Truncated P63 protein domains for enhancing human cardiac reprogramming

Abstract

The embodiments of the present disclosure include the method and composition for in situ cardiac cell regeneration, comprising transdifferentiation of cardiac cell into cardiomyocyte.In a specific embodiment, the in situ cardiac cell regeneration comprises delivery of p63-TID and one or both of Hand2 and myocardin, and in a specific embodiment, further comprises one or more of Gata4, Mef2c, and Tbx5, and / or one or more of ETV2 and VEGF.In a specific aspect of the present disclosure, adult cardiac fibroblasts are reprogrammed into cardiomyocyte using a viral vector with p63-TID and one or both of transcription factors Hand2 and myocardin.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 228,671, filed August 3, 2021, which is incorporated by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with Government support under HL152280 awarded by the National Institutes of Health. The Government has certain rights in this invention. Sequence Listing

[0003] This application contains a Sequence Listing that has been submitted electronically in XML format, which is incorporated by reference in its entirety. Said XML copy, created on August 1, 2022, is entitled BAYM_P0344WO_SequenceListing and is 51,424 bytes in size.

[0004] Embodiments of the present disclosure include at least the fields of molecular biology, cell biology, cell therapy, and medicine, including cardiac medicine. [Background technology]

[0005] Despite technological innovations in medicine and surgical procedures, heart disease remains the leading cause of death worldwide. Considering the poor regenerative capacity of the heart after myocardial infarction and the irreversible loss of cardiomyocytes, replacement of cardiomyocytes by forming induced pluripotent stem cells or stimulating direct cellular reprogramming is a promising therapeutic strategy with great promise. Both techniques are based on the idea that endogenous fibroblasts in the infarcted myocardium can be reprogrammed into functional cardiomyocytes. Several institutions have reported that fibroblasts can be reprogrammed into cardiomyocytes in vitro using a cocktail of transcription factors, most notably Gata4, Mef2c, and Tbx5. In any case, the main obstacle in the implementation of this therapy is the low reprogramming efficiency.

[0006] The present disclosure provides a solution to a long-standing need in the art for efficient and effective repair of cardiac tissue. Summary of the Invention [Means for solving the problem]

[0007] The embodiments of the present disclosure include methods and compositions for the treatment of any medical condition related to the mammalian heart. In certain embodiments, the present disclosure relates to the treatment of one or more cardiac medical conditions with a therapeutic composition that affects endogenous cells or tissues in the heart. In specific embodiments, the therapy is provided to an individual in need thereof, for example, when the individual is in need of in situ or in vivo treatment of endogenous cardiac tissue due to a cardiac medical condition or risk thereof. In certain embodiments, the individual has cardiac cell or tissue damage due to a cardiac medical condition.

[0008] In specific embodiments, delivery of certain compositions to cells in an individual in situ or in vivo allows for reprogramming of non-endogenous cardiomyocytes to become cardiomyocytes, thereby allowing for regeneration of cardiomyocytes. When a therapeutically effective amount of one or more compositions is delivered to an individual, the composition results in amelioration of the condition, at least in part, by allowing for regeneration of cardiac tissue or cells therein. In certain embodiments, the composition comprises one or more p63-transactivation inhibitory domain (p63-TID) polypeptides (or functional fragments and / or functional derivatives thereof), and / or nucleic acids encoding same, and / or other factors that will enhance the reprogrammability or plasticity of the target cells when down-regulated. In certain embodiments, the individual may also be provided with one or more cardiac cell reprogramming factors (which may or may not be transcription factors), which may or may not be provided simultaneously or in the same composition as the p63-TID. In specific embodiments, the composition comprises Hand2, myocardin, or both. In certain embodiments, the individual may also be provided with one or more chromatin destabilizing agents. In a specific embodiment, the composition comprises p63-TID, Hand2, myocardin, VEGF, and / or ETV2. In a specific embodiment, VEGF and / or ETV2 are provided to the cells prior to p63-TID, Hand2, and / or myocardin. In a specific embodiment, VEGF and / or ETV2 are provided simultaneously with p63-TID, Hand2, and / or myocardin. In certain embodiments, VEGF and / or ETV2 function synergistically with p63-TID, Hand2, and / or myocardin for cardiac cell reprogramming.

[0009] In specific embodiments of the present disclosure, p63-TID (or a functional fragment and / or derivative thereof) increases the efficiency of transdifferentiation of cardiac cells (e.g., fibroblasts) into cardiomyocytes. As described herein, provision of p63-TID (or a functional fragment and / or derivative thereof) is a novel therapeutic intervention that allows for highly efficient reprogramming of fibroblasts (for example) into cardiomyocytes. Provision of p63-TID (or a functional fragment and / or derivative thereof) is a unique therapeutic intervention that is a clinically relevant therapy for the treatment of any cardiac medical condition, including heart failure.

[0010] In certain embodiments, any p63-TID (or a functional fragment and / or derivative thereof), and / or one or more cardiac cell reprogramming factors, and / or one or more chromatin destabilizing agents, and / or one or more anti-fibrotic agents, and / or one or more angiogenic factors act synergistically with each other and are provided to an individual in need thereof, regardless of whether they are present in the same composition as the p63-TID or whether they are provided simultaneously with the p63-TID.

[0011] In certain embodiments, an individual in need thereof receives one or more anti-fibrotic agents, for example, one or more anti-Snail agents (eg, siRNA, antibodies, small molecules, such as ITD-1, etc.).

[0012] In some embodiments, there is a method of reprogramming cardiac cells in vivo, comprising providing a therapeutically effective amount of one or more compositions to the heart of an individual, wherein the one or more compositions comprise p63-TID (or a functional fragment and / or derivative thereof).

[0013] In an embodiment of the present disclosure, the method includes providing an effective amount of one or more cardiac cell reprogramming factors to an individual, which may be a polypeptide, a peptide, a nucleic acid, or a mixture thereof.In certain embodiments, the one or more cardiac cell reprogramming factors are Hand2, myocardin, Gata4, Mef2c, Tbx5, Mesoderm posterior protein 1 (Mesp1), miR-133, miR-1, Oct4, Klf4, c-myc, Sox2, Brachyury, Nkx2.5, ets variant 2 (ETS2, also referred to as ETV2), VEGF, ESRRG, Mrtf-A, MyoD, ZFPM2, miR-590, miR-208, miR-499, or a combination thereof.In certain embodiments, the one or more cardiac cell reprogramming factors are one or both of Hand2 nucleic acid or polypeptide and myocardin nucleic acid or polypeptide. In some cases, one or both of the Hand2 nucleic acid or polypeptide and the myocardin nucleic acid or polypeptide are in the same or different composition as the p63-TID (or a functional fragment and / or a functional derivative thereof). In certain embodiments, one or more compositions comprise a p63-TID (or a functional fragment and / or a functional derivative thereof) and a Hand2 nucleic acid, a p63-TID (or a functional fragment and / or a functional derivative thereof) and a myocardin nucleic acid, and / or a p63-TID (or a functional fragment and / or a functional derivative thereof), a Hand2, and a myocardin nucleic acid.

[0014] In certain embodiments, p63-TID (or a functional fragment and / or derivative thereof) is provided prior to the one or more cardiac cell reprogramming factors. In specific embodiments of the method, an effective amount of one or more chromatin destabilizing agents is provided to the individual. In specific embodiments, the one or more chromatin destabilizing agents are provided to the individual prior to p63-TID (or a functional fragment and / or derivative thereof) being provided to the individual. In some embodiments, the one or more chromatin destabilizing agents are provided to the individual prior to p63-TID (or a functional fragment and / or derivative thereof) being provided to the individual, and p63-TID (or a functional fragment and / or derivative thereof) being provided to the individual prior to the one or more cardiac cell reprogramming factors being provided to the individual.

[0015] In certain embodiments, the cardiac cells are fibroblasts, endothelial cells, myoblasts, progenitor cells, stem cells, myofibroblasts, or combinations thereof. Cardiac cells can be dividing or non-dividing cells.

[0016] In a specific embodiment, p63-TID (or a functional fragment and / or derivative thereof), comprises a nucleic acid, said nucleic acid being comprised on one or more vectors. One or more cardiac cell reprogramming factors may comprise a nucleic acid, said nucleic acid being comprised on one or more vectors. In a specific embodiment, one or more chromatin destabilizing agents comprise a nucleic acid, said nucleic acid being comprised on one or more vectors. In some embodiments, the nucleic acid is comprised on a separate vector or on the same vector. In certain cases, the vector is a viral vector or a non-viral vector, such as a nanoparticle, a plasmid, a liposome, or a combination thereof. In a specific embodiment, the viral vector is an adenovirus, a lentivirus, a retrovirus, an adeno-associated virus vector, or an episomal (non-integrating) vector. In a specific embodiment, p63-TID (or a functional fragment and / or derivative thereof), Hand2, and / or myocardin nucleic acid is comprised on a lentivirus vector, or is comprised on an adenovirus vector, or is a modified mRNA molecule. In any of the vectors encompassed by this disclosure, a cell-specific promoter, for example a fibroblast-specific promoter, may be present.

[0017] In certain embodiments, any method encompassed by the present disclosure includes a step of delivering to the individual an additional cardiac therapy, including, for example, drug therapy, surgery, ventricular assist device (VAD) implantation, ventricular endoscopy (VAT) coronary artery bypass, percutaneous coronary intervention (PCI), or a combination thereof.

[0018] Any of the compositions encompassed by this disclosure may be provided to an individual by a suitable delivery route, including systemic or local delivery, hi certain embodiments, the delivery is local to the heart, and in certain embodiments, the providing step is further defined as injecting the compound into the heart.

[0019] In certain embodiments, there is a composition comprising one or more nucleic acid vectors, the vector comprising p63-TID (or a functional fragment and / or derivative thereof) and comprising one or more cardiac cell reprogramming factors. In some cases, the vector comprising p63-TID (or a functional fragment and / or derivative thereof) is the same vector as the one comprising one or more cardiac cell reprogramming factors. In certain embodiments, the vector comprising p63-TID (or a functional fragment and / or derivative thereof) is a different vector than the vector comprising one or more cardiac cell reprogramming factors. In certain embodiments, the vector further comprises one or more chromatin destabilizing agents. The vector comprising p63-TID (or a functional fragment and / or derivative thereof) may be the same vector as the one comprising one or more chromatin destabilizing agents. The vector comprising p63-TID (or a functional fragment and / or derivative thereof) and comprising one or more cardiac cell reprogramming factors may be the same vector as the one comprising one or more chromatin destabilizing agents. The vector comprising p63-TID (or a functional fragment and / or derivative thereof) and one or more cardiac cell reprogramming factors may be a different vector from the one comprising one or more chromatin destabilizing agents. The composition comprising the vector may comprise p63-TID (or a functional fragment and / or derivative thereof) and one or both of Hand 2 and myocardin nucleic acids. In certain embodiments, the composition comprising the vector comprises p63-TID (or a functional fragment and / or derivative thereof) and Hand 2 nucleic acid. In some embodiments, the composition comprising the vector may comprise p63-TID (or a functional fragment and / or derivative thereof) and myocardin nucleic acid, or may comprise p63-TID (or a functional fragment and / or derivative thereof), Hand 2, and myocardin nucleic acid.In certain embodiments, any composition of the present disclosure may include one or more anti-fibrotic agents.

[0020] In some embodiments, there is a kit comprising a composition encompassed by the present disclosure, said composition being contained in a suitable container.

[0021] In certain embodiments, a method of treating a cardiac condition comprises providing to the heart of an individual a therapeutically effective amount of one or more compositions, wherein the one or more compositions comprise: A) a p63-transactivation inhibitory domain (p63-TID) polypeptide, and / or a functional derivative and / or functional fragment thereof, and / or a nucleotide encoding same (a p63-TID polypeptide comprises, consists essentially of, consists of, or is at least or strictly at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO:1); Provided herein are methods comprising, consisting essentially of, or consisting of A, B, C, and D. In some embodiments, the methods comprise, consist essentially of, or consist of providing A, B, C, and D. In some embodiments, the methods comprise, consist essentially of, or consist of providing A, B, C, and E. In some embodiments, D and / or E are provided on the same day as A, B, and C. In some embodiments, D and / or E are provided simultaneously with A, B, and C. In some embodiments, D and / or E are provided prior to A, B, and C.In some embodiments, D and / or E are provided at least or strictly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days, or any range derivable therein, prior to A, B, and C. In some embodiments, A, B, and C are provided prior to D and / or E. In some embodiments, A, B, and C are provided at least or strictly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days, or any range inducible therein, before D and / or E. In some embodiments, A, B, C, D, and / or E are provided in a nanoparticle, a plasmid, a liposome, a viral vector, or any combination thereof. In some embodiments, A, B, C, D, and / or E are provided in a viral vector, and the viral vector is an adenovirus, lentivirus, retrovirus, or adeno-associated viral vector. In some embodiments, the viral vector is an adenovirus vector. In certain embodiments, one or more compositions comprising A, B, C, D, and / or E are provided to cells in vitro. In certain embodiments, cells provided in vitro with A, B, C, D, and / or E are provided to an individual having a cardiac condition. In certain embodiments, cells provided in vitro with A, B, C, D, and / or E are provided directly to the heart of an individual having a cardiac condition.

[0022] The above has outlined, rather broadly, the characteristics and technical advantages of the present disclosure so that the detailed description of the disclosure that follows may be better understood. Additional characteristics and advantages of the present disclosure are described below, which form the subject matter of the claims of the present disclosure. Those skilled in the art should appreciate that the concepts and specific embodiments disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present disclosure. Those skilled in the art should also appreciate that the construction of such equivalents does not depart from the spirit and scope of the present disclosure as set forth in the appended claims. The novel features believed to be characteristic of the present disclosure, both as to its organization and method of operation, together with further objects and advantages, will be better understood from the following description when considered in conjunction with the accompanying drawings. However, each of the drawings is provided for the purpose of illustration and description only, and is not to be construed as a definition of the limits of the present disclosure.

[0023] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error of the measuring or quantification method.

[0024] When used in conjunction with the term "comprising," the use of the words "a" or "an" can mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0025] The term "and / or" means "and" or "or." By way of example, A, B, and / or C includes A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, "and / or" functions as an inclusive or.

[0026] "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") is inclusive or expansive and does not exclude additional, unrecited elements or method steps.

[0027] The compositions and methods for their use may "comprise," "consist essentially of," or "consist" of any of the components or steps disclosed throughout this specification. Compositions and methods "consist essentially of" any of the disclosed components or steps limit the scope of the claims to the specified materials or steps that do not materially affect the basic and novel characteristics of the claimed invention.

[0028] It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the invention, and vice versa. Further, a composition of the invention can be used to achieve a method of the invention.

[0029] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that this detailed description and the specific examples, while indicating particular embodiments of the present invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from the detailed description.

[0030] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief description of the drawings]

[0031] [Figure 1A-B] Figure 1 shows that p63 isoform ΔNp63α and histone deacetylase 1 (HDAC1) physically interact with each other, and p63-TID disrupts the binding between them. Figure 1A shows that ΔNp63α-FLAG and HDAC1-GFP were overexpressed (OE) in immortalized rat cardiac fibroblasts. Co-IP using anti-FLAG antibody was performed, followed by immunoblotting with antibodies against HDAC1 and p63. Figure 1B shows that overexpression of p63-TID disrupts p63 / HDAC1 interaction. HDAC1-GFP, ΔNP63α FLAG were co-expressed in human 293T cells with or without TID vector, and Co-IP was performed by using the indicated antibodies.

[0032] [Figure 2A] Figures 2A-J show the experimental design and results for differentiation of human cardiac fibroblasts. Figure 2A shows a schematic of the experimental design to determine the effect of p63-TID on iCM reprogramming in human cardiac fibroblasts (real-time quantitative reverse transcription PCR (qRT-PCR), immunofluorescence (IF), fluorescence-activated cell sorting (FACS). [Figure 2B] Figure 2B shows FACS data showing the percentage of cardiac troponin T positive (cTnT+) cells 14 days after transduction of the indicated lentiviral vectors in human cardiac fibroblasts (n=3, data presented as mean ± standard error of the mean, **p<0.01 vs. shNT, *p<0.05 vs. GMT). [Figure 2C]FIG. 2C shows the mRNA expression levels of cardiac and fibroblast marker genes in the above-mentioned treated cells assessed by qRT-PCR (n=3, error bars refer to the mean±SEM, *P<0.05 vs. GMT, **P<0.001 vs. shNT). [Figure 2D] FIG. 2D shows representative immunofluorescence staining for (DAPI) (blue), (FITC) (green), and (red) cardiomyocyte marker cTnT (bar=100 μm, images captured at 10x magnification). [Figure 2E] FIG. 2E shows representative immunofluorescence staining for (DAPI) (blue), (FITC) (green), and (red) cardiomyocyte markers, as well as α-actinin (bar=100 μm, images captured at 10x magnification). [Figure 2F] FIG. 2F shows the expression of cardiomyocyte marker genes (cTnT, Gja1, and Myh6) assessed by qRT-PCR after the indicated treatments (n=3, ****p<0.0001). [Figure 2G] FIG. 2G shows expression of fibroblast marker genes (Col1a1 and Postn) assessed by qRT-PCR after the indicated treatments (n=3, ****p<0.0001). [Figure 2H] Figure 2H shows representative flow cytometry plots of cardiac troponin T positive (cTnT+) human cardiac fibroblasts after 2 weeks of treatment with shp63 or p63-TID with or without Hand2 / myocardin (H / M) (left panel) as well as quantification of the percentage of cTnT+ cells treated as indicated, assessed by flow cytometry (right panel) (n=3, ****p<0.0001). [Figure 2I] Figure 2I shows quantification of cells expressing cardiomyocyte markers cTnT+ and α-actinin+ (e.g., imaged in Figure 2E) after 2 weeks of the indicated treatments as assessed by immunofluorescence labeling (n=3, ***p<0.001, **p<0.01). [Figure 2J]Figure 2J shows representative high magnification images of cTnT and α-actinin staining in p63-TID and H / M treated cells showing sarcomere structures, which are most clearly visible in α-actinin labeled cells. Scale bar = 25 μm (left panel) and quantification of cells with well-developed sarcomeres as a percentage of total α-actinin+ cells 4 weeks after transduction of shp63+GMT, shp63+H / M, or p63-TID+H / M (right panel) (n=3, **p<0.01, ***p<0.001).

[0033] [Diagram 3] FIG. 1 shows that p63-TID administration in rat cardiac fibroblasts resulted in better iCM reprogramming results. The mRNA expression levels of the indicated cardiac and fibroblast marker genes 2 weeks after administration of the reprogramming factors indicated above in rat cardiac fibroblasts, as assessed by qRT-PCR, are shown (n=3, all data presented as mean±standard error of the mean, *P<0.05 vs. GMT, **P<0.001 vs. shNT).

[0034] [Figure 4A] Figure 4A-B provides exemplary p63-TID sequences and exemplary construct designs of vectors containing them. Figure 4A shows an example of lentiviral vector map that contains p63-TID nucleic acid sequence (TID pp3018). Such example vectors can be used in reprogramming assays, such as qPCR, FACS, and / or immunofluorescence. [Figure 4B] FIG. 4B shows the vector utilized in the co-IP study in which the TID nucleotide sequence was cloned in the pcDNA3.1 vector backbone.

[0035] [Diagram 5]Functional efficacy of human cardiac fibroblast reprogramming after co-culture with neonatal rat cardiomyocytes. Adult human cardiac fibroblasts were treated with lentiviruses expressing GMT (left), shp63 in combination with Hand2 / myocardin (H / M) (middle), or p63-TID+Hand2 / myocardin (H / M, p63TID+H / M) (right). One week after the initial transduction, these human cardiac fibroblasts were co-cultured with (untreated) neonatal rat cardiomyocytes (GFP [green fluorescent protein] negative). The top row shows representative immunofluorescence showing (green) GFP expression by human cardiac fibroblasts treated with GMT (left), shp63+H / M (middle), or p63-TID+H / M (right) after 4 weeks of co-culture with (untransduced) neonatal rat cardiomyocytes. Bars = 100 μm. The middle and bottom panels show representative peaks reflecting contraction (top panel) and Ca2+ transients (bottom panel) from GFP+ human cardiac fibroblasts treated with GMT, shp63+H / M, and p63-TID+H / M after 4 weeks of co-culture. No contractility parameters were observed in cells treated with GMT alone. Bar=1s.

[0036] [Figure 6A] Figures 6A-D show dose-based efficacy of p63-TID in enhancing human cardiac reprogramming, comparable to shp63. Figure 6A shows FLAG co-immunoprecipitation assays in 293T cells transfected with HDAC1, p63-FLAG, and / or p63-TID vector at three different p63-TID dosages, showing increasing interference with p63-HDAC1 binding as a function of p63-TID dosage. Beta-actin was used as a loading control. IB=immunoblot, IP=immunoprecipitation. [Figure 6B]Figure 6B shows dosage screening of p63-TID in human cardiac fibroblasts, qRT-PCR analysis of cardiac troponin T (cTnT) marker 2 weeks after treatment with lentiviral vector expressing p63-TID at MOI of 20, 50, or 100 (n=3, ***p<0.001, **p<0.01). Control: lentiviral GFP vector, MOI of 20. [Figure 6C] Figure 6C shows cardiomyocyte marker gene expression in human cardiac fibroblasts assessed by qRT-PCR 2 weeks after the indicated treatments with p63-TID vector at a multiplicity of infection (MOI) of 50 (n=3, MOI of H / M=20, *p<0.05 vs. shp63+H / M). [Figure 6D] Figure 6D shows fibroblast marker gene expression in human cardiac fibroblasts assessed by qRT-PCR 2 weeks after the indicated treatments with p63-TID vector at a multiplicity of infection (MOI) of 50 (n=3, MOI of H / M=20, *p<0.05 vs. shp63+H / M).

