Prevention and treatment of cardiomyopathy-related diseases with ATF4 gene therapy

Abstract

This present disclosure provides compositions and methods of increasing activating transcription 4 (ATF4) expression in a subject, thereby preventing and / or treating cardiomyopathy-related diseases in the subject.

Description

PATENT Attorney Docket No.: 079445-015010PC-1508345 Client Reference No.: S23-405 PREVENTION AND TREATMENT OF CARDIOMYOPATHY- RELATED DISEASES WITH ATF4 GENE THERAPY CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No.63 / 676,521, filed July 29, 2024, the disclosure of which is herein incorporated by reference in its entirety for all purposes. STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with Government support under contract HL139679 awarded by the National Institutes of Health. The Government has certain rights in the invention. BACKGROUND

[0003] Heart failure (HF) affects more than 26 million people worldwide and is one of the most important threats to the sustainability of health systems in the United States. HF is a complex cardiovascular disease characterized by the inability of the heart to pump blood effectively, leading to a decline in its normal functions. Despite major improvements in the understanding of risk factors for incident HF, this knowledge has not yet been fully translated into effective interventions for the primary prevention of HF, except for blood pressure (BP) lowering medications and statins.

[0004] Dilated cardiomyopathy (DCM) is a leading cause of heart failure (HF) and the most common cause of cardiac transplantation, with a population prevalence of 1 in 400 to 1:2500 individuals. DCM is often a genetic disorder, such that genetic mutation accounts for up to half of reported cases. Nevertheless, identifying a pathogenic variant does not profoundly change clinical management. Current treatment strategies are directed toward symptoms to preserve myocardial function. Given the consistently high morbidity and mortality of DCM, there is a pressing need to develop therapies that address underlying genetic basis of the disease.

[0005] The most obvious therapeutic approach to DCM would be to correct the genetic perturbation in the individual patient. Although innovative genomic editing techniques have been devised to correct specific disease-causing variants in vitro, translating these approaches into clinical therapies is challenging and, regardless, would involve development of bespoke therapies for each of the 1000s of mutation in over 50 different genes. Instead, development of therapeutic approaches addressing common mechanisms of pathogenesis in DCM would presumably yield treatments that would be more readily and broadly clinically deployed. Despite advances in our understanding of DCM etiology, however, no mechanism-based therapeutics have been devised for DCM. A significant challenge in the field is to define common convergent pathogenic mechanisms that connect the different mutant genes to the DCM phenotype and identify therapeutic targets. SUMMARY

[0006] In one aspect, the present disclosure provides a recombinant polynucleotide comprising a nucleic acid sequence encoding an activating transcription 4 (ATF4) operably linked to a cardiac myocyte-specific promoter. In some embodiments, the ATF4 is a human ATF4. In some embodiments, the ATF4 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 1. In some embodiments, the promoter is a cardiac troponin T (cTnT) promoter. In some embodiments, the cTnT promoter comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO: 3 or 5. In some embodiments, the nucleic acid sequence has at least 80% identity to SEQ ID NO: 4 or 6.

[0007] In another aspect, the present disclosure provides a vector comprising the recombinant polynucleotide described herein. In some embodiments, the vector is an adeno- associated virus (AAV) vector. In some embodiments, the AAV vector is an AAV serotype 9 (AAV9) vector. In some embodiments, the AAV vector is a self-complementary AAV (scAAV) vector.

[0008] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising a recombinant polynucleotide described herein or a vector described herein. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0009] In one aspect, the present disclosure provides a method for improving a cardiac systolic function in a subject. In some embodiments, the method comprises administering tothe subject a therapeutically effective amount of the pharmaceutical composition described herein.

[0010] In one aspect, the present disclosure provides a method for preventing or treating a cardiovascular disease in a subject. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 11. In some embodiments, the cardiovascular disease is dilated cardiomyopathy (DCM).

[0011] In one aspect, the present disclosure provides a method for preventing a heart failure in a subject. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of the pharmaceutical composition described herein. In some embodiments, the subject is a human. In some embodiments, the subject has been diagnosed with cardiomyopathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG.1 illustrates that AAV9sc.hATF4 restores the cardiac systolic function in Tpm1 E54k transgenic (Tg) mice. The Tpm1 E54k mice are transgenic FVB / N mice expressing a Myh6-Tpm1 E54K cDNA transgene resulting in replacement of endogenous -tropomyosin with mutant protein; WT refers to nontransgenic wild-type littermates. WT and Tg littermates were injected intravenously by the tail vein with 3.5e11 vg AAV9sc.hATF4 or AAV9sc.GFP after the onset of DCM at 6-weeks of age. Cardiac function was assessed by serial Echocardiography at 2-, 6-, 8- and 16-weeks post-AAV injection; n = 7-9. Analysis of cardiac function shows that AAV9sc.hATF4 improves systolic function, including A. Ejection Fraction (EF) B. Fractional Shortening (FS); C. Cardiac Output; and D. Stroke Volume.

[0013] FIG. 2 illustrates AAV9.ATF4 halts disease progression in Tpm1E54K / + DCM mice. (A) When DCM pathology is established at 6 weeks of age, mice carrying a mutation in the Tpm1 gene (TM54) were treated with AAV9.ATF4 (or AAV.GFP control) with 2.5x1013 viral genomes per kilo (vg / kg) and followed for 8 weeks. (B) Serial Echocardiography assessment shows that AAV9.ATF4 treatment prevents cardiac functional deterioration compared to AAV9.GFP controls. No effect on wild-type littermate control animals is observed, indicating no adverse effects. (C-D) Compared to AAV9.GFP controls TM54 mice treated with AAV9.ATF4 show improvement in ejection fraction (EF, %) and preserved systolic volume after 8 weeks, whilst AAV9.GFP treated mice show significant decline inejection function and increase in systolic volume. Data represent mean se. Cardiac Ejection fraction, EF; left ventricle, LV. ***P< 0.001. The P values were computed by 2-way ANOVA repeated measures. n=8-9 per group.

[0014] FIG. 3 illustrates that AAV9.ATF4 gene therapy ameliorates fibrosis and cellular hypertrophy in TM54 DCM mice at 8 weeks post treatment. (A) Representative images of histological examination, wheat germ agglutinin staining (WGA, cell size), Picrosirius red (fibrosis), and hematoxylin and eosin (H&E) staining of heart sections of TM54 and WT heart treated with AAV9.ATF4 or AAV9.GFP control as indicated. H&E staining shows unremarkable histopathological findings in the heart. (B-C) Quantitative analyses show that AAV9.ATF4 treatment normalizes ventricular cardiomyocytes size (cross sectional area) and reduces interstitial fibrosis.

[0015] FIG. 4 illustrates that AAV9.ATF4 gene therapy has a profound impact on myocardial metabolome of the TM54 DCM hearts at 8 weeks post-treatment. (A) Principal component (PCA) analysis based on all myocardial metabolites from AAV9.ATF4 and AAV9.GFP control TM54 hearts reveals distinct clusters. (B) AAV9.ATF4 treatment significantly increases the levels of Acylcarnitines, Amino Acids and Nucleotides in the TM54 DCM hearts.

[0016] FIG.5 illustrates AAV9.ATF4 gene therapy restores the expression of key metabolic genes in the TM54 DCM heart. (A) Key genes associated with branch chain amino acid (BCAA) catabolism, fatty acid beta oxidation and mitochondria electron transport chain are upregulated in AAV9.ATF4 compared to AAV9.GFP treated DCM hearts. Symbol colors indicate an increase (red) or decrease (blue) in gene expression. These genes are downregulated in DCM compared to wild type hearts. (B) The upregulated differentially expressed genes are enriched in pathways related to cardiac metabolism and energy homeostasis.

