Use of mutant YAP to improve cardiac function

Abstract

Methods for improving cardiac function are provided. [Solution] Provided is a modified Yes-associated protein (YAP) in which the serine residues at the LATS1 / 2 phosphorylation site and in the region of YAP that binds to TEAD are substituted with alanine. Also provided are nucleic acids encoding the modified YAP, vectors containing the nucleic acid, and compositions containing the modified YAP, as well as nucleic acids encoding the modified YAP or vectors containing the nucleic acid. Also provided are methods for regenerating cardiomyocytes and treating cardiac conditions.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 444,736, filed February 10, 2023, which is incorporated herein by reference in its entirety.

[0002] Government Support Statement This invention was made with government support under Grant No. R01 HL118761 awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0003] Myocardial infarction (MI) is a leading cause of death worldwide. During an MI, contracting myocardium experiences reduced blood flow due to a decreased supply of oxygen and nutrients, ultimately leading to widespread cardiomyocyte death. Unlike nonmammalian vertebrates, such as zebrafish, whose hearts are capable of fully regenerating in response to injury throughout their lives, mature mammalian hearts possess terminally differentiated cardiomyocytes (CMs). CM loss during cardiac ischemia is irreversible and induces the activation of cardiac fibroblasts, which mediate scar formation and result in impaired myocardial contractility. However, recent studies have demonstrated that neonatal mouse hearts exhibit transient regenerative capacity within the first 7 days, suggesting a regulatory mechanism that prevents the adult heart from mounting a regenerative response to ischemia.

[0004] The Hippo signaling pathway (HSP), an evolutionarily conserved signaling pathway, was first identified in Drosophila for its regulation of organ and body size. In mammals, core components of HSPs include the sterile 20 (STE20) family protein kinases encoded by Stk4 / 3, mammalian STE20-like protein kinase 1 / 2 (MST1 / 2), large tumor suppressor kinase 1 / 2 (LATS1 / 2), which are phosphorylated and activated by MST1 / 2, and the transcriptional coactivator yes-associated protein (YAP), encoded by Yap1, which is phosphorylated and inhibited by LATS1 / 2.

[0005] Activated HSPs phosphorylate YAP via LATS1 / 2, suppressing its transcriptional activity through subsequent cytoplasmic retention and degradation. Conversely, in the unphosphorylated state, active YAP interacts with different transcription factors (TFs), such as TEA domain (TEAD) transcription factors, to regulate the expression of diverse genes involved in cell proliferation and differentiation.

[0006] Previous studies have demonstrated strong HSP activity in the postnatal heart. Deletion of the MST1 / 2 adaptor protein Salvador (SAV) in adult mouse hearts resulted in HSP deficiency and induced a repair gene program after MI injury, demonstrating the regulatory role of HSPs in cardiac regeneration. Furthermore, CM-specific YAP overexpression improved cardiac function and mouse survival by stimulating CM proliferation after MI injury, further elucidating the mechanism of HSP inhibition in CM regeneration.

[0007] To further explore YAP activity, a CM-specific YAP gain-of-function mouse model, YAP5SA, was previously developed. In the YAP5SA model, the LATS1 / 2 phosphorylation site of the YAP protein is mutated to completely bypass HSP inhibition. YAP5SA adult mice exhibit cardiac hyperplasia with thickened ventricular walls and shortened survival. In addition, YAP5SA alters the expression of multiple genes and enhances chromatin accessibility. YAP5SA also initiates both positive and negative feedback loops for CM proliferation. Therefore, improved YAP mutants that induce CM proliferation without the lethality observed in YAP5SA would be advantageous. Summary of the Invention

[0008] Some of the main aspects of the present invention are summarized below. Additional aspects are described in the Detailed Description, Examples, Figures, and Claims sections of this disclosure. The statements in each section of this disclosure are intended to be read in conjunction with the other sections. Furthermore, the various embodiments described in each section of this disclosure can be combined in various different ways, and all such combinations are intended to fall within the scope of the present invention.

[0009] Described herein is a modified YAP protein, YAP6SA, in which serine residues phosphorylated by LATS1 / 2 and in the region of YAP that binds to TEAD are substituted with alanine. These mutations bypass HSP inhibitory regulation and inhibit the interaction between YAP and TEAD. In some embodiments, YAP6SA promotes CM growth, and unlike YAP5SA, its expression is well tolerated in mammalian hearts. Additionally, YAP6SA improves cardiac repair after MI injury by reducing scar area and improving cardiac contractility.

[0010] Thus, in one embodiment, there is provided a YAP variant comprising the amino acid sequence set forth in SEQ ID NO: 3, a nucleic acid encoding the YAP variant, and / or a vector comprising the nucleic acid. In certain embodiments, the vector is a viral vector. In certain embodiments, the vector is an AAV vector, such as an AAV9 vector.

[0011] In some embodiments, the nucleic acid is operably linked to a cell- or tissue-specific promoter, e.g., a cardiac-specific promoter. In one embodiment, the promoter is a cardiomyocyte-specific promoter.

[0012] Also provided is a composition comprising a YAP mutant, a nucleic acid encoding a YAP mutant, or a vector comprising the nucleic acid, together with a carrier. In a preferred embodiment, the composition is a pharmaceutical composition.

[0013] Also provided is a method for using YAP mutant.One embodiment is a method for regenerating cardiomyocytes in a subject who needs cardiomyocyte regeneration, comprising delivering the composition of the present invention to the cardiac tissue of the subject.Another embodiment is a method for treating MI in a subject, comprising delivering the composition of the present invention to the cardiac tissue of the subject.The composition can be administered to the subject more than once.

[0014] Specific embodiments of the present invention include a method of regenerating cardiomyocytes in a subject and / or a method of treating MI in a subject, the method comprising delivering to cardiac tissue of the subject a pharmaceutical composition comprising an AAV vector, wherein the vector comprises a nucleic acid encoding SEQ ID NO:3 under the control of a cardiomyocyte-specific promoter.

[0015] Examples of subjects who would benefit from the methods of the present invention include subjects with arrhythmia, cardiomyopathy, heart failure, myocardial fibrosis, or myocarditis, and / or subjects who have experienced an MI.

[0016] It is contemplated that any aspect discussed herein can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, the compositions of the present disclosure can be used to achieve the methods of the present disclosure.

[0017] Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating specific embodiments of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0018] [Figure 1A]

[0039] Figure 1 shows phenotypic analysis of YAP6SA-overexpressing mice. A shows AAV9-GFP, AAV9-YAP5SA, and AAV9-YAP6SA expression cassettes; cTnT: cardiac troponin T. [Figure 1B] (B) Phenotype analysis of YAP6SA-overexpressing mice. (C) Immunofluorescence staining of Flag expression in mice overexpressing YAP5SA (YAP5SA OE) or YAP6SA (YAP6SA OE) on days 1 to 4 after AAV9 infection, as well as immunofluorescence staining of cTnT expression on day 4 after infection. [Figure 1C] (C) shows the results of Western blotting of Flag-YAP5SA and Flag-YAP6SA expression levels in postnatal mouse hearts. [Figure 1D] (D) Phenotype analysis of YAP6SA overexpressing mice. (E) Results of co-immunoprecipitation of interactors of YAP5SA and YAP6SA in mouse hearts. [Figure 1E] (E) Phenotype analysis of YAP6SA overexpressing mice. (E) Immunofluorescence staining of Flag expression in YAP5SA OE and YAP6SA OE mice on day 3 after AAV9 infection. [Figure 1F]Phenotype analysis of YAP6SA overexpressing mice. F shows quantification of Flag signal location in YAP5SA OE and YAP6SA OE CMs (n=6 each). [Figure 1G] (G) Phenotype analysis of YAP6SA overexpressing mice. (G) Survival curves of GFP, YAP5SA OE, and YAP6SA OE mice (n=8 each). [Figure 1H] Phenotypic analysis of YAP6SA-overexpressing mice. H shows echocardiographic results in AAV9-GFP and AAV9-YAP6SA-infected mice 4 and 8 weeks after administration. EF: ejection fraction, FS: fractional shortening. [Figure 1I]

