Compositions and methods for diagnosing and treating cardiomyopathy and heart failure

Abstract

Disclosed herein is a method for detecting cardiomyopathy and heart failure in a subject, the method involving assaying a sample from the subject for decrease in gene or protein expression of a Obg Like ATPase 1 (OLA1) and / or the presence of a Y254C mutation in OLA1, thereby detecting cardiomyopathy and heart failure in the subject. Also disclosed is a method for treating cardiomyopathy, heart failure, myocardial infarction or heart attack, ischemic heart disease, coronary heart disease and hypertrophic cardiomyopathy in a subject, the method involving administering to the subject an expression vector comprising a nucleic acid sequence encoding wild-type Obg Like ATPase 1 (OLA1) operably linked to an expression control sequence.

Description

X COMPOSITIONS AND METHODS FOR DIAGNOSING AND TREATING CARDIOMYOPATHY AND HEART FAILURE CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 707,350 filed on October 15, 2024, which is incorporated herein by reference in its entirety. CROSS-REFERENCE TO SEQUENCE LISTING

[0002] This application contains a sequence listing filed in ST.26 format entitled 222119- 2290_Sequence_Listing.xml” created on October 7, 2025, and having a file size of 12,658 bytes. The content of the sequence listing is incorporated herein in its entirety. BACKGROUND OF THE INVENTION

[0003] Cardiomyopathy is a heterogeneous group of diseases that affect the structure and function of the heart muscle, resulting in significant morbidity and mortality worldwide. Dilated cardiomyopathy (DCM) is the most common type of cardiomyopathy and is characterized by enlargement of the heart chambers, thinning of the heart muscle, and reduced pumping function. This can lead to symptoms such as shortness of breath, fatigue, and swelling in the legs and ankles. DCM is a significant contributor to heart failure, accounting for up to one- third of cases of heart failure in developed countries. DCM is more common in men than in women, and the incidence increases with age. The pathogenesis of DCM is multifactorial and involves genetic, environmental, and lifestyle factors. Inherited forms of DCM are caused by mutations in genes encoding proteins involved in the structural integrity and function of the heart muscle, such as sarcomeric proteins, cytoskeletal proteins, and ion channels. However, the majority of DCM cases are idiopathic or acquired, with no clear genetic cause. SUMMARY OF THE INVENTION

[0004] Disclosed herein is a method for detecting cardiomyopathy and heart failure in a subject, the method involving assaying a sample from the subject for decrease in gene or protein expression of a Obg Like ATPase 1 (OLA1) and / or the presence of a Y254C mutation in OLA1, thereby detecting cardiomyopathy and heart failure in the subject. In some embodiments, the sample is a cardiac tissue sample. In some embodiments, the assaying step involves assaying a DNA sample for a A5122G mutation in exon 8 of the ola1 gene. In some embodiments, the assaying step involves a PCR amplification screen to detect homologous or heterologous genotypes.

[0005] Also disclosed is a method for treating cardiomyopathy, heart failure, myocardial infarction or heart attack, ischemic heart disease, coronary heart disease and hypertrophiccardiomyopathy in a subject, the method involving administering to the subject an expression vector comprising a nucleic acid sequence encoding wild-type Obg Like ATPase 1 (OLA1) operably linked to an expression control sequence.

[0006] . In some embodiments, the expression vector is administered to the cardiac tissue of the subject. In some embodiments, the expression vector is an adeno-associated vector (AAV).. In some embodiments, the AAV is an AAV9.. In some embodiments, the nucleic acid sequence encodes the amino acid sequence SEQ ID NO:1 or 2.

[0007] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF FIGURES

[0008] FIGs.1A-1B. Human failing heart showing reduced OLA1 expression and associated increase in endoplasmic reticulum (ER) stress markers. (FIG.1A) The mRNA expression of OLA1, and ER stress markers CHOP, sXBP1, BIP was analyzed in human failing (HF) and non-failing (NF) heart tissue. The data are presented as fold change after normalization with the housekeeping 18S, n=5. The values are shown as mean ± SEM. The statistical analysis was performed using a two-tailed unpaired t-test. Significance was considered at *p<0.05, **p<0.01. (FIG.1B) A representative immunoblot of human failing and non-failing hearts showing reduced protein expression of OLA1, and increased protein expression of BIP and sXBP1. β Tubulin was used as protein loading control.

[0009] FIGs.2A-2B. Reduced OLA1 expression and increased endoplasmic reticulum (ER) stress markers in mice heart after 8-weeks of Trans-Aortic Constriction (TAC) surgery. (FIG.2A) RT-PCR showing reduced mRNA expression of OLA1 and associated increase in ER stress markers (CHOP, BIP, sXBP1) in mice heart tissue post 8 weeks of TAC surgery. The data are presented as fold change. The values are shown as mean ± SEM. The statistical analysis was performed using a two-tailed unpaired t-test. The significance levels are indicated as *p<0.05, **p<0.01, and ****p<0.0001. (FIG.2B) A representative immunoblots showing reduced OLA1 protein expression, and increased ER stress markers (CHOP and BIP) in heart tissue of TAC-operated mice. β-Tubulin and GAPDH were used as protein loading controls.

[0010] FIGs.3A-3D. (FIG.3A) Illustration of the generation of tamoxifen-inducible cardiac-specific OLA1 knockout (OLA1-cKO; - / -) mice. The study was compared to control littermate mice (control; + / +). (FIG.3B) An image of live mice that were used in the study. (FIG. 3C) Genotyping PCR data for ear punches obtained from Control littermate (+ / +, OLA1flox / flox), and heterozygous (+ / -) and homozygous (- / -) OLA1-cKO mice (OLA1flox / flox, βMyhC-Cre).(FIG.3D) An immunoblot showing the effect on OLA1 protein expression in heart tissue isolated from OLA1-cKO (- / -; OLA1flox / floxx βMyhC-Cre) and OLA1 haploinsufficiency (+ / -; OLA1flox / WT x βMHC-Cre) mice, compared to Control littermate (+ / +) mice.

[0011] Cardiac-specific deletion of OLA1 leads to dilated cardiomyopathy in mice. Echocardiogram images of OLA1-cKO (- / -; OLA1flox / floxX βMyhC-Cre) and haploinsufficiency (+ / -; OLA1flox / WTX βMyhC-Cre) mice showing left ventricular (LV) dilation as compared to Control littermate (+ / +;or βMyhC-Cre) mice at the age of 14 weeks.

[0012] FIGs.5A-5B. Cardiac-specific deletion of OLA1 leads to dilated cardiomyopathy (DCM) with aging in mice. FIG.5A. Echocardiogram images of OLA1-cKO (+ / -) mice show significant dilatation of the left ventricular (LV) chamber, when compared to Control littermate (+ / +) mice at 55 weeks of age. FIG.5B. Echocardiogram analyses reveal significantly reduced percentage ejection fraction in OLA1-cKO (- / -) mice compared to Control littermates (+ / +), suggesting cardiac dysfunction in cardiac-specific OLA1 knockout mice. ****p<0.0001.

[0013] FIG.6. Cardiac specific deletion of OLA1 leads to increased mortality in mice. Representation of the mouse survival curve for each indicated genotype. Note that lines correspond to OLA1-cKO (- / -, OLA1flox / floxX βMyhC-Cre), OLA1 haploinsufficiency (+ / - OLA1flox / WTX βMyhC-Cre) and Control littermate (+ / +) mice.

[0014] FIGs.7A-7C. FIG.7A. A representative photograph of mice heart explanted from cardiac-specific OLA1 KO (+ / -; OLA1-cKO) and Control littermate (+ / +) mice at 55 weeks of age. FIG.7B. Representative microscopic images of hematoxylin and eosin (H&E) stained heart sections from OLA1-cKO and Control littermate mouse heart collected at 55 weeks of age. The approximate LV chamber size (dotted circle) and myocardial wall thickness (blue bar) are depicted below the microscopic images. FIG.7C. Primary adult cardiomyocytes (CM) isolated from OLA1-cKO (+ / -) mice are thinner compared to CM from Control littermate (+ / +) mouse hearts at 55 weeks of age. Cells are stained with phalloidin (green), and nuclei are stained with DAPI (blue).

[0015] FIGs.8A-8B. FIG.8A. Mass-spectrophotometry data: Heatmap of top differentially expressed peptides in heart tissue of mice with OLA1 haploinsufficiency (+ / -; OLA1flox / WTx βMyhC-Cre) in comparison to Control littermate (+ / +) mice. Green indicates lower expression, while red indicates higher expression. FIG.8B. Volcano plot of differentially expressed peptides in hearts of OLA1 haploinsufficiency and Control littermate mice. Green dots indicate lower peptide expression and red indicates higher peptide expression. n=4 in each group.

[0016] FIG.9. Heat map generated from Mass spectrometry data, and gene ontology (GO) analyses reveal alteration of several key cardiac pathophysiology signaling pathways in the heart of OLA1-cKO mice compared to Control littermate mice.

[0017] FIGs.10A-10B. Immunoblot showing increased protein expression of ER stress marker like Chop (FIG.10A) and inflammatory response markers like TNF-^, IL6 and TGF-β (FIG.10B) in left ventricular tissue of cardiac-specific OLA1 mice compared to Control littermates. Tubulin and GAPDH were used as protein loading controls.

[0018] FIGs.11A-11B. Knockdown of OLA1 in cultured neonatal mouse cardiomyocytes augments tunicamycin-induced ER stress. (FIG.11A) Reduced mRNA expression of OLA1 associated with increased ER stress markers (Chop, BIP, and sXBP1) in OLA1 knockdown (siOLA1) neonatal cardiomyocytes treated with tunicamycin (TM, 2ug / ml; an ER stress inducer) for 24hrs. Data was compared to Scramble Control (Scr) siRNA-treated cells. Values are presented as mean ± SEM. *p<0.05, ***p<0.001, ****p<0.0001. Data were analyzed using one- way ANOVA test. (FIG.11B) Representative immunoblot showing protein expression of OLA1 and ER stress markers (CHOP and BIP) in neonatal cardiomyocytes following OLA1 knockdown (siOLA1) and subsequent treatment with tunicamycin for 24hrs.

[0019] FIGs.12A-12C. (FIG.12A) mRNA quantification of OLA1 gene by quantitative real-time PCR in a subset of the same hearts HF (n = 5) and nonfailing control participants (NF, n = 5). (FIG.12B) representative immunoblot showing reduced OLA1 protein expression in human hearts with severe left ventricular dysfunction (Nonischemic, Heart failure, HF) (n = 5) as compared to nonfailing hearts sourced from control individuals (Non-failing, NF; n = 5). The red square indicates sample obtained from heart that was found to carry an OLA1 variant (254Tyr> Cys). (FIG.12C) Quantification of the immunoblots. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-Tubulin were internal controls for immunoblot and the quantitative polymerase chain reaction, respectively. Data are shown in fold change and mean ± SEM. Data was analyzed using a two-tailed unpaired t-test. *p <0.05.

[0020] FIGs.13A-13D. Characterization of Human Ola1 gene. FIG.13A. mRNA of OLA1 gene showing CDS (Green, 1191bp) and 5’UTR and 3’UTR. (FIG.13B) Translated amino acids of the OLA1 gene, showing helix (Pink), coil (Brown), and strand (Yellow) in secondary structure. (FIG.13C) The tertiary structure of the OLA1 protein (FIG.13D) Phylogenetic relationship of human OLA1 gene to other species.

[0021] FIGs.14A-14B. (FIG.14A) Amplification of different OLA1 transcript variants in the human heart tissue. (FIG.14B) Expression of different OLA1 mRNA variants (202, 205,209) in failing and non-failing heart tissues. Failing heart tissue samples are coded as HTX and non-failing heart samples are coded as AOC.

[0022] FIGs.15A-15C. OLA1 Gene Mutation found in failing and nonfailing hearts from Human patients. (FIG.15A) Mutations were mapped in exons and introns of OLA1 gene. (FIG. 15B) NCBI Mapviewer showing Ola1 gene localized on human chromosome 2 Locus 2q31.1 that is associated with dilated cardiomyopathy in humans. (FIG.15C) MegaAlignment of nucleotide sequences showing OLA1 mutations (boxed) that are linked and have clear differential patterns among Failing and nonfailing heart patients. Sequences in FIG.15C represent discontinuous segments of the genome and are not read across the figure from left to right; sequences at least 10 nucleotides or longer are provided for as SEQ ID NOs.4-6 in the accompanying Sequence Listing.

[0023] FIGs.16A-16D. Non-synonymous(254Tyr>Cys) mutation in Exon 8 of OLA1 gene. (FIG.16A) Mega aliment showing heterozygous (R, A>G) variation in exon 8 of the OLA1 gene (SEQ ID NOs.7-8). (FIG.16B) Chromatogram showing homozygous AA to heterogynous A>G peak. (FIG.15C) Phyphen-2 analysis of 254 Tyr>Cys mutation showing a possible damaging effect on protein function. (FIG.16D) 3D structure of OLA1 mutation in helix.

[0024] FIGs.17A-17G. (FIG.17A) Mutation in OLA1 gene (254Tyr>Cys) that is conserved among different species. (FIG.17B) Immunoblot showing increased ubiquitination in human heart samples with OLA1 mutant (254Tyr>Cys) (SEQ ID NOs.9-11). (FIG.17C) Immunoblot showing a mutation in OLA1 leads to increased autophagy and apoptosis marker in the human heart. (FIG.17D) Chromatogram showing successful generation of a site-directed mutagenesis A>G in OLA1 gene. (FIG.17E) Microscopy image showing increased death in cells with overexpression of Mutant OLA1. GFP mRNA transfection was used as experimental control. (FIG.17F) Overexpression of mutant OLA1 (mut-OLA1) results in increased cleaved caspase-3 expression suggesting cell apoptosis. WT-OLA1 and Mut-OLA1 are tagged with Flag. (FIG.17G) Development of PCR-based assay to screen OLA1 mutation (2144A>G) in lane 2 and 8.

