Gene therapy strategy to restore electrical and cardiac function, and cardiac structure, in arrhythmogenic right ventricular cardiomyopathy

Abstract

To provide more effective therapies for patients with arrhythmogenic right ventricular cardiomyopathy, and to provide therapies that target the underlying structural nature of the disease.SOLUTION: Provided is a vector for use in a method for treating a structural cardiovascular disease or condition in a subject in need thereof, the method including administering the vector to the subject, the vector encoding a connexin 43 polypeptide sequence operably linked to a promoter that is active in myocardial tissue, and administration of an effective amount of the vector resulting in increased connexin 43 levels in at least a part of the subject's heart.SELECTED DRAWING: None

Description

[Background technology]

[0001] (Government support) The subject matter of this invention was made with government support awarded by the National Institutes of Health (Grant Number HL095780-01).

[0002] (Related Applications) This application claims the benefit of U.S. Provisional Patent Application No. 62 / 560,989, filed September 20, 2017, the entire contents of which are incorporated herein by reference.

[0003] (Background technology) Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a complex and devastating genetic cardiac disease observed in young individuals and athletes, and exhibits a wide variability in its clinical manifestations. Classical ARVC clinical symptoms include palpitations, arrhythmic syncope (or fainting dizziness), and sudden cardiac death due to ventricular arrhythmias, characteristic of primary electrical complications. However, ARVC patients also exhibit clinical symptoms associated with structural disease, which includes ventricular (right and / or left ventricular) dysfunction and / or myocardial fibrofatty replacement, as well as myocardial remodeling consisting of wall thinning and hypertrophy, characteristic of primary structural complications. The structural nature of the disease is also underpinned by the term "disease of focal adhesions," as human genetic studies have shown that 40% of patients have mutations in genes encoding components of focal adhesions (e.g., desmoplakin (DSP), plakoglobin (JUP), plakophilin 2 (PKP2), and desmoglein 2 (DSG2)), which are essential for maintaining the mechanical and structural integrity of cardiac muscle cell junctions.

[0004] Currently, there are no effective treatments for ARVC, nor are there any randomized trials of treatments, screening regimens, or ARVC-specific drugs. Consequently, treatment strategies for patients with ARVC are based on clinical expertise, the results of retrospective registry studies, and model systems and are primarily oriented toward symptomatic relief of electrophysiological disturbances. Consequently, existing treatments for ARVC patients rely on the use of antiarrhythmic medications (sotalol, amiodarone, and beta-blockers), which transition to more invasive procedures, including implantable cardioverter-defibrillators and cardiac catheter ablation, when patients become unresponsive or intolerant to antiarrhythmic treatments. However, current treatments have limited efficacy in managing the disease, as 40% of ARVC patients (younger-onset disease) die within 10–11 years of initial diagnosis, highlighting the need for the development of more effective treatments (and treatments that target the underlying structural nature of the disease) for patients with ARVC. Summary of the Invention [Means for solving the problem]

[0005] Disclosed herein are methods for treating arrhythmogenic right ventricular cardiomyopathy in a subject, the methods comprising, alternatively consisting essentially of, or even consisting of administering an effective amount of a gene therapy construct or vector comprising a connexin 43 polynucleotide sequence, wherein the administration results in increased connexin 43 levels in at least a portion of the heart. In some embodiments, the construct is an adenoviral construct. In some embodiments, the construct is an adeno-associated viral construct.

[0006] Further embodiments include a construct or vector capable of conferring expression of connexin 43 in cardiac tissue. In some embodiments, the construct or vector is a plasmid or a viral vector (such as an adenoviral construct). In some embodiments, the construct or vector is an adeno-associated viral construct.

[0007] In some embodiments, the connexin 43 polypeptide sequence is the 382 amino acid sequence of P17302 (CXA_1HUMAN, UniProtKB) (SEQ ID NO: 1). The encoding polynucleotide may have a polynucleotide sequence related to P17302 or any other nucleic acid sequence that encodes the same polypeptide sequence. In some embodiments, the encoding nucleic acid sequence is GenBank CR541660.1 (SEQ ID NO: 2).

[0008] In some embodiments, administration results in reduced electrical and structural cardiac dysfunction and improved structural integrity, in some embodiments, administration results in reduced physiological cardiac dysfunction, and in some embodiments, administration results in prolonged survival. [Brief explanation of the drawings]