[0017] FIG.6 illustrates AAV9.ATF4 restores cardiac function in Tnnt2R183W / +DCM mice. (A) Schematic diagram of AAV9.ATF4 gene therapy efficacy study in Tnnt2R183W / +DCM mouse model. When DCM pathology is evident at 6 months of age, mice were treated with AAV9.ATF4 (or AAV.GFP control) at 2.5x1013viral genomes per kilo (vg / kg) and followed for 6 months. (B) Serial Echocardiography assessment shows that AAV9.ATF4 treatment significantly improves the cardiac function in Tnnt2R183W / +mice compared to AAV9.GFP controls. No effect on wild-type littermate control animals is observed, indicating no adverse effects. (C-D) Tnnt2R183W / +mice treated with AAV9.ATF4 show improvement in ejectionfraction (EF, %) and preserved systolic volume after 6 months, whilst AAV9.GFP treated mice show significant decline in ejection function and increase in systolic volume. Data represent mean se. Cardiac Ejection fraction, EF; left ventricle, LV. ***P< 0.001. The P values were computed by 2-way ANOVA repeated measures. n=15 Tnnt2R183W / +groups; n=4, WT groups. DETAILED DESCRIPTION I. INTRODUCTION

[0018] Currently, there is no cure for DCM and HF. The existing pharmacological therapies aim at alleviating the symptoms.

[0019] Recent evidence suggests that activation of ATF4-mediated one-carbon metabolism might be cardioprotective. Recent phenotypic screens19further shows that bolstering the ATF4-mediated serine biosynthesis pathway, which branches from glycolysis, can rescue contractile dysfunction in induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) carrying DCM pathogenic mutations in genes from diverse ontologies. Conversely, inhibition of ATF4 expression exacerbates the contractility deficit of DCM iPSC-CMs. These studiespointed to ATF4 as a regulator of cardiomyocyte function and a candid novel therapeutictarget for HF.

[0020] The present disclosure provides a new approach to increase the expression of ATF4, a transcription factor, specifically in the cardiac myocytes. Unlike conventional gene therapies, our approach does not replace a faulty or missing gene. Instead, our approach aims at triggering a cardioprotective effect by bolstering the ATF4-dependent integrative stress response in the failing heart. II. DEFINITIONS

[0021] Unless specifically indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure belongs. In addition, any method or material similar or equivalent to a method or material described herein can be used in the practice of the present disclosure. For purposes of the present disclosure, the following terms are defined.

[0022] The terms “a,” “an,” or “the” as used herein not only include aspects with one member, but also include aspects with more than one member. For instance, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus,for example, reference to “a cell” includes a plurality of such cells and reference to “the agent” includes reference to one or more agents known to those skilled in the art, and so forth.

[0023] As used herein, the terms “about” and “around” indicate a close range around a numerical value when used to modify that specific value. If “X” were the value, for example, “about X” or “around X” would indicate a value from 0.9X to 1.1X, e.g., a value from 0.95X to 1.05X, or a value from 0.98X to 1.02X, or a value from 0.99X to 1.01X. Any reference to “about X” or “around X” specifically indicates at least the values X, 0.9X, 0.91X, 0.92X, 0.93X, 0.94X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X, 1.09X, and 1.1X, and values within this range.

[0024] The term “recombinant” or “engineered” when used with reference, e.g., to a nucleic acid, protein, vector, or cell, indicates that the nucleic acid, protein vector, or cell, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non- recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.

[0025] The terms “polynucleotide” and “nucleic acid” are used interchangeably and as used herein refer to both sense and anti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. In particular embodiments, a nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form of either type of nucleotide, and combinations thereof. The terms also include, but is not limited to, single- and double-stranded forms of DNA. In addition, a polynucleotide, e.g., a cDNA or mRNA, may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide linkages. Nucleic acid molecules, e.g., oligonucleotide probes or priomers, may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analogue, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.). The above term isalso intended to include any topological conformation, including single-stranded, double- stranded, partially duplexed, triplex, hairpinned, circular and padlocked conformations. A reference to a nucleic acid sequence encompasses its complement unless otherwise specified. Thus, a reference to a nucleic acid molecule having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence. The term also includes codon-optimized nucleic acids that encode the same polypeptide sequence.

[0026] The term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0027] The term "promoter," as used herein, refers to a polynucleotide sequence capable of driving transcription of a coding sequence in a cell. Thus, promoters can include cis-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and / or rate of transcription of a gene. For example, a promoter can be a cis-acting transcriptional control element, further including an enhancer, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5' and 3' untranslated regions, or an intronic sequence, which are involved in transcriptional regulation. These cis- acting sequences typically interact with proteins or other biomolecules to carry out (turn on / off, regulate, modulate, etc.) gene transcription. As disclosed herein, the promoter can be a tissue- specific promoter, such as a cardiac myocyte-specific promoter.

[0028] “Percent sequence identity” or “percent identity” or equivalents used in the context of two nucleic acids or polypeptides, refers to a sequence that has at least a specified level of identity, e.g., at least 50% sequence identity with a reference sequence (e.g., any SEQ ID NO included herein). Alternatively, percent identity can be any integer from 50% to 100%. Some embodiments include at least: 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, compared to a reference sequence using the programs described herein, e.g., BLAST using standard parameters, as described below.

[0029] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparisonalgorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0030] A "comparison window", as used herein, includes reference to a segment of any one of the numbers of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well- known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection.

[0031] Algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol.215: 403-410 and Altschul et al. (1977) Nucleic Acids Res.25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site.

[0032] The term "vector" as used herein, refers to a recombinant construct in which a nucleic acid sequence of interest is inserted into the vector. The term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. As disclosed herein, the vector can be a viral vector. For example, viral vectors can be based on adeno-associated virus (AAV), vaccinia virus, poliovirus, retrovirus, lentivirus, adenovirus, SV40, herpes simplex virus, human immunodeficiency virus, and the like. In some embodiments, viral vectors can be virus- like particles. Other useful expression vectors are known to those of skill in the art, and many are commercially available. The following exemplary vectors are provided by way of example for eukaryotic host cells: pXT1, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40. Examples of techniques that may be used to introduce a viral vector into a cell include, but not limited to, viral or bacteriophage infection, transfection, protoplast fusion, lipofection, calcium phosphateprecipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, calcium phosphate precipitation, nanoparticle- mediated nucleic acid delivery, and the like. As disclosed herein, the vector can be an adeno- associated virus (AAV) vector. The term “AAV vector” refers to any vector which comprises or derives from components of AAV and is suitable to infect mammalian cells, preferably human cells. The term AAV vector typically designates an AAV type viral particle or virion comprising at least a nucleic acid molecule encoding a therapeutic protein (such as hATF4). As described herein, the AAV vector may be derived from various serotypes or from various genomes.

[0033] By “pharmaceutically acceptable,” it is meant that the excipient is compatible with the other ingredients of the formulation and is not deleterious to the recipient thereof.

[0034] As used herein, the terms “treatment,” “treating,” and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. “Treatment,” as used herein, can include treatment resulting in inhibiting the disease, i.e., arresting its development; and relieving the disease, i.e., causing regression of the disease. For example, in the case of dilated cardiomyopathy, a response to treatment can include complete response, partial response, stable disease, progressive disease, progression free survival, or overall survival.

[0035] An “effective amount” or “therapeutically effective amount” of an interfering RNA (e.g., siRNA) is an amount sufficient to produce the desired effect, e.g., an inhibition of expression of a target sequence in comparison to the normal expression level detected in the absence of an interfering RNA. In particular embodiments, inhibition of expression of a target gene or target sequence is achieved when the value obtained with an interfering RNA relative to the control is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. Suitable assays for measuring the expression of a target gene or target sequence include, but are not limited to, examination of protein or mRNA levels using techniques known to those of skill in the art, such as, e.g., dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art.

[0036] As used herein, the term “administering” or “administration” includes any route of introducing or delivering an agent (such as a pharmaceutical composition described herein) to a subject. Administration can be carried out by any route suitable for the delivery of the agent. Thus, delivery routes can include, e.g., oral, intranasal, intravenous, intramuscular, intraperitoneal, intraarticular, intradermal, or subcutaneous.

