Adeno-associated virus vector for DWARF open reading frame

Abstract

The present invention provides compositions and methods for the treatment or prevention of heart disease (e.g., cardiomyopathy) in a subject. [Solution] A method is disclosed for treating a subject, for example, a subject having or at risk of cardiomyopathy, using an effective amount of recombinant adeno-associated virus (rAAV) virion, wherein the rAAV virion comprises an expression cassette containing an AAV capsid and a polynucleotide encoding a DWORF polypeptide functionally linked to a promoter. Related compositions and kits are also disclosed.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority under U.S. Provisional Application No. 63 / 048,743, filed July 7, 2020, the entirety of which is incorporated herein by reference.

[0002] Declaration related to federally funded research and development This invention was made with government support from the National Institutes of Health (NIH) under HL141630, HL130253, HL138426, HD087351 and AR067294. The government has certain rights with respect to this invention.

[0003] Sequence listing reference This application was filed electronically via EFS-Web and includes an electronically submitted sequence listing in .txt format. This .txt file was created on June 24, 2021, and contains a sequence listing named "UTFDP3586WO_ST25.txt" with a size of 17 kilobytes. The sequence listing contained in this .txt file is part of this specification and is incorporated herein by reference in its entirety.

[0004] Technical field This disclosure relates to compositions and methods for the treatment or prevention of cardiac diseases (e.g., cardiomyopathy) in subjects. In particular, this disclosure relates to vectors comprising a cardiac-specific promoter functionally linked to a therapeutic gene product for the treatment of cardiac diseases (e.g., cardiomyopathy). [Background technology]

[0005] background Cardiomyopathy accounts for approximately half of all cardiac-related deaths. It is estimated that between 1 in 250 and 1 in 10,000 adults suffer from some form of cardiomyopathy (McKenna et al. Circ Res. 121:722-730 (2017) (Non-patent Literature 1)). Despite considerable efforts in screening, diagnosis, and treatment strategies, the incidence of cardiomyopathy and cardiomyopathy-related deaths remain high (Brieler Am Fam Physician. 96:640-646 (2017) (Non-patent Literature 2)).

[0006] Cardiomyopathy refers to a group of cardiac conditions that occur when the heart's ability to pump blood is impaired. Impaired cardiac function, such as systolic dysfunction, can lead to myocardial infarction, heart failure, thrombosis, valvular dysfunction, and cardiac arrest. Cardiomyopathy can be classified into primary and secondary categories, resulting in a wide range of phenotypes (McKenna et al. Circ Res. 121:722-730 (2017) (Non-patent Literature 1)). Primary cardiomyopathy can have hereditary, acquired, or mixed etiologies. Hereditary cardiomyopathy is inherited and includes arrhythmogenic right ventricular dysplasia, hypertrophy, ion channel dysfunction, left ventricular noncompaction, and mitochondrial myopathy. Acquired cardiomyopathy is primarily due to non-secondary, non-hereditary factors that result in cardiac complications and includes myocarditis, peripartum, tachycardia-induced cardiomyopathy, and stress-induced cardiomyopathy. Cardiomyopathy with mixed etiology is caused by a combination of non-hereditary and hereditary factors and includes dilated cardiomyopathy and restrictive cardiomyopathy. Secondary cardiomyopathy represents heart disease caused by extracardiovascular factors. Underlying factors in secondary cardiomyopathy may be endocrine, infectious, toxin exposure, autoimmune, nutritional, and / or neuromuscular.

[0007] Cardiomyocytes play a central role in the pathology of cardiomyopathy. 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 function as a pump. Numerous mechanisms exist that impair the ability of properly functioning cardiomyocytes (Dadson et al. Clin Sci (Lond) 131:1375-1392 (2017) (Non-patent Literature 3)). In arrhythmogenic right ventricular cardiomyopathy, progressive replacement of cardiomyocytes by fibrous tissue leads to electrical isolation of cardiomyocytes and atrophy of the ventricular myocardial layer, the main structure responsible for contractile function in the heart. In mitochondrial cardiomyopathy, ATP deficiency has a direct impact on the contractile function of cardiomyocytes, which have high metabolic requirements. Cardiomyopathy also results from the loss of activity of regulatory enzymes, such as sarcoplasmic reticulum / endoplasmic reticulum calcium ATPase (SERCA), leading to a loss of normal Ca 2+ This appears as a result of abnormal contraction function caused by the loss of ion release, uptake, and sequestration processes (Lennon et al. Int J Mol Med. 7:131-41 (2001) (Non-Patent Literature 4)).

[0008] A treatment strategy for cardiomyopathy is needed. Targeting the mechanisms that control the abnormal contractile function of cardiac cells is an effective approach. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] McKenna et al. Circ Res. 121:722-730 (2017) [Non-Patent Document 2] Brieler 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) [Overview of the project]

[0010] Abstract In one aspect, the present disclosure provides a method for treating heart failure in a subject in need, comprising the step of administering an effective amount of recombinant adeno-associated virus (rAAV) virion, wherein the rAAV virion comprises an AAV capsid and an expression cassette comprising a polynucleotide encoding a DWarf open reading frame (DWORF) polypeptide functionally linked to a promoter.

[0011] In some embodiments, the subjects have or are at risk of developing cardiomyopathy. In one embodiment, the cardiomyopathy is dilated cardiomyopathy (DCM). In some embodiments, the subjects have or are at risk of developing myocardial infarction. In one embodiment, the myocardial infarction is chronic myocardial infarction. In one embodiment, the myocardial infarction is acute myocardial infarction.

[0012] In some embodiments, rAAV virions are administered by intravenous or intracoronary injection. In some embodiments, rAAV is transduced into cardiac cells. In some embodiments, rAAV is transduced into cardiomyocytes.

[0013] In some embodiments, transduction of rAAV increases DWORF polypeptide expression in the target heart.

[0014] In some embodiments, transduction of rAAV enhances SERCA activity.

[0015] In some embodiments, the rAAV virion is an rAAV virion of serotype AAV9. In some embodiments, the AAV capsid comprises a capsid protein having at least 98% identity to SEQ ID NO: 14. In some embodiments, the AAV capsid comprises a capsid protein having at least 99% identity to SEQ ID NO: 14. In some embodiments, the AAV capsid comprises a capsid protein containing the polypeptide sequence of SEQ ID NO: 14.

[0016] In some embodiments, the promoter is a chicken cardiac troponin-T (cTnT) promoter. In some embodiments, the chicken cTnT promoter includes a polynucleotide sequence having at least 95% identity to SEQ ID NO: 11. In some embodiments, the chicken cTnT promoter includes a polynucleotide sequence having at least 98% identity to SEQ ID NO: 11. In some embodiments, the chicken cTnT promoter includes a polynucleotide sequence of SEQ ID NO: 11.

[0017] In some embodiments, the DWORF polypeptide is a mouse DWORF polypeptide. In some embodiments, the DWORF polypeptide comprises a polypeptide sequence having at least 95% identity to 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 having at least 98% identity to 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.

[0018] In some embodiments, the expression cassette is adjacent to an AAV inverted terminal repeat (ITR). In some embodiments, the ITR is an AAV2 ITR. In some embodiments, the ITR contains a polynucleotide sequence with SEQ ID NO: 12 or SEQ ID NO: 13.

[0019] In some embodiments, subjects experience improvement in symptoms associated with MCD after administration. In some embodiments, symptom improvement is one or more of the following: enhanced contractility, reduced fatigue, reduced dyspnea, reduced edema, reduced chest pain, reduced arrhythmia, reduced thrombosis, improved cardiac valve function, and reduced heart murmurs.

[0020] In one aspect, the disclosure provides a recombinant adeno-associated virus (rAAV) virion comprising an AAV capsid and an expression cassette comprising a polynucleotide encoding a DWORF polypeptide functionally linked to a promoter, as well as a pharmaceutically acceptable carrier.

[0021] In one embodiment, the rAAV virion is an rAAV virion of serotype AAV9. In several embodiments, the AAV capsid comprises a capsid protein having at least 98% identity to SEQ ID NO: 14. In several embodiments, the AAV capsid comprises a capsid protein having at least 99% identity to SEQ ID NO: 14. In several embodiments, the AAV capsid comprises a capsid protein containing the polypeptide sequence of SEQ ID NO: 14.

[0022] In some embodiments, the promoter is a cardiac troponin-T (cTnT) promoter. In some embodiments, the cardiac troponin-T (cTnT) promoter contains a polynucleotide sequence having at least 95% identity to SEQ ID NO: 11. In some embodiments, the cardiac troponin-T (cTnT) promoter contains a polynucleotide sequence having at least 98% identity to SEQ ID NO: 11. In some embodiments, the cardiac troponin-T (cTnT) promoter contains a polynucleotide sequence of SEQ ID NO: 11.

[0023] In some embodiments, the DWORF polypeptide is a DWORF polypeptide. In some embodiments, the DWORF polypeptide comprises a polypeptide sequence having at least 95% identity to 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 having at least 98% identity to 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.

[0024] In some embodiments, the expression cassette is adjacent to an AAV inverted terminal repeat (ITR). In some embodiments, the ITR is an AAV2 ITR. In some embodiments, the ITR contains a polynucleotide sequence with SEQ ID NO: 12 or SEQ ID NO: 13.

