FXN (Friedreich's ataxia); CASQ2 (CPVT); FBN1 (Marfan); RAF1 and SOS1s (Noonan); SCN5A (Brugada); KCNQ1 and KCNH2s (Long QT syndrome); DMPK (Myotonic dystrophy 1); LMNA (Limb-girdle muscular dystrophy type 1B); JUP (Naxos); TGFBR2 (Lois-Dietz); EMD (X-linked EDMD); and ELN (SV aortic stenosis). In some embodiments, the polynucleotide encodes one or more of the following: cardiac troponin T (TNNT2); BAG family molecular chaperone regulatory factor 3 (BAG3); myosin heavy chain (MYH7); tropomyosin 1 (TPM1); myosin-binding protein C (MYBPC3); 5'-AMP-activated protein kinase subunit gamma-2 (PRKAG2); troponin type I 3 (TNNI3); titin (TTN); myosin, light chain 2 (MYL2); actin, alpha cardiac muscle 1 (ACTC1); potassium voltage-gated channel, KQT-like subfamily, member 1 (KCNQ1); myocyte enhancer factor 2c (MEF2C); and cardiac LIM protein (CSRP3).

[0143] In some embodiments, the transgene comprises a nucleotide sequence encoding a protein selected from DWORF, junctophilin (e.g., JPH2), BAG family molecular chaperone regulatory factor 3 (BAG3), phospholamban (PLN), alpha-crystallin B chain (CRYAB), LMNA (e.g., lamin A and lamin C isoforms), troponin type I 3 (TNNI3), lysosomal associated membrane protein 2 (LAMP2, e.g., LAMP2a, LAMP2b, and LAMP2c isoforms), desmoplakin (DSP, e.g., DPI and DPII isoforms), desmoglein 2 (DSG2), and junctional plakoglobin (JUP). In some embodiments, the transgene comprises a nucleotide sequence encoding a human protein. In some embodiments, the transgene comprises a human nucleotide sequence (human DNA sequence). In some embodiments, the transgene comprises a codon-optimized DNA sequence. In some embodiments, the transgene comprises a nucleotide sequence encoding a wild-type protein, or a functionally active fragment thereof.

[0144] In some embodiments, the transgene comprises a polynucleotide sequence encoding a DWORF polypeptide.

[0145] In some embodiments, the transgene comprises a polynucleotide sequence encoding a junctophilin 2 (JPH2) polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding a full-length JPH2 polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding an N-terminal fragment of a JPH2 polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding an N-terminal fragment of a JPH2 polypeptide that retains JPH2 activity.

[0146] In some embodiments, the transgene comprises a polynucleotide sequence encoding a BAG3 polypeptide.

[0147] In some embodiments, the transgene comprises a polynucleotide sequence encoding a CRYAB polypeptide.

[0148] In some embodiments, the transgene comprises a polynucleotide sequence encoding a LMNA polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding a Lamin A isoform of LMNA. In some embodiments, the transgene comprises a polynucleotide sequence encoding a Lamin C isoform of LMNA.

[0149] In some embodiments, the transgene comprises a polynucleotide sequence encoding a TNNI3 polypeptide.

[0150] In some embodiments, the transgene comprises a polynucleotide sequence encoding a PLN polypeptide.

[0151] In some embodiments, the transgene comprises a polynucleotide sequence encoding a LAMP2 polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding a LAMP2a isoform. In some embodiments, the transgene comprises a polynucleotide sequence encoding a LAMP2b isoform. In some embodiments, the transgene comprises a polynucleotide sequence encoding a LAMP2c isoform.

[0152] In some embodiments, the transgene comprises a polynucleotide sequence encoding a DSP polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding a DPI isoform of DSP. In some embodiments, the transgene comprises a polynucleotide sequence encoding a DPII isoform of DSP.

[0153] In some embodiments, the transgene comprises a polynucleotide sequence encoding a DSG2 polypeptide.

[0154] In some embodiments, the transgene comprises a polynucleotide sequence encoding a JUP polypeptide.

[0155] It is understood that the transgenes described herein are non-limiting and that transgenes useful for treating cardiac disease can be found for use in the expression cassettes described herein.

[0156] DWORF transgene In some embodiments, the expression cassette of the present disclosure comprises a polynucleotide sequence encoding a DWORF polypeptide. In some embodiments, the expression cassette provides increased expression of the DWORF polypeptide in cardiac cells. In some embodiments, the cardiac cells are cardiomyocytes. In some embodiments, the expression of the DWORF polypeptide may be increased by 5%, 10%, 15%, 20%, or 25% compared to the expression of the DWORF polypeptide factor in an untreated subject. In some embodiments, the expression of the DWORF polypeptide may be increased by 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold compared to the expression of the DWORF polypeptide in an untreated subject. In some embodiments, the DWORF polypeptide may be expressed at any detectable level in cardiac cells, whereas the DWORF polypeptide may not be expressed or may be expressed at an undetectable level in an untreated subject. In other words, the cardiac cells to which the rAAV virions are administered may express the DWORF polypeptide at a higher abundance than cardiac cells that have only endogenous (i.e., natural) expression of the DWORF polypeptide.

[0157] DWORF polypeptides are endogenous enhancers of SERCA calcium pump activity, which is a desirable drug target for the regulation of cardiac contractility. DWORF is also a remarkably small protein, making it a good candidate for delivery to target cells or tissues by rAAV virions. Because DWORF is an endogenous protein, expression of DWORF in humans would not be immunogenic and would allow for long-term dosing and expression. The structural features of DWORF polypeptides are as follows. First, the polypeptides may have 5 to 35 consecutive residues of the Dwarf open reading frame (DWORF), located on chromosome 3 of mammalian species, including mouse and human (see Nelson et al. Science. 351:271-275 (2016); U.S. Patent No. 10,570,183). Thus, the term "a peptide having X or fewer consecutive residues" cannot be understood to include a greater number of consecutive residues, even when it includes the term "comprises." Typically, the peptides are 35 residues or less, again including 20 or fewer consecutive residues of DWORF. The overall length can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 residues. Peptide length ranges of 5-34 / 35 residues, 6-34 / 35 residues, 7-50 residues, 7-25 residues, 5-20 residues, 6-20 residues, 7-20 residues, and 7-15 residues are contemplated. The number of consecutive DWORF residues can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Ranges of contiguous residues of 5-20 residues, 5-20 residues, 6-20 residues, 7-20 residues, and 5-15 residues, 5-15 residues, 6-15 residues, or 7-15 residues are contemplated. Exemplary DWORF sequences can be found in Table 2a.

[0158] In some embodiments, the DWORF polypeptide is a human DWORF polypeptide. In some embodiments, the expression cassette comprises a single polynucleotide sequence encoding a dwarf open reading frame (DWORF) polypeptide. In some embodiments, the polynucleotide sequence encoding DWORF is codon optimized. In some embodiments, the DWORF polypeptide comprises a polypeptide sequence that shares at least 95% identity with SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9. In some embodiments, the DWORF polypeptide comprises a polypeptide sequence that shares at least 98% identity with SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9. In some embodiments, the DWORF polypeptide comprises a polypeptide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9. [Table 2-1-1]

[0159] Other Exemplary Transgenes In some embodiments, the expression cassette of the present disclosure comprises a polynucleotide sequence encoding another gene product (not DWORF), such as a polypeptide selected from JPH2, BAG3, CRYAB, LMNA (e.g., Lamin A or Lamin C isoforms), TNNI3, PLN, LAMP2 (e.g., LAMP2a, LAMP2b, or LAMP2c isoforms), DSP (e.g., DPI or DPII isoforms), Desmoglein 2 (DSG2), and Junctional Plakoglobin (JUP). In some embodiments, the expression cassette provides increased expression of the gene product in cardiac cells. In some embodiments, the cardiac cells are cardiomyocytes. In some embodiments, expression of the polypeptide encoded by the polynucleotide sequence may be increased by 5%, 10%, 15%, 20%, or 25% compared to expression in an untreated subject. In some embodiments, expression of the polypeptide encoded by the polynucleotide sequence may be increased by 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold compared to expression in an untreated subject. In some embodiments, a polypeptide encoded by a polynucleotide sequence may be expressed at any detectable level in cardiac cells, whereas it may not be expressed or may be expressed at undetectable levels in an untreated subject. In some embodiments, cardiac cells into which a vector described herein is administered may express the polypeptide encoded by the polynucleotide sequence at a higher abundance than in cardiac cells having only endogenous (i.e., natural) expression of the polypeptide.

[0160] In some embodiments, the polypeptide is a human polypeptide. In some embodiments, the polynucleotide sequence encoding the polypeptide is codon optimized. In some embodiments, the expression cassette comprises a single polynucleotide sequence encoding the polypeptide. In some embodiments, the expression cassette comprises two polynucleotide sequences encoding the polypeptide. In some embodiments, the expression cassette comprises two polynucleotide sequences encoding the polypeptide, where at least one of the sequences is codon optimized.

[0161] In some embodiments, the polynucleotide sequence encodes JPH2, e.g., human JPH2. In some embodiments, the polynucleotide sequence has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO:201. In some embodiments, the polynucleotide sequence is a codon-optimized sequence encoding JPH2, e.g., human JPH2. In some embodiments, the gene product or polypeptide expressed using any expression construct described herein is JPH2, e.g., human JPH2. In some embodiments, the JPH2 polypeptide has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO:202.

[0162] In some embodiments, the polynucleotide sequence encodes an N-terminal fragment of JPH2, e.g., human JPH2. In some embodiments, the polynucleotide sequence of the N-terminal fragment of JPH2 has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:227. In some embodiments, the polynucleotide sequence is a codon-optimized sequence encoding an N-terminal fragment of JPH2, e.g., human JPH2. In some embodiments, the gene product or polypeptide expressed using any expression construct described herein is an N-terminal fragment of JPH2, e.g., human JPH2. In some embodiments, the N-terminal fragment of JPH2 polypeptide has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:228. The human sequence of the N-terminal fragment of JPH2 corresponds to the mouse JPH2 N-terminal peptide with amino acids 1-565, generated by calpain cleavage (see Guo et al., 2018, Science 362, doi:10.1126 / science.aan3303).

[0163] In some embodiments, the polynucleotide sequence encodes BAG3, e.g., human BAG3. In some embodiments, the polynucleotide sequence has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO:203. In some embodiments, the polynucleotide sequence is a codon-optimized sequence encoding BAG3, e.g., human BAG3. In some embodiments, the gene product or polypeptide expressed using any expression construct described herein is BAG3, e.g., human BAG3. In some embodiments, the BAG3 polypeptide has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO:204.

[0164] In some embodiments, the polynucleotide sequence encodes CRYAB, e.g., human CRYAB. In some embodiments, the polynucleotide sequence has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO:205. In some embodiments, the polynucleotide sequence is a codon-optimized sequence encoding CRYAB, e.g., human CRYAB. In some embodiments, the gene product or polypeptide expressed using any expression construct described herein is CRYAB, e.g., human CRYAB. In some embodiments, the CRYAB polypeptide has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO:206.

[0165] In some embodiments, the polynucleotide sequence encodes LMNA, e.g., human LMNA. In some embodiments, the polynucleotide sequence encodes a lamin A isoform of LMNA, e.g., human lamin A. In some embodiments, the polynucleotide sequence has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 207. In some embodiments, the polynucleotide sequence encodes a lamin C isoform of LMNA, e.g., human lamin C. In some embodiments, the polynucleotide sequence has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 209. In some embodiments, the polynucleotide sequence is a codon-optimized sequence encoding a LMNA polypeptide. In some embodiments, the polynucleotide sequence is a codon-optimized sequence encoding a lamin A or lamin C, e.g., human lamin A or lamin C. In some embodiments, the gene product or polypeptide expressed using any expression construct described herein is LMNA, e.g., human LMNA. In some embodiments, the gene product or polypeptide expressed using any expression construct described herein is a Lamin A isoform of LMNA, e.g., human. In some embodiments, the Lamin A polypeptide has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO:208. In some embodiments, the gene product or polypeptide expressed using any expression construct described herein is a Lamin C isoform of LMNA, e.g., human. In some embodiments, the Lamin C polypeptide has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO:210.

[0166] In some embodiments, the polynucleotide sequence encodes TNNI3, e.g., human TNNI3. In some embodiments, the polynucleotide sequence has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:211. In some embodiments, the polynucleotide sequence is a codon-optimized sequence encoding TNNI3, e.g., human TNNI3. In some embodiments, the gene product or polypeptide expressed using any expression construct described herein is TNNI3, e.g., human TNNI3. In some embodiments, the TNNI3 polypeptide has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:212.

[0167] In some embodiments, the polynucleotide sequence encodes PLN, e.g., human PLN. In some embodiments, the polynucleotide sequence has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 229. In some embodiments, the polynucleotide sequence is a codon-optimized sequence encoding PLN, e.g., human PLN. In some embodiments, the gene product or polypeptide expressed using any expression construct described herein is PLN, e.g., human PLN. In some embodiments, the PLN polypeptide has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 230.

[0168] In some embodiments, the polynucleotide sequence encodes LAMP2, e.g., human LAMP2. In some embodiments, the polynucleotide sequence encodes the LAMP2a isoform of LAMP2, e.g., human LAMP2a. In some embodiments, the polynucleotide sequence has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 213. In some embodiments, the polynucleotide sequence encodes the LAMP2b isoform of LAMP, e.g., human LAMP2b. In some embodiments, the polynucleotide sequence has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 215. In some embodiments, the polynucleotide sequence encodes the LAMP2c isoform of LAMP, e.g., human LAMP2c. In some embodiments, the polynucleotide sequence has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO:217. In some embodiments, the polynucleotide sequence is a codon-optimized sequence encoding LAMP2, e.g., human LAMP2. In some embodiments, the polynucleotide sequence is a codon-optimized sequence encoding LAMP2a, LAMP2b, or LAMP2c. In some embodiments, the gene product or polypeptide expressed using any expression construct described herein is a LAMP2 polypeptide, e.g., human LAMP2. In some embodiments, the gene product or polypeptide expressed using any expression construct described herein is the LAMP2a isoform of LAMP2, e.g., human. In some embodiments, the LAMP2a polypeptide has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO:214. In some embodiments, the gene product or polypeptide expressed using any of the expression constructs described herein is the LAMP2b isoform of LAMP2, e.g., human.In some embodiments, the LAMP2b polypeptide has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 216. In some embodiments, the gene product or polypeptide expressed using any expression construct described herein is a LAMP2c isoform of LAMP2, e.g., human. In some embodiments, the LAMP2c polypeptide has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 218.

[0169] In some embodiments, the polynucleotide sequence encodes a DSP, e.g., a human DSP. In some embodiments, the polynucleotide sequence encodes a DPI isoform of DSP, e.g., a human DPI. In some embodiments, the polynucleotide sequence has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 219. In some embodiments, the polynucleotide sequence encodes a DPII isoform of DSP, e.g., a human DPII. In some embodiments, the polynucleotide sequence has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 221. In some embodiments, the polynucleotide sequence is a codon-optimized sequence encoding a DSP polypeptide. In some embodiments, the polynucleotide sequence is a codon-optimized sequence encoding a DPI or DPII, e.g., a human DPI or DPII. In some embodiments, the gene product or polypeptide expressed using any expression construct described herein is a DSP, e.g., human DSP. In some embodiments, the gene product or polypeptide expressed using any expression construct described herein is a DPI isoform of DSP, e.g., human. In some embodiments, the DPI polypeptide has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 220. In some embodiments, the gene product or polypeptide expressed using any expression construct described herein is a DPII isoform of DSP, e.g., human. In some embodiments, the DPII polypeptide has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 222.

[0170] In some embodiments, the polynucleotide sequence encodes DSG2, e.g., human DSG2. In some embodiments, the polynucleotide sequence has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO:223. In some embodiments, the polynucleotide sequence is a codon-optimized sequence encoding DSG2, e.g., human DSG2. In some embodiments, the gene product or polypeptide expressed using any expression construct described herein is DSG2, e.g., human DSG2. In some embodiments, the DSG2 polypeptide has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO:224.

[0171] In some embodiments, the polynucleotide sequence encodes JUP, e.g., human JUP. In some embodiments, the polynucleotide sequence has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO:225. In some embodiments, the polynucleotide sequence is a codon-optimized sequence encoding JUP, e.g., human JUP. In some embodiments, the gene product or polypeptide expressed using any expression construct described herein is JUP, e.g., human JUP. In some embodiments, the JUP polypeptide has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO:226.

[0172] In some embodiments, any other polynucleotide sequence described herein can be used in any expression construct described herein. In some embodiments, such polynucleotide sequences encode any gene product or polypeptide described herein. The polynucleotide sequences can be sequence optimized (e.g., for expression in humans). In some embodiments, the sequences encode human polypeptides. The sequences of the polynucleotides and polypeptides described herein are known in the art. Sequences having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to such sequences are also contemplated herein. Exemplary sequences can be found in Table 2b. [Table 2-2-1] [Table 2-2-2] [Table 2-2-3] [Table 2-2-4] [Table 2-2-5] [Table 2-2-6] [Table 2-2-7] [Table 2-2-8] [Table 2-2-9] [Table 2-2-10] [Table 2-2-11] [Table 2-2-12] [Table 2-2-13] [Table 2-2-14] [Table 2-2-15] [Table 2-2-16] [Table 2-2-17] [Table 2-2-18] [Table 2-2-19] [Table 2-2-20]

[0173] Promoters and Enhancers In some embodiments, the expression cassette of the present disclosure comprises a promoter. The term "promoter" as used herein refers to a DNA sequence that directs the binding of RNA polymerase, thereby facilitating RNA synthesis. The expression of a promoter and the corresponding protein or polypeptide may be ubiquitous, meaning that it is strongly active in a wide range of cells, tissues, and species, or in a cell type-specific, tissue-specific, or species-specific manner. Examples of ubiquitous promoters include the CAG promoter and the CMB promoter (Yue et al. BioTechniques 33:672-678 (2002)). A promoter may be "constitutive", meaning that it is continuously active, or "inducible", meaning that the promoter can be activated or inactivated by the presence or absence of a biotic or abiotic factor. Also included in the nucleic acid construct or vector of the present invention are enhancer sequences that may or may not be contiguous with the promoter sequence. Enhancer sequences affect promoter-dependent gene expression and may be located in the 5' or 3' region of the native gene.

[0174] A variety of promoters may be used. The promoter may be cell type specific. Constitutive promoters may be used in the expression cassette, for example, the cytomegalovirus enhancer fused to the chicken β-actin promoter (CAG), the simian virus 40 (SV40) promoter, and the herpes simplex virus thymidine kinase (HSV-TK) promoter (Damdindorj et al. PLoS One. 9: e106472 (2014)). Other cell type specific promoters may also be used. Cardiac cell specific promoters may be, for example, the MLC2v promoter (Phillips et al. Hypertension. 39: 651-5 (2002)) and the cardiac troponin-T (cTnT) promoter (Konkalmatt et al. Circ Cardiovasc Imaging. 6: 478-486 (2013)). The transgene polynucleotide sequence in the expression cassette may be, for example, an open reading frame encoding a protein. The ITRs in the expression cassette serve as markers used for viral packaging of the expression cassette (Clark et al. Hum Gene Ther. 6:1329-41 (1995)).

[0175] Advantageously, the promoter, optionally in combination with an enhancer, allows the expression of a polynucleotide encoding a polypeptide (eg, a DWORF polypeptide), or a functional variant thereof, in a target cell.

[0176] In some embodiments, the expression cassette comprises a single promoter. In some embodiments, the expression cassette comprises at least one promoter. In some embodiments, the expression cassette comprises two promoters. In some embodiments, the expression cassette comprises a ubiquitous promoter. In some embodiments, the expression cassette comprises an inducible promoter. In some embodiments, the expression cassette comprises a cell type specific promoter. In some embodiments, the promoter specifically promotes expression of a polynucleotide encoding a polypeptide, or a functional variant thereof, in cardiac cells (e.g., cardiomyocytes). In some embodiments, the promoter specifically promotes expression of a polynucleotide encoding a DWORF polypeptide, or a functional variant thereof, in cardiac cells. In some embodiments, the promoter specifically promotes expression of a polynucleotide encoding a DWORF polypeptide, or a functional variant thereof, in cardiomyocytes. Exemplary promoter and enhancer sequences are provided in Table 3.

[0177] In some embodiments, the promoter is a chicken cardiac troponin-T (cTnT or ccTnT) promoter. In some embodiments, the chicken cTnT promoter comprises a polynucleotide sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 11. In some embodiments, the chicken cTnT promoter comprises SEQ ID NO: 11.

[0178] In some embodiments, the promoter is a human cTnT promoter. In some embodiments, the promoter is a short human cTnT promoter. In some embodiments, the short human cTnT promoter comprises a polynucleotide sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 12. In some embodiments, the short human cTnT promoter comprises SEQ ID NO: 12. In some embodiments, the promoter is a long human cTnT promoter. In some embodiments, the long human cTnT promoter comprises a polynucleotide sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 13. In some embodiments, the long human cTnT promoter comprises SEQ ID NO: 13.

[0179] An expression cassette may include one or more enhancers. The term "enhancer" as used herein refers to a DNA sequence that directs the binding of transcriptional regulatory proteins (e.g., transcription machinery) and RNA polymerase, thereby facilitating RNA synthesis. An enhancer is operably linked to a promoter and can regulate the expression of a transgene operably linked to the promoter. The presence of an enhancer can regulate transgene expression, for example, by increasing expression or decreasing expression. An enhancer can regulate transgene expression, for example, by increasing expression levels in a desired cell type, for example, cardiac cells. An enhancer can regulate transgene expression, for example, by decreasing expression levels in "off-target" cell types, or cell types in which expression is undesirable.

[0180] In some embodiments, the expression cassette comprises a single enhancer. In some embodiments, the expression cassette comprises at least one enhancer. In some embodiments, the expression cassette comprises two enhancers. In some embodiments, the expression cassette comprises three enhancers. In some embodiments, the expression cassette comprises four enhancers. In some embodiments, the expression cassette comprises an enhancer operably linked to a promoter. For example, the ACTC1 cardiac enhancer can be linked to a human cTnT promoter. In some embodiments, the expression cassette comprises an enhancer operably linked to another enhancer. For example, the ACTC1 cardiac enhancer can be operably linked to an αMHC enhancer. In some embodiments, the expression cassette comprises an enhancer operably linked to a promoter and operably linked to another enhancer.