[0039] Figure 1 shows phenotypic analysis of YAP6SA overexpressing mice. (I) H&E histological images showing cardiac structure of GFP and YAP6SA OE mice 4 weeks after AAV9 infection. [Figure 1J] (J) Phenotype analysis of YAP6SA overexpressing mice. H&E histology of cross sections of GFP and YAP6SA OE hearts 4 weeks after infection. [Figure 2A] This shows that YAP6SA promotes cardiomyocyte proliferation. A shows immunofluorescence staining of PCNA and quantification of PCNA-positive cardiomyocytes (n=6 each). [Figure 2B] Figure 2 shows that YAP6SA promotes cardiomyocyte proliferation. B shows immunofluorescence staining of pHH3 and quantification of pHH3-positive cardiomyocytes (n=6 each). [Figure 2C] This shows that YAP6SA promotes cardiomyocyte proliferation. C shows immunofluorescence staining of Aurora B and quantification of Aurora B-positive cardiomyocytes (n=6 each). **: p<0.01, ***: p<0.001. [Figure 2D] (D) Immunofluorescence staining of cardiac sections from Confetti mice and quantification of all clones (n=6 each) shows that YAP6SA promotes cardiomyocyte proliferation. [Figure 3A] YAP6SA regulates the expression of multiple genes in cardiomyocytes. A shows the bulk RNA-seq experimental pipeline. [Figure 3B](B) Heatmap of differential gene expression in AAV9-GFP CM, AAV9-YAP5SA OE CM, and AAV9-YAP6SA OE CM. [Figure 3C] Figure 1 shows that YAP6SA regulates the expression of multiple genes in cardiomyocytes.C shows split violin plots showing the average fold change of each gene cluster in YAP5SA OE CM and YAP6SA OE CM. [Figure 3D] (D) Gene ontology analysis of differentially expressed genes (DEGs) in YAP5SA OE CM and YAP6SA OE CM. [Figure 3E] Figure 1 shows that YAP6SA regulates the expression of multiple genes in cardiomyocytes.E shows the predicted upstream regulators of DEGs in AAV9-YAP6SA-overexpressing CMs. [Figure 4A] Figure 1 shows that YAP6SA interacts with various protein factors in cardiomyocytes. A shows a plot of the YAP6SA interactome in CMs. [Figure 4B] A shows that YAP6SA interacts with diverse protein factors in cardiomyocytes. B shows the intersection of the established YAP, YAP5SA, and YAP6SA interactomes. [Figure 4C] Figure 1 shows that YAP6SA interacts with various protein factors in cardiomyocytes. C shows the classification of YAP6SA interactors in CMs. [Figure 4D] (D) Gene ontology analysis of DEGs in YAP6SA OE CMs, demonstrating that YAP6SA interacts with diverse protein factors in cardiomyocytes. [Figure 4E] Figure 4E shows that YAP6SA interacts with various protein factors in cardiomyocytes. Figure 4E shows the interaction of YAP6SA with MPDZ in CMs by immunoprecipitation. [Figure 4F]This shows that YAP6SA interacts with various protein factors in cardiomyocytes. F shows quantification of Rhos signal intensity in dividing CMs in YAP6SA OE hearts. [Figure 4G] This shows that YAP6SA interacts with various protein factors in cardiomyocytes. G shows the experimental design of ROCK inhibitor treatment and quantification of immunofluorescence staining results comparing pHH3-positive CM between groups (n=6 each). [Figure 4H] Figure 1 shows that YAP6SA interacts with various protein factors in cardiomyocytes.H shows a model of the role of YAP6SA in CM proliferation. [Figure 5A] Figure 1 shows that YAP6SA stimulates cardiac regenerative responses after injury. A shows a first timeline of MI surgery, AAV9 infection, and sample analysis. [Figure 5B] (B) Trichrome histology images of AAV9-GFP and AAV9-YAP6SA mice 4 weeks after MI injury, showing that YAP6SA stimulates cardiac regenerative responses after injury. [Figure 5C] Figure 1 shows that YAP6SA stimulates cardiac regenerative responses after injury.C shows fibrosis quantification. [Figure 5D] (D) Echocardiograms of GFP and YAP6SA MI mice show that YAP6SA stimulates cardiac regenerative responses after injury. [Figure 5E] E shows that YAP6SA stimulates cardiac regenerative responses after injury. Echocardiography results show cardiac function in MI-GFP and MI-YAP6SA at 4 weeks after injury. [Figure 5F] This shows that YAP6SA stimulates cardiac regeneration responses after injury. F shows cardiac remodeling in YAP6SA-treated hearts with MI. [Figure 5G] (G) A second timeline of MI surgery, AAV9 infection, and sample analysis shows that YAP6SA stimulates cardiac regenerative responses after injury. [Figure 5H](H) Trichrome histology images of AAV9-GFP and AAV9-YAP6SA mice 3 weeks after MI injury, showing that YAP6SA stimulates cardiac regenerative responses after injury. [Figure 5I] Figure 1 shows that YAP6SA stimulates cardiac regenerative responses after injury. I shows fibrosis quantification. [Figure 5J] (J) Echocardiograms and cardiac function in GFP and YAP6SA MI mice 3 weeks after injury, showing that YAP6SA stimulates cardiac regenerative responses after injury. [Figure 6A] Figure 1 shows that YAP6SA stimulates cardiac regenerative responses in adult mice. A shows the timeline of MI surgery, AAV9 infection, and analysis. [Figure 6B] (B) Trichrome histology images of AAV9-GFP and AAV9-YAP6SA mice 3 weeks after MI injury. [Figure 6C] Figure 1 shows that YAP6SA stimulates cardiac regenerative responses in adult mice.C shows fibrosis quantification. [Figure 6D] (D) Echocardiograms of GFP and YAP6SA MI mice show that YAP6SA stimulates cardiac regenerative responses in adult mice. [Figure 6E] (E) Echocardiography results show cardiac function in MI-GFP and MI-YAP6SA at 3 weeks post-injury. DETAILED DESCRIPTION OF THE INVENTION

[0019] In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout this disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0020] Any headings provided herein are not limitations of various aspects or embodiments of the invention, but can have by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined in their entirety by reference to the specification.

[0021] All references cited in this disclosure are incorporated herein by reference in their entirety. Additionally, any manufacturer's instructions or catalogs for any products cited or referred to herein are incorporated by reference. Any document incorporated herein by reference, or any teaching therein, may be used in the practice of the present invention. Documents incorporated herein by reference are not admitted to be prior art.

[0022] The phraseology or terminology used in this disclosure is for the purpose of description and not of limitation, as the phraseology or terminology used herein would be understood by one of ordinary skill in the art in light of the teachings and guidance.

[0023] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The term "a" (or "an"), and the terms "one or more" and "at least one" can be used interchangeably.

[0024] Furthermore, "and / or" should be interpreted as a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term "and / or" used in phrases such as "A and / or B" is intended to include A and B, A or B, A alone, and B alone. Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A alone; B alone; and C alone.

[0025] The words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0026] The methods and compositions of the present invention can "comprise," "consist essentially of," or "consist of" certain ingredients, components, compositions, etc. disclosed throughout this specification. With respect to the transitional phrase "consisting essentially of," in one non-limiting aspect, a basic and novel feature of the compositions and processes of the present invention is their ability to induce proliferation or regeneration of CMs or otherwise stimulate cardiac regenerative response(s) to improve cardiac function for the treatment and / or prevention of cardiac conditions.

[0027] Whenever an embodiment is described using the language "comprising," similar embodiments otherwise described using the terms "consisting of" and / or "consisting essentially of" are included. Accordingly, in any of the claims, the terms "consisting of" or "consisting essentially of" may be substituted for any of the above open-ended linking verbs in order to modify a given claim from one using an open-ended linking verb. Additionally, "wherein" may be used interchangeably with "where."

[0028] Units, prefixes, and symbols are expressed in the accepted format of the Systeme International d'Unites (SI). Numerical ranges are inclusive of the numbers defining the range, and any individual value provided herein can serve as an endpoint of a range that includes other individual values ​​provided herein. For example, a collection of values ​​such as 1, 2, 3, 8, 9, and 10 is also a disclosure of numerical ranges such as 1 to 10, 1 to 8, 3 to 9, etc. Similarly, disclosed ranges are a disclosure of each individual value (i.e., intermediate values) encompassed by the range, including integers and fractions. For example, a range of 5 to 10 is also a disclosure of 5, 6, 7, 8, 9, and 10, as well as 5.2, 7.5, 8.7, etc.

[0029] The term "about" or "approximately" is defined as being close to, as understood by one of ordinary skill in the art. In one non-limiting embodiment, the term is defined as being within 10%, within 5%, within 1%, or within 0.5%.