[0025] In another aspect, the present disclosure provides a pharmaceutical composition comprising a recombinant adeno-associated virus (rAAV) virion and a pharmaceutically acceptable carrier as described in any one of the above claims. In some embodiments, the composition comprises about 5 x 10 13 It contains billions.

[0026] In another aspect, the Disclosure provides a kit comprising a container for housing the pharmaceutical compositions described herein.

[0027] [Invention 1001] A method for treating a subject in need thereof, comprising the step of administering an effective amount of recombinant adeno-associated virus (rAAV) virion, wherein the rAAV virion comprises an expression cassette containing an AAV capsid and a polynucleotide encoding a DWORF polypeptide functionally linked to a promoter. [Invention 1002] A method according to the present invention 1001 for treating heart failure. [Invention 1003] A method according to the present invention 1001 for preventing heart failure. [Invention 1004] The method of the present invention 1001, wherein the subject is suffering from or at risk of cardiomyopathy. [Invention 1005] The method of the present invention 1001, wherein the subject is suffering from or at risk of dilated cardiomyopathy. [Invention 1006] The method of the present invention 1001, wherein the subject has a genetic risk allele for heart failure. [Invention 1007] The method of the present invention 1001, wherein heart failure is myocardial infarction. [Invention 1008] The method of the present invention 1001, wherein the heart failure is heart failure with reduced ejection fraction (HFrEF). [Invention 1009] The method of the present invention 1001, wherein the heart failure is heart failure with preserved ejection fraction (HFpEF). [Invention 1010] A method according to any of the present invention 1001 to 1009, wherein the subject is suffering from or at risk of myocardial infarction. [Invention 1011] The method of the present invention 1010, wherein the myocardial infarction is chronic myocardial infarction. [Invention 1012] The method of the present invention 1010, wherein the myocardial infarction is acute myocardial infarction. [Invention 1013] The method according to any of items 1001 to 1012 of the present invention, wherein the subject has a genetic risk allele for heart failure. [Invention 1014] A method according to any one of the present invention 1001 to 1013 for inducing the expression of a DWORF polypeptide in the target heart. [Invention 1015] Any method of the present invention 1001 to 1014 that does not result in detectable expression of DWORF polypeptide in the target muscle, excluding the heart. [Invention 1016] Any method according to item 1001 to 1015 of the present invention that does not result in detectable expression of the DWORF polypeptide in the target liver. [Invention 1017] A method according to any of items 1001 to 1016 of the present invention that induces the expression of DWORF polypeptide in cardiomyocytes. [Invention 1018] Any method according to item 1001 to 1017 of the present invention that does not result in detectable expression of DWORF polypeptide in cardiac fibroblasts. [Invention 1019] Any method of the present invention 1001 to 1018 for improving one or more indicators of cardiac function, optionally shortening rate and / or left ventricular diameter (LVID). [Invention 1020] The method of the present invention 1018, wherein improvement in cardiac function is observed between weeks 2 and 12. [Invention 1021] A method of reducing cardiac remodeling, as described in any of the present invention 1001 to 1020. [Invention 1022] A method according to any of items 1001 to 1021 of the present invention for preventing a decrease in DWORF expression in a subject suffering from myocardial infarction. [Invention 1023] A method according to any one of items 1001 to 1022 of the present invention, wherein rAAV virion is administered by intravenous or intracoronary injection. [Invention 1024] A method according to the present invention 1017 for increasing SERCA activity. [Invention 1025] A method according to any of the present invention 1001 to 1024, wherein the rAAV virion is an rAAV virion of serotype AAV9. [Invention 1026] A method according to any one of the present invention 1001 to 1025, wherein the AAV capsid contains a capsid protein having at least 98% identity with SEQ ID NO: 14. [Invention 1027] A method according to any one of the present invention 1001 to 1026, wherein the AAV capsid contains a capsid protein having at least 99% identity with SEQ ID NO: 14. [Invention 1028] A method according to any one of the present invention 1001 to 1027, wherein the AAV capsid comprises a capsid protein containing the polypeptide sequence of SEQ ID NO: 14. [Invention 1029] Any method according to invention 1001 to 1028, wherein the promoter is a cardiac troponin-T (cTnT) promoter. [Invention 1030] A method according to any one of the invention 1001 to 1029, wherein the cardiac troponin-T (cTnT) promoter comprises a polynucleotide sequence having at least 95% identity with SEQ ID NO: 11. [Invention 1031] A method according to any one of the present invention 1001 to 1030, wherein the cardiac troponin-T (cTnT) promoter comprises a polynucleotide sequence having at least 98% identity with SEQ ID NO: 11. [Invention 1032] A method according to any one of the invention 1001 to 1031, wherein the cardiac troponin-T (cTnT) promoter contains a polynucleotide sequence of SEQ ID NO: 11. [Invention 1033] Any method according to invention 1001 to 1032, wherein the DWORF polypeptide is a human DWORF polypeptide. [Invention 1034] A method according to any one of the invention 1001 to 1033, wherein the DWORF polypeptide comprises a polypeptide sequence having 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. [Invention 1035] A method according to any one of the invention 1001 to 1034, wherein the DWORF polypeptide comprises a polypeptide sequence having 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. [Invention 1036] A method according to any one of the invention 1001 to 1035, wherein the DWORF polypeptide comprises the polypeptide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9. [Invention 1037] Any method according to invention 1001 to 1036, wherein the expression cassette is adjacent to an AAV inverted terminal repeat (ITR). [Invention 1038] Any method of the present invention 1001 to 1037, wherein the ITR is an AAV2 ITR. [Invention 1039] A method according to any of the invention 1001 to 1038, wherein the ITR comprises a polynucleotide sequence of SEQ ID NO: 12 or SEQ ID NO: 13. [Invention 1040] A method according to any of items 1001 to 1039 of the present invention, wherein the subject experiences improvement in symptoms after administration. [Invention 1041] The method of the present invention 1040, wherein the improvement of symptoms is one or more of the following: enhanced contractility, reduced fatigue, reduced dyspnea, reduced edema, reduced chest pain, reduced arrhythmia, reduced thrombosis, improved cardiac valve function, and reduced heart murmur. [Invention 1042] A recombinant adeno-associated virus (rAAV) virion comprising an AAV capsid and an expression cassette containing a polynucleotide encoding a DWORF polypeptide functionally linked to a promoter. [Invention 1043] The rAAV virion of the present invention 1042, which is an rAAV virion of serotype AAV9. [Invention 1044] The rAAV virion of Invention 1042 or 1043, wherein the AAV capsid contains a capsid protein having at least 98% identity with SEQ ID NO: 14. [Invention 1045] An rAAV virion according to any of Invention 1042 to 1044, wherein the AAV capsid contains a capsid protein having at least 99% identity with SEQ ID NO: 14. [Invention 1046] An rAAV virion according to any of Invention 1042 to 1045, wherein the AAV capsid contains a capsid protein containing the polypeptide sequence SEQ ID NO: 14. [Invention 1047] An rAAV virion according to any of invention 1042 to 1046, wherein the promoter is a cardiac troponin-T (cTnT) promoter. [Invention 1048] An rAAV virion according to any of Invention 1042 to 1047, wherein the cardiac troponin-T (cTnT) promoter contains a polynucleotide sequence having at least 95% identity with SEQ ID NO: 11. [Invention 1049] An rAAV virion according to any of Invention 1042-1048, wherein the cardiac troponin-T (cTnT) promoter contains a polynucleotide sequence having at least 98% identity with SEQ ID NO: 11. [Invention 1050] An rAAV virion according to any of Invention 1042-1049, wherein the cardiac troponin-T (cTnT) promoter contains a polynucleotide sequence of SEQ ID NO: 11. [Invention 1051] The rAAV virion according to any of the inventions 1042 to 1050, wherein the DWORF polypeptide is a human DWORF polypeptide. [Invention 1052] An rAAV virion according to any of Invention 1042 to 1051, wherein the DWORF polypeptide comprises a polypeptide sequence having 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. [Invention 1053] An rAAV virion according to any of Invention 1042 to 1052, wherein the DWORF polypeptide comprises a polypeptide sequence having 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. [Invention 1054] An rAAV virion according to any of Invention 1042 to 1053, wherein the DWORF polypeptide comprises the polypeptide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9. [Invention 1055] An rAAV virion according to any of the inventions 1042 to 1054, wherein the expression cassette is adjacent to an AAV inverted terminal repeat (ITR). [Invention 1056] An rAAV billion according to any of invention 1042 to 1055, wherein the ITR is an AAV2 ITR. [Invention 1057] An rAAV virion according to any of Invention 1042-1056, wherein the ITR contains a polynucleotide sequence of SEQ ID NO: 12 or SEQ ID NO: 13. [Invention 1058] A pharmaceutical composition comprising a recombinant adeno-associated virus (rAAV) virion according to any of invention 1042 to 1057 and a pharmaceutically acceptable carrier. [Invention 1059] A kit comprising a container for containing the pharmaceutical composition of the present invention 1058. Further aspects and embodiments of the present invention will become apparent from the following detailed description. [Brief explanation of the drawing]