[0037] [Figure 7A] Figures 7A-D show experimental designs and results for differentiation of human cardiac fibroblasts. Figure 7A shows the experimental design for delayed delivery (i.e., sequential and temporally distinct delivery) of adenoviral vectors encoding GFP (adGFP, control), Ets variant 2 (ETV2), or vascular endothelial growth factor (VEGF), as well as adenoviral vectors encoding GFP (GFP, control), GMT (Gata4 (GATA binding protein 4), Mef2c (myocyte enhancer factor 2c), and Tbx5 (t-box transcription factor 5)), GMTd (Gata4, Mef2c, and TEAD1 (TEA domain family member 1), or TIDH / M (p63-TID+H / M). [Figure 7B] FIG. 7B shows the relative mRNA expression of cTnT following the experimental design outlined in FIG. 7A (n=3). [Figure 7C]FIG. 7C shows the experimental design for co-delivery (i.e., single or multiple compositions are delivered during the same setting) of adenoviral vectors encoding GFP (adGFP, control), Ets variant 2 (ETV2), or vascular endothelial growth factor (VEGF), and adenoviral vectors encoding GFP (GFP, control), GMT (Gata4, Mef2c, and Tbx5), GMTd (Gata4, Mef2c, and TEAD1), or TIDH / M (p63-TID+ H / M). [Figure 7D] FIG. 7D shows the relative mRNA expression of cTnT following the experimental design outlined in FIG. 7C (n=3). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] Some embodiments of the present disclosure may consist of, or consist essentially of, one or more elements, method steps, and / or methods of the present disclosure. It is contemplated that any method or composition described herein may be implemented in conjunction with any other method or composition described herein.

[0039] The term "cardiac medical condition," as used herein, refers to any medical condition that affects cardiac tissue, including those that affect cardiac function.

[0040] The term "chromatin destabilizing agent" refers to one or more compounds that facilitate access of one or more factors to condensed genomic DNA.

[0041] The term "cardiac cell reprogramming factor," as used herein, refers to one or more compositions that enhance or promote the transdifferentiation of differentiated cells within the heart into cardiomyocytes.

[0042] The embodiments of the present disclosure include methods and compositions for treating or preventing any cardiac medical condition in which increasing the number of cardiomyocytes in the heart would be therapeutic.In certain embodiments, in vivo cells in the heart are reprogrammed to become cardiomyocytes.In specific embodiments, this is achieved at least in part by providing an effective amount of p63-TID (or its functional fragment and / or functional derivative).In specific embodiments, nucleic acids and / or peptides and / or polypeptides are delivered directly to the heart to allow non-cardiomyocyte cells in the heart to be reprogrammed to become cardiomyocytes. I. p63-TID composition

[0043] The embodiments of the present disclosure include methods and compositions related to p63-transactivation inhibitory domain (p63-TID). In a specific embodiment, p63-TID (or a fragment and / or derivative thereof) acts as a dominant-negative inhibitor of any kind of p63 activity. In a specific embodiment, p63-TID (or a fragment and / or derivative thereof) acts as a dominant-negative inhibitor of isoforms Tap63 and / or ΔNp63. In a specific embodiment, p63-TID acts to inhibit and / or silence the epigenetic action of p63 and / or enhance the activation of cardiogenic reprogramming genes. In a specific embodiment, p63-TID promotes the downregulation of fibrogenic genes known to impede cardiac differentiation. In a specific embodiment, p63-TID provides an HDAC-guided reprogramming strategy that avoids the activation and / or silencing of genes unrelated to the desired cardiac differentiation action, including but not limited to potential oncogenes. Compositions comprising p63-TID (or fragments and / or derivatives thereof) and / or polynucleotide vectors comprising nucleic acid sequences encoding same are utilized for the effective treatment of any cardiac medical condition.

[0044] Examples of p63-TID polypeptides are as follows: MTTIYQIEHYSMDDLASLKIPEQFRHAIWKGILDHRQLHEFSSPSHLLRTPSSASTVSVGSSETRGERVIDA (SEQ ID NO: 1)

[0045] An example of a nucleic acid sequence that can encode p63-TID is as follows: ATGACCACCATCTATATCAGATTGAGCATTACTCCATGGATGATCTGGCAAGTCTGAAAATCCCTGAGCAATTTCGACATGCGATCTGGAAGGGCATCCTGGACCACCGGCAGCTCCACGAATTCTCCTCCCTTCTCATCTCTGCGGACCCCAAGCAGTGCCTCTACAGTCAGTGGGCTCCAGTGAGACCCGGGGTGAGCGTGTTATTGATGCTTAA (SEQ ID NO: 2)

[0046] p63-TID may be isolated from a human cell and therefore no longer naturally occurring, or in certain embodiments, may be recombinant. As referred to herein, when the native sequence of SEQ ID NO:1 is produced by recombinant means, the resulting polypeptide may be referred to as recombinant p63-TID. Another example of recombinant p63-TID includes a label or tag. An embodiment of p63-TID includes its functional derivatives and / or functional fragments, and a derivative or fragment may be considered functional if it has the ability to enable reprogramming of a cell when the cell is exposed to the fragment, either alone or in combination with one or more cardiac cell reprogramming factors. Such activity may be measured by any suitable means, including, for example, by qPCR, flow cytometry, immunofluorescence, and / or beating assay. In certain embodiments, p63-TID or its functional fragments and / or derivatives are soluble. p63-TID or its functional fragments and / or derivatives may or may not be included in a fusion protein.

[0047] The p63-TID protein composition can be produced by any technique known to those skilled in the art, including expression of proteins, polypeptides, or peptides by standard molecular biology techniques, isolation of proteinaceous compounds from natural sources, or chemical synthesis of proteinaceous materials. The p63-TID coding region can be amplified and / or expressed using techniques disclosed herein or known to those skilled in the art. Alternatively, various commercial preparations of proteins, polypeptides, and peptides are known to those skilled in the art.

[0048] In certain embodiments, the p63-TID (or fragments and / or derivatives thereof) protein compound may be purified. In general, "purified" refers to a particular or protein, polypeptide, or peptide composition that has been subjected to a fractionation process to remove various other proteins, polypeptides, or peptides, and the composition substantially retains its activity as would be known to one of skill in the art for the particular or desired protein, polypeptide, or peptide, as may be assessed, for example, by protein assays. Biologically functional equivalents of p63-TID, including such derivatives and fragments, may be used. Since the structure of the p63-TID polynucleotides and / or proteins according to the present invention may be modified and / or changed, while obtaining molecules with similar or improved characteristics, such biologically functional equivalents are also encompassed within the present invention.

[0049] In some embodiments, p63-TID is utilized in protein form, including as SEQ ID NO: 1. In instances where a functional derivative or fragment of p63-TID is utilized, that functional derivative or fragment of p63-TID may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more amino acid changes compared to SEQ ID NO: 1. The functional derivative or fragment of p63-TID may comprise an N-terminal truncation of SEQ ID NO:1, for example, the truncation is not more than 1, not more than 2, not more than 3, not more than 4, not more than 5, not more than 6, not more than 7, not more than 8, not more than 9, not more than 10, not more than 12, not more than 15, not more than 20, not more than 25. In some embodiments, the truncation is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids. The functional derivative or fragment of p63-TID may comprise a C-terminal truncation of SEQ ID NO:1, for example, the truncation is not more than 1, not more than 2, not more than 3, not more than 4, not more than 5, not more than 6, not more than 7, not more than 8, not more than 9, not more than 10, not more than 12, not more than 15, not more than 20, Not more than 25, not more than 30, not more than 35, not more than 40, not more than 45, not more than 50, not more than 55, or not more than 60 amino acids, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids.The functional derivative or fragment of p63-TID may contain internal deletions in SEQ ID NO:1, for example, internal deletions of not more than 1, not more than 2, not more than 3, not more than 4, not more than 5, not more than 6, not more than 7, not more than 8, not more than 9, not more than 10, not more than 12, not more than 15, not more than 20, not more than 25, no more than 30, no more than 35, no more than 40, no more than 45, no more than 50, no more than 55, or no more than 60 amino acids, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids. In certain embodiments, a functional derivative or fragment of p63-TID may comprise a sequence that is at least or strictly 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO:1.

[0050] In some embodiments, the p63-TID is utilized in the form of a nucleic acid, such as SEQ ID NO: 2. In instances where a functional derivative or fragment of a p63-TID nucleic acid is utilized, that functional derivative or fragment of p63-TID may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more differences compared to SEQ ID NO: 2, which may or may not be at wobble positions. The functional derivative or fragment of p63-TID may comprise a truncation at the 5' end of SEQ ID NO:2, for example, the truncation is not more than 1, not more than 2, not more than 3, not more than 4, not more than 5, not more than 6, not more than 7, not more than 8, not more than 9, not more than 10, not more than 12, not more than 15, not more than 20, not more than 25, not more than 30, not more than 35, not more than 40, not more than 45, not more than 50 In some embodiments, the truncation is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 90, 100, 110, 125, or more nucleotides.The functional derivative or fragment of p63-TID may include a truncation of the 3' end of SEQ ID NO:2, for example, the truncation is not more than 1, not more than 2, not more than 3, not more than 4, not more than 5, not more than 6, not more than 7, not more than 8, not more than 9, not more than 10, not more than 12, not more than 15, not more than 20, not more than 25, not more than 30, not more than 35, not more than 40, not more than 45. In some embodiments, the amino acid sequence is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 90, 100, 110, 125, or more nucleotides. The functional derivative or fragment of p63-TID may contain an internal deletion in SEQ ID NO:2, for example, the internal deletion is not more than 1, not more than 2, not more than 3, not more than 4, not more than 5, not more than 6, not more than 7, not more than 8, not more than 9, not more than 10, not more than 12, not more than 15, not more than 20, not more than 25, not more than 30, not more than 35, not more than 40, not more than 45, not more than 50 In some embodiments, the amino acid sequence may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 90, 100, 110, 125, or more nucleotides.In certain embodiments, functional derivatives or fragments of p63-TID thereof may comprise a sequence that is at least or strictly 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 2. Any functional fragment and / or functional derivative will retain the biological activity of the full-length SEQ ID NO: 1 polypeptide.

[0051] In a specific embodiment, the present disclosure encompasses the use of p63-TID as a nucleic acid contained in a recombinant vector that contains a nucleic acid sequence encoding p63-TID as a polypeptide. In a particular embodiment, the recombinant vector contains a nucleic acid sequence that encodes SEQ ID NO:1, including all consecutive amino acids therein. In a specific aspect, the recombinant vector is a viral vector (e.g., lentivirus, adenovirus, adeno-associated virus, or retrovirus), or a non-viral vector (e.g., plasmid, transposon, etc.).

[0052] Biologically functional equivalents of p63-TID can be produced from polynucleotides that have been engineered to contain different sequences while still retaining the ability to code for "wild-type" or standard proteins. This can be achieved due to the degeneracy of the genetic code, i.e., the presence of multiple codons that code for the same amino acid. In one example, one skilled in the art may wish to introduce a restriction enzyme recognition sequence into a polynucleotide without destroying the ability of the polynucleotide to code for a protein.

[0053] In another example, p63-TID polynucleotides can be (can code for) biologically functional equivalents with larger changes. Certain amino acids can be substituted for other amino acids in protein structures without obvious loss of mutual binding ability with structures such as, for example, antigen-binding regions of antibodies, binding sites on substrate molecules, receptors, etc. So-called "conservative" changes do not destroy the biological activity of proteins, since structural changes do not affect the ability of proteins to perform their designed functions. Thus, it is contemplated by the inventors that various changes may be made to the gene and protein sequences disclosed herein while still meeting the objectives of the present invention.

[0054] With respect to functional equivalents, those skilled in the art will appreciate that implicit in the definition of a "biologically functional equivalent" protein and / or polynucleotide is the concept that there are limits to the number of changes that can be made within a defined portion of the molecule while retaining the molecule with an acceptable level of equivalent biological activity. Biologically functional equivalents are thus defined herein as proteins (and polynucleotides) that can be substituted at selected amino acids (or codons).

[0055] In general, the shorter the length of a molecule, the fewer changes can be made in the molecule while retaining function. Longer domains can have a moderate number of changes. Full-length proteins will have the highest tolerance for many changes. However, it should be understood that certain molecules or domains that are highly structure-dependent may tolerate little or no modification.

[0056] Amino acid substitutions are generally based on the relative similarity of amino acid side-chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and / or type of amino acid side-chain substituents reveals that arginine, lysine, and / or histidine are all positively charged residues, alanine, glycine, and / or serine are all small in size, and / or phenylalanine, tryptophan, and / or tyrosine are all generally similar in shape. Therefore, based on these considerations, arginine, lysine, and / or histidine, alanine, glycine, and / or serine, and / or phenylalanine, tryptophan, and / or tyrosine are herein defined as biologically functional equivalents.

[0057] To achieve more quantitative changes, the hydropathic index of amino acids can be considered. Each amino acid is assigned a hydropathic index based on its hydrophobic and / or charge characteristics, and these values ​​are: isoleucine (+4.5), valine (+4.2), leucine (+3.8), phenylalanine (+2.8), cysteine ​​(+2.5), methionine (+1.9), alanine (+1.8), glycine (0.4), threonine (0.7), serine (0.8), tryptophan (0.9), tyrosine (1.3), proline (1.6), histidine (3.2), glutamic acid (3.5), glutamine (3.5), aspartic acid (3.5), asparagine (3.5), lysine (3.9), and / or arginine (4.5).

[0058] The importance of hydropathic amino acid index in conferring mutual biological functions to proteins is generally understood in the art (Kyte & Doolittle, 1982, incorporated herein by reference).It is known that certain amino acids can be substituted with other amino acids having similar hydropathic index and / or score and / or still retain similar biological activity.When making changes based on hydropathic index, substitution of amino acids with hydropathic index within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly considered.

[0059] It is also understood in the art that substitutions of similar amino acids can be made effectively on the basis of hydrophilicity, particularly when, as in certain embodiments of the present invention, the biologically functional equivalent proteins and / or peptides produced thereby are intended for use in immunological embodiments. U.S. Patent No. 4,554,101, incorporated herein by reference, states that the highest local average hydrophilicity of a protein, as determined by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and / or antigenicity, i.e., a biological property of the protein.

[0060] As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values ​​have been assigned to amino acid residues: arginine (+3.0), lysine (+3.0), aspartic acid (+3.0±1), glutamic acid (+3.0±1), serine (+0.3), asparagine (+0.2), glutamine (+0.2), glycine (0), threonine (0.4), proline (-0.5±1), alanine (0.5), histidine (0.5), cysteine ​​(1.0), methionine (1.3), valine (1.5), leucine (1.8), isoleucine (1.8), tyrosine (2.3), phenylalanine (2.5), tryptophan (3.4). When changes are made on the basis of similar hydrophilicity values, substitution of amino acids whose hydrophilicity values ​​are within ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0061] The present disclosure relies in many embodiments on the synthesis of peptides and polypeptides in cyto via transcription and translation of appropriate polynucleotides. These peptides and polypeptides will contain the 20 "natural" amino acids and their post-translationally modified forms. However, in vitro peptide synthesis allows for the use of modified and / or unconventional amino acids. Exemplary, but not limiting, modified and / or unconventional amino acids are known in the art.

[0062] In addition to the biologically functional equivalents described above, the inventors also contemplate that structurally or functionally similar compounds can be formulated that mimic key portions of the peptides or polypeptides of the invention. Such compounds, which may be referred to as peptidomimetics, can be used in the same manner as the peptides of the invention and are therefore also functional equivalents.

[0063] Certain mimetics that mimic the elements of secondary and tertiary structure of proteins are described in Johnson et al. (1993). The underlying principle behind the use of peptidomimetics is that the peptide backbone of a protein is primarily there to orient amino acid side chains in a manner that promotes molecular interactions, such as those of antibodies and / or antigens. Peptidomimetics are therefore designed to allow similar molecular interactions to natural molecules. Such peptidomimetics include compounds that do not incorporate any natural amino acids or amino acid side chains, but are designed based on the p63-TID peptide sequence and have the ability to functionally replace p63-TID. II. Pharmaceutical Preparations

[0064] The pharmaceutical compositions of the present disclosure comprise an effective amount of p63-TID (or functional fragments and / or functional derivatives thereof) dissolved or dispersed in a pharma- ceutically acceptable carrier. The phrase "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other undesirable reactions when administered to an animal, e.g., a human, as appropriate. The preparation of pharmaceutical compositions comprising at least one p63-TID (or functional fragments and / or functional derivatives thereof) will be known to those skilled in the art in light of the present disclosure, as exemplified by Remington: The Science and Practice of Pharmacy, 21st Ed. Lippincott Williams and Wilkins, 2005, which is incorporated herein by reference. In addition, it will be understood that for animal (e.g., human) administration, preparations must meet the sterility, pyrogenicity, general safety, and purity standards required by the FDA Office of Biological Standards.

[0065] As used herein, "pharmaceutical acceptable carrier" includes any and all solvents, dispersion media, coating agents, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonicity agents, absorption retardants, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and similar materials, and combinations thereof, as would be known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, which is incorporated herein by reference).Any conventional carrier is contemplated for use in pharmaceutical compositions, unless it is incompatible with the active ingredient.

[0066] The p63-TID (or functional fragment and / or derivative thereof) may comprise different types of carriers depending on whether it is administered in solid, liquid, or aerosol form and whether it needs to be sterile for the route of administration, such as injection. The present invention may be administered intramyocardially, endocardially, epicardially, and / or intracoronary, by direct, catheter, or intracoronary injection, intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, transmucosally, orally, topically, locally, by inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion of target cells directly into a bath, via a catheter, via a lavage solution, in a cream, in a lipid composition (e.g., liposomes), or by other methods or combinations of the foregoing (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, which is incorporated herein by reference).

[0067] p63-TID (or its functional fragments and / or functional derivatives) can be formulated into the composition in free base, neutral, or salt form. Pharmaceutically acceptable salts include acid addition salts, such as those formed with free amino groups of the protein composition, or those formed with inorganic acids, such as hydrochloric acid or phosphoric acid, or organic acids, such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts formed with free carboxyl groups can also be derived from inorganic bases, such as sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases, such as isopropylamine, trimethylamine, histidine, or procaine. After formulation, the solution will be administered in a manner compatible with the dosage formulation and in an amount that will be therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as those formulated for parenteral administration, such as solutions for injection, or aerosols for delivery to the lungs, or those formulated for gastrointestinal administration, such as drug release capsules.

[0068] Further, according to the present disclosure, the composition of the present disclosure suitable for administration is provided in a pharma- ceutically acceptable carrier, with or without an inert diluent. The carrier should be absorbable, including liquid, semi-solid, i.e., paste, or solid carrier. Except where any conventional vehicle, agent, diluent, or carrier is harmful to the recipient or to the therapeutic effectiveness of the composition contained therein, its use in an administrable composition for use in carrying out the method of the present invention is appropriate. Examples of carriers or diluents include fats, oils, water, saline, lipids, liposomes, resins, binders, fillers, and the like, or combinations thereof. The composition may also include various antioxidants to retard oxidation of one or more components. In addition, the action of microorganisms can be prevented by preservatives, such as various antibacterial and antifungal agents, including, but not limited to, parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof.

[0069] According to the present disclosure, the compositions are combined with the carrier in any conventional and practical manner, i.e., by solution, suspending, emulsifying, mixing, encapsulating, absorbing, etc. Such procedures are routine to those skilled in the art.

[0070] In certain embodiments of the present disclosure, the composition is combined with semi-solid or solid carrier or mixed thoroughly.Mixing can be carried out in any conventional manner, such as grinding.Stabilizers can also be added in the mixing process to protect the composition from the loss of therapeutic activity, i.e., denaturation in the stomach.Examples of stabilizers for use in the composition include buffering agents, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.

[0071] In further embodiments, the present disclosure may relate to the use of lipid vehicle pharmaceutical compositions comprising p63-TID (or its functional fragments and / or functional derivatives), one or more lipids, and an aqueous solvent. As used herein, the term "lipid" will be defined to include any of a wide variety of substances that are characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds is well known to those skilled in the art, and the term "lipid" as used herein is not limited to any particular structure. Examples include compounds that contain long chain aliphatic hydrocarbons and their derivatives. Lipids may be naturally occurring or synthetic (i.e., designed or produced by humans). However, lipids are usually biological substances. Biological lipids are well known in the art, and include, for example, neutral lipids, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulfatides, lipids with ether and ether-linked fatty acids, and polymerizable lipids, and combinations thereof. Of course, compounds that would be understood as lipids by those of skill in the art other than those specifically described herein are also encompassed by the compositions and methods of the present invention.

[0072] Those skilled in the art will be familiar with the wide range of techniques that can be used to disperse compositions in lipid vehicles.For example, p63-TID (or its functional fragment and / or functional derivative) can be dispersed in a solution that contains lipid, dissolved with lipid, emulsified with lipid, mixed with lipid, combined with lipid, covalently bound with lipid, contained as a suspension in lipid, contained with or complexed with micelles or liposomes, or otherwise associated with lipid or lipid structures by any means known to those skilled in the art.Dispersion may or may not result in the formation of liposomes.

[0073] The actual dosage of the composition of the present disclosure administered to animal patients can be determined by physical and physiological factors, such as body weight, severity of condition, type of disease being treated, previous or concurrent therapeutic intervention, patient's specific disease, and route of administration.Depending on dosage and route of administration, the preferred dosage and / or the number of administrations of effective amount can vary according to the response of the subject.The practitioner responsible for administration will in any event determine the concentration of active ingredient in the composition and the appropriate dose for each individual subject.

