Gene therapy of Hippo signaling improves cardiac function in clinically relevant models

Abstract

Improve cardiac function in a clinically relevant model by gene therapy of Hippo signaling. **SOLUTION**: Embodiments of the present disclosure include methods and compositions for doing so in an individual in need of improving cardiac function, including reducing the risk of arrhythmia. In certain embodiments, an individual in need of improving cardiac function and / or reducing the risk of arrhythmia is provided with an shRNA molecule that targets an effective amount of the Sav1 gene.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 356,673, filed Jun. 29, 2022, which is hereby incorporated by reference in its entirety.

[0002] The present disclosure relates to at least the fields of cell biology, molecular biology, and medicine.

Background Art

[0003] The Hippo pathway is activated in the heart in response to physiological inputs such as changes in the composition of the extracellular matrix (ECM) or mechanical signaling and is maladaptively upregulated in human heart failure (HF) (Leach et al., 2017; Wang et al., 2018). Core Hippo pathway components, including Mst kinase, and the adapter salvador (Sav), phosphorylate Lats kinase, which then inhibits the downstream transcriptional cofactors Yap and Taz. When Hippo pathway activity is low, Yap and Taz enter the nucleus, where they cooperate with Tead family transcription factors to activate target genes. In adult cardiomyocytes (CMs), high levels of Yap produced via transgene expression induce a shift of cells to a more fetal-like and regenerative state by altering chromatin accessibility across the genome (Monroe et al., 2019). Yap-Tead target genes in CMs include multiple cyclin genes and many genes that promote cell cycle progression, including genes that promote cytoskeletal remodeling and protrusion formation in border zone (BZ) CMs (Morikawa et al., 2015). Since Sav encodes an adapter rather than a kinase, loss of Sav function moderately inhibits Hippo signaling, which is desirable for human CM regenerative therapy (Heallen et al., 2013). Furthermore, since there is a single Sav gene in the mammalian genome, the Sav gene is an attractive target for therapy. Indeed, as suggested by tests in mice, mild Hippo pathway inhibition via Sav knockdown is a feasible strategy for safely treating human HF (Leach et al., 2017).

[0004] Mouse studies are valuable, but there are limitations to translational studies because of the clear differences in cardiovascular anatomy and physiology between rodents and humans. In addition to obvious size differences, heart rates are much faster in rodents (300 - 840 beats per minute in mice and 330 - 480 beats per minute in rats) than in humans (80 - 100 beats per minute) (Spannbauer et al., 2019; van der Velden et al., 2004).

[0005] In contrast, the pig heart shares many similarities with the human heart both in the steady state and after myocardial infarction (MI) (Spannbauer et al., 2019; van der Velden et al., 2004). For example, the pig and human hearts have similar contractility indices as determined by cardiac catheterization measurements (Stubenitsky et al., 1997; Milani-Nejad et al., 2014). Furthermore, pig and human CMs share many features in excitation-contraction coupling. Similar to human CMs, pig CMs mainly express β-myosin heavy chain, and both the stiff N2B and compliant N2BA titin isoforms are expressed in pig myocardium, similar to the human heart (Milani-Nejad et al., 2014). Pigs also share locally similar cardiac hemodynamic characteristics with humans (McCormick et al., 2016). In diseased pig and human hearts, changes in myofilament function are seen after MI (van der Velden et al., 2004), and both pig and human hearts show reduced contractility after MI, which is caused by changes in Ca2+ handling (van der Velden et al., 2004). A decrease in SERCA2a expression has been reported in both species (van der Velden et al., 2004). An increase in Ca2+ sensitivity is also a common feature of pig and human hearts after MI (Stubenitsky et al., 1997), and these two species share hemodynamic similarities in HF following an increase in afterload (Gyongyosi et al., 2017). Therefore, due to the similarities between pig and human hearts, the pig model is superior to the mouse model for testing the treatment of heart diseases.

[0006] In recent experiments with pigs, overexpression of miR199a initially improved cardiac function after MI, but the pigs ultimately died two months after viral delivery, most likely due to arrhythmia (Gabisonia et al., 2019). Experiments in mice also show that constitutive overexpression of active Yap or microRNA in the heart can be harmful (Tian et al., 2015; Monroe et al., 2019). Until this disclosure, it was unclear whether the inhibitory regulation of the Hippo pathway in CM proliferation was conserved in large mammals. The present disclosure provides methods and compositions for fulfilling the long-standing desire for therapeutic CM proliferation and for treating or avoiding arrhythmia for any reason.

Summary of the Invention

[0007] Chronic human diseases such as HF are difficult to treat, generally fatal, and associated with the aging of the population. A characteristic of non-regenerative organs such as the heart is that functional cell types such as cardiac CMs are highly specialized and have limited self-renewal ability. The present disclosure demonstrates that using gene therapy to knockdown the Hippo pathway in CMs has cardiovascular benefits in a porcine model of MI without the harmful effects of loss of function of the Hippo pathway in non-CMs such as cardiac fibroblasts (Xiao et al., 2019). This is the first demonstration that tissue regeneration therapy, an approach that induces tissue regeneration by inhibiting endogenous pathways in failing organs, is effective and safe in the translational context of a large animal model.

[0008] The examples herein show that the Hippo pathway inhibits CM proliferation in pigs and that a similar effect occurs in mammalian hearts, including the human heart. These findings demonstrate that, for example, AAV9-Sav-shRNA therapy induces a reliable improvement in cardiac function in a safe and effective manner for the treatment of heart disease, particularly in human patients who have experienced myocardial infarction and / or myocardial fibrosis.

[0009] In certain embodiments, an individual in need of treatment for a heart condition is provided with an effective amount of one or more nucleic acids, or cells comprising one or more nucleic acids, which directly or indirectly provide a therapeutic benefit to the individual. In certain embodiments, the nucleic acid is in a form that directly or indirectly provides RNA interference, including at least shRNA. In certain embodiments, the shRNA composition targets a member of the Hippo pathway. It can target any member of the Hippo pathway, but in certain embodiments, the shRNA targets Salvador (Sav1).

[0010] In embodiments of the present disclosure, there are nucleic acid compositions that target human Sav1, and in certain embodiments, the nucleic acid composition comprises shRNA molecules. In certain embodiments, there are therapeutic compositions comprising shRNA molecules that comprise one or more of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. The composition may or may not be included in a vector, including a viral vector or a non-viral vector. In certain embodiments, the shRNA sequence is utilized in a non-integrating vector.

[0011] In some embodiments, there are isolated synthetic nucleic acid compositions comprising a derivative nucleic acid that comprises at least 80% identity to one of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 and / or to one of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. The derivative nucleic acid can be at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to one of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0012] In certain embodiments, the nucleic acid comprises the sequence of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, and further comprises the antisense sequence of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, respectively, and when the sequence and the antisense sequence hybridize together to form a double-stranded structure, the sequence and the antisense sequence are separated by a loop structure.

[0013] In certain embodiments, the nucleic acid is at least 43 nucleotides in length or 137 nucleotides in length or less. In some embodiments, the loop structure is 5 to 19 nucleotides in length. In certain embodiments, the derivative nucleic acid has 1, 2, 3, 4, or 5 mismatches compared to SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, respectively.

[0014] In some embodiments of the composition, the nucleic acid or derivative nucleic acid is contained in a vector such as a viral vector or a non-viral vector. The vector can be a non-integrating vector. The vector can be a non-integrating lentiviral vector. In some embodiments of the vector, two or more of the nucleic acids containing SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 are present on the same vector.

[0015] In certain embodiments, the expression of the nucleic acid is controlled by a tissue-specific or cell-specific promoter such as a cardiomyocyte-specific promoter (e.g., rat ventricular-specific cardiac myosin light chain 2 (MLC-2v) promoter; cardiac muscle-specific alpha myosin heavy chain (MHC) gene promoter; cardiac cell-specific minimal promoter of -137 to +85 of the NCX1 promoter; chicken cardiac troponin T (cTNT), or a combination thereof).

[0016] In certain embodiments, two or more of the nucleic acids containing SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 are present on the same vector. In certain cases, two or more nucleic acids are regulated by the same regulatory sequence or by different regulatory sequences.

[0017] The methods of the present invention include improving systolic function in human patients who have experienced myocardial infarction, reducing arrhythmias in human patients with myocardial fibrosis, and promoting angiogenesis in heart tissue in human patients in need thereof. The methods include providing an individual with an effective amount of a composition encompassed by the present disclosure. The composition may be provided to the individual multiple times. The composition may be provided to the individual systemically or locally. In certain embodiments, the individual is provided with additional therapy for the cardiac condition.

[0018] In certain embodiments, there is a kit comprising a composition encompassed by the present disclosure, the composition being contained within a suitable container.

[0019] In certain embodiments, the methods of the present invention include providing an individual with an shRNA that targets a therapeutically effective amount of Salvadore (Sav1). In certain embodiments, the shRNA is provided to the individual with an AAV9 vector.

[0020] The foregoing has outlined, in general terms, the features and technical advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described hereinafter which form the subject matter of the claims of the invention. It should be appreciated by those skilled in the art that the concepts and specific embodiments disclosed herein may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. It should also be understood by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the present invention as set forth in the appended claims. The novel features believed to be characteristic of the invention both as to its construction and method of operation, together with further objects and advantages, will be better understood from the following description when considered in connection with the accompanying drawings. It is to be expressly understood, however, that each of the drawings is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present invention.

[0021] For a more complete understanding of the present disclosure, reference is made to the following description taken in conjunction with the accompanying drawings.

Brief Description of the Drawings

[0022]

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Mode for Carrying Out the Invention

[0023] Exemplary Definitions In the context of the present invention, especially in the context of the following claims, the use of the terms "a", "an", "the", and similar designators should be construed to include both the singular and the plural, unless otherwise specified herein or clearly contradicted by the context. The terms "comprising", "having", "including", and "containing" should be construed as open-ended terms (i.e., meaning "including but not limited to") unless otherwise stated. The recitation of a range of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by the context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is merely intended to better illuminate the invention and does not impose a limitation on the scope of the invention unless otherwise claimed. No representation herein should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0024] Throughout this specification, unless the context requires otherwise, the word "comprise", "comprises" or "comprising" means including the stated step or element or group of steps or elements but not meaning to exclude any other step or element or group of steps or elements. "Consisting of" means including and limited to all that comes after the phrase "consisting of". Thus, the phrase "consisting of" indicates that the recited elements are necessary or essential and that no other elements may be present. "Consisting essentially of" means including any elements recited after this phrase, and with respect to other elements, limited to those that do not interfere with or contribute to the activity or action specified in the disclosure of the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are necessary or essential, but that other elements are optional and may or may not be present depending on whether or not they affect the activity or action of the recited elements.

[0025] References throughout this specification to "one embodiment", "an embodiment", "a certain embodiment", "related embodiments", "some embodiments", "additional embodiments", or "further embodiments", or combinations thereof, mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Thus, the appearances of the above phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0026] As used herein, the terms "or" and "and / or" are used in combination with each other or to describe multiple components that do not include each other. For example, "x, y, and / or z" may refer to "x" only, "y" only, "z" only, "x, y, and z", "(x and y) or z", "x or (y and z)", or "x or y or z". It is specifically contemplated that x, y, or z may be specifically excluded from the embodiments.

[0027] Throughout this application, the term "about" is used in accordance with its plain and ordinary meaning in the fields of cell and molecular biology, indicating that a value includes the standard deviation of error of the apparatus or method used to determine the value.

[0028] As used herein, the "complementary nucleotide sequence", also known as the "antisense sequence", refers to a nucleic acid sequence that is completely complementary to the sequence of a "sense" nucleic acid encoding a protein (e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence). As used herein, nucleic acid molecules are provided that include sequences that are complementary to at least, at most, exactly, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. An antisense nucleic acid sequence or oligonucleotide can be a sequence of DNA or RNA that can bind to a target "sense" sequence via base pairing. In some cases, the sense sequence is a nucleic acid encoding a protein, whereby the antisense nucleic acid is complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. The antisense sequence can be completely or partially complementary to the sense sequence. The antisense nucleotide sequence can be designed to specifically hybridize to a particular region of a desired target protein or mRNA to interfere with replication, transcription, or translation. The antisense sequence can be of any length, for example, complementary to a sense sequence of at least, at most, exactly, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.