[0028] The accompanying drawings form part of this specification and are included to further illustrate certain aspects of this disclosure. This disclosure may be better understood by referring to one or more of these drawings in conjunction with the detailed description of the specific embodiments shown herein. [Figure 1] This is a schematic diagram of an exemplary embodiment of an expression cassette containing a cTnT promoter adjacent to an AAV inverted terminal repeat and a polynucleotide encoding a DWORF polypeptide. [Figure 2A] This shows Western blot analysis of tissue lysis products from AAV-tdTomato or AAV-DWORF treated mice 4 weeks after AAV delivery. tdTomato expression was assessed using an antibody against red fluorescent protein (RFP). Quad, quadriceps femoris; GP, plantar gastrocnemius. [Figure 2B] This shows echocardiographic analysis of cardiac function and dimensions in 8-week-old mice. Systolic (s) and diastolic (d) left ventricular diameter (LVID) were measured. Data are expressed as mean ± SD for n=8–12 mice. P-values ​​are **p<0.01 or ***p<0.005 compared to MLP KO / AAV-tdTomato. [Figure 2C]Representative hematoxylin and eosin (H&E) staining of histological sections from mice of the indicated genotype and treatment is shown. [Figure 2D] This shows Western blot analysis of cardiac lysis products from AAV-tdTomato or AAV-DWORF treated Siamese or MI mice 12 weeks after surgery. [Figure 2E] Cardiac function and dimensions are shown as evaluated by echocardiography at baseline (0 weeks) and at 1, 2, 4, 8, and 12 weeks post-sham or MI surgery. Data are expressed as mean ± SD in n=4 Siamese mice or n=6-8 MI mice. P values ​​for MI / AAV-tdTomato are *p<0.05, **p<0.01, or ***p<0.005. [Figure 2F] This image shows Masson's trichrome staining of serial cardiac sections from mice 12 weeks after sham or MI procedures. Mice were treated with AAV-tdTomato or AAV-DWORF as shown. Sections were collected in 0.5 μm increments. [Modes for carrying out the invention]

[0029] Detailed explanation overview Abnormal calcium handling is a common feature of cardiomyopathy, and decreased activity of sarcoplasmic reticulum / endoplasmic reticulum calcium ATPase (SERCA) plays a central role in both the onset and progression of this disease. SERCA is involved in the calcium handling of cardiac cells, such as cardiomyocytes. 2+ SERCA is a calcium pump that promotes ion uptake, maintenance, and circulation. SERCA activity is regulated by the inhibitory peptide phospholamban. Increasing SERCA activity by increasing the amount of a polypeptide called DWarf open reading frame (DWORF), which enhances SERCA activity through the direct elimination of this SERCA inhibitory peptide phospholamban, has attracted considerable attention. Contacting DWORF with SERCA is a strategy for increasing SERCA activity in cells.

[0030] This disclosure provides recombinant adeno-associated virus (rAAV) virions comprising polynucleotides encoding the DWORF polypeptide or a functional variant thereof, as well as methods for using the same. In some embodiments, the rAAV virions described herein can be transduced into cardiac cells by introducing, for example, a polynucleotide comprising a sequence encoding the DWORF polypeptide functionally linked to a cardiac cell-specific promoter region into the host cell genome. In some embodiments, the targeted cardiac cells may express the DWORF polypeptide and have increased SERCA activity. Pharmaceutical compositions comprising the rAAV virions described herein are also provided in this disclosure. In one aspect, this disclosure provides methods for treating subjects diagnosed with or at risk of cardiomyopathy using the rAAV virions and pharmaceutical compositions of this disclosure.

[0031] Expression Cassette The rAAV virions of this disclosure may include an expression cassette (Figure 1). The expression cassette may optionally include a polynucleotide encoding a DWORF polypeptide or a functional variant thereof, functionally linked to a promoter, optionally a polyadenylation signal, and optionally a transcription termination signal. The expression cassette may be flanked by an inverted terminal repeat (ITR). These elements provide the function of expressing the transgene after the host cell has been targeted by this rAAV virion. The promoter sequence, if present, controls the expression of the polynucleotide encoding the DWORF polypeptide or a functional variant thereof. The promoter may be cell type specific. Constitutive promoters may be used in the expression cassette, and may include, for example, a cytomegalovirus enhancer (CAG), Simian virus 40 (SV40) promoter, and herpes simplex virus thymidine kinase (HSV-TK) promoter fused to a chicken β-actin promoter (Damdindorj et al. PLoS One. 9:e106472 (2014)). Other cell type-specific promoters may also be used. Cardiac cell-specific promoters may include, 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 within the expression cassette may be, for example, an open reading frame encoding a protein. The ITR within the expression cassette serves as a marker used for viral packaging of the expression cassette (Clark et al. Hum Gene Ther. 6:1329-41 (1995)).

[0032] In some embodiments, the expression cassette has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to SEQ ID NO: 16.

[0033] (Table 1) Sequence of expression cassettes TIFF2026143554000002.tif91156

[0034] In some embodiments, the expression cassette of the 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 untreated subjects. In some embodiments, the expression of the DWORF polypeptide may be increased 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold compared to the expression of the DWORF polypeptide in untreated subjects. In some embodiments, the DWORF polypeptide may be expressed at any detectable level in cardiac cells, while the DWORF polypeptide may not be expressed or may be expressed at an undetectable level in untreated subjects. In other words, cardiac cells administered with rAAV virion may express more DWORF polypeptide than cardiac cells having only endogenous (i.e., native) expression of the DWORF polypeptide.

[0035] DWORF polypeptides are endogenous enhancers of SERCA calcium pump activity, a desirable drug target in regulating cardiac contractility. DWORF is also a very small protein, which makes it a good candidate for delivery to target cells or tissues by rAAV virions. Because DWORF is an endogenous protein, its expression in humans is not immunogenic, and therefore long-term administration and expression are considered possible. The structural characteristics of DWORF polypeptides are as follows: Firstly, this polypeptide may have a DWarf open reading frame (DWORF) of 5-35 consecutive residues located on chromosome 3 of mammalian species, including mice and humans (Nelson et al. Science. 351: 271-275 (2016); U.S. Patent No. 10,570,183). Therefore, the term "peptide having X or fewer consecutive residues" cannot be understood as containing a larger number of consecutive residues, even if the term "contains" is included. Again, peptides containing 20 or fewer consecutive DWORF residues will typically be 35 residues or less. Their total length may 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 assumed. The number of consecutive DWORF residues may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. The expected ranges are 5-20 residues, 5-20 residues, 6-20 residues, 7-20 residues, and consecutive residues of 5-15 residues, 5-15 residues, 6-15 residues, or 7-15 residues.

[0036] In some embodiments, the DWORF polypeptide is a human DWORF polypeptide. In some embodiments, the DWORF polypeptide comprises a polypeptide sequence having at least 95% identity to 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 having at least 98% identity to 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.

[0037] (Table 2) DWORF array TIFF2026143554000003.tif81156

[0038] In some embodiments, the expression cassettes of this disclosure include promoters. The term “promoter,” as used herein, refers to a DNA sequence that conjugates RNA polymerase and thereby promotes RNA synthesis, i.e., the smallest sequence sufficient to perform transcription. The expression of promoters and corresponding proteins or polypeptides may be ubiquitous, meaning they have strong activity across a wide range of cells, tissues, and species, or they may be cell-type specific, tissue-specific, or species-specific. Promoters may be “constitutive,” meaning they are continuously active, or they may be “inducible,” meaning they can be activated or deactivated by the presence or absence of biofactors or non-biological factors. Enhancer sequences, which may or may not be contiguous with promoter sequences, are also included in the nucleic acid constructs or vectors of the present invention. Enhancer sequences may influence promoter-dependent gene expression and may be located in the 5' or 3' region of native genes.

[0039] Various promoters may be used. Advantageously, the promoter, optionally with an enhancer, promotes the expression of a polynucleotide encoding a DWORF polypeptide or a functional variant in target cells. In some embodiments, the expression cassette includes a cell type-specific promoter. In some embodiments, the promoter specifically promotes the expression of a polynucleotide encoding a DWORF polypeptide or a functional variant in cardiac cells. In some embodiments, the promoter specifically promotes the expression of a polynucleotide encoding a DWORF polypeptide or a functional variant in cardiomyocytes.

[0040] In some embodiments, the promoter is a chicken cardiac troponin-T (cTnT) promoter. In some embodiments, the chicken cTnT promoter comprises a polynucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the chicken cTnT promoter (SEQ ID NO:11).

[0041] (Table 3) Chicken cTnT promoter sequences TIFF2026143554000004.tif27156

[0042] In some embodiments, the expression cassette is adjacent to an AAV2 inverted terminal repeat (ITR). In some embodiments, the ITR comprises a polynucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:12 and / or SEQ ID NO:13.

[0043] (Table 4) ITR sequence TIFF2026143554000005.tif20156

[0044] In some embodiments, the expression cassette includes a polyadenylation (poly(A)) signal. In some embodiments, the poly(A) signal includes a polynucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:17.

[0045] (Table 5) Polyadenylated sequences TIFF2026143554000006.tif12156

[0046] Recombinant AAV billion In several aspects of this disclosure, rAAV virions are used to deliver expression cassettes described herein to target cardiac cells, for example, to treat cardiomyopathy. Accordingly, this disclosure provides rAAV virions comprising an expression cassette containing a polynucleotide encoding a DWORF polypeptide functionally linked to an AAV capsid and promoter, as well as a pharmaceutically acceptable carrier.

[0047] The rAAV virions of this disclosure include a capsid protein. The capsid protein is a structural protein that constitutes the assembled packaging of the icosahedron of the rAAV virion, which includes the expression cassette. The capsid protein is classified by its serotype. Wild-type capsid serotypes in rAAV virions may be, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 (Naso et al. BioDrugs 31:317-334 (2017)). Modified 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 into specific tissues or cell types (Liu et al. Curr Pharm Des. 21:3248-56 (2015)).