[0030] Human YAP1 has the amino acid sequence set forth in SEQ ID NO: 1. LATS phosphorylation sites are underlined. The serine residues at positions 61, 109, 127, 128, 131, 163, 164, and 381, shown in bold and italic, are substituted with alanine in YAP5SA.

[0031] Compared to YAP5SA, YAP6SA contains an additional serine-to-alanine substitution at position 94 in the YAP / TEAD interface. The ability of YAP6SA to function in the absence of its interaction with TEAD was unexpected, based on literature reports that this is an obligate interaction for YAP function in the heart (Zhao et al. 2008; Li et al. 2010; von Gise et al. 2012). As demonstrated in the Examples, YAP6SA induces CM proliferation, improves cardiac function, and, unlike YAP5SA, is safe and well-tolerated. [ka]

[0032] Thus, human YAP5SA has the amino acid sequence shown in SEQ ID NO: 2, and human YAP6SA has the amino acid sequence shown in SEQ ID NO: 3. Substitutions in the wild-type sequence are shown in bold italics. [ka] [ka]

[0033] The corresponding mouse YAP5SA and YAP6SA amino acid sequences are set forth in SEQ ID NO: 4 and SEQ ID NO: 5, respectively. Substitutions in the wild-type sequence are shown in bold italics. [ka] [ka]

[0034] Provided herein are YAP variants comprising the amino acid sequences set forth in SEQ ID NO:3 or SEQ ID NO:5, along with nucleic acids encoding these sequences. As used herein, the term "nucleic acid" refers to a polymer of DNA or RNA having a combination of purine and pyrimidine bases, sugars, and covalent internucleoside linkages comprising phosphate groups in phosphodiester linkages. Nucleic acids can be single- or double-stranded and optionally contain synthetic, non-natural, or modified nucleotide bases that can be incorporated into the DNA or RNA polymer.

[0035] The nucleic acid can be operably linked to an expression control sequence such that the coding sequence is under the transcriptional control of the expression control sequence. The expression control sequence is preferably a promoter, for example, a tissue-specific promoter. Examples of promoters specific for expression in cardiac tissue or cardiomyocytes include cardiac troponin T promoters such as chicken cardiac troponin T (cTnT) promoter, myosin light chain 2 (MLC-2v) promoter, alpha myosin heavy chain (MHC) promoter, and the minimal promoter of the NCX1 promoter from -137 to +85.

[0036] Also provided is a vector comprising a nucleic acid encoding a YAP mutant of the present invention, preferably under the transcriptional control of a promoter. Suitable vectors include, for example, viral vectors, non-viral vectors, and non-integrating vectors. Viral vectors can be adenoviral vectors, adeno-associated viral (AAV) vectors, or retroviral vectors such as lentiviral vectors. In a specific embodiment, the vector is an AAV vector. AAV vectors 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, cosmids, phages, bacterial, yeast, and cellular vectors.

[0037] In certain embodiments, the YAP mutant, the nucleic acid encoding the YAP mutant, or the vector comprising the nucleic acid encoding the YAP mutant is included in a composition, such as a pharmaceutical composition. In some aspects, the YAP mutant, the nucleic acid encoding the YAP mutant, the vector comprising the nucleic acid encoding the YAP mutant, or any of the aforementioned compositions is administered to a subject (e.g., a mammalian subject, such as a human subject). In some aspects, the nucleic acid encoding the YAP mutant is translated in vivo to produce the YAP mutant. The nucleic acid encoding the YAP mutant can be induced for translation of the YAP mutant in a cell, tissue, or organism. In exemplary embodiments, such translation occurs in vivo, for example, in cardiac tissue (e.g., myocardium) or cardiac cells (e.g., CM), although embodiments in which such translation occurs ex vivo, in culture, or in vitro can be envisioned. In exemplary embodiments, a cell, tissue, or organism is contacted with an effective amount of the YAP mutant, the nucleic acid encoding the YAP mutant, or the vector comprising the nucleic acid encoding the YAP mutant.

[0038] The term "pharmaceutical composition" refers to a preparation that is in a form that allows the biological activity of the active ingredient to be effective and does not contain additional components that produce adverse, allergic, or other undesirable reactions or that are unacceptably toxic to the subject to which the composition is administered. Pharmaceutical compositions typically contain a pharmaceutically acceptable carrier and may include one or more of the following: buffers (e.g., acetate, phosphate, or citrate buffers), surfactants (e.g., polysorbates), stabilizers (e.g., human albumin), preservatives (e.g., benzyl alcohol), and / or other conventional solubilizing or dispersing agents. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. To the extent that any conventional media or agent is incompatible with the active ingredient, its use in the compositions disclosed herein is contemplated. Supplementary active ingredients, such as other treatments for cardiac conditions, can also be incorporated into the compositions. The choice of dosage form and excipients depends on the active agent to be delivered and the disease or disorder to be treated or prevented, and is routine to those skilled in the art.

[0039] The pharmaceutical compositions disclosed herein are suitable for parenteral administration. Parenteral administration routes include intravenous, intramuscular, intraperitoneal, intrathecal, and subcutaneous routes. In a preferred embodiment, the pharmaceutical composition is suitable for local administration to the cardiac tissue of a patient. "Local administration" means that the pharmaceutical composition is administered directly to the location where its effect is desired (e.g., at or near the site of injury or symptom). One administration route is to inject the pharmaceutical composition directly into cardiac tissue, e.g., myocardium. It is within the understanding of one skilled in the art to formulate pharmaceutical compositions suitable for their intended administration route. The pharmaceutical compositions of the present invention can be administered to a patient once or more than once.

[0040] An "effective amount" of a composition disclosed herein is an amount sufficient to accomplish a specifically stated purpose. An "effective amount" can be empirically determined in relation to the stated purpose, route of administration, and dosage form. In some embodiments, an "effective amount" is an amount sufficient to ameliorate at least one symptom, behavior, or event associated with a pathological, abnormal, or otherwise undesirable condition, or to prevent or reduce the likelihood of such a condition occurring or recurring, or to delay the worsening of such a condition. For example, in some embodiments, an effective amount refers to an amount of a YAP mutant, a nucleic acid encoding a YAP mutant, a vector comprising a nucleic acid encoding a YAP mutant, or any of the aforementioned compositions capable of inducing CM proliferation or regeneration or otherwise stimulating a cardiac regenerative response to improve cardiac function for the treatment and / or prevention of a cardiac condition in a subject.

[0041] "Subject" or "individual" or "patient" means a mammalian subject for whom diagnosis, prognosis, or therapy is desired. In a preferred embodiment, the mammalian subject is a human.

[0042] The YAP mutants of the present invention, nucleic acids encoding the YAP mutants, vectors comprising nucleic acids encoding the YAP mutants, and compositions of the present invention can be used in methods for regenerating cardiomyocytes in a subject; methods for treating arrhythmia, cardiomyopathy, heart failure, myocardial fibrosis, myocardial infarction, or myocarditis in a subject; methods for producing a pharmaceutical for regenerating cardiomyocytes in a subject; and / or methods for producing a pharmaceutical for treating arrhythmia, cardiomyopathy, heart failure, myocardial fibrosis, myocardial infarction, or myocarditis in a subject.

[0043] Thus, one embodiment is a method for regenerating cardiomyocytes or treating a cardiac condition in a subject in need thereof, the method comprising administering to cardiac tissue of the subject a composition comprising YAP6SA, a nucleic acid encoding YAP6SA, or a vector comprising a nucleic acid encoding YAP6SA. A further embodiment is a method for treating arrhythmia, cardiomyopathy, heart failure, myocardial fibrosis, myocardial infarction, or myocardium in a subject in need thereof, the method comprising administering to cardiac tissue of the subject a composition comprising YAP6SA, a nucleic acid encoding YAP6SA, or a vector comprising a nucleic acid encoding YAP6SA. In some embodiments, the composition is administered to CM in the subject.

[0044] In some embodiments, a subject in need of a composition or method of the present invention has a cardiac condition that would be improved by the composition and method of the present invention, for example, the cardiac condition can be improved by regenerating cardiomyocytes in the subject. In some embodiments, the cardiac condition includes arrhythmia; heart failure; cardiomyopathy, such as age-related cardiomyopathy, diabetic cardiomyopathy, dilated cardiomyopathy, or ischemic cardiomyopathy; myocardial fibrosis; myocardial necrosis; myocarditis; or myocardial infarction. In some embodiments, the subject has arrhythmia; heart failure; cardiomyopathy, such as age-related cardiomyopathy, dilated cardiomyopathy, or ischemic cardiomyopathy; myocardial fibrosis; myocardial necrosis; or myocarditis. In some embodiments, the subject has experienced an MI.