[0074] In certain embodiments, the pharmaceutical composition may contain, for example, at least about 0.1% of the active compound. In other embodiments, the active compound may comprise about 2% to about 75% of the weight of the unit, or, for example, about 25% to about 60%, and any range derivable therein. Naturally, the amount of active compound in each therapeutically useful composition may be prepared in such a way that a suitable dosage will be obtained for any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations will be contemplated by those skilled in the art of preparing such pharmaceutical formulations, and therefore various dosages and treatment regimes may be desirable.

[0075] In other non-limiting examples, dosages can include, per administration, about 1 microgram / kg / body weight, about 5 micrograms / kg / body weight, about 10 micrograms / kg / body weight, about 50 micrograms / kg / body weight, about 100 micrograms / kg / body weight, about 200 micrograms / kg / body weight, about 350 micrograms / kg / body weight, about 500 micrograms / kg / body weight, about 1 milligram / kg / body weight, about 5 milligrams / kg / body weight, about 10 milligrams / kg / body weight, about 50 milligrams / kg / body weight, about 100 milligrams / kg / body weight, about 200 milligrams / kg / body weight, about 350 milligrams / kg / body weight, about 500 milligrams / kg / body weight, to about 1000 mg / kg / body weight or more, and any range derivable therein. In non-limiting examples of ranges derivable from the numbers recited herein, ranges such as about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 micrograms / kg / body weight to about 500 milligrams / kg / body weight, may be administered based on the above numbers. A. Gastrointestinal Compositions and Formulations

[0076] In an embodiment of the present disclosure, p63-TID (or its functional fragment and / or functional derivative) is formulated to be administered via the gastrointestinal route. The gastrointestinal route includes all possible routes of administration in which the composition comes into direct contact with the gastrointestinal tract. Specifically, the pharmaceutical compositions disclosed herein may be administered directly to the heart, although in alternative embodiments, the compositions are delivered orally, bucally, rectally, or sublingually. As such, these compositions may be formulated with an inert diluent or an assimilable edible carrier, or they may be encapsulated in a hard or soft shell gelatin capsule, or they may be compressed into tablets, or they may be directly incorporated with dietary food.

[0077] In certain embodiments, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (Mathiowitz et al., 1997; Hwang et al., 1998; U.S. Pat. Nos. 5,641,515, 5,580,579, and 5,792,451, each of which is specifically incorporated by reference herein in its entirety). Tablets, troches, pills, capsules, etc. may also contain: binders, such as, for example, gum tragacanth, acacia, corn starch, gelatin, or combinations thereof; excipients, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, or combinations thereof; disintegrants, such as, for example, corn starch, potato starch, alginic acid, or combinations thereof; lubricants, such as, for example, magnesium stearate; sweeteners, such as, for example, sucrose, lactose, saccharin, or combinations thereof; flavorings, such as, for example, peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc. When the dosage unit form is a capsule, it may contain a liquid carrier in addition to materials of the above type. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both. When the dosage form is a capsule, it may contain carriers, such as liquid carriers, in addition to the above-mentioned types of materials. Gelatin capsules, tablets, or pills may be enteric coated. Enteric coatings prevent the composition from denaturing in the stomach or upper digestive tract, where pH is acidic. For example, see U.S. Patent No. 5,629,001. When it reaches the small intestine, the basic pH there dissolves the coating, releasing the composition, allowing absorption by specialized cells, such as epithelial intestinal cells and Peyer's patch M cells.Syrup of elixir may contain active compound, sucrose as sweetener, methyl and propyl parabens as preservatives, dye, and flavoring agent, such as cherry or orange flavor.Of course, any material used in preparing any dosage unit form must be pharma-ceutically pure or substantially non-toxic in the amount utilized.In addition, active compound may be incorporated into sustained release preparations and formulations.

[0078] For oral administration, the composition of the present disclosure may alternatively be incorporated into the form of mouthwash, dentifrice, buccal tablet, mouth spray, or sublingual oral administration preparation together with one or more excipients.For example, mouthwash may be prepared by incorporating the active ingredient in the required amount into a suitable solvent, for example, sodium borate solution (Dobell's solution).Alternatively, the active ingredient may be incorporated into oral solution, for example, one that contains sodium borate, glycerin, and potassium bicarbonate, or dispersed in dentifrice, or may be added to a composition that may contain water, binder, abrasive, flavoring agent, effervescent agent, and humectant in a therapeutically effective amount.Alternatively, the composition may be in the form of a tablet or solution that can be placed under the tongue or otherwise dissolved in the mouth.

[0079] Additional formulations suitable for other digestive tract administration modes include suppositories. Suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum. After insertion, the suppository softens and melts or dissolves in intestinal fluids. In general, for suppositories, conventional carriers can include, for example, polyalkylene glycols, triglycerides, or combinations thereof. In certain embodiments, suppositories can be formed from mixtures containing, for example, about 0.5% to about 10% and preferably about 1% to about 2% of active ingredient. B. Parenteral Compositions and Formulations

[0080] In further embodiments, p63-TID (or its functional fragment and / or functional derivative) can be administered via parenteral route. As used herein, the term "parenteral" includes a route that bypasses the digestive tract. Specifically, the pharmaceutical compositions disclosed herein can be administered, for example, but not limited to, intramyocardially, endocardially, epicardially, and / or into coronary arteries, by direct catheter or intracoronary injection, intravenously, intradermally, intramuscularly, intraarterially, intraarachnoidally, subcutaneously, or intraperitoneally. See U.S. Patent Nos. 6,7537,514, 6,613,308, 5,466,468, 5,543,158, 5,641,515, and 5,399,363 (each of which is specifically incorporated herein by reference in its entirety).

[0081] Solutions of the active compounds as free bases or physiologically acceptable salts can be prepared in water suitably mixed with a surfactant, for example, hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. Pharmaceutical forms suitable for use in injection include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Patent No. 5,466,468, specifically incorporated herein by reference in its entirety). In all cases, the form must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage, and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium, including water, ethanol, polyol (i.e., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by using surfactants.Protection from the action of microorganisms can be provided by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.In many cases, it will be preferable to include an isotonic agent, for example, sugar or sodium chloride.Prolonged absorption of the injectable composition can be achieved by using an agent that delays absorption, for example, aluminum monostearate and gelatin, in the composition.

[0082] For parenteral administration in aqueous solution, for example, the solution should be suitably buffered if necessary, and the liquid diluent should first be rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, sterile aqueous media that can be used will be known to those of skill in the art in light of the present disclosure. For example, a dosage may be dissolved in an isotonic NaCl solution and added to a subcutaneous drip or injected at the proposed site of infusion (see, for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will occur as necessary depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by the FDA Office of Biological Standards.

[0083] Sterile injectable solution is prepared by incorporating active compound in the required amount in suitable solvent with various other ingredients as listed above as necessary, followed by filtration sterilization.Generally, dispersion is prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains basic dispersion medium and other necessary ingredients other than those mentioned above.In the case of sterile powder for preparing sterile injectable solution, the preferred method of preparation is vacuum drying and freeze-drying technique, which obtains powder of active ingredient plus any additional desired ingredients from a solution that has been previously sterilized and filtered.Powder composition is mixed with liquid carrier, such as water or saline, with or without sterilizing agent. C. Other Pharmaceutical Compositions and Formulations

[0084] In other specific embodiments of the present disclosure, the active compound p63-TID (or functional fragment and / or functional derivative thereof) may be formulated for administration via a variety of other routes, for example, topical (i.e., transdermal), transmucosal (intranasal, intravaginal, etc.), and / or inhalation.

[0085] Pharmaceutical compositions for topical administration may include active compounds formulated for pharmaceutical application, such as ointments, pastes, creams, or powders. Ointments include all oil-based, adsorption, emulsion, and water-soluble base compositions for topical application, while creams and lotions are compositions that include only emulsion bases. Topically administered medicaments may include a penetration enhancer to promote the adsorption of the active ingredient through the skin. Suitable penetration enhancers include glycerin, alcohol, alkyl methyl sulfoxides, pyrrolidone, and laurocapram. Possible bases for compositions for topical application include polyethylene glycol, lanolin, cold cream, and petrolatum, as well as any other suitable absorption, emulsion, or water-soluble ointment base. Topical preparations may also include emulsifiers, gelling agents, and antimicrobial preservatives as necessary to preserve the active ingredient and provide a homogeneous mixture. Transdermal administration of the present invention may also include the use of a "patch". For example, a patch may deliver one or more active agents in a continuous manner at a predetermined rate and for a fixed period of time.

[0086] In certain embodiments, pharmaceutical compositions can be delivered by eye drops, nasal spray, inhalation, and / or other aerosol delivery vehicles.Methods for directly delivering compositions into the lungs via nasal aerosol spray are described, for example, in U.S. Patent Nos. 5,756,353 and 5,804,212 (each of which is specifically incorporated herein by reference in its entirety).Similarly, drug delivery using nasal microparticle resins (Takenaga et al., 1998) and lysophosphatidyl-glycerol compounds (U.S. Patent No. 5,725,871, which is specifically incorporated herein by reference in its entirety) are also well known in the pharmaceutical arts.Similarly, transmucosal drug delivery in the form of polytetrafluoroethylene support matrix is ​​described in U.S. Patent No. 5,780,045 (which is specifically incorporated herein by reference in its entirety).

[0087] The term aerosol refers to a colloidal system in which finely divided solids of liquid particles are dispersed in a liquefied or pressurized gas propellant. The typical aerosol of the present invention for inhalation will consist of a suspension of active ingredient in a liquid propellant or a mixture of a liquid propellant and a suitable solvent. Suitable propellants include hydrocarbons and hydrocarbon ethers. Suitable containers will vary according to the pressure requirements of the propellant. The administration of aerosol will vary according to the age, weight, and severity and response of the subject. III. Method of Treatment Embodiments

[0088] The embodiments of the present disclosure are directed to methods and / or compositions related to the treatment and / or prevention of one or more cardiac-related medical conditions.The embodiments of the present disclosure relate to the regeneration of tissues, including muscle tissues, for example, cardiac tissues, through the reprogramming of non-cardiomyocyte cells present in the heart.Certain embodiments relate to the reversal (or improvement of at least one symptom) of cardiac medical conditions, including at least, for example, cardiac disease, cardiomyopathy, cardiac toxicity, congestive heart failure, ischemic heart disease, myocardial infarction, coronary artery disease, pulmonary heart, inflammatory heart disease, inflammatory cardiac hypertrophy, myocarditis, congenital heart disease, rheumatic heart disease, cardiac systolic dysfunction, cardiac diastolic dysfunction, angina, dilated cardiomyopathy, idiopathic cardiomyopathy, or other conditions that lead to cardiac fibrosis.

[0089] In a specific embodiment of the present disclosure, cardiomyopathy is the medical condition of the heart that is to be treated.The medical condition of the heart (including, for example, cardiomyopathy) can be caused by one or more of various characteristics, including, for example, long-term high blood pressure, heart valve problems, heart tissue damage (for example, one or more previous heart attacks or chronic or acute and / or recurrent episodes or sequelae of ischemic heart disease), chronic high heart rate, metabolic disorder, for example, thyroid disease or diabetes, nutritional deficiency of essential vitamins or minerals, for example, thiamine (vitamin B-1), selenium, calcium and / or magnesium, pregnancy, alcohol abuse, drug abuse, including narcotics or prescription drugs, for example, cocaine or antidepressants, for example, tricyclic antidepressants, use of some chemotherapeutic drugs to treat cancer (including adriamycin), certain viral infections, hemochromatosis, and / or unknown or undetected causes, i.e., idiopathic cardiomyopathy.

[0090] In some cases, the disclosed methods and compositions are used for the treatment or prevention of one or more cardiac medical conditions, or delay the onset of one or more cardiac medical conditions, or reduce the severity of one or more symptoms of one or more cardiac medical conditions.In specific cases, such prevention, delay or onset, or reduction of the severity of one or more symptoms occurs in individuals who are at risk for cardiac medical conditions.Exemplary risk factors include one or more of the following: age, sex (male, but also occurs in females), high blood pressure, high serum cholesterol level, smoking, excessive alcohol consumption, sugar consumption, family or personal medical history, obesity, lack of physical activity, psychosocial factors, diabetes mellitus, overweight, genetic predisposition, and / or exposure to air pollution.

[0091] A specific aspect of the present disclosure relates to the delivery of at least one polynucleotide or polypeptide to cardiac tissue for the transdifferentiation of certain cells in the tissue. In certain embodiments, the nucleic acid is the active agent, while in some embodiments, the polypeptide produced from the nucleic acid is the active agent. The tissue can be of any type, but in certain cases, it is cardiac muscle and / or scar tissue. In a specific embodiment, the method and composition of the present disclosure allows the differentiation of cardiac progenitor cells present in adults into cardiac muscle cells, and / or the transdifferentiation of differentiated cells that are not cardiac muscle cells, such as fibroblasts.

[0092] The embodiments of the present disclosure include delivery of one or more polynucleotides (which may also be referred to herein as nucleic acids) or polypeptides produced therefrom that stimulate transdifferentiation or induce reprogramming of cells (e.g., muscle cells, including cardiomyocytes) and / or tissues (including cardiac tissues). Specific aspects of such embodiments result in the reversal of one or more cardiac medical conditions. Certain aspects of such embodiments result in the amelioration of at least one symptom of a cardiac medical condition. In an exemplary embodiment, the cardiac medical condition is heart failure. Heart failure can be the result of one or more causes, including coronary artery disease and heart attack, high blood pressure, heart valve failure, cardiomyopathy (e.g., caused by disease, infection, alcohol abuse, and the toxic effects of drugs, such as cocaine or some drugs used in chemotherapy), idiopathic cardiomyopathy, and / or genetic factors.

[0093] In certain embodiments, the one or more polynucleotides are comprised in a viral vector. In certain embodiments, the viral vector comprises at least or exactly 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 108, 109, 109, 102, 103, 104, 105, 106, 107, 108, 109, 110, 3, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88 , 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, In certain embodiments, the viral vector is provided at a multiplicity of infection of 5, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150, or any range derivable therein. In certain embodiments, the viral vector is provided at a multiplicity of infection of 20, 50, or 100. In certain embodiments, the viral vector is provided at a multiplicity of infection of 50.

[0094] Specific but exemplary indications of embodiments of the present disclosure include at least 1) heart failure, including congestive heart failure, 2) prevention of ventricular remodeling, and / or 3) application to cardiomyopathy. Other indications may also include coronary artery disease, ischemic heart disease, valvular heart disease, and the like. In certain embodiments, the methods and compositions of the present disclosure provide cardiomyocyte regeneration sufficient to reverse established cardiomyopathy, congestive heart failure, and prevention of ventricular remodeling.

[0095] In cases where an individual has cardiomyopathy, the cardiomyopathy may be ischemic or non-ischemic. Cardiomyopathy may be caused by long-term high blood pressure, heart valve problems, heart tissue damage from a previous heart attack, chronically high heart rate, metabolic disorders, nutritional deficiencies, pregnancy, alcohol abuse, drug abuse, chemotherapy drugs, viral infections, hemochromatosis, genetic conditions, elevated cholesterol levels, or a combination thereof. Cardiomyopathy may also be idiopathic, i.e., no cause has been identified.

[0096] In certain embodiments, there are methods of regenerating cells at a desired location in an individual, comprising delivering to the location an effective amount of at least one molecule of p63-TID (or a functional fragment and / or derivative thereof). In some cases, the method comprises a mechanical interaction of p63-TID with, for example, HDAC. In certain embodiments, the molecule is delivered in a nucleic acid form, while in certain embodiments, one or more of the compositions of p63-TID (or a functional fragment and / or derivative thereof) of the present disclosure are polypeptides. In specific embodiments, the delivery location of the composition is the region of the heart. The delivery step may include, for example, injection directly into the heart, including intravenous perfusion, intracoronary myocardial perfusion, intra-arterial organ perfusion by catheter, or coronary sinus perfusion catheter, directly into the region of the damaged tissue.

[0097] The embodiments of the present disclosure include, for example, methods and / or compositions for myocardial regeneration and reversal of myocardial ischemic injury.In a specific embodiment, there is a method for reprogramming cardiac scar cells (fibroblasts) in mammalian heart into adult cardiomyocytes in individuals who have had cardiac medical conditions, for example, acute or chronic ischemic injury.In certain embodiments, such methods are achieved using a composition that includes at least p63-TID (or its functional fragment and / or functional derivative), and in some cases, one or more cardiac cell reprogramming factors, for example, Hand2 and / or myocardin.

[0098] In a specific embodiment, the individual is treated in an in vivo or in situ manner, while in an alternative embodiment, the individual is treated with the composition encompassed by the present disclosure in an ex vivo manner. In such an embodiment, the cells to be subjected to the nucleic acid composition of the present disclosure are either obtained from the individual or obtained from another individual. Such cells are subjected to the nucleic acid composition in vitro so that the nucleic acid composition is taken up by the cells, and the cells are then delivered to the individual to be treated.

[0099] In a specific embodiment, the individual is provided with a treatment for an additional cardiac medical condition. IV. Cardiac Cell Reprogramming Factors and Chromatin Destabilizing Agents Embodiments

[0100] Certain embodiments of the present disclosure relate to nucleic acids, and some embodiments relate to polypeptides or peptides. In certain aspects, the nucleic acid comprises p63-TID (or functional fragments and / or functional derivatives thereof). In specific embodiments, any form of p63-TID (or functional fragments and / or functional derivatives thereof) may or may not be utilized with one or more cardiac cell reprogramming factors, and may or may not be utilized with one or more chromatin destabilizing agents. A. Cardiac Cell Reprogramming Factors

[0101] In certain embodiments, one or more cardiac cell reprogramming factors are used in the methods of the present disclosure, which may or may not be provided simultaneously with the p63-TID (or a functional fragment and / or derivative thereof). In certain embodiments, the factors are provided after the individual has received the p63-TID (or a functional fragment and / or derivative thereof), although in some cases they are provided prior to or simultaneously with the agent.

[0102] Cardiac cell reprogramming factor may or may not be a transcription factor.While standard methods can be used to test whether a particular compound is effective as a cardiac cell reprogramming factor, in certain embodiments, the factor is Hand2, myocardin, Gata4, Mef2c, Tbx5, mesoderm posterior protein 1 (Mesp1), miR-133, miR-1, Oct4, Klf4, c-myc, Sox2, Brachyury, Nkx2.5, ETS2, ESRRG, Mrtf-A, MyoD, ZFPM2, or a combination thereof.Whether a compound acts as a cardiac cell reprogramming factor can be tested by administering it to fibroblasts (e.g., using lentivirus) and performing FACS for cTnT as exemplified elsewhere herein.The factor can be used as a nucleic acid, polypeptide, peptide, or a combination thereof of a particular domain of the factor. In certain embodiments, Hand2 and / or myocardin are used, including as nucleic acids. In specific embodiments, at least for Hand2 and / or myocardin, the nucleic acid encodes or comprises a transcribed nucleic acid. In other embodiments, the Hand2 and / or myocardin nucleic acid comprises a nucleic acid segment of Hand2 and / or myocardin, respectively, or a biologically functional equivalent thereof. In specific embodiments, the Hand2 and / or myocardin nucleic acid encodes a protein, polypeptide, or peptide. An exemplary human Hand2 nucleic acid is found in the GENBANK® database of the National Center for Biotechnology Information, accession number NM_021973, which is incorporated herein by reference in its entirety. An exemplary human myocardin nucleic acid is found in GENBANK® accession number AY764180, which is incorporated herein by reference in its entirety.

[0103] In certain embodiments, functional fragments of cardiac cell reprogramming factor nucleic acid or polypeptide are used instead of whole factor nucleic acid or polypeptide.Hand2 or myocardin functional fragments (for example) are sufficient to allow cell reprogramming when exposed to the fragment, either p63-TID (or functional fragments and / or functional derivatives thereof) alone or in combination with Hand2 or myocardin and p63-TID (or functional fragments and / or functional derivatives thereof).In certain embodiments, the functional fragment of Hand2 nucleic acid encodes at least 200, 180, 175, 160, 150, 140, 125, 110, 100, 90, 80, 75, 70, 60, 55, 50, 40, 30, 25, or 19 amino acids of Hand2 polypeptide. In certain embodiments, the functional fragment of the myocardin nucleic acid encodes at least 900, 800, 700, 600, 500, 400, 300, 200, 100, or 50 amino acids of the myocardin polypeptide. In some embodiments, the Hand2 polypeptide and the myocardin polypeptide are encoded on the same nucleic acid construct and / or vector. In some embodiments, the Hand2 polypeptide and the myocardin polypeptide are separated by a 2A element. In certain embodiments, the Hand2 polypeptide and the myocardin polypeptide are separated by a P2A element. In certain embodiments, the exemplary Hand2 polynucleotide sequence is and / or is included in SEQ ID NO:3. In certain embodiments, the exemplary myocardin polynucleotide sequence is and / or is included in SEQ ID NO:4. ATGTCTCTCGTGGGCGGATTTCCTCACCACCCTGTGGTGCACCATGAGGGCTATCCTTTTGCTGCCGCTGCCGCAGCCGCCGCTGCTGCTGCAGCTAGTAGATGTAGCCACGAGGAAAACCCCTACTTCCACGGCTGGCTGATCGGCCACCCTGAGATGAGCCCTCCAGATTACAGCATGGCCCTGAGCTACAGCCCTGAGTATGCTTCTGGAGCCGCTGGACTGGATCACTCTCATTATGGCGGAGTGCCTCCAGGCGCTGGACCTCCTGGACTGGGAGGACCTAGACCTGTGAAGAGAAGAGGCACCGCCAACCGGAAAGAGCGGAGAAGAACCCAGAGCATCAATAGCGCCTTCGCCGAGCTGAGAGAATGCATCCCTAATGTGCCCGCCGACACCAAGCTGAGCAAGATCAAAACCCTGCGGCTGGCCACCAGCTATATCGCCTATCTGATGGACCTGCTGGCCAAGGACGATCAGAATGGCGAGGCCGAGGCCTTCAAGGCCGAGATCAAGAAAACCGACGTGAAAGAGGAAAAGCGCAAGAAAGAGCTGAACGAGATCCTGAAGTCCACCGTGTCCAGCAACGACAAAAAGACCAAGGGCAGAACCGGCTGGCCTCAGCATGTGTGGGCTCTGGAACTGAAACAGGGCAGCGGC(SEQ ID NO: 3)

[0104] In specific embodiments, some or all of SEQ ID NO: 3 and / or 4 are utilized in the methods of the disclosure. In certain embodiments, polynucleotides having specific sequence identity to SEQ ID NO: 3 and / or 4 are utilized in the methods of the disclosure. In certain cases, functional fragments of SEQ ID NO: 3 and / or 4 are utilized, the term "functional" fragment as used herein referring to a polynucleotide that encodes a polypeptide having an activity capable of converting fibroblasts into endothelial or endothelial-like cells. In certain cases, the fragment comprises at least about 2900, 2800, 2700, 2600, 2500, 2400, 2300, 2200, 2100, 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1375, 1350, 1325, 1300, 1275, 1250, 1225, 1200, 1175, 1150, 1125, 1100, 1075, 1050, 1025, 1000, 975, 950, 925, 900, 875, 850, 825, 800, 775, 750, 725, 700, 675, 650, 625, 600, 575, 550, 525, 500, 475, 450, 425, 400, 375, 350, 325, 300, 275, 250, 225, 200, 175, 150, 125 or more, or 100 or more contiguous nucleotides in length. In addition, fragments may have sequence identity of at least or exactly 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 80, 75, or 70% identity to the corresponding regions in SEQ ID NOs: 3 and / or 4. Polynucleotides having certain sequence identity to SEQ ID NOs: 3 and / or 4, including at least or exactly 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 80, 75, or 70% identity to SEQ ID NOs: 3 and / or 4, may be used.