[0048] Any capsid protein capable of promoting transduction of rAAV virions into cardiac cells for the delivery of the transgenes described herein may be used. Capsid proteins used in rAAV virions for transgene delivery into cardiac cells resulting in high expression include AAV4, AAV6, AAV7, AAV8, and AAV9 (Zincarelli et al. Mol. Ther. 16:P1073-1080 (2008)).

[0049] In some embodiments, the rAAV virion is an rAAV virion of serotype AAV9. In some embodiments, the AAV capsid comprises a capsid protein having at least 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NO:14. In some embodiments, the polynucleotide encoding the AAV capsid has at least 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NO:15. In some embodiments, the AAV capsid comprises a capsid protein containing the polypeptide sequence of SEQ ID NO:14.

[0050] (Table 6) AAV capsid sequences TIFF2026143554000007.tif80151TIFF2026143554000008.tif170151

[0051] In some embodiments, rAAV is replication-deficient in that its rAAV virions cannot independently further replicate and package their genomes. For example, when cardiac cells are targeted by rAAV virions, the DWORF polypeptide is expressed in the targeted cardiac cells, but rAAV cannot replicate because the targeted cardiac cells lack AAV rep and cap genes as well as accessory function genes.

[0052] In some embodiments, the rAAV virions of this disclosure, encapsulating the expression cassette described herein, can be produced using helper-free production. rAAV is a replication-deficient virus and typically requires components from a live helper virus, such as an adenovirus, within a host cell for packaging infectious rAAV virions. The rAAV helper-free production system enables the production of infectious rAAV virions without the use of a live helper virus. In this helper-free system, a host packaging cell line is simultaneously transfected with three plasmids. The first plasmid may contain adenovirus gene products required for packaging rAAV virions (e.g., E2A, E4, and VA RNA genes). The second plasmid may contain required AAV genes (e.g., REP and CAP genes). The third plasmid contains a polynucleotide sequence encoding a protein of interest flanked by the ITR and a promoter. The host packaging cell line may be, for example, an AAV-293 host cell. A suitable host cell contains additional components required to package infectious rAAV virions not supplied by these plasmids. In some embodiments, the CAP gene may encode, for example, an AAV capsid protein as described herein. In some embodiments, the promoter is a promoter sequence as described herein. In some embodiments, the promoter sequence is a cTnT promoter sequence. In some embodiments, the polypeptide of interest is a DWORF polypeptide.

[0053] How to use In one aspect, rAAV virions can be used to treat diseases (Wang et al. Nat Rev Drug Discov. 18:358-378 (2019)). rAAV virions can deliver transgenes to cells within a target, which are then expressed within the cells. Transgenes delivered by rAAV virions can be integrated into the genome of the targeted cells, thereby enabling potentially long-term expression of the transgene product. Compared to other viral transgene delivery systems, such as adenoviruses, 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 (U.S. Patent No. 10,308,957; U.S. Patent No. 9,803,218). rAAV virions can contain genomes up to approximately 5.2 kilobases (kb), and the size of polynucleotides that can be incorporated into host cells is limited to approximately 4.4 kb (Choi et al. Mol Brain. 7:1 (2014)). In terms of 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)).

[0054] Various strategies for treating heart failure using rAAV-based transgene delivery are being studied in vivo. In a porcine model of heart failure, the β-adrenergic receptor, a regulator of contractility, was targeted by delivery of the small peptide βARKct, which 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 human clinical trials, rAAV-based delivery of SERCA2a, an isoform of the SERCA calcium pump to the heart, has been tested as a treatment for heart failure. SERCA, or sarcoplasmic reticulum / endoplasmic reticulum Ca 2+ -ATPase or SR Ca 2+ -ATPase is a calcium ATPase-type P-ATPase. SERCA is present in the sarcoplasmic reticulum (SR) within muscle cells. During muscle relaxation, it hydrolyzes ATP from the cytosol of the cell to the lumen of the SR via Ca ATP hydrolysis. 2+ Ca that transfers 2+ SERCA is an ATPase. SERCA activity is required for proper cardiac contractile function. However, direct substitution of SERCA activity by rAAV-based SERCA2a isoform delivery did not show significant efficacy in clinical trials (Bass-Stringer et al. Heart, Lung and Circulation. 27:1285-1300 (2018)). Enhancement of SERCA activity using alternative strategies is desired for the treatment of cardiac diseases, such as heart failure and cardiomyopathy.

[0055] On the cytoplasmic side of SERCA, there are three main domains: a phosphorylation domain and a nucleotide-binding domain that form the catalytic site, and an actuator domain that is involved in the transition of major conformational changes. SERCA crosses the SR membrane and Ca 2+The rate of movement can be controlled by the regulatory protein phospholamban (PLB / PLN). SERCA is normally inhibited by PLB, which binds tightly to it. Increased β-adrenergic stimulation reduces the binding affinity between SERCA and PLB via phosphorylation of PLB by PKA. When PLB is bound to SERCA, Ca 2+ 2+ movement rate decreases, and dissociation of PLB increases Ca 2+ 2+ movement.

[0056] An alternative strategy to enhance SERCA activity by delivering the SERCA2a isoform is to enhance the activity of natively expressed SERCA by removing PLB. The interaction between SERCA and the DWORF polypeptide detailed herein can displace PLB and enhance SERCA activity.

[0057] In one aspect, the present disclosure provides a method of treating heart failure in a subject in need thereof, comprising the step of administering an effective amount of a recombinant adeno-associated virus (rAAV) virion comprising an AAV capsid and an expression cassette comprising a polynucleotide encoding a DWORF polypeptide operably linked to a promoter.

[0058] In the methods for treating subjects described herein, “treat” or “treat a condition or subject requiring treatment” means (1) performing a procedure to obtain a beneficial or desired outcome, including a clinical outcome such as a reduction in symptoms; (2) preventing a disease, for example, preventing the progression of the clinical symptoms of the disease in a patient who may have a predisposition to the disease but has not yet experienced or shown symptoms of the disease; (3) suppressing the disease, for example, stopping or reducing the progression of the disease or its clinical symptoms; (4) alleviating the disease, for example, leading to remission of the disease or its clinical symptoms; or (5) delaying the disease. Beneficial or desired clinical outcomes for the purposes of the methods described herein 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.

[0059] Subjects requiring treatment with the compositions and methods of this disclosure include, but are not limited to, subjects suffering from or at risk of heart failure. In some embodiments, the methods described herein are useful, for example, for treating cardiomyopathy. In some embodiments, the methods described herein are useful, for example, for treating dilated cardiomyopathy. In some embodiments, subjects are suffering from or at risk of cardiomyopathy. In one embodiment, cardiomyopathy is dilated cardiomyopathy (DCM). In some embodiments, subjects are suffering from or at risk of myocardial infarction. In some embodiments, myocardial infarction is chronic myocardial infarction. In some embodiments, myocardial infarction is acute myocardial infarction.

[0060] In some aspects, the methods described herein alleviate one or more symptoms of heart disease compared to the symptoms of heart disease before administration of rAAV virion. Heart disease in these methods includes, but is not limited to, heart failure, cardiomyopathy, dilated cardiomyopathy, myocardial infarction, chronic myocardial infarction, and acute myocardial infarction. As used herein, “symptoms” includes any diagnostic criteria or symptoms associated with the heart disease described herein. The severity and changes in symptoms and diagnostic outcomes are determined by a healthcare professional qualified to give an assessment and analyze the results of such assessment. In some aspects of this disclosure, symptoms are alleviated after administration of the rAAV and compositions of this disclosure.

[0061] Common symptoms in subjects with or at risk of developing heart disease include fatigue, dyspnea, edema, chest pain, arrhythmia, thrombosis, impaired heart valve function, and heart murmurs. In some embodiments, subjects experience a reduction in the heart disease-related symptoms described herein after administration of the rAAV virions and compositions of this disclosure. In some embodiments of the methods described herein, the improvement in symptoms is one or more of the following: enhanced contractility, reduced fatigue, reduced dyspnea, reduced edema, reduced chest pain, reduced arrhythmia, reduced thrombosis, improved heart valve function, and reduced heart murmurs.

[0062] Assessment of myocardial contractility can be used to evaluate acute and chronic forms of heart failure. Myocardial contractility can be monitored using invasive hemodynamic monitoring, continuous ECG monitoring, central venous pressure, renal function, pulse oximetry, arterial pressure monitoring, pulmonary artery catheterization, and / or transesophageal echocardiography (Kuhn C, Werdan K. Surgical Treatment: Evidence-Based and Problem-Oriented. Munich: Zuckschwerdt; 2001. Available from: https: / / www.ncbi.nlm.nih.gov / books / NBK6895 / ).

[0063] The dyspnea and fatigue associated with heart disease described herein may be measured using questionnaires. For example, the Modified Pulmonary Functional Status and Dyspnea Questionnaire (PFSDQ-M) 10th edition (Huang et al. Am J Crit Care. 17:436-442 (2008)) and the Minnesota Living with Heart Failure Questionnaire (MLHFQ) 11th edition (Bilbao et al. Health Qual Life Outcomes. 14:23 (2016)) may be used to measure subjects with the heart disease described herein. These questionnaires are self-administered and provide scores used to assess the severity of symptoms related to dyspnea, fatigue, and other symptoms related to heart health.