[0009] [Figure 1A-B]Figures 1A-1G show that restoration of connexin 43 protein levels near wild-type levels can rescue physiological abnormalities in ARVC-plakophilin 2 mutant (PKP2mut) hiPSC-derived cardiomyocytes. Figures 1A-1B show representative field potential traces for non-ARVC-hiPSC-derived cardiomyocytes and ARVC-PKP2mut (also referred to as ARVC-constitutive (S))-hiPSC-derived cardiomyocytes infected with green fluorescent protein-containing adenovirus (Ad-GFP) or connexin 43-containing adenovirus (Ad43) for 48 hours under basal (Figure 1A) and isoproterenol (ISO) (Figure 1B) stimulation (which mimics stress / exercise-related conditions). Note the frequency and pattern of spikes within the traces. The vertical scale bar represents field potential amplitude (μV), and the horizontal scale bar represents time (1 s). [Figure 1C-D] Figures 1C-1D show representative impedance (contractility) traces for non-ARVC-hiPSC-derived cardiomyocytes and ARVC-PKP2mut (ARVC-S)-hiPSC-derived cardiomyocytes at basal levels (Figure 1C) and under ISO stimulation (Figure 1D) following 48 h of infection with Ad-GFP or Ad43 (note the frequency and shape of spikes within the traces). The vertical scale bar represents cell index, and the horizontal scale bar represents time (1 s). [Figure 1E] Figure 1E shows the quantification of the basal firing irregularity index in non-ARVC-iPSC-derived cardiomyocytes and ARVC-PKP2mut-iPSC-derived cardiomyocytes infected with Ad-GFP or Ad43. Mean values ​​with standard errors are shown, and ** indicates p<0.01, n = 6 (non-ARVC) and n = 7 (ARVC-PKP2mut). Two-sample t-test was performed. [Figure 1F] Figure 1F shows the quantitative results of the firing irregularity index under ISO stimulation in non-ARVC and ARVC-PKP2mut (ARVC-S) cells infected with Ad-GFP or Ad43. Mean values ​​with standard errors are shown, and *** indicates p<0.001, n = 6 (non-ARVC), n = 7 (ARVC-PKP2mut), by two-sample t-test. [Figure 1G] Figure 1G shows the expression levels of connexin 43 (Cx43), desmoplakin (DPS), desmoglein 2 (DSG2), plakoglobin (JUP), and plakophilin 2 (PKP2), as well as the expression levels of α-myosin heavy chain (α-MHC) (cardiomyocyte loading control) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (loading control) in non-ARVC, ARVC-PKP2mut (ARVC-S), and ARVC-desmoglein 2 mutant (DSG2mut, also referred to as ARVC-electrical (E)) hiPSC-derived cardiomyocytes following 48-hour infection with Ad-GFP or Ad43. [Figure 2A-B] Figures 2A-2B show that physiological abnormalities in ARVC-S cardiomyocytes can be rescued in a dose-dependent manner by Cx43 overexpression. Representative field potentials from non-ARVC-hiPSC-derived cardiomyocytes (Figure 2A) and ARVC-PKP2mut-hiPSC-derived cardiomyocytes (Figure 2B) after dose-dependent Ad43 infection are shown. Note that the irregular spike frequency and pattern in ARVC-PKP2mut-hiPSC-derived cardiomyocytes gradually become more regular with increasing Cx43 doses (MOI 5, MOI 10, and MOI 20). The vertical and horizontal scale bars represent field potential amplitude (μV) and time (1 s), respectively. MOI is the multiplicity of infection. [Figure 3A-B] Figures 3A-3F show that restoration of connexin 43 protein levels can rescue physiological abnormalities in ARVC-DSG2mut (ARVC-E) hiPSC-derived cardiomyocytes under catecholamine (ISO) stimulation to mimic stress / exercise conditions. Figures 3A-3B show representative field potential traces for non-ARVC-hiPSC-derived cardiomyocytes and ARVC-DSG2mut-hiPSC-derived cardiomyocytes infected with Ad-GFP or Ad43 for 48 hours at basal levels (Figure 3A) and under ISO stimulation (Figure 3B). The vertical scale bar represents field potential amplitude (μV), and the horizontal scale bar represents time (1 s). [Figure 3C-D]Figures 3C-3D show representative impedance (contractility) traces for non-ARVC-hiPSC-derived and ARVC-DSG2mut-hiPSC-derived cardiomyocytes at basal levels (Figure 3C) and under ISO stimulation (Figure 3D) following 48 h of infection with Ad-GFP or Ad43. The vertical scale bar represents cell index, and the horizontal scale bar represents time (1 s). [Figure 3E] Figure 3E shows the quantification of the basal firing irregularity index in non-ARVC-hiPSC-derived cardiomyocytes and ARVC-DSG2mut-hiPSC-derived cardiomyocytes infected with Ad-GFP or Ad43. Mean values ​​with standard errors are shown, n = 6 (non-ARVC), n = 6 (ARVC-DSG2mut), and a two-sample t-test was performed. [Figure 3F] Figure 3F shows the quantitative results of the firing irregularity index under ISO stimulation in non-ARVC and ARVC-DSG2mut (ARVC-E) cells infected with Ad-GFP or Ad43. Mean values ​​with standard errors are shown, and * indicates p<0.05, n=6 (non-ARVC) and n=6 (ARVC-DSG2mut), respectively. Two-sample t-test was performed. Note that in Figures 3A-3F, the irregular spike frequency and pattern in ARVC-DSG2mut-hiPSC-derived cardiomyocytes was observed only under ISO conditions, and restoration of Cx43 may make the spike frequency and pattern more regular. [Figure 4A] Figures 4A-4G show that restoring connexin 43 protein levels can improve cardiac rhythm and function in an ARVC mouse model (cardiac-specific desmoplakin knockout model (DSP-cKO)). Figure 4A shows the experimental strategy for injection of adeno-associated virus tagged with green fluorescent protein (AAV-GFP) / adeno-associated virus carrying connexin 43 and tagged with GFP (AAV-Cx43) and electrophysiological cardiac function analysis. [Figure 4B]Figure 4B shows that echocardiography of control and DSP-cKO mice (DSP-Flox mice, Cre positive) 6 weeks before adeno-associated virus (AAV) injection showed a significant decrease in left ventricular fractional shortening (%FS) in DSP-cKO mice compared with controls (DSP-Flox mice, Cre negative). [Figure 4C] Figure 4C shows that echocardiography of control and DSP-cKO mice 4 weeks (10 weeks of age) and 5 weeks (11 weeks of age) after AAV injection revealed an improvement in %FS (from 6% to 15%) in DSP-cKO mice injected with AAV-Cx43. No significant change in %FS was observed in control mice injected with AAV-Cx43. No significant differences in heart rate were observed between the control and DSP-cKO mouse groups. [Figure 4D] Figure 4D shows representative surface ECG traces of control and DSP-cKO mice 4 weeks (10 weeks of age) and 5 weeks (11 weeks of age) after AAV-Cx43 injection. At 4 weeks after AAV-Cx43 injection, DSP-cKO mice exhibited severe arrhythmias (premature ventricular contractions (PVCs)—extra beats that did not conform to a regular pattern) and a series of QRS complex inversions (spikes pointing downward instead of upward) indicative of bundle branch block). At 5 weeks after AAV-Cx43 injection, DSP-cKO mice exhibited improved cardiac rhythm (1.5 times fewer PVCs, limited QRS complex inversions (a series of 10 in the previous week (10 weeks) but only two in the next week (11 weeks))) and the appearance of normal sinus rhythm (regular frequency and rhythmic beats). [Figure 4E] Figure 4E shows the quantification of PVCs in DSP-cKO mice 4 weeks (10 weeks of age) and 5 weeks (11 weeks of age) after AAV-Cx43 injection, revealing a 1.5-fold decrease in the number of PVCs 5 weeks after AAV-Cx43 injection. [Figure 4F]Figures 4F-4H show that survival (Figure 4H), Western blot analysis (Figure 4F), and GFP immunostaining analysis (Figure 4G) revealed recovery of Cx43 protein in surviving DSP-cKO mice 5 weeks after AAV-Cx43 injection (DSP-cKO2-AAV-Cx43, green). In contrast, no recovery of Cx43 protein was observed in deceased DSP-cKO mice 3 weeks after AAV-Cx43 injection (DSP-cKO1-AAV-Cx43, red). [Figure 4G] See legend to Figure 4F. [Figure 4H] See legend to Figure 4F. [Figure 5] FIG. 5 depicts the vector map of Ad43 (SEQ ID NO: 7). [Figure 6] FIG. 6 depicts the vector map of AAV-Cx43 (SEQ ID NO: 8). DETAILED DESCRIPTION OF THE INVENTION

[0010] Disclosed herein is a gene therapy strategy for rescuing / treating electrical and structural cardiac dysfunction and extending lifespan in arrhythmogenic right ventricular cardiomyopathy, an arrhythmogenic disorder, via targeted restoration of Connexin 43 in cardiomyocytes using an adenovirus-associated strategy. While Connexin 43 is known to affect cardiac electrical function, we demonstrate that restoration of Connexin 43 can also extend lifespan and improve contractile (structural) function in a genetic mouse model harboring a severe structural form of ARVC late in the disease (lifespan). Furthermore, studies in an in vitro human ARVC stem cell-based model exhibiting severe structural damage (as evidenced by disrupted focal adhesion junctions observable in transmission electron microscopy assays) revealed that Connexin 43 restoration was sufficient to restore electrical and contractile (structural) dysfunction in these diseased cells. Furthermore, this restoration occurred rapidly. Because ARVC is a rare and fatal disease with no treatment, this approach represents the first opportunity to test a treatment approach in this highly vulnerable population, and this strategy may also have application to other cardiac diseases with underlying structural defects associated with connexin43 deletion.