[0105] In some embodiments, the Hand2 and / or myocardin polypeptide is delivered to an individual in need thereof regardless of the form in which it is present, such as on a vector, associated with a carrier, in a cell (including in a cell on a vector), etc. In certain embodiments, the Hand2 and / or myocardin polypeptide is a mammalian Hand2 and / or myocardin polypeptide, including human, mouse, rat, etc. In a specific embodiment, an example of a Hand2 polypeptide sequence is and / or is included in SEQ ID NO:5. In a specific embodiment, an example of a myocardin polypeptide sequence is and / or is included in SEQ ID NO:6. MSLVGGFPHHPVVHHEGYPFAAAAAAAAAAAASRCSHEENPYFHGWLIGHPEMSPPDYSMALSYSPEYASGAAGLDHSHYGGVPPGAGPPGLGGPRPVKRRGTANRKERRRTQSINSAFAELRECIPNVPADTKLSKIKTLRLATSYIAYLMDLLAKDDQNGEAEAFKAEIKKTDVKEEKRKKELNEILKSTVSSNDKKTKGRTGWPQHVWALELKQGSG (SEQ ID NO: 5) (query number 6)

[0106] In specific embodiments, some or all of SEQ ID NO:5 and / or 6 are utilized in the methods of the disclosure. In certain embodiments, polypeptides having specific sequence identity to SEQ ID NO:5 and / or 6 are utilized in the methods of the disclosure. In certain cases, functional fragments of SEQ ID NO:5 and / or 6 are utilized, the term "functional" fragment as used herein referring to a polypeptide having activity capable of converting fibroblasts into endothelial or endothelial-like cells. In certain cases, the fragment comprises at least about 975 or less of SEQ ID NO:5 and / or SEQ ID NO:6, 950, 925, 900, 875, 850, 825, 800, 775, 750, 725, 700, 675, 650, 625, 600, 575, 550, 525, 500, 475, 450, 425, 400, 375, 350, 325, 300, 275, 250, 240, 235, 230, 225, 220, 215, 210, 205, 200, 195, 190, 185, 180, 175, 170, 165, 160, 155, 150, 145, 140, 135, 130, 125, 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25 or less contiguous nucleotides in length, or 20 or less contiguous nucleotides in length. B. Chromatin Destabilizing Agents

[0107] In specific embodiments, one or more chromatin destabilizing agents are utilized in conjunction with the p63-TID (or a functional fragment and / or derivative thereof). The one or more chromatin destabilizing agents may be provided to an individual simultaneously with the p63-TID (or a functional fragment and / or derivative thereof), although in certain embodiments, the one or more chromatin destabilizing agents are utilized prior to or after the p63-TID (or a functional fragment and / or derivative thereof).

[0108] Standard methods can be used to test whether a particular compound will be effective as a chromatin destabilizing agent, but in certain embodiments, the chromatin destabilizing agent is Oct4, DZNep, Sall4, SOX2, KLF4, MYC, SB431542, PD0325901, Parnate, CHIR99021, A-83-01, NaB, PS48, forskolin (FSK), 2-methyl-5-hydroxytryptamine (2-Me-5HT), D4476, VPA, CHIR99021 (CHIR), 616452, tranylcypromine, prostaglandin E2, rolipram, 3-deazaneplanocin A (DZNep), 5-azacytidine, sodium butyrate, RG108, or combinations thereof. V. General Nucleic Acids

[0109] The term "nucleic acid" is well known in the art. "Nucleic acid", as used herein, generally refers to a molecule (i.e., a chain) of DNA, RNA, or derivatives or analogs thereof, that includes a nucleobase. Nucleic acid bases include, for example, naturally occurring purine or pyrimidine bases found in DNA (e.g., adenine "A", guanine "G", thymine "T", or cytosine "C") or RNA (e.g., A, G, uracil "U", or C). The term "nucleic acid" encompasses the terms "oligonucleotide" and "polynucleotide", respectively, as subgenus of the term "nucleic acid". The term "oligonucleotide" refers to a molecule of about 3 to about 100 nucleobases in length. The term "polynucleotide" refers to at least one molecule of more than about 100 nucleobases in length, at least in some cases.

[0110] These definitions generally refer to single-stranded molecules, but in certain embodiments will also encompass additional strands that are partially, substantially, or completely complementary to the single-stranded molecule. Thus, a nucleic acid can encompass a double-stranded or triple-stranded molecule that includes one or more complementary strands or "complements" of a particular sequence that constitutes the molecule. As used herein, a single-stranded nucleic acid may be designated by the prefix "ss", a double-stranded nucleic acid may be designated by the prefix "ds", and a triple-stranded nucleic acid may be designated by the prefix "ts".

[0111] As used herein, "wild type" refers to the naturally occurring sequence of a nucleic acid at a locus in the genome of an organism, or a sequence transcribed or translated from such a nucleic acid. Thus, the term "wild type" can also refer to the amino acid sequence encoded by a nucleic acid. Since a locus may have more than one sequence or allele in a population of individuals, the term "wild type" encompasses all such naturally occurring loci. As used herein, the term "polymorphism" means that there is variability at a locus in individuals within a population (i.e., there are two or more alleles). As used herein, the term "mutant" refers to a change in the sequence of a nucleic acid or its encoded protein, polypeptide, or peptide that is the result of human intervention.

[0112] The present disclosure also relates to the isolation or production of recombinant constructs or recombinant host cells through the application of recombinant nucleic acid techniques known to those of skill in the art or described herein. The recombinant constructs or host cells may comprise a nucleic acid and may express a protein, peptide, or peptide, or at least one biologically functional equivalent thereof.

[0113] As used herein, in certain embodiments, "gene" refers to a nucleic acid that is transcribed. In certain aspects, a gene includes regulatory sequences involved in transcription or message production or composition. In specific embodiments, a gene includes a transcribed sequence that codes for a protein, polypeptide, or peptide. As will be understood by those skilled in the art, this functional term "gene" includes both genomic sequences, RNA, or cDNA sequences, or smaller engineered nucleic acid segments, including nucleic acid segments of the non-transcribed parts of a gene, including but not limited to the non-transcribed promoter or enhancer regions of the gene. Smaller engineered gene nucleic acid segments can express or be adapted to express proteins, polypeptides, domains, peptides, fusion proteins, mutants, and / or the like using nucleic acid engineering techniques.

[0114] "Substantially isolated from other coding sequences" means that the gene of interest or a fragment thereof forms the majority of the coding region of the nucleic acid, or that the nucleic acid is free of the majority of naturally occurring coding nucleic acids, such as large chromosomal fragments, other functional genes, RNA, or cDNA coding regions. Of course, this refers to the nucleic acid as originally isolated, and does not exclude genes or coding regions that are later added to the nucleic acid by the hand of man.

[0115] The nucleic acids of the present disclosure, regardless of the length of the sequence itself, may be combined with other nucleic acid sequences, including but not limited to promoters, enhancers, polyadenylation signals, restriction enzyme sites, multiple cloning sites, coding segments, and the like, to create one or more nucleic acid constructs. As used herein, the term "nucleic acid construct" is a nucleic acid that has been manipulated or modified by the human hand, and generally includes one or more nucleic acid sequences assembled by the human hand.

[0116] In a non-limiting example, one or more nucleic acid constructs can be prepared that contain a contiguous stretch of nucleotides identical or complementary (at least in part) to p63. The nucleic acid constructs can be about 3, about 5, about 8, about 10 to about 14, or about 15, about 20, about 30, about 40, about 50, about 100, about 115, about 200, about 500, about 600, or about 650 nucleotides in length, as well as constructs of larger sizes up to vector size (including all intermediate lengths and intermediate ranges). "Intermediate length" and "intermediate range" as used herein refer to any length or range including or between the recited values ​​(i.e., all integers between and including such values). Non-limiting examples of intermediate lengths include about 11, about 12, about 13, about 16, about 17, about 18, about 19, etc., about 21, about 22, about 23, etc., about 31, about 32, etc., about 51, about 52, about 53, etc., about 101, about 102, about 103, etc., about 151, about 152, about 153, etc., about 600, about 601, about 605, about 610. Non-limiting examples of intermediate ranges include about 3 to about 32, about 150 to about 750, etc.

[0117] In certain embodiments, the nucleic acid construct is a recombinant vector.In a specific embodiment, the present disclosure relates to one or more recombinant vectors that comprise the nucleic acid sequence that codes for Hand2 and myocardin protein, polypeptide or peptide.In a specific embodiment, the recombinant vector is a DNA vector.

[0118] The term "biologically functional equivalent" is well understood in the art and is further defined in detail herein. Thus, sequences in which 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acids are identical or functionally equivalent to those encoded by Hand2 and Myocardin nucleic acids, respectively, retain the biological activity of the protein, polypeptide, or peptide.

[0119] In certain other embodiments, the disclosure relates to at least one recombinant vector comprising within its sequence a nucleic acid sequence that expresses a p63-TID (or a functional fragment and / or derivative thereof). In certain embodiments, the disclosure relates to at least one recombinant vector comprising within its sequence a nucleic acid sequence that expresses a Hand2 nucleic acid. In another embodiment, there is at least one recombinant vector comprising within its sequence a nucleic acid sequence that expresses a myocardin nucleic acid.

[0120] The term "functionally equivalent codon" refers to the codon that codes for the same amino acid, such as the six codons for arginine and serine, and also refers to the codon that codes for biologically equivalent amino acids.The codon usage of various organisms and organelles can be found in the literature.Therefore, it is contemplated that codon usage can be optimized for other animals, as well as other organisms, such as prokaryotes (e.g., eubacteria, archaea), eukaryotes (e.g., protists, plants, fungi, animals), viruses, etc., and organelles that contain nucleic acid, such as mitochondria, chloroplasts, etc., based on preferred codon usage, as known to those skilled in the art.

[0121] It is understood that the amino acid or nucleic acid sequence may include additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, or various combinations thereof, while still being essentially as described in one of the sequences disclosed herein, provided that the sequence meets the above criteria, including maintaining the biological protein, polypeptide, or peptide activity to which expression of the protein composition pertains. The addition of terminal sequences applies specifically to nucleic acid sequences, which may, for example, include various non-coding sequences adjacent to either the 5' and / or 3' portions of the coding region, or may include various internal sequences, i.e., introns, that are known to occur within genes.

[0122] Nucleic acid sequences having from about 70% to about 79%, or more preferably from about 80% to about 89%, or even more specifically from about 90% to about 99%, nucleotides identical to the nucleotides of the recited GENBANK® sequences disclosed herein, excluding introns and flanking regions, and allowing for the degeneracy of the genetic code, are encompassed by the present disclosure.

[0123] Recombinant vectors and isolated nucleic acid segments may therefore, in a variety of ways, contain the Hand2 or myocardin coding region itself, coding regions with selected changes or modifications within the basic coding region, which may in any event encode a larger polypeptide or peptide that includes such coding region, or may encode a biologically functional equivalent protein, polypeptide, or peptide having a variant amino acid sequence.

[0124] The nucleic acid of the present disclosure may include biologically functional equivalent coding sequences of Hand2 or myocardin proteins, polypeptides, or peptides. Such sequences may arise as a result of codon redundancy or functional equivalence known to occur naturally within nucleic acid sequences or encoded proteins, polypeptides, or peptides. Alternatively, functionally equivalent proteins, polypeptides, or peptides may be produced through the application of recombinant DNA technology, which allows changes in the structure of proteins, polypeptides, or peptides to be engineered based on the consideration of the properties of the amino acids being exchanged. Human-designed changes may be introduced, for example, through the application of site-directed mutagenesis techniques discussed herein below, for example, to introduce improvements or changes in the antigenicity of proteins, polypeptides, or peptides, or to test mutants to examine the activity of proteins, polypeptides, or peptides at the molecular level. A. Nucleic acid base

[0125] As used herein, "nucleobase" refers to a heterocyclic base, such as, for example, a naturally occurring nucleobase (i.e., A, T, G, C, or U) found in at least one naturally occurring nucleic acid (i.e., DNA and RNA), as well as naturally occurring or non-naturally occurring derivatives and analogs of such nucleobases. A nucleobase generally forms one or more hydrogen bonds ("anneals" or "hybridizes") with at least one naturally occurring nucleobase in a manner that can replace naturally occurring nucleobase pairing (e.g., hydrogen bonds between A and T, G and C, and A and U).

[0126] "Purine" and / or "pyrimidine" nucleobases encompass naturally occurring purine and / or pyrimidine nucleobases, as well as derivatives and analogs thereof, including, but not limited to, purines or pyrimidines substituted with one or more of alkyl, carboxyalkyl, amino, hydroxyl, halogen (i.e., fluoro, chloro, bromo, or iodo), thiol, or alkylthiol moieties. Preferred alkyl (e.g., alkyl, carboxyalkyl, etc.) moieties are composed of about 1, about 2, about 3, about 4, about 5, to about 6 carbon atoms. Other non-limiting examples of purines or pyrimidines include deazapurines, 2,6-diaminopurine, 5-fluorouracil, xanthine, hypoxanthine, 8-bromoguanine, 8-chloroguanine, bromothymine, 8-aminoguanine, 8-hydroxyguanine, 8-methylguanine, 8-thioguanine, azaguanine, 2-aminopurine, 5-ethylcytosine, 5-methylcytosine, 5-bromouracil, 5-ethyluracil, 5-iodouracil, 5-chlorouracil, 5-propyluracil, thiouracil, 2-methyladenine, methylthioadenine, N,N-dimethyladenine, azaadenine, 8-bromoadenine, 8-hydroxyadenine, 6-hydroxyaminopurine, 6-thiopurine, 4-(6-aminohexyl / cytosine), and the like.

[0127] The nucleobase may be included in a nucleoside or nucleotide, using any chemical or natural synthesis method described herein or known to one of skill in the art. B. Nucleosides

[0128] As used herein, "nucleoside" refers to an individual chemical unit that includes a nucleobase covalently linked to a nucleobase linker moiety. Non-limiting examples of "nucleobase linker moieties" are sugars that contain five carbon atoms (i.e., "pentoses"), including, but not limited to, deoxyribose, ribose, arabinose, or derivatives or analogs of pentoses. Non-limiting examples of derivatives or analogs of pentoses include 2'-fluoro-2'-deoxyribose, or carbocyclic sugars in which a carbon is substituted for an oxygen in the sugar ring.

[0129] Other types of covalent linkages of nucleobases to nucleobase linker moieties are known in the art. As a non-limiting example, nucleosides containing purine (i.e., A or G) or 7-deazapurine nucleobases are typically covalently linked at the 9-position of the purine or 7-deazapurine to the 1'-position of the pentose sugar. In another non-limiting example, nucleosides containing pyrimidine nucleobases (i.e., C, T or U) are typically covalently linked at the 1-position of the pyrimidine to the 1'-position of the pentose sugar (Kornberg and Baker, 1992). C. Nucleotides

[0130] As used herein, "nucleotide" refers to a nucleoside that further comprises a "backbone moiety." The backbone moiety generally covalently binds the nucleotide to another molecule that comprises a nucleotide or to another nucleotide to form a nucleic acid. The "backbone moiety" in naturally occurring nucleotides typically comprises a phosphorus moiety, which is covalently bound to the pentose sugar. The attachment of the backbone moiety typically occurs at either the 3' or 5' position of the pentose sugar. However, other types of linkages are known in the art, particularly when the nucleotide comprises a derivative or analog of the naturally occurring pentose or phosphorus moiety. D. Nucleic Acid Analogs

[0131] Nucleic acid may comprise or be entirely composed of nucleobase derivatives or analogs, nucleobase linker moieties, and / or backbone moieties that may be present in naturally occurring nucleic acids.As used herein, "derivative" refers to a chemically modified or altered form of a naturally occurring molecule, while the term "mimetic" or "analog" refers to a molecule that may or may not be structurally similar to a naturally occurring molecule or moiety, but has a similar function.As used herein, "moiety" generally refers to a smaller chemical or molecular component within a larger chemical or molecular structure.Nucleobase, nucleoside, and nucleotide analogs or derivatives are well known and described in the art (see, for example, Scheit, 1980, which is incorporated herein by reference).

[0132] Additional non-limiting examples of nucleosides, nucleotides, or nucleic acids containing pentose and / or backbone moiety derivatives or analogs include those in U.S. Pat. No. 5,681,947, which describes oligonucleotides containing purine derivatives that form triple helices and / or prevent expression of dsDNA; those in U.S. Pat. Nos. 5,652,099 and 5,763,167, which describe nucleic acids incorporating fluorescent analogs of nucleosides found in DNA or RNA, particularly nucleic acids for use as fluorescent nucleic acid probes; those in U.S. Pat. No. 5,614,617, which describes oligonucleotide analogs having substitutions in the pyrimidine ring with enhanced nuclease stability; those in U.S. Pat. Nos. 5,670,663, 5,872,232, and 5,859,221, which describe oligonucleotide analogs with modified pentoses (i.e., modified 2'-deoxyfuranosyl moieties) for use in nucleic acid detection; and those that can be used in hybridization assays. No. 5,446,137, which describes oligonucleotides containing at least one pentose moiety substituted with a substituent other than hydrogen at the 4' position, such that the 5' carbon atom can be substituted with a substituent other than hydrogen; U.S. Pat. No. 5,886,165, which describes oligonucleotides containing both deoxyribonucleotides with 3'-5' internucleotide linkages and ribonucleotides with 2'-5' internucleotide linkages; U.S. Pat. No. 5,714,606, which describes modified internucleotide linkages in which the oxygen at the 3' position of the internucleotide linkage is replaced by carbon to enhance the nuclease resistance of the nucleic acid; U.S. Pat. No. 5,672,697, which describes oligonucleotides containing one or more 5' methylene phosphonate internucleotide linkages that enhance nuclease resistance; U.S. Pat. Nos. 5,466,786 and 5,792, which describe the attachment of a substituent moiety, which may include a drug or a label, to the 2' carbon of an oligonucleotide to provide enhanced nuclease stability and the ability to deliver a drug or detection moiety.in U.S. Pat. No. 5,223,618, which describes oligonucleotide analogs having two or three carbon backbone linkages attached to the 4' and 3' positions of adjacent pentose sugar moieties for enhanced cellular uptake, resistance to nucleases, and hybridization to target RNA; in U.S. Pat. No. 5,470,967, which describes oligonucleotides containing at least one sulfamate or sulfamide internucleotide linkage useful as nucleic acid hybridization probes; and in U.S. Pat. No. 5,470,967, which describes oligonucleotides containing three or more phosphodiester backbone moieties that are used for improved nuclease resistance, cellular uptake, and modulation of RNA expression. in U.S. Pat. Nos. 5,378,825, 5,777,092, 5,623,070, 5,610,289, and 5,602,240, which describe oligonucleotides having four-atom linker moieties; in U.S. Pat. No. 5,858,988, which describes hydrophobic carrier agents attached to the 2'-O position of oligonucleotides for enhanced membrane permeability and stability; in U.S. Pat. No. 5,214,136, which describes oligonucleotides conjugated to anthraquinones at the 5' end with enhanced hybridization to DNA or RNA; and in U.S. Pat. No. 5,214,136, which describes oligonucleotides conjugated to anthraquinones at the 5' end with enhanced nuclease resistance, binding affinity, and RNase activity. No. 5,700,922, which describes PNA-DNA-PNA chimeras in which the DNA contains 2'-deoxy-erythro-pentofuranosyl nucleotides for their ability to activate H, and U.S. Pat. No. 5,708,154, which describes RNA linked to DNA to form a DNA-RNA hybrid. E. Polyether and Peptide Nucleic Acids

[0133] In certain embodiments, it is contemplated that the nucleic acid comprising nucleoside or nucleotide derivative or analogue can be used in the disclosed method and composition.A non-limiting example is the "polyether nucleic acid" described in U.S. Patent No. 5,908,845, which is incorporated herein by reference.In polyether nucleic acid, one or more nucleobases are linked to asymmetric carbon atoms in polyether backbone.