[0064] Cardiomyopathy, myocardial infarction, and cardiac valve function may be assessed using one or more of the following: exercise stress tests, electrocardiograms, echocardiograms, chest X-rays, cardiac CT scans, or angiography, in conjunction with cardiac catheterization, cardiac MRI, serum natriuretic peptide (BNP) levels, and / or genetic screening. Further testing is required to diagnose the specific type of cardiomyopathy, myocardial infarction, or cardiac valve dysfunction.

[0065] Dilated cardiomyopathy (DCM) is a progressive myocardial disease characterized by chamber enlargement and left ventricular systolic dysfunction in the absence of chronic pressure and / or volume load. DCM is primarily diagnosed using echocardiography.

[0066] Echocardiography using PLAX images in 2D / M mode is used to measure various parameters, including LVIDd / s, IVSd, LVPWd, and shortening ratio. These parameters are used to evaluate left ventricular lumen size, wall thickness, and short-axis (radial) function. Diagnostic criteria for dilated ventricle (DCM) include LVIDd / s >112% (2 SD) adjusted for age and body surface area (BSA). A shortening ratio of less than 25% is a diagnostic criterion for DCM in the presence of dilated ventricle (Mathew et al. Echo Res Pract. 4:G1-G13 (2017)).

[0067] Quantitative assessment of the structure and function of the left and right ventricles, with particular reference to short-axis and long-axis (longitudinal) function and focal wall motion abnormalities, is performed by echocardiography in 2D mode using apical four-chamber (A4C) images. Ejection fraction (EF) is estimated using the two-view Simpsons method. An EF of less than 45% is a diagnostic criterion for dilated ventricle comorbidity (DCM) in the presence of dilated ventricles (Mathew et al. Echo Res Pract. 4: G1-G13 (2017)).

[0068] Administration The rAAV virions and compositions of this disclosure may be administered systemically to subjects in need, for example, by intravenous, intra-arterial, or intraperitoneal delivery of the vector, as demonstrated in animal models (Katz et al., Gene Ther 19:659-669 (2012)). In some embodiments, the rAAV virions and compositions of this disclosure treat or prevent heart failure. In some embodiments, cardiomyopathy, where the vector is administered systemically. In some embodiments, the rAAV virions are administered by intravenous or intra-coronary injection.

[0069] In some embodiments, rAAV transduces cardiac cells. In some embodiments, rAAV virions transduce cardiomyocytes.

[0070] In some embodiments, transduction of rAAV increases DWORF polypeptide expression in the target heart. "Increased DWORF polypeptide expression" typically represents an expression of at least 5%, 10%, 15%, 20%, or higher compared to a control subject or tissue not treated with the vector. In some embodiments, detectable expression means an expression 1.5, 2, 2.5, or 3 times higher than that of a vector-free control. Expression can be assessed by Western blotting, enzyme-coupled immunosorbent assay (ELISA), or other methods known in the art, as described in the following examples. In some examples, expression is measured quantitatively using a standard curve. The standard curve can be prepared using purified protein, e.g., purified DWORF polypeptide, as described in the examples or by methods known in the art. Alternatively, the expression of the therapeutic gene product can be assessed by quantification of the corresponding mRNA.

[0071] In some aspects, increased DWORF expression in cardiac tissue is 3 × 10 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 x 10 13 vg / kg or less, 6 x 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 × 10⁻⁶ 13 This occurs at a vector genome count (vg) per kilogram of body weight (kg) of the subject, or less than vg / kg.

[0072] Pharmaceutical compositions and kits The rAAV virions of this disclosure are typically delivered to the target as a pharmaceutical composition. The pharmaceutical composition comprises a pharmaceutically acceptable solvent (e.g., water) and one or more excipients. In some embodiments, the pharmaceutical composition comprises a buffer at approximately neutral pH (pH 5, 6, 7, 8, or 9). In some embodiments, the pharmaceutical composition comprises phosphate-buffered saline (e.g., PBS at pH about 7). The pharmaceutical composition may comprise a pharmaceutically acceptable salt. The concentration of the salt may be selected to ensure that the pharmaceutical composition is isotonic or nearly isotonic to the target tissue.

[0073] In various embodiments, the compositions described herein comprise pharmaceutically acceptable vehicles (e.g., carriers, diluents, and excipients) for injectable formulations. These may particularly be isotonic, sterile saline solutions (monosodium or disodium phosphate, sodium chloride, potassium, calcium or magnesium, etc., or mixtures of such salts), or dry, particularly lyophilized, compositions to which sterile water or physiological saline, as appropriate, will constitute an injectable solution. Exemplary pharmaceutically appropriate forms for injection use include, for example, sterile aqueous solutions or dispersions, formulations comprising sesame oil, peanut oil, or aqueous propylene glycol, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions.

[0074] In various embodiments, the pharmaceutical compositions of this disclosure contain approximately 1 x 10⁶ per milliliter. 8 Genome copy (GC / mL), approximately 5 x 10 8 GC / mL, approximately 1 x 10 9 GC / mL, approximately 5 x 10 9 GC / mL, approximately 1 x 10 10 GC / mL, approximately 5 x 10 10 GC / mL, approximately 1 x 10 11 GC / mL, approximately 5 x 10 11 GC / mL, approximately 1 x 10 12 GC / mL, approximately 5 x 10 12 GC / mL, approximately 5 x 10 13 GC / mL, or approximately 1 x 1014 The GC / mL viral vector (e.g., rAAV virion) is included. In various embodiments, the pharmaceutical composition of this disclosure contains about 1 x 10¹⁶ per milliliter. 8 Genome copy (GC / mL), approximately 5 x 10 8 GC / mL ~ approx. 1 x 10 9 GC / mL, approximately 1 x 10 9 GC / mL ~ approx. 5 x 10 9 GC / mL, approximately 5 x 10 9 GC / mL ~ approx. 1 x 10 10 GC / mL, approximately 1 x 10 10 GC / mL ~ approx. 5 x 10 10 GC / mL, approximately 5 x 10 10 GC / mL ~ approx. 1 x 10 11 GC / mL, approximately 1 x 10 11 GC / mL ~ approx. 5 x 10 11 GC / mL, approximately 5 x 10 11 GC / mL ~ approx. 1 x 10 12 GC / mL, approximately 1 x 10 12 GC / mL ~ approx. 5 x 10 12 GC / mL, approximately 5 x 10 12 GC / mL ~ approx. 5 x 10 13 GC / mL, or approximately 5 x 10 13 GC / mL ~ approx. 1 x 10 14 The GC / mL viral vector (e.g., rAAV virion) is included. In various further embodiments, the pharmaceutically acceptable composition of this disclosure is approximately 5 x 10 8 GC / mL ~ approx. 5 x 10 9 GC / mL, approximately 5 x 10 9 GC / mL ~ approx. 5 x 10 10 GC / mL, approximately 5 x 10 10 GC / mL ~ approx. 5 x 10 11 GC / mL, approximately 5 x 10 11 GC / mL ~ approx. 5 x 10 12 GC / mL, or approximately 5 x 10 12 GC / mL ~ approx. 1 x 10 14It contains a GC / mL viral vector (e.g., rAAV virion). In a further embodiment, the pharmaceutical composition of this disclosure is approximately 5 x 10 8 GC / mL ~ approx. 5 x 10 10 GC / mL, approximately 5 x 10 10 GC / mL ~ approx. 5 x 10 12 GC / mL, or approximately 5 x 10 12 GC / mL ~ approx. 1 x 10 14 Contains a GC / mL viral vector (e.g., rAAV virion).

[0075] In some embodiments, the pharmaceutical compositions of the present disclosure are administered in a total volume of about 10 μL, about 20 μL, about 30 μL, about 40 μL, about 50 μL, about 60 μL, about 70 μL, about 80 μL, about 90 μL, about 100 μL, about 110 μL, about 120 μL, about 130 μL, about 140 μL, about 150 μL, about 160 μL, about 170 μL, about 180 μL, about 190 μL, or about 200 μL. In some embodiments, the pharmaceutical compositions of the present disclosure are in volumes of approximately 10 μL to approximately 20 μL, approximately 20 μL to approximately 30 μL, approximately 30 μL to approximately 40 μL, approximately 40 μL to approximately 50 μL, approximately 50 μL to approximately 60 μL, approximately 60 μL to approximately 70 μL, approximately 70 μL to approximately 80 μL, approximately 80 μL to approximately 90 μL, approximately 90 μL to approximately 100 μL, and approximately 100 μL to approximately 11 It is administered in a total volume of 0 μL, approximately 110 μL to 120 μL, approximately 120 μL to 130 μL, approximately 130 μL to 140 μL, approximately 140 μL to 150 μL, approximately 150 μL to 160 μL, approximately 160 μL to 170 μL, approximately 170 μL to 180 μL, approximately 180 μL to 190 μL, or approximately 190 μL to 200 μL.

[0076] The genome copy number per milliliter can be determined by quantitative polymerase chain reaction (qPCR) using a standard curve created with a reference sample containing the polynucleotide genome of this virus at known concentrations. In the case of AAV, the reference sample used is often the transfer plasmid used in the generation of rAAV virions, but other reference samples may also be used.