[0045] Terms such as "inhibiting" or "reducing" or "preventing" or "avoiding" or any variation thereof, as used in the claims and / or herein, refer to an approach to preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of a disease or condition, e.g., a cardiac condition, and include any measurable reduction (e.g., at least, maximally, exactly, or between any two of a 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% reduction) in the intensity, effects, symptoms, and / or burden of the disease or condition or complete (i.e., 100%) inhibition thereof. It also refers to delaying the onset or recurrence of the disease or condition, or delaying the onset or recurrence of symptoms of the disease or condition. As used herein, "prevention" and similar words also include reducing the intensity, effects, symptoms, and / or burden of a disease or condition prior to the onset or recurrence of the disease or condition.

[0046] Terms such as "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" refer to therapeutic measures that cure, slow, alleviate symptoms, and / or halt the progression of a diagnosed pathological condition or disorder. Treatment can serve to achieve one or more of a variety of desired results, including, for example, preventing the onset or recurrence of the disease, alleviating or reducing the severity of symptoms, and attenuating any direct or indirect pathological consequences of the disease, preventing the spread of the disease, slowing the rate of disease progression, ameliorating or alleviating the disease state, and achieving remission or improving prognosis. Thus, those in need of treatment include those already with the disorder. In certain embodiments, a subject is successfully "treated" for a disease or disorder in accordance with the methods provided herein if the patient, for example, exhibits total, partial, or temporary alleviation or elimination of symptoms associated with the disease or disorder. Thus, "treatment" does not necessarily indicate complete eradication or cure of the disease or condition or its associated symptoms.

[0047] In this context, successful treatment can include, for example, improved cardiac function, such as improved contractile function, improved capillary formation in cardiac tissue, and / or reduced arrhythmia. The improvement in cardiac function in an individual treated with a composition or method of the present invention is relative to the cardiac function in the individual before treatment with a composition or method of the present invention. Cardiac function can be assessed, for example, by measuring ejection fraction, fractional shortening level, diastolic volume, systolic volume, left ventricular end-diastolic volume, left ventricular end-systolic volume, left ventricular systolic function, diastolic function, stroke volume, cardiac rhythm, or a combination thereof.

[0048] The reduction in arrhythmias in a patient is assessed as the frequency and / or number of arrhythmias in a patient treated with a composition or method of the present invention relative to the frequency and / or number of arrhythmias in the patient before treatment with a composition or method of the present invention.

[0049] Subjects treated with the compositions and methods of the present invention can experience a reduction in cardiac tissue fibrosis. Fibrosis can be assessed by measuring cardiac function and / or visually, for example, by computed tomography, echocardiography, endomyocardial biopsy, and / or magnetic resonance imaging. The reduction in fibrosis in individuals treated with the compositions or methods of the present invention is relative to the fibrosis in the individual before treatment with the compositions or methods of the present invention.

[0050] Embodiments of the present disclosure can be further defined by reference to the following non-limiting examples. The examples are included to demonstrate aspects of the present disclosure. The examples are provided for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way. It should be understood by those of skill in the art that the techniques disclosed in the examples below represent techniques discovered by the inventors to work well in practicing the present disclosure. However, it will be apparent to those skilled in the art that many modifications to both materials and methods can be made without departing from the scope of the present disclosure and still obtain similar or similar results. Although efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), some experimental error and deviation should, of course, be allowed for. [Example]

[0051] Example 1. Generation of a YAP6SA gain-of-function expression cassette We generated a YAP6SA expression cassette in which YAP6SA is tagged with Flag protein and transcribed by the cardiomyocyte-specific cardiac troponin T promoter (cTnT) (Figure 1A). YAP6SA has nine point mutations, eight of which are identical to those in YAP5SA. In both YAP5SA and YAP6SA, the LATS1 / 2 phosphorylation site is mutated from serine to alanine. YAP6SA has an additional serine to alanine mutation in the YAP / TEAD interface region.

[0052] YAP5SA and YAP6SA expression cassettes were packaged into adeno-associated virus serotype 9 (AAV9) vectors, and 6-day-old mice (P6) were infected with the AAV9 vectors. To confirm the expression of YAP5SA and YAP6SA in cardiomyocytes (CMs), immunofluorescence (IF) was performed to detect Flag protein signals at different time points after AAV9 infection, from day 1 to day 7. Cardiac troponin T (cTnT) was used as a CM marker.

[0053] YAP5SA and YAP6SA were widely expressed on day 3 (Figure 1B). Blotting results demonstrated that YAP5SA and YAP6SA showed similar expression levels in postnatal mouse hearts after AAV9 delivery (Figure 1C). Co-immunoprecipitation confirmed disruption of the interaction between TEAD and YAP6SA (Figure 1D). Both Flag-YAP5SA and Flag-YAP6SA were specifically expressed in CMs, whereas YAP6SA showed a decreased nuclear localization ratio and an increased cytoplasmic ratio (Figures 1E and 1F). These results suggest that YAP / TEAD interaction regulates YAP nuclear translocation.

[0054] Example 2. YAP6SA overexpression is safely tolerated in vivo. To determine the effect of YAP6SA overexpression (OE) on cardiac development, we examined mouse survival among the AAV9-GFP, AAV9-YAP5SA, and AAV9-YAP6SA OE groups. All YAP6SA OE and control (GFP) mice survived for at least 60 days after administration, indicating that YAP6SA was well tolerated in vivo. In contrast, YAP5SA was lethal in all YAP5SA OE mice by day 17 after administration. Survival curves are shown in Figure 1G.

[0055] To determine whether YAP6SA affects cardiac function in mice, echocardiography was performed to measure the ejection fraction (EF) and fractional shortening (FS) levels of GFP and YAP6SA OE hearts 4 and 8 weeks after AAV9 delivery. YAP6SA OE mice had similar FS (approximately 40%) and EF (approximately 75%) levels and echocardiograms (Figure 1H) to AAV9-GFP control mice. Furthermore, histological cardiac sections demonstrated that YAP6SA did not alter cardiac structure 4 weeks after AAV9 infection (Figures 1I and 1J). Collectively, these data indicate that YAP6SA OE is well tolerated in vivo and does not impair cardiac structure and function in mammals.

[0056] Example 3. Cardiac-specific YAP6SA promotes cardiomyocyte proliferation To investigate whether YAP6SA promotes CM division, immunofluorescence (IF) analysis of mouse hearts was performed 3 days after AAV9 infection to examine the CM cell cycle status using three cell cycle markers: proliferating cell nuclear antigen (PCNA), phosphorylated histone H3-Ser10 (pHH3), and Aurora B. PCNA plays an essential role in DNA synthesis and is highly expressed during S phase. During M phase, pHH3 is expressed during chromosome condensation (prophase), persists through interphase, and then declines during anaphase. The chromosomal passenger protein Aurora B is more widely detected throughout cytokinesis.

[0057] IF results showed increased CM proliferation in YAP6SA OE hearts compared with AAV9-GFP controls, with approximately 10% PCNA-positive CMs (Figure 2A), 0.6% pHH3-positive CMs (Figure 2B), and 0.2% Aurora B-positive CMs (Figure 2C) per area in the heart. In addition, YAP6SA OE hearts had a similar number of CM divisions compared with YAP5SA OE hearts.

[0058] To further confirm the enhanced CM proliferation, YAP6SA OE hearts were collected 1 week after AAV9 infection and analyzed via histology to compare the ventricular wall thickness between GFP control hearts and YAP6SA OE hearts. YAP6SA OE hearts had thicker ventricular walls (Figure 2D). These results indicate that YAP6SA promotes CM cell cycle progression.

[0059] Example 4. YAP6SA alters the expression of various genes in cardiomyocytes To investigate the mechanism of YAP6SA function, gene expression in YAP6SA OE CM was compared with that in GFP control and YAP5SA OE CM. CM nuclei were harvested 3 days after AAV9 infection, and RNA was extracted for transcriptional profiling (Figure 3A). Results for YAP6SA OE CM showed five distinct clusters of differentially expressed genes (DEGs) between GFP, YAP5SA, and YAP6SA OE CM. YAP6SA OE CM had fewer DEGs compared to YAP5SA OE CM, including 209 up-regulated genes and 96 down-regulated genes (FDR < 0.05) (Figures 3B and 3C). These data indicate reduced transcriptional activity in YAP6SA OE CM compared to YAP5SA OE CM.