[0029] In certain embodiments, when an antisense nucleotide (nucleic acid) or small interfering RNA (siRNA) (processed from short hairpin RNA (shRNA)) binds to a target sequence, the particular antisense or small interfering RNA (siRNA) sequence is substantially complementary to the target sequence and thus specifically binds to a portion of the mRNA encoding the polypeptide. Thus, typically, the sequences of those nucleic acids are highly complementary to the mRNA target sequence and will have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer base mismatches throughout the sequence. In many cases, it is desirable for the sequences of the nucleic acids to be exactly matched, i.e., completely complementary to the sequence to which the oligonucleotide specifically binds, and thus it may be desirable to have zero mismatches along the complementary regions. Highly complementary sequences typically bind very specifically to the target sequence region of the mRNA and thus will be more efficient at reducing and / or further inhibiting the translation of the target mRNA sequence into the polypeptide product. See, e.g., U.S. Patent No. 7,416,849.

[0030] Substantially complementary oligonucleotide sequences can have a complementarity (or percent identity) of greater than about 80% to the corresponding mRNA target sequence to which the oligonucleotide specifically binds, more preferably greater than about 85% complementary to the corresponding mRNA target sequence to which the oligonucleotide specifically binds. In certain embodiments, as described above, it is desirable to have an even more substantially complementary oligonucleotide sequence for use in the practice of the invention, and in such cases, the oligonucleotide sequence is greater than about 90% complementary to the corresponding mRNA target sequence to which the oligonucleotide specifically binds, and in certain embodiments, can be greater than about 95% complementary to the corresponding mRNA target sequence to which the oligonucleotide specifically binds, and can be complementary to the target mRNA to which the designed oligonucleotide specifically binds at up to, at least, exactly 96%, 97%, 98%, 99%, or between any two of these and 100% identity. See, for example, U.S. Patent No. 7,416,849. The percent similarity or complementarity of any nucleic acid sequence can be determined, for example, by utilizing any computer program well known in the art.

[0031] The terms "inhibitory nucleic acid" and "inhibitory oligonucleotide" are used synonymously and refer to molecules that knockdown the expression of a target gene by preventing the translation of the corresponding mRNA. As described above, expression is inhibited by the sequence-specific binding of the inhibitory nucleic acid to its target. Certain inhibitory RNAs, such as short hairpin RNA (shRNA) and short interfering RNA (siRNA), utilize sequence complementarity to target mRNA for destruction. When appropriately targeted to a specific mRNA within a cell via its nucleotide sequence, the inhibitory RNA specifically suppresses the expression of the target gene, reduces the cellular level of the corresponding target mRNA, and decreases the level of the protein encoded by such mRNA.

[0032] The inhibitory nucleic acid can be single-stranded or double-stranded. Examples of inhibitory nucleic acids include antisense DNA and RNA oligonucleotides, siRNA, shRNA, and microRNA. As used herein, the term "knockdown" or "knockdown technology" refers to a gene silencing technology in which the expression of a target gene or a gene of interest is reduced as compared to the gene expression prior to the introduction of an inhibitory RNA such as shRNA that can lead to inhibition of the production of the target gene product. For example, the expression can be reduced by at least, up to, exactly 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or can be reduced between any two of these. The expression can be reduced by any amount (%) within these intervals, for example, 2-4, 11-14, 16-19, 21-24, 26-29, 31-34, 36-39, 41-44, 46-49, 51-54, 56-59, 61-64, 66-69, 71-74, 76-79, 81-84, 86-89, 91-94, 96, 97, 98, or 99. The reduction in gene expression can be statistically significant as compared to the unchanged or wild-type gene expression when measured by, for example, Student's T-test or other known statistical methods. The knockdown of gene expression can be directed by techniques well known in the art, for example, by the use of inhibitory RNA or by the use of genome editing, for example, by CRISPR or TALEN.

[0033] As used herein, the terms "knockdown" or "knockdown technology" refer to gene silencing technologies in which the expression of a target gene or gene of interest is reduced compared to the gene expression prior to the introduction of shRNA that can lead to inhibition of the production of the target gene product. The term "reduced" is used herein to indicate that the expression of the target gene is decreased by 0.1 - 100%. For example, the expression can be reduced by at least, at most, exactly 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or can be reduced between any two of these. The expression can be reduced by any amount (%) within these intervals, for example, 2 - 4, 11 - 14, 16 - 19, 21 - 24, 26 - 29, 31 - 34, 36 - 39, 41 - 44, 46 - 49, 51 - 54, 56 - 59, 61 - 64, 66 - 69, 71 - 74, 76 - 79, 81 - 84, 86 - 89, 91 - 94, 96, 97, 98, or 99. Knockdown of gene expression can be directed by the use of siRNA or shRNA.

[0034] As used herein, the terms "nucleotide sequence" or "nucleic acid" refer to a polymer of DNA or RNA having a combination of covalent bonds between nucleosides that includes purine and pyrimidine bases, sugars, and phosphate groups in phosphodiester bonds. Nucleic acids can be single-stranded or double-stranded and optionally contain synthetic, non-natural, or modified nucleotide bases that can be incorporated into DNA or RNA polymers. The term "polynucleotide" is used synonymously with the term "oligonucleotide". The terms "nucleotide sequence" and "nucleic acid sequence" refer to the sequence of nucleotides in a polynucleotide molecule. The term "nucleotide sequence" is synonymous with "nucleic acid sequence" unless expressly stated otherwise. It should be understood that when a sequence containing thymine is provided, the corresponding RNA sequence contains uracil at the positions shown as thymine.

[0035] In some cases, nucleic acid analogs may have alternative backbones or non-natural internucleoside linkages, including, for example, modified phosphorus-containing backbones and non-phosphorus backbones, such as morpholino backbones; siloxane, sulfide, sulfoxide, sulfone, sulfonate, sulfonamide, and sulfamate backbones; formacetyl and thioformacetyl backbones; alkene-containing backbones; methyleneimino and methylenehydrazino backbones; amide backbones, and the like. See, e.g., U.S. Patent No. 7,410,944. Examples of modified phosphorus-containing backbones include phosphoramide, phosphorothioate, phosphorodithioate, chiral phosphorothioate, O-methylphosphoramidite phosphotriester, aminoalkyl phosphotriester, alkyl phosphonate, thionoalkyl phosphonate, phosphinate, phosphoramidate, thionophosphoramidate, thionoalkyl phosphotriester, boranophosphate, and their various salt forms. Examples of the above-described non-phosphorus-containing backbones are well known in the art and are described, for example, in U.S. Patent No. 5,677,439, each of which is incorporated herein by reference. Other analog nucleic acids include those having positive backbones, non-ionic backbones, and non-ribose backbones, including those described in U.S. Patent Nos. 5,235,033 and 5,034,506. Modification of the ribose-phosphate backbone can facilitate the addition of moieties such as labels or improve the stability and half-life of the molecule in physiological environments.

[0036] The nucleic acid can contain a substituted or modified sugar moiety, such as a 2'-O-methoxyethyl sugar moiety or a carbocyclic sugar. The nucleic acid can also contain modified nucleosides (nucleoside analogs), i.e., modified purine or pyrimidine bases, such as 5-substituted pyrimidines, 6-azapyrimidines, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxybenzene, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridines (e.g., 5-bromouridine), or 6-azapyrimidines or 6-alkylpyrimidines (e.g., 6-methyluridine), 2-thiouridine, 4-thiouridine, 5-(carboxyhydroxymethyl)uridine, 5'-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 4-acetylcytidine, 3-methylcytidine, propin, kethoxal, wybutosine, wybutoxosine, beta-D-galactosylqueosine, N-2, N-6, and O-substituted purines, inosine, 1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, 2-methylthio-N-6-isopentenyladenosine, beta-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine, threonine derivatives, etc. See, e.g., U.S. Patent No. 7,410,944.

[0037] As used herein, the term "operably linked" refers to the association of nucleic acid sequences on a polynucleotide such that the function of one of the sequences is affected by another. For example, a regulatory DNA sequence is said to be "operably linked" to a DNA sequence encoding RNA (an "RNA coding sequence" or "shRNA coding sequence") or a polypeptide if the two sequences are positioned such that the regulatory DNA sequence affects the expression of the coding DNA sequence (i.e., the coding sequence or functional RNA is under the transcriptional control of a promoter). The coding sequence can be operably linked to the regulatory sequence in the sense or antisense direction. An RNA coding sequence refers to a nucleic acid that can function as a template for the synthesis of RNA molecules such as siRNA and shRNA. Preferably, the RNA coding region is a DNA sequence.

[0038] As used herein, the term "pharmaceutically acceptable" means that the compound is physiologically tolerable and does not cause an adverse reaction and does not exert an inhibitory effect on the action of the active ingredient when administered to a patient.

[0039] As used herein, the term "promoter" normally refers to a nucleotide sequence that is upstream (5') of its coding sequence and that directs and / or controls the expression of the coding sequence by providing recognition by RNA polymerase and other factors necessary for proper transcription. "Promoter" includes a minimal promoter, which is a short DNA sequence consisting of a TATA box and other sequences that help identify the transcription start site and to which regulatory elements are added to control expression. "Promoter" also refers to a nucleotide sequence that includes a minimal promoter and regulatory elements capable of controlling the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal upstream elements, the latter of which are often called enhancers. Thus, an "enhancer" is a DNA sequence that stimulates promoter activity and can be either an indigenous element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of the promoter. It can operate in both directions (sense or antisense) and function even when moved either upstream or downstream of the promoter. Both enhancers and other upstream promoter elements bind to sequence-specific DNA-binding proteins that mediate their effects. A promoter can be composed entirely of elements derived from a natural gene, or of different elements derived from different promoters found in nature, or even of synthetic DNA segments. A promoter can also contain DNA sequences that participate in the binding of protein factors that control the effectiveness of transcription initiation in response to physiological or developmental conditions. Any promoter well known in the art for regulating the expression of shRNA or an RNA coding sequence is contemplated for use in the practice of the present invention.

[0040] As used herein, the terms "reporter element" or "marker" mean a polynucleotide encoding a polypeptide that can be detected in a screening assay. Examples of polypeptides encoded by reporter elements include, but are not limited to, lacZ, GFP, luciferase, and chloramphenicol acetyltransferase. See, e.g., U.S. Patent No. 7,416,849. Many reporter elements and marker genes are well known in the art and are contemplated for use in the inventions disclosed herein.

[0041] As used herein, the term "RNA transcript" refers to the product resulting from the transcription of a DNA sequence catalyzed by RNA polymerase. "Messenger RNA transcript" ("mRNA") refers to RNA that does not contain introns and can be translated into protein by a cell.

[0042] As used herein, the terms "small interfering RNA" or "short interfering RNA" or "siRNA" refer to an RNA duplex of nucleotides that can be targeted to a desired gene and inhibit the expression of a gene sharing homology therewith. The RNA duplex comprises at least, at most, or exactly two complementary single-stranded RNAs of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides, which form at least, at most, or exactly 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 base pairs and have 3' overhangs of two nucleotides. The RNA double strand is formed by complementary pairing between two regions of the RNA molecule. siRNA "targets" a gene in which the nucleotide sequence of the double-stranded portion of the siRNA is complementary to the nucleotide sequence of its targeted gene. In some embodiments, the length of the double strand of siRNA is less than 30 nucleotides. The double strand can be at least, at most, or exactly 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 nucleotides in length. The length of the double strand can be 17-25 nucleotides in length. The double-stranded RNA can be expressed intracellularly from a single construct.

[0043] As used herein, the term "shRNA" (short hairpin RNA) refers to an RNA duplex in which part of the siRNA is part of a hairpin structure (shRNA). In addition to the duplex portion, the hairpin structure may contain a loop portion located between the two sequences that form the duplex. The length of the loop can vary. In some embodiments, the loop is at least, at most, or exactly 5, 6, 7, 8, 9, 10, 11, 12, or 13 nucleotides in length. The hairpin structure may also contain a 3' or 5' overhang portion. In some aspects, the overhang is a 3' or 5 overhang and is at least, at most, or exactly 0, 1, 2, 3, 4, or 5 nucleotides in length. In one aspect of the invention, the nucleotide sequence of the vector functions as a template for the expression of a short hairpin RNA comprising a sense region, a loop region, and an antisense region. After expression, the sense and antisense regions form a duplex. This duplex forms the shRNA, which hybridizes, for example, to Sav1 mRNA and reduces the expression of Sav1.