[0077] Alternatively or additionally, the concentration of the viral vector can be determined by measuring the titer of the vector against the cell line. Viral titer is typically expressed as the number of viral particles (vp) per unit volume (e.g., vp / mL). In various aspects, the pharmaceutical composition of the present disclosure comprises about 1 x 10 8 viral particles (vp / mL), about 5 x 10 8 vp / mL, about 1 x 10 9 vp / mL, about 5 x 10 9 vp / mL, about 1 x 10 10 vp / mL, about 5 x 10 10 vp / mL, about 1 x 10 11 vp / mL, about 5 x 10 11 vp / mL, about 1 x 10 12 vp / mL, about 5 x 10 12 vp / mL, about 5 x 10 13 vp / mL, or about 1 x 10 14 vp / mL of viral vector (e.g., rAAV virions). In various further aspects, the pharmaceutical composition of the present disclosure comprises from about 1 x 10 8 viral particles (vp / mL) to about 5 x 10 8 vp / mL, from about 5 x 10 8 vp / mL to about 1 x 10 9 vp / mL, from about 1 x 10 9 vp / mL to about 5 x 10 9 vp / mL, from about 5 x 10 9 vp / mL to about 1 x 10 10 vp / mL, from about 1 x 10 10 vp / mL to about 5 x 10 10 vp / mL, from about 5 x 10 10 vp / mL to about 1 x 10 11 vp / mL, from about 1 x 10 11 vp / mL to about 5 x 10 11 vp / mL, from about 5 x 10 11 vp / mL to about 1 x 10 12 vp / mL, from about 1 x 10 12 vp / mL to about 5 x 10 12vp / mL, approximately 5 x 10 12 vp / mL ~ approx. 5 x 10 13 vp / mL, or approximately 5 x 10 13 vp / mL ~ approx. 1 x 10 14 Contains a vp / mL viral vector (e.g., rAAV virion).

[0078] In one embodiment, the present disclosure provides a kit comprising a container for housing the pharmaceutical compositions described herein. [Examples]

[0079] The following embodiments and drawings are included to illustrate preferred embodiments of the Disclosure. Those skilled in the art will understand that the techniques disclosed in these embodiments or drawings are techniques found by the inventors to function well in carrying out the Disclosure and can therefore be considered to constitute a preferred form of implementation. However, those skilled in the art will understand that many modifications can be made in light of the Disclosure without departing from the spirit and scope of the Disclosure, and similar or comparable results can still be obtained.

[0080] Example 1 Results. To investigate the therapeutic capacity of DWORF gene therapy in heart failure, we constructed a safe and effective adeno-associated virus (AAV) approach for in vivo gene delivery (Lin et al. Circ Res. 115:354-63 (2014)). AAV serotype 9 (AAV9) was selected due to its cardiac tropism, and a cardiac troponin-T (cTnT) promoter was used to drive cardiomyocyte-specific expression (Addgene plasmid #69915) (Lin et al. Circ Res. 115:354-63 (2014)). AAV9-cTnT-DWORF (AAV-DWORF) and control AAV9-cTnT-tdTomato (AAV-tdTomato) viruses were introduced at 5x10⁻¹⁰ days postnatally (P5). 13The viral genome / kilogram was administered via intraperitoneal injection to mice for validation. Protein expression was assessed by Western blot analysis after 4 weeks, and cardiac-specific overexpression of DWORF (16.9 ± 2.4 times) and tdTomato was observed (Figure 2A). The efficacy of AAV-DWORF gene therapy was evaluated in a mouse model of DCM caused by gene deletion of muscle-specific LIM protein (MLP, encoded by the Cspr3 gene). Similar to the protective effects previously observed through transgenic overexpression of DWORF in MLP knockout (KO) mice (Makarewich et al. Elife. 7 (2018)), echocardiography of MLP KO mice treated with AAV-DWORF at P5 showed a significant improvement in cardiac function compared to 8-week-old control MLP KO / AAV-tdTomato mice (Figure 2B). Furthermore, adverse cardiac remodeling, characterized by ventricular wall thinning, chamber dilation, and increased heart weight to tibia length measurements, was mitigated in MLP KO / AAV-DWORF mice compared to MLP KO / AAV-tdTomato animals (Figures 2B and 2C). The degree of cardioprotection observed in MLP KO / AAV-DWORF mice was less than that in MLP KO / DWORF Tg mice (Makarewich et al. Elife. 7 (2018)), which is thought to be due to the lower level of DWORF overexpression achieved by AAV delivery (16.9 ± 2.4 times) compared to DWORF Tg overexpression (58.5 ± 14.7 times) (Figure 2A). Nevertheless, these results indicate that enhancement of SERCA activity through DWORF gene therapy is an effective and promising therapeutic strategy.

[0081] Next, we tested the ability of DWORF gene therapy to improve cardiac outcomes in a myocardial infarction (MI) model of heart failure. Mice received either AAV-DWORF or AAV-tdTomato gene therapy at P5 and were subjected to MI by sham surgery or permanent ligation of the left coronary artery at 8 weeks of age, and the induction and progression of heart failure were monitored for 12 weeks. Similar to previous observations in other models of heart failure (Makarewich et al. Elife. 7 (2018); Nelson et al. Science. 351;271-275 (2016)), endogenous DWORF protein expression decreased in the heart in response to MI (a 3.4 ± 1.0-fold decrease) (Figure 2D), as detected by Western blot analysis, which is thought to contribute to the reduction in SERCA activity that causes heart failure. Western blot analysis also showed overexpression of DWORF via AAV in both sham (14.9±1.0x) and MI samples (17.0±4.8x) at the final 12-week postoperative point (Figure 2D). Cardiac function was assessed by echocardiography in mice at baseline (pre-surgery) and after MI (Figure 2E). Compared to MI / AAV-tdTomato mice, MI / AAV-DWORF mice showed a significant improvement in ventricular function as measured by shortening rate (Figure 2D), and also a marked reduction in cardiac dilation (Figures 2E and 2F). Histological analysis of the heart by Masson's trichrome staining did not show significant differences in infarct size between the groups (Figure 2F). The inability of AAV-DWORF to fully restore cardiac function is thought to reflect the permanent loss of cardiomyocytes due to ischemia, and therefore the beneficial effects of DWORF are limited to living cardiomyocytes.

[0082] Discussion. Compared to previous SERCA gene therapy approaches used in heart failure clinical trials (Penny et al. Hum Gene Ther. 28:378-384 (2017)), AAV-DWORF may offer therapeutic advantages for several reasons. Firstly, the small size of the DWORF micropeptide (34 amino acids) allows it to be translated more efficiently compared to SERCA, a much larger (nearly 1,000 amino acids) multi-pass transmembrane protein. Furthermore, previous studies suggest that DWORF has a higher apparent affinity for SERCA than the inhibitory peptide phospholamban, and can respond to hyperinhibition of SERCA in phospholamban transgenic mice. Therefore, overexpression of DWORF is thought to reduce SERCA inhibition in heart failure induced by an increased phospholamban-to-SERCA ratio (Kranias et al. Circ Res. 110:1646-1660 (2012)). Furthermore, DWORF expression itself is reduced in various mouse models of human heart failure and hereditary and acquired cardiomyopathy (Makarewich et al. Elife. 7 (2018); Nelson et al. Science. 351;271-275 (2016)), which directly contributes to calcium dysregulation, and therefore increasing DWORF expression may be a crucial element in restoring calcium homeostasis in the disease. This example characterizes DWORF as a molecular inotropic agent that may be able to enhance SERCA activity and cardiomyocyte contractility, providing further evidence of its potential clinical validity as a therapeutic target in heart disease. In summary, the data presented herein indicate that DWORF gene therapy has potential as a novel heart failure therapy and represents a new approach compared to previous SERCA-level manipulations.

[0083] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes will be suggested to those skilled in the art in light of them, and that these should be incorporated into the spirit and scope of this application and the appended claims. All publications, patents, and patent applications referenced herein are incorporated herein by reference in their entirety for all purposes.