[0060] Furthermore, gene ontology (GO) analysis revealed that YAP5SA-specific upregulated genes were primarily involved in cell cycle progression, while downregulated genes were related to oxidative metabolism, while YAP6SA-specific upregulated genes were involved in the TCA cycle and CM differentiation. Notably, YAP5SA and YAP6SA OE CM shared several DEGs related to cytoskeleton organization. GO analysis of YAP6SA OE relative to GFP CM confirmed increased expression of actin- and microtubule-related genes, such as Acta1, Actg1, Myl9, and Rhoa. Furthermore, several upregulated genes, including Myh8 and Srf, mediate myocyte differentiation and development, and several genes, such as Pdk4, are involved in the tricarboxylic acid (TCA) metabolic cycle (Figure 3D). Furthermore, YAP6SA-repressed genes, such as Csf1 and Arel1, are involved in immune response.

[0061] Predicted upstream regulators of DEGs in YAP6SA OE CMs included YAP (Figure 3E), confirming the RNA-seq results. These data suggest a TEAD-independent role for YAP6SA in reorganizing CM cytoskeletal structure.

[0062] Example 5. YAP6SA interacts with multiple protein factors in cardiomyocytes YAP6SA interactors in CM were further identified by applying an anti-Flag antibody to pull down Flag-tagged YAP6SA and its interacting proteins from mouse hearts 3 days after AAV9 infection for mass spectrometry analysis. The results showed that YAP6SA interacted with various protein factors (Figures 4A and 4B). For example, YAP6SA bound to the classical Hippo pathway components WW domain-containing protein / kidney and brain-expressed protein (WWC1 / KIBRA), angiomotin-like protein 2 (AMOTL2), and neurofibromatosis type 2 protein (NF2), which regulate LATS1 / 2 and YAP / transcriptional coactivator with PDZ-binding motif protein (TAZ) activity.

[0063] YAP6SA also cooperates with retinoblastoma binding protein 8 (RBBP8), proteasome 26S non-ATPase subunit 14 (PSMD14), protein phosphatase 1 catalytic subunit alpha (PPP1CA), and transcription cofactor four and a half LIM domains 2 (FHL2), all of which are associated with cell cycle progression and CM development. Interestingly, YAP6SA forms complexes with mitochondrial ribosomal protein S33 (MRPS33), heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1), RNA-binding motif protein X-linked (RBMX), eukaryotic elongation factor-1 gamma (EEF1G), phosphoribosyl pyrophosphate synthase 1 (PRPS1), and phosphoribosyl pyrophosphate synthase 1-like 3 (PRPS1L3), all of which are involved in RNA transcription and translation, suggesting alternative functions for YAP6SA. Proteins involved in mitochondrial oxidative metabolism, such as cytochrome C oxidase subunit 7A2 (COX7A2), NADH-ubiquinone oxidoreductase subunit A8 (NDUFA8), and Parkinson's disease protein 7 (PARK7), were also YAP6SA interactors. Furthermore, multiple PDZ domain protein (MPDZ), membrane protein palmitoylation 5 (MPP5), myristoylated alanine-rich c-kinase substrate (MARCKS), αE-catenin (CTNNA1), and zyxin (ZYX), which have been shown to regulate cytoskeleton and cell junction organization, were significant YAP6SA partners (Figure 4C).

[0064] The combined mass spectrometry and RNA-seq results indicated that YAP6SA has multiple TEAD-independent functions in regulating various CM activities, and according to the increased ratio of YAP6SA localization in the cytoplasm, YAPSA appears to have a major role in regulating CM cytoskeletal structure and providing a suitable environment for CM cell cycle progression.

[0065] Example 6. YAP6SA promotes cardiomyocyte proliferation by activating Rho GTPases Mass spectrometry results demonstrated that the MPDZ protein has a strong binding affinity for YAP6SA. MPDZ, also known as MUPP1, contains 13 PDZ domains and serves as a scaffolding protein that organizes higher-order protein complexes and helps maintain cell polarity. MPDZ has also been shown to activate Rho GTPases in cooperation with Rho guanine nucleotide exchange factors (GEFs) in endothelial cell migration and near synapses. Rho family proteins control nearly all fundamental cellular processes in eukaryotes and regulate cytokinesis in certain cell types.

[0066] To determine whether YAP6SA stimulates CM cell cycle reentry by increasing Rho GTPase activity, we analyzed co-immunoprecipitation of YAP6SA-MPDZ in CMs (Figure 4E) and the expression levels of Rho family genes among GFP, YAP5SA OE, and YAP6SA OE groups. Indeed, Rho genes, particularly Rhobtb1 and Rhoa, were upregulated in the YAP6SA OE group. CM division in YAP6SA OE hearts increased Rho protein activity (Figure 4F).

[0067] To investigate whether inhibition of Rho GTPase activity disrupts YAP6SA function, we delivered the ROCK inhibitor Y-27632 to the hearts of YAP6SA OE mice and measured the number of pHH3-positive CMs 2 days later (Figure 4G). Indeed, pHH3-positive CMs were significantly reduced in YAP6SA OE hearts treated with Y-27632 compared with DMSO-treated YAP6SA OE hearts. The data indicate that YAP6SA enhances CM proliferation by activating Rho-GTPases (Figure 4H).

[0068] Example 7. YAP6SA OE hearts have improved regenerative capacity after MI Fully developed mammalian CMs lose their self-renewal capacity, which leads to scar formation and cardiac dysfunction after MI injury. Because YAP6SA promoted CM re-entry into the cell cycle, we investigated whether YAP6SA could induce a cardiac regenerative response to ischemic injury in mouse hearts.

[0069] Seven-day-old (P7) mice were infected with AAV9-GFP or AAV9-YAP6SA and underwent surgically induced MI by permanent occlusion of the left anterior descending coronary artery 3 days after AAV9 infection. Hearts were analyzed 4 weeks after surgery (Figure 5A). YAP6SA MI mice showed significantly reduced scar area compared with control MI mice (Figures 5B, 5C). YAP6SA OE hearts also showed enhanced cardiac ejection fraction and fractional shortening levels after MI, suggesting that YAP6SA improved cardiac function after injury (Figures 5D, 5E). Furthermore, cardiac remodeling was reduced in YAP6SA MI hearts, demonstrating YAP6SA-induced CM regrowth under ischemic stress (Figure 5F).

[0070] In another experiment, MI injury was induced 6 hours before AAV9 viral infection, and MI hearts were analyzed 3 weeks after injury (Figure 5G). The results were consistent, demonstrating that post-MI YAP6SA delivery reduced cardiac fibrosis and restored cardiac contractility compared to the GFP control group (Figures 5H-5J). Furthermore, MI surgery was performed in 8-week-old mice infected with AAV9-GFP or AAV9-YAP6SA. YAP6SA enhanced cardiac repair in adult mouse hearts 4 weeks after infection (Figures 6A-6E).

[0071] These findings support the conclusion that YAP6SA improves cardiac repair after ischemic injury in mammals.

[0072] Example 8. Materials and Methods animal Mice were housed and maintained in accordance with the recommendations set out in the National Institutes of Health's Guide for the Care and Use of Laboratory Animals. All animal protocols were approved by the Baylor College of Medicine Institutional Animal Care and Use Committee (IACUC). Male and female mice were used for all experiments except for adult mouse MI surgery, in which all mice were male. Mice were maintained on an FVB or ICR background. All control animals were littermates or age-matched if littermates were unavailable.

[0073] Surgical procedure To induce myocardial infarction in 8-10 week-old mice, the left anterior descending artery was permanently ligated as previously described. Briefly, mice were anesthetized with 2% isoflurane and then intubated. The heart was exposed by performing a thoracotomy through the fourth or fifth intercostal space. An 8-0 nylon suture was tied around the left anterior descending artery. The clinical definition of heart failure is a 20% reduction in left ventricular ejection fraction (i.e., an ejection fraction of >50% to <40% in humans) as demonstrated by echocardiography.

[0074] To induce myocardial infarction in P8 postnatal mouse hearts, the left anterior descending artery was permanently ligated as previously described. Briefly, mice were anesthetized with hypothermia, the heart was exposed via thoracotomy through the fourth or fifth intercostal space, and an 8-0 nylon suture was tied around the left anterior descending coronary artery.