[0025] FIGs.18A-18C. Protein expression and localization of OLA1 in mice tissue. (FIG. 18A) Protein expression of OLA1 in different mice tissues SK-Skeletal Muscles, Adi. T- Adipose tissue. (FIG.18B) Densitometry analyses of above immunoblot. β-Tubulin was used as protein loading control. (FIG.18C) OLA1 protein localization in nuclear and cytoplasmic fractions of heart tissue lysates. GAPDH is loading control for cytoplasmic fraction and Lamin B1 is loading control for nuclear fraction.

[0026] FIGs.19A-19B. (FIG.19A) Genomic organization of OLA1 gene, Ex (Exon), UTR (Untranslated Region), CDS (Coding DNA sequence). (FIG.19B) PCR amplification of different fragments on the OLA1 genome covering exonic region along with adjacent introns. DETAILED DESCRIPTION

[0027] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0028] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0029] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0030] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

[0031] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Anyrecited method can be carried out in the order of events recited or in any other order that is logically possible.

[0032] Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of chemistry, biology, and the like, which are within the skill of the art.

[0033] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

[0034] Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible. Definitions

[0035] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0036] The terms “polypeptides,” “proteins” and “peptides” are used interchangeably herein. The “polypeptides,” “proteins” and “peptides” encoded by the “polynucleotide sequences,” include full-length native sequences, as with naturally occurring proteins, as well as functional subsequences, modified forms or sequence variants so long as the subsequence, modified form or variant retains some degree of functionality of the native full-length protein. Such polypeptides, proteins and peptides encoded by the polynucleotide sequences can be but are not required to be identical to an endogenous protein in the treated patient.

[0037] The terms “nucleic acid” and “polynucleotide” are used interchangeably herein to refer to all forms of nucleic acid, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids include genomic DNA, cDNA, and antisense DNA, and spliced or unspliced mRNA, rRNA tRNA and inhibitory DNA or RNA (RNAi, e.g., small or short hairpin (sh)RNA, microRNA (miRNA), small or short interfering (si)RNA, trans-splicing RNA, or antisense RNA). Nucleic acids include naturally occurring, synthetic, and intentionally modifiedor altered polynucleotides. Nucleic acids can be single, double, or triplex, linear or circular, and can be of any length. In discussing nucleic acids, a sequence or structure of a particular polynucleotide may be described herein according to the convention of providing the sequence in the 5' to 3' direction.

[0038] A “heterologous” polynucleotide or nucleic acid sequence refers to a polynucleotide inserted into a plasmid or vector for purposes of vector mediated transfer / delivery of the polynucleotide into a cell. Heterologous nucleic acid sequences are distinct from viral nucleic acid, i.e., are non-native with respect to viral nucleic acid. Once transferred / delivered into the cell, a heterologous nucleic acid sequence, contained within the vector, can be expressed e.g., transcribed, and translated if appropriate). Alternatively, a transferred / delivered heterologous polynucleotide in a cell, contained within the vector, need not be expressed. Although the term “heterologous” is not always used herein in reference to nucleic acid sequences and polynucleotides, reference to a nucleic acid sequence or polynucleotide even in the absence of the modifier “heterologous” is intended to include heterologous nucleic acid sequences and polynucleotides in spite of the omission.

[0039] The term “expression vector” as used herein refers to a vector containing a nucleic acid sequence (e.g., BAG3) coding for all or at least part of a gene capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules, siRNA, ribozymes, and the like. Expression vectors can contain a variety of control sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operatively linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well.

[0040] A “promoter” as used herein can refer to a DNA sequence that is typically located adjacent to a nucleic acid sequence (e.g., BAG3). A promoter typically increases an amount of nucleic acid sequence (e.g., BAG3) expressed compared to an amount expressed when no promoter exists.

[0041] An “enhancer” as used herein can refer to a sequence that is located adjacent to the nucleic acid sequence (e.g., BAG3). Enhancer elements are typically located upstream of a promoter element but also function and can be located downstream of or within a nucleic acid sequence (e.g., BAG3). Hence, an enhancer element can be located 100 base pairs, 200 base pairs, or 300 or more base pairs upstream or downstream of a nucleic acid sequence (e.g.,BAG3). Enhancer elements typically increase expression of a nucleic acid sequence (e.g., BAG3) above increased expression afforded by a promoter element.

[0042] The term “recombinant,” as a modifier of a viral vector, such as a recombinant AAV (rAAV) vector, as well as a modifier of sequences such as recombinant polynucleotides and polypeptides, means that compositions have been manipulated (i.e., engineered) in a fashion that generally does not occur in nature. A “recombinant viral vector” therefore refers to a viral vector comprising one or more heterologous gene products or sequences.

[0043] A “vector genome,” which may be abbreviated as “vg,” refers to the portion of the recombinant plasmid sequence that is ultimately packaged or encapsidated to form a rAAV particle. In cases where recombinant plasmids are used to construct or manufacture recombinant AAV vectors, the AAV vector genome does not include the portion of the “plasmid” that does not correspond to the vector genome sequence of the recombinant plasmid. This non- vector genome portion of the recombinant plasmid is referred to as the “plasmid backbone,” which is important for cloning and amplification of the plasmid, a process that is needed for propagation and recombinant AAV vector production, but is not itself packaged or encapsidated into rAAV particles. Thus, a “vector genome” refers to the nucleic acid that is packaged or encapsidated by rAAV.

[0044] As used herein, the term “serotype” in reference to an AAV vector means a capsid that is serologically distinct from other AAV serotypes. Serologic distinctiveness is determined on the basis of lack of cross-reactivity between antibodies to one AAV as compared to another AAV. Cross-reactivity differences are usually due to differences in capsid protein sequences / antigenic determinants (e.g., due to VP1, VP2, and / or VP3 sequence differences of AAV serotypes). An antibody to one AAV may cross-react with one or more other AAV serotypes due to homology of capsid protein sequence.

[0045] An “effective amount,” “sufficient amount” or “therapeutically effective amount” refers to an amount that provides, in single or multiple doses, alone or in combination, with one or more other compositions, treatments, protocols, or therapeutic regimens agents, a detectable response of any duration of time (long or short term), an expected or desired outcome in or a benefit to a patient of any measurable or detectable degree or for any duration of time (e.g., for minutes, hours, days, months, years, or cured). The doses of an “effective amount” or “sufficient amount” for treatment of the condition, disorder or disease (e.g., to ameliorate or to provide a therapeutic benefit or improvement of the condition, disorder or disease) typically are effective to provide a response to one, multiple or all adverse symptoms, consequences or complications of the disease, one or more adverse symptoms, disorders, illnesses, pathologies, orcomplications of the condition, disorder or disease, for example, caused by or associated with the condition, disorder or disease, to a measurable extent, although decreasing, reducing, inhibiting, suppressing, limiting or controlling progression or worsening of the condition, disorder or disease is a satisfactory outcome.

[0046] An effective amount or a sufficient amount can but need not be provided in a single formulation or administration, may require multiple administrations, and can but need not be, administered alone or in combination with another composition (e.g., agent), treatment, protocol, or therapeutic regimen. For example, the amount may be proportionally increased as indicated by the need of the patient, type, status and severity of the condition, disorder or disease treated or side effects (if any) of treatment. In addition, an effective amount or a sufficient amount need not be effective or sufficient if given in single or multiple doses without a second composition (e.g., another drug or agent), treatment, protocol or therapeutic regimen, since additional doses, amounts or duration above and beyond such doses, or additional compositions (e.g., drugs or agents), treatments, protocols or therapeutic regimens may be included in order to be considered effective or sufficient in a given patient. Amounts considered effective also include amounts that result in a reduction of the use of another treatment, therapeutic regimen, or protocol.

[0047] “Treatment” is an intervention performed with the intention of preventing the development, altering the pathology or one or more symptoms of a condition, disorder, or disease, or delaying progression or worsening of a condition, disorder, or disease. Accordingly, “treatment” refers to both therapeutic treatment and prophylactic or preventative measures. “Treatment” may also be specified as palliative care.

[0048] "Prophylaxis" and grammatical variations thereof mean a method in accordance with the invention in which contact, administration or in vivo delivery to a subject is prior to manifestation or onset of a condition, disorder or disease (or an associated symptom or physiological or psychological response), such that it can eliminate, prevent, inhibit, decrease or reduce the probability, susceptibility, onset or frequency of having a condition, disorder or disease, or an associated symptom. Target patients for prophylaxis can be one of increased risk (probability or susceptibility) of contracting a condition, disorder or disease, such as heart failure, heart failure with reduced ejection fraction, heart failure with preserved ejection fraction, or an associated symptom, or recurrence of a previously diagnosed condition, disorder or disease, or an associated symptom, as set forth herein.

[0049] By the term “modulate,” it is meant that any of the mentioned activities of the compounds embodied herein, are, e.g., increased, enhanced, increased, promoted, agonized(acts as an agonist), decreased, reduced, inhibited, suppressed, blocked or antagonized (acts as an antagonist). Modulate can reduce or decrease its activity below baseline values, e.g., a reduction or decrease of 1 to 5 fold, 1 to 10 fold, 5 to 10 fold, 10 to 20 fold, 20 to 30 fold, 40 to 50 fold, or the like, or at least 1-fold, 2-fold, 3-fold, 5-fold, 10-fold, 20 fold, 50 fold 100-fold, or the like Modulate also can increase or enhance activity over baseline values, e.g., an increase or enhancement of 1 to 5 fold, 1 to 10 fold, 5 to 10 fold, 10 to 20 fold, 20 to 30 fold, 40 to 50 fold, or the like, or at least 1- fold, 2-fold, 3-fold, 5-fold, 10-fold, 20 fold, 50 fold 100-fold, or the like.

[0050] The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

[0051] A “lipid nanoparticle” or “LNP” refers to a lipid-based vesicle useful for administration or delivery of nucleic acids, expression vectors including viral vectors having dimensions on the nanoscale, i.e., from about 10 nm to about 1000 nm, or from about 50 to about 500 nm, or from about 75 to about 127 nm. Without being bound by theory, LNP is believed to provide nucleic acid, expression vector or recombinant viral vector with partial or complete shielding from the immune system. Shielding allows delivery of the nucleic acid, expression vector or viral vector to a tissue or cell while avoiding inducing a substantial immune response against the nucleic acid, expression vector or viral vector in vivo. Shielding may also allow repeated administration without inducing a substantial immune response. Shielding may also improve or increase delivery efficiency, duration of therapeutic effect and / or therapeutic efficacy in vivo.

[0052] The terms “cationic lipid” and “amino lipid” are used interchangeably herein to include those lipids and salts thereof having one, two, three, or more fatty acid or fatty alkyl chains and a pH-titratable amino group (e.g., an alkylamino or dialkylamino group). The cationic lipid is typically protonated (i.e., positively charged) at a pH below the pKa of the cationic lipid and is substantially neutral at a pH above the pKa. The cationic lipids may also be titratable cationic lipids. In certain embodiments, the cationic lipids comprise: a protonatable tertiary amine (e.g., pH-titratable) group; Cl 8 alkyl chains, wherein each alkyl chain independently has 0 to 3 (e.g., 0, 1, 2, or 3) double bonds; and ether, ester, or ketal linkages between the head group and alkyl chains.

[0053] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, or up to 10%, or up to 5% within a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, for example within 5-fold, 4-fold, 3 -fold, 2-fold, or within 1-fold, of a given value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be understood.

[0054] All numerical values or numerical ranges include integers within such ranges and fractions of the values or the integers within ranges unless the context clearly indicates otherwise. Thus, to illustrate, reference to reduction of 95% or more includes 95%, 96%, 97%, 98%, 99%, 100% or the like, as well as 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, or the like, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, or the like, and so forth. Thus, to also illustrate, reference to a numerical range, such as “1-4” includes 2, 3, as well as 1.1, 1.2, 1.3, 1.4, or the like, and so forth. For example, “1 to 4 weeks” includes 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days.

[0055] Reference to an integer with more (greater) or less than includes any number greater or less than the reference number, respectively. Thus, for example, reference to more than 2 includes 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or the like, and so forth. For example, administration of a recombinant viral vector “two or more” times includes 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more times.

[0056] “Optional” or “optionally” means that the subsequently described circumstance may or may not occur, such that the description includes instances where the circumstance occurs and instances where it does not.

[0057] As used herein, the terms “comprising,” “comprise” or “comprised,” and variations thereof, in reference to defined or described elements of an item, composition, formulation, method, process, system, or the like are meant to be inclusive or open ended, permitting additional elements, thereby indicating that the defined or described item, composition, formulation, method, process, system, or the like includes those specified elements — or, as appropriate, equivalents thereof — and that other elements can be included and still fall within the scope / definition of the defined item, composition, formulation, method, process, system, or the like.

[0058] The “control elements” or “regulatory sequences” are those non-translated regions of the vector—enhancers, promoters, 5' and 3' untranslated regions—which interactwith host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity.

[0059] A “promoter” is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard to the transcription start site. A “promoter” contains core elements required for basic interaction of RNA polymerase and transcription factors and can contain upstream elements and response elements.