[0084] Sequence information SEQUENCE LISTING <110> THE BOARD OF REGENTS OF THE UNIVERSITY OF TEXAS SYSTEM <120> ADENO-ASSOCIATED VIRUS VECTOR FOR DWARF OPEN READING FRAME <150> US 63 / 048,743 <151> 2020-07-07 <160> 17 <170> PatentIn version 3.5 <210> 1 <211> 34 <212> PRT <213> Mus Musculus <400> 1 Met Ala Glu Lys Glu Ser Thr Ser Pro His Leu Met Val Pro Ile Leu 1 5 10 15 Leu Leu Val Gly Trp Ile Val Gly Cys Ile Ile Val Ile Tyr Ile Val 20 25 30 Phe Phe <210> 2 <211> 105 <212> DNA <213> Mus Musculus <400> 2 atggctgaga aagagtcaac atcaccacac ctcatggttc ccattcttct cctggttgga 60 tggattgtag gctgcatcat cgttatttac attgtcttct tctaa 105 <210> 3 <211> 35 <212> PRT <213> Homo Sapiens <400> 3 Put Ala Glu Lys Ala Gly Ser Thr Phe Ser His Leu Leu Val Pro Ile 1 5 10 15 Leu Leu Leu Ile Gly Trp Ile Val Gly Cys Ile Ile Met Ile Tyr Val 20 25 30 Val Phe Ser 35 <210> 4 <211> 108 <212> DNA <213> Homo Sapiens <400> 4 atggctgaaa aagcggggtc tacattttca caccttctgg ttcctattct tctcctgatt 60 ggctggattg tgggctgcat cataatgatt tatgttgtct tctcttag 108 <210> 5 <211> 35 <212> PRT <213> Artificial Sequence <220> <223> DWORF variant <400> 5 Met Ala Glu Lys Ala Glu Ser Thr Ser Pro His Leu Met Val Pro Ile 1 5 10 15 Leu Lion Leo Val Gly Trp Ile Val Gly Ile Ile Val Ile Tyr Ile 20 25 30 Val Phe Phe 35 <210> 6 <211> 108 <212> DNA <213> Artificial Sequence <220> <223> Variant DWORF <400> 6 atggctgaga aagcagagtc aacatcacca cacctcatgg ttcccattct tctcctggtt 60 ggatggattg taggctgcat catcgttatt tacattgtct tcttctaa 108 <210> 7 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> Variant DWORF <400> 7 Met Ala Glu Lys Glu Ser Thr Ser Pro His Leu Ile Val Pro Ile Leu 1 5 10 15 Leu Leu Val Gly Trp Ile Val Gly Cys Ile Ile Val Ile Tyr Ile Val 20 25 30 Where Where <210> 8 <211> 105 <212> DNA <213> Artificial Sequence <220> <223> DWORF variant <400> 8 atggctgaga aagagtcaac atcaccacac ctcattgttc ccattcttct cctggttgga 60 tggattgtag gctgcatcat cgttatttac attgtcttct tctaa 105 <210> 9 <211> 35 <212> PRT <213> Artificial Sequence <220> <223> DWORF variant <400> 9 Met Ala Glu Lys Ala Glu Ser Thr Ser Pro His Leu Ile Val Pro Ile 1 5 10 15 Leu Leu Leu Val Gly Trp Ile Val Gly Cys Ile Ile Val Ile Tyr Ile 20 25 30 Val Phe Phe 35 <210> 10 <211> 108 <212> DNA <213> Artificial Sequence <220> <223> DWORF variant <400> 10 atggctgaga aagcagagtc aacatcacca cacctcattg ttcccattct tctcctggtt 60 ggatggattg taggctgcat catcgttatt tacattgtct tcttctaa 108 <210> 11 <211> 413 <212> DNA <213> Artificial Sequence <220> <223> cTnT promoter <400> 11 gggataaaag cagtctgggc tttcacatga cagcatctgg ggctgcggca gagggtcggg 60 tccgaagcgc tgccttatca gcgtccccag ccctgggagg tgacagctgg ctggcttgtg 120 tcagcccctc gggcactcac gtatctccgt ccgacgggtt taaaatagca aaactctgag 180 gccacacaat agcttgggct tatatgggct cctgtggggg aagggggagc acggaggggg 240 ccggggccgc tgctgccaaa atagcagctc acaagtgttg cattcctctc tgggcgccgg 300 gcacattcct gctggctctg cccgccccgg ggtgggcgcc ggggggacct taaagcctct 360 gccccccaag gagcccttcc cagacagccg ccggcaccca ccgctccgtg gga 413 <210> 12 <211> 129 <212> DNA <213> Artificial Sequence <220> <223> AAV Inverted terminal repeat sequence <400> 12 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120 aggggttcc 129 <210> 13 <211> 129 <212> DNA <213> Artificial Sequence <220> <223> AAV Inverted terminal repeat sequence <400> 13 ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc 60 cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg 120 agcgcgcag 129 <210> 14 <211> 736 <212> PRT <213> Artificial Sequence <220> <223> AAV9 capsid polypeptide <400> 14 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser 1 5 10 15 Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro 20 25 30 Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly 145 150 155 160 Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro 180 185 190 Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly 195 200 205 Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser 210 215 220 Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile 225 230 235 240 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255 Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn 260 265 270 Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg 275 280 285 Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn 290 295 300 Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile 305 310 315 320 Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn 325 330 335 Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu 340 345 350 Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro 355 360 365 Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp 370 375 380 Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe 385 390 395 400 Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu 405 410 415 Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu 420 425 430 Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser 435 440 445 Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser 450 455 460 Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro 465 470 475 480 Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn 485 490 495 Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn 500 505 510 Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys 515 520 525 Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly 530 535 540 Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile 545 550 555 560 Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser 565 570 575 Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Gln Ala Gln 580 585 590 Thr Gly Trp Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp Gln 595 600 605 Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His 610 615 620 Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Met 625 630 635 640 Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala 645 650 655 Asp Pro Pro Thr Ala Phe Asn Lys Asp Lys Leu Asn Ser Phe Ile Thr 660 665 670 Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln 675 680 685 Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn 690 695 700 Tyr Tyr Lys Ser Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly Val 705 710 715 720 Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu 725 730 735 <210> 15 <211> 2209 <212> DNA <213> Artificial Sequence <220> <223> AAV9 nucleic acid <400> 15 atggctgccg atggttatct tccagattgg ctcgaggaca accttagtga aggaattcgc 60 gagtggtggg ctttgaaacc tggagcccct caacccaagg caaatcaaca acatcaagac 120 aacgctcgag gtcttgtgct tccgggttac aaataccttg gacccggcaa cggactcgac 180 aaggggagc cggtcaacgc agcagacgcg gcggccctcg agcacgacaa ggcctacgac 240 cagcagctca aggccggaga caacccgtac ctcaagtaca accacgccga cgccgagttc 300 caggagcggc tcaaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360 gccaaaaaga ggcttcttga acctcttggt ctggttgagg aagcggctaa gacggctcct 420 ggaaagaaga ggcctgtaga gcagtctcct caggaaccgg actctcccgc gggtattggc 480 aaatcgggtg cacagcccgc taaaaagaga ctcaatttcg gtcagactgg cgacacagag 540 tcagtcccag accctcaacc aatcggagaa cctcccgcag ccccctcagg tgtgggatct 600 cttacaatgg cttcaggtgg tggcgcacca gtggcagaca ataacgaagg tgccgatgga 660 gtgggtagtt cctcgggaaa ttggcattgc gattcccaat ggctggggga cagagtcatc 720 accaccagca cccgaacctg ggccctgccc acctacaaca atcacctcta caagcaaatc 780 tccaacagca catctggagg atcttcaaat gacaacgcct acttcggcta cagcacccc 840 tggggtatt ttgacttcaa cagattccac tgccacttct caccacgtga ctggcagcga 900 ctcatcaaca acaactgggg attccggcct aagcgactca acttcaagct cttcaacatt 960 caggtcaaag aggttacgga caacaatgga gtcaagacca tcgccaataa ccttaccagc 1020 acggtccagg tcttcacgga ctcagactat cagctcccgt acgtgctcgg gtcggctcac 1080 gagggctgcc tcccgccgtt cccagcggac gttttcatga ttcctcagta cgggtatctg 1140 acgcttaatg atggaagcca ggccgtgggt cgttcgtcct tttactgcct ggaatatttc 1200 ccgtcgcaaa tgctaagaac gggtaacaac ttccagttca gctacgagtt tgagaacgta 1260 cctttccata gcagctacgc tcacagccaa agcctggacc gactaatgaa tccactcatc 1320 gaccaatact tgtactatct ctcaaagact attaacggtt ctggacagaa tcaacaaacg 1380 ctaaaattca gtgtggccgg acccagcaac atggctgtcc agggaagaaa ctacatacct 1440 ggacccagct accgacaaca acgtgtctca accactgtga ctcaaaacaa caacagcgaa 1500 tttgcttggc ctggagcttc ttcttgggct ctcaatggac gtaatagctt gatgaatcct 1560 ggacctgcta tggccagcca caaagaagga gaggaccgtt tctttccttt gtctggatct 1620 ttaatttttg gcaaacaagg aactggaaga gacaacgtgg atgcggacaa agtcatgata 1680 accaacgaag aagaaattaa aactactaac ccggtagcaa cggagtccta tggacaagtg 1740 gccacaaacc accagagtgc ccaagcacag gcgcagaccg gctgggttca aaaccaagga 1800 atacttccgg gtatggtttg gcaggacaga gatgtgtacc tgcaaggacc catttgggcc 1860 aaaattcctc acacggacgg caactttcac ccttctccgc tgatgggagg gtttggaatg 1920 aagcacccgc ctcctcagat cctcatcaaa aacacacctg tacctgcgga tcctccaacg 1980 gccttcaaca aggacaagct gaactctttc atcacccagt attctactgg ccaagtcagc 2040 gtggagatcg agtgggagct gcagaaggaa aacagcaagc gctggaaccc ggagatccag 2100 tacacttcca actattacaa gtctaataat gttgaatttg ctgttaatac tgaaggtgta 2160 tatagtgaac cccgccccat tggcaccaga tacctgactc gtaatctgt 2209 <210> 16 <211> 1684 <212> DNA <213> Artificial Sequence <220> <223> Expression cassette <400> 16 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120 aggggttcct tgtagttaat gattaacccg ccatgctact tatctaccag ggtaatgggg 180 atcctctaga actatagcta gaattcgccc ttacgggccc cccctcgagg tcgggataaa 240 agcagtctgg gctttcacat gacagcatct ggggctgcgg cagagggtcg ggtccgaagc 300 gctgccttat cagcgtcccc agccctggga ggtgacagct ggctggcttg tgtcagcccc 360 tcgggcactc acgtatctcc gtccgacggg tttaaaatag caaaactctg aggccacaca 420 atagcttggg cttatatggg ctcctgtggg ggaaggggga gcacggaggg ggccggggcc 480 gctgctgcca aaatagcagc tcacaagtgt tgcattcctc tctgggcgcc gggcacattc 540 ctgctggctc tgcccgcccc ggggtgggcg ccggggggac cttaaagcct ctgcccccca 600 aggagccctt cccagacagc cgccggcacc caccgctccg tgggacgatc cccgaagctc 660 tagagcttta ttgcggtagt ttatcacagt taaattgcta acgcagtcag tgcttctgac 720 780. acaacagtct cgaacttaag ctgcagaagt tggtcgtgag gcactgggca ggtaagtatc aaggttacaa gacaggttta aggagaccaa tagaaactgg gcttgtcgag acagagaaga ctcttgcgtt tctgataggc acctattggt cttactgaca tccactttgc ctttctctcc 900 acaggtgtcc actcccagtt caattacagc tcttaaggct agagtactta attackca ctataggcta gccgccacca tggctgagaa agagtcaaca tcaccacacc tcatggttcc cattcttc ctggttggat ggattgtagg ctgcatcatc gttatttaca ttgtcttctt 1080 ctaacggccg cgcggatcca gacatgata gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt agtcgacccg ggcggcctcg aggacggggt 1320 gaactacgcc tgaggatccg atctttttcc ctctgccaaa aattatgggg acatcatgaa gccccttgag catctgactt ctggctaata aaggaaattt attttcattg caatagtgtg ttggaattt ttgtgtctct cactcggaag caattcgttg atctgaattt cgaccacca 1500 tatacccat taccctggta gataagtagc atggcgggtt aatcattaac tacaaggaac 1560 ccctagtgat ggagttggcc actccctctc tgcgcgctg ctcgctcact gaggccgggc 1620 gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc 1680 gcag 1684 <210> 17 <211> 134 <212> DNA <213> Artificial Sequence <220> <223> polyadenylation sequence <400> 17 gatccagaca tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga 60 aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc 120 tgcaataaac augt 134