[0044] The terms "subject" and "individual" and "patient" are used synonymously and typically include humans.

[0045] As used herein, the phrase "a subject in need thereof" or "an individual in need thereof" refers to a subject or individual who is, for example, suffering from, or at risk of suffering from (e.g., predisposed to, e.g., genetically predisposed to, or exposed to environmental conditions that predispose to) a cardiac condition, disease, or disorder. In certain embodiments, a subject is successfully "treated" for a disease or disorder according to the methods provided herein if the subject exhibits a complete, partial, or transient alleviation or disappearance of one or more symptoms associated with the disease or disorder.

[0046] As used herein, the term "treating" refers to ameliorating, curing, and / or preventing the onset of at least one symptom of a disease or disorder, where the disease or disorder is, for example but not limited to, a cardiac condition including arrhythmia, myocardial infarction, ischemic heart disease, heart failure, cardiomyopathy, etc.

[0047] As used herein, the term "vector" refers to any viral or non-viral vector, as well as any double-stranded or single-stranded linear or circular plasmid, cosmid, phage, or binary vector that can be self-transmissible or mobilizable or non-self-transmissible or non-mobilizable, and that can transform prokaryotic or eukaryotic host cells either by integration into the cell genome or by existing episomally (e.g., an autonomously replicating plasmid having an origin of replication). Any vector well known in the art is contemplated for use in the practice of the present invention.

[0048] General embodiments Embodiments of the present disclosure relate to methods and compositions for improving cardiac function in an individual in need thereof. For example, the individual may have cardiomyopathy that can lead to HF. Cardiomyopathy results from excessive fibroblast activity and extracellular matrix accumulation in cardiac tissue. Cardiomyopathy can be caused, for example, by amyloidosis, hypertensive heart disease, hypertrophic cardiomyopathy, idiopathic dilated cardiomyopathy, myocardial infarction, myocarditis, and sarcoidosis. In certain embodiments, the individual has an arrhythmia associated with myocardial infarction.

[0049] Examples provided herein demonstrate cardiovascular benefits without adverse effects, including at least improvement in systolic function, promotion of angiogenesis, and treatment of arrhythmias associated with MI. In certain embodiments, CMs are regenerated in these treated hearts without loss of function of non-CM cells or subsequent loss.

[0050] A unique but exemplary set of three short hairpin RNAs (shRNAs) that specifically target Salvador (Sav1), a member of the Hippo pathway, is demonstrated herein. In certain embodiments, the shRNAs result in a selective reduction of Sav1 mRNA levels. In certain embodiments, the shRNAs can be delivered using a vector having tropism for the heart, such as an AAV9 (adeno-associated virus serotype 9) vector having tropism for the heart.

[0051] Salvador In a specific embodiment, Salvador (Salvador family WW domain-containing protein 1), a member of the Hippo pathway, is targeted by shRNAs in the treatment of heart disease conditions. The gene may be referred to as Salvador homolog 1, Salv, SAV1, SAV, WW45, or WWP4. Representative nucleic acids are provided by GenBank® accession number CR457297.1, and a representative protein sequence is provided by GenBank® accession number Q9H4B6.

[0052] This gene encodes a protein containing two WW domains (modular protein domains that mediate specific interactions with protein ligands) and a coiled-coil region. It is ubiquitously expressed in adult tissues. It also contains a SARAH (Sav / Rassf / Hpo) domain at the C-terminus (three classes of eukaryotic tumor suppressors that give the name to this domain). In the Sav (Salvador) family and the Hpo (Hippo) family, the SARAH domain mediates signaling from Hpo to downstream component Wts (Warts) via the Sav scaffold protein. Phosphorylation of Wt by Hpo induces cell cycle arrest and apoptosis by downregulating cyclin E, Diap 1, and other targets. The SARAH domain may also be involved in dimerization.

[0053] Examples of methods of treatment In embodiments of the present disclosure, a method for improving cardiac function in an individual is provided. The individual may have experienced myocardial infarction, arrhythmia, heart failure, myocardial fibrosis, cardiomyopathy, ischemic cardiomyopathy, myocardial necrosis, dilated cardiomyopathy, diabetic cardiomyopathy, age-related cardiomyopathy, combinations thereof, and the like.

[0054] In certain embodiments, the individual has an arrhythmia. In such cases, the arrhythmia can be tachycardic or bradycardic. The arrhythmia can be atrial or ventricular, and the individual may or may not have experienced a myocardial infarction. Tachycardias that can be treated include atrial fibrillation, atrial flutter, ventricular tachycardia, ventricular fibrillation, or ventricular tachycardia. Bradycardias that can be treated include sinoatrial node syndrome or conduction block. Symptoms of arrhythmia include palpitations, rapid heartbeat, slow heartbeat, chest pain, shortness of breath, anxiety, fatigue, dizziness, lightheadedness, sweating, and / or fainting.

[0055] Embodiments of the present disclosure include methods of improving a contractile function, the method including delivering to the heart tissue of a human patient who has experienced a myocardial infarction a composition comprising an effective amount of at least one inhibitory nucleic acid that targets salbutamol. Embodiments of the present disclosure include methods of reducing arrhythmia in a human patient having cardiomyopathy, the method including delivering to the heart tissue of the human patient a composition comprising an effective amount of at least one inhibitory nucleic acid that targets salbutamol. Embodiments of the present disclosure include methods of promoting angiogenesis in heart tissue, the method including delivering to the heart tissue of an individual in need thereof a composition comprising an effective amount of at least one inhibitory nucleic acid that targets salbutamol. Embodiments of the present disclosure include methods of improving the outcome in an individual who has suffered a myocardial infarction, the method including delivering to the heart tissue of a human patient a composition comprising an effective amount of at least one inhibitory nucleic acid that targets salbutamol. Embodiments of the present disclosure include methods of restoring normal sinus rhythm in an individual who has suffered a myocardial infarction, the method including delivering to the heart tissue of a human patient a composition comprising an effective amount of at least one inhibitory nucleic acid that targets salbutamol. Embodiments of the present disclosure include methods of reducing the risk of, delaying the onset of, or reducing the severity of arrhythmia in an individual having or having had a myocardial infarction, the method including delivering to the heart tissue of the individual a composition comprising an effective amount of at least one inhibitory nucleic acid that targets salbutamol. Embodiments of the present disclosure include methods of reducing the risk of any type of arrhythmia including post-infarction arrhythmia, or at least refractory ventricular arrhythmia, the method including delivering to the heart tissue of an individual a composition comprising an effective amount of at least one inhibitory nucleic acid that targets salbutamol. Embodiments of the present disclosure include methods of reducing the frequency of ventricular arrhythmia after myocardial infarction, the method including delivering to the heart tissue of an individual a composition comprising an effective amount of at least one inhibitory nucleic acid that targets salbutamol.

[0056] Embodiments of the present disclosure include methods for treating, preventing, or reducing the risk of arrhythmia in a human patient, the method comprising: (a) identifying a need for treating, preventing, or reducing the risk of arrhythmia in a human patient; and (b) delivering to the heart tissue of the human patient a composition comprising an effective amount of at least one inhibitory nucleic acid, the inhibitory nucleic acid targeting salvador. In certain embodiments, the human patient has experienced or is at risk of myocardial infarction.

[0057] In the methods encompassed herein, the nucleic acid can be provided to the individual one or more times. The nucleic acid compositions of the present disclosure are provided to the individual systemically or locally, including by injection into or near the heart. Delivery can be performed at the time of diagnosis of a need for improvement of cardiac function, such as improvement of systolic function, promotion of angiogenesis in cardiac tissue, and / or reduction of arrhythmia. Improvement of cardiac function in an individual subjected to treatment by the method of the present invention is compared to the cardiac function in the individual prior to treatment by the method of the present invention. Cardiac function can be evaluated, for example, by measuring ejection fraction, diastolic volume, systolic volume, left ventricular end-diastolic volume, left ventricular end-systolic volume, left ventricular systolic function, diastolic function, stroke volume, heart rhythm, or a combination thereof.

[0058] In a treatment context, reduction of arrhythmia in a patient is evaluated as the frequency and / or number of arrhythmias in the patient subjected to treatment by the method of the present invention compared to the frequency and / or number of arrhythmias in the patient prior to treatment by the method of the present invention. In a prevention context, reduction of arrhythmia in a patient is evaluated as the frequency and / or number of arrhythmias in the patient subjected to treatment by the method of the present invention compared to the frequency and / or number of arrhythmias in an individual with comparable cardiac function, the individual not having been subjected to treatment by the method of the present invention.

[0059] In some embodiments, the individual is at risk of arrhythmia, such as an individual at risk of arrhythmia during and / or after myocardial infarction. In certain embodiments, the individual is at risk compared to the general population, for example, has a history of arrhythmia, is elderly, has an overall health condition, has one or more genetic factors, has a history of self and / or familial heart disease, is at risk due to a personal history of arrhythmia, a family history of arrhythmia, etc. In certain embodiments, the individual is at risk of having an arrhythmia due to having cardiomyopathy and / or having experienced a myocardial infarction.

[0060] In certain embodiments of the method, the inhibitory nucleic acid has or is encoded by a sequence having at least 80% identity to a nucleotide sequence selected from the group consisting of SEQ ID NO: 2 (aagtacgtga agaaggagac g), SEQ ID NO: 3 (aagatttacc ccttcctcct g), and SEQ ID NO: 4 (aattcctgac tggcttcagg t). In a particular method, the composition comprises (i) an inhibitory nucleic acid having or encoded by a sequence having at least 80% identity to SEQ ID NO: 2, (ii) an inhibitory nucleic acid having or encoded by a sequence having at least 80% identity to SEQ ID NO: 3, or (iii) an inhibitory nucleic acid having or encoded by a sequence having at least 80% identity to SEQ ID NO: 4. The inhibitory nucleic acid can have or be encoded by a sequence having at least, up to, and strictly 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity, or identity between any two of these, to a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. The inhibitory nucleic acid can have or be encoded by a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. In certain embodiments, a mixture of inhibitory nucleic acids having or encoded by a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 can be utilized. In some embodiments, the composition comprises derivative nucleic acid SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 having at least, up to, or strictly 1, 2, 3, 4, or 5 mismatches compared to each respective SEQ ID NO.

[0061] In certain methods of the present disclosure, the inhibitory nucleic acid can be of any type, for example, an antisense DNA molecule, RNA, or short hairpin RNA (shRNA). The inhibitory nucleic acid may or may not be of a specific length, for example, at least, at most, or exactly 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides, or longer. In certain embodiments, the inhibitory nucleic acid is an shRNA having a length of at least 43 nucleotides. The inhibitory nucleic acid may or may not be less than a certain number of nucleotides in length, for example, less than 150, 149, 148, 147, 146, 145, 144, 143, 142, 141, 140, 139, 138, 137, 136, 135, 134, 133, 132, 131, 130, 129, 128, 127, 126, 125 nucleotides, etc. In certain cases, the inhibitory nucleic acid is an shRNA having a length of less than 138 nucleotides.

[0062] In embodiments of the method in which shRNA is used as the inhibitory nucleic acid, the shRNA may include a loop structure of a specific length. In certain cases, the shRNA may include a loop structure having a length of about 5 to about 19 nucleotides. The loop structure can be at least, at most, or exactly 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 nucleotides in length.

[0063] In certain embodiments of the method, the inhibitory nucleic acid or the nucleotide sequence encoding the inhibitory nucleic acid may be included in a nucleic acid construct. In certain embodiments, a nucleotide sequence encoding an inhibitory nucleic acid as RNA is utilized. In certain embodiments, the nucleotide sequence encoding RNA is expressed in cardiomyocytes, such as a nucleotide sequence encoding RNA operably linked to a tissue-specific promoter. One specific promoter includes the cardiac troponin T promoter; the rat ventricular-specific myosin light chain 2 (MLC-2v) promoter; the cardiac-specific alpha myosin heavy chain (MHC) gene promoter; the cardiac cell-specific minimal promoter of -137 to +85 of the NCX1 promoter; chicken cardiac troponin T (cTNT); the Nppa promoter (for atrial cardiomyocyte-specific expression, etc.), or a combination thereof. In certain embodiments of the method, the nucleotide sequence encoding RNA may or may not include a post-transcriptional control element such as the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and is included in a nucleic acid construct. In some embodiments, the nucleic acid construct may include sequences encoding the 3'microRNA-30 sequence and the 5'microRNA-30 sequence. The nucleic acid construct may, in certain embodiments, include 5' and 3' inverted terminal repeats.