[0181] In some embodiments, the enhancer comprises an ACTC1 cardiac enhancer (ACTC1e). In some embodiments, the ACTC1 cardiac enhancer shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 78. In some embodiments, the ACTC1 cardiac enhancer comprises SEQ ID NO: 78. In some embodiments, the enhancer comprises an αMHC enhancer (αMHCe). In some embodiments, the αMHC enhancer shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 79. In some embodiments, the αMHC enhancer comprises SEQ ID NO: 79. [Table 3-1] [Table 3-2]

[0182] Introns The expression cassette may include an intron sequence, e.g., a synthetic intron sequence or a chimeric intron sequence. Intron sequences can be used to adjust the length (i.e., size) of the expression cassette to improve recombinant AAV packaging. Intron sequences can be used to improve the efficiency of transgene expression (i.e., mRNA production or transcription) in a host cell that includes the expression cassette. In some embodiments, the expression cassette includes an intron. In some embodiments, the intron includes a CMV intron (CMVint). In some embodiments, the CMV intron shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:80. In some embodiments, the CMV intron includes SEQ ID NO:80. In some embodiments, the intron includes a chimeric intron. In some embodiments, the chimeric intron shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:81. In some embodiments, the chimeric intron includes SEQ ID NO:81. [Table 9]

[0183] WPRE sequences and other post-transcriptional elements In some embodiments, the expression cassette comprises a post-transcriptional regulatory element.

[0184] In some embodiments, the expression cassette comprises a Woodchuck Hepatitis Virus Post-Transcriptional Element (WPRE). The WPRE sequence can be inserted proximal to the 3' end of a transgene in a viral vector, for example, to optimize gene expression in the viral vector (Lee et al. Exp Physiol. 90:33-37 (2005)). In some embodiments, the WPRE comprises a polynucleotide sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:26. In some embodiments, the WPRE comprises SEQ ID NO:26. [Table 4]

[0185] Polyadenylation sequence In some embodiments, the expression cassette comprises a poly(A) signal sequence. In some embodiments, the poly(A) signal is a BGH poly(A) sequence. In some embodiments, the BGH poly(A) signal sequence comprises a polynucleotide sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:27. In some embodiments, the poly(A) signal is an SV40 poly(A) signal. In some embodiments, the SV40 poly(A) signal sequence comprises a polynucleotide sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:28. [Table 5]

[0186] Inverted terminal repeats In some embodiments, the expression cassette is flanked by AAV inverted terminal repeats (ITRs). In some embodiments, the ITRs comprise a polynucleotide sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:14 and / or SEQ ID NO:15. [Table 6]

[0187] Exemplary Expression Cassettes The present disclosure provides expression cassettes comprising a polynucleotide comprising a 5' to 3' arrangement (sometimes referred to as orientation) of elements. In some embodiments, the elements comprise one or more promoters; optionally, one or more enhancers; optionally, one or more introns; one or more transgenes; optionally, one or more WPRE sequences; and optionally, one or more polyadenylation sequences (p(A)). Exemplary orders of elements in a polynucleotide are shown in Figure 1, Figure 7A, Figure 7B, Figure 7C, and Table 1. Exemplary orientations of elements on a polynucleotide are also shown in Figure 1, Figure 7A, Figure 7B, Figure 7C, and Table 1. In some embodiments, the 5' to 3' arrangement of elements is selected from the following:

[0188] 5'-promoter-transgene-WPRE-p(A)-3';

[0189] 5'-promoter-intron-transgene-WPRE-p(A)-3';

[0190] 5'-promoter-transgene-WPRE-p(A)-promoter-transgene-WPRE-p(A);

[0191] 5'-enhancer-promoter-transgene-WPRE-p(A)-3';

[0192] 5'-enhancer-promoter-intron-transgene-WPRE-p(A)-3';

[0193] 5'-enhancer-enhancer-promoter-transgene-WPRE-p(A)-3';

[0194] 5'-enhancer-enhancer-promoter-intron-transgene-WPRE-p(A)-3';

[0195] 5'-enhancer-promoter-intron-transgene-WPRE-p(A)-p(A)-transgene-intron-promoter-enhancer-3';

[0196] 5'-enhancer-promoter-intron-transgene-WPRE-p(A)-enhancer-promoter-intron-transgene-p(A)-3';

[0197] 5'-p(A)-WPRE-transgene-intron-promoter-enhancer-enhancer-promoter-intron-transgene-p(A)-3';

[0198] 5'-promoter-intron-transgene-WPRE-p(A)-p(A)-transgene-intron-promoter-3';

[0199] 5'-promoter-intron-transgene-WPRE-p(A)-promoter-intron-transgene-p(A)-3'; and

[0200] 5'-p(A)-WPRE-transgene-intron-promoter-promoter-intron-transgene-p(A)-3'.

[0201] In some embodiments, the expression cassettes described herein achieve increased expression levels of a transgene compared to a second expression cassette comprising a polynucleotide having a 5' to 3' arrangement of elements comprising: 5'-promoter-transgene-WPRE-p(A)-3'. In some embodiments, the expression level is increased between about 1.5-fold and about 150-fold compared to the second expression cassette.

[0202] In some embodiments, the expression cassettes provided herein include the following elements (wherein the elements may be as described herein, e.g., the sequences of which are provided herein):

[0203] 5'-promoter-transgene-WPRE-p(A)-3';

[0204] 5'-promoter-intron-transgene-WPRE-p(A)-3';

[0205] 5'-promoter-transgene-WPRE-p(A)-promoter-transgene-WPRE-p(A);

[0206] 5'-enhancer-promoter-transgene-WPRE-p(A)-3';

[0207] 5'-enhancer-promoter-intron-transgene-WPRE-p(A)-3';

[0208] 5'-enhancer-enhancer-promoter-transgene-WPRE-p(A)-3';

[0209] 5'-enhancer-enhancer-promoter-intron-transgene-WPRE-p(A)-3';

[0210] 5'-enhancer-promoter-intron-transgene-WPRE-p(A)-p(A)-transgene-intron-promoter-enhancer-3';

[0211] 5'-enhancer-promoter-intron-transgene-WPRE-p(A)-enhancer-promoter-intron-transgene-p(A)-3';

[0212] 5'-p(A)-WPRE-transgene-intron-promoter-enhancer-enhancer-promoter-intron-transgene-p(A)-3';

[0213] 5'-promoter-intron-transgene-WPRE-p(A)-p(A)-transgene-intron-promoter-3';

[0214] 5'-promoter-intron-transgene-WPRE-p(A)-promoter-intron-transgene-p(A)-3'; or

[0215] 5'-p(A)-WPRE-transgene-intron-promoter-promoter-intron-transgene-p(A)-3'.

[0216] In the expression cassettes described herein (e.g., such as those listed above), the orientation of the promoters, enhancers, transgenes, and poly(A) elements may be forward or reverse (e.g., when there are two or more promoters, one promoter, optionally an enhancer, and an operably linked transgene, another promoter, optionally an enhancer, and an operably linked transgene may be oriented in the reverse direction).

[0217] In some embodiments, the expression cassettes provided herein contain the following elements:

[0218] 5'-cardiac-specific promoter-transgene-WPRE-p(A)-3';

[0219] 5'-cardiac-specific promoter-intron (e.g., chimeric intron)-transgene-WPRE-p(A)-3';

[0220] 5'-cardiac-specific promoter-transgene-WPRE-p(A)-promoter-transgene-WPRE-p(A), where both the promoter and transgene sequences are in the same forward orientation;

[0221] 5'-cardiac-specific promoter-transgene-WPRE-p(A)(e.g., bGHpA)-3';

[0222] 5'-cardiac-specific promoter-intron (e.g., CMV intron)-transgene-WPRE-p(A) (e.g., bGHpA)-3';

[0223] 5'-enhancer (e.g., ACTC1e)-cardiac-specific promoter-transgene-WPRE-p(A) (e.g., bGHpA)-3';

[0224] 5'-enhancer (e.g., αMHCe)-cardiac-specific promoter-transgene-WPRE-p(A) (e.g., bGHpA)-3';

[0225] 5'-enhancer (e.g., ACTC1e)-cardiac-specific promoter-intron (e.g., CMV intron)-transgene-WPRE-p(A) (e.g., bGHpA)-3';

[0226] 5'-enhancer (e.g., αMHCe)-cardiac-specific promoter-intron (e.g., CMV intron)-transgene-WPRE-p(A) (e.g., bGHpA)-3';

[0227] 5'-enhancer (e.g., ACTC1e)-enhancer (e.g., αMHCe)-cardiac-specific promoter-transgene-WPRE-p(A) (e.g., bGHpA)-3';

[0228] 5'-enhancer (e.g., αMHCe)-enhancer (e.g., ACTC1e)-cardiac-specific promoter-transgene-WPRE-p(A) (e.g., bGHpA)-3';

[0229] 5'-enhancer (e.g., ACTC1e)-enhancer (e.g., αMHCe)-cardiac-specific promoter-intron (e.g., CMV intron)-transgene-WPRE-p(A) (e.g., bGHpA)-3';

[0230] 5'-enhancer (e.g., αMHCe)-enhancer (e.g., ACTC1e)-cardiac-specific promoter-intron (e.g., CMV intron)-transgene-WPRE-p(A) (e.g., bGHpA)-3';

[0231] 5'-cardiac-specific promoter-transgene with codon-optimized polynucleotide sequence-WPRE-p(A) (e.g., bGHpA)-3';

[0232] 5'-enhancer (e.g., αMHCe)-cardiac-specific promoter-intron (e.g., CMV intron)-transgene-WPRE-p(A) (e.g., bGHpA)-p(A) (e.g., SV40pA) transgene (e.g., with codon-optimized polynucleotide sequence)-intron (e.g., chimeric intron) cardiac-specific promoter enhancer (e.g., ACTC1e)-3', optionally with a first order transgene and its operably linked promoter / enhancer sequence in a forward orientation and a second order transgene and its operably linked promoter / enhancer sequence in a reverse orientation;

[0233] 5'-enhancer (e.g., αMHCe)-cardiac-specific promoter-intron (e.g., CMV intron)-transgene-WPRE-p(A) (e.g., bGHpA)-enhancer (e.g., ACTC1e)-cardiac-specific promoter-intron (e.g., chimeric intron)-transgene (e.g., with codon-optimized polynucleotide sequence)-p(A) (e.g., SV40pA)-3', optionally with both the first and second order transgenes and promoter / enhancer sequences operably linked thereto in a forward orientation;

[0234] 5'-p(A) (e.g., bGHpA)-WPRE-transgene-intron (e.g., CMV intron)-cardiac-specific promoter enhancer (e.g., αMHCe) enhancer (e.g., ACTC1e)-cardiac-specific promoter-intron (e.g., chimeric intron) transgene (e.g., with codon-optimized polynucleotide sequence)-p(A) (e.g., SV40pA)-3', optionally with a first order transgene and its operably linked promoter / enhancer sequence in a reverse orientation and a second order transgene and its operably linked promoter / enhancer sequence in a forward orientation;

[0235] 5'-cardiac specific promoter-intron (e.g., CMV intron)-transgene-WPRE-p(A) (e.g., bGHpA)-p(A) (e.g., SV40pA)-transgene (e.g., with codon-optimized polynucleotide sequence)-intron (e.g., chimeric intron)-cardiac specific promoter-3', optionally with a first order transgene and a promoter / enhancer sequence operably linked thereto in a forward orientation and a second order transgene and a promoter / enhancer sequence operably linked thereto in a reverse orientation;

[0236] 5'-cardiac specific promoter-intron (e.g., CMV intron)-transgene-WPRE-p(A) (e.g., bGHpA)-cardiac specific promoter-intron (e.g., chimeric intron)-transgene (e.g., with codon-optimized polynucleotide sequence)-p(A) (e.g., SV40pA)-3', optionally with both the first and second order transgenes and promoter / enhancer sequences operably linked thereto in a forward orientation; or

[0237] 5'-p(A) (e.g., bGHpA)-WPRE-transgene-intron (e.g., CMV intron)-cardiac-specific promoter-cardiac-specific promoter-intron (e.g., chimeric intron) transgene (e.g., with codon-optimized polynucleotide sequence)-p(A) (e.g., SV40pA)-3', optionally with the first order transgene and its operably linked promoter / enhancer sequence in reverse orientation and the second order transgene and its operably linked promoter / enhancer sequence in forward orientation.

[0238] In the expression cassettes described herein (such as those listed above), the cardiac-specific promoter may be a short human cTnT promoter (such as hcTnTp) or a chicken cTnT promoter (such as ccTnTp). More specific examples of the expression cassettes described above can be found, for example, in Figure 1, Figure 7A, Figure 7B, Figure 7C, and Table 1.

[0239] In some embodiments, the expression cassettes described herein allow for increased expression levels of a transgene as compared to a second expression cassette comprising a polynucleotide having a 5' to 3' arrangement of elements comprising: 5'-promoter-transgene-WPRE-p(A)-3'. In some embodiments, the expression level is increased between about 1.5-fold and about 150-fold as compared to the second expression cassette.

[0240] In some embodiments, one or more (eg, one, two, three, or four) elements of the expression cassette described herein can be omitted.

[0241] In some embodiments, one or more (e.g., one, two, three, or four) elements of the expression cassettes described herein can be replaced with other elements, such as functionally equivalent elements.

[0242] In some embodiments of the expression cassettes provided herein, the WPRE element is replaced by any other post-transcriptional regulatory element known in the art. In some embodiments, the expression cassettes provided herein include any post-transcriptional regulatory element known in the art. In some embodiments, the expression cassettes provided herein do not include a post-transcriptional regulatory element (e.g., do not include a WPRE element). In some embodiments, the expression cassettes provided herein include a WPRE.

[0243] In some embodiments of the expression cassettes provided herein, the bGHpA and / or SV40pA poly(A) elements are replaced by any other poly(A) elements known in the art. In some embodiments, the expression cassettes provided herein comprise any poly(A) element known in the art. In some embodiments, the expression cassettes provided herein do not comprise a poly(A) element. In some embodiments, the expression cassettes provided herein do not comprise bGHpA. In some embodiments, the expression cassettes provided herein do not comprise SV40pA. In some embodiments, the expression cassettes provided herein do not comprise bGHpA or SV40pA. In some embodiments, the expression cassettes provided herein comprise one or both of bGHpA and SV40pA.

[0244] In some embodiments of the expression cassettes provided herein, the CMV intron and / or chimeric intron element is replaced by any other intron element known in the art. In some embodiments, the expression cassettes provided herein include any intron element known in the art. In some embodiments, the expression cassettes provided herein do not include an intron. In some embodiments, the expression cassettes provided herein do not include a CMV intron. In some embodiments, the expression cassettes provided herein do not include a chimeric intron (e.g., do not include a Chim int). In some embodiments, the expression cassettes provided herein do not include a CMV intron or a Chim int. In some embodiments, the expression cassettes provided herein include one or both of a CMV intron and a Chim int.

[0245] It should be understood that the exemplary orientation of the expression cassette may include flanking inverted terminal repeat (ITR) sequences at the 5' and 3' ends of the expression cassette. It should be understood that the ITR sequences may be optional. In some embodiments, the expression cassettes described herein do not include ITR sequences (e.g., non-AAV, such as DNA plasmid-based expression cassettes).

[0246] Operably linked elements, such as those in the above illustrative orientations, can be on one or both strands of the polynucleotide.

[0247] In some embodiments, the expression cassette comprises one copy of the sequence encoding the polypeptide (i.e., one copy of the transgene). In some embodiments, the expression cassette comprises two copies of the sequence encoding the polypeptide (i.e., two copies of the transgene). In some embodiments, when the expression cassette comprises two copies of the sequence encoding the polypeptide, the two "copies" are not identical. Without being bound by any theory, by using two sequences encoding the polypeptide that are not identical, DNA recombination within the vector may be prevented. In some embodiments, the expression cassette comprises one copy having a native DNA sequence encoding the polypeptide and one copy having a codon-optimized DNA sequence encoding the polypeptide. In some embodiments, when the expression cassette comprises two copies of the sequence encoding the polypeptide, the two copies are identical.

[0248] In some embodiments, the expression cassette comprises one or more promoters described herein (with or without one or more enhancers described herein) driving one or more copies of a transgene (such as any transgene described herein). In some embodiments, the expression cassette does not comprise an enhancer (e.g., αMHCe and / or ACTC1e). In some embodiments, the expression cassette comprises one or more enhancers, such as a cardiac specific enhancer (e.g., αMHCe and / or ACTC1e). In some embodiments, the expression cassette comprises an αMHCe enhancer (and optionally does not comprise an ACTC1e enhancer). In some embodiments, the expression cassette comprises an ACTC1e enhancer (and optionally does not comprise an αMHCe enhancer). In some embodiments, the expression cassette comprises at least two enhancers in the order first αMHCe and then ACTC1e. In some embodiments, the expression cassette comprises at least two enhancers in the order first ACTC1e and then αMHCe. In some embodiments, the expression cassette comprises an intron element, e.g., a CMV intron element and / or a chimeric intron (such as, e.g., Chim int, as described herein). In some embodiments, the expression cassette comprises an intron element, but does not comprise an enhancer. In some embodiments, the expression cassette comprises an intron element (e.g., a CMV intron and / or a chimeric intron) and further comprises an enhancer (e.g., αMHCe and / or ACTC1e). In some embodiments, the expression cassette comprises a transgene with a codon-optimized polynucleotide sequence. In some embodiments, the expression cassette comprises a transgene with a codon-optimized polynucleotide sequence, but does not comprise an enhancer. In some embodiments, the expression cassette comprises a transgene with a codon-optimized polynucleotide sequence and further comprises an enhancer (e.g., αMHCe and / or ACTC1e).In some embodiments, the expression cassette comprises one or more promoters described herein and one, two or more enhancers described herein (e.g., comprising an αMHCe and / or ACTC1e enhancer) driving expression of one or more copies of the transgene (with or without CMV intron elements or chimeric intron elements). In some embodiments, the promoter is a cardiac specific promoter, such as a human cTnT promoter (e.g., the short human promoter, hcTnTp, etc.) and / or a chicken cTnT promoter (e.g., ccTnTp, etc.). In some embodiments, the enhancer is a cardiac specific enhancer, such as αMHCe and / or ACTC1e. In some embodiments, two or more cardiac specific enhancers are used, and two or more of the enhancers can be the same or different (e.g., both or all of αMHCe, both or all of ACTC1e, or at least one αMHCe and at least one ACTC1e). In some embodiments, two cardiac-specific enhancers are used, and the two enhancers can be the same or different (e.g., both αMHCe, both ACTC1e, or one αMHCe and one ACTC1e). In some embodiments, the transgene comprises a non-codon optimized polynucleotide sequence that encodes a gene product. In some embodiments, the order of elements is as shown in any of the expression cassettes depicted in Figure 1, Figure 7A, Figure 7B, Figure 7C, and Table 1.For example, (i) the order of the promoter and transgene elements can be as shown in FIG. 1, FIG. 7A, FIG. 7B, FIG. 7C, or Table 1; (ii) the order of the promoter, enhancer, and transgene elements can be as shown in FIG. 1, FIG. 7A, FIG. 7B, FIG. 7C, or Table 1; (iii) the order of the promoter, transgene, WPRE, and poly(A) elements can be as shown in FIG. 1, FIG. 7A, FIG. 7B, FIG. 7C, or Table 1, optionally with or without enhancer elements in the same order as shown in FIG. 1, FIG. 7A, FIG. 7B, FIG. 7C, or Table 1; or (iv) the order of the promoter, transgene, WPRE, poly(A), CMV intron (such as CMVint) elements can be as shown in FIG. 1, FIG. 7A, FIG. 7B, FIG. 7C, or Table 1, optionally with or without enhancer elements in the same order as shown in FIG. 1, FIG. 7A, FIG. 7B, FIG. 7C, or Table 1. In some embodiments, the orientation of any of the elements is as shown in any of the expression cassettes depicted in Figures 1, 7A, 7B, 7C and Table 1. The sequences of the individual expression cassette elements discussed herein (e.g., promoters, enhancers, transgenes, WPREs, poly(A), CMV introns, chimeric introns, etc.) can be any of the sequences of such elements provided herein, or any sequence with at least, e.g., 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity thereto.