[0037] The terms “dose” and “dosage” are used interchangeably herein. A dose refers to the amount of active ingredient given to an individual at each administration. The dose will vary depending on a number of factors, including frequency of administration; size and tolerance of the individual; severity of the condition; risk of side effects; the route of administration; and the imaging modality of the detectable moiety (if present). One of skill in the art will recognize that the dose can be modified depending on the above factors or based on therapeutic progress. The term “dosage form” refers to the particular format of the pharmaceutical, and depends on the route of administration. For example, a dosage form can be in a liquid, e.g., a saline solution for injection.

[0038] “Subject,” “patient,” “individual” and like terms are used interchangeably and refer to, except where indicated, mammals such as humans and non-human primates, as well as rabbits, rats, mice, goats, pigs, dogs, cats, and other mammalian species. The term does not necessarily indicate that the subject has been diagnosed with a particular disease, but typically refers to an individual under medical supervision. A patient can be an individual that is seeking treatment, monitoring, adjustment or modification of an existing therapeutic regimen, etc. III. DESCRIPTION OF THE EMBODIMENTS

[0039] This present disclosure provides compositions comprising a recombinant polynucleotide that comprises a nucleic acid sequence encoding an activating transcription 4 (ATF4) operably linked to a cardiac myocyte-specific promoter. In some embodiments, cardiac myocyte-specific promoter is a cardiac troponin T (cTnT) promoter. Such compositions can be used to increase activating transcription 4 (ATF4) expression in a host cell, such as a cardiac myocyte. Cardiac myocytes, also called “cardiac muscle cells” or “cardiomyocytes”, are the contractile myocytes of the cardiac muscle. A cardiac myocyte is generally defined as a myocyte that 1) resides in the heart 2) is responsible for the heart’s contraction 3) develops from myoblasts 4) has a central nucleus 5) is smaller than skeletal myocytes and 6) has abundant sarcoplasm (See, Keepers B, Liu J, Qian L. Biochim Biophys Acta Mol Cell Res. 2020;1867(3):118464). In some embodiments, the recombinantpolynucleotide comprising the ATF4 expression cassette can be delivered into a host cell (e.g., a cardiac myocyte), through a vector. In some embodiments, the vector is a self- complementary, adeno-associated virus serotype 9 (scAAV9) vector. Such compositions can be also used to prevent and / or treat cardiomyopathy-related diseases, such as a dilated cardiomyopathy (DCM) or a heat failure, in a subject. 1. Activating Transcription 4 (ATF4)

[0040] Activating transcription factor 4 (ATF4) belongs to the ATF / CREB (activating transcription factor / cyclic AMP response element binding protein) family of basic region- leucine zipper (bZip) transcription factors, which have the consensus binding site cAMP responsive element (CRE). ATF4 is induced by stress signals including anoxia / hypoxia, endoplasmic reticulum stress, amino acid deprivation, and oxidative stress. ATF4 regulates the expression of genes involved in oxidative stress, amino acid synthesis, differentiation, metastasis, and angiogenesis. See, Ameri K, Harris AL. Int J Biochem Cell Biol. 2008;40(1):14-21. ATF4 also plays a central role in mitochondrial function, including amino acid synthesis and energy homeostasis, under physiological and pathophysiological conditions.1-3

[0041] The present disclosure provides a recombinant polynucleotide comprising a nucleic acid sequence encoding an activating transcription 4 (ATF4). In some embodiments, the ATF4 is a human ATF4 (Protein Accession: P18848 in UniProtKB / Swiss-Prot). In some embodiments, the ATF4 comprises an amino acid sequence having at least 80%, 85%, or 90% identical to SEQ ID NO: 1. In some embodiments, the ATF4 comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1; or comprises an amino acid sequence of SEQ ID NO: 1.

[0042] In some embodiments, the nucleic acid sequence encoding an ATF4 comprises a nucleic acid sequence having at least 80%, 85%, or 90% identical to SEQ ID NO: 2. In some embodiments, the nucleic acid sequence encoding an ATF4 comprises a sequence having at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 2; or comprises a sequence of SEQ ID NO: 2. 2. Promoters

[0043] In some embodiments, the nucleic acid sequence encoding an ATF4 is operably linked to a promoter. In some embodiments, the promoter is a tissue-specific promoter thatonly be activated in certain cell types. In some embodiments, the promoter is a cardiac myocyte-specific promoter, thereby the ATF4 can be specifically expressed in a cardiomyocyte. Non-limiting examples of the cardiac myocyte-specific promoter include cardiac troponin T (cTnT) promoter, myosin binding protein C3 (MYBPC3) promoter, troponin T2 (TNNT2) promoter, alpha cardiac actin (ACTC) promoter, and Trans-2,3-Enoyl- CoA Reductase Like (TECRL) promoter.

[0044] In some embodiments, the promoter is a cardiac troponin T (cTnT) promoter. In some embodiments, the cTnT promoter is a human cTnT promoter. In some embodiments, the cTnT promoter comprises a nucleic acid sequence having at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% identical to SEQ ID NO: 5. In some embodiments, the cTnT promoter comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 5; or comprises a sequence of SEQ ID NO: 5. In some embodiments, the cTnT promoter is a chicken cTnT promoter. In some embodiments, the cTnT promoter comprises a nucleic acid sequence having at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, or 90% identical to SEQ ID NO: 3. In some embodiments, the cTnT promoter comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 3; or comprises a sequence of SEQ ID NO: 3. In some embodiments, the nucleic acid sequence further comprises an enhancer.

[0045] As disclosed herein, the present disclosure provides a recombinant polynucleotide comprising a nucleic acid sequence encoding an activating transcription 4 (ATF4) operably linked to a cardiac myocyte-specific promoter. In some embodiments, the recombinant polynucleotide comprises a nucleic acid sequence having at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, or 90% identical to SEQ ID NO: 4 or 6. In some embodiments, the recombinant polynucleotide comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 4 or 6; or comprises a sequence of SEQ ID NO: 4 or 6. 3. Adeno-Associated Virus (AAV) Vectors

[0046] The present disclosure also provides a vector comprising the recombinant polynucleotide described herein. Such vector can deliver the recombinant polynucleotide encoding an ATF4 into a cell (e.g. a cardiac myocyte), thereby upregulating ATF4 expression in the cell (e.g. a cardiac myocyte).

[0047] In some embodiments, the vector is a viral vector. In some embodiments, the vector is an adeno-associated virus (AAV) vector. Adeno-associated virus (AAV) is a dependent parvovirus, of approximately twenty nanometers in size. Like other parvoviruses, AAV is a single-stranded, non-enveloped DNA virus, having a genome of about 5000 nucleotides in length, containing two open reading frames. The left-hand open reading frame codes for the proteins responsible for replication (Rep), while the right-hand open reading frame encodes the structural proteins of the capsid (Cap). The open reading frames are flanked by two ITR sequences, which serve as the origin of replication of the viral genome. Furthermore, the genome also contains a packaging sequence, allowing packaging of the viral genome into an AAV capsid.

[0048] AAV requires co-helper functions (which may be provided e.g. by an adenovirus, or by suitable packaging cells or helper plasmids) to undergo a productive infection in cultured cells. In the absence of such helper functions, the AAV virions essentially enter the cells, migrate to the nucleus as a single-stranded DNA molecule, and integrate into the cell genomes. AAV has a broad host range for infectivity, including human cells, is ubiquitous in humans, and is completely non-pathogenic.

[0049] AAV vectors have been designed, produced and used to mediate gene delivery in human subjects, including for therapeutic purposes. Clinical trials are presently ongoing in various countries using AAV vectors. Typically, AAV vectors for use in gene transfer comprise a replication defective AAV genome lacking functional Rep and Cap coding viral sequences. Such replication defective AAV vectors more preferably lack most or all of the Rep and Cap coding sequences, and essentially retain one or two AAV ITR sequences and a packaging sequence.