[0064] In certain embodiments of the method, the nucleotide sequence encoding the inhibitory nucleic acid is included in any type of vector, including non-viral vectors, non-integrating vectors, viral vectors, etc. The viral vector can be, for example, an adenovirus, an adeno-associated virus (AAV), a lentiviral vector, or a retrovirus. In certain embodiments, the inhibitory nucleic acid is included in an AAV vector. The AAV vector can be of any serotype, including, for example, AAV2, AAV6, AAV7, AAV8, and AAV9. In one embodiment, an AAV9 vector is used. Non-viral vectors include plasmids, transposons, and the like.

[0065] In certain embodiments of the method, the method utilizes a composition comprising a nucleic acid construct comprising (i) a nucleic acid having the nucleotide sequence set forth in SEQ ID NO: 2, (ii) a nucleic acid having the nucleotide sequence set forth in SEQ ID NO: 3, and (iii) a nucleic acid having the nucleotide sequence set forth in SEQ ID NO: 4, wherein the nucleic acids (i)-(iii) are operably linked to a promoter. The nucleotide sequences encoding two or more inhibitory nucleic acids may or may not be included in a single nucleic acid construct. In embodiments where two or more inhibitory nucleic acids are included in a single nucleic acid construct, they may or may not be regulated by a single promoter.

[0066] In some embodiments of the method, an individual is provided with an effective amount of a therapy other than the therapies encompassed herein. In certain embodiments, the additional therapies can include antiarrhythmic agents (such as amiodarone, flecainide, ibutilide, lidocaine, procainamide, propafenone, quinidine, and / or tocainide), calcium channel blockers, beta blockers, and / or anticoagulants.

[0067] Nucleic acids targeting Sav1 In certain embodiments, one or more nucleic acids targeting Sav1 are present such that the expression of Sav1 is detectably reduced. Nucleic acids targeting Sav1 can be considered inhibitory nucleic acids. The nucleic acid can be DNA or RNA, but in certain embodiments, the nucleic acid is RNA such as shRNA.

[0068] In some embodiments, the target nucleic acid targets human Sav1. An example of human Sav1 is provided below and is described in GenBank® accession number NM_021818.4.

[0069] In one embodiment, the inhibitory RNA has or is encoded by the nucleotide sequence (SEQ ID NO: 2). In one embodiment, the inhibitory RNA has or is encoded by the nucleotide sequence (SEQ ID NO: 3). In one embodiment, the inhibitory RNA has or is encoded by the nucleotide sequence (SEQ ID NO: 4).

[0070] In one embodiment, the shRNA is a "hairpin" or stem-loop RNA molecule that includes a sense region, a loop region, and an antisense region complementary to the sense region. In other embodiments, the inhibitory RNA is a siRNA that includes two separate complementary RNA molecules (strands) that associate non-covalently via base pairing to form a duplex. See, e.g., U.S. Patent No. 7,195,916.

[0071] In certain cases, the inhibitory RNA is shRNA. In certain cases, shRNA is a single-stranded RNA molecule that forms a stem-loop structure in vivo and can be about 40 to 135 nucleotides in length. In certain embodiments, the length is about 40 to 135, 40 to 120, 40 to 100, 40 to 80, 40 to 75, 40 to 50, 50 to 135, 50 to 120, 50 to 100, 50 to 80, 50 to 75, 50 to 60, 75 to 135, 75 to 120, 75 to 100, 75 to 90, 75 to 80, 90 to 135, 90 to 120, 90 to 100, 100 to 135, 110 to 135, 110 to 125, or 125 to 135 nucleotides in length. shRNA is a single-stranded RNA molecule that forms a stem-loop structure in vivo and is at least, at most, precisely, or about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides in length to 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, or 140 (nt) in length. In certain embodiments, the double-stranded portion of the stem-loop structure is less than 30 nucleotides in length, for example, at least, at most, or precisely 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 nucleotides in length (including ranges within these lengths). In at least certain cases, a 5 to 19 nucleotide loop (including 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 nucleotides) joins two complementary 19 to 29 nucleotide long RNA fragments (including 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 nucleotides) that create a double-stranded stem by base pairing. shRNA can further include an overhang region. Such an overhang can be a 3' overhang region or a 5' overhang region. The overhang region can be, for example, at least, at most, or precisely 1, 2, 3, 4, 5, or 6 nucleotides in length.In vivo transcription and synthesis of shRNA is directed by a Pol III promoter, and then the resulting shRNA is cleaved by Dicer, an RNase III enzyme, to generate mature siRNA. The mature siRNA enters the RISC complex. Thus, in certain embodiments, the shRNA for inhibition of Sav1 expression according to the present disclosure contains both sense and antisense nucleotide sequences.

[0072] In certain embodiments, the nucleic acid comprises the sequence of SEQ ID NO: 2 (or SEQ ID NO: 3 or 4, respectively), further comprises the antisense sequence of SEQ ID NO: 2 (or SEQ ID NO: 3 or 4, respectively), and when the sequence and the antisense sequence hybridize together to form a double-stranded structure, the sequence and the antisense sequence are separated by a loop structure.

[0073] In one embodiment, the nucleic acid construct comprises a polynucleotide sequence encoding an shRNA operably linked to a promoter. In one embodiment, the shRNA comprises a first segment, a second segment located immediately 3' to the first segment, and a third segment located immediately 3' to the second segment, wherein the first and third segments can each be less than 30 base pairs in length and can each be more than 10 base pairs in length. The first segment and the third segment are complementary to each other, and with respect to the target sequence, one contains an antisense sequence and the other contains a sense sequence. The second segment located immediately 3' to the first segment encodes a loop structure.

[0074] In certain embodiments, the inhibitory nucleic acid has, or is encoded by, a sequence having at least, at most, exactly 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or identity between any two of these, to a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. In some embodiments, the composition comprises derivative nucleic acids of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4 having one, two, three, four, or five mismatches as compared to each respective SEQ ID NO.

[0075] When appropriately targeted to a specific mRNA in a cell via a nucleotide sequence, the shRNA specifically suppresses the gene expression of Sav1. In at least some cases, the shRNA can reduce the cellular level of a specific mRNA and the level of the protein encoded by such an mRNA. The shRNA targets the mRNA for destruction using sequence complementarity and is sequence specific. Thus, they can be highly target specific and have been shown to target mRNAs encoded by different alleles of the same gene in mammals.

[0076] In certain embodiments, an shRNA corresponding to a region of a target gene to be downregulated or knocked down is expressed intracellularly. The shRNA duplex has a sequence that is substantially identical (e.g., at least, at most, exactly, or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to the sequence of the gene targeted for downregulation. In certain embodiments, there are 5 or fewer mismatches between the shRNA sequence and the target Sav1 sequence. In certain embodiments, a minimum of 18 bp of homology is utilized in the region of complementarity between the shRNA sequence and its target. In certain embodiments, a particular assay is utilized to test for suitable mismatches for the shRNA and its target. In certain embodiments, an algorithm can be used to identify suitable mismatches for the shRNA and its target.

[0077] It should be noted that, therefore, perfect complementarity between the target sequence and the shRNA is not required. That is, the resulting antisense siRNA (after the process of the shRNA) is sufficiently complementary to the target sequence. The sense strand is substantially complementary to the antisense strand in order to anneal (hybridize) to the antisense strand under biological conditions.

[0078] In particular, the complementary polynucleotide sequence of the shRNA can be designed to specifically hybridize to a specific region of the desired target protein or mRNA and interfere with replication, transcription, or translation. The term "hybridize" or variations thereof refers to a degree of complementarity or pairing sufficient for stable and specific binding to occur between the antisense nucleotide sequence and the target DNA or mRNA. In particular, 100% complementarity or pairing is desirable but not essential. Specific hybridization occurs when, under predetermined conditions, such as for in vivo therapeutic purposes, preferably under physiological conditions, sufficient hybridization occurs between the antisense nucleotide sequence and its intended target nucleic acid with substantially no non-specific binding of the antisense nucleotide sequence to non-target sequences. Preferably, specific hybridization results in interference with the normal expression of the gene product encoded by the target DNA or mRNA. In the context of nucleic acids, the terms "hybridize", "bind", "target", or variations thereof refer to a degree of complementarity or base pairing sufficient between the complementary or inhibitory nucleic acid sequence and the target DNA or mRNA such that a stable and specific interaction occurs between them. Specific hybridization occurs when, under predetermined conditions, preferably under physical or physiological conditions, there is substantially no non-specific binding of the complementary or inhibitory nucleotide sequence to non-specific sequences and sufficient interaction occurs between the complementary or inhibitory nucleotide sequence and its intended target nucleic acid. Preferably, specific hybridization results in inhibition, i.e., interference with the normal expression of the gene product encoded by the target DNA or mRNA. Complete complementarity between the target sequence and the inhibitory RNA is not required. An inhibitory nucleotide sequence, such as a single-stranded antisense oligonucleotide or the antisense strand of a double-stranded inhibitory RNA, is sufficiently complementary if it binds to the target sequence under predetermined conditions and inhibits target gene expression.For example, the antisense nucleotide sequence can be designed to specifically hybridize to the replication or transcription control region of the target gene, or the translation control region such as the translation initiation region and exon / intron junction, or the coding region of the target mRNA.

[0079] For example, the antisense nucleotide sequence can be designed to specifically hybridize to the replication or transcription control region of the target gene, or the translation control region such as the translation initiation region and exon / intron junction, or the coding region of the target mRNA. In certain embodiments, the shRNA targets a sequence encoding the N-terminal region of the Sav1 protein, a sequence encoding the middle of the Sav1 protein, or a sequence encoding the C-terminal region of the Sav1 protein.

[0080] shRNA: synthetic As is generally known in the art, commonly used oligonucleotides are oligomers or polymers of ribonucleic acid or deoxyribonucleic acid having a combination of covalent bonds between nucleosides including naturally occurring purine and pyrimidine bases, sugars, and phosphate groups in the phosphodiester bond. However, it should be noted that the term "oligonucleotide" also encompasses various unnatural mimics and derivatives of naturally occurring oligonucleotides as described below, i.e., modified forms.

[0081] The shRNA molecules of the present disclosure can be prepared by any method well known in the art for the synthesis of DNA and RNA molecules. These include, for example, techniques for chemically synthesizing oligodeoxy-ribonucleotides and oligo-ribonucleotides well known in the art, such as solid-phase phosphoramidite chemical synthesis. Alternatively, RNA molecules can be generated by in vitro and in vivo transcription of DNA sequences encoding the shRNA molecules. Such DNA sequences can be incorporated into a variety of vectors incorporating appropriate RNA polymerase promoters, such as T7 or SP6 polymerase promoters. Alternatively, depending on the promoter used, antisense cDNA constructs that constitutively or inducibly synthesize shRNA can be stably introduced into cell lines.

[0082] The shRNA molecules can be chemically synthesized using appropriately protected ribonucleoside phosphoramidites and conventional DNA / RNA synthesizers. Custom shRNA synthesis services are available from commercial vendors such as Ambion (Austin, Tex., USA) and Dharmacon Research (Lafayette, Colo., USA). See, for example, U.S. Patent No. 7,410,944.

[0083] A variety of well-known modifications to DNA molecules can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of adjacent sequences of ribonucleotides or deoxynucleotides to the 5' and / or 3' ends of the molecule, or the use of phosphorothioates or 2'O-methyls instead of phosphodiester bonds within the oligodeoxyribonucleotide backbone. The antisense nucleic acids of the present invention can be constructed using procedures well known in the art, using chemical synthesis or enzymatic ligation reactions. Antisense oligonucleotides can be composed of naturally occurring nucleotides, or various modified nucleotides designed to increase the biological stability of the molecule, or to increase the physical stability of the duplex formed between the antisense nucleic acid and the sense nucleic acid (e.g., phosphorothioate derivatives and acridine-substituted nucleotides can be used).