[0060] “Enhancer” generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5' or 3' to the transcription unit. Furthermore, enhancers can be within an intron as well as within the coding sequence itself. They are usually between 10 and 300 bp in length, and they function in cis. Enhancers function to increase transcription from nearby promoters. Enhancers, like promoters, also often contain response elements that mediate the regulation of transcription. Enhancers often determine the regulation of expression.

[0061] An “endogenous” enhancer / promoter is one which is naturally linked with a given gene in the genome. An “exogenous” or “heterologous” enhancer / promoter is one which is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques) such that transcription of that gene is directed by the linked enhancer / promoter. Genetic Screening Assay

[0062] Disclosed herein is a method for detecting cardiomyopathy and heart failure in a subject, the method comprising assaying a sample from the subject for a Y254C mutation in Obg Like ATPase 1 (OLA1), thereby detecting cardiomyopathy and heart failure in the subject.

[0063] In some embodiments, the wildtype amino acid sequence for OLA1 (NCBI Gene bank accession. No ON073791.1 or ON073790.1) is MPPKKGGDGIKPPPIIGRFGTSLKIGIVGLPNVGKSTFFNVLTNSQASAENFPFCTIDPNESRVP VPDERFDFLCQYHKPASKIPAFLNVVDIAGLVKGAHNGQGLGNAFLSHISACDGIFHLTRAFED DDITHVEGSVDPIRDIEIIHEELQLKDEEMIGPIIDKLEKVAVRGGDKKLKPEYDIMCKVKSWVIDQ KKPVRFYHDWNDKEIEVLNKHLFLTSKPMVYLVNLSEKDYIRKKNKWLIKIKEWVDKYDPGALVI PFSGALELKLQELSAEERQKYLEANMTQSALPKIIKAGFAALQLEYFFTAGPDEVRAWTIRKGTK APQAAGKIHTDFEKGFIMAEVMKYEDFKEEGSENAVKAAGKYRQQGRNYIVEDGDIIFFKFNTP QQPKKK (SEQ ID NO:1).

[0064] In some embodiments, the amino acid sequence for mutant OLA1 is MPPKKGGDGIKPPPIIGRFGTSLKIGIVGLPNVGKSTFFNVLTNSQASAENFPFCTIDPNESRVP VPDERFDFLCQYHKPASKIPAFLNVVDIAGLVKGAHNGQGLGNAFLSHISACDGIFHLTRAFED DDITHVEGSVDPIRDIEIIHEELQLKDEEMIGPIIDKLEKVAVRGGDKKLKPEYDIMCKVKSWVIDQKKPVRFYHDWNDKEIEVLNKHLFLTSKPMVYLVNLSEKDYIRKKNKWLIKIKEWVDKCDPGALVI PFSGALELKLQELSAEERQKYLEANMTQSALPKIIKAGFAALQLEYFFTAGPDEVRAWTIRKGTK APQAAGKIHTDFEKGFIMAEVMKYEDFKEEGSENAVKAAGKYRQQGRNYIVEDGDIIFFKFNTP QQPKKK (SEQ ID NO:2).

[0065] In some embodiments, the method involves assaying a DNA sample for a mutation encoding the Y254C mutation in OLA1. For example, the mutation can be a A5144G single nucleotide polymorphism (SNP) mutation in exon 8 of the ola1 gene (NCBI-Gene Bank accession no ON073791.1). In some embodiments, the method also involves confirming whether the mutation is homozygous (GG or AA) or heterozygous (i.e., GA).

[0066] Any of the well-known methods for genetic screening by genotyping such SNPs (e.g., DNA sequencing), hybridization techniques, PCR based assays, fluorescent dye and quenching agent-based PCR assay (Taqman PCR detection system), RFLP-based techniques, single strand conformational polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), chemical mismatch cleavage (CMC), heteroduplex analysis based system, techniques based on mass spectroscopy, invasive cleavage assay, polymorphism ratio sequencing (PRS), microarrays, a rolling circle extension assay, HPLC-based techniques, DHPLC-based techniques, oligonucleotide extension assays (OLA), extension based assays (Amplification Refractory Mutation System), ALEX (Amplification Refractory Mutation Linear Extension), SBCE (Single base chain extension), a molecular beacon assay, invader (Third wave technologies), a ligase chain reaction assay, 5'-nuclease assay-based techniques, hybridization capillary array electrophoresis (CAE), pyrosequencing, protein truncation assay (PTT), haplotype analysis, and solid phase hybridization (dot blot, reverse dot blot, chips) are very well known in the art and may be used in the methods. Expression Vectors

[0067] Disclosed herein are viral and non-viral expression vectors encoding OLA1 for use in the disclosed gene therapy methods. In certain embodiments, viral vectors include, for example and without limitation, retroviral, adeno associated virus (AAV), adenoviral, helper- dependent adenoviral, hybrid adenoviral, herpes simplex virus, lentiviral, poxvirus, Epstein-Barr virus, vaccinia virus, and human cytomegalovirus vectors, including recombinant versions thereof.

[0068] The disclosed expression vectors comprise a nucleic acid sequence encoding OLA1. Therefore, in some embodiments the expression vectors comprise a nucleic acid ATGCCCCCTAAAAAGGGAGGTGATGGAATTAAACCACCCCCAATCATTGGAAGATTTGGAA CCTCACTGAAAATTGGTATTGTTGGATTGCCAAATGTTGGGAAATCTACTTTCTTCAATGTGTTAACCAATAGTCAGGCTTCAGCAGAAAACTTCCCGTTCTGCACTATTGATCCTAATGAGAG CAGAGTACCTGTGCCAGATGAAAGGTTTGACTTTCTTTGTCAATACCACAAACCAGCAAGC AAAATTCCTGCCTTTCTAAATGTGGTGGATATTGCTGGCCTTGTGAAAGGAGCTCACAATG GGCAGGGCCTGGGGAATGCTTTTTTATCTCATATTAGTGCCTGTGATGGCATCTTTCATCTA ACACGTGCTTTTGAAGATGATGATATCACGCACGTTGAAGGAAGTGTAGATCCTATTCGAG ATATAGAAATAATACATGAAGAGCTTCAGCTTAAAGATGAGGAAATGATTGGGCCCATTATA GATAAACTAGAAAAGGTGGCTGTGAGAGGAGGAGATAAAAAACTAAAACCTGAATATGATA TAATGTGCAAAGTAAAATCCTGGGTTATAGATCAAAAGAAACCTGTTCGCTTCTATCATGAT TGGAATGACAAAGAGATTGAAGTGTTGAATAAACACTTATTTTTGACTTCAAAACCAATGGT CTACTTGGTTAATCTTTCTGAAAAAGACTACATTAGAAAGAAAAACAAATGGTTGATAAAAAT TAAAGAGTGGGTGGACAAGTATGACCCAGGTGCTTTGGTCATTCCTTTTAGTGGGGCCTTG GAACTCAAGTTGCAAGAATTGAGTGCTGAGGAGAGACAGAAGTATCTGGAAGCGAACATG ACACAAAGTGCTTTGCCAAAGATCATTAAGGCTGGGTTTGCAGCACTCCAACTAGAATACT TTTTCACTGCAGGCCCAGATGAAGTGCGTGCATGGACCATCAGGAAAGGGACTAAGGCTC CTCAGGCTGCAGGAAAGATTCACACAGATTTTGAAAAGGGATTCATTATGGCTGAAGTAAT GAAATACGAAGATTTTAAAGAGGAAGGTTCTGAAAATGCAGTCAAGGCTGCTGGAAAGTAC AGACAACAAGGCAGAAATTATATTGTTGAAGATGGAGATATTATCTTCTTCAAATTTAACACA CCTCAACAACCGAAGAAGAAATAA (SEQ ID NO:3).

[0069] Since many viral vectors exhibit size-constraints associated with packaging, the heterologous gene products or sequences are typically introduced by replacing one or more portions of the viral genome. Such viruses may become replication-defective, requiring the deleted function(s) to be provided in trans (i.e., “helper” function) during viral replication and encapsidation (by using, e.g., a helper virus or a packaging cell line carrying gene products necessary for replication and / or encapsidation, such as AAV rep, AAV cap, human adenoviral E4 and adenoviral VA RNA). Modified viral vectors in which a polynucleotide to be delivered is carried on the outside of the viral particle have also been described (see, e.g., Curiel, D T, et al., PNAS 88:8850-8854, 1991).

[0070] A particular example of a recombinant adeno associated virus (rAAV) vector would be where a nucleic acid that is not normally present in a wild-type AAV genome (heterologous polynucleotide) is inserted within the AAV genome. An example of which would be where a nucleic acid e.g., gene) encoding a therapeutic protein or polynucleotide sequence is cloned into a vector, with or without 5’, 3’ and / or intron regions that the gene is normally associated within the AAV genome. Although the term “recombinant” is not always used herein in reference to an AAV vector, as well as sequences such as polynucleotides, recombinantforms including AAV vectors, polynucleotides, or the like, are expressly included in spite of any such omission.

[0071] A “rAAV vector,” for example, is derived from a wild-type genome of AAV by using molecular methods to remove all or a part of a wild-type AAV genome, and replacing with a non-native (heterologous) nucleic acid, such as a nucleic acid encoding a therapeutic protein or polynucleotide sequence. Typically, for a rAAV vector one or both inverted terminal repeat (ITR) sequences of AAV genome are retained. A rAAV is distinguished from an AAV genome since all or a part of an AAV genome has been replaced with a non-native sequence with respect to the AAV genomic nucleic acid, such as with a heterologous nucleic acid encoding a therapeutic protein or polynucleotide sequence. Incorporation of a non-native (heterologous) sequence therefore defines an AAV as a “recombinant” AAV vector, which can be referred to as a “rAAV vector.”

[0072] A recombinant AAV vector sequence (or genome) can be packaged- referred to herein as a “particle” for subsequent infection (transduction) of a cell, ex vivo, in vitro, or in vivo. Where a recombinant vector sequence is encapsidated or packaged into an AAV particle, the particle can also be referred to as a “rAAV,” “rAAV particle” and / or “rAAV virion.” Such rAAV, rAAV particles and rAAV virions include proteins that encapsidate or package a vector genome. Particular examples include in the case of AAV, capsid proteins.

[0073] Under the traditional definition, a serotype means that the virus of interest has been tested against serum specific for all existing and characterized serotypes for neutralizing activity and no antibodies have been found that neutralize the virus of interest. As more naturally occurring virus isolates are discovered and / or capsid mutants generated, there may or may not be serological differences with any of the currently existing serotypes. Thus, in cases where the new virus (e.g., AAV) has no serological difference, this new virus (e.g., AAV) would be a subgroup or variant of the corresponding serotype. In many cases, serology testing for neutralizing activity has yet to be performed on mutant viruses with capsid sequence modifications to determine if they are of another serotype according to the traditional definition of serotype. Accordingly, for the sake of convenience and to avoid repetition, the term “serotype” broadly refers to both serologically distinct viruses (e.g., AAV) as well as viruses (e.g., AAV) that are not serologically distinct that may be within a subgroup or a variant of a given serotype.

[0074] rAAV vectors include any viral strain or serotype. For example and without limitation, a rAAV vector genome or particle (capsid, such as VP1, VP2 and / or VP3) can be based upon any AAV serotype, such as AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, AAV3Bor AAV-2i8, for example. Such vectors can be based on the same strain or serotype (or subgroup or variant), or be different from each other. For example and without limitation, a rAAV plasmid or vector genome or particle (capsid) based upon one serotype genome can be identical to one or more of the capsid proteins that package the vector. In addition, a rAAV plasmid or vector genome can be based upon an AAV serotype genome distinct from one or more of the capsid proteins that package the vector genome, in which case at least one of the three capsid proteins could be a different AAV serotype, e.g., AAV1, AAV2, AAV3, AAV3B, AAV-2i8 (AAV2 / AAV8 chimera), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or variant thereof, for example. More specifically, a rAAV2 vector genome can comprise AAV2 ITRs but capsids from a different serotype, such as AAV1, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, or variant thereof, for example. Accordingly, rAAV vectors include gene / protein sequences identical to gene / protein sequences characteristic for a particular serotype, as well as “mixed” serotypes, which also can be referred to as “pseudotypes.”

[0075] In certain embodiments, a rAAV vector includes or consists of a capsid sequence at least 70% or more (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or the like) identical to one or more AAV1, AAV2, AAV3, AAV3B, AAV-2i8, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 capsid proteins (VP1, VP2, and / or VP3 sequences). In certain embodiments, a rAAV vector includes or consists of a sequence at least 70% or more (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or the like) identical to one or more AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 ITR(s).

[0076] In certain embodiments, rAAV vectors include AAV1, AAV2, AAV3, AAV3B, AAV- 2i8, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 variants (e.g., ITR and capsid variants, such as amino acid insertions, additions, substitutions and deletions) thereof, for example, as set forth in WO 2013 / 158879 (International Application PCT / US2013 / 037170), WO 2015 / 013313 (International Application PCT / US2014 / 047670) and US 2013 / 0059732 (US Application No.13 / 594,773).

[0077] rAAV, such as AAV1, AAV2, AAV3, AAV3B, AAV-2i8, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 and variants, hybrids and chimeric sequences, can be constructed using recombinant techniques that are known to a skilled artisan, to include one or more heterologous polynucleotide sequences (transgenes) flanked with one or more functional AAV ITR sequences. Such AAV vectors typically retain at least one functional flanking ITR sequence(s), as necessary for the rescue, replication, and packaging of the recombinantvector into a rAAV vector particle. A rAAV vector genome would therefore include sequences required in cis for replication and packaging (e.g., functional ITR sequences).