[0050] Methods of producing such AAV vectors have been disclosed in the literature, including using packaging cells, auxiliary viruses or plasmids, and / or baculovirus systems (Samulski et al., (1989) J. Virology 63, 3822; Xiao et al., (1998) J. Virology 72, 2224; Inoue et al., (1998) J. Virol. 72, 7024; WO98 / 22607; WO2005 / 072364). It should be noted that several of these methods relates to helper-free AAV production, which is a preferred production method within the scope of the present invention. Methods of producing pseudotyped AAV vectors have also been reported (e.g., WO00 / 28004), as well as various modifications or formulations of AAV vectors, to reduce their immunogenicity upon in vivo administration (see e.g., WO01 / 23001; WO00 / 73316; WO04 / 112727; WO05 / 005610; WO99 / 06562).

[0051] In one aspect, the present disclosure provides a self-complementary AAV (scAAV) comprising the recombinant polynucleotide described herein. ScAAV vectors are generated by deleting the terminal resolution site (trs) from one of the AAV terminal repeats. These modified vectors, whose replicating genome is half the length of the wild type have the tendency to package DNA dimers (McCarty et al., Gene Therapy, 2003). Briefly, during the replication cycle of AAV, Rep endonuclease nicks the trs to initiate a second DNA replication process generating monomeric genomes. Dimeric genomes of scAAV are generated when Rep fails to nick the trs (McCarty, Molecular Therapy 2008). Replication continues through the ITR to generate a dimeric template which initiates a new round of DNA synthesis, producing a dimeric single-strand genome (dimeric inverted repeat genomes). Both strands are thus packaged into the AAV virion as a single molecule. The two halves of the single-strand DNA molecule can then fold and base pair to form a dsDNA molecule. The above-mentioned articles of McCarty et al. describe in detail the production process of an scAAV and may be followed to obtain such scAAV vector described herein.

[0052] AAV vectors may be prepared or derived from various serotypes of AAVs. In a particular embodiment, the scAAV vector for use in the present disclosure is derived from a human AAV virus. Such a human AAV (capsid and ITR) may be derived from any known serotype, e.g. from any one of serotypes 1-11, preferably from AAV2, AAV4, AAV6, AAV8 and AAV9, more preferably from AAV6, AAV8 and AAV9, even more preferably from AAV9. Specific examples of such AAV vectors are vectors comprising an AAV2-derived genome (a nucleic acid molecule comprising an AAV2-derived ITR and an AAV2-derived packaging sequence, operatively linked to a nucleic acid encoding a therapeutic protein, preferably two AAV2-derived ITR flanking an AAV2-derived packaging sequence and a nucleic acid encoding a therapeutic protein) in an AAV2-derived capsid; vectors comprising an AAV4-derived genome in an AAV4-derived capsid; vectors comprising an AAV6-derived genome in an AAV6-derived capsid; vectors comprising an AAV8-derived genome in an AAV8-derived capsid; vectors comprising an AAV9-derived genome in an AAV9-derived capsid.

[0053] In some embodiments, the AAV vector is an AAV serotype 9 (AAV9) vector. In some embodiments, the AAV vector is a self-complementary AAV (scAAV) vector. In some embodiments, the AAV vector is a scAAV9 vector.

[0054] As disclosed herein, a scAVV vector is engineered from the naturally occurring adeno-associated virus (AAV) as its coding region forms an intra-molecular double-stranded DNA template. As such, upon entering into a host cell, the two complementary halves of scAAV can associate to form one double stranded DNA (dsDNA) for immediate replication and transcription. An scAAV can increase and prolong transgene expression in vitro and in vivo, as well as "higher in vivo DNA stability and more effective circularization. 4. Pharmaceutical Compositions

[0055] The present disclosure also provides a composition comprising a recombinant polynucleotide or vector described herein. In some embodiments, the composition is a cell comprising a recombinant polynucleotide or vector described herein. In some embodiments, the cell is a cardiac myocyte.

[0056] In some embodiments, the composition is a pharmaceutical composition which further comprises a pharmaceutically acceptable carrier. The pharmaceutical composition may be administered in any suitable form, either as a liquid solution or suspension, as a solid form suitable for solution or suspension in liquid prior to injection, as a gel or as an emulsion. The pharmaceutical composition is typically formulated with any appropriate and pharmaceutically acceptable excipient, carrier, adjuvant, diluent, etc. For injection, the excipient may be a liquid, isotonic solution, buffer, such as sterile and pyrogen-free water or a sterile and pyrogen-free phosphate-buffered saline solution. For inhalation, the excipient may be in particulate form. 5. Administration

[0057] The present disclosure provides compositions and methods for preventing or treating cardiomyopathy-related diseases in a subject. The methods comprise administering to the subject a therapeutically effective amount of a pharmaceutical composition. As disclosed herein, the pharmaceutical composition comprises a recombinant polynucleotide encoding ATF4 or a vector comprising such recombinant polynucleotide.

[0058] As disclosed herein, the administration can be any administration route to introduce the pharmaceutical composition into the subject. In some embodiments, the administration includes, without limitation, oral, intranasal, intravenous, intraperitoneal, intramuscular, intraarticular, intralesional, subcutaneous, and intradermal. More particularly, the administration includes a systemic injection of the the pharmaceutical composition (e.g., comprising a recombinant scAAV vector), such as intramuscular (i.m.), intravascular, i.e. intra-arterial (i.a.) or intravenous (i.v.), intraperitoneal (i.p.), sub-cutaneous or transdermic injections. Peripheral administration also includes oral administration of AAV vectors (WO96 / 40954), delivery using implants (WO01 / 91803), or administration by instillation through the respiratory system (for example by the intranasal route), e.g., using sprays, aerosols or any other appropriate formulations. Most preferred systemic administration include the systemic injection of the pharmaceutical composition, most preferably via an i.m., i.p., i.a. or i.v. injection. Most preferably, the pharmaceutical composition is administered via an i.v. injection.

[0059] The pharmaceutical composition is typically administered in a “therapeutically- effective” amount, i.e., an amount that is sufficient to alleviate (e.g., decrease, reduce) at least one of the symptoms associated with the disease state, or to provide improvement in the condition of the subject. It should be pointed out that repeated administrations may be performed, if required, using either the same or different systemic administration routes (and / or the same or distinct scAAV serotypes). Alternatively, a single administration of the pharmaceutical composition may also be performed.

[0060] Naturally, the amount of pharmaceutical composition in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

[0061] The amount of pharmaceutical composition administered will depend upon the ratio of therapeutic composition (e.g., the recombinant polynucleotide or the vector described herein) to carrier, the particular therapeutic composition used, the disease or disorder being treated, the age, weight, and condition of the patient, and the judgment of the clinician. In some embodiments, the pharmaceutical composition can be delivered with high penetrance to the heart by antegrade coronary infusion. In some embodiments, the pharmaceutical composition can be delivered with high penetrance to the heart by intravenous infusion. In some embodiments, the pharmaceutical composition can be delivered to the heart at a dose of 108, 109, 1010, 1011, 1012, 1013, 1014, 1015,1016, or 1017viral genomes (vg) / kg body weight, preferably from about 1011to 1016vg / kg body weight, most preferably from 1012to 1014. In some embodiments, the pharmaceutical composition can be delivered to the heart at a dose of1x, 2x, 3x, 4x, or 5x 1013vg / kg body weight. As disclosed herein, the range of doses shall not cause toxicity. In some embodiments, one dose of administration can confer days, months, or even years of therapeutic benefit. 6. Methods a) Methods of increasing expression of ATF4 in a cell

[0062] In one aspect, the present disclosure provides a method of increasing / upregulating expression of ATF4 in a cell. In some embodiments, the method comprises introducing the recombinant polynucleotide or the vector described herein into the cell. In some embodiments, the cell is a cardiac myocyte. b) Methods for improving cardiac systolic function in a subject

[0063] In another aspect, the present disclosure provides a method for improving a cardiac systolic function in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition described herein.