[0249] In some embodiments, an expression cassette comprises a promoter as described herein (with or without one or more enhancers as described herein) driving a single copy of a transgene (such as, for example, any of the transgenes described herein). In some embodiments, an expression cassette comprises a promoter as described herein without any enhancers (e.g., without any enhancers as described herein, e.g., without αMHCe and / or without ACTC1e) driving a single copy of a transgene, and optionally such expression cassettes comprise a transgene that includes intron elements, e.g., CMV intron elements and / or chimeric introns (such as, for example, Chimint as described herein), and / or with a codon-optimized polynucleotide sequence. In some embodiments, the expression cassette comprises a promoter described herein without any enhancers driving a single copy of the transgene (e.g., without any enhancers described herein, e.g., without αMHCe and / or without ACTC1e), and further comprises a CMV intron and / or a chimeric intron (e.g., Chimint, etc.). In some embodiments, the expression cassette comprises a promoter described herein without any enhancers driving a single copy of the transgene (e.g., without any enhancers described herein, e.g., without αMHCe and / or without ACTC1e), and further comprises a CMV intron. In some embodiments, the expression cassette comprises a promoter described herein without any enhancers driving a single copy of the transgene (e.g., without any enhancers described herein, e.g., without αMHCe and / or without ACTC1e), and further comprises a CMV intron. In some embodiments, the expression cassette comprises a promoter described herein and one, two or more enhancers described herein (e.g., including the αMHCe and / or ACTC1e enhancers) driving expression of one copy of the transgene.In some embodiments, the expression cassette comprises one promoter described herein and one enhancer described herein (e.g., an αMHCe or ACTC1e enhancer) driving expression of one copy of the transgene. In some embodiments, the expression cassette comprises one promoter described herein and two enhancers described herein (e.g., both αMHCe, both ACTC1e, or one αMHCe and one ACTC1e) operably linked to one copy of the transgene. In some embodiments, the promoter is a cardiac specific promoter, such as a human cTnT promoter (e.g., the short human promoter, hcTnTp, etc.) and / or a chicken cTnT promoter (e.g., ccTnTp, etc.). In some embodiments where one or more enhancers are used, the enhancer is a cardiac specific enhancer, such as αMHCe and / or ACTC1e. In some embodiments, two or more cardiac-specific enhancers are used, and the two or more of the enhancers can be the same or different (e.g., both or all αMHCe, both or all ACTC1e, or at least one αMHCe and at least one ACTC1e). In some embodiments, two cardiac-specific enhancers are used, and the two enhancers can be the same or different (e.g., both αMHCe, both ACTC1e, or one αMHCe and one ACTC1e). In some embodiments, the expression cassette comprises at least two enhancers in the order of first αMHCe and then ACTC1e. In some embodiments, the expression cassette comprises at least two enhancers in the order of first ACTC1e and then αMHCe. In some embodiments, the transgene comprises a non-codon optimized polynucleotide sequence that encodes a gene product. In some embodiments, the transgene comprises a codon optimized polynucleotide sequence that encodes a gene product. In some embodiments, one or more intron elements are also used in addition to the promoter and enhancer elements. In some embodiments, a CMV intron element is used. In some embodiments, chimeric intron elements (Chim int) are used.In some embodiments, both a CMV intron and a chimeric intron (Chim int) are used. In some embodiments where one promoter is used, the order of the elements is as shown in any of the expression cassettes depicted in FIG. 7B. For example, (i) the order of the promoter and transgene elements can be as shown in FIG. 7B, (ii) the order of the promoter, enhancer, and transgene elements can be as shown in FIG. 7B, (iii) the order of the promoter, transgene, WPRE, and poly(A) elements can be as shown in FIG. 7B, optionally with or without an enhancer element in the same order as shown in FIG. 7B, or (iv) the order of the promoter, transgene, WPRE, poly(A), CMV intron (such as CMVint) elements can be as shown in FIG. 7B, optionally with or without an enhancer element in the same order as shown in FIG. 7B. In some embodiments where one promoter is used, the orientation of any of the elements is as shown in any of the expression cassettes depicted in FIG. 7B. In some embodiments, the orientation of the elements is forward. In some embodiments, the orientation of the elements is reverse. The sequences of the individual expression cassette elements discussed herein (such as promoters, enhancers, transgenes, WPREs, poly(A), CMV introns, and chimeric introns) can be any of the sequences of such elements provided herein, or any sequence with at least, e.g., 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity thereto.

[0250] In some embodiments, the expression cassette comprises two promoters described herein (with or without one or more enhancers described herein) driving expression of two copies of a transgene (such as, for example, any transgene described herein). In some embodiments, the expression cassette comprises two promoters described herein (with or without one or more enhancers described herein), each promoter operably linked to one copy of a transgene (such as, for example, any transgene described herein). In some embodiments, the expression cassette comprises two promoters described herein without any enhancers driving expression of two copies of a transgene (e.g., without any enhancers described herein, e.g., without αMHCe and / or without ACTC1e). In some embodiments, the expression cassette comprises two promoters described herein and one, two or more enhancers described herein (e.g., including αMHCe and / or ACTC1e enhancers) driving expression of two copies of a transgene. In some embodiments, the expression cassette comprises two promoters as described herein and two enhancers as described herein (e.g., including the αMHCe and / or ACTC1e enhancers) operably linked to two copies of a transgene, each transgene being operably linked to one promoter and one enhancer. In some embodiments, the promoter is a cardiac-specific promoter, such as a human cTnT promoter (e.g., the short human promoter, hcTnTp, etc.) and / or a chicken cTnT promoter (e.g., ccTnTp, etc.). In embodiments in which two promoters are used, the two promoters can be the same or different. In some embodiments, when two promoters drive expression of two copies of a transgene (each promoter drives expression of one copy of a transgene), both promoters can be cardiac-specific promoters, the same cardiac-specific promoter, or can be different from each other.In some embodiments, both promoters can be human cTnT promoters (e.g., both can be short human promoters, hcTnTp). In some embodiments, both promoters can be chicken cTnT promoters (e.g., ccTnT, etc.). In some embodiments, the two promoters are different, e.g., one is a human cTnT promoter (e.g., short human cTnT promoter, hcTnTp, etc.) and one is a chicken cTnT promoter (e.g., ccTnT, etc.). In some embodiments where two promoters and two transgenes are used, the two transgenes can be the same or different (e.g., the same or different variants of the same transgene, etc.). For example, the first copy of the transgene can be a non-codon optimized polynucleotide sequence that encodes a gene product, and the second copy of the transgene can be a codon optimized polynucleotide sequence that encodes a gene product. In some embodiments, both copies of the transgene used in the expression cassette are the same. In some embodiments where one or more enhancers are used, the enhancer is a cardiac-specific enhancer, e.g., αMHCe and / or ACTC1e. In some embodiments, two or more cardiac-specific enhancers are used, where the two or more enhancers can be the same or different (e.g., both or all αMHCe, both or all ACTC1e, or at least one αMHCe and at least one ACTC1e). In some embodiments, two cardiac-specific enhancers are used, where the two enhancers can be the same or different (e.g., both αMHCe, both ACTC1e, or one αMHCe and one ACTC1e). In some embodiments where two promoters are used, two cardiac-specific enhancers operably linked to the transgene are also used, optionally one enhancer is αMHCe and another is ACTC1e. In some embodiments, the expression cassette comprises at least two enhancers in the order first αMHCe and then ACTC1e. In some embodiments, the expression cassette comprises at least two enhancers, first ACTC1e and then αMHCe, in that order.In some embodiments where two promoters are used, one or more intron elements are also used. In some embodiments where two promoters are used, a CMV intron element is also used. In some embodiments where two promoters are used, a chimeric intron element (Chim int) is also used. In some embodiments where two promoters are used, a CMV intron and a chimeric intron (Chim int) are used. In some embodiments where two promoters are used, the order of elements is as shown in any of the expression cassettes depicted in Figure 7C. For example, (i) the order of the promoter and transgene elements can be as shown in Figure 7C, (ii) the order of the promoter, enhancer, and transgene elements can be as shown in Figure 7C, (iii) the order of the promoter, transgene, WPRE, and poly(A) elements can be as shown in Figure 7C, optionally with or without enhancer elements in the same order as shown in Figure 7C, or (iv) the order of the promoter, transgene, WPRE, poly(A), CMV intron (such as CMVint), and chimeric intron (such as Chim int) elements can be as shown in Figure 7C, optionally with or without enhancer elements in the same order as shown in Figure 7C. In some embodiments where two promoters are used, the first promoter and associated transgene (and optionally enhancer) are oriented in a forward 5' to 3' direction and the second promoter and associated transgene (and optionally enhancer) are oriented in a reverse direction. In some embodiments where two promoters are used, the first promoter and associated transgene (and optionally enhancer) are oriented in a reverse 5' to 3' direction and the second promoter and associated transgene (and optionally enhancer) are oriented in a forward direction. In some embodiments where two promoters are used, both the promoter and associated transgene (and optionally enhancer) are oriented in a forward 5' to 3' direction.In some embodiments where two promoters are used, both the promoter and associated transgene (and, optionally, the enhancer) are oriented in opposite directions relative to the 5' to 3' direction. In some embodiments where two promoters are used, the orientation of any of the elements (e.g., forward or reverse orientation in the 5' to 3' direction) is as shown in any of the expression cassettes depicted in Figure 7C. In some embodiments where two promoters are used, the orientation of the promoter, enhancer (if any), transgene, WPRE, poly(A), CMV intron, and chimeric intron elements (e.g., forward or reverse orientation in the 5' to 3' direction) is as shown in any of the expression cassettes depicted in Figure 7C. The sequences of the individual expression cassette elements discussed herein (e.g., promoters, enhancers, transgenes, WPREs, poly(A), CMV introns, and chimeric introns, etc.) can be any of the sequences of such elements provided herein, or any sequence with at least, e.g., 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity thereto.

[0251] Expression cassette sequences of the present disclosure can be found, but are not limited to, in Table 1. In some embodiments, the expression cassette comprises about 3.2 kilobases (kb), 3.3 kb, 3.4 kb, 3.5 kb, 3.6 kb, 3.7 kb, or less. In some embodiments, the expression cassette comprises about 1.9 kb, 2.1 kb, 2.2 kb, 2.3 kb, 2.4 kb, 2.5 kb, 2.6 kb, 2.7 kb, 2.8 kb, 2.9 kb, 3.0 kb, 3.1 kb, 3.2 kb, or more.

[0252] In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NOs: 20-24 or 45-63. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NOs: 64-75. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 61. In some embodiments, the expression cassette comprises SEQ ID NO: 61. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 62. In some embodiments, the expression cassette comprises SEQ ID NO: 62. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 63. In some embodiments, the expression cassette comprises SEQ ID NO: 63. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 49. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 51. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 55. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 56.In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 57. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 58. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 59. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 60. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 67. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 69. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 74. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 75. In some of these embodiments, the sequence encoding DWORF (DWORF open reading frame) may be replaced with a sequence encoding another polypeptide described herein, and the sequence identity referenced above does not take into account the portion of the polynucleotide sequence encoding DWORF (DWORF open reading frame).

[0253] In some embodiments, the transgene in the expression cassette encodes a polypeptide for use in treating or preventing a cardiac disease or disorder. In some embodiments, the transgene in the expression cassette encodes a polypeptide selected from DWORF, junctophilin (e.g., JPH2), BAG family molecular chaperone regulator 3 (BAG3), alpha-crystallin B chain (CRYAB), LMNA (e.g., lamin A and lamin C isoforms), troponin type I 3 (TNNI3), phospholamban (PLN), lysosomal associated membrane protein 2 (LAMP2, e.g., LAMP2a, LAMP2b, and LAMP2c isoforms), desmoplakin (DSP, e.g., DPI and DPII isoforms), desmoglein 2 (DSG2), and junctional plakoglobin (JUP), or a variant of any of these polypeptides (e.g., having at least 75%, at least 85%, at least 95%, at least 97%, or at least 99% sequence identity thereto). In some embodiments, the transgene in the expression cassette encodes DWORF (or a variant thereof). In some embodiments, the transgene in the expression cassette encodes JPH2 (e.g., full length JPH2 or an N-terminal fragment of JPH2) (or a variant thereof). In some embodiments, the transgene in the expression cassette encodes BAG3 (or a variant thereof). In some embodiments, the transgene in the expression cassette encodes CRYAB (or a variant thereof). In some embodiments, the transgene in the expression cassette encodes a LMNA lamin A isoform (or a variant thereof). In some embodiments, the transgene in the expression cassette encodes a LMNA lamin C isoform (or a variant thereof). In some embodiments, the transgene in the expression cassette encodes TNNI3 (or a variant thereof). In some embodiments, the transgene in the expression cassette encodes PLN (or a variant thereof). In some embodiments, the transgene in the expression cassette encodes LAMP2a (or a variant thereof). In some embodiments, the transgene in the expression cassette encodes LAMP2b (or a variant thereof).In some embodiments, the transgene in the expression cassette encodes LAMP2c (or a variant thereof). In some embodiments, the transgene in the expression cassette encodes a DSP DPI isoform (or a variant thereof). In some embodiments, the transgene in the expression cassette encodes a DSP DPII isoform (or a variant thereof). In some embodiments, the transgene in the expression cassette encodes a DSG2 (or a variant thereof). In some embodiments, the transgene in the expression cassette encodes a JUP (or a variant thereof). In some embodiments, the transgene in the expression cassette encodes a human polypeptide (such as any of the human polypeptides described herein).

[0254] In some embodiments, the expression cassettes described herein direct cardiac cell-specific expression of the transgene. In some embodiments, the expression cassettes described herein direct cardiomyocyte-specific expression of the transgene. In some embodiments, the expression cassettes described herein allow for high expression of the transgene in cardiac cells (e.g., cardiomyocytes) and low or no expression in other cells (e.g., low or no expression in liver cells, low or no expression in muscle cells other than cardiac muscle cells, low or no expression in cardiac fibroblasts). In some embodiments, the expression cassettes described herein allow for high expression of the transgene in cardiac tissue of a subject (e.g., human heart). In some embodiments, the expression cassettes described herein allow for no or low expression of the transgene in tissues of a subject other than the heart (e.g., in the liver or in muscle other than that of the heart). "High" and "low" may be relative to each other, for example, expression of a transgene in cardiac cells (e.g., cardiomyocytes) and / or cardiac tissue may be at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 50-fold, 100-fold, 150-fold, or 200-fold higher than its expression in other cells and tissues (e.g., liver, muscle excluding heart). [Table 1-1] [Table 1-2]

Table 1-3

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

Table 1-21

Table 1-22

Table 1-23

Table 1-24

Table 1-25

Table 1-26

Table 1-27

Table 1-28

Table 1-29

Table 1-30

Table 1-31

Table 1-32

Table 1-33

Table 1-34

Table 1-35

Table 1-36

[0255] vector In some aspects, the present disclosure provides a vector comprising an expression cassette provided herein. The vector may be any viral vector or any non-viral vector known in the art or described herein.

[0256] In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adeno-associated viral vector (AAV), an adenoviral vector (AV), a lentiviral vector (LV), a retroviral vector (RV), a herpes simplex viral vector (HSV), or a pox viral vector.

[0257] In some embodiments, provided herein is an AAV comprising any of the expression cassettes described herein. In some embodiments, provided herein is an AV comprising any of the expression cassettes described herein. In some embodiments, provided herein is an LV comprising any of the expression cassettes described herein. In some embodiments, provided herein is an RV comprising any of the expression cassettes described herein. In some embodiments, provided herein is an HSV comprising any of the expression cassettes described herein. In some embodiments, provided herein is a box virus-based vector comprising any of the expression cassettes described herein.

[0258] In some embodiments, the vector is a non-viral vector. In some embodiments, the non-viral vector is naked DNA (e.g., a DNA plasmid). In some embodiments, the non-viral vector is a plasmid. In some embodiments, the non-viral vector is a liposome or lipid vector comprising plasmid DNA and a lipid solution.

[0259] For example, viral and non-viral vectors and delivery systems are described in Sung & Kim 2019, Biomaterials Research 23:8; Mali, 2013, Indian Journal of Human Genetics, 19(1):3-8; Hardee et al., 2017, Genes 8:65; Bulcha et al., 2020, Signal Transduction and Targeted Therapy; Ghosh et al., 2020, Applied Bio Safety: Journal of ABSA International 25(1):7-18, the disclosures of each of which are hereby incorporated by reference in their entireties.

[0260] In some embodiments, the vector is a recombinant vector.

[0261] In some embodiments, the vectors described herein comprise an expression cassette comprising a polynucleotide encoding any of the gene products described herein. In some embodiments, the expression cassette comprises a sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NOs: 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225.

[0262] In some embodiments, the vectors described herein comprise an expression cassette comprising a polynucleotide encoding DWORF. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NOs:20-24 and 45-75.

[0263] In some aspects of the disclosure, the vector is used to deliver an expression cassette described herein to a cardiac cell of a subject, for example, to treat cardiomyopathy. In some embodiments, the disclosure provides a viral vector comprising an expression cassette comprising a polynucleotide encoding a gene product (such as any gene product described herein, e.g., a DWORF polypeptide) operably linked to a promoter and a pharma- tically acceptable carrier. In some embodiments, the disclosure provides a virion comprising a capsid and an expression cassette comprising a polynucleotide encoding a gene product (such as any gene product described herein, e.g., a DWORF polypeptide) operably linked to a promoter and a pharma- tically acceptable carrier. In some embodiments, the disclosure provides a plasmid comprising an expression cassette comprising a polynucleotide encoding a gene product (such as any gene product described herein, e.g., a DWORF polypeptide) operably linked to a promoter and a pharma- tically acceptable carrier.

[0264] In some embodiments, the viral vector described herein is replication-deficient in that it cannot independently replicate and package its genome further.For example, when cardiac cells are targeted with virions, transgenes are expressed in the targeted cardiac cells, but due to the fact that the targeted cardiac cells lack packaging and accessory function genes, the virions cannot replicate.In some embodiments, the viral vector described herein is replication-competent.

[0265] In some embodiments, the vectors described herein are capable of being delivered to both dividing and non-dividing cells. In some embodiments, the vectors described herein are capable of being delivered to non-dividing cells. In some embodiments, the vectors described herein are capable of being delivered to dividing cells.

[0266] In some embodiments, vectors comprising the expression cassettes described herein direct cardiac cell-specific expression of a transgene. In some embodiments, vectors comprising the expression cassettes described herein direct cardiomyocyte-specific expression of a transgene. In some embodiments, vectors comprising the expression cassettes described herein allow high expression of a transgene in cardiac cells (e.g., cardiomyocytes) and low or no expression in other cells (e.g., low or no expression in liver cells, low or no expression in muscle cells other than cardiac muscle cells, low or no expression in cardiac fibroblasts). In some embodiments, vectors comprising the expression cassettes described herein allow high expression of a transgene in cardiac tissue of a subject (e.g., in a human heart). In some embodiments, vectors comprising the expression cassettes described herein allow no or low expression of a transgene in tissues of a subject other than the heart (e.g., in the liver or in muscle other than that of the heart). "High" and "low" may be relative to each other, for example, expression of a transgene in cardiac cells (e.g., cardiomyocytes) and / or cardiac tissue may be at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 50-fold, 100-fold, 150-fold, or 200-fold higher than its expression in other cells and tissues (e.g., liver, muscle excluding heart).

[0267] Recombinant AAV virions In some aspects, the present disclosure provides a recombinant AAV (rAAV) virion comprising an expression cassette provided herein. In some embodiments, the rAAV virion comprises a capsid protein and an expression cassette. In some embodiments, the expression cassette comprises a polynucleotide encoding any of the gene products described herein.

[0268] In some embodiments, the expression cassette comprises a polynucleotide encoding DWORF. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NOs:20-24 or 45-63. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:61. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:62. In some embodiments, the expression cassette comprises SEQ ID NO:62. In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 63. In some embodiments, the expression cassette comprises SEQ ID NO:63.

[0269] In some aspects of the present disclosure, the rAAV virion is used to deliver the expression cassette described herein to cardiac cells of a subject, for example, to treat cardiomyopathy. Thus, the present disclosure provides an rAAV virion comprising an rAAV virion, an AAV capsid, and an expression cassette comprising a polynucleotide encoding a DWORF polypeptide operably linked to a promoter, and a pharma- ceutically acceptable carrier.

[0270] The rAAV virions of the present disclosure include capsid proteins. Capsid proteins are structural proteins that constitute the assembled icosahedral package of the rAAV virion that contains the expression cassette. Capsid proteins are classified by serotype. The wild-type capsid serotype in the rAAV virion can be, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 (Naso et al. BioDrugs 31:317-334(2017)). Engineered capsid types include chimeric capsids and mosaic capsids (Choi et al. Curr Gene Ther. 5: 299-310(2005)). Capsids are selected for rAAV virions based on their ability to transduce specific tissues or cell types (Liu et al. Curr Pharm Des. 21:3248-56 (2015)).

[0271] Any capsid protein that can promote rAAV virion transduction into cardiac cells can be used for the delivery of transgenes described herein. Capsid proteins used in rAAV virions for transgene delivery to cardiac cells resulting in high expression can be, for example, AAV4, AAV6, AAV7, AAV8, and AAV9 (Zincarelli et al. Mol. Ther. 16: P1073-1080 (2008)). Artificial capsids, such as chimeric capsids generated through composite libraries, can also be used for transgene delivery to cardiac cells resulting in high expression (see U.S. Patent No. 63 / 012,703, the contents of which are incorporated herein by reference). Other capsid proteins with different characteristics can also be used in the rAAV virions of the present disclosure. AAV vectors and capsids can be found in U.S. Patent Application Publication Nos. US10011640B2; US7892809B2, US8632764B2, US8889641B2, US9475845B2, US10889833B2, US10480011B2, and US10894949B2, the contents of which are incorporated by reference herein; and International Patent Publication Nos. WO2020198737A1, WO20 No. 19028306A2, WO2016054554A1, WO2018152333A1, WO2017106236A1, WO2008124724A1, WO2017212019A1, WO2020117898A1, WO2017192750A1, WO2020191300A1, and WO2017100671A1, the contents of which are incorporated herein by reference.

[0272] In some embodiments, the rAAV virions of the present disclosure comprise engineered capsid proteins. The engineered capsid proteins can be derived from a parent, e.g., wild-type, capsid, but can include, for example, a variant polypeptide sequence with respect to the parent capsid sequence at one or more sites. For example, the variant sites of the parent capsid can occur at the VR-IV site, the VR-V site, the VR-VII site, and / or the VR-VIII site (see, e.g., Buning and Srivastava. Mol Ther Methods Clin Dev. 12:248-265 (2019)).

[0273] In some embodiments, the capsid protein is an AAV5 / AAV9 chimeric capsid protein. In some embodiments, the chimeric capsid protein comprises at least one, two, three, four, five or more polypeptide segments derived from an AAV5 capsid protein (SEQ ID NO: 144). In some embodiments, the chimeric capsid protein comprises at least one, two, three, four, five or more polypeptide segments derived from an AAV9 capsid protein (SEQ ID NO: 143). In some embodiments, at least one polypeptide segment is derived from an AAV5 capsid protein and at least one polypeptide segment is derived from an AAV9 capsid protein.

[0274] In some embodiments, the capsid protein is a combined capsid protein. As used herein, "combined capsid protein" refers to an AAV5 / AAV9 chimeric capsid protein, which further comprises an amino acid variation relative to the chimeric parent sequence at one or more sites. In some embodiments, the one or more sites of the chimeric parent sequence are selected from sites equivalent to the VR-IV, VR-V, VR-VII, and VR-VIII sites of the AAV9 capsid protein.

[0275] In some embodiments, the rAAV virion comprises an engineered capsid protein selected from Table 7. [Table 7]

[0276] In some embodiments, rAAV is replication-deficient, in that rAAV virion cannot independently replicate and package its genome.For example, when cardiac cells are targeted with rAAV virion, transgenes are expressed in targeted cardiac cells, but rAAV cannot replicate due to the fact that targeted cardiac cells lack AAV rep and cap genes and accessory function genes.