[0075] AAV Constructs containing GFP, YAP5SA, or YAP6SA gene sequences were cloned into the pENN.AAV.cTNT vector, transcribed under the cTnT promoter. All vectors were packaged in the myotrophic serotype AAV9 by the Intellectual and Developmental Disabilities Research Center Neuroconnectivity Core at Baylor College of Medicine. After titration, the virus was aliquoted, immediately frozen, and placed at -80°C for long-term storage. Each aliquot was diluted to a volume of 50 μl in saline for subcutaneous injection into postnatal mice. A total of 1 × 10 virions were administered to each mouse. 11 Each aliquot was diluted in saline to a volume of 100 ul for retro-orbital injection into adult MI mouse models. A total of 3 x 10 viral genomes were delivered to each mouse. 11 The viral genome was delivered.

[0076] Co-immunoprecipitation Mouse whole hearts were homogenized and lysed using RIPA lysis buffer [10 mM Tris-Cl, pH 8.0, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 1x protease inhibitor cocktail, and 1x phosphatase inhibitor (Roche)]. The lysates were centrifuged at 12,000 rpm for 20 minutes, and the supernatants were collected for immunoprecipitation. YAP5SA and YAP6SA and their interacting proteins were purified using anti-FLAG M2 magnetic beads (Sigma) for 4 hours of rotational incubation at 4°C. The beads were washed three times for 10 minutes each using RIPA lysis buffer and boiled for 10 minutes in elution buffer (four times loading:RIPA=1:3). The antibodies used for immunoblotting in this context were rabbit anti-YAP (1:2000), Novus Biologicals catalog number NB110-583538, rabbit anti-TEAD (D3F7L) (1:1000), Cell Signaling Technology catalog number 13295, and rabbit anti-MUPP1 / MPDZ (1:1000), Invitrogen catalog number 42-2700.

[0077] Western blotting Western blotting was performed using standard methods with lysates prepared by homogenizing hearts in RIPA buffer. After boiling for 5 minutes in reducing Tris-based sodium dodecyl sulfate (SDS) sample buffer, the lysates were loaded onto an acrylamide gel and run at 120 volts for sufficient time to achieve separation. Proteins were then transferred to PVDF membranes and imaged using an Amersham Imager 680 system (GE Healthcare). Primary antibodies were as follows: rabbit anti-YAP (1:1000), Novus Biologicals, catalog number NB110-583538; rabbit anti-DYKDDDDK tag (D6W5B) (1:2000), Cell Signaling Technology, catalog number 14973; rabbit anti-GAPDH (1:3000), Abcam; and mouse anti-β-actin (C4) (1:3000), Santa Cruz Biotechnology, catalog number sc-47778. The HRP-conjugated secondary antibodies were goat anti-rabbit IgG (H+L) and goat anti-mouse IgG (H+L) (1:5000), Jackson ImmunoResearch catalog number 111-035-003. Quantification was performed using the gel analysis feature in Fiji (ImageJ) (National Institutes of Health, Bethesda, MD, USA).

[0078] Ultrasound echocardiography M-mode and B-mode parasternal echocardiography of the left ventricle was performed according to protocols established at the Baylor College of Medicine Mouse Phenotyping Core using an MS550S transducer operating at 40 MHz on a VisualSonics Vevo 2100 system and analyzed using Vevolab5.7 software (Fujifilm VisualSonics).

[0079] Histology and immunofluorescence Freshly dissected hearts were imaged for GFP fluorescence using a Zeiss LSM 780 confocal microscope. For fixation, hearts were retrogradely perfused with cardioplegic 20 mM KCl-PBS, then perfused with 10% neutral buffered formalin and subsequently embedded in paraffin. Cross sections (7 microns) were cut and mounted on charged polylysine slides. Sections were stained with Masson's trichrome or H&E stain. Immunohistochemistry was performed by first deparaffinizing and rehydrating the sections, followed by antigen retrieval and permeabilization in 0.5% Triton-X in PBS. Sections were blocked (10% donkey serum, 0.1% Triton-X in phosphate-buffered saline [PBS]) and then incubated with primary antibodies overnight at 4°C and secondary antibodies for 1 hour at room temperature before imaging. (Figure 2A: anti-PCNA-Alexa-488, Santa Cruz Biotechnology catalog number sc-56; mouse anti-cTnT-Alexa-647 conjugate, BD Pharmingen catalog number 565744; Figure 2B: rat anti-pHH3, Abcam catalog number ab10543; anti-rat Alexa 488, Thermo Fisher Scientific catalog number A-21208; Figure 2C: rabbit anti-Aurora B, Abcam catalog number ab2254; anti-rabbit Alexa 488, Thermo Fisher Scientific catalog number A-21206.) Nuclei were stained with DAPI (4',6-diamidino-2-phenylindole) (Thermo Fisher Scientific catalog number 62248). Rhodamine-conjugated WGA was from Vector labs catalog number RL-1022. All imaging was performed on a Zeiss LSM 780 confocal microscope at the Optical Imaging and Vital Microscopy Core at Baylor College of Medicine (Houston, TX, USA).

[0080] For frozen sections (Figures 1B and 1E), hearts were dehydrated in 15% and then 30% sucrose-PBS solutions, then placed in Tissue-Tek optical cutting temperature (OCT) compound (VWR catalog no. 25608-930) and frozen on dry ice. Sections (10 microns) were cut and mounted on glass slides. For immunofluorescence staining, sections were fixed, permeabilized, and then incubated with primary and secondary antibodies before imaging. (Figure 1E: Rabbit DYKDDDDK tag antibody, Cell signaling technology catalog no. 14793; mouse anti-cTnT-Alexa-647 conjugate, BD Pharmingen catalog no. 565744.) Rhodamine-conjugated WGA was from Vector Labs catalog no. RL-1022. Nuclei were stained with DAPI. All imaging was performed on a Zeiss LSM 780 confocal microscope at the Optical Imaging and Vital Microscopy Core at Baylor College of Medicine.

[0081] Nuclei isolation for sequencing Nuclei isolation was performed as previously described. Briefly, fresh cardiac tissue was harvested on ice and immediately cut into fine pieces, followed by Dounce homogenization in NP40 lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl, 0.1% NP-40, 1 mM DTT, and RNase inhibitor). The homogenized solution was filtered, and the homogenate was mixed 1:1 with 50% iodoxinal (5 volumes of Optiprep [Sigma-Aldrich, catalog no. D1556] containing 1 volume of diluent [20 mM MgCl; 60 mM Tris-Cl pH 7.4; 50 mM NaCl; 6% BSA; 6 mM DTT, and RNase inhibitor]). Nuclei were isolated via density gradient centrifugation using Optiprep density gradient medium. After centrifugation at 10,000 g for 12 minutes, all nuclei isolated from the 30%–40% interface were precleared with Protein-G Dynabeads (Thermo Fisher Scientific, catalog no. 10003D). Nuclei were then immunoprecipitated with anti-PCM1 (Sigma-Aldrich, catalog no. HPA023370) antibody and Protein-G Dynabeads (washed twice with wash buffer [10 mM Tris-HCl pH 7.4; 10 mM NaCl; 3 mM MgCl2; 1% BSA; 0.1% Tween-20; 1 mM DTT, and RNase inhibitor]) to enrich for CM nuclei as described above.

[0082] RNA sequencing RNA was collected from bead-bound PCM-1(+) nuclei using the RNEasy Plus Micro kit (Qiagen, Hilden, Germany). Nuclear RNA sequencing (RNA-seq) libraries were constructed using a stranded RNA-seq kit (Kapa Biosystems Inc.) containing Ribo-Erase with custom Y-shaped adapters (Kapa Biosystems Inc.). Paired-end 2 × 75 bp sequencing was performed on the RNA-seq libraries using an Illumina Nextseq instrument (DNA Link). Reads were first mapped to the mouse genome (mm10) using STAR (Dobin et al., 2013). Differential expression analysis was then performed using DESeq2 (Love et al., 2014). Gene ontology analysis was performed using Metascape (Tripathi et al., 2015) and displayed using GOplot (Walter et al., 2015). Gene set enrichment analysis using publicly available data ( Uosaki et al., 2015 ) was performed by examining the top 200 most enriched transcripts in either the embryonic (E12–14) versus adult heart or the embryonic versus adult heart against our RNA-seq dataset (enrichment scores compared to control) ( Mootha et al., 2003 ; Subramanian et al., 2005 ; Uosaki et al., 2015 ).