[0078] In certain embodiments, a lentivirus used in the invention may be a human immunodeficiency- 1 (HIV-1), human immunodeficiency-2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Jembrana Disease Virus (JDV), equine infectious anemia virus (EIAV), or caprine arthritis encephalitis virus (CAEV). Lentiviral vectors are capable of providing efficient delivery, integration, and long- term expression of heterologous polynucleotide sequences into non-dividing cells both in vitro and in vivo. A variety of lentiviral vectors are known in the art, see Naldini el al. (Proc. Natl. Acad. Sci. USA, 93: 11382-11388 (1996); Science, 272: 263-267 (1996)), Zufferey et al., (Nat. Biotechnol., 15:871-875, 1997), Dull et al., (J Virol.1998 Nov;72(l l):8463-71, 1998), U.S. Pat. Nos.6,013,516 and 5,994,136, any of which may be a suitable viral vector for use in the invention.

[0079] Recombinant viral vector doses can be formulated, administered, or delivered at any appropriate dose. Generally, doses will range from at least 1x108vector genomes per kilogram (vg / kg), or more, for example, 1x109, 1x1010, 1x1011, 1x1012, 1x1013or 1x1014, or more, vector genomes per kilogram (vg / kg) of the weight of the patient, to achieve an effect. rAAV doses in the range of 1x10l0- 1x1011vg / kg in mice, and 1x1012-1x1013vg / kg in dogs have been effective. More particularly, a dose from about 1x1011vg / kg to about 5x1014vg / kg inclusive, or from about 5x10nvg / kg to about 1x1014vg / kg inclusive, or from about 5x10nvg / kg to about 5x1013vg / kg inclusive, or from about 5x10nvg / kg to about 1x1013vg / kg inclusive, or from about 5x10nvg / kg or about 5x1012vg / kg inclusive, or from about 5x10nvg / kg to about 1x1012vg / kg inclusive. Doses can be, for example, about 5xl014vg / kg, or less than about 5x1014vg / kg, such as a dose from about 2x10nto about 2x1014vg / kg inclusive, in particular, for example, about 2x1012vg / kg, about 6x1012vg / kg, or about 2x1013vg / kg.

[0080] An effective amount or a sufficient amount need not be effective in each and every patient treated, nor a majority of treated patients in a given group or population. An effective amount or a sufficient amount means effectiveness or sufficiency in a particular patient, not a group or the general population. As is typical for such methods, some patients will exhibit a greater response, or less or no response to a given treatment method or use.

[0081] Methods to construct expression vectors containing genetic sequences and appropriate transcriptional and translational control elements are well known in the art. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook et al., Molecular Cloning, ALaboratory Manual (Cold Spring Harbor Press, Plainview, N.Y., 1989), and Ausubel et al., Current Protocols in Molecular Biology (John Wiley & Sons, New York, N.Y., 1989).

[0082] Expression vectors generally contain regulatory sequences necessary elements for the translation and / or transcription of the inserted coding sequence. For example, the coding sequence is preferably operably linked to a promoter and / or enhancer to help control the expression of the desired gene product.

[0083] Promoters used in biotechnology are of different types according to the intended type of control of gene expression. They can be generally divided into constitutive promoters, tissue-specific or development-stage-specific promoters, inducible promoters, and synthetic promoters.

[0084] Constitutive promoters direct expression in virtually all tissues and are largely, if not entirely, independent of environmental and developmental factors. As their expression is normally not conditioned by endogenous factors, constitutive promoters are usually active across species and even across kingdoms. Examples of constitutive promoters include CMV, EF1a, SV40, PGK1, Ubc, Human beta actin, Human Troponin I, and CAG.

[0085] Tissue-specific or development-stage-specific promoters direct the expression of a gene in specific tissue(s) or at certain stages of development. For plants, promoter elements that are expressed or affect the expression of genes in the vascular system, photosynthetic tissues, tubers, roots and other vegetative organs, or seeds and other reproductive organs can be found in heterologous systems (e.g. distantly related species or even other kingdoms) but the most specificity is generally achieved with homologous promoters (i.e. from the same species, genus or family). This is probably because the coordinate expression of transcription factors is necessary for regulation of the promoter's activity.

[0086] The performance of inducible promoters is not conditioned to endogenous factors but to environmental conditions and external stimuli that can be artificially controlled. Within this group, there are promoters modulated by abiotic factors such as light, oxygen levels, heat, cold and wounding. Since some of these factors are difficult to control outside an experimental setting, promoters that respond to chemical compounds, not found naturally in the organism of interest, are of particular interest. Along those lines, promoters that respond to antibiotics, copper, alcohol, steroids, and herbicides, among other compounds, have been adapted and refined to allow the induction of gene activity at will and independently of other biotic or abiotic factors.

[0087] The two most commonly used inducible expression systems for research of eukaryote cell biology are named Tet-Off and Tet-On. The Tet-Off system makes use of thetetracycline transactivator (tTA) protein, which is created by fusing one protein, TetR (tetracycline repressor), found in Escherichia coli bacteria, with the activation domain of another protein, VP16, found in the Herpes Simplex Virus. The resulting tTA protein is able to bind to DNA at specific TetO operator sequences. In most Tet-Off systems, several repeats of such TetO sequences are placed upstream of a minimal promoter such as the CMV promoter. The entirety of several TetO sequences with a minimal promoter is called a tetracycline response element (TRE), because it responds to binding of the tetracycline transactivator protein tTA by increased expression of the gene or genes downstream of its promoter. In a Tet-Off system, expression of TRE-controlled genes can be repressed by tetracycline and its derivatives. They bind tTA and render it incapable of binding to TRE sequences, thereby preventing transactivation of TRE-controlled genes. A Tet-On system works similarly, but in the opposite fashion. While in a Tet-Off system, tTA is capable of binding the operator only if not bound to tetracycline or one of its derivatives, such as doxycycline, in a Tet-On system, the rtTA protein is capable of binding the operator only if bound by a tetracycline. Thus the introduction of doxycycline to the system initiates the transcription of the genetic product. The Tet-On system is sometimes preferred over Tet-Off for its faster responsiveness.

[0088] In some embodiments, the nucleic acid sequences encoding OLA1 are operably linked to the same expression control sequence. Alternatively, internal ribosome entry sites (IRES) elements can be used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites. IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter / enhancer to transcribe a single message. Pharmaceutical Compositions

[0089] The compositions disclosed can be used therapeutically in combination with a pharmaceutically acceptable carrier. By “pharmaceutically acceptable” is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.

[0090] The materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles such as “stealth” and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214- 6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).

[0091] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled inthe art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.

[0092] Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

[0093] Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like.

[0094] Preparations for parenteral administration include sterile aqueous or non- aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

[0095] Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.

[0096] The herein disclosed compositions, including pharmaceutical composition, may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. For example, the disclosed compositions can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally. The compositions may be administered orally, parenterally (e.g., intravenously),by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, ophthalmically, vaginally, rectally, intranasally, topically or the like, including topical intranasal administration or administration by inhalant. Method of Treatment

[0097] Disclosed herein are methods of treating cardiomyopathy, heart failure, myocardial infarction or heart attack, ischemic heart disease, coronary heart disease and hypertrophic cardiomyopathy in a subject that involve heterologous expression of OLA1 in the heart of the subject. For example, the methods can involve administering to the subject a viral or non-viral expression vector encoding OLA1 either indirectly or directly to the heart of the subject. For example, in some embodiments the method includes administering to a subject the expression vector via retrograde coronary venous or sinus delivery thereby delivering the expression vector to the heart. In other embodiments, mRNA is delivered to cells within the heart using nanotransfection or a suitable carrier, such as a liposome or extracellular vesicle (EV).

[0098] Methods, uses and formulations of the invention therefore include providing a detectable or measurable beneficial effect to a patient, or any objective or subjective transient or temporary, or longer-term improvement (e.g., cure) in the condition, disorder, or disease. Thus, a satisfactory clinical endpoint is achieved when there is an incremental improvement in the patient’s condition, disorder or disease or a partial reduction in severity, frequency, duration or progression of one or more associated adverse symptoms or complications of the condition, disorder or disease, or inhibition, reduction, elimination, prevention or reversal of one or more of the physiological, biochemical or cellular manifestations or characteristics of the condition, disorder or disease. A therapeutic benefit or improvement ("ameliorate" is used synonymously) therefore need not be complete ablation of any or all adverse symptoms or complications associated with the condition, disorder or disease but is any measurable or detectable, objectively, or subjectively, meaningful improvement in the condition, disorder, or disease. For example, inhibiting a worsening or progression of the condition, disorder or disease, or an associated symptom (e.g., slowing progression or stabilizing one or more symptoms, complications or physiological or psychological effects or responses), even if only for a few days, weeks or months, even if complete ablation of the condition, disorder or disease, or an associated adverse symptom is not achieved is considered to be a beneficial effect.

[0099] Formulations can be administered one from one or more times per day; once every other day; one or more times per week; one or more times per month; one or more times per year; or 1-2 times over the patient’s lifetime. The skilled artisan will appreciate that certainfactors can influence the dosage and timing required to treat a patient, including but not limited to the severity of the condition, disorder or disease, desired outcome, previous treatments, the general health and / or age of the patient, and other diseases present. Moreover, treatment of a patient with a therapeutically effective amount in accordance with the invention can include a single treatment or multiple treatments, such as a series of treatments.

[0100] Formulations, compositions, and pharmaceutical compositions of the invention include compositions wherein the active agent is contained in an effective amount to achieve the intended therapeutic purpose. Determining an effective dose is well within the capability of a skilled medical practitioner using techniques and guidance known in the art and using the teachings provided herein.

[0101] Formulations, such as pharmaceutical compositions, may be delivered to a patient, so as to allow nucleic acid transcription and translation of encoded protein. In certain embodiments, formulations, such as pharmaceutical compositions, comprise sufficient genetic material to enable production of a therapeutically effective amount of OLA1 in the patient to treat a condition, disorder, or disease.

[0102] Compositions and formulations may be sterile and methods and uses may be practiced using sterile technique, and optionally with sterile compositions and formulations. Compositions may be formulated with or be administered in any biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions may be formulated, administered, or delivered to a patient alone, or in combination with other agents, which influence dosage amount, administration frequency and / or therapeutic efficacy.

[0103] Formulations, methods, and uses of the invention include delivery and administration systemically, regionally, or locally (e.g., to a particular region, tissue, organ, or cell), or by any route, for example, by injection or infusion. Administration or delivery of the compositions, formulations and pharmaceutical compositions in vivo may generally be accomplished via injection using a conventional syringe or catheter, although other delivery methods are envisioned. For example, formulations and compositions may be administered to a patient via retrograde coronary venous or sinus delivery.

[0104] Also in accordance with the invention, nucleic acids, expression vectors including viral vectors and viral particles may be encapsulated or complexed with liposomes, nanoparticles, lipid nanoparticles, polymers, microparticles, microcapsules, micelles, or extracellular vesicles.

[0105] The AAV surface carries a slight negative charge. As such it may be beneficial for the LNP to comprise a cationic lipid such as, for example, an amino lipid. Exemplary amino lipids have been described in U.S. Patent Nos.9,352,042, 9,220,683, 9,186,325, 9,139,554, 9,126,9669,018,187, 8,999,351, 8,722,082, 8,642,076, 8,569,256, 8,466,122, and 7,745,651 and U.S. Patent Publication Nos.2016 / 0213785, 2016 / 0199485, 2015 / 0265708, 2014 / 0288146, 2013 / 0123338, 2013 / 0116307, 2013 / 0064894, 2012 / 0172411 and 2010 / 0117125.

[0106] In certain embodiments, cationic lipid may be present in an amount from about 10% by weight of the LNP to about 85% by weight of the lipid nanoparticle, or from about 50 % by weight of the LNP to about 75% by weight of the LNP.

[0117] LNP can comprise a neutral lipid. Neutral lipids may comprise any lipid species which exists either in an uncharged or neutral zwitterionic form at physiological pH. Such lipids include, without limitation, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. The selection of neutral lipids is generally guided by consideration of, inter alia, particle size and the requisite stability. In certain embodiments, the neutral lipid component may be a lipid having two acyl groups (e.g., diacylphosphatidylcholine and diacylphosphatidylethanolamine).

[0107] In certain embodiments, the neutral lipid may be present in an amount from about 0.1% by weight of the lipid nanoparticle to about 75% by weight of the LNP, or from about 5% by weight of the LNP to about 15% by weight of the LNP. Kits

[0108] Disclosed are kits that include packaging material and one or more components therein. A kit typically includes a label or packaging insert including a description of the components or instructions for use in vitro, in vivo, or ex vivo, of the components therein. A kit can contain a collection of such components, e.g., a nucleic acid, recombinant vector, virus (e.g., AAV, lentivirus) vector, or virus particle.

[0109] A kit refers to a physical structure housing one or more components of the kit. Packaging material can maintain the components sterilely and can be made of material commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules, vials, tubes, or the like).

[0110] Labels or inserts can include identifying information of one or more components therein, dose amounts, clinical pharmacology of the active ingredient(s) including mechanism of action, pharmacokinetics, and pharmacodynamics. Labels or inserts can include information identifying manufacturer, lot numbers, manufacture location and date, expiration dates. Labels or inserts can include information identifying manufacturer information, lot numbers,manufacturer location and date. Labels or inserts can include information on a condition, disorder, or disease for which a kit component may be used. Labels or inserts can include instructions for the clinician or patient for using one or more of the kit components in a method, use, or treatment protocol or therapeutic regimen. Instructions can include dosage amounts, frequency or duration, and instructions for practicing any of the methods, uses, treatment protocols or prophylactic or therapeutic regimes described herein.