[0134] Another non-limiting example is "peptide nucleic acid", also known as "PNA", "peptide-based nucleic acid analog", or "PENAM", described in U.S. Patent Nos. 5,786,461, 5891,625, 5,773,571, 5,766,855, 5,736,336, 5,719,262, 5,714,331, 5,539,082, and International Publication No. WO 92 / 20702, each of which is incorporated herein by reference.Peptide nucleic acid generally has enhanced sequence specificity, binding properties, and resistance to enzymatic degradation compared to molecules such as DNA and RNA (see, for example, Egholm et al., 1993, PCT / EP / 01219). Peptide nucleic acids generally comprise one or more nucleotides or nucleosides, including a nucleic acid base portion, a nucleic acid base linker portion that is not a pentose sugar, and / or a backbone portion that is not a phosphate backbone portion. Examples of nucleic acid base linker portions described for PNA include aza nitrogen atoms, amides, and / or ureido tethers (see, for example, U.S. Patent No. 5,539,082). Examples of backbone portions described for PNA include aminoethylglycine, polyamide, polyethyl, polythioamide, polysulfinamide, or polysulfonamide backbone portions.

[0135] In certain embodiments, nucleic acid analogs, such as peptide nucleic acids, can be used to inhibit nucleic acid amplification, such as in PCR, to reduce false positives and distinguish between single base mutants, as described in U.S. Pat. No. 5,891,625.Other modifications and uses of nucleic acid analogs are known in the art and are encompassed herein.In a non-limiting example, U.S. Pat. No. 5,786,461 describes PNAs with amino acid side chains attached to the PNA backbone to enhance the solubility of the molecule.In another example, the cellular uptake properties of PNAs are increased by the attachment of lipophilic groups.U.S. Application No. 117,363 describes some alkylamino moieties that are used to enhance the cellular uptake of PNAs. Further examples are described in U.S. Pat. Nos. 5,766,855, 5,719,262, 5,714,331, and 5,736,336, which describe PNAs containing naturally occurring and non-naturally occurring nucleobases and alkylamine side chains that provide improved sequence specificity, solubility, and / or binding affinity compared to naturally occurring nucleic acids. F. Preparation of Nucleic Acids

[0136] Nucleic acid can be produced by any technique known to those skilled in the art, for example, chemical synthesis, enzymatic production, or biological production.Non-limiting examples of synthetic nucleic acid (e.g., synthetic oligonucleotide) include the nucleic acid produced by in vitro chemical synthesis using phosphotriester, phosphite, or phosphoramidite chemical reaction and solid phase technology, for example, the nucleic acid produced by the one described in European Patent No. EP 266,032, which is incorporated herein by reference, or the nucleic acid produced through deoxynucleoside H-phosphonate intermediates described by Froehler et al., 1986 and US Patent No. 5,705,629, each of which is incorporated herein by reference.In the method of the present disclosure, one or more oligonucleotides can be used. A variety of different mechanisms of oligonucleotide synthesis are disclosed, for example, in U.S. Pat. Nos. 4,659,774, 4,816,571, 5,141,813, 5,264,566, 4,959,463, 5,428,148, 5,554,744, 5,574,146, and 5,602,244, each of which is incorporated herein by reference.

[0137] Non-limiting examples of enzymatically produced nucleic acids include those produced by enzymes in amplification reactions such as PCR™ (see, e.g., U.S. Pat. Nos. 4,683,202 and 4,682,195, each of which is incorporated herein by reference), or the synthesis of oligonucleotides as described in U.S. Pat. No. 5,645,897, which is incorporated herein by reference. Non-limiting examples of biologically produced nucleic acids include recombinant nucleic acids produced (i.e., replicated) in living cells, such as recombinant DNA vectors replicated in bacteria (see, e.g., Sambrook et al. 1989, which is incorporated herein by reference). G. Nucleic Acid Purification

[0138] Nucleic acids can be purified by polyacrylamide gels, cesium chloride centrifugation gradients, or any other means known to those of skill in the art (see, e.g., Sambrook et al., 1989, which is incorporated herein by reference).

[0139] In certain aspects, the present disclosure relates to nucleic acid that is isolated nucleic acid.As used herein, the term "isolated nucleic acid" refers to a nucleic acid molecule (e.g., RNA or DNA molecule) that is isolated or otherwise free from the majority of the total genomic and transcribed nucleic acids of one or more cells.In certain embodiments, "isolated nucleic acid" refers to a nucleic acid that is isolated or otherwise free from the majority of cellular components or in vitro reaction components, such as, for example, macromolecules, such as lipids or proteins, biological small molecules. H. Nucleic Acid Segments

[0140] In certain embodiments, the nucleic acid is a nucleic acid segment. As used herein, the term "nucleic acid segment" refers to a small fragment of a nucleic acid, such as, but not limited to, one that codes for only a portion of a peptide or polypeptide sequence. Thus, a "nucleic acid segment" can include any portion of a gene sequence, from about 2 nucleotides to the full length of a peptide or polypeptide coding region.

[0141] Various nucleic acid segments can be designed based on a particular nucleic acid sequence and can be of any length. For example, an algorithm can be created that defines all nucleic acid segments by assigning numerical values ​​to the sequence, such as 1 for the first residue, 2 for the second residue, etc.

[0142] From n to n+y

[0143] Here, n is an integer from 1 to the last number of the sequence, y is the length of the nucleic acid segment minus 1, and n+y does not exceed the last number of the sequence. Thus, for a 10-mer, the nucleic acid segment corresponds to bases 1 to 10, 2 to 11, 3 to 12, etc. For a 15-mer, the nucleic acid segment corresponds to bases 1 to 15, 2 to 16, 3 to 17, etc. For a 20-mer, the nucleic acid segment corresponds to bases 1 to 20, 2 to 21, 3 to 22, etc. In certain embodiments, the nucleic acid segment can be a probe or primer. As used herein, a "probe" generally refers to a nucleic acid used in a detection method or composition. As used herein, a "primer" generally refers to a nucleic acid used in an extension or amplification method or composition. VI. Nucleic Acid-Based Expression Systems

[0144] In specific embodiments of the disclosure, p63-TID (or a functional fragment and / or derivative thereof) (e.g., a polypeptide or nucleic acid), in some cases one or more cardiac cell reprogramming factors (e.g., Hand2 and / or myocardin), and in some cases one or more destabilizing agents and / or anti-fibrotic agents and / or angiogenic factors are provided to an individual in need thereof in the form of a nucleic acid. In some cases, the nucleic acid is not contained on a vector, but in specific embodiments, the nucleic acid is present on one or more vectors. In specific embodiments, the different nucleic acids are present on the same vector, while in other cases they are present on two or three separate vectors. The vectors may be viral or non-viral. Figures 4A-B provide illustrations of embodiments of vectors for use in the methods of the disclosure.

[0145] The vectors utilized in the embodiments of the present disclosure may have one or more means for targeted delivery to cardiac tissue and / or targeted expression in certain cells. In some cases, the vector is provided to an individual with local delivery to the heart, while in other cases, the vector is provided to an individual systemically with means for targeted delivery to cardiac tissue and / or targeted expression in certain cells, such as cardiac fibroblasts, for example. In certain embodiments, p63-TID (or its functional fragment and / or functional derivative) and one or both of cardiac cell reprogramming factor (e.g., Hand2 and myocardin) polynucleotides are on the same molecule, while in some embodiments, p63-TID (or its functional fragment and / or functional derivative) and one or both of cardiac cell reprogramming factor polynucleotides are on different molecules. When p63-TID (or its functional fragment and / or functional derivative) and one or both of cardiac cell reprogramming factor are expressed from the same polynucleotide, they may have the same or different regulatory regions for their expression. In certain embodiments, the p63-TID (or a functional fragment and / or functional derivative thereof) and the one or more chromatin destabilizing agent polynucleotides are on the same or different molecules.

[0146] In a specific embodiment, an expression vector for use in the present disclosure may include one or more suitable restriction enzyme digestion sequences, initiation codons, termination codons, nuclear localization signals, protease cleavage codons, selectable markers, replication origins, regulatory regions, multiple cloning sites, and combinations thereof. Such moieties may be positioned in any suitable order in the expression vector. A. Vector

[0147] The term "vector" is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell, where it can be replicated. A nucleic acid sequence can be "exogenous", meaning that it is foreign to the cell into which the vector is introduced, or that the sequence is homologous to the sequence in the cell, but is at a location in the host cell nucleic acid where the sequence is not found in nature. Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). Those skilled in the art will be well versed in constructing vectors through standard recombinant techniques (see, e.g., Maniatis et al., 1988 and Ausubel et al., 1994, both of which are incorporated herein by reference).

[0148] The term "expression vector" refers to any kind of genetic construct that contains a nucleic acid that codes for an RNA that can be transcribed. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, such as in the case of the production of antisense molecules or ribozymes. Expression vectors can contain various "control sequences," which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host cell. In addition to control sequences that govern transcription and translation, vectors and expression vectors can contain nucleic acid sequences, described below, that perform other functions as well. 1. Promoters and Enhancers

[0149] A "promoter" is a control sequence that is a region of a nucleic acid sequence where the initiation and rate of transcription are controlled. It can include genetic elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, bind to initiate specific transcription of a nucleic acid sequence. The phrases "operably positioned," "operably linked," "under control," and "under transcriptional control" mean that the promoter is in the correct functional location and / or orientation relative to the nucleic acid sequence so as to control the initiation and / or expression of the transcription of the nucleic acid sequence.

[0150] In embodiments of the present disclosure, a CMV promoter or a tissue-specific promoter may be used. The tissue-specific promoter may be a cardiac tissue-specific promoter. Examples of cardiac tissue-specific promoters include, but are not limited to, ventricular specific myosin light chain-2 (mlc-2v) and / or alpha-myosin heavy chain (α-MHC). In certain embodiments, a fibroblast-specific promoter is utilized. Examples of fibroblast-specific promoters include, but are not limited to, Fsp1 and / or periostin.

[0151] Promoters generally contain sequences that function to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters that lack a TATA box, such as the promoters of the mammalian terminal deoxynucleotidyl transferase gene and the promoters of the SV40 late genes, separate elements that overlap the start site itself help to anchor the start location. Additional promoter elements regulate the frequency of transcription initiation. Typically, these are located in the region 30-110 bp upstream of the start site, but some promoters have been shown to contain functional elements downstream of the start site as well. To bring a coding sequence "under the control" of a promoter, the 5' end of the transcription start site of the transcriptional reading frame is positioned "downstream" (i.e., 3') of the selected promoter. The "upstream" promoter stimulates transcription of the DNA and promotes expression of the encoded RNA.

[0152] In many cases, the spacing between promoter elements is flexible, so that promoter function is maintained even when elements are inverted or moved relative to one another. In the case of the tk promoter, the spacing between promoter elements can be increased to 50 bp apart, after which activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription. Promoters may or may not be used in conjunction with "enhancers," which refer to cis-acting regulatory sequences involved in the transcriptional activation of a nucleic acid sequence.

[0153] A promoter may be one that is naturally associated with a nucleic acid sequence, such as can be obtained by isolating the 5' non-coding sequence located upstream of a coding segment and / or exon. Such a promoter may be referred to as "endogenous". Similarly, an enhancer may be one that is naturally associated with a nucleic acid sequence, located either downstream or upstream of the nucleic acid sequence. Alternatively, certain advantages may be obtained by placing the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer that is not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, as well as promoters or enhancers isolated from any other virus or prokaryotic or eukaryotic cell, as well as promoters or enhancers that are not "naturally occurring", i.e., that contain different elements of different transcriptional regulatory regions and / or mutations that alter expression. For example, promoters most commonly used in recombinant DNA construction include the β-lactamase (penicillinase), lactose, and tryptophan (trp) promoter systems. In addition to producing promoter and enhancer nucleic acid sequences synthetically, the sequences may be produced using recombinant cloning and / or nucleic acid amplification techniques, including PCR™, in conjunction with the compositions disclosed herein (see U.S. Pat. Nos. 4,683,202 and 5,928,906, each of which is incorporated herein by reference). Furthermore, it is contemplated that control sequences that direct transcription and / or expression of sequences in non-nuclear organelles, such as mitochondria, chloroplasts, etc., can be used as well.

[0154] Naturally, it will be important to use a promoter and / or enhancer that effectively induces the expression of the DNA segment in the organelle, cell type, tissue, organ, or organism selected for expression. Those skilled in the art of molecular biology generally understand the use of promoter, enhancer, and cell type combinations for protein expression (see, for example, Sambrook et al. 1989, which is incorporated herein by reference). The promoter used may be constitutive, tissue-specific, inducible, and / or useful for inducing high-level expression of the introduced DNA segment under appropriate conditions, for example, beneficial in large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

[0155] In addition, any promoter / enhancer combination (e.g., as found in the Eukaryotic Promoter Database EPDB, http: / / www.epd.isb-sib.ch / ) can also be used to drive expression. The use of T3, T7, or SP6 cytoplasmic expression systems is another possible embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional gene expression construct. 2. Initiation signals and internal ribosome binding sites

[0156] Specific initiation signals may also be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. Those skilled in the art will be able to easily determine this and provide the necessary signals. It is well known that the initiation codon must be in frame with the reading frame of the desired coding sequence to ensure translation of the entire insert. Exogenous translational control signals and initiation codons may be either natural or synthetic. Expression efficiency can be enhanced by the inclusion of appropriate transcriptional enhancer elements.

[0157] In certain embodiments of the present disclosure, the use of internal ribosome entry site (IRES) elements is used to create multigene or polycistronic messages. IRES elements can bypass the ribosome scanning model of translation that relies on 5' methylated Cap and initiate translation at internal sites (see, e.g., Pelletier and Sonenberg, 1988). IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as well as IRES from mammalian messages (see, e.g., Macejak and Sarnow, 1991). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, to create polycistronic messages. By utilizing an IRES element, each open reading frame can be accessed by ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter / enhancer to transcribe a single message (see U.S. Pat. Nos. 5,925,565 and 5,935,819, each of which is incorporated herein by reference). 3. Multiple Cloning Site

[0158] A vector may contain a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used with standard recombinant techniques to digest the vector (see, e.g., Carbonelli et al., 1999; Levenson et al., 1998; and Cocea, 1997, which are incorporated herein by reference). "Restriction enzyme digestion" refers to the catalytic cleavage of a nucleic acid molecule with an enzyme that functions only at a specific location within the nucleic acid molecule. Many of these restriction enzymes are commercially available. The use of such enzymes is well understood by those of skill in the art. Often, vectors are linearized or fragmented using a restriction enzyme that cuts within the MCS so that exogenous sequences can be ligated into the vector. "Ligation" refers to the process of forming phosphodiester bonds between two nucleic acid fragments, which may or may not be contiguous with each other. Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology. 4. Splicing Sites

[0159] Most transcribed eukaryotic RNA molecules will undergo RNA splicing to remove introns from the primary transcript. Vectors containing genomic eukaryotic sequences may require donor and / or acceptor splice sites to ensure proper processing of the transcript for protein expression (see, e.g., Chandler et al., 1997, which is incorporated herein by reference). 5. Termination signal

[0160] The vector or construct of the present disclosure will generally include at least one termination signal. "Termination signal" or "terminator" is composed of the DNA sequence that is involved in the specific termination of RNA transcript by RNA polymerase. Thus, in certain embodiments, a termination signal that ends the production of RNA transcript is contemplated. Terminator may be required in vivo to achieve desired message level.

[0161] In eukaryotic systems, the terminator region may also contain specific DNA sequences that allow site-specific cleavage of the new transcript to expose a polyadenylation site. This signals a specialized endogenous polymerase to add a stretch of about 200 A residues (polyA) to the 3' end of the transcript. RNA molecules modified with this polyA tail are believed to be more stable and more efficiently translated. Thus, in other embodiments involving eukaryotes, it is preferred that the terminator contains a signal for cleavage of the RNA, and more preferred that the terminator signal promotes polyadenylation of the message. The terminator and / or polyadenylation site elements may function to enhance message levels and minimize read-through from the cassette to other sequences.

[0162] Terminators contemplated for use in the present disclosure include any known terminator described herein or known to those skilled in the art, including, but not limited to, gene termination sequences, such as, for example, the bovine growth hormone terminator, or viral modified sequences, such as, for example, the SV40 terminator. In certain embodiments, the termination signal may be absent from a transcribable or translatable sequence, such as those resulting from sequence truncation. 6. Polyadenylation Signal

[0163] During expression, especially during eukaryotic expression, a polyadenylation signal will typically be included to achieve proper polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be critical to the successful implementation of the present disclosure, and any such sequence can be used. Preferred embodiments include the SV40 polyadenylation site signal or the bovine growth hormone polyadenylation signal, which are conventional and known to function well in various target cells. Polyadenylation can increase the stability of the transcript or facilitate cytoplasmic transport. 7. Origin of replication

[0164] In order for a vector to propagate in a host cell, it may contain one or more origins of replication (often referred to as "ori"), which are specific nucleic acid sequences at which replication is initiated. Alternatively, when the host cell is yeast, an autonomously replicating sequence (ARS) can be used. 8. Selectable and Screenable Markers

[0165] In certain embodiments of the present disclosure, cells containing the nucleic acid constructs of the present disclosure can be identified in vitro or in vivo by including a marker in the expression vector. Such a marker will confer an identifiable change to the cell, allowing easy identification of cells containing the expression vector. Generally, a selectable marker is one that confers a property that allows selection. A positive selectable marker is one whose presence allows its selection, while a negative selectable marker is one whose presence prevents its selection. An example of a positive selectable marker is a drug resistance marker.

[0166] Typically, the inclusion of a drug selection marker aids in cloning and identification of transformants; for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol are useful selectable markers. In addition to markers that confer a phenotype that allows for differentiation of transformants based on state implementation, other types of markers are also contemplated, including screenable markers such as GFP based on colorimetric analysis. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized. Those skilled in the art will also know how to use immunogenic markers, possibly with FACS analysis. The marker used is not believed to be critical, so long as it can be expressed simultaneously with the nucleic acid encoding the gene product. Further examples of selectable and screenable markers are well known to those skilled in the art. 9. Additional Characteristics

[0167] In some embodiments, the vector and / or nucleic acid construct of the present disclosure may include a 2A element or sequence. In some embodiments, the vector and / or nucleic acid construct of the present disclosure may include one or more cloning sites. In some such embodiments, the cloning site may not be completely removed before preparing for administration to a subject. In some embodiments, the cloning site may have a functional role, including as a linker sequence or as part of a Kozak site. As will be understood by those skilled in the art, the cloning site may vary significantly in primary sequence but retain their desired function.

[0168] In some embodiments, the 2A element is a T2A, P2A, E2A, and / or F2A element. In some embodiments, the 2A sequence may include an optional 5' linker sequence, such as, but not limited to, GSG (glycine, serine, glycine). SEQ ID NO:7 - Exemplary T2A amino acid sequence EGRGSLLTCGDVEENPGP SEQ ID NO:8 - Exemplary P2A amino acid sequence ATNFSLLKQAGDVEENPGP SEQ ID NO:9 - Exemplary E2A amino acid sequence QCTNYALLKLAGDVESNPGP SEQ ID NO:10 - Exemplary F2A amino acid sequence VKQTLNFDLLKLAGDVESNPGP SEQ ID NO:11 - Exemplary P2A nucleotide sequence GCCACAAACTTCAGCCTGCTGAACAGGCTGGCGACGTGGAAGAGAACCCTGGCCCTTCTAGA B. Plasmid Vectors

[0169] In certain embodiments, plasmid vectors are intended for use in transforming host cells. In general, plasmid vectors containing replicon and control sequences derived from species compatible with the host cell are used in connection with these hosts. Vectors usually have a replication site, as well as a marking sequence that can provide phenotypic selection in transformed cells. In a non-limiting example, E. coli is often transformed using a derivative of pBR322, a plasmid derived from E. coli species. pBR322 contains genes for ampicillin and tetracycline resistance, thus providing an easy means for identifying transformed cells. pBR plasmids or other microbial plasmids or phages must also contain, or be modified to contain, promoters that the microorganism can use to express its own proteins, for example.

[0170] In addition, phage vectors containing replicon and control sequences compatible with host microorganisms can be used as transformation vectors in connection with these hosts. For example, phage lambda GEM™-11 can be utilized in the production of recombinant phage vectors that can be used to transform host cells such as, for example, E. coli LE392.

[0171] Genome-integrating plasmids, such as piggyBac or Sleeping Beauty transposon gene delivery plasmids, can be used for long-term transgenic expression of nucleic acids in the heart or other organs.

[0172] Additional useful plasmid vectors include the pIN vectors (Inouye et al., 1985), as well as the pGEX vectors for use in generating glutathione S-transferase (GST) soluble fusion proteins for subsequent purification and isolation or cleavage. Other suitable fusion proteins are those with β-galactosidase, ubiquitin, and the like.