[0011] Human induced pluripotent stem cells (hiPSCs) have shown great potential for in vitro disease modeling of inherited cardiac diseases. To this end, the inventors of the present disclosure generated a panel of hiPSC lines without gene insertions from genetically, biochemically, and physiologically characterized patients with arrhythmogenic right ventricular cardiomyopathy (ARVC), a cardiac disorder of focal adhesion cell junctions that manifests across a range of symptom severity and is known to cause sudden death in young individuals. Through ultrastructural, molecular, and real-time physiological assays using the xCelligence® RTCA CardioECR system, the inventors demonstrate that cardiac cell phenotypes derived from ARVC hiPSCs can essentially capture the altered cardiac disease phenotypes (electrical vs. structural) observed in donor ARVC patient cardiac tissue. Through a candidate genetic approach utilizing the xCelligence® RTCA CardioECR system in our ARVC hiPSC line, we have uncovered novel therapeutic agents that can commonly reverse cardiac physiological defects underlying both electrical and structural forms of ARVC.

[0012] The present invention builds on the seminal discovery that restoration of Connexin 43 alone (to levels similar to those of healthy controls) in structurally weakened hearts (via one-shot cardiac-targeted gene therapy) is sufficient for positive treatment outcomes. The studies described herein suggest that Connexin 43 is a key driver of the progression of structural heart disease, in which the structural integrity of cardiomyocytes is weakened. Because Connexin 43 is found between cardiomyocytes and cardiac macrophages as well as between cardiomyocytes and cardiac fibroblasts, structural connections to these other related cell types may also be weakened in structurally weakened hearts, and therefore, restoring Connexin 43 may also restore the structural integrity of these cell-cell interactions. This is an unexpected finding, as Connexin 43 has not previously been considered a therapeutic agent for structurally weakened hearts.

[0013] In a specific example, the treatment methods described herein primarily target arrhythmogenic right ventricular cardiomyopathy, a rare arrhythmogenic heart disease characterized by disruption or loss of focal adhesion mechanical cell-cell junctions, with Connexin 43 being an early hallmark of the disease. It is believed that Connexin 43 restoration may only alleviate early electrical damage in electrically impaired ARVC-hiPSC-derived cardiomyocytes. The results presented herein were surprising in the late structural model of ARVC described herein. Using ARVC-hiPSC-derived cardiomyocytes with structural features of the disease (human in vitro model) and a genetic mouse model mimicking the severe structural morphology of ARVC (DSP cKO mouse in vivo model), data showed that Connexin 43 gene therapy was effective in improving survival, cardiac rhythm, and function in the (late) structural stage of ARVC disease, even when key mechanical (focal adhesion) cell-cell junction genes were disrupted or mutated and other related focal adhesion cell-cell junction proteins were disrupted or lost. Furthermore, since there is evidence in the literature that connexin 43 is also lost at later stages in these diseases, when fatal arrhythmias and the structural integrity of the myocardium (and the accompanying cellular interactions) are most weakened, it is believed that the methods of the present invention can now be extended to other structurally weakened forms of cardiac disease (such as hypertrophic cardiomyopathy and congestive heart failure).

[0014] ARVC is a primarily genetic heart disease characterized by myocardial fibrofatty replacement and right ventricular dysfunction (but recently also left ventricular dysfunction), resulting in fatal / severe ventricular arrhythmias that can cause sudden cardiac death in young people and athletes. ARVC accounts for 10% of sudden cardiac deaths in people under 65 years of age and 24% of sudden cardiac deaths in people under 30 years of age. ARVC is considered a rare disease, occurring in 1 in 1,000–5,000 individuals, although the prevalence may be higher because some patients are undiagnosed or misdiagnosed due to a lack of diagnostic markers. Furthermore, growing evidence reveals earlier onset of the disease, highlighting the critical need for earlier patient identification and treatment, as the pediatric population, ranging from infants to teenagers, is particularly vulnerable to ARVC.

[0015] Currently, there are no effective treatments for ARVC, nor are there any randomized trials of treatments, screening regimens, or ARVC-specific drugs. Consequently, treatment strategies for patients with ARVC are based on clinical expertise, the results of retrospective registry studies, and the results of model system studies, and are directed toward symptomatic relief of electrophysiological disturbances. Consequently, existing treatments for patients with ARVC rely on the use of antiarrhythmic medications (sotalol, amiodarone, and beta-blockers), which transition to more invasive procedures, including implantable cardioverter-defibrillators and cardiac catheter ablation, when patients become unresponsive or intolerant to antiarrhythmic treatments. However, current treatments have limited efficacy in managing the disease, as 40% of patients with ARVC die within 10–11 years of initial diagnosis, highlighting the need for the development of more effective treatments for patients with ARVC.

[0016] Disclosed herein is a gene therapy strategy for rescuing / treating electrical and physiological cardiac dysfunction in ARVC, an arrhythmogenic disorder, via targeted overexpression of Connexin 43 polypeptide in cardiomyocytes using a vector delivery strategy. In one example described herein, one-time viral-mediated delivery of a Connexin 43 polynucleotide encoding a Connexin 43 polypeptide to cardiac myocytes of two ARVC hiPSC patient lines in vitro and to cardiomyocytes of a novel mouse model of ARVC in vivo was sufficient to substantially reverse the electrical and physiological cardiac dysfunction associated with ARVC. Furthermore, in vivo studies using the DSP-cKO mouse model of ARVC suggest that a Connexin 43 gene therapy treatment strategy could be utilized during late-stage disease to extend lifespan by avoiding sudden death associated with ARVC. This strategy may also have therapeutic applications in other genetic disorders resulting in sudden arrhythmic death (e.g., hypertrophic cardiomyopathy) and late-stage heart failure, where deletion of Connexin 43 polypeptide has been reported and carries an increased risk of arrhythmia and death. Furthermore, this study demonstrates that connexin 43 polypeptides may have roles beyond electrical function and may also act as scaffolds bridging structural connections between cells (independent of classical structural proteins such as focal adhesion proteins), contrary to the conventional view of the role of this protein in the field, which is classically associated only with electrical function. Furthermore, this study demonstrates that connexin 43 polypeptides may have roles beyond electrical function and may also act as scaffolds bridging structural connections between cells (independent of classical structural proteins such as focal adhesion proteins). This study also demonstrates that connexin 43 polypeptides may have roles beyond electrical function and may also act as scaffolds bridging structural connections between cells (independent of classical structural proteins such as focal adhesion proteins). This study demonstrates that connexin 43 polypeptides may also have roles beyond electrical function and may also act as scaffolds bridging structural connections between cells (independent of classical structural proteins such as focal adhesion proteins). mut ) and a mouse model of ARVC), which also improved cardiac function.

[0017] As used herein, the term "overexpression" refers to an increased level of expression relative to a pathological condition, and in certain embodiments, an increased level of expression relative to a normal, healthy, or wild-type state, but relative to minimal or absent levels of connexin 43 polypeptide expression seen in a pathological condition. Restoration of connexin 43 polypeptide expression levels to at least about 5% of normal is beneficial, although it does not necessarily result in complete restoration of cardiac function. Restoration of connexin 43 expression levels to 5%, 10%, 15%, 20%, 25% or more, or even to as much as 50% or more of normal levels is effective in restoring normal cardiac function.