[0111] Labels or inserts can include information on any benefit that a component may provide, such as a prophylactic or therapeutic benefit. Labels or inserts can include information on potential adverse side effects, complications, or reactions, such as warnings to the patient or clinician regarding situations where it would not be appropriate to use a particular composition. Adverse side effects or complications could also occur when the patient has, will be or is currently taking one or more other medications that may be incompatible with the composition, or the patient has, will be or is currently undergoing another treatment protocol or therapeutic regimen which would be incompatible with the composition and, therefore, instructions could include information regarding such incompatibilities.

[0112] Labels or inserts include “printed matter,” e.g., paper or cardboard, or separate or affixed to a component, a kit or packing material (e.g., a box), or attached to an ampule, tube or vial containing a kit component.

[0113] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. EXAMPLES Example 1: Loss of Obg-like ATPase 1 leads to spontaneous development of dilated cardiomyopathy, dysfunction and left ventricular wall rupture Introduction

[0114] The pathogenesis of DCM is multifactorial and involves genetic, environmental, and lifestyle factors. Inherited forms of DCM are caused by mutations in genes encoding proteins involved in the structural integrity and function of the heart muscle, such as sarcomeric proteins, cytoskeletal proteins, and ion channels. However, the majority of DCM cases are idiopathic or acquired, with no clear genetic cause.

[0115] The identification and characterization of novel genes that contribute to cardiac pathophysiology are crucial for understanding the development of DCM and designing effective therapeutic strategies. Several studies have reported genetic variants associated with DCM,including titin, MYH7, and LMNA. However, the genetic etiology of DCM remains incompletely understood.

[0116] OBG-like ATPase 1 (OLA1) is a translation-factor-related (TRAFAC) class, YchF subfamily of P-loop GTPases, and obg family gene. It possesses both GTPase and ATPase activities and is involved in the translational regulation of cell proliferation, growth, and protein synthesis in mammals and yeast. Studies have shown that OLA1 is critical for normal mammalian development and that its null mice exhibit growth retardation and developmental delay due to reduced cell proliferation rates. OLA1 modulates the hypertrophic response of cardiomyocytes through the GSK-beta / beta-catenin signaling pathway. However, its role in cardiac pathophysiology is unknown.

[0117] The significance of this study lies in the potential of the OLA1 gene as a therapeutic target for DCM and heart failure. Currently, there are limited treatment options for DCM, and heart transplantation is often the only option for end-stage disease. Thus, the identification of novel therapeutic targets is essential to improve outcomes for patients with DCM and heart failure.

[0118] In this study, the role of a novel gene, Obg-like ATPase 1 (OLA1), in cardiac pathophysiology was investigated using a cardiac-specific knockout mouse model. The protein levels of OLA1 were significantly reduced in heart tissue samples from human patients with heart failure and in mice with an experimental cardiac stress model. Furthermore, cardiac- specific OLA1 knockout mice spontaneously developed severe left ventricular (LV) dilatation, thinning of the LV wall, reduced LV function, and, in some cases, ventricular wall rupture and death. These findings indicate that OLA1 is critical for cardiac homeostasis and that its deficiency leads to DCM and dysfunction. In addition, mass spectrophotometry analyses of heart tissue proteins isolated from cardiac-specific OLA1 knockout mice revealed significant changes in protein expression, including those involved in mitochondrial function, contractile machinery, and ATP production and metabolism. Furthermore, deficiency of OLA1 in human AC16 ventricular cells lead to increased expression of endoplasmic reticulum (ER) stress response proteins such as CHOP, BIP, XBP1, and GADD34.

[0119] In conclusion, this study highlights the potential of the OLA1 gene as a therapeutic target for DCM and heart failure. The findings suggest that OLA1 is critical for cardiac homeostasis, and its deficiency leads to DCM and dysfunction. Further studies are warranted to elucidate the precise molecular mechanisms underlying the role of OLA1 in cardiac pathophysiology and to develop targeted therapies for DCM and heart failure.Materials and methods Patient samples

[0120] Heart tissue was obtained from failing human hearts at the time of transplantation. Non-failing heart tissue, which was unsuitable for transplantation, was collected as a control (n=5 in each group). The study protocols were approved by the UAB Institutional Review Board. Transgenic Mouse and genotyping

[0121] Mouse colonies were maintained in a standard vivarium with a 12 h light / 12 h dark cycle and provided with food and water ad libitum. To generate cardiac-specific OLA1 knockout mouse, OLA1fl / fl(B6(SJL)-Ola1tm1.1Zshi / J, Jackson laboratories, stock no.021431) mice were crossed with alpha-myosin-Cre (B6.FVB-Tg(Myh6-cre)2182Mds / J, ^MyhC-cre; Jackson laboratories, stock no.011038) mice (Kanisicak, et al. Nat. Commun.20167). The OLA1fl / fl / Cre+ / −(+ / -) and (OLA1flox / flox, Myh6-Cre; - / -) mice were used as cardiac-specific OLA1 knockouts (KO), whereas littermates OLA1fl / flmice were designated as controls (CT). Mice were born healthy and were fertile. Genotyping was performed using primers suggested by Jackson Laboratory. The animals were observed for 55 weeks. Mortality was recorded. Heart function was evaluated by echocardiography and mouse were euthanized at the end of 52-55 weeks. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Alabama at Birmingham. Echocardiography

[0122] Mice were anesthetized with a mixture of 1.5% isoflurane and oxygen (1 L / min) and transthoracic two-dimensional echocardiography was performed using Vevo2100 (VisualSonics, Canada) as described previously (Patil, et al. Circ. Res.2021129 :1006–1020). Left ventricular (LV) internal dimensions (LVIDs and LVIDd), ejection fraction (EF), and fractional shortening (FS) were recorded at baseline and eight weeks after TAC. Histological analysis

[0123] At endpoint, animals were euthanized and the heart was immediately removed and fixed in 10% neutral-buffered formalin. Heart sections of 5 μm thickness were prepared, stained with hematoxylin and eosin and Masson’s trichrome, and imaged under a microscope (Nikon Eclipse E200 microscope with NIS-Elements software version 4.60) and recorded as previously described (Patil, et al. Circ. Res.2021129 :1006–1020; Govindappa, et al. FASEB J. 202034:2238–2251).Cell culture, RNA interference, and treatments

[0124] Heart was excised from mouse neonates (1-3 days), washed several times with washing media (chilled PBS supplemented with 20mM 2, 3-Butanedione Monoxime, BDM), atria and blood were removed and incubated overnight at 4°C on rocker in isolation media (20mM BDM, 0.25% trypsin into HBSS). The tissue was further incubated at 37°C in digestion media (20 mM BDM in L-15 supplement with Collagenase / Dispase Enzyme) on a rocker. After centrifugation, cell pellet was resuspending in plating media (DMEM, M-199 supplement with horse and bovine serum with pen strep antibiotic) and plated onto 10cm dishes for 3 hours for attachment of fibroblast or the like Non-adherent cells were counted and plated in 24wells and 6 well plate (corning Primaria plates). After 24 hrs, cells were cultured in maintaining media (DMEM, M-199 supplemented with horse serum and antibiotic). After 48hrs, cells were used for further experimentation. For RNA interference studies, CMs were transfected either with OLA1 siRNA (siHuR, Silencer® Select, Thermo Fisher Scientific) or scramble control (siControl, Silencer® Select Negative Control#1, Thermo Fisher Scientific) using LipofectamineTM RNAiMAX reagent (Cat# 13778150, Thermo Fisher Scientific) as per the manufacturer's instructions. OLA1 knockdown was confirmed by qPCR and Western blotting. CMs were serum- starved (medium containing 0.1% BSA) overnight before treatment with tunicamycin (10 ng / ml, Peprotech) for indicated time points. RNA isolation and Quantitative Real-Time PCR

[0125] The hearts of control and knockout (KO) mice were dissected in phosphate buffered saline (PBS) immediately following euthanasia, and washed free of blood via aortic injection of PBS. Tissues were flash frozen in liquid nitrogen after dissection and stored at −80°C for subsequent RNA or protein extraction. Total RNA was extracted from approximately 50 mg of human failing and non-failing heart tissues using the TRIzol method (Invitrogen), followed by purification with the Qiagen RNA extraction kit (cat# 74106, Qiagen), in accordance with both the manufacturer's instructions and previously published protocols (Dubey, et al. Mol Cell Biochem.2022477(1):129-141; Dubey, et al. J. Cell. Physiol.2022237:2169–2182; Patil, et al. Circ. Res.2021129:1006–1020). RNA quality and quantity were evaluated using Nanodrop, and 1 μg of RNA from each sample was reverse-transcribed using the RevertAid First Strand cDNA Synthesis Kit (cat# K1691, ThermoFisher Scientific). qPCR analyses were performed on a QuantStudio 3 system (Applied Biosystems, ThermoFisher Scientific) using PowerUp™ SYBR™ Green Master Mix (cat# A25778, ThermoFisher Scientific) and gene- specific primers. Target gene expression was normalized to housekeeping genes (18S rRNA or β-actin or GAPDH) and presented as fold change versus control.Western Blotting

[0126] Whole cell lysates for protein extraction were prepared in RIPA buffer supplemented with a protease inhibitor cocktail. The concentration of proteins was determined using the Bio-Rad protein assay dye (Cat# 5000006, Bio-Rad), following the manufacturer's protocol. Equal amounts of protein were denatured in 4x Laemmli buffer, separated on denaturing SDS-PAGE gels (4%-20%, Mini-PROTEAN® TGX™ stain-free precast gels, Bio- Rad), and then transferred to a PVDF membrane (Trans-Blot® Turbo™ PVDF transfer system, Bio-Rad). The membranes were blocked using 5% non-fat dry milk in TBS-T and incubated with primary antibodies, followed by horseradish peroxidase (HRP)-conjugated secondary antibodies. The visualization of blots was achieved through the use of the enhanced chemiluminescence (Pierce) detection system. Blot images were captured using the ChemiDoc Touch Imaging System (Bio-Rad), and densitometric analysis was performed using ImageJ (NIH) software. Mass spectrometry, protein identification and bioinformatics analysis

[0127] Mass spectrometry was employed for the analysis of the digested peptides in each sample. Eight samples were analyzed, consisting of four samples for OLA1-cKO and matched littermate control groups. Tissue samples (100mg) were lysed in a buffer with RIPA protease inhibitor cocktail and 1 mM PMSF, and protein quantification was performed using a BCA assay kit. Acetone precipitation was used to precipitate 100 μg protein from each sample, which was then subjected to tryptic digestion, TMT labeling, SDC cleaning, and peptide desalting for base-reversed-phase (RP) fractionation. For quantitative proteomic analysis of cardiac tissues, samples were extracted, digested with trypsin, and labeled with TMT reagents. Labeled samples were mixed, desalted, and fractionated into eight fractions using a C18 column. Peptides were separated and analyzed with mass spectrometer. Data-dependent acquisition was performed with an Orbitrap analyzer.

[0128] Principal component analysis (PCA) was used to analyze overall protein expression patterns between OLA1-cKO and control mice. Raw MS files were processed with MaxQuant (Version 1.5.6.0) using a UniProt mouse protein sequence database with a mass tolerance of 10 ppm for precursor and fragment ions. Quantification used n-plex TMT labeling with specific modifications considered, and only unmodified unique peptides with an FDR <0.01 were used. Proteins with at least one unique peptide match and a TMT ratio meeting a ≥1.20- or ≤0.83-fold cutoff value with a p-value of <0.05 were considered upregulated or downregulated.

[0129] The GO project is a major bioinformatics initiative to unify the representation of gene and gene product attributes across all species. It provides an ontology of defined termsrepresenting gene product properties. To probe the main biological process (BPs), cellular components (CCs), and molecular functions (MFs) of differentially abundant proteins, the responsive proteins were further summarized based on GO terms. Results Myocardial OLA1 protein levels are reduced in human heart failure and mice with pressure-overload injury.

[0130] The objective of this study was to investigate whether changes in the protein levels of OLA1 are associated with cardiac disease. For this purpose, Western blot analysis was performed on samples of explanted human hearts obtained from patients diagnosed with heart failure undergoing cardiac transplantation, with donor hearts without cardiac disease ailment serving as controls. As shown in FIGs.1A-1B, the levels of OLA1 mRNA and protein were found to be significantly reduced in heart failure samples as compared to control hearts. In addition, there was a corresponding increase in mRNA and protein expression of endoplasmic reticulum (ER) stress markers such as BIP, CHOP and sXBP1.

[0131] To further explore the role of OLA1 in cardiac disease, the expression levels of OLA1 was examined in a mouse model of pressure overload-induced dilated cardiomyopathy, a condition characterized by compensatory hypertrophy and eventual left ventricular dilatation and failure. Transaortic constriction (TAC) surgery was performed on the mice and assessed the expression of OLA1 protein in the myocardium (LV) at 8 weeks post-surgery. Results demonstrated a significant decrease in the levels of OLA1 mRNA and protein expression in mouse hearts with TAC-induced cardiac stress, with a corresponding increase in expression of ER stress related markers (FIGs.2A-2B).