[0277] In some embodiments, the rAAV virions of the present disclosure that encapsulate the expression cassettes described herein can be produced using helper-free production. rAAV is a replication-defective virus that typically requires components from a live helper virus, such as adenovirus, in a host cell for packaging of infectious rAAV virions. The rAAV helper-free production system allows for the production of infectious rAAV virions without the use of a live helper virus. In the helper-free system, a host packaging cell line is co-transfected with three plasmids. The first plasmid can contain adenovirus gene products (e.g., E2A, E4, and VA RNA genes) required for packaging of rAAV virions. The second plasmid can contain the required AAV genes (e.g., REP and CAP genes). The third plasmid contains a polynucleotide sequence encoding the transgene of interest and a promoter flanked by ITRs. The host cell packaging cell line can be, for example, an AAV-293 host cell. Suitable host cells contain additional components required for packaging of infectious rAAV virions that are not supplied by the plasmid, hi some embodiments, the CAP gene can encode, for example, the AAV capsid proteins described herein.

[0278] In some embodiments, the CAP gene can code for, for example, the AAV capsid protein described herein. In some embodiments, the promoter is a promoter sequence described herein. In some embodiments, the promoter sequence is a cTnT promoter sequence. In some embodiments, the polypeptide of interest is a DWORF polypeptide.

[0279] The expression cassettes, enhancers, and / or promoters described herein with respect to AAV virions need not be limited to their use in AAV virions, but may be incorporated into essentially any other construct in which expression of a polynucleotide encoding a gene product is desired.

[0280] rAAV virions can deliver transgenes to cells in a subject, which are then expressed in the cells. The transgenes delivered by rAAV virions can be integrated into the genome of targeted cells, allowing for potential long-term expression of transgene products. Compared with other viral transgene delivery systems, such as adenovirus, rAAV virions have the advantage of low immunogenicity. rAAV virions can be used to transduce and deliver transgenes to many cell types, including eye, blood, liver, heart, joint tissue, muscle, brain kidney, or lung cells (US Pat. No. 10,308,957; US Pat. No. 9,803,218). rAAV virions can contain genomes up to about 5.2 kilobases (kb), limiting the size of polynucleotides that can be integrated into host cells to about 4.4 kb (Choi et al. Mol Brain. 7:1 (2014)).

[0281] How to use Methods for increasing polypeptide expression The present disclosure provides methods of increasing polypeptide expression in a cell, comprising contacting the cell with any of the vectors or virions (e.g., rAAV virions) described herein. In some embodiments, the cell is a cardiac cell. In some embodiments, the cell is a cardiomyocyte. In some embodiments, the contacting is in vitro. In some embodiments, the contacting is in vivo. In some embodiments, the polypeptide is any polypeptide for use in treating or preventing cardiac disease. In some embodiments, the polypeptide is any polypeptide described herein. In some embodiments, the polypeptide is encoded by any of the transgenes described herein.

[0282] The present disclosure provides methods of increasing polypeptide expression in a tissue, comprising contacting the tissue with any of the vectors or virions (e.g., rAAV virions) described herein. In some embodiments, the tissue is cardiac tissue. In some embodiments, the contacting is in vitro. In some embodiments, the contacting is in vivo.

[0283] The disclosure provides methods of increasing polypeptide expression in an organ comprising contacting the organ with any of the vectors or virions (e.g., rAAV virions) described herein. In some embodiments, the organ is a heart. In some embodiments, the heart is diseased or at risk of disease. In some embodiments, the heart has a borderline or reduced ejection fraction. In some embodiments, the heart has a normal ejection fraction. In some embodiments, the heart comprises a genetic mutation associated with heart disease. In some embodiments, the genetic mutation is a PLN mutation. In some embodiments, the heart has low or undetectable polypeptide expression compared to a healthy heart. In some embodiments, the contacting is in vitro. In some embodiments, the contacting is in vivo.

[0284] The present disclosure provides a method of increasing polypeptide expression in a subject, comprising administering to the subject any of the vectors or virions (e.g., rAAV virions) described herein. In some embodiments, the subject is an animal. The animal may be, but is not limited to, a mouse, a rat, a dog, or a non-human primate. In some embodiments, the subject is a human. In some embodiments, the increased polypeptide expression is in the heart of the subject. In some embodiments, the subject has or is at risk for heart disease. In some embodiments, the subject has a borderline or reduced ejection fraction. In some embodiments, the subject has a normal ejection fraction. In some embodiments, the subject has a genetic mutation associated with heart disease. In some embodiments, the genetic mutation is a PLN mutation. In some embodiments, the subject has a low or undetectable level of DWORF expression compared to healthy subjects.

[0285] In some embodiments, the polypeptide is expressed in a cell, tissue, organ, or subject at a desired expression level. A "desired expression level" may be selected such that the level of expression is relative to the polypeptide expression in a healthy or diseased cell, tissue, organ, or subject. For example, an increased level in polypeptide expression compared to diseased cardiac cells, cardiac tissue, heart, or a subject with a cardiac disease or disorder may be a desired expression level. A desired expression level may be expressed relative to the difference in expression achieved between different vectors or virions (e.g., rAAV virions) that contain different expression cassettes. For example, a vector or virion (e.g., rAAV virion) that contains an expression cassette that includes a promoter and an enhancer may achieve a desired expression level compared to a vector or virion (e.g., rAAV virion) that contains only a promoter.

[0286] The polypeptide expression level achieved by any vector or virion (e.g., rAAV virion) containing an expression cassette may be described as a "fold" change (i.e., increase or decrease) compared to basal polypeptide expression. The polypeptide expression level achieved by a vector or virion (e.g., rAAV virion) containing an expression cassette may be described as a "fold change" compared to polypeptide expression achieved by an expression cassette containing a single promoter, no enhancer, and a sequence encoding the polypeptide. Fold change is a relative amount, such that the level of expression between the expression level achieved by the expression cassette and the reference expression level is expressed as a ratio. When describing a fold change in polypeptide expression, it is understood that "about" refers to ±0.5 fold. Polypeptide expression levels can be classified as "low expression," "moderate expression," or "high expression." "Low expression" is meant to include expression levels between about a 1.5-fold and 20-fold increase in polypeptide expression. "Moderate expression" is meant to include expression levels between about a 20-fold increase and about a 60-fold increase in polypeptide expression. "High expression" is meant to include expression levels of between about a 60-fold and a 140-fold increase in polypeptide expression.

[0287] In some embodiments, the polypeptide expression level is increased by between about 1.5-fold and 150-fold. In some embodiments, the polypeptide expression level is increased by at least about 1.5-fold, about 3.5-fold, about 5.5-fold, about 7.5-fold, about 9.5-fold, about 11.5-fold, about 13.5-fold, about 15.5-fold, about 17.5-fold, about 19.5-fold, about 21.5-fold, about 23.5-fold, about 25.5-fold, about 27.5-fold, about 29.5-fold, about 31.5-fold, about 33.5-fold, about 34.5-fold, about 35.5-fold, about 36.5-fold, about 37.5-fold, about 38.5-fold, about 39.5-fold, about 40.5-fold, about 41.5-fold, about 42.5-fold, about 43.5-fold, about 44.5-fold, about 45.5-fold, about 46.5-fold, about 47.5-fold, about 48.5-fold, about 49.5-fold, about 50.5-fold, about 51.5-fold, about 52.5-fold, about 53.5-fold, about 54.5-fold, about 55.5-fold, about 56.5-fold, about 57.5-fold, about 58.5-fold, about 59.5-fold, about 60.5-fold, about 61.5-fold, about 62.5-fold, about 63.5-fold, about 64.5-fold, about 65.5-fold, about 66.5-fold, about 67.5-fold, about 68.5-fold, about 69.5-fold 5.5 times, approximately 37.5 times, approximately 39.5 times, approximately 41.5 times, approximately 43.5 times, approximately 45.5 times, approximately 47.5 times, approximately 49.5 times, approximately 51.5 times, approximately 53.5 times, approximately 55. 5 times, approximately 57.5 times, approximately 59.5 times, approximately 61.5 times, approximately 63.5 times, approximately 65.5 times, approximately 67.5 times, approximately 69.5 times, approximately 71.5 times, approximately 73.5 times, approximately 75.5 times, Approximately 77.5 times, approximately 79.5 times, approximately 81.5 times, approximately 83.5 times, approximately 85.5 times, approximately 87.5 times, approximately 89.5 times, approximately 91.5 times, approximately 93.5 times, approximately 95.5 times, approximately 9 7.5 times, approximately 99.5 times, approximately 101.5 times, approximately 103.5 times, approximately 105.5 times, approximately 107.5 times, approximately 109.5 times, approximately 111.5 times, approximately 113.5 times, approximately 115. 5-fold, about 117.5-fold, about 119.5-fold, about 121.5-fold, about 123.5-fold, about 125.5-fold, about 127.5-fold, about 129.5-fold, about 131.5-fold, about 133.5-fold, about 135.5-fold, about 137.5-fold, about 139.5-fold, about 141.5-fold, about 143.5-fold, about 145.5-fold, about 147.5-fold, or about 149.5-fold increase.

[0288] In some embodiments, the polypeptide expression level is increased by at least about 5-fold or more. In some embodiments, the polypeptide expression level is increased by at least about 10-fold or more. In some embodiments, the polypeptide expression level is increased by at least about 15-fold or more. In some embodiments, the polypeptide expression level is increased by at least about 25-fold or more. In some embodiments, the polypeptide expression level is increased by at least about 35-fold or more. In some embodiments, the polypeptide expression level is increased by at least about 50-fold or more. In some embodiments, the polypeptide expression level is increased by at least about 60-fold or more. In some embodiments, the polypeptide expression level is increased by at least about 75-fold or more. In some embodiments, the polypeptide expression level is increased by at least about 85-fold or more. In some embodiments, the polypeptide expression level is increased by at least about 100-fold or more. In some embodiments, the polypeptide expression level is increased by at least about 110-fold or more. In some embodiments, the polypeptide expression level is increased by at least about 125-fold or more.

[0289] In some embodiments, the fold increase is relative to an expression cassette comprising a single promoter, no enhancer, and a sequence encoding the polypeptide. In some embodiments, the fold increase is relative to a healthy cell, tissue, organ, or subject. In some embodiments, the fold increase is relative to a diseased cell, tissue, organ, or subject.

[0290] Methods for increasing DWORF expression The present disclosure provides a method for increasing DWORF expression in a cell, comprising contacting the cell with the rAAV virion described herein.In some embodiments, the cell is a cardiac cell.In some embodiments, the cell is a cardiomyocyte.In some embodiments, the contact is in vitro.In some embodiments, the contact is in vivo.

[0291] The present disclosure provides a method for increasing DWORF expression in a tissue, comprising contacting the tissue with the rAAV virion described herein.In some embodiments, the tissue is cardiac tissue.In some embodiments, the contacting is in vitro.In some embodiments, the contacting is in vivo.

[0292] The present disclosure provides a method of increasing DWORF expression in an organ, comprising contacting the organ with a rAAV virion described herein. In some embodiments, the organ is a heart. In some embodiments, the heart is diseased or at risk of disease. In some embodiments, the heart has a borderline or reduced ejection fraction. In some embodiments, the heart has a normal ejection fraction. In some embodiments, the heart comprises a genetic mutation associated with heart disease. In some embodiments, the genetic mutation is a PLN mutation. In some embodiments, the heart has low or undetectable DWORF expression compared to a healthy heart. In some embodiments, the contacting is in vitro. In some embodiments, the contacting is in vivo.

[0293] The present disclosure provides a method of increasing DWORF expression in a subject, comprising administering to the subject a rAAV virion as described herein. In some embodiments, the subject is an animal. The animal may be, but is not limited to, a mouse, a rat, a dog, or a non-human primate. In some embodiments, the subject is a human. In some embodiments, the increased DWORF expression is in the heart of the subject. In some embodiments, the subject has or is at risk for heart disease. In some embodiments, the subject has a borderline or reduced ejection fraction. In some embodiments, the subject has a normal ejection fraction. In some embodiments, the subject has a genetic mutation associated with heart disease. In some embodiments, the genetic mutation is a PLN mutation. In some embodiments, the subject has a low or undetectable level of DWORF expression compared to a healthy subject.

[0294] In some embodiments, DWORF is expressed in a cell, tissue, organ, or subject at a desired expression level. The "desired expression level" may be selected such that the level of expression is relative to DWORF expression in a healthy or diseased cell, tissue, organ, or subject. For example, an increased level of DWORF expression compared to diseased cardiac cells, cardiac tissue, heart, or a subject with a cardiac disease or disorder may be a desired expression level. The desired expression level may be expressed relative to the difference in expression achieved between different rAAV virions containing different expression cassettes. For example, a rAAV virion containing an expression cassette containing a promoter and an enhancer may achieve a desired expression level compared to a rAAV virion containing only a promoter.

[0295] The DWORF expression level achieved by a rAAV virion containing an expression cassette may be described as a "fold" change (i.e., increase or decrease) compared to basal DWORF expression. The DWORF expression level achieved by a rAAV virion containing an expression cassette may be described as a "fold change" compared to DWORF expression achieved by an expression cassette containing a single promoter, no enhancer, and a sequence encoding DWORF. The fold change is a relative amount, such that the level of expression between the expression level achieved by the expression cassette and the reference expression level is expressed as a ratio. When describing a fold change in DWORF expression, it is understood that "about" refers to ±0.5-fold. DWORF expression levels can be classified as "low expression," "moderate expression," or "high expression." "Low expression" is meant to include expression levels between about a 1.5-fold and 20-fold increase in DWORF expression. "Moderate expression" is meant to include expression levels between about a 20-fold increase and about a 60-fold increase in DWORF expression. "High expression" is meant to include expression levels between about a 60-fold and a 140-fold increase in DWORF expression.

[0296] In some embodiments, the DWORF expression level is between about a 1.5-fold and 150-fold increase. In some embodiments, the DWORF expression level is between about a 1.5-fold, about a 3.5-fold, about a 5.5-fold, about a 7.5-fold, about a 9.5-fold, about a 11.5-fold, about a 13.5-fold, about a 15.5-fold, about a 17.5-fold, about a 19.5-fold, about a 21.5-fold, about a 23.5-fold, about a 25.5-fold, about a 27.5-fold, about a 29.5-fold, about a 31.5-fold, about a 33.5-fold, about a 35.5-fold, or about a 40-fold increase. Approximately 37.5 times, approximately 39.5 times, approximately 41.5 times, approximately 43.5 times, approximately 45.5 times, approximately 47.5 times, approximately 49.5 times, approximately 51.5 times, approximately 53.5 times, approximately 55.5 times, approximately 57.5 times, approximately 59.5 times, approximately 61.5 times, approximately 63.5 times, approximately 65.5 times, approximately 67.5 times, approximately 69.5 times, approximately 71.5 times, approximately 73.5 times, approximately 75.5 times, approximately 77 .5 times, approximately 79.5 times, approximately 81.5 times, approximately 83.5 times, approximately 85.5 times, approximately 87.5 times, approximately 89.5 times, approximately 91.5 times, approximately 93.5 times, approximately 95.5 times, approximately 97. 5 times, approximately 99.5 times, approximately 101.5 times, approximately 103.5 times, approximately 105.5 times, approximately 107.5 times, approximately 109.5 times, approximately 111.5 times, approximately 113.5 times, approximately 115.5 fold, about 117.5-fold, about 119.5-fold, about 121.5-fold, about 123.5-fold, about 125.5-fold, about 127.5-fold, about 129.5-fold, about 131.5-fold, about 133.5-fold, about 135.5-fold, about 137.5-fold, about 139.5-fold, about 141.5-fold, about 143.5-fold, about 145.5-fold, about 147.5-fold, or about 149.5-fold.

[0297] In some embodiments, the DWORF expression level is increased by at least about 5-fold or more. In some embodiments, the DWORF expression level is increased by at least about 10-fold or more. In some embodiments, the DWORF expression level is increased by at least about 15-fold or more. In some embodiments, the DWORF expression level is increased by at least about 25-fold or more. In some embodiments, the DWORF expression level is increased by at least about 35-fold or more. In some embodiments, the DWORF expression level is increased by at least about 50-fold or more. In some embodiments, the DWORF expression level is increased by at least about 60-fold or more. In some embodiments, the DWORF expression level is increased by at least about 75-fold or more. In some embodiments, the DWORF expression level is increased by at least about 85-fold or more. In some embodiments, the DWORF expression level is increased by at least about 100-fold or more. In some embodiments, the DWORF expression level is increased by at least about 110-fold or more. In some embodiments, the DWORF expression level is increased by at least about 125 fold or more.

[0298] In some embodiments, the fold increase is relative to an expression cassette that includes a single promoter, no enhancer, and a sequence encoding a DWORF. In some embodiments, the fold increase is relative to a healthy cell, tissue, organ, or subject. In some embodiments, the fold increase is relative to a diseased cell, tissue, organ, or subject.

[0299] Treatment methods In one aspect, any of the vectors containing the expression cassettes described herein can be used to treat diseases, such as cardiac diseases.

[0300] In one aspect, rAAV virions containing the expression cassettes described herein can be used to treat disease (Wang et al. Nat Rev Drug Discov. 18:358-378 (2019)). For treatment, rAAV virions have been used to deliver transgenes encoding polypeptides such as microdystrophin (Chamberlain et al. Mol Ther. 25:1125-1131 (2017)), glial cell line-derived neurotrophic factor (McFarthing et al. J Parkinsons Dis. 9:251-264 (2019)), and factor IX (Nathwani et al. N Engl J Med. 371:1994-2004 (2014)).

[0301] Various strategies to treat heart failure using rAAV-based delivery of transgenes have been pursued in vivo. In a porcine model of heart failure, the β-adrenergic receptor has been targeted by delivery of βARKct, a small polypeptide that is a regulator of contractility and indirectly prevents disruption of β-adrenergic receptor signaling (Raake et al. Eur Heart J. 34:1437-47 (2013)). In a canine model, cardiomyocyte viability was enhanced by rAAV-based delivery of vascular endothelial growth factor (VEGF) isoforms. In a human clinical trial, rAAV-based delivery of an isoform of the SERCA calcium pump, SERCA2a, to the heart was tested as a treatment for heart failure. SERCA, or sarco / endoplasmic reticulum Ca 2+ -ATPase, or SR Ca 2+ SERCA is a calcium ATPase-type P-ATPase. SERCA is present in the sarcoplasmic reticulum (SR) in muscle cells. It converts Ca at the expense of ATP hydrolysis during muscle relaxation. 2+ The transport of Ca from the cell cytosol into the lumen of the SR is 2+SERCA is an ATPase. SERCA activity is required for proper contractile function of the heart. However, direct replacement of SERCA activity by rAAV-based delivery of the SERCA2a isoform showed no significant effect in clinical trials (Bass-Stringer et al. Heart, Lung and Circulation. 27:1285-1300 (2018)). Enhancing SERCA activity using alternative strategies is desirable for treating cardiac diseases, such as heart failure and cardiomyopathies.

[0302] The cytoplasmic face of SERCA contains three major domains: the phosphorylation domain and the nucleotide-binding domain that form the catalytic site, and the actuator domain that is involved in transmitting the major conformational change. SERCA transduces Ca across the SR membrane. 2+ The rate at which SERCA moves can be controlled by the regulatory protein phospholamban (PLN). SERCA is normally inhibited by PLN, with which it is tightly associated. Increased β-adrenergic stimulation reduces the association between SERCA and PLN due to phosphorylation of PLN by PKA. When PLN associates with SERCA, it inhibits Ca 2+ Upon dissociation of pLN, Ca 2+ Increased movement.

[0303] An alternative strategy to enhance SERCA activity by delivering the SERCA2a isoform is to enhance the activity of naturally expressed SERCA by replacing PLN. Contacting SERCA with a DWORF polypeptide, as described in detail above, can replace PLN and enhance SERCA activity.

[0304] In some embodiments, the disclosure provides a method of treating a cardiac disease or disorder in a subject in need thereof, the method comprising administering an effective amount of a vector comprising an expression cassette comprising a polynucleotide encoding a therapeutic polypeptide operably linked to a promoter, the therapeutic polypeptide can be any polypeptide useful for treating a cardiac disease. As described herein, the vector can be any viral or non-viral vector.

[0305] In some embodiments, the disclosure provides a method of treating a cardiac disease or disorder in a subject in need thereof, the method comprising administering an effective amount of a recombinant adeno-associated virus (rAAV) virion, the rAAV virion comprising an AAV capsid and an expression cassette comprising a polynucleotide encoding a DWORF polypeptide operably linked to a promoter.

[0306] In the methods of treating a subject as described herein, "treating" or "treatment of a condition or subject in need thereof" refers to (1) taking steps to obtain a beneficial or desired result, including a clinical outcome, e.g., a reduction in symptoms; (2) inhibiting the disease, e.g., halting or reducing the occurrence of the disease or its clinical symptoms; (3) relieving the disease, e.g., causing regression of the disease or its clinical symptoms; or (4) slowing the disease. For purposes of the methods described herein, beneficial or desired clinical results include, but are not limited to, a reduction in symptoms associated with heart failure, cardiomyopathy, dilated cardiomyopathy, myocardial infarction, acute myocardial infarction, and chronic myocardial infarction.

[0307] In another aspect, the disclosure provides a method of preventing a cardiac disease or disorder in a subject in need thereof, the method comprising administering an effective amount of a vector comprising an expression cassette comprising a polynucleotide encoding a therapeutic polypeptide operably linked to a promoter, the therapeutic polypeptide can be any polypeptide useful for preventing cardiac disease. As described herein, the vector can be any viral or non-viral vector. In some embodiments, prevention of the disease prevents clinical symptoms of the disease from occurring in a patient who may be predisposed to the disease but has not yet experienced or exhibited symptoms of the disease.

[0308] Subjects in need of treatment using the compositions and methods of the present disclosure include, but are not limited to, subjects suffering from or at risk for heart failure. A subject "suffering from" heart failure is considered to have symptoms associated with any of the cardiac diseases described herein, or a confirmed diagnosis of any of them. A subject "at risk" for heart failure is considered to have one or more risk factors associated with any of the cardiac diseases described herein.

[0309] In some embodiments, the methods described herein are useful for treating cardiac diseases or disorders associated with reduced ejection fraction (HFrEF). In some embodiments, the methods described herein are useful for treating cardiac diseases or disorders associated with preserved ejection fraction (HFpEF).

[0310] In some embodiments, the methods described herein are useful for treating a cardiomyopathy. In some embodiments, the methods described herein are useful for treating dilated cardiomyopathy. In some embodiments, the subject is suffering from or at risk for a cardiomyopathy. In some embodiments, the cardiomyopathy is dilated cardiomyopathy (DCM). In some embodiments, the DCM is inherited DCM (e.g., DCM associated with a PLN mutation in the treated subject). In some embodiments, the methods described herein are useful for treating a PLN mutation-associated cardiomyopathy. In some embodiments, the DCM is non-inherited DCM. In some embodiments, the subject is suffering from or at risk for a myocardial infarction. In some embodiments, the myocardial infarction is a chronic myocardial infarction. In some embodiments, the myocardial infarction is an acute myocardial infarction.