[0064] In some embodiments, the administration is selected from the group consisting of oral, intranasal, intravenous, intraperitoneal, intramuscular, intraarticular, intralesional, subcutaneous, and intradermal. In some embodiments, the subject is a human. In some embodiments, the subject has been diagnosed with cardiomyopathy. c) Methods for preventing or treating cardiovascular diseases in a subject

[0065] In another aspect, the present disclosure provides a method for preventing or treating a cardiovascular disease in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition described herein. Non-limiting examples of cardiovascular diseases include heart muscle disease (cardiomyopathy), abnormal heart rhythms (arrhythmias), Aorta disease and Marfan syndrome, congenital heart disease, coronary artery disease (narrowing of the arteries), deep vein thrombosis and pulmonary embolism, heart attack, heart failure, heart valve disease, pericardial disease, peripheral vascular disease, rheumatic heart disease, stroke, and vascular disease (blood vessel disease). In some embodiments, the cardiomyopathy can be dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular dysplasia, or transthyretin amyloid cardiomyopathy (ATTR-CM). In some embodiments, the heart failurecan be heart failure with preserved ejection fraction (HFpEF), heart failure with reduced ejection (HFrEF), right ventricular heart failure, or congestive heart failure (CHF).

[0066] In some embodiments, the cardiovascular disease is dilated cardiomyopathy (DCM), eccentric cardiac hypertrophy, ventricular dilation, or other pathological cardiac remodeling. In some embodiments, the cardiovascular disease is dilated cardiomyopathy (DCM).

[0067] In some embodiments, the administration is selected from the group consisting of oral, intranasal, intravenous, intraperitoneal, intramuscular, intraarticular, intralesional, subcutaneous, and intradermal. In some embodiments, the subject is a human. In some embodiments, the subject has been diagnosed with cardiomyopathy. d) Methods for preventing or treating a heart failure in a subject

[0068] In another aspect, the present disclosure provides a method for preventing or treating a heart failure in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition described herein. In some embodiments, the method can be used to prevent or treat a heart failure with reduced ejection fraction in the subject. In some embodiments, the heart failure is caused by ischemic cardiomyopathy. In some embodiments, the heart failure is caused by nonischemic cardiomyopathy.

[0069] In some embodiments, the administration is selected from the group consisting of oral, intranasal, intravenous, intraperitoneal, intramuscular, intraarticular, intralesional, subcutaneous, and intradermal. In some embodiments, the subject is a human. In some embodiments, the subject has been diagnosed with cardiomyopathy. In some embodiments, the subject has reduced ejection fraction (HFrEF). In some embodiments, the subject has ischemic cardiomyopathy. In some embodiments, the subject has nonischemic cardiomyopathy. IV. EXAMPLES

[0070] The subject matter of the present disclosure will be described in greater detail by way of specific examples. The following examples are provided to illustrate, but not to limit, the claimed subject matter. Example 1. AAV9.ATF4 Biologic Drug

[0071] The example illustrates a self-complementary, adeno-associated virus serotype 9 (AAV9) gene therapy vector that expresses the activating transcription 4 (ATF4) under thecontrol of the cardiac myocyte-specific cardiac troponin T (cTnT) promoter. ATF4 is a basic leucine zipper (bZIP) transcription factor, which plays a central role in mitochondrial function, including amino acid synthesis and transport and energy homeostasis.1-3Our data indicate that AAV9.ATF4 may rewire the cardiac metabolism and restore the failing heart’s metabolic inflexibility, leading to improvements in heart structure and function. Introduction

[0072] More than two decades after its designation as an emerging epidemic, HF remains a clinical and public health problem of major proportion associated with significant mortality and morbidity.7HF is a complex clinical syndrome resulting from structural or functional impairment of ventricular filling or ejection of blood. Our collective understanding of the HF syndrome remains incomplete, which hinders therapeutic progress and contributes to fuel the epidemic. Current treatments focus on managing symptoms to preserve cardiac function, though they are mainly ineffective as recent data suggest that mortality from HF has been increasing over the past few years. Research is urgently needed to discover new targets and develop new therapies.

[0073] The etiology of HF is diverse. A wide range of cardiac conditions, hereditary defects, and systemic diseases can result in HF. Accumulating evidence suggest the failing heart is ‘an engine out of fuel’ that undergoes profound metabolic changes irrespective of the etiology. Cardiac metabolism in the failing heart is characterized by a metabolic remodeling that includes alterations in energy substrate preference and utilization, mitochondrial dysfunction, and changes in metabolic signaling pathways. The healthy adult heart has high metabolic flexibility, with fatty acids being the predominant substrate for ATP production (85%), followed by lactate, ketone bodies, glucose, and branched-chain amino acids.8In contrast, the failing heart undergoes profound metabolic changes. It loses its metabolic flexibility due to remodeling, becomes defective in the catabolism of fatty acids and shift toward a greater reliance on glycolysis and ketone body oxidation as a source of energy.8-11These metabolic shifts are not merely consequences of heart failure but are thought to be actively contribute to disease progression and severity.

[0074] In this study, we propose a novel concept of metabolic engineering to restore the failing heart metabolic flexibility to improve cardiac function and outcome in HF. We will test this concept using a novel gene therapy approach based on targeting the Activating transcription factor 4 (ATF4), a pleiotropic transcription factor. If successful, our studies willpotentially lead to the discovery of a new biologic, AAV9.ATF4, which could become a paradigm-shifting therapy that may benefit all HF irrespective of the underlying etiology.

[0075] ATF4 is a basic leucine zipper (bZIP) transcription factor that is activated in response to cellular stress – an adaptive program known as the integrated stress response (ISR).12ATF4 also plays a central role in mitochondrial function, including amino acid synthesis and energy homeostasis, under physiological and pathophysiological conditions.1-3ATF4 is known to modulate the expression of one carbon metabolism genes comprise parallel cytosolic and mitochondrial pathways connected by the one-carbon donors serine, glycine and formate. For example, ATF4 regulates genes involved in the synthesis of serine from glucose. Serine is a non-essential amino acid synthesized from a branch of glycolysis and is converted to glycine, providing carbon units for one-carbon metabolism. Serine synthesis and metabolism provides diverse outputs, such as the biosynthesis of lipids, nucleosides, proteins, the maintenance of redox status, and the substrates for methylation reactions.13-15

[0076] In the context of HF, recent evidence suggests that activation of ATF4-mediated one- carbon metabolism might be cardioprotective. For example, the shunting of glycolytic intermediates into the one-carbon metabolism has been shown to correlate with the myocardial recovery of the failing human heart after mechanical unloading. This likely supports mitochondrial bioenergetics and maintains the redox status of cardiomyocytes.16Moreover, ATF4-mediated induction of the one-carbon metabolism has a beneficial effect on the hypertrophic heart by promoting the production of antioxidant metabolites, such as reduced glutathione (GSH).17Additionally, reactive aldehydes trigger the ATF4-dependent one carbon metabolism in the heart, eliciting a cytoprotective response against the ischemic reperfusion injury of the heart.18

[0077] Supporting the notion that modulation of ATF4-dependent signaling may be cardioprotective, in recent phenotypic screens,19we showed that bolstering the ATF4-mediated serine biosynthesis pathway, which branches from glycolysis, rescued contractile dysfunction in induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) carrying DCM pathogenic mutations in genes from diverse ontologies.19Conversely, inhibition of ATF4 expression exacerbates the contractility deficit of DCM iPSC-CMs. These studies pointed toATF4 as a regulator of cardiomyocyte function and a candid novel therapeutic target forHF.

[0078] We have translated this concept into a preclinical animal model of DCM. Specifically, we developed a new gene therapy vector expressing a constitutive active form of the human ATF4 (hATF4), controlled by a chicken cardiac-specific promoter (cardiac troponin T, cTnT). We tested this construct via an AAV-based gene transfer approach using the cardiotropic adeno-associated virus serotype-920(AAV9.ATF4) in the TM54 DCM mouse – a well- established transgenic mouse that expresses a mutant tropomyosin gene under the control ofthe cardiac myocyte-specific -myosin heavy chain ( MHC) promoter, Tpm1 p. E54K(TM54).21By 6 weeks of age, this mouse model demonstrates noticeable systolic dysfunction and ventricular dilation, and increased mortality starting at about 4 months of age.