[0018] As used herein, the term "vector" has its ordinary meaning in the art, including the understanding that it is capable of introducing a nucleic acid sequence into a target cell. For example, a vector may contain a coding sequence capable of being expressed in a target cell. For purposes of this disclosure, "vector construct," "expression vector," and "gene transfer vector" generally refer to any nucleic acid construct capable of directing the expression of a gene of interest and useful for introducing the gene of interest into a target cell. Thus, the term includes cloning vectors and expression vectors, in addition to integrating vectors.

[0019] As used herein, the term "adeno-associated virus" (AAV) has its ordinary meaning in the art, including, but not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV2i8 (Asokan et al., Nat Biotechnol. (1):79-82, 2010, incorporated herein by reference in its entirety), avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV, as well as other AAVs now known or yet to be discovered. Many additional AAV serotypes and clades have been identified and are also encompassed by the term "AAV."

[0020] The genomic sequences of various AAV and autonomous parvoviruses, as well as the sequences of inverted terminal repeats (ITRs), Rep proteins, and capsid subunits, are known in the art and can be found in the literature or public databases, such as the GenBank® database.

[0021] As used herein, the term "cardiomyocyte" (or "cardiac myocyte") has its ordinary meaning in the art and includes specialized muscle cells that primarily form the cardiac myocardium. Cardiomyocytes may have five major components: (1) a cell membrane (sarcolemma) and T-tubules for conduction of impulses; (2) a sarcoplasmic reticulum, which is a calcium reservoir necessary for contraction; (3) contractile elements; (4) mitochondria; and (5) a nucleus. Cardiomyocytes can be classified into subtypes, including, but not limited to, atrial cardiomyocytes, ventricular cardiomyocytes, sinoatrial (SA) node cardiomyocytes, peri-SA node cardiomyocytes, and central SA node cardiomyocytes. Stem cells can be propagated to mimic the physiological function of cardiomyocytes or, alternatively, can differentiate into cardiomyocytes. Differentiation can be detected by the use of markers selected from, but not limited to, myosin heavy chain, myosin light chain, actinin, troponin, tropomyosin, GATA4, myocyte enhancer factor (Mef)2c, and Nkx-2.5.

[0022] As used herein, the term "control" has its ordinary meaning in the art and includes a representative control or sample used in an experiment for comparison purposes. A control can be "positive" or "negative." For example, if the purpose of an experiment is to determine the correlation between altered expression levels of a gene and a particular phenotype, it is usually preferable to use a positive control (a sample from a subject that has such an alteration and expresses the desired phenotype) and a negative control (a subject or sample from a subject that lacks the altered expression or phenotype).

[0023] As used herein, the term "stem cell" has its ordinary meaning in the art and includes cells that are undifferentiated or partially differentiated and have the capacity to self-renew and / or produce differentiated progeny. Self-renewal is defined as the ability of a stem cell to proliferate and give rise to more such stem cells while maintaining its developmental potential (i.e., totipotency, pluripotency, multipotency, etc.). As used herein, the term "somatic stem cell" has its ordinary meaning in the art and includes any stem cell derived from non-embryonic tissue, including fetal, juvenile, and adult tissue. Naturally occurring somatic stem cells have been isolated from a wide range of adult tissues, including blood, bone marrow, brain, olfactory epithelium, skin, pancreas, skeletal muscle, and cardiac muscle. Exemplary naturally occurring somatic stem cells include, but are not limited to, mesenchymal stem cells (MSCs) and neural stem cells (NSCs). In some embodiments, the stem or progenitor cell is an embryonic stem cell. As used herein, the term "embryonic stem cells" has its ordinary meaning in the art and includes stem cells derived from tissues formed after fertilization and before the end of gestation (including pre-embryonic tissue (e.g., blastocysts), embryonic tissue, or fetal (fetal) tissue obtained any time during gestation, typically, but not necessarily, before about 10 to 12 weeks of gestation). Most frequently, embryonic stem cells are pluripotent cells derived from early embryos or early blastocysts. Embryonic stem cells can be obtained directly from any suitable tissue, including, but not limited to, human tissue, or from established embryonic cell lines. As used herein, the term "embryonic-like stem cells" has its ordinary meaning in the art and includes cells that share one or more (but not all) of the characteristics of embryonic stem cells.

[0024] As used herein, the term "pluripotent cell" has its ordinary meaning in the art and includes a less differentiated cell that can give rise to at least two distinct (genotypically and / or phenotypically) types of more differentiated progeny cells. In some embodiments, "pluripotent cells" include induced pluripotent stem cells (iPSCs), which are stem cells artificially derived from non-pluripotent cells, typically adult somatic cells, by inducing the expression of one or more stem cell-specific genes. Such stem cell-specific genes include, but are not limited to, the family of octamer transcription factors (e.g., Octomer-binding transcription factor binding (Oct)-3 / 4), the family of SRY Box (Sox) genes (e.g., Sox1, Sox2, Sox3, Sox15, and Sox18), the family of Krüppel-like transcription factor (Klf) genes (e.g., Klf1, Klf2, Klf4, and Klf5), the family of Myc genes (e.g., c-myc and L-myc), the family of Nanog genes (e.g., OCT4, NANOG, and REX1), or LIN28.

[0025] As used herein, the term "induced pluripotent cells" has its usual meaning in the art and includes embryonic-like cells that have been reprogrammed from adult cells to an immature phenotype, and human iPSCs express stem cell markers and have the ability to generate characteristic cells of all three germ layers.

[0026] As used herein, the term "effective amount" has its ordinary meaning in the art and includes a concentration or amount of a reagent or composition (such as a composition described herein), viral vector, or other vector that is effective to produce an intended result, including cell growth and / or differentiation, in vitro or in vivo, or that is effective for treating a condition described herein. It is understood that the amount of viral vector or other vector administered will depend on the specifics of the disorder being treated, including, but not limited to, the size or total volume / surface area to be treated, and the proximity of the administration site to the location of the area to be treated, among other factors well known to medicinal biologists.

[0027] As used herein, the term "arrhythmogenic right ventricular cardiomyopathy" (ARVC) has its ordinary meaning in the art and includes an inherited, progressive disorder that usually affects the right side of the heart but can affect both sides. The walls of the ventricles become thin and stretched. ARVC can also cause arrhythmias.

[0028] As used herein, the term "hypertrophic cardiomyopathy" has its ordinary meaning in the art and includes conditions in which cardiac muscle cells become enlarged, causing the ventricles to thicken.

[0029] As used herein, the term "connexin 43" has its ordinary meaning in the art and includes gap junction proteins. Gap junctions may be essential for many physiological processes, such as coordinated depolarization of cardiac cells, proper embryonic development, and microvascular conduction responses. For this reason, mutations in genes encoding connexins can result in functional and developmental abnormalities. The amino acid sequence of the relevant human protein can be found under Genbank accession number AAA52131. The amino acid sequence of the relevant mouse protein can be found under accession number AAA37444. The amino acid sequence of the relevant dog protein can be found under accession number AAR25626. The amino acid sequence of the relevant horse protein can be found under accession number NP_001296155.