Claims

1. A method for treating a subject in need thereof, comprising the step of administering an effective amount of recombinant adeno-associated virus (rAAV) virion, wherein the rAAV virion comprises an expression cassette containing an AAV capsid and a polynucleotide encoding a DWORF polypeptide functionally linked to a promoter.

2. A method for treating heart failure according to claim 1.

3. A method for preventing heart failure as described in claim 1.

4. The method according to claim 1, wherein the subject is suffering from or at risk of cardiomyopathy.

5. The method according to claim 1, wherein the subject is suffering from or at risk of dilated cardiomyopathy.

6. The method according to claim 1, wherein the subject has an allele with a genetic risk of heart failure.

7. The method according to claim 1, wherein the heart failure is myocardial infarction.

8. The method according to claim 1, wherein the heart failure is heart failure with reduced ejection fraction (HFrEF).

9. The method according to claim 1, wherein the heart failure is heart failure with preserved ejection fraction (HFpEF).

10. The method according to any one of claims 1 to 9, wherein the subject is suffering from or at risk of myocardial infarction.

11. The method according to claim 10, wherein the myocardial infarction is chronic myocardial infarction.

12. The method according to claim 10, wherein the myocardial infarction is acute myocardial infarction.

13. The method according to any one of claims 1 to 12, wherein the subject has an allele with a genetic risk of heart failure.

14. A method according to any one of claims 1 to 13, which brings about the expression of a DWORF polypeptide in the target heart.

15. The method according to any one of claims 1 to 14, wherein no detectable expression of the DWORF polypeptide occurs in the target muscle, excluding the heart.

16. The method according to any one of claims 1 to 15, wherein no detectable expression of the DWORF polypeptide is produced in the target liver.

17. A method according to any one of claims 1 to 16, which brings about the expression of a DWORF polypeptide in cardiomyocytes.

18. The method according to any one of claims 1 to 17, which does not result in detectable expression of the DWORF polypeptide in cardiac fibroblasts.

19. A method according to any one of claims 1 to 18, for improving one or more indicators of cardiac function, optionally shortening rate and / or left ventricular diameter (LVID).

20. The method according to claim 18, wherein improvement in cardiac function is observed between 2 and 12 weeks.

21. A method according to any one of claims 1 to 20 for reducing cardiac remodeling.

22. A method according to any one of claims 1 to 21 for preventing a decrease in DWORF expression in a subject suffering from myocardial infarction.

23. The method according to any one of claims 1 to 22, wherein rAAV virion is administered by intravenous or intracoronary injection.

24. The method according to claim 17, which increases SERCA activity.

25. The method according to any one of claims 1 to 24, wherein the rAAV virion is an rAAV virion of serotype AAV9.

26. The method according to any one of claims 1 to 25, wherein the AAV capsid comprises a capsid protein having at least 98% identity with SEQ ID NO:

14.

27. The method according to any one of claims 1 to 26, wherein the AAV capsid comprises a capsid protein having at least 99% identity with SEQ ID NO:

14.

28. The method according to any one of claims 1 to 27, wherein the AAV capsid comprises a capsid protein containing a polypeptide sequence of SEQ ID NO:

14.

29. The method according to any one of claims 1 to 28, wherein the promoter is a cardiac troponin-T (cTnT) promoter.

30. The method according to any one of claims 1 to 29, wherein the cardiac troponin-T (cTnT) promoter comprises a polynucleotide sequence having at least 95% identity with SEQ ID NO:

11.

31. The method according to any one of claims 1 to 30, wherein the cardiac troponin-T (cTnT) promoter comprises a polynucleotide sequence having at least 98% identity with SEQ ID NO:

11.

32. The method according to any one of claims 1 to 31, wherein the cardiac troponin-T (cTnT) promoter comprises a polynucleotide sequence of SEQ ID NO:

11.

33. The method according to any one of claims 1 to 32, wherein the DWORF polypeptide is a human DWORF polypeptide.

34. The method according to any one of claims 1 to 33, wherein the DWORF polypeptide comprises a polypeptide sequence having 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.

35. The method according to any one of claims 1 to 34, wherein the DWORF polypeptide comprises a polypeptide sequence having 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.

36. The method according to any one of claims 1 to 35, wherein 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.

37. The method according to any one of claims 1 to 36, wherein the expression cassette is adjacent to an AAV inverted terminal repeat (ITR).

38. The method according to any one of claims 1 to 37, wherein the ITR is an AAV2 ITR.

39. The method according to any one of claims 1 to 38, wherein the ITR comprises a polynucleotide sequence of SEQ ID NO: 12 or SEQ ID NO:

13.

40. The method according to any one of claims 1 to 39, wherein the subject experiences improvement in symptoms after administration.

41. The method according to claim 40, wherein the improvement in symptoms is one or more of the following: enhanced contractility, reduced fatigue, reduced dyspnea, reduced edema, reduced chest pain, reduced arrhythmia, reduced thrombosis, improved cardiac valve function, and reduced heart murmur.

42. A recombinant adeno-associated virus (rAAV) virion comprising an AAV capsid and an expression cassette containing a polynucleotide encoding a DWORF polypeptide functionally linked to a promoter.

43. The rAAV virion according to claim 42, which is an rAAV virion of serotype AAV9.

44. The rAAV virion according to claim 42 or 43, wherein the AAV capsid comprises a capsid protein having at least 98% identity with SEQ ID NO:

14.

45. The rAAV virion according to any one of claims 42 to 44, wherein the AAV capsid comprises a capsid protein having at least 99% identity with SEQ ID NO:

14.

46. The rAAV virion according to any one of claims 42 to 45, wherein the AAV capsid comprises a capsid protein containing the polypeptide sequence SEQ ID NO:

14.

47. The rAAV virion according to any one of claims 42 to 46, wherein the promoter is a cardiac troponin-T (cTnT) promoter.

48. The rAAV virion according to any one of claims 42 to 47, wherein the cardiac troponin-T (cTnT) promoter comprises a polynucleotide sequence having at least 95% identity with SEQ ID NO:

11.

49. The rAAV virion according to any one of claims 42 to 48, wherein the cardiac troponin-T (cTnT) promoter comprises a polynucleotide sequence having at least 98% identity with SEQ ID NO:

11.

50. The rAAV virion according to any one of claims 42 to 49, wherein the cardiac troponin-T (cTnT) promoter comprises a polynucleotide sequence of SEQ ID NO:

11.

51. The rAAV virion according to any one of claims 42 to 50, wherein the DWORF polypeptide is a human DWORF polypeptide.

52. The rAAV virion according to any one of claims 42 to 51, wherein the DWORF polypeptide comprises a polypeptide sequence having 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.

53. The rAAV virion according to any one of claims 42 to 52, wherein the DWORF polypeptide comprises a polypeptide sequence having at least 98% identity with respect to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO:

9.

54. The rAAV virion according to any one of claims 42 to 53, wherein 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.

55. The rAAV virion according to any one of claims 42 to 54, wherein the expression cassette is adjacent to an AAV inverted terminal repeat (ITR).

56. The rAAV billion according to any one of claims 42 to 55, wherein the ITR is an AAV2 ITR.

57. The rAAV virion according to any one of claims 42 to 56, wherein the ITR comprises a polynucleotide sequence of SEQ ID NO: 12 or SEQ ID NO:

13.

58. A pharmaceutical composition comprising a recombinant adeno-associated virus (rAAV) virion according to any one of claims 42 to 57 and a pharmaceutically acceptable carrier.

59. A kit comprising a container for containing the pharmaceutical composition according to claim 58.

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