[0083] In vivo drug treatment For in vivo treatment experiments, ROCK inhibitor Y-27632 (StemCell Technologies, Inc. 72304) was diluted in mineral oil (Sigma, lot number MKCH0156) and administered at a dose of 10 μg / g via subcutaneous injection to P9 mice. Mouse hearts were collected on day 2 after drug treatment and analyzed via pHH3 immunofluorescence followed by quantification.

[0084] Scar quantification Scar quantification was performed as previously described. Briefly, quantification was based on Masson's trichrome staining performed on representative serial cardiac sections throughout the heart. In each section, the percentage of scar (blue) was expressed as the angle (degrees, out of 360°) of fibrous tissue measured using a protractor (using the center of the heart as the center). The percentage of scarring was then averaged across all sections of the heart, spanning from the beginning to the apex of the ventricle.

[0085] Quantification and statistical analysis Statistical tests, error bars, P values, and n numbers are reported in the corresponding figure legends. Sample sizes were not predetermined but were selected based on previous publications. A pre-determined decision was made to exclude mice only if they had obvious anatomical or health abnormalities before the experimental procedure. To address randomness, any available (mutant or control) mice were included in the study. Control mice were AAV9-GFP-infected mice, AAV9-rtTA-infected mice, or mice not infected with the AAV9 virus, as indicated in the figure legends. Controls were littermates or age-matched with the experimental mice. In the reported experiments, no differences in variance were detected between any groups. One-way, two-tailed analysis of variance (ANOVA) followed by post-hoc tests was calculated using Origin Pro (OriginLab Corporation). Fisher's exact test, chi-square test, and Mantel-Cox test were performed using Prism 5 (GraphPad). All graphs were generated in R or Microsoft Excel and presented using Prism. Cartoons were created using Biorender.

[0086] References [1] Galvan, FR, V. Barranco, JCGalvan, S. Batlle, Sebastian FeliuFajardo, and Garcia, “epidemiology of myocardial infarction,” Intech, vol.i, no.tourism, p.13, 2016. [2]K.Thygesen,J.S.Alpert,and H.D.White,“Universal definition of myocardial infarction,”Circulation,vol.116,no.22,pp.2634-2653,2007. [3]K.D.Poss,L.G.Wilson,and M.T.Keating,“Heart regeneration in zebrafish,”Science(80-.).,vol.298,no.5601,pp.2188-2190,2002. [4]A.Abbate,R.Bussani,M.S.Amin,G.W.Vetrovec,and A.Baldi,“Acute myocardial infarction and heart failure:Role of apoptosis,”Int.J.Biochem.Cell Biol.,vol.38,no.11,pp.1834-1840,2006. [5]J.H.Van Berlo andJ.D.Molkentin,“An emerging consensus on cardiac regeneration,”Nat.Med.,vol.20,no.12,pp.1386-1393,2014. [6]M.Dobaczewski,C.Gonzalez-Quesada,and N.G.Frangogiannis,“The extracellular matrix as a modulator of the inflammatory and reparative response following myocardial infarction,”J.Mol.Cell.Cardiol.,vol.48,no.3,pp.504-511,2010. [7]E.R.Porrello et al.,“Transient regenerative potential of the neonatal mouse heart,”Science(80-.).,vol.331,no.6020,pp.1078-1080,2011. [8]F.X.Yu and K.L.Guan,“The Hippo pathway:Regulators and regulations,”Genes Dev.,vol.27,no.4,pp.355-371,2013. [9]Z.Meng,T.Moroishi,and K.L.Guan,“Mechanisms of Hippo pathway regulation,”Genes Dev.,vol.30,no.1,pp.1-17,2016.

[10] J.Wang,S.Liu,T.Heallen,andJ.F.Martin,“The Hippo pathway in the heart:pivotal roles in development,disease,and regeneration,”Nat.Rev.Cardiol.,vol.15,no.11,pp.672-684,2018.

[11] T.Heallen et al.,“Hippo pathway deficiency reverses systolic heart failure after infarction,”2017.

[12] Z.Lin et al.,“Cardiac-specific YAP activation improves cardiac function and survival in an experimental murine MI model,”Circ.Res.,vol.115,no.3,pp.354-363,2014.

[13] M.Xin et al.,“Hippo pathway effector Yap promotes cardiac regeneration,”Proc.Natl.Acad.Sci.U.S.A.,vol.110,no.34,pp.13839-13844,2013.

[14] A.Von Gise et al.,“YAP1,the nuclear target of Hippo signaling,stimulates heart growth through cardiomyocyte proliferation but not hypertrophy,”Proc.Natl.Acad.Sci.U.S.A.,vol.109,no.7,pp.2394-2399,2012.

[15] T.O.Monroe et al.,“YAP Partially Reprograms Chromatin Accessibility to Directly Induce Adult Cardiogenesis In Vivo,”Dev.Cell,vol.48,no.6,pp.765-779.e7,2019.

[16] Z.Li et al.,“Structural insights into the YAP and TEAD complex,”Genes Dev.,vol.24,no.3,pp.235-240,2010.

[17] R.Forough,C.Scarcello,and M.Perkins,“Cardiac biomarkers:A focus on cardiac regeneration,”J.Tehran Univ.Hear.Cent.,vol.6,no.4,pp.179-186,2011.

[18] P.Kurki,M.Vanderlaan,F.Dolbeare,J.Gray,and E.M.Tan,“Expression of proliferating cell nuclear antigen(PCNA) / cyclin during the cell cycle,”Exp.Cell Res.,vol.166,no.1,pp.209-219,1986.

[19] A.Sawicka and C.Seiser,“Histone H3 phosphorylation -A versatile chromatin modification for different occasions,”Biochimie,vol.94,no.11,pp.2193-2201,2012.

[20] J.Livet et al.,“Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system,”Nature,vol.450,no.7166,pp.56-62,2007.

[21] A.Loumaye,P.Lause,X.Zhong,T.A.Zimmers,L.B.Bindels,andJ.Thissen,“Activin A Causes Muscle Atrophy through MEF2C-Dependent Impaired Myogenesis,”2022.

[22] R.K.P.Natl,A.Sci,M.Light,and C.Isoform,“Characterization and Differential Expression of Human Vascular Smooth Muscle,”vol.2,no.1981,pp.4027-4035,1989.

[23] T.Aksel et al.,“Ensemble Force Changes that Result from Human Cardiac Myosin Mutations and a Small-Molecule Article Ensemble Force Changes that Result from Human Cardiac Myosin Mutations and a Small-Molecule Effector,”pp.910-920,2015.

[24] D.Kwon et al.,“SRF is a nonhistone methylation target of KDM2B and SET7 in the regulation of skeletal muscle differentiation,”Exp.Mol.Med.,pp.250-263,2021.

[25] Y.Yatsuka et al.,“A homozygous variant in NDUFA8 is associated with developmental delay,microcephaly,and epilepsy due to mitochondrial complex I deficiency,”Clin.Genet.,vol.98,no.2,pp.155-165,2020.

[26] X.Song et al.,“Article PDK4 dictates metabolic resistance to ferroptosis by suppressing pyruvate oxidation and fatty acid synthesis ll ll PDK4 dictates metabolic resistance to ferroptosis by suppressing pyruvate oxidation and fatty acid synthesis,”CellReports,vol.34,no.8,p.108767,2021.

[27] V.Chitu and E.R.Stanley,“Colony-stimulating factor-1 in immunity and inflammation,”vol.1,no.Il,pp.39-48,2006.

[28] T.B.Lear et al.,“degradation KIAA0317 regulates pulmonary inflammation through SOCS2 degradation,”vol.4,no.19,2019.

[29] G.S.Park et al.,“An evolutionarily conserved negative feedback mechanism in the Hippo pathway reflects functional difference between LATS1 and LATS2,”Oncotarget,vol.7,no.17,pp.24063-24075,2016.

[30] B.Zhao et al.,“Angiomotin is a novel Hippo pathway component that inhibits YAP oncoprotein,”Genes Dev.,vol.25,no.1,pp.51-63,2011.

[31] T.Matsuda et al.,“NF2 Activates Hippo Signaling and Promotes Ischemia / Reperfusion Injury in the Heart,”Circ.Res.,vol.119,no.5,pp.596-606,2016.