[0030] The methods disclosed herein comprise viable treatment strategies that have implications for extending lifespan by rescuing electrical and physiological cardiac dysfunction associated with arrhythmogenic disorders such as ARVC. To date, there are no existing treatments utilizing connexin 43-based or gene therapy treatments for ARVC patients. Currently disclosed connexin 43-based strategies focus on the use of peptidomimetics and small molecule drugs, which primarily serve to relocate existing connexin 43 from cells to the "correct location," and these strategies rely on having existing connexin 43 protein in the heart. However, ARVC patients and patients with end-stage heart failure have extremely low or no connexin 43 protein in cardiac myocytes, and therefore, the aforementioned approaches do not work in these situations.

[0031] Gene therapy has been shown to be a safe and effective approach to circumventing cardiac disease, and the experiments presented herein provide evidence that this approach can restore connexin 43 expression in the heart and circumvent the electrical and structural disorders associated with ARVC that lead to sudden death. Because ARVC is a rare and fatal disease with no treatment, this approach provides the first opportunity to test a treatment approach in this highly vulnerable population, and this strategy may also have application to other cardiac diseases with potential structural and arrhythmogenic disorders associated with loss of connexin 43 polypeptide.

[0032] Thus, in some embodiments, connexin 43 polypeptide is targeted for clinical therapy for ARVC patients by creating a cardiac troponin T promoter-driven adeno-associated viral vector (serotype 9) containing human connexin 43 cDNA, amplifying clinical-grade virus, and delivering it to ARVC patients as a means to restore connexin 43 protein levels and rescue electrical and contractile dysfunction. However, in some embodiments, other vectors are used. In some embodiments, the vector uses a different promoter. The promoter must be active in cardiac tissue, but it may also be active only or primarily in cardiac tissue. In some embodiments, the vector is an adeno-associated viral vector. In some embodiments, the vector is based on a different virus. In some embodiments, the vector is non-viral.

[0033] In some cases, the vector is a viral vector. In some cases, the viral vector is based on or derived from a replication-deficient virus. Non-limiting examples of viral vectors suitable for delivering the nucleic acid molecules of the present disclosure to a subject include those derived from adenovirus, retrovirus (e.g., lentivirus), adeno-associated virus (AAV), and herpes simplex-1 (HSV-1). In certain cases, the viral vector is derived from AAV.

[0034] In some embodiments, the vector has a tropism for cardiac tissue, such as adeno-associated virus serotype 9 (AAV9), and in other embodiments, the vector is not specific for cardiac tissue. In some embodiments, connexin 43 expression is restricted to cardiac tissue as a result of one or both of a tissue-specific promoter and a cardiac tropic vector. In some embodiments, the vector confers long-term expression of the connexin 43 polypeptide. In some embodiments, the vector is a non-integrating vector.

[0035] In some embodiments, effective treatment is achieved with a single administration of the gene therapy vector. In such embodiments, weak or non-immunogenicity is of reduced importance compared to vectors that may or will be administered more than once. In some embodiments, the vector is one against which the recipient does not have a pre-existing immune response, e.g., an anti-vector antibody response.

[0036] This strategy may also have therapeutic applications in other genetic disorders resulting in sudden arrhythmic death (e.g., hypertrophic cardiomyopathy) and late-stage heart failure, in which there is an increased risk of arrhythmia and death and in which loss of connexin 43 has been reported.

[0037] Provided herein are methods for treating a disease or disorder in a subject in need thereof, the subject suffering from one or more of arrhythmogenic right ventricular cardiomyopathy, right and / or left ventricular dysfunction, myocardial fibrofatty replacement, hypertrophic cardiomyopathy, and electrical and physiological cardiac dysfunction in arrhythmogenic disease. The method comprises, alternatively consists essentially of, or even consists of administering to the subject a vector encoding a connexin 43 polypeptide sequence, wherein administration of an effective amount of the vector results in increased connexin 43 levels in at least a portion of the subject's heart. Increased connexin 43 polypeptide is typically indicative of symptomatic improvement. In some embodiments, symptomatic improvement can be seen within days of increased connexin 43 expression or within days, e.g., 1, 2, 3, or 4 days, of administering a connexin 43 gene therapy vector.

[0038] In some embodiments, the connexin 43 polypeptide comprises at least the 382 amino acid sequence of P17302 (CXA_1HUMAN, UniProtKB) (SEQ ID NO: 1), or a biological equivalent thereof. At least four smaller truncated isoforms of connexin 43 exist in the human heart, which may function as functional fragments, including 32 kDa (100-382 AA, SEQ ID NO: 3), 29 kDa (125-382 AA, SEQ ID NO: 4), 26 kDa (147-382 AA, SEQ ID NO: 5), and 20 kDa (213-382 AA, SEQ ID NO: 6) ("Autoregulation of connexin 43 gap junction formation by internally translated isoforms," ​​Smith & Shaw, Cell Rep., 5(3):611-8, 2013, the entire contents of which are incorporated herein by reference). In some embodiments, the three fragments are encoded by SEQ ID NOs: 9-12, respectively.

[0039] The polypeptide can be delivered in a gene delivery vehicle or construct comprising a polynucleotide encoding connexin 43 operably linked to sequences for expression of the polynucleotide in vivo. Non-limiting examples of these include the cardiac troponin T promoter, cardiac myosin light chain promoter, cardiac myosin heavy chain promoter, and the cardiac alpha actin enhancer associated with the elongation factor 1 alpha promoter. Cardiomyocyte-specific, spatially restricted promoters include control sequences derived from genes such as, but not limited to, myosin light chain 2, alpha myosin heavy chain, AE3, cardiac troponin C, and cardiac actin.

[0040] The construct can also be contained within a viral vector. Non-limiting examples of such vectors include adenoviral vectors, adeno-associated vectors (AAV), or lentiviral vectors. The viral vector can be selected for its tissue tropism to the heart; for example, the AAV vector can be selected from the group of AAV1, AAV2, AAV2i8, or AAV9 serotypes (see, for example, "An Emerging Adeno-Associated Viral Vector Pipeline for Cardiac Gene Therapy" (Asokan and Samulski, Hum Gene Ther., 24(11):906-913, 2013) and "Systemic Gene Transfer to Skeletal Muscle Using Reengineered AAV Vectors" (Phillips et al., Methods Mol Biol., 709:141-51, 2011), each of which is incorporated herein by reference in its entirety for all of its teachings regarding in vivo cardiac gene transfer). In addition, AAV vectors can be chimeric, further enhancing the tissue tropism of the vector. Non-limiting examples of such include the AAV1 / 2 vectors described in "AAV Vectors for Cardiac Gene Transfer: Experimental Tools and Clinical Opportunities" (Mol. Ther., 19(9):1582-1590, 2011). This document is incorporated herein by reference in its entirety for all of its teachings regarding in vivo cardiac gene transfer.