[0132] Collectively, these findings suggest that OLA1 may play a crucial role in the regulation of cardiac function and pathology. Generation and confirmation of Cardiac-specific OLA1 knockout mice (OLA1-cKO).

[0133] To investigate the role of OLA1 in cardiac pathophysiology in vivo, OLA1-cKO mice were created by crossing OLA1 flox / flox mice with transgenic mice expressing mouse cardiac specific α-myosin heavy chain (α-MyHC) promoter-driven Cre recombinase (FIGs.3A- 3D). The resultant OLA1-cKO (α-MyHC-Cre; OLA1flox / flox; - / -) mice exhibited normal viability and were born at the expected Mendelian ratio. Activation of Cre and OLA1 deletion by PCR-based genotyping of DNA and immunoblotting of proteins isolated from heart tissue. To enhance the translational relevance of the studies, haploinsufficiency mouse (+ / -) were also generated where only one copy of the OLA1 gene is deleted, therefore the remaining functional copy of the gene is not adequate to produce the needed levels of protein to preserve normal cardiac function.This is in contrast to complete knockout (- / -), where both the copy of gene is deleted (which is typically not the scenario expected in all human individuals). The partial and full deletion is shown in FIG.3D.

[0134] At 6-8 weeks of age, no discernible differences in body weight, the ratio of whole heart or left ventricular (LV) weight to body weight or tibia length between OLA1-cKO and littermate control mice was observed. Additionally, there were no significant differences in echocardiographic parameters such as end-diastolic / systolic LV internal dimension (LVIDd / LVIDs), ejection fraction (EF) or fractional shortening (FS), interventricular septal (IVS) thickness, and LV posterior wall (LVPW) thickness between the two groups of mice.

[0135] Taken together, these results indicate that there were no significant differences in the overall cardiac structure and function between OLA1-cKO and littermate control mice at the age of 6-8 weeks. Loss of OLA1 gene in mice leads to dilated cardiomyopathy (DCM), left ventricular (LV) wall thinning and dysfunction.

[0136] In order to understand whether OLA1 affects cardiac function and structural remodeling in vivo, LV function was assessed using echocardiography in both, littermate control and OLA1-cKO mice. The mice were allowed to age naturally without any cardiac stress until 14 weeks and 60 weeks. Findings indicate that the mice deficient in OLA1 progressively developed severe ventricular dilation by 14 weeks, as evidenced by an increase in LV chamber diameter even in haploinsufficiency OLA1-cKO mice. There was a more pronounced phenotype in homozygous (- / -) KO mice (FIG.4). The dilation became more remarkable at 55 weeks and a notable reduction in cardiac function as evidenced by reduced % Ejection fraction (%EF) in OLA1-cKO mice (FIGs.5A-5B). Furthermore, interestingly, the homozygous knockout of OLA1 (100% knockout) is detrimental to the survival of mice. Also, the survival was significantly lower in haploinsufficiency (50% OLA1 knockout) mice as compared to littermate control mice (FIG. 6). Also, importantly, rupture of the LV wall in the some of the dead mouse was observed.

[0137] Pathological examination of the heart from OLA1-cKO mice revealed enlargement of the heart (FIG.7A). Histology, H&E staining, and evaluation showed thinning of the LV wall and increase in LV chamber size or dilatation in OLA1-cKO mice (FIG.7B). Interestingly, cardiomyocytes isolated from the heart tissue showed thinner cardiomyocytes in OLA1-cKO mice as compared to littermate control mice (FIG.7C). These data suggest that loss of OLA1 leads to spontaneous development of dilated cardiomyopathy and adversely affects cardiac function and survival.

[0138] Effect of OLA1 ablation on changes in cardiac proteome and signaling OLA1- cKO mice showed spontaneous development of dilated cardiomyopathy and dysfunction. Also, OLA1 has been shown to play an important role in protein translation. Thus, it was hypothesized that OLA1 plays an important role in defining the cardiac proteome profile. To determine the consequence of OLA1 deficiency on cardiac protein expression in the heart, protein expression was compared in Control littermate and OLA1 cKO hearts at 58 weeks of age by mass spectrophotometry on proteins extracted from left-ventricular tissues. A total of 1777 proteins were detected, among them 262 differentially expressed abundant proteins were used for further analysis. A hierarchical cluster heat map and volcano plot (FIGs.8A-8B) shows changes in relative protein abundances across OLA1-deficient mice and their matched littermates. Among the differentially expressed proteins, 155 proteins showed higher expression and 117 showed lower expression in OLA1-cKO mice than in controls. Among differentially expressed proteins included proteins related to cytoskeletal structure and function, calcium signaling, ER stress and mitochondrial function (FIG.9). Protein included Destrin (DEST), Dynamin-2 (DYN2), Plectin (PLEC), Moesin (MOES), Profilin-1 (PROF1), Cofilin-2 (COF2), Calreticulin (CALR), Myozenin-2 (MYOZ2), Protein Ahnak, Reticulon-4 (RTN4), Protein disulfide-isomerase (PDIA1), Protein disulfide-isomerase A6 (PDIA1), Protein disulfide-isomerase A3 (PDIA3), Mitochondrial antiviral-signaling protein (MAVS), BolA-like protein 1 (Bola1) , Vimentin (VIM) or the like significantly increased, whereas proteins including Tropomyosin alpha-1 chain (TPM1), Dystrophin (DMD), Delta-sarcoglycan (SGCD), Sarcalumenin (SRCA), Ryanodine receptor 2 (RYR2), Calsequestrin-2 (CASQ2), ATP synthase subunit epsilon, mitochondrial (Atp5f1e), Perilipin-5 (Plin5), Mitochondrial glutamate carrier 1 (Slc25a22), NADH dehydrogenase [ubiquinone] 1 alpha subcomplex assembly factor 5 (Ndufaf5) significantly decreased. GO analysis of proteins induced by cardiac-specific OLA1 knockout

[0139] The differentially expressed proteins identified in cardiac OLA1-deficient mice versus control littermate mice were further categorized using GO annotation. The responsive proteins were classified based on GO terms: cellular components (CCs), molecular functions (MFs), and biological processes (BPs). The differentially expressed proteins corresponded to 132, 241, and 176 terms for BPs, CCs, and MFs, respectively. According to the percentage of genes (49.8%) in GO enrichment, the BP analysis revealed that most of the proteins with altered expression belong to 11 major processes TCA cycle, Stress response, ER stress, Protein biosynthesis, a (FIG.9). The CC analysis indicated that these proteins were mainly located in the mitochondria, cytoskeleton, ER, proteasome, Sarcoplasmic reticulum, or the like,whereas molecular function analysis showed that the identified proteins were mainly involved in Actin binding, chaperone, initiation factor, and the like (FIG.9).

[0140] To validate some of the proteins that might be perturbed by the signaling pathways identified in GO analyses, ER stress and inflammatory response markers were measured in Cardiac-specific depletion of OLA1 by α-MHC-MerCreMer strategy and in control mouse heart tissue. Data showed that cardiac-specific OLA1 deficiency increases Chop, a marker of ER stress, expression in isolated hearts (FIG.10A), indicating increased ER stress. There was an increase in the expression of inflammatory markers such as TNF-alpha, IL-6, and TGF-beta in the OLA1-cKO hearts compared to the control hearts (FIG.10B). Collectively, the findings underscore the crucial role of OLA1 in regulating ER stress and inflammation in the heart, and suggest that OLA1-mediated cardiac protection might be due to modulation of ER stress and inflammation. Notably, the increase in Chop expression and inflammatory markers in the CM-OLA1 knockout hearts implies that OLA1 deficiency heightens the susceptibility to cardiac stress and dysfunction. In vitro, OLA1 deficiency enhances endoplasmic reticulum (ER) stress response in mouse primary cardiomyocyte.

[0141] The cardiac-specific OLA1 knockout mice spontaneously developed dilated cardiomyopathy. Previous reports have shown that OLA1 plays an essential role in protein translation. During cardiac stress / injury, the rate of protein synthesis in the heart increases, which stimulates the adaptive ER stress response to protect cells and maintain ER homeostasis. The adaptive ER stress response can activate the unfolded protein response (UPR), leading to an acute decrease in global protein synthesis while increasing the synthesis of only protective proteins essential for preserving ER homeostasis and cell survival. Prolonged endoplasmic reticulum (ER) stress is a contributing factor in the development of pathological cardiac remodeling and heart failure in humans. In order to evaluate the influence of OLA1 on ER stress, a knock-down approach was employed to assess the effect of OLA1 deficiency on tunicamycin-induced ER stress in cardiomyocytes isolated from neonatal mouse hearts aged 1- 3 days. Specifically, OLA1 siRNA transfection was utilized for 48 hours followed by treatment with tunicamycin (2μg / ml), an established ER stress inducer, for 24 hours, and the expression of ER stress markers such as CHOP and BIP was subsequently evaluated through Western blotting and RT-PCR. The efficacy of OLA1 knockdown was assessed through mRNA and protein expression analysis, which is presented in FIGs.11A-11B, respectively. Notably, tunicamycin-induced protein expression of CHOP and BIP was found to be increased in OLA1 knockdown cells as compared to scramble siRNA-treated cardiomyocytes (FIGs.11A-11B).Taken together, these data suggest that OLA1 is required to maintain ER homeostasis in cardiomyocytes, and that OLA1 deficiency perturbs ER stress-related signaling. These findings provide a basis for further studies to elucidate the molecular mechanisms underlying the role of OLA1 in protein translation, it is interaction with structural protein, calcium signaling pathways, ER stress and the development of dilated cardiomyopathy. Discussion

[0142] Heart failure remains a major health burden affecting more than 26 million individuals worldwide with an increasing prevalence. Under physiological stress, the heart increases in size to reduce wall tension and maintain cardiac function. Sustained cardiac stress, however, leads to a pathological growth of the heart progressing to dilated cardiomyopathy and heart failure. The major findings of the current study include: i) OLA1 expression was significantly reduced in heart tissue samples from human patients with heart failure; ii) mice with an experimental cardiac stress (pressure-overload induced) showed reduced OLA1 protein expression; iii) cardiac-specific OLA1 knockout (OLA1-cKO) mice spontaneously developed severe left ventricular (LV) dilatation, thinning of the LV wall, reduced LV function, and, in some cases, ventricular wall rupture and death; and most importantly, iv) loss of OLA1 resulted in altered expression of several key proteins involved in mitochondrial function, contractile machinery, ER stress and calcium signaling, the critical signaling components that maintain heart function and structure.

[0143] OBG-like ATPase 1 (OLA1) has been shown to be involved in the translational regulation of cell proliferation, growth and protein synthesis in both mammals and yeast. There is no previous report of OLA1 role and its regulation in cardiac diseases. In the present study- i) a reduction in OLA1 RNA and protein expression in the heart samples of patients with diagnosed heart failure was observed, and ii) mouse subjected to pressure overload induced cardiac stress showed decrease in OLA1 expression in the heart. A previous study showed that upon ANG II administration in mouse, initially, OLA1 protein expression in the myocardium gradually increased at 3d and 14d (compensatory hypertrophic response), followed by a subsequent decline of OLA1 expression at 28 days (decompensatory phase). These studies suggest that OLA1 is essential for adaptive response during the initial stages of heart disease, while a decrease in OLA1 could be a cause / effect of molecular switch from the initial protective compensatory hypertrophic response transitioning into maladaptive response. Therefore, the objective of this study was to determine the consequence of cardiac-specific ablation of OLA1 on cardiac remodeling and function in vivo. OLA1 is essential for normal progression of mammalian development. In a previous report, OLA1 null mice showed growth retardation anddevelopmental delay due to reduced rate of cell proliferation through translational regulation of p21. In contrast, in the present study it was observed that cardiac-specific OLA1 knockout mice are viable and have no obvious morphological and functional abnormality before 6 weeks. However, OLA1-cKO mice spontaneously developed cardiomyopathy as the mice aged. Cardiac specific OLA1 gene ablation in mouse resulted in spontaneous development of severe dilatation of the left ventricle, thinning of the LV wall, reduced LV function and ventricular wall rupture in some cases. In another on-going study a mutation was identified in OLA1 gene in heart tissue of patients with heart failure and the experimental expression of mutant OLA1 in cardiomyocyte cell line showed pathological cellular effects. These data suggest that OLA1 is critical for cardiac homeostasis and its deficiency adversely affects heart function.

[0144] The role of OLA1 in heart disease is not known so far. It is not clear what signaling mechanisms are perturbed due to OLA1 decrease (observed in heart tissue from heart failure patients) or during loss of OLA1 (such as in the cardiac-specific knockout mouse model). Previous studies have shown that OLA1 plays an important role in protein translation. A study demonstrated that OLA1 modulates invitro response to ANGII treatment via GSK3β / β-Catenin pathway. In the present study, LC-MS analyses of heart tissue proteins isolated from cardiac- specific OLA1-cKO mice identified 136 differentially abundant proteins in cardiac tissues, including proteins involved in mitochondrial function, contractile machinery, ATP production and metabolism, ER stress and calcium signaling. This suggests that the loss of OLA1 results in a global dysregulation of cardiac protein expression and function. The differentially abundant proteins included profilin 1, tubulin beta 6 class V, Xin actin-binding repeat-containing protein, Myosin Heavy Chain 9, destrin, dynamin-2, calreticulin, desmoyokin, Coatomer subunit beta, Protein Disulfide Isomerase Family A Member 3, BolA Family Member 1, Gamma- Glutamylcyclotransferase, Creatine Kinase B, dystrophin, delta-sarcoglycan, sarcoplasmic reticulum Ca2+-ATPase, COX Assembly Mitochondrial Protein Homolog, calsequestrin, Ryanodine Receptor 2, Eenoyl-CoA hydratase 1, iron-sulfur cluster assembly 1, mitochondrial ATP synthase, Solute carrier family 25 member 22, mitochondrial ribosomal protein L28, Coenzyme Q8A. These differentially expressed proteins suggest that OLA1 is involved in regulating multiple biological pathways essential for maintaining cardiac function, including mitochondrial function, sarcomere organization, calcium signaling, and protein folding. This study provides new insights into the crucial role of OLA1 in maintaining cardiac homeostasis and that its loss would result in the development of dilated cardiomyopathy.