[0311] Cardiomyopathy phenotypes can manifest in subjects through a number of molecular mechanisms. Transgenic animals have been developed to study the molecular pathophysiology of specific mechanisms and the efficacy of potential therapeutic and preventive strategies for cardiomyopathy phenotypes (Law et al. J Clin Med. 9:520 (2020)). These animal models can be used to evaluate aspects of the rAAV viral genome, rAAV virion, and their compositions described herein. MLP - / - Transgenic mouse models recapitulate the dilated cardiomyopathy phenotype, for example, by knocking out muscle LIM proteins, which are positive regulators of myogenic differentiation associated with the actin-based cytoskeleton. Absence of LIM proteins results in disrupted cytoskeletal structure and reduced Ca 2+ (Minamisawa et al. Cell. 99:313-22 (1999)). Artificial replacement with a phosphomimetic PLN transgene delivered by rAAV reduced DCM symptoms in animals, including improved ejection fraction (Iwanaga Y et al. J Clin Invest. 113:727-736 (2004). MLP - / -While the model reproduces the DCM phenotype in a general way, other transgenic mouse models of DCM are more appropriate for specific DCM phenotypes, such as those driven by mutations in the PLN gene. For example, a transgenic mouse model has been developed to reproduce the clinically observed PLN-R14Del mutation (Haghighi et al. Proc. Natl. Acad. Sci. USA 103:1388-1393 (2006)). Mouse models with different transgenic modifications to induce DCM phenotypes are not interchangeable for the purpose of evaluating the efficacy of a given therapeutic or preventive strategy, and each model is expected to provide different information about the translation of treatment.

[0312] In some embodiments, the subject in need of treatment has an inherited risk allele (i.e., a mutation) for a cardiac disease or disorder. The risk allele can be, for example, a mutation to the PLN gene. Mutations to the PLN gene can cause a dysfunctional inhibitory effect on SERCA activity. Clinically observable mutations in the PLN gene and protein include mutations in the PLN promoter, PLNL L39stop These include truncations resulting in mutants, aberrant R9C, R9L, and R9H mutations, PLN gene duplications, and a deletion of arginine 14 (R14del) in the regulatory domain of PLN. Each of these mutations has been directly associated with dilated, hypertrophic, or arrhythmogenic right ventricular cardiomyopathy (Table 8) (Landstrom et al. Am Heart J. 161:165-171 (2011), Lee et al. Cardiol Young. 24:953-954 (2014); Haghighi et al. J. Clin. Invest. 111:869-876 (2003); Schmitt et al. Science 299:1410-1413 (2003); Haghighi et al. Proc. Natl. Acad. Sci. USA 103:1388-1393 (2006); Medeiros A et al. Am. Heart J. 162:1088-1095 (2011)). [Table 8]

[0313] Various mutations in PLN have different mechanisms of inducing cardiomyopathy phenotype. For example, R9C mutation indirectly blocks phosphorylation of PLN by PKA, preventing the formation of monomeric PLN that can bind to SERCA. In some embodiments, the subject in need of treatment has a PLN promoter mutation. In some embodiments, the subject in need of treatment has a PLN promoter mutation. L39stop In some embodiments, the subject in need of treatment has a R9C mutation. In some embodiments, the subject in need of treatment has a R9L mutation. In some embodiments, the subject in need of treatment has a R9H mutation. In some embodiments, the subject in need of treatment has a PLN gene duplication. In some embodiments, the subject in need of treatment has a R14del mutation.

[0314] Mutations can be detected by many types of genetic analysis known in the art. Genetic analysis can be, for example, direct sequencing, fluorescent in situ hybridization assay, polymerase chain reaction-based assay, nucleotide microarray assay, or any other technique known in the art to determine sequence characteristics of polynucleotides sampled from subjects. For example, DNA was isolated from peripheral blood samples of patients diagnosed with either dilated cardiomyopathy (DCM) or arrhythmogenic right ventricular cardiomyopathy (ARVC). The coding region of the PLN gene in the isolated DNA was sequenced using BigDye Terminator DNA Sequencing Kit (version 2.0) on a 3730 Genetic analyzer (Applied Biosystems, Foster City, CA, USA). Both patients diagnosed with either DCM or ARVC carried the PLN R14del mutation (van der Zwaag et al. Eur J Heart Fail. 14:1199-1207 (2012)).

[0315] Methods for reducing, ameliorating, and preventing symptoms In some embodiments, the disclosure provides a method of reducing one or more symptoms of a cardiac disease or disorder in a subject, comprising administering any vector comprising an expression cassette described herein. In some embodiments, the symptoms are reduced compared to the symptoms of the cardiac disease or disorder prior to administration of a vector comprising an expression cassette described herein to the subject. In some embodiments, the disclosure provides a method of reducing one or more symptoms of a cardiac disease or disorder in a subject, comprising administering a rAAV virion described herein. In some embodiments, the symptoms are reduced compared to the symptoms of the cardiac disease or disorder prior to administration of the rAAV virion to the subject. In some embodiments, the cardiac disease or disorder is heart failure. In some embodiments, the cardiac disease or disorder is cardiomyopathy. In some embodiments, the cardiac disease or disorder is dilated cardiomyopathy. In some embodiments, the cardiac disease or disorder is myocardial infarction. In some embodiments, the cardiac disease or disorder is chronic myocardial infarction. In some embodiments, the cardiac disease or disorder is acute myocardial infarction.

[0316] In some embodiments, the disclosure provides a method of improving one or more symptoms of a cardiac disease or disorder in a subject, comprising administering a vector comprising an expression cassette described herein. In some embodiments, the symptoms are improved compared to the symptoms of the cardiac disease or disorder prior to administration of the vector to the subject. In some embodiments, the disclosure provides a method of improving one or more symptoms of a cardiac disease or disorder in a subject, comprising administering a rAAV virion described herein. In some embodiments, the symptoms are improved compared to the symptoms of the cardiac disease or disorder prior to administration of the rAAV virion to the subject. In some embodiments, the cardiac disease or disorder is heart failure. In some embodiments, the cardiac disease or disorder is cardiomyopathy. In some embodiments, the cardiac disease or disorder is dilated cardiomyopathy. In some embodiments, the cardiac disease or disorder is myocardial infarction. In some embodiments, the cardiac disease or disorder is chronic myocardial infarction. In some embodiments, the cardiac disease or disorder is acute myocardial infarction.

[0317] In some embodiments, the disclosure provides a method of preventing one or more symptoms of a cardiac disease or disorder in a subject, comprising administering a vector comprising an expression cassette described herein. In some embodiments, the disclosure provides a method of preventing one or more symptoms of a cardiac disease or disorder in a subject, comprising administering a rAAV virion described herein. In some embodiments, the symptoms are prevented in a subject believed to be at risk for a cardiac disease or disorder. In some embodiments, the cardiac disease or disorder is heart failure. In some embodiments, the cardiac disease or disorder is cardiomyopathy. In some embodiments, the cardiac disease or disorder is dilated cardiomyopathy. In some embodiments, the cardiac disease or disorder is myocardial infarction. In some embodiments, the cardiac disease or disorder is chronic myocardial infarction. In some embodiments, the cardiac disease or disorder is acute myocardial infarction.

[0318] In some embodiments, the symptoms are reduced compared to the symptoms of the cardiac disease or disorder prior to administration of the vector to the subject. In some embodiments, the symptoms are reduced compared to the symptoms of the cardiac disease or disorder prior to administration of the rAAV virion to the subject. In some embodiments, the cardiac disease or disorder is heart failure. In some embodiments, the cardiac disease or disorder is cardiomyopathy. In some embodiments, the cardiac disease or disorder is dilated cardiomyopathy. In some embodiments, the cardiac disease or disorder is myocardial infarction. In some embodiments, the cardiac disease or disorder is chronic myocardial infarction. In some embodiments, the cardiac disease or disorder is acute myocardial infarction.

[0319] As used herein, a "condition" includes, for example, any of the diagnostic criteria or symptoms associated with cardiac disease described herein. The severity and progression of conditions and diagnoses are determined by a medical professional qualified to perform the evaluation and analyze the results of such evaluation.

[0320] Common symptoms in subjects with or at risk of developing cardiac disease are fatigue, dyspnea, edema, chest pain, arrhythmia, blood clots, cardiac valve dysfunction, and heart murmur. In some embodiments, the subject experiences reduced symptoms associated with cardiac disease described herein after administration of a vector, rAAV virion, or composition of the present disclosure. In some embodiments, the improved symptoms are one or more of: enhanced contractility; reduced fatigue; reduced dyspnea; reduced edema; reduced chest pain; reduced arrhythmia; reduced blood clots; improved cardiac valve function; and reduced heart murmur. In some embodiments, the symptom is a change in 6-minute walk distance. In some embodiments, the symptom is determined by the Minnesota Heart Failure Questionnaire. In some embodiments, the symptom is an abnormal level of B-type natriuretic peptide (i.e., BNP, NT-proBNP). In some embodiments, the severity of the symptom is determined by measuring LV remodeling. Some embodiments of the methods described herein improve one or more measures of cardiac function. In some embodiments, the cardiac function measure comprises fractional shortening and / or left ventricular internal diameter (LVID). In some embodiments, the cardiac function measure comprises left ventricular end systolic volume (LVESV). In some embodiments, the improvement in cardiac function is ejection fraction. In some embodiments, the improvement in cardiac function is observed at weeks 2-12. In some embodiments, the method reduces cardiac remodeling. In some embodiments, the method counteracts a decrease in DWORF expression in a subject suffering from myocardial infarction.

[0321] Ejection fraction is a measure of the percentage of blood that leaves the heart with each contraction. Ejection fraction is determined using stroke volume (SV) and end diastolic volume (EDV) and is calculated as EF(%)=(SV / EDV)×100. Ejection fraction can be measured in a subject using imaging tests, including echocardiogram, cardiac catheterization, magnetic resonance imaging (MRI), computed tomography (CT), and / or nuclear medicine scan. A normal ejection fraction is between about 50% and about 75%. A borderline ejection fraction can range between about 41% and about 50%. A reduced ejection fraction is less than about 41%. A borderline or reduced ejection fraction can be used as a symptom in diagnosing a heart disease or disorder. It is understood that the cutoff values ​​between normal, borderline, and reduced ejection fractions are approximate and are ultimately determined by one skilled in the art, e.g., a cardiologist.

[0322] In some embodiments of the methods provided herein, it may be desirable to improve the ejection fraction. The ejection fraction may be considered to be improved if the percentage of the ejection fraction increases. In some embodiments, the ejection fraction

[0323] In some embodiments of the methods provided herein, it may be desirable to preserve ejection fraction, which can be used to prevent the onset of a cardiac disease or disorder in a subject at risk thereof, to prevent the progression of a cardiac disease or disorder, or to prevent the worsening of symptoms associated with a cardiac disease or disorder in a subject at risk of or suffering from a cardiac disease or disorder.

[0324] The present disclosure provides a method of improving ejection fraction in a subject at risk for or suffering from a cardiac disease or disorder. In some embodiments, the ejection fraction is improved (i.e., increased) in a subject after administration of a vector comprising an expression cassette described herein. In some embodiments, the ejection fraction is improved (i.e., increased) in a subject after administration of a vector or rAAV virion described herein. In some embodiments, the ejection fraction is improved about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 22 weeks, or about 24 weeks after administration of the vector or rAAV virion to the subject. In some embodiments, the ejection fraction is improved by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% following administration of the vector or rAAV virion to a subject.

[0325] The present disclosure provides a method of preserving ejection fraction in a subject at risk for or suffering from a cardiac disease or disorder. For example, the subject may maintain an ejection fraction that would otherwise be expected to be reduced in the absence of a vector or rAAV virion or pharmaceutical composition of the present disclosure. In some embodiments, the ejection fraction is preserved in the subject after administration of a vector or rAAV virion of the present disclosure. In some embodiments, the ejection fraction is preserved for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 22 weeks, or about 24 weeks after administration of a vector or rAAV virion to the subject. In some embodiments, the ejection fraction is preserved by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% following administration of the vector or rAAV virion to a subject.

[0326] Assessment of cardiac contractility can be used to assess acute and chronic forms of heart failure.Cardiac contractility can be monitored by using invasive hemodynamic monitoring, continuous ECG monitoring, central venous pressure, renal function, pulse oximetry, arterial pressure monitoring, pulmonary artery catheter, and / or transesophageal echocardiogram (Kuhn C, Werdan K.Surgical Treatment: Evidence-Based and Problem-Oriented.Munich: Zuckschwerdt;2001.Available at https: / / www.ncbi.nlm.nih.gov / books / NBK6895 / ).

[0327] Dyspnea and fatigue associated with the cardiac diseases described herein can be measured using questionnaires. The modified Pulmonary Function Status and Dyspnea Questionnaire (PFSDQ-M)10 (Huang et al. Am J Crit Care. 17: 436-442 (2008)) and the Minnesota Heart Failure Questionnaire (MLHFQ)11 (Bilbao et al. Health Qual Life Outcomes. 14: 23 (2016)) can be used to measure subjects with cardiac diseases described herein, for example. The questionnaires are self-administered and derive scores that are used to assess the severity of symptoms for dyspnea, fatigue, and other cardiac health-related symptoms.

[0328] Cardiomyopathy, myocardial infarction, and heart valve function may be evaluated using one or more of an exercise stress test, an electrocardiogram, an echocardiogram, a chest x-ray, a cardiac CT scan or an angiogram with cardiac catheterization, a cardiac MRI, B-type natriuretic peptide (BNP) levels in the blood, and / or genetic screening. Further tests may be required to diagnose a particular type of cardiomyopathy, myocardial infarction, or heart valve dysfunction.

[0329] In some aspects, administration of a vector comprising an expression cassette described herein to a subject results in an improvement in the subject's athletic performance (e.g., improvement in distance run and / or time to exhaustion). In some aspects, administration of a rAAV comprising an expression cassette described herein encoding DWORF to a subject results in an improvement in the subject's athletic performance (e.g., improvement in distance run and / or time to exhaustion).

[0330] Dilated cardiomyopathy (DCM) is a progressive disease of the myocardium characterized by left ventricular chamber enlargement and systolic dysfunction in the absence of chronic pressure and / or volume overload. DCM is primarily diagnosed using echocardiography.

[0331] Echocardiograms with PHE view in 2D / M mode are used to measure several parameters including ejection fraction, LVIDd / s, IVSd, LVPWd, and shortening fraction. These parameters are used to evaluate left ventricular cavity size, wall thickness, and radial function. Diagnostic criteria for DCM include LVIDd / s greater than 112% (2S.D) corrected for age and body surface area (BSA). A shortening fraction of less than 25% is the criterion for the diagnosis of DCM in the presence of dilated ventricles (Mathew et al. Echo Res Pract. 4:G1-G13 (2017)).

[0332] Qualitative assessment of left and right ventricular structure and function with particular reference to radial and longitudinal function and regional wall motion abnormalities is assessed by echocardiography in the apical four-chamber (A4C) view in 2D mode. Ejection fraction (EF) can be estimated, for example, using the biplane Simpsons method. An EF of less than 45% is a diagnostic criterion for DCM in the presence of dilated ventricles (Mathew et al. Echo Res Pract. 4:G1-G13 (2017)).

[0333] Administration In some embodiments, the vectors and compositions of the present disclosure can be administered to a subject in need thereof by systemic application, for example, by intravenous, intraarterial, or intraperitoneal delivery. In some embodiments, the rAAV virions and compositions of the present disclosure can be administered to a subject in need thereof by systemic application, for example, by intravenous, intraarterial, or intraperitoneal delivery of the vector similar to that shown in animal models (Katz et al., Gene Ther 19:659-669 (2012)). In some embodiments, the vectors, rAAV virions, and compositions of the present disclosure treat or prevent heart failure. In some embodiments, cardiomyopathy, the vector is administered systemically. In some embodiments, the rAAV virions are administered by intravenous or intracoronary injection.

[0334] The present disclosure provides a method for expressing a polypeptide in a cell in vitro, ex vivo, or in vivo. In some embodiments, the present disclosure provides a method for expressing a DWORF polypeptide in a cell in vitro, ex vivo, or in vivo. The method includes, for example, exposing a target cell to a vector, rAAV virion, or pharmaceutical composition described herein. The target cell can be, for example, but not limited to, a cardiac cell, a muscle cell, an induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM), and / or a cardiomyocyte. In some embodiments, the method for expressing a polypeptide (e.g., a DWORF polypeptide) in a cell includes transfecting or transducing (alternatively referred to as "infecting") a target cell or a population of target cells with a vector described herein. In some embodiments, the method for expressing a polypeptide (e.g., a DWORF polypeptide) in a cell includes transducing (alternatively referred to as "infecting") a target cell or a population of target cells with a rAAV virion or pharmaceutical composition described herein. In some embodiments, rAAV transduces cardiac cells. In some embodiments, rAAV transduces cardiomyocytes. In some embodiments, rAAV transduces induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs).

[0335] In some embodiments, vector transfection or transduction increases polypeptide expression in the subject's heart. In some embodiments, rAAV transduction increases DWORF polypeptide expression in the subject's heart. "Increased polypeptide expression" typically refers to at least 5%, 10%, 15%, 20% or more expression compared to a control subject or tissue not treated with the vector. "Increased DWORF polypeptide expression" typically refers to at least 5%, 10%, 15%, 20% or more expression compared to a control subject or tissue not treated with the vector. In some embodiments, detectable expression means 1.5-fold, 2-fold, 2.5-fold, or 3-fold greater expression than a no vector control. Expression can be assessed by Western blot, or enzyme-linked immunosorbent assay (ELISA), or other methods known in the art, as described in the Examples that follow. In some cases, expression is quantitatively measured using a standard curve. A standard curve can be generated using purified proteins, such as purified DWORF polypeptides, as described in the Examples or as known in the art. Alternatively, expression of a therapeutic gene product can be assessed by quantification of the corresponding mRNA. In some embodiments, the method results in expression of a polypeptide (eg, a DWORF polypeptide) in the heart of the subject.

[0336] In some embodiments, the method does not result in detectable expression of the polypeptide in muscle of the subject except the heart, in the liver of the subject, and / or in cardiac fibroblasts, hi some embodiments, the method results in expression of the polypeptide in cardiomyocytes.

[0337] In some embodiments, the method does not cause detectable expression of DWORF polypeptide in muscle of the subject, except heart.In some embodiments, the method does not cause detectable expression of DWORF polypeptide in liver of the subject.In some embodiments, the method causes expression of DWORF polypeptide in cardiomyocyte.In some embodiments, the method does not cause detectable expression of DWORF polypeptide in cardiac fibroblast.

[0338] In some embodiments, increased polypeptide expression in cardiac tissue is greater than or equal to 3×10 vector genomes per kilogram (kg) of subject body weight. 14 vg / kg or less, 2×10 14 vg / kg or less, 1×10 14 vg / kg or less, 9 x 10 13 vg / kg or less, 8 x 10 13 vg / kg or less, 7×10 13 vg / kg or less, 6×10 13 vg / kg or less, 5×10 13 vg / kg or less, 4×10 13 vg / kg or less, 3×10 13 vg / kg or less, 2×10 13 vg / kg or less, or 1×10 13 This occurs at doses of vg / kg or less.

[0339] In some embodiments, increased DWORF expression in cardiac tissue is greater than or equal to 3×10 vector genomes per kilogram (kg) of subject body weight. 14 vg / kg or less, 2×10 14 vg / kg or less, 1×10 14 vg / kg or less, 9 x 10 13 vg / kg or less, 8 x 10 13 vg / kg or less, 7×10 13 vg / kg or less, 6×10 13 vg / kg or less, 5×10 13 vg / kg or less, 4×10 13vg / kg or less, 3 × 10 13 vg / kg or less, 2×10 13 vg / kg or less, or 1×10 13 occurs at doses of ≤ 500 mg / kg.

[0340] Pharmaceutical Compositions and Kits The vectors of the present disclosure are generally delivered to a subject as a pharmaceutical composition. In some embodiments, the rAAV virions of the present disclosure are delivered to a subject as a pharmaceutical composition. The pharmaceutical composition comprises a pharma- ceutical acceptable solvent (e.g., water, etc.) and one or more excipients. In some embodiments, the pharmaceutical composition comprises a buffer at about neutral pH (pH 5, 6, 7, 8, or 9). In some embodiments, the pharmaceutical composition comprises phosphate buffered saline (e.g., PBS at a pH of about 7). The pharmaceutical composition may include a pharma- ceutical acceptable salt. The concentration of the salt may be selected to ensure that the pharmaceutical composition is isotonic with respect to the target tissue, or is nearly isotonic with respect to the target tissue.

[0341] In various embodiments, the compositions described herein contain pharma- ceutically acceptable solvents (e.g., carriers, diluents, and excipients) for formulations that can be injected. These may be, in particular, isotonic sterile saline solutions (monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride, or magnesium chloride, and the like, or mixtures of such salts), or may be dry, in particular lyophilized compositions, which, upon addition, allow the constitution of an injectable solution, depending on the case of sterile water or saline. Illustrative pharmaceutical forms suitable for injection use include, for example, sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.

[0342] In various embodiments, the pharmaceutical compositions of the present disclosure comprise about 1×10 8 Genome copies / milliliter (GC / mL), approximately 5 × 10 8 GC / mL, approximately 1×10 9 GC / mL, approximately 5×109 GC / mL, approximately 1×10 10 GC / mL, approximately 5×10 10 GC / mL, approximately 1×10 11 GC / mL, approximately 5×10 11 GC / mL, approximately 1×10 12 GC / mL, approximately 5×10 12 GC / mL, approximately 5×10 13 GC / mL, approximately 1×10 14 GC / mL, or approximately 5 x 10 14 GC / mL of viral vector (e.g., rAAV virion).