[0041] The vector containing the connexin 43 polynucleotide is administered locally or systemically.

[0042] The methods are useful for treating mammals, such as human patients. As will be appreciated by those skilled in the art, the proteins and / or polynucleotides should be derived from the same species as the subject being treated.

[0043] An effective amount of polynucleotide and / or vector may be, for example, about 2×10 per kg of subject body weight. 11 to approximately 2 × 10 14 The vector should be delivered in a pharmaceutically acceptable carrier.

[0044] Also provided are methods for treating a subject in need thereof, the subject suffering from one or more of arrhythmogenic right ventricular cardiomyopathy, right and / or left ventricular dysfunction, myocardial fibrofatty replacement, hypertrophic cardiomyopathy, and electrical and physiological cardiac dysfunction in arrhythmogenic disease. The methods comprise, alternatively consist essentially of, or even consist of administering to the subject an effective amount of vector-delivered expression of a connexin 43 polypeptide as described herein. Those skilled in the art can determine whether connexin 43 levels are increased in a model system by immunohistochemistry, Western blot, affinity chromatography, or indirectly based on detection of mRNA via Northern blot or reverse transcriptase polymerase chain reaction (RT-PCR). In actual patients, detection of increased connexin 43 polypeptide is typically indicative of improvement in symptomatology.

[0045] In some embodiments, the connexin 43 polypeptide comprises at least the 382 amino acid sequence of P17302 (CXA_1HUMAN, UniProtKB) (SEQ ID NO: 1), or a biological equivalent thereof.

[0046] Some embodiments are pharmaceutical compositions comprising a vector capable of conferring connexin 43 polypeptide expression and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier in such products is typically a sterile aqueous solution. In various aspects of these embodiments, the pharmaceutically acceptable carrier may comprise culture medium, phosphate-buffered saline, or HEPES-buffered saline. In some embodiments, the vector is supplied as a liquid formulation (which may be stored frozen) for immediate use. In another embodiment, the vector is supplied in lyophilized form and is reconstituted with water for injection or a sterile aqueous solution immediately prior to administration.

[0047] Some embodiments of the present disclosure involve administration to a mammal, e.g., a human, and constitute methods of treatment. As used herein, the terms "treat" or "treatment," collectively or in individual embodiments, broadly include any type of therapeutic activity, including the diagnosis, mitigation, or prevention of disease in humans or other animals, or any activity that otherwise affects the structure or any function of the human or other animal body. Treatment can include achieving a desired pharmacological and / or physiological effect. This effect can be prophylactic, meaning that a disorder or its signs or symptoms are completely or partially prevented, and / or therapeutic, meaning that a disorder and / or adverse effects resulting from the disorder are partially or completely cured. Examples of "treatment" include, but are not limited to, preventing the onset of a disorder in a subject who has not yet been diagnosed with the disorder but may be susceptible to the disorder, inhibiting the disorder, i.e., preventing its development, and / or reducing or ameliorating symptoms of the disorder (e.g., cardiac arrhythmia). As will be understood by those skilled in the art, "treatment" can include the systemic amelioration of symptoms associated with the pathology and / or delaying the onset of symptoms, such as chest pain. Clinical or subclinical evidence of "treatment" will vary by pathology, individual, and procedure. Treatment activities include the administration of medicaments, dosage forms, and pharmaceutical compositions described herein to a patient, whether by a medical professional, the patient themselves, or another person, particularly in accordance with the various treatment methods described herein. Treatment activities include the orders, instructions, and advice of a medical professional (e.g., a surgeon, surgical assistant, nurse practitioner), which are then carried out by another person, including another medical professional or the patient themselves. In some embodiments, treatment activities can also include encouraging, inducing, or coercing the selection of a particular medication or combination of medications for the treatment of a condition (and their actual use), as might be done by insurance companies, pharmacy benefit management companies, and the like, by approving coverage for the medication, denying coverage for alternative medications, including or excluding the medication from an adopted formulary, or providing financial incentives for the use of the medication.In some embodiments, treatment activities can also include encouraging, guiding, or mandating, by policy or practice standards, that a particular medication be selected for the treatment of a condition (and that the medication be used), as might be done by a hospital, clinic, health maintenance organization, medical practice, or physician group.

[0048] In some embodiments disclosed herein, a connexin 43 polypeptide expression vector is prepared for administration to or administered to a mammal. In some embodiments, the mammal is a human. In other embodiments, the mammal is a domestic pet, such as a cat or dog. In some embodiments, the mammal is an agricultural animal, such as a horse, cow, sheep, or pig. In other embodiments, the mammal is a laboratory animal, such as a mouse, rat, hamster, or rabbit.

[0049] In related aspects, the therapeutic compositions of the invention are administered to a subject as a prophylactic or palliative measure, where "palliative" as used herein means improving or reducing the symptoms associated with the disorder in the subject, including curing such disorder.

[0050] It should be understood that, if desired, compositions as described herein may be administered in combination with other agents, such as, for example, other proteins or polypeptides or various pharmaceutically active agents. Indeed, there is virtually no limit to the other components that may be included, provided that the additional agents do not cause significant adverse effects upon contact with target cells or host tissues. Compositions may therefore be delivered with a variety of other agents as required in a particular case. Such compositions may be purified from host cells or other biological sources, or alternatively, may be chemically synthesized as described herein.

[0051] Administration of gene therapy vectors is typically by injection or infusion. In some embodiments, intravenous administration is used. In other embodiments, the vector is administered to a tissue, organ, or body cavity at or in communication with the site where treatment will be effective, such as the heart itself or the pericardial space.

[0052] An appropriate dose of the vector can be administered to a subject in need thereof. Non-limiting examples of administration methods include subcutaneous administration, intravenous administration, intramuscular administration, intradermal administration, intraperitoneal administration, oral administration, infusion, intracranial administration, intrathecal administration, intranasal administration, and intra-arterial administration. In some cases, administration can involve injection of a formulation of the vector composition.

[0053] Also, continuous and discontinuous administration schedules by any method include dosing schedules in which the dose of vector is adjusted throughout the effective period, for example, such that the dose is low at the beginning of the connexin 43 administration period and increased throughout the connexin administration period, such that the dose is initially high and decreased throughout the administration period, such that the dose is initially low and increased to a peak level and then decreased toward the end of the administration period, and any combination thereof. Dosing schedules may also be performed using methods standard in the art, such as catheter systems.

[0054] Recombinant AAV (rAAV) virions or in vitro transduced cells may be delivered directly to muscle by injection with a needle, catheter, or related device using techniques known in the art. For in vivo delivery, rAAV virions may be formulated into a pharmaceutical composition and administered directly in one or more doses as directed. A therapeutically effective amount is about 10 8 / kg to 10 16 / kg, more preferably 10 10 / kg to 10 14 / kg, and even more preferably about 10 11 / kg to 10 13 / kg of rAAV virions (or viral genomes, also called "vg" or "vg") or any value between these ranges.