[0173] Bacterial host cells, such as E. coli, containing the expression vector are grown in any of a number of suitable media, such as LB. Expression of the recombinant protein in a particular vector can be induced by contacting the host cells with an agent specific for a particular promoter, such as adding IPTG to the medium or switching the incubation to a higher temperature, as will be understood by those skilled in the art. After culturing the bacteria for an additional period, typically 2-24 hours, the cells are harvested by centrifugation and washed to remove residual medium. C. Viral Vectors

[0174] The ability of certain viruses to infect or enter cells by receptor-mediated endocytosis, as well as the ability to integrate into host cell genome and express viral genes stably and efficiently, makes them attractive candidates for transferring foreign nucleic acid into cells (e.g., mammalian cells).The non-limiting examples of viral vectors that can be used to deliver the nucleic acid of the present disclosure are described below. 1. Adenovirus Vector

[0175] A specific method for delivery of nucleic acids involves the use of adenoviral expression vectors. Adenoviral vectors are known to have a low capacity for integration into genomic DNA, but this property is balanced by the high efficiency of gene transfer afforded by these vectors. "Adenoviral expression vector" means to include a construct that contains sufficient adenoviral sequences to (a) aid in packaging the construct and (b) ultimately express the tissue- or cell-specific construct cloned therein. Knowledge of the genetic organization of adenovirus, a 36 kb linear double-stranded DNA virus, allows replacement of large pieces of adenoviral DNA with foreign sequences up to 7 kb in length (Grunhaus and Horwitz, 1992). 2. AAV Vectors

[0176] Nucleic acid may be introduced into cells using adenovirus-assisted transfection. Increased transfection efficiency has been reported in cell lines using adenovirus-linked systems (Kelleher and Vos, 1994; Cotten et al., 1992; Curiel, 1994). Adeno-associated virus (AAV) is a promising vector system for use in embodiments of the present disclosure, since it has a high integration frequency and can infect non-dividing cells, making it useful for delivering genes to mammalian cells, for example, in tissue culture (Muzyczka, 1992) or in vivo. AAV has a wide host range for infection (Tratschin et al., 1984; Laughlin et al., 1986; Lebkowski et al., 1988; McLaughlin et al., 1988). Details regarding the generation and use of rAAV vectors are described in U.S. Patent Nos. 5,139,941 and 4,797,368, each of which is incorporated herein by reference. In certain embodiments, AAV vectors (including, for example, rAAV vectors) are specific and / or have increased specificity for muscle cells and / or cardiomyocytes compared to a suitable control vector. 3. Retroviral Vectors

[0177] Retroviruses are promising delivery vectors due to their ability to integrate genes into the host genome, transfer large amounts of foreign genetic material, infect a wide range of species and cell types, and be packaged in specialized cell lines (Miller, 1992).

[0178] To construct retroviral vectors, nucleic acids are inserted into the viral genome in place of certain viral sequences to produce viruses that are replication-defective. To produce virions, packaging cell lines are constructed that contain gag, pol, and env genes but lack LTR and packaging components (Mann et al., 1983). When recombinant plasmids containing cDNA are introduced into special cell lines together with retroviral LTRs and packaging sequences (e.g., by calcium phosphate precipitation, for example), the packaging sequences allow the RNA transcripts of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture medium (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The medium containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors can infect a wide range of cell types. However, integration and stable expression require the division of host cells (Paskind et al., 1975).

[0179] Lentivirus is a complex retrovirus that contains other genes with regulatory or structural functions in addition to the general retroviral genes gag, pol, and env.Lentivirus vectors are well known in the art (see, for example, Naldini et al., 1996, Zufferey et al., 1997, Blomer et al., 1997, U.S. Pat. Nos. 6,013,516 and 5,994,136).Some examples of lentivirus include human immunodeficiency virus HIV-1, HIV-2, and simian immunodeficiency virus SIV.Lentivirus vectors have been produced by multiple attenuation of HIV toxic genes, for example, genes env, vif, vpr, vpu, and nef are deleted to make the vector biologically safe.

[0180] Recombinant lentiviral vectors can infect non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression of nucleic acid sequences. For example, recombinant lentiviruses capable of infecting non-dividing cells, transfecting suitable host cells with two or more vectors having packaging functions, i.e., gag, pol, and env, and rev and tat, are described, for example, in U.S. Patent No. 5,994,136, which is incorporated herein by reference. Recombinant viruses can be targeted by linking the envelope protein with an antibody or a specific ligand for targeting to a receptor of a specific cell type. For example, the vector becomes target-specific by inserting the sequence of interest (including the regulatory region) into the viral vector together with another gene that codes for a ligand for a receptor on a specific target cell. 4. Other viral vectors

[0181] Other viral vectors can be used as vaccine constructs in the present disclosure.Vector derived from viruses, such as vaccinia virus (see, for example, Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988), Sindbis virus, cytomegalovirus, and herpes simplex virus can be used.They offer several attractive properties for various mammalian cells (see, for example, Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990). D. Delivery Using Modified Viruses

[0182] The nucleic acid to be delivered may be housed in an infectious virus that has been engineered to express a specific binding ligand. The viral particle will then specifically bind to the cognate receptor of the target cell and deliver its contents to the cell. A novel approach designed to allow specific targeting of retroviral vectors has been developed based on the chemical modification of retroviruses by chemical addition of lactose residues to the viral envelope. This modification may allow specific infection of liver cells via sialoglycoprotein receptors.

[0183] Another approach for targeting recombinant retroviruses has been designed, using biotinylated antibodies against retroviral envelope proteins and specific cell receptors. The antibodies are linked through the biotin moiety by using streptavidin (see, for example, Roux et al., 1989). Using antibodies against major histocompatibility complex class I and class II antigens, they demonstrated the infection of various human cells bearing these surface antigens by ecotropic viruses in vitro (see, for example, Roux et al., 1989). E. Vector Delivery and Cell Transformation

[0184] Methods suitable for nucleic acid delivery for transformation of an organelle, cell, tissue, or organism for use in the present disclosure are contemplated to include virtually any method by which a nucleic acid (e.g., DNA) can be introduced into an organelle, cell, tissue, or organism, as described herein or known to those of skill in the art. Such methods include direct delivery of DNA, e.g., by ex vivo transfection (see, e.g., Wilson et al., 1989; Nabel et al, 1989), microinjection (see, e.g., Harlan and Weintraub, Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466, and 5,580,859, each of which is incorporated herein by reference), including by injection (see, e.g., U.S. Pat. Nos. 5,384,253, Tur-Kaspa et al., 1986, Potter et al., 1984, which are incorporated herein by reference), by calcium phosphate precipitation (see, e.g., Graham and Van Der Eb, 1973, Chen and Okayama, 1987, Rippe et al., 1990), DEAE-dextran followed by polyethylene glycol (see, e.g., Gopal, 1985), by direct sonic loading (see, e.g., Fechheimer et al., 1987), by liposome-mediated transfection (see, e.g., Nicolau and Sene, 1982, Fraley et al., 1979, Nicolau et al., 1987, Wong et al., 1980, Kaneda et al., 1989, Kato et al., 1991) and receptor-mediated transfection (see, e.g., Wu and Wu, 1987; Wu and Wu, 1988), by particle bombardment (see, e.g., PCT Application Nos. WO 94 / 09699 and 95 / 06128; U.S. Pat. Nos. 5,610,042; 5,322,783; 5,563,055; 5,550,318; 5,538,877; and 5,538,880, each of which is incorporated herein by reference), by agitation with silicon carbide fibers (see, e.g., Kaeppler et al., 1990, U.S. Patent Nos. 5,302,523 and 5,464,765, each of which is incorporated herein by reference), by Agrobacterium-mediated transformation (e.g., U.S. Patent Nos. 5,591,616 and 5,563,055, each of which is incorporated herein by reference), by PEG-mediated protoplast transformation (e.g., Omirulleh et al., 1993, U.S. Patent Nos. 4,684,611 and 4,952,500, each of which is incorporated herein by reference), by desiccation / inhibition-mediated DNA uptake (e.g., Potrykus et al., 1985), and any combination of such methods. Through the application of such techniques, organelles, cells, tissues, or organisms can be stably or transiently transformed. 1. Ex Vivo Transformation

[0185] Methods for transfecting vascular cells and tissues removed from an organism in an ex vivo environment are known to those skilled in the art. For example, canine endothelial cells have been genetically modified by retroviral gene transfer in vitro and transplanted into a dog (see, for example, Wilson et al., 1989). In another example, Yucatan miniature pig endothelial cells have been transfected by retrovirus in vitro and transplanted into an artery using a double balloon catheter (see, for example, Nabel et al., 1989). Thus, it is contemplated that cells or tissues can be removed and transfected ex vivo using the nucleic acid of the present disclosure. In a specific embodiment, the transplanted cells or tissues can be placed into an organism. In a preferred aspect, the nucleic acid is expressed in the transplanted cells or tissues. 2. Injection

[0186] In certain embodiments, the nucleic acid can be delivered to an organelle, cell, tissue, or organism by one or more injections (i.e., needle injections), for example, intramyocardially, endocardially, epicardially, intracoronary, by direct injection or intracoronary injection, subcutaneously, intradermally, intramuscularly, intravenously, intraperitoneally, etc. In certain embodiments, p63-TID is delivered directly to the heart by injection. Methods of injection are well known to those skilled in the art (e.g., injection of a composition containing saline). Further embodiments of the present disclosure include the introduction of nucleic acid by direct microinjection. 3. Electroporation

[0187] In certain embodiments of the present disclosure, nucleic acid is introduced into organelles, cells, tissues, or organisms by electroporation. Electroporation involves exposing a suspension of cells and DNA to high-voltage discharge. In some variations of this method, certain cell wall degrading enzymes, such as pectin degrading enzymes, are used to make target recipient cells more susceptible to transformation by electroporation than untreated cells (see, for example, U.S. Patent No. 5,384,253, which is incorporated herein by reference). Alternatively, recipient cells can be made more susceptible to transformation by mechanical damage.

[0188] Transfection of eukaryotic cells using electroporation has been extremely successful: mouse pre-B lymphocytes have been transfected with the human kappa immunoglobulin gene (see, e.g., Potter et al., 1984) and rat hepatocytes with chloramphenicol acetyltransferase (see, e.g., Tur-Kaspa et al., 1986) in this manner.

[0189] To achieve transformation by electroporation in cells, such as, for example, plant cells, friable tissues, such as cell suspension cultures or embryonic calli, may be utilized, or alternatively, immature embryos or other organized tissues may be transformed directly. In this technique, the cell walls of selected cells are partially degraded by exposing them to pectin-degrading enzymes (pectolyases) or by mechanically damaging them in a controlled manner. Examples of some species that have been transformed by electroporation of intact cells include corn (see, e.g., U.S. Pat. No. 5,384,253; Rhodes et al., 1995; D'Halluin et al., 1992), wheat (see, e.g., Zhou et al., 1993), tomato (see, e.g., Hou and Lin, 1996), soybean (see, e.g., Christou et al., 1987), and tobacco (see, e.g., Lee et al., 1989).

[0190] Protoplasts may also be used for electroporation transformation of plant cells (see, e.g., Bates, 1994; Lazzeri, 1995). For example, the generation of transgenic soybean plants by electroporation of cotyledon-derived protoplasts is described by Dhir and Widholm in International Patent Application No. WO 9217598, which is incorporated herein by reference. Other examples of species for which protoplast transformation has been described include barley (see, e.g., Lazerri, 1995), sorghum (see, e.g., Battraw et al., 1991), maize (see, e.g., Bhattacharjee et al., 1997), wheat (see, e.g., He et al., 1994), and tomato (see, e.g., Tsukada, 1989). 4. Calcium phosphate

[0191] In another embodiment of the present disclosure, nucleic acid is introduced into cells using calcium phosphate precipitation.Using this technique, human KB cells have been transfected with adenovirus 5 DNA (see, for example, Graham and Van Der Eb, 1973).Also, in this manner, mouse L(A9), mouse C127, CHO, CV-1, BHK, NIH3T3 and HeLa cells have been transfected with neomycin marker gene (see, for example, Chen and Okayama, 1987), and rat hepatocytes have been transfected with various marker genes (see, for example, Rippe et al., 1990). 5. DEAE-Dextran

[0192] In another embodiment, the nucleic acid is delivered to the cells using DEAE-dextran followed by polyethylene glycol. In this manner, reporter plasmids have been introduced into mouse myeloma and erythroleukemia cells (see, e.g., Gopal, 1985). 6. Sonication Loading

[0193] Additional embodiments of the present disclosure include the introduction of nucleic acids by direct sonication loading. LTK-fibroblasts have been transfected with the thymidine kinase gene by sonication loading (see, e.g., Fechheimer et al., 1987). 7. Liposome-mediated Transfection

[0194] In further embodiments of the present disclosure, nucleic acid may be encapsulated in lipid complexes, such as liposomes, for example. Liposomes are vesicular structures characterized by a bilayer membrane of phospholipids and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. Self-rearrangement of lipid components occurs, followed by the formation of closed structures, encapsulating water and dissolved solutes between the lipid bilayers (see, for example, Ghosh and Bachhawat, 1991). Nucleic acid complexed with Lipofectamine (Gibco BRL) or Superfect (Qiagen) is also contemplated.

[0195] Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful (see, for example, Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987). The feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa, and hepatoma cells has also been demonstrated (see, for example, Wong et al., 1980).

[0196] In certain embodiments of the present disclosure, the liposome may be complexed with hemagglutinating virus (HVJ). This has been shown to promote fusion with cell membranes and facilitate cell entry of DNA encapsulated in liposomes (see, e.g., Kaneda et al., 1989). In other embodiments, the liposome may be complexed with or used together with nuclear non-histone chromosomal protein (HMG-1) (see, e.g., Kato et al., 1991). In yet further embodiments, the liposome may be complexed with or used together with both HVJ and HMG-1. In other embodiments, the delivery vehicle may include a ligand and a liposome. 8. Receptor-mediated Transfection

[0197] Still further, nucleic acids may be delivered to target cells via receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis that may occur in target cells. Given the cell type-specific distribution of various receptors, this delivery method adds an additional degree of specificity to the present disclosure.

[0198] Certain receptor-mediated gene targeting vehicles include cell receptor-specific ligands and nucleic acid binding agents. Others include cell receptor-specific ligands to which the nucleic acid to be delivered is operably linked. Several ligands have been used in receptor-mediated gene transfer (see, for example, Wu and Wu, 1987; Wagner et al., 1990; Perales et al., 1994; Myers, EPO 0273085), which establishes the operability of this technique. Specific delivery in the context of other mammalian cell types has been described (see, for example, Wu and Wu, 1993, which is incorporated herein by reference). In certain embodiments of the present disclosure, the ligand will be selected to correspond to a receptor that is specifically expressed in the target cell population.

[0199] In other embodiments, the nucleic acid delivery vehicle component of the cell-specific nucleic acid targeting vehicle may include a specific binding ligand in combination with a liposome. The nucleic acid to be delivered is housed within the liposome, and the specific binding ligand is functionally incorporated into the liposome membrane. The liposome will then specifically bind to the receptor of the target cell and deliver its contents to the cell.

[0200] In yet another embodiment, the nucleic acid delivery vehicle component of the targeted delivery vehicle may be a liposome itself, which will preferably contain one or more lipids or glycoproteins that induce cell-specific binding.For example, lactosyl-ceramide, a galactose-terminal asialganglioside, has been incorporated into liposomes, and increased uptake of insulin gene by hepatocytes has been observed (see, for example, Nicolau et al., 1987).It is contemplated that the tissue-specific transformation construct of the present disclosure can be specifically delivered to target cells in a similar manner. 9. Microparticle gun

[0201] Nucleic acid can be introduced into at least one organelle, cell, tissue, or organism using biolistic techniques (see, for example, U.S. Patent No. 5,550,318, U.S. Patent No. 5,538,880, U.S. Patent No. 5,610,042, and PCT Application No. WO 94 / 09699, each of which is incorporated herein by reference). This method relies on the ability to accelerate DNA-coated microprojectiles to high velocities to allow them to pierce cell membranes and enter cells without killing the cells (see, for example, Klein et al., 1987). There are a wide variety of biolistic techniques known in the art, many of which are applicable to the present disclosure.

[0202] Microprojectile bombardment can be used to transform a variety of cells, tissues, or organisms, such as, for example, any plant species. Examples of species that have been transformed by biolistic bombardment include monocotyledonous species such as maize (see, e.g., PCT Application No. WO 95 / 06128), barley (see, e.g., Ritala et al., 1994; Hensgens et al., 1993), wheat (see, e.g., U.S. Pat. No. 5,563,055, which is incorporated herein by reference), rice (see, e.g., Hensgens et al., 1993), oats (see, e.g., Torbet et al., 1995; Torbet et al., 1998), rye (see, e.g., Hensgens et al., 1993), sugarcane (see, e.g., Bower et al., 1992), and sorghum (see, e.g., Casas et al., 1993; Hagio et al., 1991), as well as several dicotyledonous plants including tobacco (see, e.g., Tomes et al., 1990; Buising and Benbow, 1994), soybean (see, e.g., U.S. Pat. No. 5,322,783, which is incorporated herein by reference), sunflower (see, e.g., Knittel et al. 1994), peanut (see, e.g., Singsit et al., 1997), cotton (see, e.g., McCabe and Martinell, 1993), tomato (see, e.g., VanEck et al. 1995), and common legumes (see, e.g., U.S. Pat. No. 5,563,055, which is incorporated herein by reference).

[0203] In this microprojectile, one or more particles may be coated with at least one nucleic acid and delivered to cells by propelling force. Several devices have been developed to accelerate small particles. One such device relies on high voltage discharge to generate an electric current, which provides the motive force (see, for example, Yang et al., 1990). The microprojectiles used are made of biologically inert materials, such as tungsten or gold particles or beads. Exemplary particles include those made of tungsten, platinum, and preferably gold. It is contemplated that in some cases, DNA precipitation onto metal particles will not be necessary for DNA delivery to recipient cells using microprojectiles. However, it is contemplated that particles may contain DNA rather than be coated with DNA. Although DNA-coated particles may increase the level of DNA delivery by microprojectiles, it is not necessary in itself.

[0204] For bombardment, the cells in suspension are concentrated on a filter or solid culture medium. Alternatively, immature embryos or other target cells may be placed on solid culture medium. The cells to be bombarded are positioned at an appropriate distance below the microprojectile stopping plate.

[0205] An exemplary embodiment of a method for delivering DNA to cells (e.g., plant cells) by acceleration is the Biolistics particle delivery system, which can be used to propel particles coated with DNA or cells through a screen, e.g., a stainless steel or Nytex screen, onto a filter surface covered with cells, e.g., monocotyledonous plant cells cultured in suspension. The screen disperses the particles so that they are not delivered to the recipient cells in large aggregates. It is believed that the screen interposed between the projectile and the cells to be shot reduces the size of the projectile aggregates, which may contribute to a higher frequency of transformation by reducing the damage that oversized projectiles cause to the recipient cells. VII. Proteins, Polypeptides, and Peptides

[0206] In some cases, embodiments may utilize p63-TID as a polypeptide, and optionally utilize one or more purified cardiac cell programming factors, such as Hand2, myocardin, Gata4, Mef2c, or Tbx5 proteins, polypeptides, or peptides, or one or more chromatin destabilizer proteins, polypeptides, or peptides, or other proteins, polypeptides, or peptides, in addition to or as an alternative to utilizing the respective nucleic acid forms. The term "purified protein, polypeptide, or peptide" as used herein refers to a protein composition that can be isolated from a mammalian cell or a recombinant host cell, in which at least one protein, polypeptide, or peptide has been purified to any degree relative to its naturally available state, i.e., relative to its purity in a cell extract. A purified protein, polypeptide, or peptide therefore also refers to a wild-type or mutant protein, polypeptide, or peptide that is free from the environment in which it naturally occurs.

[0207] Nucleotide, as well as protein, polypeptide, and peptide sequences of various genes have been previously disclosed and can be found in computerized databases known to those skilled in the art. One such database is the GENBANK® and GENPEPT® databases of the National Center for Biotechnology Information. The coding regions of these known genes can be amplified and / or expressed using the techniques disclosed herein or by any technique that would be known to those skilled in the art. In addition, peptide sequences can be synthesized by methods known to those skilled in the art, such as peptide synthesis using an automated peptide synthesizer, such as those available from Applied Biosystems (Foster City, Calif.).

[0208] Generally, "purified" refers to a particular protein, polypeptide, or peptide composition that has been subjected to a fractionation procedure to remove various other proteins, polypeptides, or peptides, and which composition substantially retains its activity as may be assessed, for example, by protein assays, as described below, or as known to those of skill in the art for the desired protein, polypeptide, or peptide.

[0209] When the term "substantially purified" is used, it will refer to a composition in which a particular protein, polypeptide, or peptide forms a major component of the composition, e.g., constitutes about 50% or more of the protein in the composition. In preferred embodiments, a substantially purified protein will constitute more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 99%, or even more of the protein in the composition.

[0210] As applied to this disclosure, a peptide, polypeptide, or protein "purified to homogeneity" means that the peptide, polypeptide, or protein has a level of purity such that the peptide, polypeptide, or protein is substantially free of other proteins and biological components. For example, a purified peptide, polypeptide, or protein will often be sufficiently free of other protein components that degradative sequencing can be successfully performed.

[0211] Various methods for quantifying the degree of purification of a protein, polypeptide, or peptide will be known to one of skill in the art in light of the present disclosure, including, for example, determining the specific protein activity of a fraction or assessing the number of polypeptides within a fraction by gel electrophoresis.

[0212] In order to purify a desired protein, polypeptide, or peptide, a natural or recombinant composition containing at least some of the particular protein, polypeptide, or peptide is subjected to fractionation to remove various other components from the composition. In addition to the techniques described in detail herein below, various other techniques suitable for use in protein purification will be known to those skilled in the art. These include, for example, precipitation with ammonium sulfate, PEG, antibodies, etc., or heat denaturation, followed by centrifugation, chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite, lectin affinity, and other affinity chromatography steps, isoelectric focusing, gel electrophoresis, and combinations of these and other techniques.

[0213] Another example is the purification of a particular fusion protein using a specific binding partner. Such purification methods are conventional in the art. Since the present disclosure provides the DNA sequence of a particular protein, any fusion protein purification method can be implemented here. This is exemplified by the generation of a particular protein-glutathione S-transferase fusion protein, expression in E. coli, and isolation to homogeneity using affinity chromatography on glutathione-agarose, or the generation of a polyhistidine tag on the N- or C-terminus of the protein, and then purification using Ni affinity chromatography. However, given that a large number of DNAs and proteins are known or can be identified and amplified using the methods described herein, any purification method can be utilized here.