[0145] Maladaptive cardiac remodeling is known to be regulated by various signaling pathways that control protein synthesis and degradation. The Endoplasmic reticulum (ER) playsa crucial role in protein synthesis, folding, and trafficking, and any disturbances in this process can result in ER stress. ER stress occurs due to the accumulation of unfolded proteins, which triggers the unfolded protein response (UPR) under physiological conditions. The UPR activates cytoprotective pathways, including decreasing protein synthesis, increasing protein folding capacity, and enhancing the degradation of misfolded proteins, to maintain ER homeostasis. However, persistent ER stress (caused by genetic mutations, ischemia, or oxidative stress) can lead to sustained activation of the UPR, resulting in maladaptive processes like cardiomyopathies.

[0146] A current study demonstrated that cultured primary mouse cardiomyocytes lacking OLA1 expression exhibit increased expression of ER stress response proteins such as CHOP, BIP, XBP1, and GADD34. Similar effects were also observed in hearts of OLA1-cKO mice. These findings suggest that the loss of OLA1 may activate ER stress, which is known to contribute to the pathogenesis of cardiomyopathy.

[0147] While ER stress could be one reason for the observed pathological phenotype following OLA1 deficiency, it is acknowledged that OLA1-mediated effects could also occur through critical signaling mechanisms such as mitochondrial function, ATP production, calcium signaling / sarcomere structure and its impact on contractility. The proteomics analyses support these possibilities.

[0148] Overall, these findings highlight the crucial role of OLA1 in the development of cardiomyopathy. Targeting this pathway could offer a promising therapeutic approach for dilated cardiomyopathy and heart failure, which are significant clinical challenges. By understanding the complex mechanisms underlying these conditions, new treatments can be developed that address the underlying causes of these diseases, ultimately leading to improved patient outcomes.

[0149] In conclusion, this study provides evidence that OLA1 plays a critical role in cardiac pathophysiology and that its deficiency leads to dilated cardiomyopathy and dysfunction. These findings provide important new insights into the potential molecular mechanisms underlying cardiac pathophysiology and suggests that therapies targeting the OLA1 gene (like modified-RNA or AAV9 virus) may have the potential to treat dilated cardiomyopathy and heart failure.Example 2: OLA1 Tyr254Cys genetic variant identified in human failing heart is associated with pathological cellular effects Materials and methods Patient samples

[0150] Heart tissue from failing human hearts were obtained at the time of transplantation. Non-failing heart tissue, which was unsuitable for transplantation, was collected as a control (n=5 in each group). The study protocols were approved by the UAB Institutional Review Board. RNA isolation and cDNA synthesis

[0151] Total RNA was isolated from ~50 mg of human failing and non-failing heart tissues using the TRIzol method (Invitrogen) and purified with the Qiagen RNA extraction kit (cat# 74106, Qiagen) according to manufacturer's instructions and a published protocol. RNA quality and quantity were assessed with Nanodrop, and 1 μg of RNA from each sample was reverse-transcribed using the RevertAid First Strand cDNA Synthesis Kit (cat# K1691, ThermoFisher Scientific). Gene-specific primers were used for performing qPCR analyses on a QuantStudio 3 system (Applied Biosystems, ThermoFisher Scientific) using the PowerUp™ SYBR™ Green Master Mix (cat# A25778, ThermoFisher Scientific). Target gene expression was normalized to housekeeping genes (18S rRNA or β-actin or GAPDH) and presented as fold change versus control. DNA isolation

[0152] Total DNA from human failing and non-failing heart tissues (~10 mg) was isolated using the Qiagen DNAeasy kit (cat# 69504, Qiagen) according to the manufacturer's instructions. The quality and quantity of the isolated DNA were measured using Nanodrop. Primers designing and PCR amplification

[0153] To screen for polymorphisms in the OLA1 gene, different fragments of PCR products covering all exons along with partial introns were amplified from isolated genomic DNA from both failing and non-failing patient hearts. Nine sets of primers were designed using the Primer Blast server of NCBI based on available human OLA1 gene sequences from the ensemble genome browser (Transcript: ENST00000284719.8). Before sequencing, all PCR products were optimized for amplification and sequencing performance to ensure reliable screening for mutations in the OLA1 gene. For each PCR reaction, a total volume of 20 μl containing 20-50ng genomic DNA, 10 pmole of each primer, along with 2X PCR Master Mix (Cat.no. PRM7505, Promega) was used. After an initial denaturation step (95°C for 3 min), samples were subjected to 32 cycles of PCR consisting of 94°C for 30s, primer-specificannealing temperature for 30 sec, and 72°C for 90 sec, followed by a final extension for 7 min at 72°C. Amplified products were then checked on a 1.5% agarose gel, and images were captured using Bio-Rad Gel doc with a UV transilluminator. DNA Sequencing and Analyses

[0154] Prior to Sanger sequencing, the amplified PCR products were enzymatically cleaned up using ExoSAP-IT™ PCR Product Cleanup Reagent (Cat no.78200.200.UL, Thermofisher). Subsequently, the cleaned PCR products were sent for Sanger sequencing at the UAB core facility. Each PCR product was sequenced with its respective PCR primer using the fluorescent dideoxy terminator method of cycle sequencing (ABI Prism BigDye Terminator v3.1 Cycle Sequencing kits using 1 / 16 of the “standard” protocol). The sequencing reactions were purified using the Applied Biosystems X Terminator purification system as per the manufacturer's protocols. After purification, sequencing reactions were run on a 3730xl DNA Analyzer (Applied Biosystems Division, Foster City, CA) following Applied Biosystems' protocols. The sequence of each DNA sample was manually checked using the Chromas Lite program and further subjected to multiple alignments to identify nucleotide variations, using the MegAlign program of the Lasergene software. The different fragments of human OLA1 gene sequences were assembled using the SeqMan program of Lasergene and finally submitted to NCBI GenBank. Genetic screening for OLA1 point mutation

[0155] For genotyping of the non-synonymous Exon8 SNP A751G in the OLA1 gene that changes amino acid Y254C, a tetra-primers ARMS-PCR-based screening protocol was developed using four sets of primers following previously described protocols (Dubey, et al. J Appl Anim Res.201240:17–19; Goyal, et al. Gene.2014540:238–245; Ye, et al. Nucleic Acids Res.200129:e88). The primers were designed from the human OLA1 sequence (accession no. ON073791.1) using web-based software and synthesized through IDT. Further, nucleotide- specific amplification of the genotype protocol was standardized and validated by Sanger sequencing. PCR was performed on isolated genomic DNA samples in a 20 μl reaction volume containing approximately 25-50 ng of template DNA, 0.5 μl of 10 pmol of each inner primer, 0.1 μl of 10 pmol of each outer primer, and 2X PCR Master Mix (Cat.no. PRM7505, Promega). Amplification was carried out using the following PCR conditions: 95°C for 3 min, followed by 32 cycles of 94°C for 30 sec, annealing at 58°C for 30 sec, and 72°C for 1 min, followed by a final extension at 72°C for 5 min. Samples were checked on ethidium bromide-stained 2% ultra-pure agarose gel, and genotypes were recorded.Plasmid construction

[0156] The Human flag tagged OLA1 gene was amplified using synthesized cDNA derived from human mRNA. The resulting amplicon was cloned into a mammalian expression vector, pcDNA3.1 TOPO TA expression kit (Thermo Fisher Scientific), following the manufacturer's instructions. Generation of WT and mutant human OLA1 vector.

[0157] To introduce a point mutation in the OLA1 gene at SNP A754G resulting in the Y254C substitution, site-directed mutagenesis primers were designed for the G allele. The primers were designed using the Q5® Site-Directed Mutagenesis protocol and synthesized by Integrated DNA technology, USA. The A751G point mutation was generated in the WT OLA1 vector using the Q5® Site-Directed Mutagenesis Kit (NEB) according to the manufacturer's instructions. Positive clones were further validated by Tetra Arms PCR genotyping and Sanger sequencing. Overexpression of WT and Mutant OLA1 in human cardiomyocytes

[0158] Ac16 (Human cardiomyocyte cell line, cat# SCC109) was purchased from Merk Sigma and grown in cultured in Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12 (DMEM / F12) with 2 mM L-Glutamine, (Cat# 11320082, Thermo scientific Life Technologies, Grand Island, NY) supplemented with 12.5% fetal bovine serum (FBS, Corning, USA) and 1% Penicillin-Streptomycin (Life Technologies, USA) in a humidified incubator at 37°C with 5% CO2. AC16 cells were seeded into 6 well tissue culture plates for 70–80% confluency and transfected with either with GFP, WT Human OLA1 or Mut Human OLA1 expressing vectors for 24hrs, and protein were isolated. Protein extraction and Western blot analysis

[0159] Protein lysates from human failing and non-failing hearts, and human cardiomyocytes cells (Ac16) were prepared into RIPA Cell Lysis Buffer (cat# J63324, Alfa Aesar) supplemented with HaltTM protease inhibitor (cat# 87786, ThermoFisher Scientific) cocktails and phosphatase inhibitor (cat# P5726, Millipore Sigma) following a published protocol (Henderson et al., 2021; Singh et al., 2022). Equal amounts of proteins were loaded on AnyKD Mini-PROTEAN TGX stain-free protein gels (cat# 4569033, Bio-Rad Laboratories) and transferred to polyvinylidene difluoride (PVDF) membranes using Trans-Blot Turbo transfer system (Bio-Rad Laboratories). The immunoblot blots were quickly washed with water and blocked with 5% fat free milk for an hour. After washing membrane incubated with primary antibodies against Flag (cat# 80010-1-RR, Proteintech), GFP (cat# 50430-2-AP, Proteintech), OLA1 (cat# 16371-1-AP, Proteintech), Ubiquitin (cat# 10201-2-AP, Proteintech), XIAP (cat#14334, Cell signaling), Diablo (cat# 60004-1-Ig, Novous biotech), Cleaved caspase3 (cat# 9664, cell signalling), Laminb1 (cat# 12987-1-AP, Proteintech), and GAPDH (cat# 60004-1-Ig, Proteintech). After washing immune blots were incubated with secondary HRP-conjugated antibodies against mouse (cat# SA00001-1, Proteintech) and rabbit (cat# SA00001-2, Proteintech) at room temperature for 1 hour on shaker. Images were acquired using a ChemiDoc™ Touch Imaging System (Bio-Rad, USA) using an enhanced chemiluminescence (Pierce) detection system. Results Human heart failing leads to downregulation of OLA1

[0160] OLA1 has been shown to play a critical role in the progression of cancer and heart diseases (Lu S, et al. J Hematol OncolJ Hematol Oncol.202114:188; Narasimhan G, et al. Clin Exp Pharmacol Physiol.201946:743–751; Schuldt M, et al. Circ Heart Fail.2021 14:e007022.). Both mRNA and protein expression of OLA1 gene was checked in human failing and non-failing heart. Interestingly both mRNA and protein level of OLA1 was significantly downregulated in failing heart as compared to non-failing (FIGs.12A-12C).

[0161] Further to see tissue specific expression of OLA1 gene and OLA1 protein localization expression of OLA1 was checked in different mice tissues (Brain, Adipose Tissue, lung, Kidney, Spleen, Liver, Skeletal muscle, and Heart) and found maximum expression in Skeletal muscle, Heart, and lowest expression in brain (FIGs.18A-18B). Fractionation of cytoplasmic and nuclear revealed OLA1 mainly localized into cytoplasm and little in nuclear, suggestions OLA1 playing important role in cytosol and energy demanding tissue such as Skeletal muscle and heart. Characterization of human OLA1 gene

[0162] To characterize the human OLA1 gene at both mRNA and genomic levels, the OLA gene was amplified from mRNA and DNA isolated from human heart tissue. Complete ORF of OLA1 mRNA was amplified and cloned into the pCDNA3.1 TA cloning system and sequenced. Sequences analysis of human OLA1 mRNA submitted 236 to NCBI data bank (accession numbers ON073790.1).