[32] X.Yu andJ.Chen,“DNA Damage-Induced Cell Cycle Checkpoint Control Requires CtIP,a Phosphorylation-Dependent Binding Partner of BRCA1 C-Terminal Domains,”Mol.Cell.Biol.,vol.24,no.21,pp.9478-9486,2004.

[33] J.Li,Y.Li,F.Xu,B.Sun,L.Yang,and H.Wang,“Deubiquitinating enzyme PSMD14 facilitates gastric carcinogenesis through stabilizing PTBP1,”Exp.Cell Res.,vol.415,no.2,2022.

[34] Y.Ma et al.,“LACTB suppresses melanoma progression by attenuating PP1A and YAP interaction,”Cancer Lett.,vol.506,no.639,pp.67-82,2021.

[35] M.K.Tran,K.Kurakula,D.S.Koenis,and C.J.M.de Vries,“Protein-protein interactions of the LIM-only protein FHL2 and functional implication of the interactions relevant in cardiovascular disease,”Biochim.Biophys.Acta-Mol.Cell Res.,vol.1863,no.2,pp.219-228,2016.

[36] B.E.Christian and L.L.Spremulli,“Mechanism of protein biosynthesis in mammalian mitochondria,”Biochim.Biophys.Acta-Gene Regul.Mech.,vol.1819,no.9-10,pp.1035-1054,2012.

[37] K.Takahashi et al.,“Heterogenous nuclear ribonucleoprotein H1 promotes colorectal cancer progression through the stabilization of mRNA of sphingosine-1-phosphate lyase 1,”Int.J.Mol.Sci.,vol.21,no.12,pp.1-17,2020.

[38] Y.Cho,T.Ideue,M.Nagayama,N.Araki,and T.Tani,“RBMX is a component of the centromere noncoding RNP complex involved in cohesion regulation,”Genes to Cells,vol.23,no.3,pp.172-184,2018.

[39] G.Palacios et al.,“Novel ALK fusion in anaplastic large cell lymphoma involving EEF1G,a subunit of the eukaryotic elongation factor-1 complex,”Leukemia,vol.31,no.3,pp.743-747,2017.

[40] X.Jing et al.,“Cell-cycle-dependent phosphorylation of PrPS1 fuels nucleotide synthesis and promotes tumorigenesis,”Cancer Res.,vol.79,no.18,pp.4650-4664,2019.

[41] S.Deng et al.,“Overexpression of COX7A2 is associated with a good prognosis in patients with glioma,”J.Neurooncol.,vol.136,no.1,pp.41-50,2018.

[42] T.Taira,Y.Saito,T.Niki,S.M.M.Iguchi-Ariga,K.Takahashi,and H.Ariga,“DJ-1 has a role in antioxidative stress to prevent cell death,”EMBO Rep.,vol.5,no.2,pp.213-218,2004.

[43] E.Assemat,E.Crost,M.Ponserre,J.Wijnholds,A.Le Bivic,and D.Massey-Harroche,“The multi-PDZ domain protein-1(MUPP-1)expression regulates cellular levels of the PALS-1 / PATJ polarity complex,”Exp.Cell Res.,vol.319,no.17,pp.2514-2525,2013.

[44] A.Kantardzhieva,S.Alexeeva,I.Versteeg,andJ.Wijnholds,“MPP3 is recruited to the MPP5 protein scaffold at the retinal outer limiting membrane,”FEBS J.,vol.273,no.6,pp.1152-1165,2006.

[45] D.Yu et al.,“Myristoylated alanine-rich protein kinase substrate(MARCKS)regulates small GTPase rac1 and cdc42 activity and is a critical mediator of vascular smooth muscle cell migration in intimal hyperplasia formation,”J.Am.Heart Assoc.,vol.4,no.10,pp.1-18,2015.

[46] J.Huelsken,W.Birchmeier,andJ.Behrens,“E-cadherin and APC compete for the interaction with β-catenin and the cytoskeleton,”J.Cell Biol.,vol.127,no.6 II,pp.2061-2069,1994.

[47] H.Hirata,H.Tatsumi,and M.Sokabe,“Mechanical forces facilitate actin polymerization at focal adhesions in a zyxin-dependent manner,”J.Cell Sci.,vol.121,no.17,pp.2795-2804,2008.

[48] C.Ullmer,K.Schmuck,A.Figge,and H.Luebbert,“Cloning and characterization of MUPP1,a novel PDZ domain protein,”FEBS Lett.,vol.424,no.1-2,pp.63-68,1998.

[49] M.Ernkvist et al.,“The Amot / Patj / Syx signaling complex spatially controls RhoA GTPase activity in migrating endothelial cells,”Blood,vol.113,no.1,pp.244-253,2009.

[50] M.A.Estevez et al.,“The neuronal RhoA GEF,Tech,interacts with the synaptic multi-PDZ-domain-containing protein,MUPP1,”J.Neurochem.,vol.106,no.3,pp.1287-1297,2008.

[51] N.Mosaddeghzadeh and M.R.Ahmadian,“The rho family gtpases:Mechanisms of regulation and signaling,”Cells,vol.10,no.7,2021.

[52] A.Piekny,M.Werner,and M.Glotzer,“Cytokinesis:Welcome to the Rho zone,”Trends Cell Biol.,vol.15,no.12,pp.651-658,2005.

[53] O. Kanisicak et al., “Genetic lineage tracing defines myofibroblast origin and function in the injured heart,” Nat.Commun., vol.7, 2016.

[54] T. Heallen et al., “Hippo signaling impedes adult heart regeneration,” Dev., vol. 140, no. 23, pp. 4683-4690, 2013.

[55] R. Gilsbach et al., “Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and disease,” Nat.Commun., vol. 5, no. May, 2014.

[56] A. Aharonov et al., “ERBB2 drives YAP activation and EMT-like processes during cardiac regeneration,” Nat. Cell Biol., vol. 22, no. 11, pp. 1346-1356, 2020.

[0087] The foregoing description of specific embodiments fully reveals the general nature of the present invention such that others, applying knowledge within the purview of those skilled in the art, may readily modify and / or adapt such specific embodiments for various applications without undue experimentation and without departing from the general concept of the present invention. Accordingly, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology used herein is for purposes of description and not of limitation, as would be understood by one of ordinary skill in the art in light of the teaching and guidance. The present invention is further described by the following claims.

Claims

1. A Yes-associated protein (YAP) variant comprising SEQ ID NO:

3.

2. A nucleic acid encoding the YAP mutant of claim 1.

3. A vector comprising the nucleic acid of claim 2.

4. The vector of claim 3 , wherein the nucleic acid is operably linked to a cell-specific or tissue-specific promoter.

5. The vector of claim 4 , wherein the tissue-specific promoter is a cardiomyocyte-specific promoter.

6. The vector of claim 3 , which is a viral vector.

7. The vector of claim 6 , wherein the vector is an adeno-associated virus (AAV) vector.

8. A composition comprising (i) the vector of claim 3 and (ii) a carrier.

9. The composition of claim 8 which is a pharmaceutical composition.

10. 10. The composition of claim 9 for use in a method for regenerating cardiomyocytes in an individual in need thereof.

11. 10. The composition of claim 9 for use in a method for treating myocardial infarction in an individual.

12. 10. A method for regenerating cardiomyocytes in a subject in need thereof, the method comprising delivering the composition of claim 9 to cardiac tissue of the subject.

13. 10. A method of treating myocardial infarction (MI) in a subject, the method comprising delivering the composition of claim 9 to cardiac tissue of the subject.

14. The method of claim 12 or 13, wherein the composition comprises the vector of claim 5.

15. 15. The method of claim 14, wherein the vector is an adeno-associated virus vector.

16. 13. The method of claim 12, wherein the subject has arrhythmia, cardiomyopathy, heart failure, myocardial fibrosis, or myocarditis.

17. 13. The method of claim 12, wherein the subject has experienced an MI.

18. The method of any one of claims 12 to 17, wherein the composition is administered to the subject two or more times.

19. 1. A method for regenerating cardiomyocytes in a subject in need thereof, the method comprising delivering to cardiac tissue of the subject an AAV vector comprising the nucleic acid encoding the YAP mutant of claim 1 operably linked to a cardiomyocyte-specific promoter.

20. 10. A method for treating MI in a subject, comprising delivering to cardiac tissue of the subject an AAV vector comprising the nucleic acid encoding the YAP mutant of claim 1 operably linked to a cardiomyocyte-specific promoter.

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