[0214] Although contemplated for use in certain embodiments, there is no general requirement that a protein, polypeptide, or peptide always be provided in its most purified state. Indeed, it is contemplated that proteins, polypeptides, or peptides that are substantially less purified than their native state, but which are nonetheless enriched in a desired protein composition, will have utility in certain embodiments.

[0215] Methods exhibiting a lower degree of relative purity may have advantages in overall recovery of protein product, or in maintaining activity of the expressed protein. Inactive products also have utility in certain embodiments, for example, when determining antigenicity via antibody generation. VIII.Host cells

[0216] In some embodiments, the nucleic acids of the disclosure are provided directly to cardiac tissue and taken up by cells within the tissue, while in some embodiments the nucleic acids are first produced and engineered in cells ex vivo, e.g., by utilizing conventional recombinant technology methods.

[0217] As used herein, the terms "cell", "cell line", and "cell culture" may be used interchangeably. All of these terms include their progeny, any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. In the context of expressing heterologous nucleic acid sequences, "host cell" refers to a prokaryotic or eukaryotic cell, including any transformable organism capable of replicating a vector and / or expressing a heterologous gene encoded by the vector. A host cell can be and has been used as a recipient of a vector. A host cell can be "transfected" or "transformed", which refers to the process of transferring or introducing an exogenous nucleic acid into a host cell. A transformed cell includes the primary subject cell and its progeny. As used herein, the terms "engineered" and "recombinant" cells or host cells are intended to refer to a cell into which an exogenous nucleic acid sequence, such as, for example, a vector, has been introduced. Thus, a recombinant cell can be distinguished from a naturally occurring cell that does not contain a recombinantly introduced nucleic acid.

[0218] In certain embodiments, RNA or protein sequences can be co-expressed with other selected RNA or protein sequences in the same host cell. Co-expression can be achieved by co-transfecting two or more different recombinant vectors into host cells. Alternatively, a single recombinant vector can be constructed to contain multiple different coding regions of RNA or DNA (as active agent) or polypeptide (as active agent), which can then be expressed in a host cell transfected with a single vector.

[0219] The tissue may comprise a host cell or cells transformed with a polynucleotide or nucleic acid encoding p63-TID (or a functional fragment and / or derivative thereof), one or more cardiac cell reprogramming factors, and / or one or more chromatin destabilizing agents. In certain embodiments, the cells may have a polynucleotide encoding p63-TID (or a functional fragment and / or derivative thereof), Hand2, and / or myocardin. The tissue may be part of or separated from an organism. In certain embodiments, tissues may include, but are not limited to, muscle cells, adipocytes, alveoli, ameloblasts, axons, basal cells, blood (e.g., lymphocytes), blood vessels, bone, bone marrow, brain, breast, heart, cartilage, cervix, colon, cornea, embryo, endometrium, endothelium, epithelium, esophagus, facia, fibroblasts, follicles, ganglion cells, glial cells, goblet cells, kidney, liver, lung, lymph node, muscle, neurons, ovaries, pancreas, peripheral blood, prostate, skin, skin, small intestine, spleen, stem cells, stomach, testes, and the like.

[0220] In certain embodiments, the host cell or tissue can be contained in at least one organism. In certain embodiments, the organism can be a prokaryote (e.g., eubacteria, archaea) or a eukaryote, as will be understood by those skilled in the art (see, for example, the webpage http: / / phylogeny.arizona.edu / tree / phylogeny.html).

[0221] Numerous cell lines and cultures are available for use as host cells and are available through the American Type Culture Collection (ATCC), an organization that serves as a record of living cultures and genetic material. An appropriate host can be determined by one of skill in the art based on the vector backbone and the desired results. Plasmids or cosmids, for example, can be introduced into prokaryotic host cells for replication of many vectors. Cell types available for replication and / or expression of vectors include, but are not limited to, bacteria such as E. coli (e.g., E. coli strains RR1, E. coli LE392, E. coli B, E. coli X1776 (ATCC No. 31537), and E. coli W3110 (F-, lambda-, prototrophic, ATCC No. 273325), DH5α, JM109, and KC8, bacilli such as Bacillus subtilis, as well as other Enterobacter species such as Salmonella typhimurium, Serratia marcescens, various Pseudomonas species, and several commercially available bacterial hosts such as SURE® Competent Cells and SOLOPACK™ Gold Cells (STRATAGENE®, La Jolla). In certain embodiments, bacterial cells such as E. coli LE392 are specifically contemplated as host cells for phage viruses.

[0222] Examples of eukaryotic host cells for replicating and / or expressing vector include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos and PC12.Many host cells from various cell types and organisms are available and will be known to those skilled in the art.Similarly, viral vectors can be used with either eukaryotic or prokaryotic host cells, particularly those that allow vectors to replicate or express.

[0223] Some vectors may utilize control sequences that allow them to replicate and / or express in both prokaryotic and eukaryotic cells.Those skilled in the art will further understand the conditions for incubating all of the above-mentioned host cells to maintain them and allow vector replication.The techniques and conditions that will allow large-scale production of vectors and the production of nucleic acids, their homologous polypeptides, proteins, or peptides encoded by vectors are also understood and known. IX. Combination Therapy

[0224] In certain cases, the therapeutic methods of the present disclosure are utilized in conjunction with one or more other therapeutic methods for cardiac medical conditions. p63-TID (or its functional fragments and / or derivatives) can be used in conjunction with one or more cardiac cell reprogramming factors, and / or with one or more chromatin destabilizing agents and / or with one or more anti-fibrotic or angiogenic factors. In certain embodiments, p63-TID (or its functional fragments and / or derivatives) is used in conjunction with Hand2 and / or myocardin gene therapy, but it may also be used in conjunction with other genes or gene products, including Gata4, Mef2c, Tbx5, miR-133, miR-1, Oct4, Klf4, c-myc, Sox2, Mesp1, Brachyury, Nkx2.5, ETS2, ESRRG, Mrtf-A, MyoD, and / or ZFPM2 (in certain embodiments, in the form of nucleic acid or polypeptide or peptide). The one or more other therapies may be directly or indirectly related to the cardiac medical condition (examples of indirectly related therapies include those for pain or infection). In certain embodiments, the additional therapies related to the cardiac medical condition are drug therapy, surgery, ventricular assist device (VAD) implantation, thoracoscopic surgery (VAT), coronary artery bypass, or a combination thereof.

[0225] In certain embodiments, one or more agents that prevent fibrosis and / or enhance or promote angiogenesis may be used as adjuncts to the embodiments of the present disclosure. They may be provided to an individual in localized areas of the heart, including areas with tissue damage, cardiomyocyte loss, scar tissue, etc., or may be provided systemically. The one or more agents may be any composition suitable for promoting angiogenesis in the desired area. In certain embodiments, the agent may be a protein, peptide, small molecule, nucleic acid, etc. Embodiments such as those described in US Application No. US2003 / 0103943 or US2001 / 0041679 may be used with the methods of the present disclosure. Embodiments such as those described in US Application No. US2018 / 0066252A1 and US2022 / 0143142A1 (each of which is incorporated herein in their entirety for the purposes described herein) may be used with the methods of the present disclosure. Particular embodiments include fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), Ets variant 2 (ETV2), angiopoietin, Ang1 and Ang2, matrix metalloproteinase (MMP), delta-like ligand 4 (DII4), or peptides thereof, or combinations thereof. ITD-1 is a small molecule that inhibits TGF-beta and thus inhibits fibrosis and cardiac remodeling (Willems E, Cabral-Teixeira J, Schade D, et al. Cell Stem Cell. 2012. pp. 242-252.), which may be utilized.

[0226] In certain embodiments, the agent that enhances angiogenesis is VEGF. In certain embodiments, the agent that enhances angiogenesis is ETV2. In certain embodiments, VEGF and / or ETV2 are administered using any method described herein, including but not limited to, protein / peptide, vector, plasmid vector, and / or viral vector. In certain embodiments, VEGF and / or ETV2 are administered using an adenoviral vector. In certain embodiments, VEGF and / or ETV2 are administered simultaneously with p63-TID and H / M. In certain embodiments, VEGF and / or ETV2 are administered prior to administration of p63-TID and H / M. In certain embodiments, administration of VEGF and / or ETV2 with p63-TID and H / M results in superior cardiac cell reprogramming when compared to administration of VEGF and / or ETV2 with control, GMT, and / or GMTd treatment.

[0227] In certain embodiments, an exemplary ETV2 polynucleotide sequence is provided in GENBANK® Accession No. NM_001300974 (SEQ ID NO: 12). is and / or is contained therein.

[0228] In certain embodiments, an exemplary VEGF polynucleotide sequence is GENBANK® Accession No. AY047581 (SEQ ID NO: 13). (SEQ ID NO:13) is and / or is contained therein.

[0229] In specific embodiments, some or all of SEQ ID NO: 12 and / or 13 are utilized in the methods of the disclosure. In certain embodiments, polynucleotides having specific sequence identity to SEQ ID NO: 12 and / or 13 are utilized in the methods of the disclosure. In certain cases, functional fragments of SEQ ID NO: 12 and / or 13 are utilized, and the term "functional" fragment as used herein refers to a polynucleotide that encodes a polypeptide having an activity that can convert fibroblasts into endothelial cells or endothelial-like cells. In certain cases, the fragments are at least about 1375 or no more than 1350, 1325, 1300, 1275, 1250, 1225, 1200, 1175, 1150, 1125, 1100, 1075, 1050, 1025, 1000, 975, 950, 925, 900, 875, 850, 825 ... In addition, fragments may have a length of at least or exactly 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 80, 75, or 70% identity with the corresponding region in SEQ ID NO: 12 and / or 13. Polynucleotides having a certain sequence identity to SEQ ID NOs: 12 and / or 13, including at least or exactly 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 80, 75, or 70% identity to SEQ ID NOs: 12 and / or 13, may be used.

[0230] In some embodiments, the ETV2 and / or VEGF polypeptide is delivered to an individual in need thereof regardless of the form in which it is present, such as on a vector, associated with a carrier, in a cell (including in a cell on a vector), etc. In certain embodiments, the ETV2 and / or VEGF polypeptide is a mammalian ETV2 and / or VEGF polypeptide, including human, mouse, rat, etc. In a specific embodiment, one example of an ETV2 polypeptide sequence is set forth in GENBANK® Accession No. NP_001287903 (SEQ ID NO: 14). ACTAWDSWSGASQTLGPAPLGPGPIPAAGSEGAAGQNCVPVAGEATSWSRAQAAGSNTSWDCSVGPDGDTYWGSGLGGEPRTDCTISWGGPAGPDCTTSWNPGLHAGGTTSLKRYQSSALTVCSEPSPQSDRASLARCPKTNHRGPIQLWQFLLELLHDGARSSCIRWTGNSREFQLCDPKEVARLWGERKRKPGMNYEKLSRGLRYYYRRDIVRKSGGRKYTYRFGGRVPSLAYPDCAGGGRGAETQ (SEQ ID NO: 14) is and / or is contained therein.

[0231] In a specific embodiment, one exemplary VEGF polypeptide sequence is GENBANK® Accession No. AAK95847 (SEQ ID NO: 15). MNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKDRARQENPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRR (SEQ ID NO: 15) is and / or is contained therein.

[0232] In specific embodiments, part or all of SEQ ID NO: 14 and / or 15 are utilized in the methods of the disclosure. In certain embodiments, polypeptides having specific sequence identity to SEQ ID NO: 14 and / or 15 are utilized in the methods of the disclosure. In certain cases, functional fragments of SEQ ID NO: 14 and / or 15 are utilized, the term "functional" fragment as used herein referring to a polypeptide having activity capable of converting fibroblasts into endothelial or endothelial-like cells. In certain cases, the fragment has a length of at least about 245 or less, 240, 235, 230, 225, 220, 215, 210, 205, 200, 195, 190, 185, 180, 175, 170, 165, 160, 155, 150, 145, 140, 135, 130, 125, 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25 or less, or 20 or less consecutive amino acids of SEQ ID NO: 14 and / or 15.

[0233] The agent that enhances angiogenesis can be referred to as angiogenesis factor.This agent can be provided to an individual before the individual receives p63-TID (or its functional fragment and / or functional derivative), and / or before cardiac cell reprogramming factor, and / or before chromatin destabilizing agent.In certain embodiments, more than one agent is utilized.

[0234] The disclosed therapy may precede or follow the treatment of the other agent by intervals ranging from minutes to hours, days, weeks, or months. In embodiments in which the other agent and the disclosed therapy are applied to an individual separately, one will generally ensure that no significant period of time passes between the respective delivery times, so that the disclosed therapy and the additional therapy can still exert a beneficial combined effect on the individual. In such cases, it is contemplated that the individual may be contacted with both modalities simultaneously, or within minutes of each other, or within about 1-12, 6-12, or 12-24 hours of each other. In some circumstances, however, it may be desirable to extend the duration of treatment significantly, in which case several days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks) pass between the respective administrations.

[0235] In certain embodiments, the disclosed therapeutic method and the additional therapeutic method are provided at the same time or at different times. The separate entities may be in the same composition, or they may be included in separate compositions. In the case where the disclosed therapeutic method and the second therapeutic method are provided at different times, they may be separated by any suitable time range, such as minutes, hours, days, or weeks. In the embodiment where they are provided separately, the order of the two (or more) therapeutic methods may be any suitable order, including delivering p63-TID (or its functional fragment and / or functional derivative) together with Hand2 and / or myocardin before or after another therapeutic method.

[0236] Examples of other treatments contemplated for use in conjunction with the therapeutic methods of the present disclosure include one or more of the following: ACE inhibitors, aldosterone inhibitors, angiotensin II receptor blockers (ARBs), beta blockers, calcium channel blockers, cholesterol-lowering drugs, digoxin, diuretics, inotropic therapy, potassium or magnesium, vasodilators, anticoagulants, aspirin, surgery, VAD implantation, VAT, coronary artery bypass, percutaneous coronary intervention (PCI), or combinations thereof. X. Kits of the Present Disclosure

[0237] Any of the compositions described herein can be included in the kit. In a non-limiting example, p63-TID (or its functional fragment and / or functional derivative), one or more cardiac cell reprogramming factors, and / or one or more chromatin destabilizing agents, or other polynucleotides or primers for its amplification can be included in the kit. In a specific embodiment, the kit includes p63-TID (or its functional fragment and / or functional derivative), with or without Hand2 and / or myocardin polypeptide or peptide. One or more reagents for generating p63-TID (or its functional fragment and / or functional derivative) or one or more of the other mentioned factors, such as specific primers for amplifying the desired sequence, can be included in the kit. In such a case, the kit may or may not include standard reagents for such methods, such as nucleotides, buffers, etc. The kit may additionally include additional agents for the treatment of cardiac medical conditions.

[0238] The components of the kit may be packaged in either aqueous media or lyophilized form. The container means of the kit will generally include at least one vial, test tube, flask, bottle, syringe, or other container means into which the components may be placed and preferably suitably aliquoted. If there is more than one component in the kit, the kit will also generally include a second, third, or other additional container into which the additional components may be separately placed. However, various combinations of components may be included in one vial. The kit of the present disclosure will also typically include a means for containing the p63-TID (or its functional fragment and / or functional derivative) and one or more compositions in tightly sealed condition for commercial sale. Such containers may include injection-molded or blow-molded containers into which the desired vials are retained.

[0239] When the components of the kit are provided in one and / or multiple solutions, the solution is an aqueous solution, with sterile aqueous solutions being specifically contemplated. The p63-TID (or functional fragment and / or derivative thereof) composition may also be formulated into a syringe-injectable composition. In that case, the container means may itself be a syringe, pipette, and / or such a similar device, from which the formulation may be applied to the infected area of ​​the body, injected into an animal, and / or even applied to and / or mixed with other components of the kit. However, the components of the kit may also be provided as dry powders. When the reagents and / or components are provided as dry powders, the powders can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.

[0240] The kits of the present disclosure will also typically include a means for containing the vials in tightly sealed condition for commercial sale, such as, for example, injection- or blow-molded plastic containers into which the desired vials are retained.

[0241] In a specific embodiment, the kit includes a reagent and / or a tool for determining that an individual has a cardiac medical condition. In some embodiments, the kit includes one or more additional therapies for cardiac-related medical conditions, such as one or more of ACE inhibitors, aldosterone inhibitors, angiotensin II receptor blockers (ARBs), beta blockers, calcium channel blockers, cholesterol-lowering drugs, digoxin, diuretics, inotropic therapy, potassium, magnesium, vasodilators, anticoagulants, aspirin, TGF-beta inhibitors, and combinations thereof. In certain embodiments, the individual receives an angiogenesis therapeutic agent before, during, or after the disclosed therapy. Examples of angiogenesis therapeutic agents include fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), Ets variant 2 (ETV2), angiopoietin, Ang1 and Ang2, matrix metalloproteinase (MMP), delta-like ligand 4 (DII4), or peptides thereof, or combinations thereof. EXAMPLES

[0242] The following examples are included to illustrate preferred embodiments of the present invention. Those skilled in the art should understand that the techniques disclosed in the following examples represent techniques that the inventors have discovered to work well in the practice of the present invention, and therefore can be considered to constitute preferred modes for its practice. However, those skilled in the art should understand, in light of this disclosure, that many changes may be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the present invention. method

[0243] Unless otherwise indicated, the experiments and procedures described herein were carried out as follows. Tissue harvesting and cardiac fibroblast isolation

[0244] Neonatal and adult cardiac fibroblasts were harvested from rats aged 0-3 days and 6-8 weeks, respectively, using standard cell isolation techniques (Harlan Sprague Dawley Inc, Indianapolis, IN) (see, e.g., refs. 9, 10, and 27, each of which is incorporated herein by reference for purposes described herein). All experimental animals were approved by the Institutional Animal Care and Use Committee (IACUC) at Baylor College of Medicine, and all methods were performed in accordance with NIH guidelines (Guide for the Care and Use of Laboratory Animals) under protocol AN-6223. These studies are conducted and reported in compliance with the relevant elements of the ARRIVE guidelines.

[0245] Adult human cardiac fibroblasts were isolated from ventricular myocardial tissue obtained from explants of heart failure patients undergoing placement of mechanical assist devices or heart transplantation at Baylor St. Luke's Medical Center using standard isolation techniques (see, e.g., citations 9 and 10, each of which is incorporated herein by reference for the purposes described herein). Written informed consent was obtained from all subjects and / or their legal guardians prior to tissue acquisition. All experimental methods were performed under protocols approved by the Baylor College of Medicine Institutional Review Board (IRB H-33421) in accordance with relevant guidelines and regulations. Briefly, explanted tissues were ground and then cultured in DMEM, 10% fetal bovine serum (FBS), and 1% penicillin / streptomycin. Fibroblasts were thereby allowed to migrate from these explants over a period of two weeks, after which they were passaged three times in M106 medium (M106500, Thermo Fisher Scientific), 10% FBS, and LSGS kit supplement (S-003-K, Thermo Fisher Scientific). Cell Reprogramming

[0246] Lentiviral vectors encoding Gata4, Mef2, or Tbx5 (GMT), Hand2 / myocardin (H / M), non-targeting (NT) shRNA, p63 short hairpin RNA (Origene, Rockville, MD), p63-transactivation inhibitory domain tagged with green fluorescent protein (GFP) (Vectorbuilder, Chicago, IL), or a GFP control vector were prepared from associated plasmids by the Baylor College Of Medicine Gene Vector Core as previously described (see, e.g., refs. 9, 10, 27, and 28, each of which is incorporated herein by reference for purposes described herein).

[0247] Rat and human cardiac fibroblasts isolated as described above were seeded into 6-cm or 10-cm culture dishes for fluorescence-activated cell sorting [FACS] analysis, into 6-well plates for quantitative reverse transcription polymerase chain reaction [qRT-PCR] analysis, or into 24-well dishes pre-coated with Surecoat (SC-9035, Cellutron Life Technologies) for immunocytochemistry analysis. 24 h after cells were 70%–80% confluent, lentiviral vectors at a multiplicity of infection (MOI) of 20 (unless otherwise indicated) were added to the cell culture plates in a mixture with polybrene at a final concentration of 5 μg / μL. Two days after cell culture treatment with the relevant reprogramming factors, the initial transfer medium (DMEM / 199 [4:1], 10% FBS, and 1% penicillin / streptomycin) was replaced with induction medium (iCM medium), as described previously. The medium was replaced with fresh induction medium every two days until the cells were harvested (see, e.g., references 9 and 10, each of which is incorporated herein by reference for the purposes described herein).