[0343] In various embodiments, the pharmaceutical compositions of the present disclosure comprise about 1×10 8 Viral genomes / milliliter (vg / mL), 5 x 10 8 vg / mL, approximately 1×10 9 vg / mL, approximately 5×10 9 vg / mL, approximately 1×10 10 vg / mL, approximately 5×10 10 vg / mL, approximately 1×10 11 vg / mL, approximately 5×10 11 vg / mL, approximately 1×10 12 vg / mL, approximately 5×10 12 vg / mL, approximately 5×10 13 vg / mL, approximately 1×10 14 vg / mL, or approximately 5 × 10 14 vg / mL of viral vector (e.g., rAAV virion).

[0344] In some embodiments, the pharmaceutical compositions of the present disclosure comprise a volume of about 1 mL, 5 mL, 10 mL, about 20 mL, about 25 mL, about 30 mL, about 35 mL, about 40 mL, about 45 mL, about 50 mL, about 55 mL, about 60 mL, 65 mL, about 70 mL, about 75 mL, about 80 mL, about 85 mL, about 90 mL, about 95 mL, about 100 mL, about 105 mL, about 110 mL, about 115 mL, or about 120 mL. L, about 120 mL, about 125 mL, about 130 mL, about 135 mL, about 140 mL, about 145 mL, about 150 mL, about 155 mL, about 160 mL, about 165 mL, about 170 mL, about 175 mL, about 180 mL, about 185 mL, about 190 mL, about 200 mL, about 205 mL, about 210 mL, about 215 mL, or about 220 mL.

[0345] Genome copies per milliliter can be determined by quantitative polymerase chain reaction (qPCR) using a standard curve generated with a reference sample with a known concentration of the viral polynucleotide genome. For AAV, the reference sample is often the transfer plasmid used to generate rAAV virions, although other reference samples may be used.

[0346] Alternatively, or additionally, the concentration of the viral vector can be determined by measuring the titer of the vector in the cell line. Viral titers are generally expressed as viral particles per unit volume (vp) (e.g., vp / mL). In various embodiments, the pharmaceutical compositions of the present disclosure contain a concentration of about 1×10 8 Virus particles / milliliter (vp / mL), approximately 5 x 10 8 vp / mL, approximately 1×10 9 vp / mL, approximately 5×10 9 vp / mL, approximately 1×10 10 vp / mL, approximately 5×10 10 vp / mL, approximately 1×10 11 vp / mL, approximately 5×10 11 vp / mL, approximately 1×10 12 vp / mL, approximately 5×10 12 vp / mL, approximately 5×10 13 vp / mL, or approximately 1 × 10 14vp / mL, or approximately 5 × 10 14 viral vectors (e.g., rAAV virions).

[0347] In one embodiment, the present disclosure provides a kit comprising a container housing a pharmaceutical composition described herein.

[0348] Numbered embodiment I of the present invention Embodiment 1: A recombinant adeno-associated virus (rAAV) virion comprising a viral genome comprising an expression cassette comprising a polynucleotide sequence encoding a capsid protein and a dwarf open reading frame (DWORF) polypeptide operably linked to a promoter, wherein the expression cassette is flanked by inverted terminal repeats.

[0349] Embodiment 2: The rAAV virion of embodiment 1, wherein the DWORF polypeptide shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with a sequence selected from SEQ ID NOs: 1, 3, 4, 7, 9, 23, and 43.

[0350] Embodiment 3: The rAAV virion of embodiment 1, wherein the DWORF polypeptide is selected from SEQ ID NOs: 1, 3, 4, 7, 9, 23, and 43.

[0351] Embodiment 4: The rAAV virion of embodiment 1 or 2, wherein the promoter is a chicken cTnT promoter.

[0352] Embodiment 5: The rAAV virion of embodiment 4, wherein the chicken cTnT promoter shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:11.

[0353] Embodiment 6: The rAAV virion of embodiment 4, wherein the chicken cTnT promoter comprises SEQ ID NO:11.

[0354] Embodiment 7: The rAAV virion of embodiment 1 or 2, wherein the promoter is a human cTnT promoter.

[0355] Embodiment 8: The rAAV virion of embodiment 7, wherein the human cTnT promoter shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:12 or SEQ ID NO:13.

[0356] Embodiment 9: The rAAV virion of embodiment 7, wherein the human cTnT promoter comprises SEQ ID NO:12 or SEQ ID NO:13.

[0357] Embodiment 10: The rAAV virion of any one of embodiments 1-9, wherein the expression cassette further comprises one or more enhancers.

[0358] Embodiment 11: The rAAV virion of embodiment 10, wherein the enhancer, or the one or more enhancers, is selected from an ACTC1 cardiac enhancer and an αMHC enhancer.

[0359] Embodiment 12: The rAAV virion of embodiment 11, wherein the ACTC1 cardiac enhancer shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with sequence number 78.

[0360] Embodiment 13: The rAAV virion of embodiment 11, wherein the ACTC1 cardiac enhancer comprises sequence number 78.

[0361] Embodiment 14: The rAAV virion of embodiment 11, wherein the αMHC enhancer shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:79.

[0362] Embodiment 15: The rAAV virion of embodiment 11, wherein the αMHC enhancer comprises sequence number 79.

[0363] Embodiment 16: 16. The rAAV virion of any one of embodiments 1-15, wherein the expression cassette further comprises an intron.

[0364] Embodiment 17: The rAAV virion of embodiment 16, wherein the intron is selected from a CMV intron and a chimeric intron.

[0365] Embodiment 18: The rAAV virion of embodiment 17, wherein the CMV intron shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:80.

[0366] Embodiment 19: The rAAV virion of embodiment 17, wherein the CMV intron comprises SEQ ID NO:80.

[0367] Embodiment 20: The rAAV virion of embodiment 17, wherein the chimeric intron shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:81.

[0368] Embodiment 21: The rAAV virion of embodiment 17, wherein the chimeric intron comprises SEQ ID NO:81.

[0369] Embodiment 22: The rAAV virion of any one of embodiments 1-21, wherein the expression cassette further comprises a WPRE sequence.

[0370] Embodiment 23: The rAAV virion of embodiment 22, wherein the WPRE sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:26.

[0371] Embodiment 24: The rAAV virion of embodiment 22, wherein the WPRE sequence comprises sequence number 26.

[0372] Embodiment 25: The rAAV virion of any one of embodiments 1-24, wherein the expression cassette further comprises a polyadenylation sequence.

[0373] Embodiment 26: The rAAV virion of embodiment 25, wherein the polyadenylation sequence is selected from the BGH polyadenylation sequence and the SV40 polyadenylation sequence.

[0374] Embodiment 27: The rAAV virion of embodiment 26, wherein the BGH polyadenylation sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:27.

[0375] Embodiment 28: The rAAV virion of embodiment 26, wherein the BGH polyadenylation sequence comprises SEQ ID NO:27.

[0376] Embodiment 29: The rAAV virion of embodiment 26, wherein the SV40 polyadenylation sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:28.

[0377] Embodiment 30: The rAAV virion of embodiment 26, wherein the SV40 polyadenylation sequence comprises SEQ ID NO:28.

[0378] Embodiment 31: The rAAV virion of any one of embodiments 1-30, wherein the expression cassette is flanked by ITRs.

[0379] Embodiment 32: The rAAV virion of embodiment 31, wherein the ITRs share at least 90%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO:14 and SEQ ID NO:15.

[0380] Embodiment 33: The rAAV virion of embodiment 31, wherein the ITRs comprise one or more of SEQ ID NO:14 and SEQ ID NO:15.

[0381] Embodiment 34: The rAAV virion of any one of embodiments 1-33, wherein the expression cassette comprises a single promoter.

[0382] Embodiment 35: The rAAV virion of any one of embodiments 1-33, wherein the expression cassette comprises two promoters.

[0383] Embodiment 36: 36. The rAAV virion of any one of embodiments 1 to 35, wherein the expression cassette comprises a single copy of a sequence encoding a DWORF polypeptide.

[0384] Embodiment 37: 36. The rAAV virion of any one of embodiments 1 to 35, wherein the expression cassette comprises two copies of a sequence encoding a DWORF polypeptide.

[0385] Embodiment 38: The rAAV virion of any one of embodiments 1-37, wherein the expression cassette comprises one, two, three, or four enhancers.

[0386] Embodiment 39: The rAAV virion of any one of embodiments 1-38, wherein the expression cassette comprises one or two introns.

[0387] Embodiment 40: The rAAV virion of any one of embodiments 1 to 39, wherein the expression cassette comprises one or two WPRE sequences.

[0388] Embodiment 41: The rAAV virion of any one of embodiments 1 to 40, wherein the expression cassette comprises one or two polyadenylation sequences.

[0389] Embodiment 42: 42. The rAAV virion of any one of embodiments 1-41, wherein the expression cassette comprises about 3.2 kb, about, about 3.3 kb, about 3.4 kb, about 3.5 kb, about 3.6 kb, about 3.7 kb, or less.

[0390] Embodiment 43: 42. The rAAV virion of any one of embodiments 1-41, wherein the expression cassette comprises about 1.9 kb, about 2.1 kb, about 2.2 kb, about 2.3 kb, about 2.4 kb, about 2.5 kb, about 2.6 kb, about 2.7 kb, about 2.8 kb, about 2.9 kb, about 3.0 kb, about 3.1 kb, about 3.2 kb, or more.

[0391] Embodiment 44: The rAAV virion of embodiment 1, wherein the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 20-24 or SEQ ID NOs: 45-75.

[0392] Embodiment 45: The rAAV virion of embodiment 1, wherein the expression cassette comprises any one of SEQ ID NOs: 20 to 24 or SEQ ID NOs: 45 to 75.

[0393] Embodiment 46: The rAAV virion of embodiment 1, wherein the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:61.

[0394] Embodiment 47: The rAAV virion of embodiment 1, wherein the expression cassette comprises SEQ ID NO:61.

[0395] Embodiment 48: The rAAV virion of embodiment 1, wherein the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:62.

[0396] Embodiment 49: The rAAV virion of embodiment 1, wherein the expression cassette comprises SEQ ID NO:62.

[0397] Embodiment 50: The rAAV virion of embodiment 1, wherein the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:63.

[0398] Embodiment 51: The rAAV virion of embodiment 1, wherein the expression cassette comprises SEQ ID NO:63.

[0399] Embodiment 52: The rAAV virion of any one of embodiments 1-51, wherein the capsid protein shares at least 98%, at least 99%, or 100% identity with the AAV9 capsid protein (SEQ ID NO: 143).

[0400] Embodiment 53: The rAAV virion of any one of embodiments 1-51, wherein the capsid protein shares at least 98%, at least 99%, or 100% identity with the AAV5 capsid protein (SEQ ID NO: 144).

[0401] Embodiment 54: The rAAV virion of any one of embodiments 1-51, wherein the capsid protein is a chimeric capsid protein.

[0402] Embodiment 55: The rAAV virion of any one of embodiments 1-51, wherein the capsid protein is an AAV5 / AAV9 chimeric capsid protein.

[0403] Embodiment 56: The rAAV virion of any one of embodiments 1-51, wherein the capsid protein is selected from any one of SEQ ID NOs: 145-200.

[0404] Embodiment 57: An expression cassette comprising a polynucleotide sequence encoding a dwarf open reading frame (DWORF) polypeptide operably linked to a promoter.

[0405] Embodiment 58: 58. The expression cassette of embodiment 57, wherein the DWORF polypeptide is selected from SEQ ID NOs: 1, 3, 4, 7, 9, 23, and 43.

[0406] Embodiment 59: 59. The expression cassette of embodiment 57 or 58, wherein the promoter is a chicken cTnT promoter.

[0407] Embodiment 60: 60. The expression cassette of embodiment 59, wherein the chicken cTnT promoter shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:11.

[0408] Embodiment 61: 60. The expression cassette of embodiment 59, wherein the chicken cTnT promoter comprises SEQ ID NO:11.

[0409] Embodiment 62: 59. The expression cassette of embodiment 57 or 58, wherein the promoter is a human cTnT promoter.

[0410] Embodiment 63: 63. The expression cassette of embodiment 62, wherein the human cTnT promoter shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:12 or SEQ ID NO:13.

[0411] Embodiment 64: 63. The expression cassette of embodiment 62, wherein the human cTnT promoter comprises SEQ ID NO:12 or SEQ ID NO:13.

[0412] Embodiment 65: The expression cassette of any one of embodiments 57 to 64, wherein the expression cassette further comprises one or more enhancers.

[0413] Embodiment 66: 66. The expression cassette of embodiment 65, wherein the one or more enhancers are selected from an ACTC1 cardiac enhancer and an αMHC enhancer.

[0414] Embodiment 67: 67. The expression cassette of embodiment 66, wherein the ACTC1 cardiac enhancer shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:78.

[0415] Embodiment 68: 67. The expression cassette of embodiment 66, wherein the ACTC1 cardiac enhancer comprises sequence number 78.

[0416] 69. 67. The expression cassette of embodiment 66, wherein the αMHC enhancer shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:79.

[0417] Embodiment 70: 67. The expression cassette of embodiment 66, wherein the αMHC enhancer comprises SEQ ID NO:79.

[0418] Embodiment 71: The expression cassette of any one of embodiments 57 to 70, wherein the expression cassette further comprises an intron.

[0419] Embodiment 72: 71. The expression cassette of any one of embodiments 57-70, wherein the intron is selected from a CMV intron and a chimeric intron.

[0420] Embodiment 73: 73. The expression cassette of embodiment 72, wherein the CMV intron shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:80.

[0421] EMBODIMENT 74: 73. The expression cassette of embodiment 72, wherein the CMV intron comprises SEQ ID NO:80.

[0422] Embodiment 75: 73. The expression cassette of embodiment 72, wherein the chimeric intron shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:81.

[0423] EMBODIMENT 76: 73. The expression cassette of embodiment 72, wherein the chimeric intron comprises SEQ ID NO:81.

[0424] EMBODIMENT 77: The expression cassette of any one of embodiments 57 to 76, wherein the expression cassette further comprises a WPRE sequence.

[0425] EMBODIMENT 78: 78. The expression cassette of embodiment 77, wherein the WPRE sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:26.

[0426] EMBODIMENT 79: 78. The expression cassette of embodiment 77, wherein the WPRE sequence comprises SEQ ID NO:26.

[0427] Embodiment 80: 80. The expression cassette of any one of embodiments 57 to 79, wherein the expression cassette further comprises a polyadenylation sequence.

[0428] Embodiment 81: 81. The expression cassette of embodiment 80, wherein the polyadenylation sequence is selected from the BGH polyadenylation sequence and the SV40 polyadenylation sequence.

[0429] Embodiment 82: 82. The expression cassette of embodiment 81, wherein the BGH polyadenylation sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:27.

[0430] Embodiment 83: 82. The expression cassette of embodiment 81, wherein the BGH polyadenylation sequence comprises SEQ ID NO:27.

[0431] EMBODIMENT 84: 82. The expression cassette of embodiment 81, wherein the SV40 polyadenylation sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:28.

[0432] Embodiment 85: 82. The expression cassette of embodiment 81, wherein the SV40 polyadenylation sequence comprises SEQ ID NO:28.

[0433] EMBODIMENT 86: 86. The expression cassette of any one of embodiments 57 to 85, wherein the expression cassette is flanked by ITRs.

[0434] Embodiment 87: 87. The expression cassette of embodiment 86, wherein the ITRs share at least 90%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO:14 and SEQ ID NO:15.

[0435] EMBODIMENT 88: 87. The expression cassette of embodiment 86, wherein the ITRs comprise one or more of SEQ ID NO:14 and SEQ ID NO:15.

[0436] EMBODIMENT 89: 89. The expression cassette of any one of embodiments 57 to 88, comprising a single promoter.

[0437] Embodiment 90: 89. The expression cassette of any one of embodiments 57 to 88, comprising two promoters.

[0438] EMBODIMENT 91: 91. The expression cassette of any one of embodiments 57 to 90, comprising a single copy of the sequence encoding the DWORF polypeptide.

[0439] EMBODIMENT 92: 91. The expression cassette of any one of embodiments 57 to 90, comprising two copies of the sequence encoding the DWORF polypeptide.

[0440] EMBODIMENT 93: 93. The expression cassette of any one of embodiments 57 to 92, comprising one, two, three, or four enhancers.

[0441] EMBODIMENT 94: 94. The expression cassette of any one of embodiments 57 to 93, comprising one or two introns.

[0442] EMBODIMENT 95: The expression cassette of any one of embodiments 57 to 94, comprising one or two WPRE sequences.

[0443] EMBODIMENT 96: 96. The expression cassette of any one of embodiments 57 to 95, comprising one or two polyadenylation sequences.

[0444] EMBODIMENT 97: 97. The expression cassette of any one of embodiments 57-96, comprising about 3.2 kb, about, about 3.3 kb, about 3.4 kb, about 3.5 kb, about 3.6 kb, about 3.7 kb, or less.

[0445] EMBODIMENT 98: 97. The expression cassette of any one of embodiments 57-96, comprising about 1.9 kb, about 2.1 kb, about 2.2 kb, about 2.3 kb, about 2.4 kb, about 2.5 kb, about 2.6 kb, about 2.7 kb, about 2.8 kb, about 2.9 kb, about 3.0 kb, about 3.1 kb, about 3.2 kb, or more.

[0446] EMBODIMENT 99: 2. The expression cassette of embodiment 1, comprising a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NOs: 20-24 or 45-75.

[0447] EMBODIMENT 100: 58. The expression cassette of embodiment 57, comprising any one of SEQ ID NOs: 20-24 or 45-75.

[0448] EMBODIMENT 101: 58. The expression cassette of embodiment 57, comprising a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:61.

[0449] EMBODIMENT 102: 58. The expression cassette of embodiment 57, comprising SEQ ID NO:61.

[0450] EMBODIMENT 103: 58. The expression cassette of embodiment 57, comprising a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:62.

[0451] EMBODIMENT 104: 58. The expression cassette of embodiment 57, comprising SEQ ID NO:62.

[0452] EMBODIMENT 105: 58. The expression cassette of embodiment 57, comprising a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:63.

[0453] EMBODIMENT 106: 58. The expression cassette of embodiment 57, comprising SEQ ID NO:63.

[0454] EMBODIMENT 107: 99. The expression cassette of any one of embodiments 57 to 98, further comprising a 5' inverted terminal repeat and a 3' inverted terminal repeat.

[0455] EMBODIMENT 108: A pharmaceutical composition comprising the rAAV virion of any one of embodiments 1 to 56 and a pharma- ceutical acceptable diluent.

[0456] EMBODIMENT 109: A kit comprising the pharmaceutical composition of embodiment 108.

[0457] EMBODIMENT 110: A method for increasing DWORF expression in a cell, comprising contacting the cell with a rAAV virion of any one of embodiments 1 to 56 or the composition of embodiment 108.

[0458] EMBODIMENT 111: The method of embodiment 110, wherein the cell is a cardiac cell.

[0459] EMBODIMENT 112: 112. The method of embodiment 111, wherein the cardiac cells are cardiomyocytes.

[0460] EMBODIMENT 113: The method of any one of embodiments 110-112, wherein DWORF expression is increased between about 1.5-fold and 150-fold.

[0461] EMBODIMENT 114: The method of any one of embodiments 110-113, wherein the contacting is in vitro.

[0462] EMBODIMENT 115: The method of any one of embodiments 110-113, wherein the contacting is in vivo.

[0463] EMBODIMENT 116: A method for increasing DWORF expression in a tissue, comprising contacting the tissue with a rAAV virion of any one of embodiments 1 to 56 or the composition of embodiment 108.

[0464] EMBODIMENT 117: The method of embodiment 116, wherein the tissue is cardiac tissue.

[0465] EMBODIMENT 118: The method of embodiment 116 or 117, wherein DWORF expression is increased between about 1.5-fold and 150-fold.

[0466] EMBODIMENT 119: The method of any one of embodiments 116-118, wherein the contacting is in vitro.

[0467] EMBODIMENT 120: The method of embodiment 116 or 118, wherein the contacting is in vivo.

[0468] EMBODIMENT 121: A method for increasing DWORF expression in an organ, comprising contacting the organ with a rAAV virion of any one of embodiments 1 to 56 or the composition of embodiment 108.

[0469] EMBODIMENT 122: The method of embodiment 121, wherein the organ is the heart.

[0470] EMBODIMENT 123: The method of embodiment 122, wherein the heart is diseased or at risk of disease.

[0471] EMBODIMENT 124: The method of embodiment 122 or embodiment 123, wherein the heart has a reduced or borderline ejection fraction.

[0472] EMBODIMENT 125: The method of embodiment 122 or embodiment 123, wherein the heart has a normal ejection fraction.

[0473] EMBODIMENT 126: The method of any one of embodiments 122-125, wherein the heart comprises a genetic mutation associated with heart disease.

[0474] EMBODIMENT 127: The method of embodiment 126, wherein the genetic mutation is a PLN mutation.

[0475] EMBODIMENT 128: The method of any one of embodiments 121-127, wherein the heart has low or undetectable DWORF expression compared to a healthy heart.

[0476] EMBODIMENT 129: The method of any one of embodiments 121-128, wherein DWORF expression is increased between about 1.5-fold and 150-fold.

[0477] EMBODIMENT 130: The method of any one of embodiments 121-129, wherein the contacting is in vitro.

[0478] EMBODIMENT 131: The method of any one of embodiments 121-129, wherein the contacting is in vivo.

[0479] EMBODIMENT 132: A method for increasing DWORF expression in a subject, comprising administering to the subject a rAAV virion of any one of embodiments 1 to 56 or the composition of embodiment 108.

[0480] EMBODIMENT 133: The method of embodiment 132, wherein the subject is an animal.

[0481] EMBODIMENT 134: The method of embodiment 132, wherein the subject is a human.

[0482] EMBODIMENT 135: The method of any one of embodiments 132-134, wherein DWORF expression is increased in the heart of the subject.

[0483] EMBODIMENT 136: The method of any one of embodiments 132-135, wherein the subject has or is at risk for heart disease.

[0484] EMBODIMENT 137: The method of any one of embodiments 132-136, wherein the subject has a borderline or reduced ejection fraction.

[0485] EMBODIMENT 138: The method of any one of embodiments 132-136, wherein the subject has a normal ejection fraction.

[0486] EMBODIMENT 139: The method of any one of embodiments 132-138, wherein the subject has a genetic mutation associated with heart disease.

[0487] Embodiment 140: 140. The method of embodiment 139, wherein the genetic mutation is a PLN mutation.

[0488] EMBODIMENT 141: The method of any one of embodiments 132-140, wherein the subject has low or undetectable levels of DWORF expression compared to healthy subjects.