[0084] The shRNA molecules of the present invention can be various modified equivalents of the structure of any Sav1 shRNA. "Modified equivalent" means a modified form of a particular siRNA molecule that has the same target specificity (i.e., recognizes the same mRNA molecule that is complementary to the unmodified particular siRNA molecule). Thus, a modified equivalent of an unmodified siRNA molecule can have modified ribonucleotides, i.e., ribonucleotides that contain modifications to the chemical structure of the unmodified nucleotide base, sugar, and / or phosphate (or phosphodiester bond). See, e.g., U.S. Patent No. 7,410,944.

[0085] Preferably, the modified shRNA molecules contain a modified backbone or non-natural internucleoside linkages, such as modified phosphorus-containing backbones and non-phosphorus backbones, e.g., morpholino backbones; siloxane, sulfide, sulfoxide, sulfone, sulfonate, sulfonamide, and sulfamate backbones; formacetyl and thioformacetyl backbones; alkene-containing backbones; methyleneimino and methylenehydrazino backbones; amide backbones, etc. See, e.g., U.S. Patent No. 7,410,944.

[0086] Examples of modified phosphorus-containing backbones include, but are not limited to, phosphorothioate, phosphorodithioate, chiral phosphorothioate, phosphotriester, aminoalkyl phosphotriester, alkyl phosphonate, thionoalkyl phosphonate, phosphinate, phosphoramidate, thionophosphoramidate, thionoalkyl phosphotriester, and boranophosphate, as well as various salt forms thereof. See, for example, U.S. Patent No. 7,410,944.

[0087] Examples of the above-described non-phosphorus-containing backbones are well known in the art and are described, for example, in U.S. Patent No. 5,677,439, each of which is incorporated herein by reference. See, for example, U.S. Patent No. 7,410,944.

[0088] Modified forms of the shRNA compounds can also contain modified nucleosides (nucleoside analogs), i.e., modified purine or pyrimidine bases such as 5-substituted pyrimidines, 6-azapyrimidines, pyridin-4-ones, pyridin-2-ones, phenyl, pseudouracil, 2,4,6-trimethoxybenzene, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridines (e.g., 5-bromouridine), or 6-azapyrimidines or 6-alkylpyrimidines (e.g., 6-methyluridine), 2-thiouridine, 4-thiouridine, 5-(carboxyhydroxymethyl)uridine, 5'-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 4-acetylcytidine, 3-methylcytidine, propin, kethoxal, wybutosine, wybutoxosine, beta-D-galactosylqueosine, N-2, N-6, and O-substituted purines, inosine, 1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, 2-methylthio-N-6-isopentenyladenosine, beta-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine, threonine derivatives, etc. See, e.g., U.S. Patent No. 7,410,944.

[0089] In addition, the modified shRNA compounds can also have substituted or modified sugar moieties, such as 2'-O-methoxyethyl sugar moieties. See, e.g., U.S. Patent No. 7,410,944.

[0090] shRNA: Administration The present disclosure provides a composition of a polymer or excipient and one or more vectors encoding one or more shRNA molecules. The vectors can be formulated into a pharmaceutical composition using a suitable carrier and administered to a patient using a suitable route of administration.

[0091] In particular, the shRNA compounds can be administered systemically, for example, via parenteral administration. They can also be delivered directly to a particular organ or tissue by any suitable method of local administration, such as direct injection into the target tissue.

[0092] In certain embodiments, the shRNA molecules are included in vectors that include viral or non-viral vectors. In certain embodiments, the vectors are non-integrating, while in other embodiments, the vectors are integrating. Viral vectors can be, for example, lentivirus, adeno-associated virus, and retrovirus. Examples of non-viral vectors include plasmids. In certain embodiments, an AAV9 vector (Piras et al., 2013) is used. The vectors can be delivered systemically or locally to an individual. In certain specific embodiments, the vectors utilize a tissue-specific or cell-specific promoter, such as a cardiomyocyte-specific promoter. In certain embodiments, the vectors are delivered by local injection.

[0093] One route of administration of the shRNA molecules of the present disclosure includes, for example, direct injection of the vector into cardiac tissue such as the myocardium.

[0094] Generally, what is included in the present invention is a vector comprising a polynucleotide sequence, an isolated nucleic acid sequence encoding a first segment, a second segment located immediately 3' to the first segment, and a third segment located immediately 3' to the second segment, wherein the first segment and the third segment each have a length of less than 30 base pairs and each have a length of more than 10 base pairs, and the sequence of the third segment is the complement of the sequence of the first segment. The second segment located immediately 3' to the first segment encodes a loop structure containing 4 to 10 nucleotides (i.e., at least, at most, or exactly 4, 5, 6, 7, 8, 9, 10 nucleotides). The nucleic acid sequence is expressed as siRNA and functions as a small hairpin RNA molecule (shRNA) targeted to the designated nucleic acid sequence.

[0095] More specifically, the present disclosure includes compositions and methods for selectively reducing the expression of the gene product from Sav1. The present invention provides a vector comprising a polynucleotide sequence comprising a nucleic acid sequence encoding an shRNA targeted to Sav1. The shRNA forms a hairpin structure comprising a double-stranded structure and a loop structure. The loop structure may contain 4 to 10 nucleotides, such as 4, 5, or 6 nucleotides. The double strand has a length of less than 30 nucleotides, such as 10 to 27 nucleotides in length. The shRNA may further comprise an overhang region. Such an overhang may be a 3' overhang region or a 5' overhang region. The overhang region may be, for example, at least, at most, or exactly 1, 2, 3, 4, 5, or 6 nucleotides in length.

[0096] In one aspect of the invention, a plurality of vectors, each encoding a different shRNA (targeting different regions of the Sav1 nucleic acid sequence), can be administered to a patient simultaneously or sequentially. An individual vector can encode multiple shRNAs targeting different regions of the same gene; i.e., it can contain two or more of the shRNAs comprising SEQ ID NO: 2, and the shRNA comprising SEQ ID NO: 3, and the shRNA comprising SEQ ID NO: 4. In another aspect, an individual vector can encode multiple copies of the shRNA comprising SEQ ID NO: 2, or multiple copies of the shRNA comprising SEQ ID NO: 3, or multiple copies of the shRNA comprising SEQ ID NO: 4, in any ratio.

[0097] The vectors of the disclosure can further comprise a promoter. Examples of promoters include inducible promoters and constitutive promoters. For example, the promoter can be a CMV or RSV promoter. The vector can further comprise a polyadenylation signal, such as a synthetic minimal polyadenylation signal. Many such promoters are well known in the art and are contemplated for use in the present invention. In other cases, the promoter can be a tissue-specific promoter, such as a heart tissue-specific promoter.

[0098] The vector may further comprise one or more marker genes or reporter genes. Many marker genes and reporter genes are well known in the art. The present invention contemplates the use of one or more marker genes and / or reporter genes well known in the art in the practice of the present invention. Marker genes or reporter genes provide a way to track the expression of one or more linked genes. Marker genes or reporter genes, upon expression in a cell, provide a product, usually a protein, that is detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. Gene expression products, whether from the gene of interest, the marker gene, or the reporter gene, can also be detected by labeling. Labels contemplated for use in the present invention include fluorescent dyes, high electron density reagents, enzymes (such as those widely used in ELISA), biotin, digoxigenin, or haptens, and proteins that can be made detectable, for example, by introducing a radioactive label into a peptide, or that detect an antibody that specifically reacts with a peptide. See, for example, U.S. Patent No. 7,419,779.

[0099] shRNA: Pharmaceutical Composition The nucleic acids encoding the shRNAs of the present disclosure can be formulated in a pharmaceutical composition. The pharmaceutical compositions of the present invention comprise a vector encoding a therapeutically effective amount of the shRNA. These compositions can include, in addition to the vector, pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials. Such materials should be non-toxic and should not interfere with the effectiveness of the active ingredient. The carrier can take a variety of forms depending on the form of preparation desired for administration.

[0100] When the vectors of the present disclosure are prepared for administration, they can be combined with pharmaceutically acceptable carriers, diluents, or excipients to form a pharmaceutical preparation, or unit dosage form.

[0101] For example, the vector can be formulated in a buffer such as phosphate buffered saline, saline, or water. Other pharmaceutically acceptable carriers, excipients, stabilizers, and / or preservatives can be included in the formulation.

[0102] It will be appreciated that the selection of a pharmaceutically acceptable carrier, which may include physiologically acceptable compounds, depends, for example, on the route of administration of the composition.

[0103] The use of formulations of vectors containing cationic lipids can facilitate the transfection of vectors into isolated cells. For example, cationic lipids such as lipofectin, cationic glycerol derivatives, and polycationic molecules such as polylysine can be used. Suitable lipids include, for example, oligofectamine and lipofectamine (Life Technologies), which can be used according to the manufacturer's instructions.

[0104] The shRNA or a composition containing it can be administered in a therapeutically effective amount. "Effective amount" or "therapeutically effective amount" is an amount sufficient to effect the specifically recited purpose, such as improvement of cardiac function, improvement of systolic function, reduction of arrhythmia, and / or promotion of angiogenesis in cardiac tissue.

[0105] shRNA: Gene Therapy siRNA can also be delivered to mammalian cells, particularly human cells, by gene therapy approaches using, for example, DNA vectors into which siRNA compounds of the small hairpin type (shRNA) can be directly transcribed. Recent studies have demonstrated that although double-stranded siRNA is very effective in mediating RNAi, short single-stranded hairpin RNAs can also mediate RNAi, presumably because they fold into intramolecular duplexes that are processed by cellular enzymes into double-stranded siRNA. The production of such shRNAs can be readily achieved in vivo by transfecting cells or tissues with DNA vectors having short inverted repeat sequences separated by a small number (e.g., 3, 4, 5, 6, 7, 8, 9) of nucleotides that direct the transcription of such small hairpin RNAs, and this discovery has important and far-reaching implications. Furthermore, if mechanisms are included to direct the integration of the vector or vector segment into the host cell genome or to ensure the stability of the transcription vector, the RNAi induced by the encoded shRNA can be made stable and heritable.

[0106] Gene therapy is performed according to generally accepted methods well known in the art. See, for example, U.S. Pat. Nos. 5,837,492 and 5,800,998, and the references cited therein. A vector in the context of gene therapy means a polynucleotide sequence that contains a sequence sufficient to express the polynucleotide encoded therein. When the polynucleotide encodes shRNA, an antisense polynucleotide sequence is produced upon expression. Thus, in this context, expression does not require the synthesis of a protein product. In addition to the shRNA encoded within the vector, the vector also includes a promoter that functions in eukaryotic cells. The shRNA sequence is under the control of this promoter. Suitable eukaryotic promoters are described elsewhere herein and are well known in the art. The expression vector may also include sequences such as selectable markers, reporter genes, and other regulatory sequences conventionally used.

[0107] Thus, the amount of shRNA produced in situ is regulated by controlling factors such as the nature of the promoter used to direct transcription of the nucleic acid sequence (i.e., whether the promoter is constitutive or regulatable, strong or weak), and the copy number of the nucleic acid sequence encoding the shRNA sequence within the cell.

[0108] In the case of the expression of Sav1 shRNA, the promoter is operably linked to the shRNA sequence. As used herein, the term "promoter" refers to a DNA sequence that controls the expression of a target gene sequence operably linked to the promoter sequence in a particular host cell. The terms "operably linked" or "operably connected" mean that one nucleic acid fragment is linked to another nucleic acid fragment such that its function or expression is affected by the other nucleic acid fragment. The expression cassette of the present invention may further comprise various expression regulatory sequences such as an optional operator sequence for controlling transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for controlling the termination of transcription and translation. The promoter used in the present invention may be a constitutive promoter that constitutively induces the expression of the target gene, or an inducible promoter that induces the expression of the target gene at a given position and time point. Specific examples of the promoter include U6 promoter, CMV (cytomegalovirus) promoter, SV40 promoter, CAG promoter (Hitoshi Niwa et al., Gene, 108: 193-199, 1991; and Monahan et al., Gene Therapy, 7: 24-30, 2000), CaMV 35S promoter (Odell et al., Nature 313: 810-812, 1985), Rsyn7 promoter (U.S. Patent Application No. 08 / 991,601), ubiquitin promoter (Christensen et al., Plant Mol. Biol. 12: 619-632, 1989), ALS promoter (U.S. Patent Application No. 08 / 409,297), and the like. Also usable promoters are disclosed in U.S. Patent Nos. 5,608,149, 5,608,144, 5,604,121, 5,569,597, 5,466,785, 5,399,680, 5,268,463, 5,608,142, and the like.