[0079] AAV9sc.hATF4 restores the cardiac systolic function in Tpm1 E54k transgenic (Tg) mice. The Tpm1 E54k mice are transgenic FVB / N mice expressing a Myh6-Tpm1 E54K cDNA transgene resulting in replacement of endogenous -tropomyosin with mutant protein; WT refers to nontransgenic wild-type littermates. WT and Tg littermates were injected intravenously by the tail vein with 3.5e11 vg AAV9sc.hATF4 or AAV9sc.GFP after the onset of DCM at 6-weeks of age. Cardiac function was assessed by serial Echocardiography at 2-, 6- , 8- and 16-weeks post-AAV injection; n = 7-9. Analysis of cardiac function shows that AAV9sc.hATF4 improves systolic function, including A. Ejection Fraction (EF) B. Fractional Shortening (FS); C. Cardiac Output; and D. Stroke Volume.

[0080] We delivered the AAV9.ATF4 systemically in the TM54 DCM mice at 6 weeks of age when DCM is established and assessed cardiac function by echocardiography at 2, 4 and 8 weeks after gene delivery. A vector expressing GFP (AAV9.GFP) was used as a control. Cardiac function was assessed by serial Echocardiography at 2-, 6-, 8- and 16-weeks post-AAV injection. As shown in FIG. 1, AAV9sc.hATF4 significantly improved systolic function as demonstrated in Ejection Fraction (EF, FIG. 1A), Fractional Shortening (FS, FIG. 1B); Cardiac Output (FIG.1C); and Stroke Volume (FIG.1D) of the Tg mice, in comparison to the AAV9.GFP control.

[0081] At 8 weeks post-gene transfer, our analyses show that AAV9.ATF4 improves the heart’s systolic function and prevents dilatation compared to AAV9.GFP control treated TM54 mice. Importantly, the AAV9.ATF4 treatment halts the disease progression. The WT littermate controls mice treated with either AAV9.ATF4 or AAV9.GFP show no adverse effects from the treatment (FIG.2).

[0082] In addition, we observe a significantly reduction in LV interstitial fibrosis and normalization of cardiomyocyte size in the AAV9.ATF4 treated hearts compared to controls AAV9.ATF4 in the TM54 mice, and histopathological findings are unremarkable, indicating no adverse effects in the heart of TM54 or WT littermate controls (FIG.3).

[0083] Next, we investigated the cardiac metabolic and transcriptional changes in the hearts after AAV9.ATF4 treatment. Our findings indicate that the functional and structural improvements in the TM54 DCM hearts correlate with a significant impact on the cardiac transcriptome and metabolome, as evaluated by RNAseq and untargeted metabolomic analysis, respectively (FIG.4 & FIG.5).

[0084] Our metabolic analyses reveal that treatment with AAV9.ATF4 has a substantial impact on the cardiac metabolome (FIG. 4). Eight weeks post-treatment, a comparison with AAV9.GFP-treated TM54 hearts demonstrates significant changes in the metabolic profile of the myocardial tissue (FIG. 4). Primary observations include: 1. Increased concentrations of myocardial lipid intermediates. Notably, there is a rise in both medium- and long-chain acylcarnitines, the primary types of energetic lipid substrates utilized in mitochondrial fatty acid oxidation. This suggests that AAV9.ATF4 treatment may enhance the heart's capacity to generate energy from fatty acids.2. Elevated levels of nucleotides, encompassing both purines (adenine, adenosine, and inosine) and pyrimidines (uridine, cytosine, cytidine). This indicates an increase in nucleotide metabolism, an essential process for cellular energy production and signaling.3. Increased concentrations of certain amino acids such as valine (a branched-chain amino acid), phenylalanine, and histidine. An increase in these amino acids might indicate changes in protein metabolism or heightened availability of these amino acids for other metabolic processes.

[0085] Consistent with the metabolomic changes, transcriptomics analyses reveal significant changes in the cardiac transcriptome (FIG. 5). Specifically, our differential gene expression analyses of AAV9.ATF4 compared to AAV9.GFP treated hearts show that the myocardial expression of key regulators of FA -oxidation, such as Cpt2, which generate acylcarnitines and is considered a rate limiting step of fatty acid oxidation; and Acadl and Acads encoding enzymes that catalyze the initial step of mitochondrial beta-oxidation of short and long-chain fatty acid. Similarly, genes that encode key enzymes responsible for branch chain amino acid catabolism (BCAA), such as Bckdhb and Bckdha, were upregulated together with several other genes encoding critical proteins of mitochondrial electron transport chain, such as Sdha andSdhb (FIG. 5A). Accordingly, the upregulated genes are significantly enriched for pathways related to cardiac metabolism, such as fatty acid oxidation, branch chain amino acids and ATP synthesis (FIG.5B).

[0086] Collectively, our analyses indicate that AAV9.ATF4 treatment rewires the metabolism of the failing heart, which correlates with improvements in the systolic function. Given that the failing myocardium is thought to be defective in fatty acid and BCAA catabolism and nucleotide metabolism,22these findings are consistent with the notion that AAV9.ATF4 treatment might induce a beneficial metabolic rewiring, potentially restoring its metabolic flexibility to produce energy and meet energetic demands.

[0087] Finally, as metabolic remodeling is a common phenotype independent of the underlying etiology, we hypothesize that AAV9.ATF4 treatment may rescue the HF phenotype in other DCM models. To test this hypothesis, we generated a new knock-in model that carries a pathogenic mutation in the cardiac troponin T gene (Tnnt2 p.R183W) mutation. Hemizygous mice develop ventricular dilatation and systolic dysfunction at ~4 months of age. We delivered the AAV9.ATF4 systemically in the Tnnt2R183W / + DCM mice at 6 months of age when the DCM is established and assessed cardiac function after gene transfer by echocardiography and at 2, 4 and 6 months after delivery. At 6 months post-gene transfer, our analyses show that AAV9.ATF4 improves the systolic function and prevents dilatation compared to AAV9.GFP control treated mice. The WT littermate controls mice treated with either AAV9.ATF4 or AAV9.GFP show no change in heart function parameters, indicating no adverse effects from the treatment (FIG.6). Example 2. Efficacy

[0088] In future clinical trials, we expect that there will be sustained improvement or stabilization in cardiovascular pathophysiology, as assessed by cardiopulmonary exercise testing, heart failure functional class assessment (NYHA), patient-reported outcomes using Kansas City Cardiomyopathy Questionnaire (KCCQ), echocardiographic evaluation of cardiac structure and function, and biomarkers for heart failure (NT-proBNP and high-sensitivity troponin). In addition, we anticipate that there will be an improvement in the overall survival, as well as a decrease in the percentage of patients who require a heart transplant, left ventricular assist device (LVAD), or subsequent hospitalizations for heart failure.Example 3. Safety

[0089] When administered at extremely high doses, AAV9 biologics have been known to cause liver toxicity and may induce immune responses that could lead to cell death. However, these effects should not be an issue at the doses considered and are expected to be minimized by use of a cardiac myocyte-specific promoter. Nevertheless, people with liver disease, elevated liver function tests, active sepsis, or an infectious disease would be advised against treatment with AAV9.ATF4.