[0248] For cell contractility co-culture studies, cardiomyocytes were isolated from 0-3 day old rat pups under protocol AN-6223 as previously described (see, e.g., refs. 28-30, each of which is incorporated herein by reference for purposes described herein). Human cardiac fibroblasts were treated with GFP-labeled reprogramming factors (e.g., GMT, shp63+ H / M, p63-TID+ H / M) and after 1 week of treatment as described above, cells were harvested and reseeded onto neonatal rat cardiomyocytes at a ratio of 1:10 and cultured in DMEM / M-199 / 10% FBS medium (see, e.g., ref. 31, which is incorporated herein by reference for purposes described herein). Flow cytometry

[0249] Fluorescence-activated cell sorting (FACS) was performed as previously described (see, e.g., refs. 8-10, 27, and 28, each of which is incorporated herein by reference for purposes described herein). Briefly, cells attached to culture dishes were first washed with DPBS and trypsinized with 0.25% trypsin / EDTA. Cells were then fixed with fixation buffer (BD Biosciences), washed with Perm / Wash buffer (BD Biosciences), and then incubated with mouse monoclonal anti-cardiac troponin T (cTnT) antibody (ab8295, Abcam) in Perm / Wash buffer. The cells were then incubated with donkey anti-mouse Alexa Fluor 647 (ab150107, INVITROGEN™), washed again three times with Perm / Wash buffer, and further analyzed for cTnT expression using an LSR Fortessa cell sorter (BD Biosciences) with FlowJo software (FlowJo, LLC, Ashland, Ore) and Diva software (version 6.0). Immunocytochemistry

[0250] Immunofluorescence (IF) staining was performed using cells fixed in 4% paraformaldehyde and permeabilized with 0.5% Triton-X solution as previously described (see, e.g., refs. 8-10 and 28, each of which is incorporated herein by reference for purposes described herein). After blocking the cells with 10% goat serum, they were incubated with primary antibodies against cTnT (1:300 dilution, Thermo Fisher Scientific) or α-actinin (1:300 dilution, Sigma-Aldrich), followed by incubation with the appropriate Alexa luminescent secondary antibody (INVITROGEN™). 4',6-diamidino-2-phenylindole (DAPI, INVITROGEN™) was used to stain nuclei. For quantification of cTnT and α-actinin positive cells, the ratio of cells expressing the relevant IF marker relative to the total cells marked by DAPI was calculated in five random images selected by an examiner blinded to the treatment group. qRT-PCR

[0251] Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed by first extracting total RNA using the TRIzol method (INVITROGEN™) as previously described (see, e.g., 8-10, 28, 32). Relative quantification of RNA was performed using SYBR green detection of PCR products in real time using an ABI ViiA 7 (Applied Biosystems Inc). The primers for qRT-PCR used in this study are shown in Table 1 (cardiac isoform of troponin T (cTnT), ryanodine receptor (RyR), phospholamban (Pln), actin alpha cardiac 1 (Actc1), collagen type 1 alpha 1 chain (Col1a1), periostin (Postn), glyceraldehyde-3-phosphate dehydrogenase (Gapdh), delta N isoform of tumor protein p63 (ΔNp63), transactivation (TA) isoform of tumor protein p63 (TAp63), myosin heavy chain 6 (Myh6), and Gap junction alpha-1 (Gja1)). The mRNA levels were normalized relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by the comparable ΔΔCT method. [Table 1]

[0252] Co-immunoprecipitation (Co-IP) and Western analysis For Western analysis and Co-IP, 293T cells were transfected with plasmid vectors pcDNA3.1, ΔNp63 α-FLAG (p63-FLAG, #26979, Addgene), HDAC1-GFP (#11054, Addgene), or p63-TID-HA (GenScriptR) in LIPOFECTAMINE™ 3000 Transfection Reagent (L3000008, Thermo Fisher Scientific). Cell lysates were harvested and homogenized in cell lysis buffer. Proteins were quantified using Pierce BCA Protein Assay Kit (23227, Thermo Fisher Scientific), and Co-IP was performed on quantified proteins using Immunoprecipitation Kit (10007D, INVITROGEN™) according to the manufacturer's protocol. As a final step, samples were loaded onto SDS-PAGE and after separation, protein bands were transferred to a nitrocellulose membrane (IB301001, INVITROGEN™).

[0253] Immunodetection was performed with the following primary antibodies: FLAG tag (F1804-200UG, Sigma-Aldrich), HDAC1 (sc-7872, Santa Cruz Biotechnology, Inc), β-actin (sc-47778, Sigma-Aldrich), HA tag (sc-57592, Santa Cruz Biotechnology, Inc,), or TP63 (GTX 102425, GeneTex), followed by the appropriate HRP-conjugated secondary antibodies (Millipore, Billerica, Mass.). Membranes were then washed with 1x Tris-buffered saline with Tween 20 and visualized by chemiluminescence detection (WBLUF0500, Millipore Sigma). Measurement of contractility and calcium transients

[0254] Cell contractility (cell shortening) and calcium transients in coculture studies were measured at room temperature (22-23 °C). To perform these studies, cells were placed in a Plexiglas chamber, which was placed on the stage of an inverted epifluorescence microscope (Nikon Diaphot 2000) and buffered with 1.8 mmol / L of CaCl containing (in mmol / L): NaCl 140, KCl 5.4, MgCl2 1, CaCl2 1.8, HEPES 5, and glucose 10, pH 7.4. 2+ -Tyrode's solution was perfused. Cells that had been pre-treated with reprogramming factors were identified by GFP fluorescence.

[0255] 1.8 mM Ca 2+ Field stimulation was provided by a Grass S5 stimulator using platinum electrodes placed alongside the cell culture chamber containing Ca, delivering bipolar pulses at a voltage 50% above the myocyte stimulation threshold. Contractions of iCMs from the random fields were videotaped and digitally processed by computer. 2+ For signal measurement, cells were loaded with 3 μmol / L Fura-2 / AM (Life Technologies) and alternately excited at 340 and 380 nm at 0.5 Hz using a Delta Scan dual-beam spectrofluorometer (Photon Technology International, Edison, NJ). 2+ Transients were expressed as the ratio of 340 / 380 nm to the resulting 510 nm emission. Data were analyzed using Felix software (Photon Technology International) (see, e.g., refs. 8, 9, and 28-30, each of which is incorporated herein by reference for purposes described herein). statistical analysis

[0256] Every study was performed with each measurement in technical triplicate of at least three independent biological replicates. All data are expressed as mean ± standard error (SEM). Statistical analysis was performed using SAS, version 9.4. Student's t-test was used to determine significant differences between two groups. One-way ANOVA was used to determine significant differences when comparing more than two groups.

[0257] Example 1 Truncated p63 protein domains enhanced human cardiac reprogramming Direct reprogramming represents a promising new strategy for treating heart failure by inducing in situ transdifferentiation of cardiac fibroblasts into functional cardiomyocyte-like cells (iCMs). Notably, the combination of three cardiac transcription factors Gata4, Mef2c, and Tbx5 can convert fibroblasts into iCMs. Recent findings, however, suggest that human cells are resistant to reprogramming compared to rodent cells, possibly due to epigenetic restrictions on reprogramming gene activation. The present disclosure relates to enhancing cardiac reprogramming efficiency and maturation of iCMs by modulation (e.g., inhibition, e.g., silencing epigenetic effects) of the epigenetic regulatory gene p63, which has been shown to enhance pluripotent stem cell differentiation. Data show that p63 transactivation inhibitory domain (TID, p63-TID) exerts reprogramming benefits.

[0258] The family of histone deacetylases (HDACs) removes acetylated groups, resulting in the formation of condensed and transcriptionally more silenced chromatin in cardiac transdifferentiation genes.In fact, there is evidence that HDAC inhibitors enhance cardiac reprogramming.In this disclosure, we consider whether p63-HDAC1 complex plays a major role in affecting the gene transcription of cardiac transcription factors.

[0259] As shown in Figure 1A, we verified p63-HDAC1 interaction by co-immunoprecipitation (co-IP) assay. In addition, we investigated whether overexpressed p63-TID acts to compete with the protein-protein interaction between p63 and HDAC1, and whether the resulting epigenetic interaction can be specifically targeted by using p63-TID. As shown in Figure 1B, overexpression of p63-TID fragment reduced the binding between ΔNP63α and HDAC1 by competitive binding to HDAC1, as shown by co-IP pull-down assay.

[0260] An exemplary p63-TID peptide (e.g., SEQ ID NO:1) nucleotide coding sequence (SEQ ID NO:2) including an optional tag (e.g., an HA tag) and a nucleotide vector containing the same were obtained and are shown in Figures 4A-B.

[0261] We then demonstrated the effect of p63-TID on cardiac reprogramming by treating human cardiac fibroblasts with lentivirus encoding p63-TID±Hand2 / myocardin. After 14 days of culture, cells were assessed for changes in cardiomyocyte-specific properties using qRT-PCR, flow cytometry, and immunofluorescence assays. Lentivirus-mediated p63-TID overexpression combined with Hand2 / myocardin administration to human cardiac fibroblasts upregulated the same cardiac gene panel as seen with p63 shRNA+Hand2 / myocardin ((+H / M), Figure 2A-J), and we observed that p63-TID overexpression similarly increased the percentage of human cardiac fibroblasts expressing cTnT and α-actinin (Figure 2A-J). Cells treated with p63-TID+H / M showed increased expression of a panel of cardiomyocyte marker genes (cTnT, Gja1, Myh6) similar to that induced by shp63+H / M (Figure 2I), and reduced expression of fibroblast marker genes (col1a1, Postn) comparable to that achieved with shp63+H / M treatment (Figure 2G). Interestingly, p63-TID overexpression alone upregulated the expression of a panel of genes favorable for cardiac differentiation (e.g., CTnT, Gja1, α-actinin, and Myh6) and similarly downregulated genes associated with a fibroblast signature (e.g., col1a1 and Postn).

[0262] Also, an exemplary p63-TID (e.g., SEQ ID NO: 1) was administered to rat cardiac fibroblasts, and good iCM reprogramming results were obtained compared to the control. As shown in Figure 3, the mRNA expression levels of the indicated cardiac markers (e.g., cTnT, RyR, Pln, and Actc1) and the mRNA expression of fibroblast marker genes (e.g., col1a1) were determined by qRT-PCR two weeks after administration of the reprogramming factors. A significant increase in cardiogenic gene expression levels was observed with p63-TID+H / M administration, and a concomitant decrease in the fibroblast marker col1a1 was also observed.

[0263] In addition, FACS analysis showed that the percentage of human cardiac fibroblasts expressing cTnT was similarly increased after treatment with p63-TID+H / M compared to shp63+H / M treatment (12.5% ​​± 0.9% and 15.2% ± 1.1%, Fig. 2H). Immunofluorescence studies also showed a similar 3-fold increase in the number of cells expressing the cardiomyocyte markers cTnT and α-actinin after treatment with p63-TID+ H / M or shp63+H / M compared to cells treated with shp63+GMT (p<0.001, Fig. 2D, E, and I). Four weeks after reprogramming factor treatment, three times more p63-TID+H / M-treated cells showed α-sarcomeric actinin+ expression compared to shp63 and GMT-treated cells, and p63-TID+H / M-treated cells showed advanced sarcomere organization (Figure 2J).

[0264] We then determined that p63 silencing induced iCM contractility. Human cardiac fibroblasts treated with shp63+H / M or p63-TID+H / M were not observed to contract independently, but approximately 5% of human cardiac fibroblasts treated with shp63+H / M or TID+H / M contracted synchronously with surrounding neonatal rat cardiomyocytes after 4 weeks of co-culture, as verified by their GFP expression (FIG. 5). In comparison, human cardiac fibroblasts treated with GMT with or without shp63 were unable to contract in co-culture experiments (see, e.g., Pinnamaneni et al., p63 silencing induces epigenetic modulation to enhance human cardiac fibroblast to cardiomyocyte-like differentiation. Scientific Reports (2022)12:11416, Supplemental Videos S1, S2, and / or S3, which are incorporated by reference in their entirety for purposes described herein). Cells treated with shp63+H / M or p63-TID+H / M also exhibited calcium transients upon electrical stimulation synchronous with contractile function, whereas no calcium transients were observed following stimulation of cells treated with GMT with or without shp63 (Figure 5, middle and bottom rows, and Pinnamaneni et al., 2022, Supplemental Videos S1, S2, S3, which are incorporated by reference in their entireties for the purposes described herein).

[0265] We then determined p63-TID dose response and identified the enhanced potency of p63-TID compared to shp63 in enhancing human cardiac differentiation. To determine whether p63-TID was more potent than shp63 in enhancing cardiac differentiation, we used Co-IP analysis to generate a dose-response analysis of p63 binding to HDAC1 as a function of p63-TID overexpression (Figure 6A). qRT-PCR analysis of human cardiac fibroblasts treated with p63-TID at MOI of 20, 50, or 100 showed an increase in cTnT expression in a dose-dependent manner, with an MOI of 50 resulting in the highest cTnT expression without cytotoxicity (p<0.001, Figure 6B). Using qRT-PCR of human cardiac fibroblasts treated at an MOI of 50, we were therefore able to show significantly greater changes in the expression of cardiogenic and fibrogenic genes after p63-TID+H / M treatment compared to shp63+ H / M treatment (p<0.05, Figures 6C-D).

[0266] As shown herein and in Pinnamaneni et al., 2022, the inventors have shown that rodent and human cardiac fibroblasts can be converted into contractile iCMs through a reprogramming strategy mediated by silencing the epigenetic effects of p63. In particular, the inventors have shown that shp63 in combination with cardiac differentiation factors Hand2 and myocardin (H / M) resulted in enhanced neonatal, adult rat, and adult human cardiac fibroblast differentiation compared to treatment with a standard reprogramming cocktail (i.e., Gata4, Mef2c, and Tbx5 [GMT]) alone (see, e.g., citations 5, 10, and 34, each of which is incorporated herein by reference for the purposes described herein). In comparison, neither shp63 nor H / M exerted significant reprogramming effects alone.

[0267] The focus on p63 described in these studies was based on the observation that the p53 family of epigenetic regulator proteins plays a key role in disrupting induced pluripotent stem cell (iPSC) reprogramming (see, e.g., refs. 18-20, 23, 35, and 36, which are incorporated by reference for purposes described herein). The inventors hypothesized that silencing p63, which appears to play a similar role to p53 in suppressing iPSC reprogramming without its oncogenic effects, may be an ideal reprogramming agent for enhancing cardiac cell transdifferentiation and iCM generation (see, e.g., refs. 24, 37-43, each of which is incorporated by reference for purposes described herein). The results described herein and in Pinnamaneni et al., 2022 confirmed our hypothesis and, in particular, identified the interaction of p63 with the epigenetic repressor HDAC1 as a possible mechanism of action underlying this effect. The discovery that p63 interacts with HDAC1 to initiate epigenetic repatterning and modulate cardiac differentiation gene promoters confirmed the role of epigenetic modulation as a key regulator of human cellular cardiac reprogramming (see, e.g., references 44 and 45, each of which is incorporated by reference herein for purposes described herein).

[0268] The C-terminus of both of the two major isoforms of p63 (TAp63, ΔNp63) contains a transactivation inhibitory domain (TID) that has been reported to play an important role in gene regulation through its interaction with HDAC1 (see, e.g., citation 26, which is incorporated herein by reference for purposes described herein). The inventors subsequently hypothesized that overexpression of TID could replace the use of shRNA to inhibit the epigenetic action of p63 and the associated inhibition of cellular reprogramming. The results provided herein showed that p63-TID could be used in this manner and could enhance cardiogenic reprogramming gene activation. Without being limited by theory, the efficacy of the p63 silencing strategy in inducing contractile iCMs compared to the use of standard reprogramming cocktails could be related to its observed effect in affecting the regulation of a diverse panel of associated cardiogenic and fibrogenic genes. In comparison, standard reprogramming cocktails required administration of each of these constitutive reprogramming factors to achieve efficacy (see, e.g., refs. 11, 31, 34, and 46-48, each of which is incorporated herein by reference for purposes described herein). In this context, without being limited by theory, the addition of H / M as an adjunct to p63 silencing is likely related to the status of H / M as a "missing element" that compensates for key cardiac differentiation factors, such as GMT, that were originally upregulated by the described p63 silencing strategy. It is equally interesting that p63 silencing resulted in the downregulation of fibrogenic genes known to disrupt cardiac differentiation, which was addressed by adding additional potentially undesirable anti-fibrogenic factors to the reprogramming cocktail (see, e.g., refs. 9, 10, and 46-48, each of which is incorporated herein by reference for purposes described herein).

[0269] Taken together, the results described and presented herein showed that overexpression of p63-transactivation inhibitory domain (TID), a p63 motif responsible for binding to epigenetic regulator histone deacetylase 1 (HDAC1), was a strong alternative to shp63 in enhancing human cardiac differentiation.Furthermore, the results presented herein suggest that p63 acts as an epigenetic barrier to human cardiac reprogramming, and that p63-TID provides a new promising strategy to target epigenetic regulation of cardiogenic gene activation as a means to enhance human cardiac reprogramming and / or treat and / or prevent diseases described herein (e.g., but not limited to, cardiac-related indications).

[0270] Example 2 Combination of p63-TID with Hand2 / myocardin and ETV2 or VEGF enhanced human cardiac reprogramming As shown in Figure 7A-B, human cardiac fibroblasts were treated with adenoviruses encoding GFP (adGFP), ETV2 (adETV2), or VEGF (adVEGF), and 7 days later, cells were treated with adenoviruses encoding GFP, GMT, GMTd, or TIDH / M for 14 days. Results showed that cTnT marker gene expression was significantly increased in the TIDH / M-treated group compared to controls, as assessed by qRT-PCR after the indicated treatments (n=3). Data are expressed as fold change, and the internal control was GAPDH.

[0271] As shown in Figure 7C-D, human cardiac fibroblasts were treated with adenoviruses encoding GFP (adGFP), ETV2 (adETV2), or VEGF (adVEGF) with or without co-treatment with adenoviruses encoding GFP, GMT, GMTd, or TIDH / M. Two weeks later, cTnT marker gene expression was assessed by qRT-PCR after the indicated treatments (n=3), and the results showed that cTnT marker gene expression was significantly increased in the TIDH / M-treated group compared to the control. Data are expressed as fold change, and the internal control was GAPDH.

[0272] Collectively, these results demonstrated that simultaneous and / or delayed administration of TIDH / M with ETV2 or VEGF significantly improved myocardial reprogramming. References

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[0274] All of the methods disclosed and claimed herein can be made and performed without undue experimentation in light of this disclosure. Although the compositions and methods of the present invention have been described with reference to preferred embodiments, it will be apparent to those skilled in the art that variations may be applied to the methods described herein and to the steps or sequence of steps of the methods without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while achieving the same or similar results. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims

1. A composition comprising a recombinant protein and / or a recombinant nucleic acid encoding the same, wherein the recombinant protein comprises a p63-transactivation inhibitory domain (p63-TID) polypeptide, and the p63-TID polypeptide comprises the sequence of SEQ ID NO:

1.

2. The composition of claim 1 , wherein the composition comprises a pharmaceutically acceptable carrier.

3. The composition of claim 1 , wherein the recombinant polypeptide is labeled and / or one or more of the amino acids of the recombinant polypeptide comprises a post-translational modification.

4. The composition of claim 3 , wherein the post-translational modification comprises phosphorylation, acetylation, ubiquitination, acylation, methylation, or a combination thereof.

5. The composition described in claim 1, comprising the recombinant nucleic acid.

6. The composition of claim 5 , wherein the recombinant nucleic acid is DNA or RNA.

7. The composition of claim 5 , wherein the recombinant nucleic acid is contained in one or more viral vectors.

8. 8. The composition of claim 7, wherein the viral vector is a lentiviral vector, an adenoviral vector, or an adeno-associated viral (AAV) viral vector.

9. The composition of claim 5 , wherein the recombinant nucleic acid is contained in a non-viral vector.

10. The composition described in claim 1, comprising one or more nucleic acid vectors encoding the recombinant polypeptide, comprising one or more cardiac cell reprogramming factors, and / or comprising one or more chromatin destabilizing agents.

11. The composition described in claim 10, comprising a vector encoding (a) the recombinant polypeptide and one or more cardiac cell reprogramming factors, (b) the recombinant polypeptide and one or more chromatin destabilizing agents, or (c) the recombinant polypeptide, one or more cardiac cell reprogramming factors, and one or more chromatin destabilizing agents.

12. The composition described in claim 10, comprising a vector encoding the recombinant polypeptide and a different vector encoding one or more cardiac cell reprogramming factors and / or chromatin destabilizing agents.

13. The composition described in claim 11, comprising a vector encoding the recombinant polypeptide and one or more cardiac cell reprogramming factors, and a different vector encoding one or more chromatin destabilizing agents.

14. The composition described in claim 1, further comprising: (a) one or both of a nucleic acid encoding Hand2 and a nucleic acid encoding myocardin; (b) one or more antifibrotic agents; (c) a nucleic acid encoding Hand2, a nucleic acid encoding myocardin, and a nucleic acid encoding Ets variant 2 (ETV2) and / or vascular endothelial growth factor (VEGF); or (d) any of the above or a combination thereof.

15. The composition of claim 14, wherein the nucleic acid encoding ETV2 and / or VEGF is deliverable by one or more viral vectors.

16. 16. The composition of claim 15, wherein the one or more viral vectors comprise a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a retroviral vector.

17. A kit comprising the composition of claim 1, wherein the composition is contained in a suitable container.

18. 1. A composition for treating a cardiac condition, comprising: recombinant proteins and / or recombinant nucleic acids encoding the same, the recombinant protein comprises a p63-transactivation inhibitory domain (p63-TID) polypeptide, or a functional derivative and / or functional fragment thereof; A composition wherein the p63-TID polypeptide comprises a sequence that is at least or exactly 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO:

1.

19. 1. A composition for treating a cardiac condition, comprising: A) p63-transactivation inhibitory domain (p63-TID) polypeptides and / or functional derivatives and / or functional fragments thereof, and / or nucleotides encoding same, wherein the p63-TID polypeptide comprises a sequence that is at least or exactly 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 1; B) Hand2 polypeptides and / or functional derivatives and / or functional fragments thereof, and / or nucleotides encoding same; C) myocardin polypeptides and / or functional derivatives and / or functional fragments thereof, and / or nucleotides encoding same; D) ETV2 polypeptides and / or functional derivatives and / or functional fragments thereof, and / or nucleotides encoding same, and / or E) VEGF polypeptides and / or functional derivatives and / or functional fragments thereof, and / or nucleotides encoding same; A composition comprising:

20. The composition described in claim 5, wherein the recombinant nucleic acid encoding the recombinant polypeptide comprises sequence number 2.

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