[0489] EMBODIMENT 142: A method for treating a cardiac disease or disorder in a subject in need thereof, comprising administering to the subject a rAAV virion of any one of embodiments 1 to 56 or the composition of embodiment 108.

[0490] EMBODIMENT 143: The method of embodiment 142, wherein the subject has a cardiac disease or disorder.

[0491] EMBODIMENT 144: The method of embodiment 142, wherein the subject is at risk of developing a cardiac disease or disorder.

[0492] EMBODIMENT 145: The method of any one of embodiments 142-144, wherein the cardiac disease or disorder is cardiomyopathy.

[0493] EMBODIMENT 146: The method of any one of embodiments 142-144, wherein the cardiac disease or disorder is dilated cardiomyopathy.

[0494] EMBODIMENT 147: The method of any one of embodiments 142-144, wherein the cardiac disease or disorder is myocardial infarction.

[0495] EMBODIMENT 148: The method of any one of embodiments 142-144, wherein the cardiac disease or disorder is chronic myocardial infarction.

[0496] EMBODIMENT 149: The method of any one of embodiments 142-144, wherein the cardiac disease or disorder is acute myocardial infarction.

[0497] EMBODIMENT 150: The method of any one of embodiments 142-149, wherein the subject has an inherited risk allele for a cardiac disease or disorder.

[0498] EMBODIMENT 151: The method of any one of embodiments 142-150, wherein the inherited risk allele comprises a mutation to the PLN gene.

[0499] EMBODIMENT 152: The method of embodiment 151, wherein the mutation to the PLN gene is a PLN promoter mutation.

[0500] EMBODIMENT 153: Mutations in the PLN gene cause PLN L39stop The method of embodiment 151, wherein the mutation is

[0501] EMBODIMENT 154: The method of embodiment 151, wherein the mutation to the PLN gene is an RC9 mutation.

[0502] EMBODIMENT 155: The method of embodiment 151, wherein the mutation to the PLN gene is an R9L mutation.

[0503] EMBODIMENT 156: The method of embodiment 151, wherein the mutation to the PLN gene is a PLN gene duplication.

[0504] EMBODIMENT 157: The method of embodiment 151, wherein the mutation to the PLN gene is an R14del mutation.

[0505] EMBODIMENT 158: The method of any one of embodiments 142-157, wherein the cardiac disease or disorder is associated with reduced ejection fraction (HFrEF).

[0506] EMBODIMENT 159: The method of any one of embodiments 142-157, wherein the impaired cardiac disease is accompanied by preserved ejection fraction (HFpEF).

[0507] EMBODIMENT 160: The method of any one of embodiments 142-159, wherein the method causes expression of the DWORF polypeptide in the heart of the subject.

[0508] EMBODIMENT 161: The method of any one of embodiments 142-160, wherein the method causes expression of the DWORF polypeptide in cardiomyocytes.

[0509] EMBODIMENT 162: The method of any one of embodiments 142-161, wherein the method does not result in detectable expression of the DWORF polypeptide in muscles of the subject, except for the heart.

[0510] EMBODIMENT 163: The method of any one of embodiments 142-162, wherein the method does not result in detectable expression of the DWORF polypeptide in the liver of the subject.

[0511] EMBODIMENT 164: The method of any one of embodiments 142-163, wherein the method does not result in detectable expression of DWORF polypeptide in cardiac fibroblasts.

[0512] EMBODIMENT 165: The method of any one of embodiments 142 to 164, wherein the method improves one or more measures of cardiac function, optionally fractional shortening and / or left ventricular internal diameter (LVID).

[0513] EMBODIMENT 166: The method of any one of embodiments 142-165, wherein an improvement in cardiac function is observed at 2 weeks to 16 weeks.

[0514] EMBODIMENT 167: The method of any one of embodiments 142-166, wherein the method reduces cardiac remodeling.

[0515] EMBODIMENT 168: The method of any one of embodiments 142-166, wherein the method combats a decrease in DWORF expression in a subject suffering from or at risk for cardiac disease.

[0516] EMBODIMENT 169: The method of any one of embodiments 142-168, wherein the rAAV virion is administered by systemic administration.

[0517] EMBODIMENT 170: The method of embodiment 169, wherein the systemic administration is selected from intravenous injection or intracoronary injection.

[0518] EMBODIMENT 171: The method of embodiment 169 or 170, wherein the rAAV is administered as a unit dose.

[0519] EMBODIMENT 172: The unit dose is approximately 3 x 10 14 vg / kg or less, approximately 2×10 14 vg / kg or less, approximately 1×10 14 vg / kg or less, approximately 9 × 10 13 vg / kg or less, approximately 8 × 10 13 vg / kg or less, approximately 7 × 10 13 vg / kg or less, approximately 6×10 13vg / kg or less, approximately 5 × 10 13 vg / kg or less, approximately 4 × 10 13 vg / kg or less, approximately 3 × 10 13 vg / kg or less, approximately 2×10 13 vg / kg or less, or approximately 1 × 10 13 172. The method of embodiment 171, comprising 17.5 vg / kg or less.

[0520] EMBODIMENT 173: A method for alleviating one or more symptoms of a cardiac disease or disorder in a subject in need thereof, comprising administering a rAAV virion of any one of embodiments 1 to 56 or the composition of embodiment 108.

[0521] EMBODIMENT 174: A method for ameliorating one or more symptoms of a cardiac disease or disorder in a subject in need thereof, comprising administering a rAAV virion of any one of embodiments 1 to 56 or the composition of embodiment 108.

[0522] EMBODIMENT 175: A method for preventing one or more symptoms of a cardiac disease or disorder in a subject in need thereof, comprising administering a rAAV virion of any one of embodiments 1 to 56 or the composition of embodiment 108.

[0523] EMBODIMENT 176: 1. An expression cassette comprising a polynucleotide comprising a 5' to 3' arrangement of elements, the elements being: i. one or more promoters; ii. optionally, one or more enhancers; iii. optionally, one or more introns; iv. one or more transgenes; v. optionally, one or more WPRE sequences; and vi. An expression cassette, optionally including one or more polyadenylation sequences, p(A).

[0524] EMBODIMENT 177: 177. The expression cassette of embodiment 176, wherein the 5' to 3' arrangement of elements is selected from the following: i.5'-promoter-intron-transgene-WPRE-p(A)-3'; ii.5'-enhancer-promoter-transgene-WPRE-p(A)-3'; iii.5'-enhancer-enhancer-promoter-transgene-WPRE-p(A)-3'; iv.5'-enhancer-enhancer-promoter-intron-transgene-WPRE-p(A)-3'; v.5'-enhancer-enhancer-promoter-intron-transgene-WPRE-p(A)-3'; vi. 5'-enhancer-promoter-intron-transgene-WPRE-p(A)-p(A)-transgene-intron-promoter-enhancer-3'; vii. 5'-enhancer-promoter-intron-transgene-WPRE-p(A)-enhancer-promoter-intron-transgene-p(A)-3'; viii. 5'-p(A)-WPRE-transgene-intron-promoter-enhancer-enhancer-promoter-intron-transgene-p(A)-3'; ix. 5'-promoter-intron-transgene-WPRE-p(A)-p(A)-transgene-intron-promoter-3'; x.5'-promoter-intron-transgene-WPRE-p(A)-promoter-intron-transgene-p(A)-3'; and xi. 5'-p(A)-WPRE-transgene-intron-promoter-promoter-intron-transgene-p(A)-3'.

[0525] EMBODIMENT 178: The expression cassette of embodiment 176 or embodiment 177, wherein the transgene has an increased expression level compared to a second expression cassette comprising a polynucleotide having a 5' to 3' element arrangement comprising: 5'-promoter-transgene-WPRE-p(A)-3'.

[0526] EMBODIMENT 179: 179. The expression cassette of embodiment 178, wherein the increased expression level is between about 1.5-fold and about 150-fold compared to the second expression cassette.

[0527] Embodiment 180: A recombinant adeno-associated virus (rAAV) virion comprising a viral genome comprising a capsid protein and an expression cassette of any one of embodiments 176 to 179, wherein the expression cassette is flanked by inverted terminal repeats.

[0528] Embodiment 181: The rAAV of embodiment 180, wherein the expression cassette comprises a transgene, the transgene encoding a polypeptide used for treating or preventing cardiac disease or for alleviating symptoms associated with cardiac disease.

[0529] EMBODIMENT 182: The rAAV of embodiment 180 or embodiment 181, wherein the capsid protein is selected from any one of SEQ ID NOs: 145 to 200.

[0530] Numbered embodiment II of the present invention Embodiment 1: A recombinant adeno-associated virus (rAAV) virion comprising a viral genome comprising an expression cassette comprising a capsid protein and a polynucleotide sequence encoding a polypeptide operably linked to a promoter, the expression cassette being flanked by inverted terminal repeats, and optionally the polypeptide being for expression in a cardiac cell or tissue and / or for use in treating or preventing cardiac disease.

[0531] Embodiment 2: 2. The rAAV virion of embodiment 1, wherein the polypeptide is selected from DWORF, JPH2, BAG3, CRYAB, the lamin A isoform of LMNA, the lamin C isoform of LMNA, TNNI3, PLN, LAMP2a, LAMP2b, LAMP2c, the DPI isoform of DSP, the DPII isoform of DSP, DSG2, and JUP.

[0532] Embodiment 3: The rAAV virion of embodiment 1 or 2, wherein the polypeptide shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from the polypeptide sequences in Tables 2a and 2b.

[0533] Embodiment 4: The rAAV virion of any one of embodiments 1-3, wherein the promoter is a chicken cTnT promoter.

[0534] Embodiment 5: The rAAV virion of embodiment 4, wherein the chicken cTnT promoter shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:11.

[0535] Embodiment 6: The rAAV virion of embodiment 4, wherein the chicken cTnT promoter comprises SEQ ID NO:11.

[0536] Embodiment 7: The rAAV virion of any one of embodiments 1-3, wherein the promoter is a human cTnT promoter.

[0537] Embodiment 8: The rAAV virion of embodiment 7, wherein the human cTnT promoter shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:12 or SEQ ID NO:13.

[0538] Embodiment 9: The rAAV virion of embodiment 7, wherein the human cTnT promoter comprises SEQ ID NO:12 or SEQ ID NO:13.

[0539] Embodiment 10: The rAAV virion of any one of embodiments 1-9, wherein the expression cassette further comprises one or more enhancers.

[0540] Embodiment 11: The rAAV virion of embodiment 10, wherein the enhancer, or the one or more enhancers, is selected from an ACTC1 cardiac enhancer and an αMHC enhancer.

[0541] Embodiment 12: The rAAV virion of embodiment 11, wherein the ACTC1 cardiac enhancer shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with sequence number 78.

[0542] Embodiment 13: The rAAV virion of embodiment 11, wherein the ACTC1 cardiac enhancer comprises sequence number 78.

[0543] Embodiment 14: The rAAV virion of embodiment 11, wherein the αMHC enhancer shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:79.

[0544] Embodiment 15: The rAAV virion of embodiment 11, wherein the αMHC enhancer comprises sequence number 79.

[0545] Embodiment 16: 16. The rAAV virion of any one of embodiments 1-15, wherein the expression cassette further comprises an intron.

[0546] Embodiment 17: The rAAV virion of embodiment 16, wherein the intron is selected from a CMV intron and a chimeric intron.

[0547] Embodiment 18: The rAAV virion of embodiment 17, wherein the CMV intron shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:80.

[0548] Embodiment 19: The rAAV virion of embodiment 17, wherein the CMV intron comprises SEQ ID NO:80.

[0549] Embodiment 20: The rAAV virion of embodiment 17, wherein the chimeric intron shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:81.

[0550] Embodiment 21: The rAAV virion of embodiment 17, wherein the chimeric intron comprises SEQ ID NO:81.

[0551] Embodiment 22: The rAAV virion of any one of embodiments 1-21, wherein the expression cassette further comprises a WPRE sequence.

[0552] Embodiment 23: The rAAV virion of embodiment 22, wherein the WPRE sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:26.

[0553] Embodiment 24: The rAAV virion of embodiment 22, wherein the WPRE sequence comprises sequence number 26.

[0554] Embodiment 25: The rAAV virion of any one of embodiments 0-24, wherein the expression cassette further comprises a polyadenylation sequence.

[0555] Embodiment 26: The rAAV virion of embodiment 25, wherein the polyadenylation sequence is selected from the BGH polyadenylation sequence and the SV40 polyadenylation sequence.

[0556] Embodiment 27: The rAAV virion of embodiment 26, wherein the BGH polyadenylation sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:27.

[0557] Embodiment 28: The rAAV virion of embodiment 26, wherein the BGH polyadenylation sequence comprises SEQ ID NO:27.

[0558] Embodiment 29: The rAAV virion of embodiment 26, wherein the SV40 polyadenylation sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:28.

[0559] Embodiment 30: The rAAV virion of embodiment 26, wherein the SV40 polyadenylation sequence comprises SEQ ID NO:28.

[0560] Embodiment 31: The rAAV virion of any one of embodiments 1-30, wherein the expression cassette is flanked by ITRs.

[0561] Embodiment 32: The rAAV virion of embodiment 31, wherein the ITRs share at least 90%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO:14 and SEQ ID NO:15.

[0562] Embodiment 33: The rAAV virion of embodiment 31, wherein the ITRs comprise one or more of SEQ ID NO:14 and SEQ ID NO:15.

[0563] Embodiment 34: The rAAV virion of any one of embodiments 1-33, wherein the expression cassette comprises a single promoter.

[0564] Embodiment 35: The rAAV virion of any one of embodiments 1-33, wherein the expression cassette comprises two promoters.

[0565] Embodiment 36: 36. The rAAV virion of any one of embodiments 1-35, wherein the expression cassette comprises a single copy of the sequence encoding the polypeptide.

[0566] Embodiment 37: 36. The rAAV virion of any one of embodiments 1 to 35, wherein the expression cassette comprises two copies of the sequence encoding the polypeptide.

[0567] Embodiment 38: The rAAV virion of any one of embodiments 1-37, wherein the expression cassette comprises one, two, three, or four enhancers.

[0568] Embodiment 39: The rAAV virion of any one of embodiments 1-38, wherein the expression cassette comprises one or two introns.

[0569] Embodiment 40: The rAAV virion of any one of embodiments 1 to 39, wherein the expression cassette comprises one or two WPRE sequences.

[0570] Embodiment 41: The rAAV virion of any one of embodiments 1 to 40, wherein the expression cassette comprises one or two polyadenylation sequences.

[0571] Embodiment 42: 42. The rAAV virion of any one of embodiments 1-41, wherein the expression cassette comprises about 3.2 kb, about, about 3.3 kb, about 3.4 kb, about 3.5 kb, about 3.6 kb, about 3.7 kb, or less.

[0572] Embodiment 43: 43. The rAAV virion of any one of embodiments 1-42, wherein the expression cassette comprises about 1.9 kb, about 2.1 kb, about 2.2 kb, about 2.3 kb, about 2.4 kb, about 2.5 kb, about 2.6 kb, about 2.7 kb, about 2.8 kb, about 2.9 kb, about 3.0 kb, about 3.1 kb, about 3.2 kb, or more.

[0573] Embodiment 44: 4. The rAAV virion of any one of embodiments 1 to 3, wherein the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NOs: 20 to 24 or SEQ ID NOs: 45 to 75, optionally without a sequence or sequences encoding DWORF (open reading frame or open reading frame encoding DWORF). In some embodiments, when the expression cassette is for expression of a polypeptide other than DWORF, the polynucleotide sequence shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a sequence within any one of SEQ ID NOs:20-24 or 45-75, where a sequence or sequence encoding DWORF (an open reading frame or open reading frame encoding DWORF) is replaced with a sequence or sequence encoding a polypeptide other than DWORF (e.g., any polypeptide described herein, such as any polypeptide listed in Table 2b, which also provides sequences of such polypeptides).

[0574] Embodiment 45: The rAAV virion of any one of embodiments 1-3, wherein the expression cassette comprises any one of SEQ ID NOs:20-24 or 45-75, optionally without a sequence or sequence encoding DWORF (an open reading frame or open reading frame encoding DWORF). In some embodiments, when the expression cassette is for expression of a polypeptide other than DWORF, the polynucleotide sequence comprises a sequence within any one of SEQ ID NOs:20-24 or 45-75, wherein the sequence or sequence encoding DWORF (an open reading frame or open reading frame encoding DWORF) is replaced by a sequence or sequence encoding a polypeptide other than DWORF (e.g., any polypeptide described herein, such as any polypeptide listed in Table 2b, which also provides sequences of such polypeptides).

[0575] Embodiment 46: 4. The rAAV virion of any one of embodiments 1 to 3, wherein the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, and SEQ ID NO:60, optionally without a sequence or sequence encoding DWORF (an open reading frame or open reading frame encoding DWORF). In some embodiments, when the expression cassette is for expression of a polypeptide other than DWORF, the polynucleotide sequence comprises a sequence sharing at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, and SEQ ID NO:60, in which a sequence or sequence encoding DWORF (an open reading frame or open reading frame encoding DWORF) is replaced with a sequence or sequence encoding a polypeptide other than DWORF (e.g., any polypeptide described herein, such as any polypeptide listed in Table 2b, which also provides sequences of such polypeptides).

[0576] Embodiment 47: The rAAV virion of any one of embodiments 1-3, wherein the expression cassette comprises any one of SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, and SEQ ID NO:60, optionally without a sequence or sequence encoding DWORF (an open reading frame or open reading frame encoding DWORF). In some embodiments, when the expression cassette is for expression of a polypeptide other than DWORF, the polynucleotide sequence comprises a sequence of SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, and SEQ ID NO:60, in which the sequence or sequence encoding DWORF (an open reading frame or open reading frame encoding DWORF) is replaced by a sequence or sequence encoding a polypeptide other than DWORF (e.g., any polypeptide described herein, such as, for example, any polypeptide listed in Table 2b, which also provides sequences of such polypeptides).

[0577] Embodiment 48: 4. The rAAV virion of any one of embodiments 1 to 3, wherein the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58, optionally without a sequence or sequence encoding DWORF (an open reading frame or open reading frame encoding DWORF). In some embodiments, when the expression cassette is for expression of a polypeptide other than DWORF, the polynucleotide sequence comprises a sequence sharing at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58, in which a sequence or sequence encoding DWORF (an open reading frame or open reading frame encoding DWORF) is replaced with a sequence or sequence encoding a polypeptide other than DWORF (e.g., any polypeptide described herein, such as any polypeptide listed in Table 2b, which also provides sequences of such polypeptides).

[0578] Embodiment 49: The rAAV virion of any one of embodiments 1-3, wherein the expression cassette comprises any one of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58, optionally without a sequence or sequence encoding DWORF (an open reading frame or open reading frame encoding DWORF). In some embodiments, when the expression cassette is for expression of a polypeptide other than DWORF, the polynucleotide sequence comprises any one of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58, in which the sequence or sequence encoding DWORF (an open reading frame or open reading frame encoding DWORF) is replaced by a sequence or sequence encoding a polypeptide other than DWORF (e.g., any polypeptide described herein, such as, for example, any polypeptide listed in Table 2b, which also provides sequences of such polypeptides).

[0579] Embodiment 50: The rAAV virion of any one of embodiments 1 to 3, wherein the expression cassette comprises a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:74, and SEQ ID NO:75, optionally without a sequence or sequence encoding DWORF (an open reading frame or open reading frame encoding DWORF). In some embodiments, when the expression cassette is for expression of a polypeptide other than DWORF, the polynucleotide sequence comprises a sequence sharing at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:74, and SEQ ID NO:75, in which a sequence or sequence encoding DWORF (an open reading frame or open reading frame encoding DWORF) is replaced with a sequence or sequence encoding a polypeptide other than DWORF (e.g., any polypeptide described herein, such as any polypeptide listed in Table 2b, which also provides sequences of such polypeptides).

[0580] Embodiment 51: The rAAV virion of any one of embodiments 1-3, wherein the expression cassette comprises any one of SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:74, and SEQ ID NO:75, optionally without a sequence or sequence encoding DWORF (an open reading frame or open reading frame encoding DWORF). In some embodiments, when the expression cassette is for expression of a polypeptide other than DWORF, the polynucleotide sequence comprises any one of SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:74, and SEQ ID NO:75, in which the sequence or sequence encoding DWORF (an open reading frame or open reading frame encoding DWORF) is replaced by a sequence or sequence encoding a polypeptide other than DWORF (e.g., any polypeptide described herein, such as, for example, any polypeptide listed in Table 2b, which also provides sequences of such polypeptides).

[0581] Embodiment 52: The rAAV virion of any one of embodiments 1-51, wherein the capsid protein shares at least 98%, at least 99%, or 100% identity with the AAV9 capsid protein (SEQ ID NO: 143).

[0582] Embodiment 53: The rAAV virion of any one of embodiments 1-51, wherein the capsid protein shares at least 98%, at least 99%, or 100% identity with the AAV5 capsid protein (SEQ ID NO: 144).

[0583] Embodiment 54: The rAAV virion of any one of embodiments 1-51, wherein the capsid protein is a chimeric capsid protein.

[0584] Embodiment 55: The rAAV virion of any one of embodiments 1-51, wherein the capsid protein is an AAV5 / AAV9 chimeric capsid protein.

[0585] Embodiment 56: The rAAV virion of any one of embodiments 1-51, wherein the capsid protein is selected from any one of SEQ ID NOs: 145-200.

[0586] Embodiment 57: An expression cassette comprising a polynucleotide sequence encoding a polypeptide operably linked to a promoter, optionally wherein the polypeptide is for expression in a cardiac cell or tissue and / or for use in treating or preventing cardiac disease.

[0587] Embodiment 58: 58. The expression cassette of embodiment 57, wherein the polypeptide is selected from DWORF, JPH2, BAG3, CRYAB, Lamin A isoform of LMNA, Lamin C isoform of LMNA, TNNI3, PLN, LAMP2a, LAMP2b, LAMP2c, DPI isoform of DSP, DPII isoform of DSP, DSG2, and JUP; optionally, the polypeptide shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from the polypeptide sequences in Tables 2a and 2b.

[0588] Embodiment 59: 59. The expression cassette of embodiment 57 or 58, wherein the promoter is a chicken cTnT promoter.

[0589] Embodiment 60: 60. The expression cassette of embodiment 59, wherein the chicken cTnT promoter shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:11.