[0109] The recombinant vectors of the present disclosure can be introduced into isolated host cells using conventional methods well known in the art. The host cells can be used for manipulation of the vectors or as a means for transferring the vectors into an individual. Preferably, the intracellular integration of the vectors into the host cells can be carried out by conventional methods well known in the art, such as calcium chloride, microprojectile bombardment, electroporation, PEG-mediated fusion, microinjection, liposome-mediated methods, copper, etc.

[0110] Examples of isolated host cells that can be used in the present disclosure include prokaryotic cells such as Escherichia coli, Bacillus subtilis, Streptomyces, Pseudomonas, Proteus mirabilis, and Staphylococcus, fungi (e.g., Aspergillus), yeasts (e.g., Pichia pastoris), lower eukaryotic cells such as Saccharomyces cerevisiae, Schizosaccharomyces, and Neurospora crassa, and higher eukaryotic cells such as insect cells, plant cells, mammalian cells, etc., but are not limited thereto. Preferably, the host cells can be human cells.

[0111] On the one hand, the standard recombinant DNA and molecular cloning techniques used in the present disclosure are well known in the art and can be found in the following references: Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989); Silhavy, T.J., Bennan, M.L. and Enquist, L.W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1984); and Ausubel, F.M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987).

Example

[0112] The following examples are included to demonstrate specific non-limiting embodiments of the invention. It should be understood by those skilled in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the invention. However, those skilled in the art will understand that, in light of the present disclosure, many modifications may be made to the specific embodiments disclosed without departing from the spirit and scope of the invention and still obtain a like or similar result.

[0113] Example 1 Viral delivery of sav shRNA improves systolic function in a porcine model of myocardial infarction Using adeno-associated virus 9 (AAV9)-based gene therapy, Hippo pathway gene Sav was locally knocked down in border zone (BZ) cardiomyocytes (CMs) in a porcine model of ischemia-reperfusion-induced myocardial infarction (MI). Two weeks after MI, when the pigs showed left ventricular (LV) systolic dysfunction, AAV9-Sav-shRNA or a control virus was directly administered to BZ CMs via catheter-mediated endocardial injection. Three months after injection, hearts treated with high-dose AAV9-Sav-shRNA showed a 14.3% improvement in ejection fraction (a measure of LV systolic function), evidence of CM proliferation, and a reduction in scar size compared to controls. None of the animals died during the 3-month follow-up period, nor did they show uncontrolled CM proliferation or immaturity.

[0114] More specifically, to determine whether AAV9-Sav-shRNA promotes cardiac regeneration after MI, a balloon angioplasty was used to temporarily occlude the left anterior descending artery (LAD) in the hearts of pigs approximately 3 months old for 90 minutes and then reperfused to induce MI via ischemia / reperfusion (I / R) (Figure 1A). Two weeks after MI, echocardiography revealed that the left ventricular ejection fraction (EF) was reduced to less than 40% in both pig groups (AAV9-GFP, EF: 36.2 ± 2.0% [mean ± SEM]; AAV9-Sav-shRNA, EF: 36.5 ± 1.7% [mean ± SEM]) (Figure 1B, Table 1), indicating a significant decline in cardiac function. [Table 1]

[0115] Furthermore, a transmural scar in the anterior wall was prominent, indicating that MI was successfully induced by I / R treatment. NOGA mapping was used to inject AAV9-GFP or AAV9-Sav-shRNA into the border zone (BZ) myocardium to identify the injection site, and echocardiography was performed 20, 40, 60, and 90 days after virus injection to longitudinally monitor the cardiac function of each pig. (Figure 1A).

[0116] Notably, longitudinal analysis of AAV9-GFP-injected porcine hearts revealed that LV function was definitely deteriorated, consistent with progressive pathological remodeling. In contrast, in AAV9-Sav-shRNA-injected hearts, LV function gradually improved over time (Figs. 1B-1F, 2A). In AAV9-GFP-injected hearts, EF further decreased by 6.3±1.9% during the 3-month follow-up period (Figs. 1C, 1E, and 1F), while in low-dose AAV9-Sav-shRNA-injected hearts, EF increased by 4.1±2% (1×10 13 viral genome copies / experimental group) (Figs. 1C, 1D, and 1F). Three pigs administered high-dose AAV9-Sav-shRNA (4×10 13 viral genome copies) showed a strong improvement in EF (8.0±0.5%) on day 104 compared to day 14 (Figs. 1E and 1F). The difference in EF between the high and low viral doses did not reach statistical significance, probably due to the limited number of pigs (Figs. 1E and 1F). AAV9-GFP control-treated pigs showed an increase in absolute heart weight and the ratio of heart weight to body weight (HW / BW) compared to pigs treated with AAV9-Sav-shRNA, suggesting that the control pigs suffered from cardiac hypertrophy (Fig. 2B).

[0117] Importantly, AAV9-Sav-shRNA treatment improved LV systolic function over time, with only a minor effect on diastolic function, indicating that the improvement in EF was mainly due to the recovery of LV systolic function (Figure 1G, 1H; Figure 2C, 2D). Consistent with this, AAV9-Sav-shRNA-injected hearts showed an improvement in stroke volume compared to AAV9-GFP-injected hearts (Figure 1I, Figure 2E). Three months after virus injection, all pigs were euthanized and the hearts were carefully analyzed. Transmural scars were observed in all hearts and were significantly smaller in AAV9-Sav-shRNA-injected hearts than in control hearts (Figure 1J, Table 2). After slicing the ventricles into seven slices, the infarct area was measured in each slice. A significant reduction in scarring was observed in AAV9-Sav-shRNA-injected hearts compared to control hearts (Figure 1J). Collectively, these data indicate that AAV9-Sav-shRNA improves LV systolic cardiac function and reduces scar formation in a porcine model of MI.

Table 2

[0118] Notably, blood test results revealed normal white and red blood cell counts, as well as normal liver and kidney function 45 days after virus injection. Upon careful examination of liver and lung tissues three months after injection, no tumors were observed, indicating that local infection with AAV9-Sav-shRNA is safely tolerated.

[0119] Example 2 Viral delivery of sav shRNA promotes angiogenesis in the hearts of pigs after myocardial infarction The hearts treated with AAV9-Sav-shRNA showed an increase in capillary density and a decrease in the CM fold. In particular, to determine whether AAV9-Sav-shRNA infection promotes angiogenesis in the hearts of pigs after MI, IF staining was performed for the endothelial marker isolectin B4. By tile image analysis, more capillary formation was revealed in the low-dose AAV9-Sav-shRNA-injected hearts than in the control hearts. Endothelial cells were observed in the infarcted tissue of the pig hearts 90 days after virus injection. Quantification is shown in Figure 3A.

[0120] To determine the number of CD45-positive leukocytes in the BZ of the hearts after MI, IF was used to investigate the inflammatory response. In the IF staining of CD45, there was no difference in the number of leukocytes in the BZ of the AAV9-GFP-injected hearts and the control hearts, which is likely due to the attenuation of the immune response three months after MI. Leukocytes were observed in the infarcted tissue of the pig hearts 90 days after virus injection. Quantification is shown in Figure 3B.

[0121] These findings indicate that AAV9-Sav-shRNA infection promotes persistent capillary formation in the hearts of pigs after MI.

[0122] Example 3 The materials and methods of Examples 1 and 2 AAV9 virus packaging. The viral vector was used as previously described (Leach et al., 2017). A construct containing a triple shRNA against Sav with adjacent miR30 sequences was cloned into the pENN.AAV.cTNT, p1967-Q vector downstream of GFP (AAV9-Sav-shRNA). An empty vector encoding GFP was used as a control (AAV9-GFP). Both vectors were packaged into the myotropic serotype AAV9 by the Intellectual and Developmental Disabilities Research Center Neuroconnectivity Core at Baylor College of Medicine. After titer determination, the virus was aliquoted (1×10 13 viral genome particles per tube), immediately frozen, and placed at -80 °C for long-term storage. Prior to each injection, each aliquot was diluted with saline to make a 3 mL injection solution.

[0123] Pigs. Animals were handled and maintained in accordance with the requirements of the Laboratory Animal Welfare Act (P.L. 89-544) and its amendments in 1970 (PL91-579), 1976 (PL94-279), and 1985 (P.L. 99-198). Pigs were fed a commercially available diet and fresh, clean water in amounts sufficient to maintain body weight and allow reasonable weight gain in growing animals. Appropriate treatment for the species was given at the discretion of the veterinarian or the requester.

[0124] When the animal underwent surgery, appropriate analgesics and antibiotics were administered. Buprenorphine (0.05 - 0.1 mg / kg) was administered intramuscularly or orally every 6 - 12 hours or as determined necessary by the attending veterinarian. Flunixin meglumine (1.1 - 2.2 mg / kg) was administered intravenously, intramuscularly, or orally every 6 - 8 hours as needed. Naxcel (3 - 5 mg / kg) or equivalent was administered once daily intramuscularly for 24 hours starting from the day after the procedure. After discontinuation of Naxcel, Baytril (enrofloxacin; 2.5 - 5.0 mg / kg) was administered orally twice daily for up to 7 days. Baytril (enrofloxacin, injection, 7.5 mg / kg body weight (3.4 mL / 100 lb)) was administered intramuscularly or subcutaneously (behind the ear). The continued use of antibiotics and analgesics was based on post-treatment monitoring unless otherwise specified. After MI treatment, nitroglycerin paste was applied topically, followed by aspirin (162.5 mg (1 / 2 of a standard 325 mg tablet)) orally once daily and ranitidine (Zantac; 150 mg) orally 1 - 3 times daily. Finally, pantoprazole (40 mg) was administered orally once daily for gastric protection.

[0125] EMM and endocardial injection. Endocardial electromechanical mapping (EMM) was performed as previously described (Vale et al., 2000). Briefly, pigs were maintained under general anesthesia, appropriately prepared, and draped to allow sterile surgical access to the penile area. Access to the thigh was achieved by percutaneous penetration. An introducer sheath was placed intra-arterially, and AAV-Sav-shRNA or AAV-GFP was delivered using a MyoStar™ injection catheter (Biosense Webster, Diamond Bar, CA, USA). The catheter was advanced into the left ventricle, and first, an electromechanical endocardial map of the heart was created. After completion of point acquisition, the map was processed using a medium filter and a manual filter to exclude inner points, points taken outside the LV cavity (e.g., atrial points or aortic points), points obtained during early ventricular contractions, or points that did not meet the standard quality criteria (e.g., loop stability > 6 mm, position stability > 4 mm, cycle length variation > 10%). After EMM was completed, the MyoStar™ injection catheter was primed with 0.1 mL of AAV-Sav-shRNA or AAV-GFP to fill the dead space before the start of the injection procedure. Appropriate delivery into the myocardium was ensured by the vertical positioning of the catheter within the LV wall and the presence of early ventricular contractions when the needle was extended into the myocardium. For the MI pig model, the infarcted area was characterized with the NOGA system according to the presence of low unipolar voltage (i.e., < 4 mV). The BZ identified by EMM was typically a 5 - 10 mm area adjacent to the scar (Bolli et al., 2018). Ten to fifteen endocardial injections were performed in the infarcted BZ (4 - 7 mV, 0.2 mL unipolar voltage value per injection). The catheter was then removed, and the artery was closed with an Angioseal (Terumo). The pigs were then transferred to the intensive care unit for monitoring.

[0126] Acute MI induced by I / R. A 6F sheath was inserted percutaneously into the common femoral artery. A 5F coronary guiding catheter was advanced through the aorta and selectively engaged into the left main coronary artery ostium. Subsequently, an angioplasty balloon (selected to match the diameter of the LAD) was placed within the LAD between the first and second diagonal branches over a floppy 0.014-inch guide wire. The balloon was inflated at the nominal pressure, and complete coronary occlusion was demonstrated by the absence of distal flow of contrast agent. After 90 minutes, the balloon was deflated to allow passive reperfusion of the distal coronary bed. The patency of the distal circulation was demonstrated by injecting contrast agent. Ventricular arrhythmias were prevented and treated with lidocaine (a bolus of 1.5 mg / kg followed by a continuous infusion), amiodarone (a bolus of 5 mg / kg followed by a continuous infusion), and electrical cardioversion as needed. During the coronary occlusion period, the activated clotting time was measured regularly, and heparin boluses were repeated as needed to maintain the ACT range between 250 and 350 seconds. After the procedure was completed, the pigs were transferred to the intensive care unit for monitoring.