[0055] One form of administration of recombinant AAV virions uses a convection-enhanced delivery (CED) system. In this manner, recombinant virions can be delivered to numerous cells across a wide area of ​​muscle. Furthermore, the delivered vector efficiently expresses the transgene within muscle cells. Any convection-enhanced delivery device may be suitable for delivering viral vectors. In a preferred embodiment, the device is an osmotic pump or an infusion pump. Both osmotic pumps and infusion pumps are commercially available from various sources, such as Alzet Corporation, Hamilton Corporation, and Alza Inc. (Palo Alto, California). Typically, viral vectors are delivered via a CED device as follows: A catheter, cannula, or other injection device is inserted into the appropriate muscle tissue (e.g., skeletal muscle) of a selected subject. For a detailed description of CED delivery, see U.S. Patent No. 6,309,634, which is incorporated herein by reference in its entirety.

[0056] Various perfusion methods are available and standard in the art, and the present invention is not limited to any one method. Any perfusion method (e.g., catheter-based) that provides the desired results is contemplated. The purpose of the perfusion method is to increase the contact time between the vector (e.g., adenoviral, AAV, or lentiviral vector) and the target cells (e.g., smooth muscle cells). Thus, the present methods encompass perfusion methods (e.g., closed-circuit perfusion) performed at body temperature and under defined conditions, e.g., at 37°C, for about 2, 5, 10, 12, 15, 30, or 60 minutes or more, or, in larger animals or humans, for about 2, 4, 6, 8, 10, or 12 hours or more, to allow viral entry into the target cells and create optimal conditions for gene expression and protein synthesis. To this end, various additives, such as natural and unnatural amino acids and growth factors, may be added to provide sufficient materials for protein synthesis.

[0057] Each treatment may be expressed as a composition(s) for use in such a medical treatment. For example, an embodiment including a connexin 43 polypeptide expression vector for use in treating an arrhythmogenic disorder. Similarly, each treatment may be expressed as a composition(s) for use in the manufacture of a medicament. For example, a connexin 43 polypeptide expression vector for use in the manufacture of a medicament for treating an arrhythmogenic disorder.

[0058] The present invention will be more particularly described in the following examples. These examples are intended to be illustrative only, as numerous modifications and variations therein will be apparent to those skilled in the art. The following examples are intended to illustrate, but not to limit, the present invention.

[0059] (Example) Example 1: Increased connexin 43 expression promoted extended lifespan and restoration of cardiac rhythm and function in a genetic model of cardiomyopathy. Connexin 43 is lost in hiPSC-derived cardiomyocytes of arrhythmogenic right ventricular cardiomyopathy, which exhibits abnormal electrical, structural, and contractile activity, and cardiac Connexin 43 is lost early in a mouse model of ARVC, which exhibits cardiac dysfunction and sudden death. Attempts to relocate existing Connexin 43 remaining in cells using the Connexin 43 carboxy-terminal mimetic α-carboxy-terminal 1 (CT1) peptide and rotigaptide failed, suggesting that there is not enough Connexin 43 and that genetic restoration of Connexin 43 may be required to restore its levels and function.

[0060] Using Cx43 YFP adenovirus, we demonstrated that connexin 43 overexpression is essential for the development of iPSC cell lines from a severely structurally abnormal human ARVC patient with PKP2 c.1171-2A>G (PKP2 mut) was sufficient to restore normal cardiomyocyte rhythm (measured via field potential) and contractile function (measured via impedance) (Figure 1A-F). Western blot analysis showed that Connexin 43 overexpression increased the expression of focal adhesion (mechanical cell-cell junction) proteins in ARVCs (PKP2) without affecting their function, which had been shown to be lost in this ARVC-hiPSC line. mut ) alone was found to be sufficient to rescue functional alterations in ARVC (Figure 1G). Despite this, in some embodiments, connexin 43 gene therapy may be used with adjunctive therapies directed at restoring focal adhesion proteins to further reinforce cell-cell junctions in ARVC.

[0061] Using Cx43 YFP adenovirus, we demonstrated that structural ARVC (PKP2 mut ) We also showed that the rescued effect of connexin 43 overexpression in hiPSC lines was dose-dependent ( Fig. 2 ).

[0062] Using Cx43 YFP adenovirus, we demonstrated that connexin 43 overexpression increased the expression of an ARVC-hiPSC line (DSG2 c.1498 C>A) carrying the DSG2 c.1498 C>A gene after isoproterenol stimulation. mut ) and rescued catecholamine-induced arrhythmias and dysfunction (Fig. 3A-F).

[0063] Using a Cx43 GFP adeno-associated virus (AAV) construct, one-time gene delivery of Cx43 GFP AAV can extend lifespan (2-fold), restore cardiac function (2.5-fold), and restore rhythm (1.5-fold) in an adult mouse model of ARVC (DSP-cKO) that suffers from premature sudden death (Figure 4A-H). The DSP-cKO mouse model is described above. Adult DSP-cKO mice injected with Cx43 GFP AAV died 3 weeks after AAV injection. However, Western blot analysis revealed that these mice did not successfully express Cx43 in the heart (presumably because they did not have enough time for expression), further supporting the idea that Cx43 overexpression was crucial for the extended lifespan and restoration of cardiac rhythm and function in surviving DSP-cKO mice (Figure 4H).

[0064] We tested our in vitro proof-of-concept prototype using human Connexin43-yellow fluorescent protein-tagged adenovirus driven by the human cytomegalovirus (CMV) immediate-early enhancer / promoter in human iPSC-derived cardiomyocytes from two ARVC patient lines exhibiting primarily electrical (catecholamine-induced impairments) and combined structural / electrical features. Restoration of Connexin43 to control levels was sufficient to rescue both catecholamine-induced electrical and contractile impairments in our electrical ARVC-hiPSC line and to rescue both basal and catecholamine-induced electrical and contractile impairments in our combined structural and electrical ARVC-hiPSC line (Figures 1-3). We also generated a cardiac troponin T-driven adeno-associated virus (cardiotropic serotype 9) carrying human connexin 43 and tagged with green fluorescent protein to demonstrate that the virus could be successfully delivered and expressed in the hearts of late-stage diseased mice with ARVC (DSP-cKO mice) using a one-time delivery method (retroorbital delivery). We estimated that optimal expression of Cx43 AAV requires 2.4 × 10 Cx43 AAVs, based on the DSP-cKO mice that survived the virus (6 weeks after AAV injection, showing robust connexin 43 protein expression) versus the DSP-cKO mice that died (3 weeks after AAV injection, showing no connexin 43 protein expression). 11 We also show that the AAV virus has no effect on cardiac electrical or contractile function in control mice (sacrificed 5 weeks after AAV injection) over the course of the study (Figure 4). We further demonstrate that DSP-cKO mice that received the virus and exhibited robust connexin 43 protein expression lived longer (2-fold) and exhibited improved cardiac function (2.5-fold) and rhythm (1.5-fold) (Figure 4). We also demonstrate that the AAV virus did not affect cardiac electrical or contractile function in control mice (sacrificed 5 weeks after AAV injection) over the course of the study (Figure 4).