[0090] Humans have AAV9 neutralizing antibodies, which precludes the use of this serotype in 18-34% of the patients.4, 5To address this, future clinical trials will exclude those with a neutralizing antibody titer higher than 1:8 two weeks and three days prior to treatment, as determined by our recently published assay.6Despite these exclusion criteria, there is still a large target population, given the high prevalence of HF in the general population. Example 4. Envisioned dosing regimen and route of delivery

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Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Mol Ther. 2008;16(6):1073-80. Epub 2008 / 04 / 17. doi: 10.1038 / mt.2008.76. PubMed PMID: 18414476. 21. Rajan S, Ahmed RP, Jagatheesan G, Petrashevskaya N, Boivin GP, Urboniene D, Arteaga GM, Wolska BM, Solaro RJ, Liggett SB, Wieczorek DF. Dilated cardiomyopathy mutant tropomyosin mice develop cardiac dysfunction with significantly decreased fractional shortening and myofilament calcium sensitivity. Circ Res. 2007;101(2):205-14. Epub 2007 / 06 / 09. doi: 10.1161 / CIRCRESAHA.107.148379. PubMed PMID: 17556658. 22. Flam E, Jang C, Murashige D, Yang Y, Morley MP, Jung S, Kantner DS, Pepper H, Bedi KC, Jr., Brandimarto J, Prosser BL, Cappola T, Snyder NW, Rabinowitz JD, Margulies KB, Arany Z. Integrated landscape of cardiac metabolism in end-stage human nonischemic dilated cardiomyopathy. Nat Cardiovasc Res. 2022;1(9):817-29. Epub 20220829. doi: 10.1038 / s44161-022-00117-6. PubMed PMID: 36776621; PMCID: PMC9910091. VI. EXEMPLARY EMBODIMENTS

[0092] Exemplary embodiments provided in accordance with the presently disclosed subject matter include, but are not limited to, the claims and the following embodiments:

[0093] Embodiment 1. A recombinant polynucleotide comprising a nucleic acid sequence encoding an activating transcription 4 (ATF4) operably linked to a cardiac myocyte- specific promoter.

[0094] Embodiment 2. The recombinant polynucleotide of embodiment 1, wherein the ATF4 is a human ATF4.

[0095] Embodiment 3. The recombinant polynucleotide of embodiment 1 or 2, wherein the ATF4 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 1.

[0096] Embodiment 4. The recombinant polynucleotide of any one of embodiments 1- 3, wherein the promoter is a cardiac troponin T (cTnT) promoter.

[0097] Embodiment 5. The recombinant polynucleotide of embodiment 4, wherein the cTnT promoter comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO: 3 or 5.

[0098] Embodiment 6. The recombinant polynucleotide of any one of embodiments 1- 5, wherein the nucleic acid sequence has at least 80% identity to SEQ ID NO: 4 or 6.

[0099] Embodiment 7. A vector comprising the recombinant polynucleotide of any one of embodiments 1-6.

[0100] Embodiment 8. The vector of embodiment 7, wherein the vector is an adeno- associated virus (AAV) vector.

[0101] Embodiment 9. The vector of embodiment 8, wherein the AAV vector is an AAV serotype 9 (AAV9) vector.

[0102] Embodiment 10. The vector of embodiment 8 or 9, wherein the AAV vector is a self-complementary AAV (scAAV) vector.

[0103] Embodiment 11. A pharmaceutical composition comprising a recombinant polynucleotide of any one of embodiments 1-6 or a vector of any one of embodiments 7-10, and a pharmaceutically acceptable carrier.

[0104] Embodiment 12. A method for improving a cardiac systolic function in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of embodiment 11.

[0105] Embodiment 13. A method for preventing or treating a cardiovascular disease in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of embodiment 11.

[0106] Embodiment 14. The method of embodiment 13, wherein the cardiovascular disease is dilated cardiomyopathy (DCM).

[0107] Embodiment 15. A method for preventing a heart failure in a subject, comprising: administering to the subject a therapeutically effective amount of the pharmaceutical composition of embodiment 11.

[0108] Embodiment 16. The method of any one of embodiments 12-15, wherein the subject is a human.

[0109] Embodiment 17. The method of any one of embodiments 12-16, wherein the subject has been diagnosed with cardiomyopathy.