[0590] Embodiment 61: 60. The expression cassette of embodiment 59, wherein the chicken cTnT promoter comprises SEQ ID NO:11.

[0591] Embodiment 62: 59. The expression cassette of embodiment 57 or 58, wherein the promoter is a human cTnT promoter.

[0592] Embodiment 63: 63. The expression cassette of embodiment 62, wherein the human cTnT promoter shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:12 or SEQ ID NO:13.

[0593] Embodiment 64: 63. The expression cassette of embodiment 62, wherein the human cTnT promoter comprises SEQ ID NO:12 or SEQ ID NO:13.

[0594] Embodiment 65: The expression cassette of any one of embodiments 57 to 64, wherein the expression cassette further comprises one or more enhancers.

[0595] Embodiment 66: 66. The expression cassette of embodiment 65, wherein the one or more enhancers are selected from an ACTC1 cardiac enhancer and an αMHC enhancer.

[0596] Embodiment 67: 67. The expression cassette of embodiment 66, wherein the ACTC1 cardiac enhancer shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:78.

[0597] Embodiment 68: 67. The expression cassette of embodiment 66, wherein the ACTC1 cardiac enhancer comprises sequence number 78.

[0598] 69. 67. The expression cassette of embodiment 66, wherein the αMHC enhancer shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:79.

[0599] Embodiment 70: 67. The expression cassette of embodiment 66, wherein the αMHC enhancer comprises SEQ ID NO:79.

[0600] Embodiment 71: The expression cassette of any one of embodiments 57 to 70, wherein the expression cassette further comprises an intron.

[0601] Embodiment 72: 71. The expression cassette of any one of embodiments 57-70, wherein the intron is selected from a CMV intron and a chimeric intron.

[0602] Embodiment 73: 73. The expression cassette of embodiment 72, wherein the CMV intron shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:80.

[0603] EMBODIMENT 74: 73. The expression cassette of embodiment 72, wherein the CMV intron comprises SEQ ID NO:80.

[0604] Embodiment 75: 73. The expression cassette of embodiment 72, wherein the chimeric intron shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:81.

[0605] EMBODIMENT 76: 73. The expression cassette of embodiment 72, wherein the chimeric intron comprises SEQ ID NO:81.

[0606] EMBODIMENT 77: The expression cassette of any one of embodiments 57 to 76, wherein the expression cassette further comprises a WPRE sequence.

[0607] EMBODIMENT 78: 78. The expression cassette of embodiment 77, wherein the WPRE sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:26.

[0608] EMBODIMENT 79: 78. The expression cassette of embodiment 77, wherein the WPRE sequence comprises SEQ ID NO:26.

[0609] Embodiment 80: 80. The expression cassette of any one of embodiments 57 to 79, wherein the expression cassette further comprises a polyadenylation sequence.

[0610] Embodiment 81: 81. The expression cassette of embodiment 80, wherein the polyadenylation sequence is selected from the BGH polyadenylation sequence and the SV40 polyadenylation sequence.

[0611] Embodiment 82: 82. The expression cassette of embodiment 81, wherein the BGH polyadenylation sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:27.

[0612] Embodiment 83: 82. The expression cassette of embodiment 81, wherein the BGH polyadenylation sequence comprises SEQ ID NO:27.

[0613] EMBODIMENT 84: 82. The expression cassette of embodiment 81, wherein the SV40 polyadenylation sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:28.

[0614] Embodiment 85: 82. The expression cassette of embodiment 81, wherein the SV40 polyadenylation sequence comprises SEQ ID NO:28.

[0615] EMBODIMENT 86: 86. The expression cassette of any one of embodiments 57 to 85, wherein the expression cassette is flanked by ITRs.

[0616] Embodiment 87: 87. The expression cassette of embodiment 86, wherein the ITRs share at least 90%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO:14 and SEQ ID NO:15.

[0617] EMBODIMENT 88: 87. The expression cassette of embodiment 86, wherein the ITRs comprise one or more of SEQ ID NO:14 and SEQ ID NO:15.

[0618] EMBODIMENT 89: 89. The expression cassette of any one of embodiments 57 to 88, comprising a single promoter.

[0619] EMBODIMENT 90: 89. The expression cassette of any one of embodiments 57 to 88, comprising two promoters.

[0620] EMBODIMENT 91: 91. The expression cassette of any one of embodiments 57 to 90, comprising a single copy of the sequence encoding the polypeptide.

[0621] EMBODIMENT 92: 91. The expression cassette of any one of embodiments 57 to 90, comprising two copies of the sequence encoding the polypeptide.

[0622] EMBODIMENT 93: 93. The expression cassette of any one of embodiments 57 to 92, comprising one, two, three, or four enhancers.

[0623] EMBODIMENT 94: 94. The expression cassette of any one of embodiments 57 to 93, comprising one or two introns.

[0624] EMBODIMENT 95: The expression cassette of any one of embodiments 57 to 94, comprising one or two WPRE sequences.

[0625] EMBODIMENT 96: 96. The expression cassette of any one of embodiments 57 to 95, comprising one or two polyadenylation sequences.

[0626] EMBODIMENT 97: 97. The expression cassette of any one of embodiments 57-96, comprising about 3.2 kb, about, about 3.3 kb, about 3.4 kb, about 3.5 kb, about 3.6 kb, about 3.7 kb, or less.

[0627] EMBODIMENT 98: 98. The expression cassette of any one of embodiments 57-97, comprising about 1.9 kb, about 2.1 kb, about 2.2 kb, about 2.3 kb, about 2.4 kb, about 2.5 kb, about 2.6 kb, about 2.7 kb, about 2.8 kb, about 2.9 kb, about 3.0 kb, about 3.1 kb, about 3.2 kb, or more.

[0628] EMBODIMENT 99: 59. The expression cassette of embodiment 57 or 58, comprising a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NOs: 20-24 or 45-75, optionally without a sequence or sequences encoding DWORF (open reading frame or open reading frame encoding DWORF). In some embodiments, when the expression cassette is for expression of a polypeptide other than DWORF, the polynucleotide sequence shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NOs:20-24 or 45-75, in which a sequence or sequence encoding DWORF (an open reading frame or open reading frame encoding DWORF) is replaced with a sequence or sequence encoding a polypeptide other than DWORF (e.g., any polypeptide described herein, such as any polypeptide listed in Table 2b, which also provides sequences of such polypeptides).

[0629] EMBODIMENT 100: The expression cassette of embodiment 57 or 58, optionally comprising the polynucleotide sequence of any one of SEQ ID NOs: 20-24 or 45-75, without a sequence or sequence encoding DWORF (open reading frame or open reading frame encoding DWORF). In some embodiments, when the expression cassette is for expression of a polypeptide other than DWORF, the polynucleotide sequence comprises any one of SEQ ID NOs: 20-24 or 45-75, in which the sequence or sequence encoding DWORF (open reading frame or open reading frame encoding DWORF) is replaced by a sequence or sequence encoding a polypeptide other than DWORF (e.g., any polypeptide described herein, such as any polypeptide listed in Table 2b, which also provides sequences of such polypeptides).

[0630] EMBODIMENT 101: 59. The expression cassette of embodiment 57 or 58, comprising a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, and SEQ ID NO:60, optionally without a sequence or sequence encoding DWORF (an open reading frame or open reading frame encoding DWORF). In some embodiments, when the expression cassette is for expression of a polypeptide other than DWORF, the polynucleotide sequence shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, and SEQ ID NO:60, in which a sequence or sequence encoding DWORF (an open reading frame or open reading frame encoding DWORF) is replaced with a sequence or sequence encoding a polypeptide other than DWORF (e.g., any polypeptide described herein, such as any polypeptide listed in Table 2b, which also provides sequences of such polypeptides).

[0631] EMBODIMENT 102: The expression cassette of embodiment 57 or 58, optionally comprising the polynucleotide sequence of any one of SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 60, without a sequence or sequence encoding DWORF (open reading frame or open reading frame encoding DWORF). In some embodiments, when the expression cassette is for expression of a polypeptide other than DWORF, the polynucleotide sequence comprises the sequence of any one of SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 60, in which the sequence or sequence encoding DWORF (open reading frame or open reading frame encoding DWORF) is replaced by a sequence or sequence encoding a polypeptide other than DWORF (e.g., any polypeptide described herein, such as any polypeptide listed in Table 2b, which also provides sequences of such polypeptides).

[0632] EMBODIMENT 103: 59. The expression cassette of embodiment 57 or 58, comprising a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58, optionally without a sequence or sequence encoding DWORF (an open reading frame or open reading frame encoding DWORF). In some embodiments, when the expression cassette is for expression of a polypeptide other than DWORF, the polynucleotide sequence shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58, in which a sequence or sequence encoding DWORF (an open reading frame or open reading frame encoding DWORF) is replaced with a sequence or sequence encoding a polypeptide other than DWORF (e.g., any polypeptide described herein, such as any polypeptide listed in Table 2b, which also provides sequences of such polypeptides).

[0633] EMBODIMENT 104: The expression cassette of embodiment 57, optionally comprising any one of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58, without a sequence or sequence encoding DWORF (open reading frame or open reading frame encoding DWORF). In some embodiments, when the expression cassette is for expression of a polypeptide other than DWORF, the polynucleotide sequence comprises any one of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58, in which the sequence or sequence encoding DWORF (open reading frame or open reading frame encoding DWORF) is replaced by a sequence or sequence encoding a polypeptide other than DWORF (e.g., any polypeptide described herein, such as any polypeptide listed in Table 2b, which also provides sequences of such polypeptides).

[0634] EMBODIMENT 105: 59. The expression cassette of embodiment 57 or 58, comprising a polynucleotide sequence that shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:74, and SEQ ID NO:75, optionally without a sequence or sequence encoding DWORF (an open reading frame or open reading frame encoding DWORF). In some embodiments, when the expression cassette is for expression of a polypeptide other than DWORF, the polynucleotide sequence comprises a sequence sharing at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:74, and SEQ ID NO:75, in which a sequence or sequence encoding DWORF (an open reading frame or open reading frame encoding DWORF) is replaced with a sequence or sequence encoding a polypeptide other than DWORF (e.g., any polypeptide described herein, such as any polypeptide listed in Table 2b, which also provides sequences of such polypeptides).

[0635] EMBODIMENT 106: The expression cassette of embodiment 57 or 58, optionally comprising any one of SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:74, and SEQ ID NO:75, without a sequence or sequence encoding DWORF (open reading frame or open reading frame encoding DWORF). In some embodiments, when the expression cassette is for expression of a polypeptide other than DWORF, the polynucleotide sequence comprises any one of SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:74, and SEQ ID NO:75, in which the sequence or sequence encoding DWORF (open reading frame or open reading frame encoding DWORF) is replaced by a sequence or sequence encoding a polypeptide other than DWORF (e.g., any polypeptide described herein, such as any polypeptide listed in Table 2b, which also provides sequences of such polypeptides).

[0636] EMBODIMENT 107: The expression cassette of any one of embodiments 99 to 106, wherein the expression cassette does not comprise a 5' inverted terminal repeat and a 3' inverted terminal repeat of the polynucleotide sequence.

[0637] EMBODIMENT 108: A pharmaceutical composition comprising the rAAV virion of any one of embodiments 1 to 56 and a pharma- ceutical acceptable carrier, or a vector comprising the expression cassette of any one of embodiments 57 to 107 and a pharma-ceutical acceptable carrier.

[0638] EMBODIMENT 109: A kit comprising the pharmaceutical composition of embodiment 108.

[0639] EMBODIMENT 110: A method for increasing polypeptide expression in a cell, comprising contacting the cell with a rAAV virion of any one of embodiments 1 to 56, a vector comprising the expression cassette of any one of embodiments 57 to 107, or a composition of embodiment 108.

[0640] EMBODIMENT 111: The method of embodiment 110, wherein the cell is a cardiac cell.

[0641] EMBODIMENT 112: The method of embodiment 111, wherein the cardiac cells are cardiomyocytes.

[0642] EMBODIMENT 113: The method of any one of embodiments 110-112, wherein polypeptide expression is increased by between about 1.5-fold and 150-fold.

[0643] EMBODIMENT 114: The method of any one of embodiments 110-113, wherein the contacting is in vitro.

[0644] EMBODIMENT 115: The method of any one of embodiments 110-113, wherein the contacting is in vivo.

[0645] EMBODIMENT 116: A method for increasing polypeptide expression in a tissue, comprising contacting the tissue with a rAAV virion of any one of embodiments 1 to 56, a vector comprising the expression cassette of any one of embodiments 57 to 107, or a composition of embodiment 108.

[0646] EMBODIMENT 117: The method of embodiment 116, wherein the tissue is cardiac tissue.

[0647] EMBODIMENT 118: 118. The method of embodiment 116 or 117, wherein polypeptide expression is increased by between about 1.5-fold and 150-fold.

[0648] EMBODIMENT 119: The method of any one of embodiments 116-118, wherein the contacting is in vitro.

[0649] EMBODIMENT 120: The method of embodiment 116 or 118, wherein the contacting is in vivo.

[0650] EMBODIMENT 121: A method for increasing polypeptide expression in an organ, comprising contacting the organ with a rAAV virion of any one of embodiments 1 to 56, a vector comprising an expression cassette of any one of embodiments 57 to 107, or a composition of embodiment 108.

[0651] EMBODIMENT 122: The method of embodiment 121, wherein the organ is the heart.

[0652] EMBODIMENT 123: The method of embodiment 122, wherein the heart is diseased or at risk of disease.

[0653] EMBODIMENT 124: The method of embodiment 122 or embodiment 123, wherein the heart has a reduced or borderline ejection fraction.

[0654] EMBODIMENT 125: The method of embodiment 122 or embodiment 123, wherein the heart has a normal ejection fraction.

[0655] EMBODIMENT 126: The method of any one of embodiments 122-125, wherein the heart comprises a genetic mutation associated with heart disease.

[0656] EMBODIMENT 127: The method of embodiment 126, wherein the genetic mutation is a PLN mutation.

[0657] EMBODIMENT 128: 128. The method of any one of embodiments 121-127, wherein the heart has low or undetectable polypeptide expression compared to a healthy heart.

[0658] EMBODIMENT 129: The method of any one of embodiments 121-128, wherein polypeptide expression is increased between about 1.5-fold and 150-fold.

[0659] EMBODIMENT 130: The method of any one of embodiments 121-129, wherein the contacting is in vitro.

[0660] EMBODIMENT 131: The method of any one of embodiments 121-129, wherein the contacting is in vivo.

[0661] EMBODIMENT 132: A method for increasing polypeptide expression in a subject, comprising administering to the subject a rAAV virion of any one of embodiments 1 to 56, a vector comprising an expression cassette of any one of embodiments 57 to 107, or a composition of embodiment 108.

[0662] EMBODIMENT 133: The method of embodiment 132, wherein the subject is an animal.

[0663] EMBODIMENT 134: The method of embodiment 132, wherein the subject is a human.

[0664] EMBODIMENT 135: The method of any one of embodiments 132-134, wherein polypeptide expression is increased in the heart of the subject.

[0665] EMBODIMENT 136: The method of any one of embodiments 132-135, wherein the subject has or is at risk for heart disease.

[0666] EMBODIMENT 137: The method of any one of embodiments 132-136, wherein the subject has a borderline or reduced ejection fraction.

[0667] EMBODIMENT 138: The method of any one of embodiments 132-136, wherein the subject has a normal ejection fraction.

[0668] EMBODIMENT 139: The method of any one of embodiments 132-138, wherein the subject has a genetic mutation associated with heart disease.

[0669] Embodiment 140: 140. The method of embodiment 139, wherein the genetic mutation is a PLN mutation.

[0670] EMBODIMENT 141: The method of any one of embodiments 132-140, wherein the subject has low or undetectable levels of polypeptide expression compared to healthy subjects.

[0671] EMBODIMENT 142: A method for treating a cardiac disease or disorder in a subject in need thereof, comprising administering to the subject a rAAV virion of any one of embodiments 1 to 56, a vector comprising an expression cassette of any one of embodiments 57 to 107, or a composition of embodiment 108.

[0672] EMBODIMENT 143: The method of embodiment 142, wherein the subject has a cardiac disease or disorder.

[0673] EMBODIMENT 144: The method of embodiment 142, wherein the subject is at risk of developing a cardiac disease or disorder.

[0674] EMBODIMENT 145: The method of any one of embodiments 142-144, wherein the cardiac disease or disorder is cardiomyopathy.

[0675] EMBODIMENT 146: The method of any one of embodiments 142-144, wherein the cardiac disease or disorder is dilated cardiomyopathy.

[0676] EMBODIMENT 147: The method of any one of embodiments 142-144, wherein the cardiac disease or disorder is myocardial infarction.

[0677] EMBODIMENT 148: The method of any one of embodiments 142-144, wherein the cardiac disease or disorder is chronic myocardial infarction.

[0678] EMBODIMENT 149: The method of any one of embodiments 142-144, wherein the cardiac disease or disorder is acute myocardial infarction.

[0679] EMBODIMENT 150: The method of any one of embodiments 142-149, wherein the subject has an inherited risk allele for a cardiac disease or disorder.

[0680] EMBODIMENT 151: The method of any one of embodiments 142-150, wherein the inherited risk allele comprises a mutation to the PLN gene.

[0681] EMBODIMENT 152: The method of embodiment 151, wherein the mutation to the PLN gene is a PLN promoter mutation.

[0682] EMBODIMENT 153: Mutations in the PLN gene cause PLN L39stop The method of embodiment 151, wherein the mutation is

[0683] EMBODIMENT 154: The method of embodiment 151, wherein the mutation to the PLN gene is an RC9 mutation.

[0684] EMBODIMENT 155: The method of embodiment 151, wherein the mutation to the PLN gene is an R9L mutation.

[0685] EMBODIMENT 156: The method of embodiment 151, wherein the mutation to the PLN gene is a PLN gene duplication.

[0686] EMBODIMENT 157: The method of embodiment 151, wherein the mutation to the PLN gene is an R14del mutation.

[0687] EMBODIMENT 158: The method of any one of embodiments 142-157, wherein the cardiac disease or disorder is associated with reduced ejection fraction (HFrEF).

[0688] EMBODIMENT 159: The method of an...

Claims

**Claim 1**: An expression cassette comprising a polynucleotide sequence encoding a dwarf open reading frame (DWORF) polypeptide operably linked to a cardiac troponin T (cTnT) promoter, actin, alpha cardiac actin 1 (ACTC1) cardiac enhancer, and an alpha myosin heavy chain (αMHC) enhancer, wherein the expression cassette further comprises a polyadenylation sequence, and the ACTC1 cardiac enhancer comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO: 78, the expression cassette further comprises a woodchuck hepatitis virus posttranscriptional element (WPRE) sequence. **Claim 2**: The expression cassette according to claim 1, wherein the DWORF polypeptide is a human DWORF polypeptide or a functional variant thereof that shares at least 80% or 90% sequence identity with SEQ ID NO: 3, and optionally, the human DWORF polypeptide comprises the amino acid sequence of SEQ ID NO:

3. **Claim 3**: The expression cassette according to claim 1, wherein the cTnT promoter is a human cTnT promoter, and optionally, the human cTnT promoter comprises a polynucleotide sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 12, or optionally, the human cTnT promoter comprises the polynucleotide sequence of SEQ ID NO:

12. **Claim 4**: The expression cassette according to claim 1, wherein the αMHC enhancer comprises a polynucleotide sequence that shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 79, or the αMHC enhancer comprises the polynucleotide sequence of SEQ ID NO:

79. **Claim 5**: The expression cassette according to claim 1, wherein the ACTC1 cardiac enhancer comprises the polynucleotide sequence of SEQ ID NO:

78. **Claim 6**: The expression cassette according to claim 1, wherein the expression cassette further comprises an intron. **Claim 7**: The intron is selected from the CMV intron and the chimeric intron. Optionally, the CMV intron comprises a polynucleotide sequence sharing at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 80, and the chimeric intron comprises a polynucleotide sequence sharing at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:

81. The expression cassette according to claim 6. **Claim 8**: The WPRE sequence comprises a polynucleotide sequence sharing at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:

26. The expression cassette according to claim 1. **Claim 9**: The polyadenylation sequence is selected from the BGH polyadenylation sequence and the SV40 polyadenylation sequence. Optionally, the BGH polyadenylation sequence comprises a polynucleotide sequence sharing at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 27, and the SV40 polyadenylation sequence comprises a polynucleotide sequence sharing at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:

28. The expression cassette according to claim 1. **Claim 10**: The expression cassette according to claim 1, comprising a single copy of the polynucleotide sequence encoding the DWORF polypeptide. **Claim 11**: The expression cassette comprises a single copy of the polynucleotide sequence encoding the human DWORF polypeptide. The polynucleotide sequence is operably linked to a human cTnT promoter, an ACTC1 heart enhancer, and an αMHC enhancer. The expression cassette further comprises a CMV intron and a BGH polyadenylation sequence. Optionally, the expression cassette comprises a polynucleotide sequence sharing at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 69, or optionally, the expression cassette comprises the polynucleotide sequence of SEQ ID NO:

69. The expression cassette according to claim 1. **Claim 12**: The expression cassette has a 5' to 3' arrangement of the ACTC1 heart enhancer, the αMHC enhancer, the human cTnT promoter, the CMV intron, the polynucleotide sequence encoding the human DWORF polypeptide, and the BGH polyadenylation sequence, wherein the ACTC1 heart enhancer, the αMHC enhancer, the human cTnT promoter, and the polynucleotide sequence encoding the human DWORF polypeptide are in the same orientation, and optionally, the expression cassette further comprises a WPRE sequence between the polynucleotide sequence encoding the human DWORF polypeptide and the BGH polyadenylation sequence, the expression cassette according to claim 11. **Claim 13**: The expression cassette is flanked by inverted terminal repeats (ITRs), and the ITRs comprise a polynucleotide sequence sharing at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 14 or SEQ ID NO: 15, the expression cassette according to claim 1. **Claim 14**: A recombinant adeno-associated virus (rAAV) virion comprising a capsid protein and an expression cassette according to any one of claims 1 to 13, wherein optionally, the capsid protein is a wild-type AAV9 capsid protein or a variant thereof, and optionally, the capsid protein shares at least 98% sequence identity with SEQ ID NO:

143. **Claim 15**: A pharmaceutical composition comprising the rAAV virion according to claim 14 and a pharmaceutically acceptable diluent.

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