[0127] Two-dimensional echocardiogram. Under general anesthesia, the pigs were placed in the left lateral decubitus position. Oxygen saturation and 12-lead electrocardiogram were monitored. Images including parasternal long-axis view, parasternal short-axis view, and apical view were acquired using a 3.5-MHz phased array transducer equipped with a Vivid 7 ultrasound device (GE Medical Systems, Milwaukee, WI, USA). Left ventricular ejection fraction, left ventricular end-systolic volume, and left ventricular end-diastolic volume were calculated using the biplane Simpson's method according to the guidelines of the American Society of Echocardiography (Lang et al., 2015). A blinded independent operator was performed in all analyses.

[0128] Summary of pathological reports. Hearts and other organs (e.g., lungs, liver, kidneys) were collected from 18 pigs that had undergone MI and reperfusion 2 weeks before receiving endocardial injection of AAV9-Sav-shRNA or AAV9-GFP virus. A total of 27 animals were included: 7 animals were sacrificed 45 days after MI, 2 animals were sacrificed 74 days after MI, and 18 animals were sacrificed 104 days after MI. Pigs with at least a 30% (relative to baseline) reduction in EF 2 weeks after MI were used for further experiments (Koudstaal et al., 2014). Two animals, P-1912 and P-1965, were excluded due to insufficient induction of MI. In P-1912, EF was 61% before MI and 44% after MI (a 28% reduction compared to baseline). In P-1965, EF was 54% before MI and 38% after MI (a 29% reduction compared to baseline).

[0129] One animal, P-1946, died immediately after receiving endocardial injection. Examination of the heart revealed a large healed MI in the LV anterior wall / LV anteroseptum from the apex to the midventricular level. Additionally, the segments injected with NOGA showed small foci of myocardial hemorrhage, 2 - 3, near the infarct border. These foci suggested the acute injection sites and correlated with the sites in the respective two-dimensional NOGA maps.

[0130] Hearts of all other pigs showed gross and microscopic findings of focal replacement fibrosis compatible with healed MI in the LAD region. All infarcted hearts showed typical LV remodeling with marked thinning of the wall in the involved area. In several of the pigs, smaller linear or patchy fibrosis areas away from the ischemic scar were considered treatment-related and suggested healed injection sites or partial needle tracks.

[0131] Furthermore, small foci of interstitial or perivascular chronic inflammatory infiltrates were observed in 14 out of 18 pigs sacrificed 74 or 104 days after MI. In most of these cases (11 out of 14 affected), this finding was mild or minimal and, in some cases, was associated with single or small groups of necrotic or vacuolar CM. However, in three animals, these areas were somewhat more extensive and diffuse (P-1895, P-1918), or showed a higher degree of chronicity and myocyte loss with associated interstitial and / or replacement fibrosis (P-1917). The infiltrated areas were mainly present in the NOGA injection segments.

[0132] There were few gross findings in other organs, which were considered incidental or secondary to the procedures. The lungs, liver, and kidneys collected at sacrifice appeared mostly normal on gross examination. The lungs typically had varying amounts of dark red discoloration dorsocaudally, which was generally due to atelectasis from dorsal recumbency, general anesthesia, and euthanasia. There were few incidental congenital findings in the kidneys, consisting of severe renal hypoplasia (P-1864) and renal cysts (P-1903, P-1947). The liver appeared particularly normal, with the gallbladder wall being thin in only one animal (P-1937).

[0133] A subset of pigs was selected for limited microscopic examination of lung and liver tissues. The subset included nine animals: seven test pigs (P-1937, P-1947, P-1949, P-1950, P-1953, P-1956, and P-1960) mixed from both test groups, and one untreated control pig (P-1989). Histological sections were randomly selected from the middle regions of each organ and stained with hematoxylin and eosin (H&E). In the lungs of all pigs except one, no significant findings were shown except for some areas of atelectasis generally caused by various procedures (general anesthesia, dorsal rest) performed before euthanasia. One pig, P-1950, had localized interstitial pneumonia in the middle lobe, which was most likely related to foreign body aspiration. No significant findings were observed in the liver except for a very small area of localized inflammatory cell aggregation in one animal, P-1960. These findings were considered to be incidental.

[0134] Heart sample treatment. The heart was excised, weighed, and photographed at the time of euthanasia. After gross evaluation of the appearance, a small amount of fresh sample was taken for cryosectioning and subsequent molecular analysis. The heart was then perfused and fixed with 10% neutral buffered formalin flowing retrograde from the ascending aorta at a pressure of approximately 100 mmHg for 20 minutes. After perfusion fixation, the heart was sliced according to a segmentation protocol designed for sampling of the heart that had received endocardial injection of biological agents (Vela et al., 2015). The heart slices and pedestals were then immersed in 10% neutral buffered formalin for 24 - 48 hours. The segments that received the most injections (as determined by each two-dimensional EMM (NOGA) map) were selected for analysis. Those segments were further sliced into 4 - 5 levels of 2 mm thickness and embedded in different paraffin blocks (usually 40 - 50 blocks per heart). One to three additional samples from remote non-injected segments were also collected. All samples were processed for paraffin embedding, cut to a thickness of 5 microns, and stained with H&E.

[0135] Infarct size calculation. The ventricle was sectioned into 7 - 8 transverse slices from the apex to the base. Each slice was photographed at its apical and basal surfaces. Digital images were analyzed using ImagePro software. The LV and infarct regions were manually traced using ImagePro software from the digital photographs of the apical and basal surfaces of each slice. For each slice plane, the infarct was calculated as a percentage of the LV. Then, the results from both surfaces were averaged and multiplied by the slice weight to calculate the infarct size. Next, the sum of the infarct sizes from all slices was divided by the sum of the LV weights from all slices and expressed as a percentage (Jones et al., 2015).

[0136] IF staining. The processed frozen and paraffin sections were used for downstream analysis. For frozen sections, after fixation, the heart was dehydrated in 15% and 30% sucrose gradients and then embedded in an appropriate cutting temperature compound (catalog number 25608 - 930, VWR International, Radnor, PA, USA) for sectioning. The slides were sectioned at 10 - μm intervals for IF staining. For paraffin sections, the samples were deparaffinized and rehydrated, treated with 3% H2O2 in EtOH, treated with antigen retrieval solution (Vector Laboratories, Inc., Burlingame, CA, USA), blocked with 10% donkey serum in phosphate - buffered saline (PBS), and then incubated with primary antibodies. The antibodies were rabbit anti - CD45 (Abcam ab10559) and isolectin B4 (FL - 1201, Vector Laboratories).

[0137] EdU incorporation assay. EdU incorporation was detected using the Click - iT™ EdU Imaging Kit (Life Technologies, Carlsbad, CA, USA). Imaging of tissue slides was performed on a Leica TCS SP5 confocal microscope, and the images were processed using Leica LAS AF software (Leica Microsystems, Wetzlar, Germany).

[0138] Tile imaging analysis and machine learning. After IF staining, tile images were captured using a Zeiss LSM880 equipped with an Airyscan FAST Confocal Microscope at the Optical Imaging & Vital Microscopy Core (OiVM) of Baylor College of Medicine. Each tile image included at least 8×8 = 64 scan fields in a 10x objective scan image and had a 20% overlap (20 - 30 mm per image) for stitching. 2 ) Then, the images were analyzed for machine learning in Fiji (Schindelin et al., 2012). Pixel - based image segmentation was generated using the Trainable Weka Segmentation (Arganda - Carreras et al., 2017) plugin, and binarization of regions larger than 300 pixels in the region of interest (ROI) of Fiji was added. DAPI staining within regions larger than 100 pixels was counted as nuclei, and the overlap between nuclei and ROI was detected with Speckle Inspector and counted as one cell (Brocher, 2014). Intensity analysis using the updated ROI was used to exclude cells (non - CM) where nuclei were attached to the cell boundary. Finally, CMs overlapping with EdU staining were counted as EdU - positive CMs.

[0139] Statistics. Throughout the study, all analyses were performed in a double - blind manner. Data are presented as mean ± standard error of the mean. Quantitative data for two groups were evaluated for significance using the Mann - Whitney U test. For comparisons between multiple groups, two - way ANOVA with Bonferroni's pairwise post - hoc test was used. A p - value of less than 0.05 was considered significant in all analyses. Significant differences between experimental groups were represented as *P < 0.05, **P < 0.01, or ***P < 0.001.

[0140] Each animal was treated with multiple individual injections into the left ventricle of the heart. The injection sites were mapped / tracked using a physical (NOGA) map and GFP reporter for virus-infected cells. Each injection site functions as an island of infected cells surrounded by non-infected cells in between. Histological segments were collected from the injection positions for analysis (not an equal number from each animal due to the number of available samples and limitations of histological preparations). Each segment has measurements. Since each injection site can be defined and the cells infected with the virus can be tracked, each tile image / segment was classified as a replicate rather than for each pig. In nested figures, the data was presented in nested graph format while retaining the individual tile images / segments as replicates, and analysis of variance with post hoc tests was used to evaluate the effect of AAV9-Sav-shRNA treatment on the cell ability to enter the cell cycle. These analyses were performed using GraphPad Prism software. For analysis of variance analysis, the chi-square test was used to test the assumption of independence, the normal QQ plot was used to test for normality, and the Brown-Forsythe test or Bartlett test was used to test for homoscedasticity. If the test results showed that the assumption of homogeneity of variance was invalid, a non-parametric Kruskal-Wallis analysis of variance test was used.

[0141] Example 4 Gene therapy knockdown of Hippo signaling resolves arrhythmic events in pigs after myocardial infarction After myocardial infarction, the heart is unable to perform significant regeneration, and ischemic / reperfusion injury causes the loss of millions of cardiomyocytes, initiates pathological fibrotic remodeling, and gradually impairs systolic function. It has been demonstrated that inducing cardiomyocyte regeneration through genetic manipulation of various developmental pathways and cell cycle pathways may restore muscle tissue and improve cardiac function. However, uncontrolled cardiomyocyte proliferation is associated with harmful effects on electrical conduction, resulting in sudden and fatal arrhythmogenic events in large animal models of myocardial infarction (Riching et al., 2021; Gabisonia et al., 2019). Reports of arrhythmogenic events have prompted the verification of cardiac electrical conduction in a porcine model of myocardial infarction using AAV-based shRNA gene therapy (Figure 4A).

[0142] Before surgically inducing MI, a subcutaneous telemetry device (easyTEL+L-EEEETA, emka Technologies) was implanted subcutaneously to monitor heart rhythm. Four leads were extended to the four limbs via a subcutaneous tunnel to obtain 6-lead ECG recordings. Then, before MI, continuous ECG recordings for 10-15 minutes were saved for each pig. To induce MI, an angioplasty balloon was positioned in the left anterior descending artery between the first and second diagonal branches and inflated to block blood flow. Complete coronary occlusion was continued for 90 minutes, and total coronary occlusion was verified by the absence of contrast agent flow distally. After 14 days of recovery, the viral vector was delivered, and ECG recordings were continued for an additional 30 days. The total ECG recording period was approximately 45 days until the animals were euthanized (Figure 4A). From each pig, all 24-hour ECG recording segments for the entire 45-day period were reviewed for electrical and mechanical interference, as well as artifacts. Since the ECG segments recorded at night had the least interference, the analysis focused on premature ventricular complexes (PVC, Figures 4B-4D) and atrial tachycardia (A-Tach, Figures 4E-4J) in the ECG segments recorded from 0:00 to 6:00 am.