[0110] Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference.INFORMAL SEQUENCE LISTING SEQ ID NO: 1 ATF4 AA MTEMSFLSSEVLVGDLMSPFDQSGLGAEESLGLLDDYLEVAKHFKPHGFSSDKAKA GSSEWLAVDGLVSPSNNSKEDAFSGTDWMLEKMDLKEFDLDALLGIDDLETMPDDL LTTLDDTCDLFAPLVQETNKQPPQTVNPIGHLPESLTKPDQVAPFTFLQPLPLSPGVLS STPDHSFSLELGSEVDITEGDRKPDYTAYVAMIPQCIKEEDTPSDNDSGICMSPESYLG SPQHSPSTRGSPNRSLPSPGVLCGSARPKPYDPPGEKMVAAKVKGEKLDKKLKKME QNKTAATRYRQKKRAEQEALTGECKELEKKNEALKERADSLAKEIQYLKDLIEEVR KARGKKRVP*gcctaggtctcttagatgattacctggaggtggccaagcacttcaaacctcatgggttctccagcgacaaggctaaggcgggctcctcc gaatggctggctgtggatgggttggtcagtccctccaacaacagcaaggaggatgccttctccgggacagattggatgttggagaaaa tggatttgaaggagttcgacttggatgccctgttgggtatagatgacctggaaaccatgccagatgaccttctgaccacgttggatgaca cttgtgatctctttgcccccctagtccaggagactaataagcagcccccccagacggtgaacccaattggccatctcccagaaagtttaa caaaacccgaccaggttgcccccttcaccttcttacaacctcttcccctttccccaggggtcctgtcctccactccagatcattcctttagtt tagagctgggcagtgaagtggatatcactgaaggagataggaagccagactacactgcttacgttgccatgatccctcagtgcataaa ggaggaagacaccccttcagataatgatagtggcatctgtatgagcccagagtcctatctggggtctcctcagcacagcccctctacca ggggctctccaaataggagcctcccatctccaggtgttctctgtgggtctgcccgtcccaaaccttacgatcctcctggagagaagatg gtagcagcaaaagtaaagggtgagaaactggataagaagctgaaaaaaatggagcaaaacaagacagcagccactaggtaccgcc agaagaagagggcggagcaggaggctcttactggtgagtgcaaagagctggaaaagaagaacgaggctctaaaagagagggcg gattccctggccaaggagatccagtacctgaaagatttgatagaagaggtccgcaaggcaagggggaagaaaagggtcccctag SEQ ID NO: 3 cTnT promoter NA gcagtctgggctttcacaagacagcatctggggctgcggcagagggtcgggtccgaagcgctgccttatcagcgtccccagccctg ggaggtgacagctggctggcttgtgtcagcccctcgggcactcacgtatctccgtccgacgggtttaaaatagcaaaactctgaggcc acacaatagcttgggcttatatgggctcctgtgggggaagggggagcacggagggggccggggccgctgctgccaaaatagcagc tcacaagtgttgcattcctctctgggcgccgggcacattcctgctggctctgcccgccccggggtgggcgccggggggaccttaaag cctctgccccccaaggagcccttcccagacagccgccggcacccaccgctccgtgggacct SEQ ID NO: 4 cTnT promoter NA + ATF4 NA >cTnT-ATF4-SV40 gcagtctgggctttcacaagacagcatctggggctgcggcagagggtcgggtccgaagcgctgccttatcagcgtccccagccctg ggaggtgacagctggctggcttgtgtcagcccctcgggcactcacgtatctccgtccgacgggtttaaaatagcaaaactctgaggcc acacaatagcttgggcttatatgggctcctgtgggggaagggggagcacggagggggccggggccgctgctgccaaaatagcagc tcacaagtgttgcattcctctctgggcgccgggcacattcctgctggctctgcccgccccggggtgggcgccggggggaccttaaag cctctgccccccaaggagcccttcccagacagccgccggcacccaccgctccgtgggacctaagcttgctagcgctaccggtcgcc accatgaccgaaatgagcttcctgagcagcgaggtgttggtgggggacttgatgtcccccttcgaccagtcgggtttgggggctgaag aaagcctaggtctcttagatgattacctggaggtggccaagcacttcaaacctcatgggttctccagcgacaaggctaaggcgggctc ctccgaatggctggctgtggatgggttggtcagtccctccaacaacagcaaggaggatgccttctccgggacagattggatgttggag aaaatggatttgaaggagttcgacttggatgccctgttgggtatagatgacctggaaaccatgccagatgaccttctgaccacgttggat gacacttgtgatctctttgcccccctagtccaggagactaataagcagcccccccagacggtgaacccaattggccatctcccagaaa gtttaacaaaacccgaccaggttgcccccttcaccttcttacaacctcttcccctttccccaggggtcctgtcctccactccagatcattcct ttagtttagagctgggcagtgaagtggatatcactgaaggagataggaagccagactacactgcttacgttgccatgatccctcagtgc ataaaggaggaagacaccccttcagataatgatagtggcatctgtatgagcccagagtcctatctggggtctcctcagcacagcccctctaccaggggctctccaaataggagcctcccatctccaggtgttctctgtgggtctgcccgtcccaaaccttacgatcctcctggagaga agatggtagcagcaaaagtaaagggtgagaaactggataagaagctgaaaaaaatggagcaaaacaagacagcagccactaggta ccgccagaagaagagggcggagcaggaggctcttactggtgagtgcaaagagctggaaaagaagaacgaggctctaaaagagag ggcggattccctggccaaggagatccagtacctgaaagatttgatagaagaggtccgcaaggcaagggggaagaaaagggtcccc taggcggccgctaggcctcacctgcgatctcgatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaata aacaagttaacaacaacaattgcattcattttatgtttcaggttcagggggaggtgtgggaggttttttaaagcaagtaaaacctctacaaa tgtggtatggctgatta SEQ ID NO: 5 HUMAN cardiac Troponin T (hcTnT) promoter NA ctcagtctcagcggggactgggtgaggcagaggatggagagggctttaagcaggcatgtgggctggggcctggtgagccagccct gcggagggaggaatgtgcgacaggggacgggtggggcagggggatggcggtgggggtggggggtgttggctgctattttggcag gtgccagggacaaggctacaggaacatgtaccccacgccatataagcccatgtggtcctccagctgctcagataagctatttaaaacc agagcagatatgcagggaacagtcatgcaacataaaccagctgtccctcttgagaatcctgataaagcagaggccagcaacccagg cctgggagggccagctgggagcagggttggggggcagaaggcaacctccaagacactccataagtctcagcaccagaatcttgga aggcagagggcaagagttatgtgctgctccacttgaactgatgctgggggtaaagacatcttccaggctactggctcctaatggactga gcagccttaggcaggttgccggctctgccagccccagtgaggacatctgcaaggtgggtcttctccatgacccccaaagccatgtgg cacaccctcctcacagggaatggactgaaactgctacaggaggccagatacaaggtagaacttccagtcaagaaccttgaacaggc gctcgttgtgtgagaggggtgatgcggactgtcaaaggtcatcccctaacggctttaaaa SEQ ID NO: 6 hcTnT promoter NA+ ATF4 NA >hcTnT-ATF4-SV40 ctcagtctcagcggggactgggtgaggcagaggatggagagggctttaagcaggcatgtgggctggggcctggtgagccagccct gcggagggaggaatgtgcgacaggggacgggtggggcagggggatggcggtgggggtggggggtgttggctgctattttggcag gtgccagggacaaggctacaggaacatgtaccccacgccatataagcccatgtggtcctccagctgctcagataagctatttaaaacc agagcagatatgcagggaacagtcatgcaacataaaccagctgtccctcttgagaatcctgataaagcagaggccagcaacccagg cctgggagggccagctgggagcagggttggggggcagaaggcaacctccaagacactccataagtctcagcaccagaatcttgga aggcagagggcaagagttatgtgctgctccacttgaactgatgctgggggtaaagacatcttccaggctactggctcctaatggactga gcagccttaggcaggttgccggctctgccagccccagtgaggacatctgcaaggtgggtcttctccatgacccccaaagccatgtgg cacaccctcctcacagggaatggactgaaactgctacaggaggccagatacaaggtagaacttccagtcaagaaccttgaacaggc gctcgttgtgtgagaggggtgatgcggactgtcaaaggtcatcccctaacggctttaaaaaagcttgctagcgctaccggtcgccacc atgaccgaaatgagcttcctgagcagcgaggtgttggtgggggacttgatgtcccccttcgaccagtcgggtttgggggctgaagaaa gcctaggtctcttagatgattacctggaggtggccaagcacttcaaacctcatgggttctccagcgacaaggctaaggcgggctcctcc gaatggctggctgtggatgggttggtcagtccctccaacaacagcaaggaggatgccttctccgggacagattggatgttggagaaaa tggatttgaaggagttcgacttggatgccctgttgggtatagatgacctggaaaccatgccagatgaccttctgaccacgttggatgaca cttgtgatctctttgcccccctagtccaggagactaataagcagcccccccagacggtgaacccaattggccatctcccagaaagtttaa caaaacccgaccaggttgcccccttcaccttcttacaacctcttcccctttccccaggggtcctgtcctccactccagatcattcctttagtt tagagctgggcagtgaagtggatatcactgaaggagataggaagccagactacactgcttacgttgccatgatccctcagtgcataaa ggaggaagacaccccttcagataatgatagtggcatctgtatgagcccagagtcctatctggggtctcctcagcacagcccctctacca ggggctctccaaataggagcctcccatctccaggtgttctctgtgggtctgcccgtcccaaaccttacgatcctcctggagagaagatg gtagcagcaaaagtaaagggtgagaaactggataagaagctgaaaaaaatggagcaaaacaagacagcagccactaggtaccgcc agaagaagagggcggagcaggaggctcttactggtgagtgcaaagagctggaaaagaagaacgaggctctaaaagagagggcg gattccctggccaaggagatccagtacctgaaagatttgatagaagaggtccgcaaggcaagggggaagaaaagggtcccctaggc ggccgctaggcctcacctgcgatctcgatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaa gttaacaacaacaattgcattcattttatgtttcaggttcagggggaggtgtgggaggttttttaaagcaagtaaaacctctacaaatgtggt atggctgatta

Claims

WHAT IS CLAIMED IS:

1. A recombinant polynucleotide comprising a nucleic acid sequence encoding an activating transcription 4 (ATF4) operably linked to a cardiac myocyte-specific promoter.

2. The recombinant polynucleotide of claim 1, wherein the ATF4 is a human ATF4.

3. The recombinant polynucleotide of claim 1 or 2, wherein the ATF4 comprises an amino acid sequence having at least 80% identity to SEQ ID NO:

1.

4. The recombinant polynucleotide of claim 1, wherein the promoter is a cardiac troponin T (cTnT) promoter.

5. The recombinant polynucleotide of claim 4, wherein the cTnT promoter comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO: 3 or 5.

6. The recombinant polynucleotide of claim 1, wherein the nucleic acid sequence has at least 80% identity to SEQ ID NO: 4 or 6.

7. A vector comprising the recombinant polynucleotide of claim 1.

8. The vector of claim 7, wherein the vector is an adeno-associated virus (AAV) vector.

9. The vector of claim 8, wherein the AAV vector is an AAV serotype 9 (AAV9) vector.

10. The vector of claim 8 or 9, wherein the AAV vector is a self-complementary AAV (scAAV) vector.

11. A pharmaceutical composition comprising a recombinant polynucleotide of claim 1 or a vector of claim 7, and a pharmaceutically acceptable carrier.

12. A method for improving a cardiac systolic function in a subject, comprising: administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 11.. A method for preventing or treating a cardiovascular disease in a subject, comprising: administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 11.

14. The method of claim 13, wherein the cardiovascular disease is dilated cardiomyopathy (DCM).

15. A method for preventing a heart failure in a subject, comprising: administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 11.

16. The method of claim 12, wherein the subject is a human.

17. The method of claim 12, wherein the subject has been diagnosed with cardiomyopathy.

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