[0163] Analyses revealed that the human OLA1 CDS (Coding DNA sequence) is 1191 base pairs long, encoding 396 amino acids, consistent with other species (FIG.13A). Comparison of human OLA1 with other species showing maximum homology among them. Further PsiPred analysis for 2D and 3D structure of human OLA1 translated protein revealed that human OLA1 protein consist of 13 strands 16 helix, and rest 23 coils (FIGs.13B-13C). Phylogenetic analysis based on both nucleotide and amino acid sequences indicated that thehuman OLA1 gene is closely related to Chimpanzees, followed by monkeys, rats, mice, sheep, pigs, and horses (FIG.13D). Notably, the horse was the most distant species in the phylogenetic tree from zebrafish compared to all other species. The findings provide insights into the genetic characteristics and evolutionary relationships of the human OLA1 gene. Genomic organization of OLA1 gene

[0164] For genomic characterization of human OLA1 gene different fragments of the human OLA1 genomic sequence were assembled (FIG.19A). To find Open reading frame (ORF) in OLA1 gene align with amplified human OLA1 mRNA sequence and further validated with online NCBI Splign program. Analysis revealed that the OLA1 gene is composed of 11 exons, with the start codon located in exon 2 and the stop codon in exon 11 (FIG.19B). The length of each exon is shown in the figure and has been submitted to the NCBI gene bank under accession number ON073791.1. Furthermore, this analysis also showed that the intronic sequence of the OLA1 gene is relatively long, spanning over 100,000 base pairs. This information is of potential importance for future studies of the regulatory mechanisms that control the expression of the OLA1 gene. Expression of OLA1 splice variant in Human heart tissue

[0165] OLA1 has been shown to play a critical role in the progression of cancer and heart diseases. The genome browser (Ensemble human genome browser and NCBI data bank) has revealed several splice variants of the gene. In order to identify splice variants in the human heart tissue samples, several sets of primers targeting each variant's exon were designed after alignment with the Ensemble human data bank. To assess the expression of these variants, real-time quantitative PCR was performed on reverse transcribed human RNA isolated from human heart samples. Analysis revealed four distinct splice variants (201, 203, 204, and 209; FIGs.14A-14B). To validate these findings, the variants were amplified and cloned into a TA cloning system for sequencing from both ends using the Sanger sequencing method. After assembly, alignment, and translation of the CDS protein, it was found that the GTPase domain of OLA1 is conserved in all four variants. These findings further highlight the importance of the OLA1 gene and its GTPase domain. Future studies may explore the specific roles of these splice variants in cancer and heart diseases. Detection of mutation in OLA1 gene

[0166] In most cases (70-80%), familial dilated cardiomyopathy is inherited as an autosomal disease, where a single copy of the altered gene is sufficient to cause the disorder and is inherited from one affected parent. A previous study showed that a locus on chromosome 2q31 is strongly associated with familial dilated cardiomyopathy in humans. This locus(CAD1G) also contains the major cytoskeletal muscle protein TITIN 20. Several studies have shown that genetic mutations in the Titin gene cause DCM due to the loss of contractile function crucial for myofibrillar elasticity and integrity. Interestingly, the OLA1 gene also maps to the same locus (chromosome 2q31) close to the Titin gene, and alterations in the OLA1 gene expression has been detected in human heart failure samples, and in mice cardiac tissue after ANGII administration. Studies in cancer have shown that mutations in the OLA1 gene are associated with disease progression and poor diagnosis and survival in humans. A point mutation (E168Q) in the OLA1 gene in breast cancer failed to bind BRCA1 for centromere function 6. A recent study in atherosclerosis patients has shown several mutations in the OLA1 gene, among which five have been strongly associated with the development of atherosclerosis in humans. To screen for polymorphisms in the OLA1 gene and their associations with heart diseases, the OLA1 gene was amplified in nine different fragments covering all exons along with adjacent partial introns from isolated DNA from human failing and non-failing heart tissue. After Sanger sequencing, the data were analyzed and mapped to the OLA1 gene. Interestingly, there was a total of 16 mutations, including 2 transversions, 2 substitutions, 1 deletion, and 11 transition mutations in the OLA1 gene (FIGs.15A-15C, Table 1). All mutations were intronic except for one non-synonymous mutation 5122A>G resulting in 254Tyr>Cys in OLA1 exon 8. Genotype and allelic frequency were analyzed for all SNPs found in the human OLA1 gene (Table 1). Association of ola1 mutation with heart failure

[0167] Furthermore, to investigate the association of OLA1 mutation with heart failure, the full OLA1 gene sequence of each individual was assembled and aligned it in MegaAlign to find variations. Each variation was then confirmed by checking it with the respective chromatogram using the Chromas Lite program. Interestingly, clear variations were found among failing and non-failing heart DNA samples and identified SNPs in OLA1 that showed a strong correlation with failing heart. The pattern of genotype and allele frequency differed significantly between failing and non-failing patient DNA samples (Table 1), and these SNPs were linked to each other (MegaAlign, FIGs.15A-15C). Most of the SNPs were intronic and showed a correlation between them. One heterozygous exonic mutation (5144 A>G, resulting in 254 Tyr >Cys) was also identified in exon 8, which has been reported in cancer patients with the same SNP ID (rs11558990) showing poor diagnosis (FIGs.16A-16D). To further test the effect of this mutation, different online servers were used to predict its possible effect. The Predictor of Human Deleterious Single Nucleotide Polymorphisms (PhD-SNP) analyses revealed that themutation in OLA1 (254 Y>C) is deleterious in nature. Similar effects of this SNP were also found by the PhyPhen2 (0.89) and SIFT (.078) analyses (FIGs.16A-16D).

[0168] A distinct pattern of OLA1 mutations was observed between failing and non- failing hearts, with some SNPs being linked to each other (FIG.4). This resulted in different haplotypes between the two conditions (Table 2). Notably, several mutations were found in intronic regions, which may play a crucial role in RNA splicing which need to be confirm. Role of OLA1 mutation (254Tyr>328 Cys) on cardiac biology

[0169] A mutation (254 Tyr vs Cys) was found in exon8 of OLA1 gene. In Human accumulated polyubiquitinated proteins is one of common feature resulting load on ubiquitin proteasome system (UPS) in DCM and heart failure. Studies in humans and animal have been shown increased polyubiquitination of proteins in failing hearts compared with control. To check does OLA1 mutation (254Tyr>Cys) also regulate ubiquitination polyubiquitination was checked in human heart with OLA1 mutation and WT OLA1. Interestingly there was increased ubiquitination which led to downregulation of antiapoptotic gene XIAP known to be activate caspase pathway for the activation of apoptosis (FIGs.17B-17C). To validate the finding, WT OLA1, Mut OLA1 and GFP as control were overexpressed in human cardiomyocytes cells (Ac16) (FIGs.17D-17E). There was increased cardiomyocytes death in overexpression of Mut- OLA1 by activation of caspase3 (FIG.17F). This finding suggests that OLA1 mutation 254Tyr>Cys in human could be a potential cause of cardiomyopathy in human heart failing, which need to be further validated. Development of PCR based screening protocol for OLA1254 Tyr>Cys mutation.

[0170] The mutation 5144A>G was found in the OLA1 gene of both failing and non- failing human heart patients, and has also been reported in a large cohort of cancer patients with poor survival. Crystallographic analysis of Mutant OLA1 showed an altered structure compared to WT OLA1. Due to the importance of the nonsynonymous SNP 5144 A>G (254Tyr>Cys) present in the GTPase domain of human OLA1, a simplified, cost-effective, and quick screening protocol was developed to detect this point mutation. To achieve this, a Tetra- ARMS PCR screening assay utilizing four different primer sets was developed. Two outer primers (forward and reverse) were common for both genotypes, amplifying a 264 bp fragment, while two inner primer sets were developed, each specific to a particular allele. The inner forward primer specific for the G allele amplified a 149 bp fragment, while the inner reverse primer specific for the A allele amplified a 171 bp fragment (see FIG.17G). This screening protocol was further validated using known genotypes to test its accuracy, and as expected, the AA genotype showed two bands (264bp and 171bp), while the heterozygous A / G genotypeshowed three bands (264bp, 358171bp, and 149 bp) (see FIG.17G). SNP data from cancer patients showed a somatic missense mutation in the OLA1 gene (chr2:q31.2.174082033 A>G) resulting in a 254 threonine to histidine substitution in cancer patients (254Thr>His). This screening protocol is also useful for detecting the OLA1 somatic mutation 254Tyr>His in-cancer patients. Discussion

[0171] OLA1 is a member of the GTPase family of proteins, possessing both GTPase and ATPase activities. It is highly expressed in cancer cells and associated with poor survival. Overexpression of OLA1 inhibits apoptosis by interacting with breast cancer associated gene 1 (BRCA1) and BRCA1-associated RING domain protein (BARD1) in cancer cells. OLA1 interacts with elf2a to form a ternary complex that regulates protein translation and cell proliferation. A recent study has shown that OLA1 is required to interact with Hsp70 for the stabilization of mitochondrial superoxide dismutase 2 (SOD2) in pulmonary smooth muscle cells in mice. Loss of OLA1 causes SOD2 deficiency, leading to increased expression of an anti-apoptotic gene, X- linked inhibitor of apoptosis (XIAP), resulting in increased proliferation of pulmonary SMCs.

[0172] In human failing hearts, the expression of OLA1 was downregulated in hypertrophic cardiomyopathy (HCM) patients' hearts. It was recently found that in a mouse model of angiotensin II (AngII)-induced cardiomyopathy, altered expression of OLA1 leads to phosphorylation of the GSK3β / β-catenin pathway, resulting in the development of cardiac hypertrophy. The OLA1 gene is required for cell survival, and genetic deletion of OLA1 in mice results in stunted growth, delayed development, and the birth of immature organs, ultimately leading to perinatal lethality. Interestingly, deletion of OLA1 leads to a significant increase in heart weight compared to body weight, suggesting that OLA1 plays an important role in the heart.

[0173] Looking importance of OLA1 gene in human diseases, the OLA1 gene is first characterized. DNA sequencing of human OLA1 gene mRNA and genomic organization of human OLA1 gene were showing like other species suggesting that conservation of OLA1 gene among of cardiomyopathy, with the same gene implicated in multiple forms of the disease. This underscores the importance of genomic context in the pathophysiology of disease-associated variants. Gene mutations are an important cause of DCM, with over 40 genes encoding components of the sarcomere, cytoskeleton, or nuclear lamina shown to cause DCM in humans. Titin (TTN) is a giant protein in the human body that plays a crucial role in the basic organization of the sarcomere structural unit, facilitating contraction of striated muscle. TTN is mapped to chromosome 2 at the locus 1q31, which is associated with familial DCM 20. Recent studieshave shown that truncation or mutation in the TTN gene is a major cause of DCM, accounting for up to 25% of cases in humans. The OLA1 gene is mapped to the locus 1q31 of chromosome 2, which is close to TTN and associated with familial dilated cardiomyopathy 20. Recent studies have also shown that mutations in OLA1 are associated with poor survival and increased risk for developing atherosclerotic diseases in humans.

[0174] However, the role of OLA1 mutations in human heart failure patients has not yet been studied. In this study, the goal was to screen for mutations in the OLA1 gene and investigate its association with human heart failure. In this study 16 mutations were found in OLA1 gene among failing and non-failing patients. All mutations found were intronic except one nonsynonymous 2144 A>G resulting amino acid change 254Tyr>Cys. This mutation also reported SNPs data bank under SNP ID rs11558990. Shown a strong correlation with disease progression and survival in cancer patients. Interestingly, in REGARDS study, same mutation also found in enrolled 30,239 Black and white participants between 2003-2007, have higher rates of stroke and related diseases that affect brain health.

[0175] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0176] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMS 1. A method for detecting cardiomyopathy and heart failure in a subject, the method comprising assaying a sample from the subject for decrease in expression of a Obg Like ATPase 1 (OLA1) relative to a sample from a healthy subject, the presence of a Y254C mutation in OLA1, or both, thereby detecting cardiomyopathy and heart failure in the subject.

2. The method of claim 1, wherein the sample is a cardiac tissue sample.

3. The method of claim 1, wherein the sample comprises autosomal DNA and wherein the method comprises assaying the sample for the presence of the Y254C mutation.

4. The method of claim 1, wherein assaying the sample further comprises assaying a DNA sample for an A5122G mutation in exon 8 of the ola1 gene.

5. The method of claim 4, wherein the method further comprises performing a PCR amplification screen to detect homologous or heterologous genotypes.

6. The method of claim 1, further comprising assaying the sample for an increase in mRNA and protein expression of one or more endoplasmic reticulum stress markers relative to a sample from a healthy subject.

7. The method of claim 6, wherein the one or more endoplasmic reticulum stress markers comprise BIP, CHOP, sXBP1, or any combination thereof.

8. A method for treating a heart condition in a subject, the method comprising administering to the subject an expression vector comprising a nucleic acid sequence encoding wild-type Obg Like ATPase 1 (OLA1).

9. The method of claim 8, wherein the heart condition comprises dilated cardiomyopathy, heart failure, myocardial infarction or heart attack, ischemic heart disease, coronary heart disease, hypertrophic cardiomyopathy, or any combination thereof.

10. The method of claim 8, wherein the expression vector is administered to cardiac tissue in the subject.

11. The method of claim 10, wherein administering the expression vector to cardiac tissue reduces or eliminates at least one side effect relative to systemic administration of the expression vector.

12. The method of claim 10, wherein the expression vector is administered by retrograde coronary venous or sinus delivery to the subject.

13. The method of claim 10, wherein the expression vector is delivered to cardiac cells using nanotransfection, in a liposome, or in an extracellular vesicle.

14. The method of claim 10, wherein the expression vector comprises mRNA.

15. The method of claim 10, wherein the nucleic acid sequence is operably linked to an expression control sequence.

16. The method of claim 10, wherein the expression vector is an adeno-associated vector (AAV).

17. The method of claim 16; wherein the AAV is an AAV9.

18. The method of claim 16, further comprising administering a helper virus or packaging cell line to the subject with the AAV.

19. The method of claim 16, wherein the AAV is delivered in a lipid nanoparticle, wherein the lipid nanoparticle comprises a cationic lipid.

20. The method of claim 19, wherein the lipid nanoparticle further comprises a neutral lipid.

21. The method of any one of claims 8-20 , wherein the nucleic acid sequence encodes the amino acid sequence SEQ ID NO:1.

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