[0143] To assist in the identification and quantification of ventricular and atrial arrhythmias, semi-automatic analysis software (ecgAUTO V3.5.5.27) was used. A library of different waveforms, including normal complex waveforms, was saved for every 6-hour ECG segment. As expected, various arrhythmia phenotypes could be identified immediately after MI treatment. Ventricular arrhythmias were seen during the test period (45 days), and the incidence decreased over time (Figures 4B - 4D). Within 3 days of MI, multiple severe ventricular arrhythmia phenotypes were present, including intraventricular conduction delay, premature ventricular complexes ([PVC], Figures 4B, 4D), bigeminal PVC, PVC couplets, and non-sustained ventricular tachycardia. One of the focused examples was potentially lethal ventricular arrhythmias, especially premature ventricular complexes. The PVC frequency per ECG complex was determined for each period from midnight to 6 am daily during the experimental timeline. As expected, the mixed-effects analysis of PVC frequency in the period from after MI to before treatment showed a significant decrease in the fixed effect of time (P = 0.0065) and no difference between treatment groups (P = 0.6400, Figure 4B). Furthermore, a single non-linear curve fit showed both datasets (P = 0.8399). After treatment, the PVC-frequency not only decreased over time (P < 0.0001) but also decreased with AAV-Sav-shRNA treatment (P = 0.025), and different curves were used between treatment groups to show non-linear fit (P < 0.0001, Figure 4C). Ten days after treatment, PVCs were confirmed in AAV-GFP (Figure 4D), but were minimal in animals treated with AAV-Sav-shRNA (Figure 4J).

[0144] Similarly, multiple atrial arrhythmias, including premature atrial complexes, atrial flutter, atrial fibrillation, and atrial tachycardia, were present (Figs. 4E - 4I). All atrial arrhythmias were paroxysmal and resolved rapidly, especially in the early stages after MI (Fig. 4I). Here, we focused on atrial tachycardia and counted the cumulative number of tachycardia events observed in a single day during the experimental period from 0:00 to 6:00 am (Figs. 4E, 4F). Before treatment, there was no distinguishable difference in the incidence of atrial tachycardia between the AVV - GFP group and the AAV - Sav - shRNA group (Fig. 4E). Approximately 2 weeks after treatment with AAV - Sav - shRNA, atrial tachycardia events were no longer detected, and thus the cumulative incidence reached a plateau (Fig. 4F). The incidence of atrial tachycardia was analyzed using a generalized linear mixed model to generate the predicted odds and 95% confidence intervals before AAV injection (AAV - GFP, 0.59 [0.44, 0.72]; AAV - Sav - shRNA, 0.77 [0.59, 0.88], Fig. 4G) and after AAV injection (AAV - GFP, 0.50 [0.40, 0.60]; AAV - Sav - shRNA, 0.30 [0.21, 0.40], Fig. 4H). The odds of observing atrial tachycardia in animals treated with AAV - Sav - shRNA were significantly reduced (P(|z|)> = 0.0066, Fig. 4H). Considering that most cardiomyocyte proliferation occurs in the early stages of treatment (30 days after virus injection), and in addition to the fact that none of the animals died during the 3 - month follow - up period or showed uncontrolled cardiomyocyte proliferation or dedifferentiation (see Example 1), the decrease in PVC frequency and the incidence of atrial tachycardia indicates that the adult cardiomyocyte regeneration approach does not inherently induce arrhythmogenicity. Finally, post - infarct arrhythmias, especially refractory ventricular arrhythmias, significantly contribute to the death of patients who survive myocardial infarction (Bhar - Amato et al., 2017), so gene therapy used for cardiomyocyte regeneration also has therapeutic value in reducing the frequency of life - threatening post - myocardial infarction ventricular arrhythmias.

[0145] References Arganda-Carreras, I. et al. Trainable Weka Segmentation: a machine learning tool for microscopy pixel classification. Bioinformatics 33, 2424-2426, doi:10.1093 / bioinformatics / btx180(2017). Bhar-Amato J, Davies W, Agarwal S. Ventricular Arrhythmia after Acute Myocardial Infarction: ‘The Perfect Storm’. Arrhythm Electrophysiol Rev. 2017 Aug;6(3):134-139. doi:10.15420 / aer.2017.24.1. PMID:29018522; PMCID:PMC5610731. Bolli, R. et al. Rationale and design of the CONCERT-HF trial (combination of mesenchymal and c-kit(+) cardiac stem cells as regenerative therapy for heart failure). Circ.Res. 122, 1703-1715, doi:10.1161 / CIRCRESAHA.118.312978(2018). Brocher, J. Qualitative and quantitative evaluation of two new histogram limiting binarization algorithms. Int.J.Image Process. 8, 30-48(2014). Gabisonia, K. et al. MicroRNA therapy stimulates uncontrolled cardiac repair after myocardial infarction in pigs. Nature 569, 418-422, doi:10.1038 / s41586-019-1191-6(2019). Gyongyosi, M. et al. Porcine model of progressive cardiac hypertrophy and fibrosis with secondary postcapillary pulmonary hypertension. J Transl Med 15, 202, doi:10.1186 / s12967-017-1299-0 (2017). Heallen, T. et al. Hippo signaling impedes adult heart regeneration. Development 140, 4683 - 4690, doi:10.1242 / dev.102798 (2013). Jones, S.P. et al. The NHLBI-sponsored Consortium for preclinicAl assESsment of cARdioprotective therapies(CAESAR): a new paradigm for rigorous, accurate, and reproducible evaluation of putative infarct-sparing interventions in mice, rabbits, and pigs. Circ Res 116, 572 - 586, doi:10.1161 / CIRCRESAHA.116.305462 (2015). Lang, R.M. et al. Recommendations for cardiac chamber quantification by echocardiography in adults: an update from the American Society of Echocardiography and the European Association of Cardiovascular Imaging. J Am Soc Echocardiogr 28, 1 - 39 e14, doi:10.1016 / j.echo.2014.10.003 (2015). Leach, J.P. et al. Hippo pathway deficiency reverses systolic heart failure after infarction. Nature 550, 260 - 264, doi:10.1038 / nature24045 (2017). Milani - Nejad, N. & Janssen, P.M. Small and large animal models in cardiac contraction research: advantages and disadvantages. Pharmacol Ther 141, 235 - 249, doi:10.1016 / j.pharmthera.2013.10.007 (2014). McCormick, M.E. et al. Integrated Regional Cardiac Hemodynamic Imaging and RNA Sequencing Reveal Corresponding Heterogeneity of Ventricular Wall Shear Stress and Endocardial Transcriptome. J Am Heart Assoc 5, e003170, doi:10.1161 / jaha.115.003170 (2016). Monroe, T.O. et al. YAP partially reprograms chromatin accessibility to directly induce adult cardiogenesis. Dev. Cell 48, 765 - 779.e767, doi:10.1016 / j.devcel.2019.01.017 (2019). Morikawa, Y. et al. Actin cytoskeletal remodeling with protrusion formation is essential for heart regeneration in Hippo - deficient mice. Sci Signal 8, ra41, doi:10.1126 / scisignal.2005781 (2015). Riching AS, Song K. Cardiac Regeneration: New Insights Into the Frontier of Ischemic Heart Failure Therapy. Frontiers Bioeng Biotechnology. 2021;8:637538. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat Methods 9, 676 - 682, doi:10.1038 / nmeth.2019 (2012). Spannbauer, A. et al. Large Animal Models of Heart Failure with Reduced Ejection Fraction (HFrEF). Front Cardiovasc Med 6, 117, doi:10.3389 / fcvm.2019.00117 (2019). Stubenitsky, R., van der Weerd, R.W., Haitsma, D.B., Verdouw, P.D. & Duncker, D.J. Cardiovascular effects of the novel Ca2+-sensitiser EMD 57033 in pigs at rest and during treadmill exercise. Br J Pharmacol 122, 1257 - 1270, doi:10.1038 / sj.bjp.0701491 (1997). Tian, Y. et al. A microRNA-Hippo pathway that promotes cardiomyocyte proliferation and cardiac regeneration in mice. Sci Transl Med 7, 279ra238, doi:10.1126 / scitranslmed.3010841 (2015). Vale,P.R.et al.Left ventricular electromechanical mapping to assess efficacy of phVEGF(165) gene transfer for therapeutic angiogenesis in chronic myocardial ischemia.Circulation 102,965-974,doi:10.1161 / 01.cir.102.9.965(2000). van der Velden,J.et al.Alterations in myofilament function contribute to left ventricular dysfunction in pigs early after myocardial infarction.Circ Res 95,e85-95,doi:10.1161 / 01.Res.0000149531.02904.09(2004). Vela,D.,Gahremanpour,A.&Buja,L.M.Method for sectioning and sampling hearts for histologic evaluation after delivery of biological agents by transendocardial injection.Cardiovasc Pathol 24,304-309,doi:10.1016 / j.carpath.2015.04.005(2015). Wang,J.,Liu,S.,Heallen,T.&Martin,J.F.The Hippo pathway in the heart:pivotal roles in development,disease,and regeneration.Nat.Rev.Cardiol 15,672-684,doi:10.1038 / s41569-018-0063-3(2018). Xiao, Y., et al. Hippo pathway deletion in adult resting cardiac fibroblasts initiates a cell state transition with spontaneous and self-sustaining fibrosis. Genes Dev 33, 1491-1505, doi:10.1101 / gad.329763.119 (2019).

[0146] Although the invention and its advantages have been described in detail, it should be understood that various changes, substitutions, and modifications can be made without departing from the spirit and scope of the invention as defined by the appended claims. Further, the scope of this application is not intended to be limited to the specific embodiments of the processes, machines, manufactures, compositions, means, methods, and steps described in the specification. As will be readily understood by those skilled in the art, existing or later-developed processes, machines, manufactures, compositions, means, methods, and steps that perform substantially the same function and achieve substantially the same result as the corresponding embodiments described herein can be utilized in accordance with the invention. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufactures, compositions, means, methods, or steps.

Claims

1. A composition for use in improving systolic function in human patients who have experienced myocardial infarction, comprising at least one inhibitory nucleic acid targeting Salvador, wherein the inhibitory nucleic acid has a sequence selected from the group consisting of Sequence ID No. 2, Sequence ID No. 3, and Sequence ID No. 4 in the sequence listing, or is encoded by such sequence.

2. A composition for use in a method of reducing arrhythmias in human patients with myocardial fibrosis, comprising at least one inhibitory nucleic acid targeting Salvador, wherein the inhibitory nucleic acid has a sequence selected from the group consisting of Sequence ID No. 2, Sequence ID No. 3, and Sequence ID No. 4 of the sequence listing, or is encoded by said sequence.

3. A composition for use in a method to promote capillary formation in the cardiac tissue of a human patient, comprising at least one inhibitory nucleic acid targeting Salvador, wherein the inhibitory nucleic acid has a sequence selected from the group consisting of Sequence ID No. 2, Sequence ID No. 3, and Sequence ID No. 4 of the sequence listing, or is encoded by said sequence.

4. The composition according to any one of claims 1 to 3, wherein the inhibitory nucleic acid is an antisense DNA molecule or RNA.

5. The composition according to claim 4, wherein the inhibitory nucleic acid is short hairpin RNA (shRNA).

6. The composition according to claim 5, wherein the nucleotide sequence encoding the shRNA is included in the nucleic acid construct, and the shRNA is expressed in cardiomyocytes.

7. The composition according to claim 6, wherein the nucleotide sequence encoding the shRNA is operably linked to a tissue-specific promoter.

8. The composition according to claim 7, wherein the promoter is a cardiac troponin T promoter.

9. The composition according to any one of claims 1 to 3, wherein the composition comprises a nucleic acid construct having (i) a nucleic acid having the nucleotide sequence represented by SEQ ID NO: 2, (ii) a nucleic acid having the nucleotide sequence represented by SEQ ID NO: 3, and (iii) a nucleic acid having the nucleotide sequence represented by SEQ ID NO: 4, wherein the nucleic acids (i) to (iii) are operably linked to a promoter.

10. The composition according to claim 9, wherein the promoter is a cardiac troponin T promoter.

11. The composition according to claim 9, wherein the nucleic acid construct comprises one or more selected from (i) sequences encoding a 3' microRNA-30 sequence and a 5' microRNA-30 sequence, (ii) a Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), and (iii) a 5' reverse terminal repeat and a 3' reverse terminal repeat.

12. The composition according to claim 11, wherein the nucleic acid construct is an adeno-associated virus (AAV) vector or a lentiviral vector.

13. The composition according to claim 9, wherein the inhibitory nucleic acid is shRNA.

14. The composition according to claim 2 or 3, wherein the human patient has experienced a myocardial infarction.

15. The composition according to claim 2, wherein the arrhythmia is an atrial arrhythmia or a ventricular arrhythmia.

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