[0065] Example 2: Increased Cx43 expression promoted extended lifespan and restoration of cardiac rhythm and function in an injury model of cardiac hypertrophy. To induce pressure overload-induced cardiac hypertrophy and heart failure, 6-8 week-old mice undergo transverse aortic coarctation for 4 weeks. After this 4-week period, mice receive Cx43 gene therapy via a one-time retro-orbital vein injection at a dose similar to that used in the ARVC model in Example 1 (above). Mice are continuously monitored for 1, 2, and 4 weeks via echocardiography and telemetry to monitor left ventricular function and cardiac rhythm, respectively. Compared to controls, AAV-Cx43-treated mice have improved cardiac function (e.g., left ventricular fractional shortening), reduced rhythm abnormalities (e.g., fewer PVCs), and improved survival. Histological analysis is performed to assess cardiac size and fibrotic infiltration into the myocardium after infection. AAV-Cx43-treated mice show reduced cardiac size (and dimensions) and less fibrosis after pressure overload compared to controls.

[0066] Unless otherwise noted, all numbers used in the specification and claims expressing quantities of ingredients, properties (e.g., molecular weight), reaction conditions, and the like, should be understood to be modified in all instances by the term "about." As used herein, the terms "about" and "approximately" mean a range of 10 to 15%, preferably 5 to 10%. Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. Without limiting the application of the doctrine of equivalents to the scope of the claims, at a minimum, each numerical parameter should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. These numerical values, however, inherently contain certain errors necessarily resulting from the standard deviations inherent in their respective testing measurements.

[0067] As used in the context of the present invention (particularly in the context of the appended claims), the singular references "a," "an," and "the" should be construed to include both the singular and the plural unless otherwise indicated and unless the context clearly contradicts. The recitation of ranges of values ​​herein merely serves as a shorthand method of individually referring to each individual value falling within that range. Unless otherwise stated herein, each of these individual values ​​is incorporated herein as if it were individually set forth herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein and unless the context clearly contradicts. The use of any examples and illustrative language (e.g., "etc.") described herein is intended merely to facilitate a clearer understanding of the present invention and does not in any way limit the scope of the claimed invention in any way other than as described in these examples and illustrative language. No language described herein should be construed as indicating any element essential to the practice of the invention but not recited in the claims.

[0068] Collections of alternative elements or embodiments of the invention described herein are not to be construed as limitations. Each collection element may be referenced or claimed individually or in combination with other collection elements or elements found herein. One or more elements of a collection may be added to or deleted from a group for reasons of convenience and / or patentability. When such additions or deletions occur, the specification shall be deemed to include the group as modified, and all mark-brush groups used in the claims shall satisfy the written description requirement.

[0069] Certain embodiments herein are described, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will be apparent to those skilled in the art in light of the foregoing description. The inventors contemplate such variations as those skilled in the art would appropriately employ, and intend that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Furthermore, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0070] Certain embodiments described herein may be further limited in the claims by the use of phrases such as "consisting of" or "consisting essentially of." When used in a claim as filed or amended, the transitional phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. The transitional phrase "consisting essentially of" limits the scope of the claim to include the specified materials or steps, plus those that do not materially affect the claim's basic and novel characteristics. Embodiments of the claimed invention are inherently or explicitly described and enabled herein.

[0071] Additionally, throughout this specification, various references are made to patents and publications. Each of the above references and publications is incorporated herein by reference in its entirety.

[0072] Finally, it is to be understood that the embodiments of the invention described herein are illustrative of the principles of the invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, and not of limitation, alternative configurations of the invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precise nature shown and described.

[0073] [Note] [Appendix 1] A method for treating a structural cardiovascular disease or condition in a subject in need thereof, comprising administering to the subject a vector encoding a connexin 43 polypeptide sequence operably linked to a promoter that is active in myocardial tissue, wherein administration of an effective amount of the vector results in increased connexin 43 levels in at least a portion of the subject's heart.

[0074] [Appendix 2] The disease or condition is Arrhythmogenic right ventricular cardiomyopathy, Right ventricular dysfunction, left ventricular dysfunction, fibrofatty replacement of the myocardium, Hypertrophic cardiomyopathy, or Electrical and physiological cardiac dysfunction in arrhythmogenic diseases, 2. The method of claim 1, wherein the method is one or more of:

[0075] [Appendix 3] 3. The method of claim 1 or 2, wherein the connexin 43 polypeptide sequence comprises the 382 amino acid sequence of P17302 (CXA_1HUMAN, UniProtKB) (SEQ ID NO: 1) or a functional fragment thereof.

[0076] [Appendix 4] The method of claim 3, wherein the sequence encoding the connexin 43 polypeptide is SEQ ID NO: 2.

[0077] [Appendix 5] 5. The method of any one of claims 1 to 4, wherein the vector is a viral vector.

[0078] [Appendix 6] The method of claim 5, wherein the viral vector is an adenoviral vector or an adeno-associated viral vector (AAV).

[0079] [Appendix 7] The method of claim 6, wherein the AAV vector is derived from the AAV1, AAV2, or AAV9 serotype group.

[0080] [Appendix 8] 8. The method of any one of claims 1 to 7, wherein the promoter is a CMV immediate early enhancer / promoter.

[0081] [Appendix 9] 8. The method of any one of claims 1 to 7, wherein the promoter is a cardiac-specific promoter.

[0082] [Appendix 10] 10. The method of claim 9, wherein the promoter is a troponin T promoter.

[0083] [Appendix 11] 10. The method of claim 9, wherein the promoter is a cardiac myosin light chain promoter, a cardiac myosin heavy chain promoter, or an elongation factor 1 alpha promoter-associated cardiac alpha actin enhancer.

[0084] [Appendix 12] 12. The method of any one of claims 1 to 11, wherein the vector is administered locally or systemically.

[0085] [Appendix 13] 13. The method of any one of claims 1 to 12, wherein the subject is a mammal.

[0086] [Appendix 14] 14. The method of claim 13, wherein the subject is a human.

[0087] [Appendix 15] 15. The method of any one of claims 1 to 14, wherein the effective amount is about 2 x 10 to about 2 x 10 viral genomes per kg of body weight of the subject.

[0088] [Appendix 16] 16. The method of any one of claims 1 to 15, wherein said administering results in reduced cardiac electrical dysfunction.

[0089] [Appendix 17] 17. The method of any one of claims 1 to 16, wherein said administering results in reduced physiological cardiac dysfunction.

[0090] [Appendix 18] 18. The method of any one of claims 1 to 17, wherein said administering results in improved structural integrity of the heart.

[0091] [Appendix 19] A vector comprising a polynucleotide comprising a promoter that is active in cardiac tissue and is operably linked to a nucleic acid sequence encoding a connexin 43 polypeptide.

[0092] [Appendix 20] 19. The vector of claim 18, wherein the connexin 43 polypeptide comprises the 382 amino acid sequence (SEQ ID NO: 1) of P17302 (CXA_1HUMAN, UniProtKB) or a functional fragment thereof.

[0093] [Appendix 21] 21. The vector of claim 19 or 20, wherein the promoter is a human CMV immediate-early enhancer / promoter.

[0094] [Appendix 22] 21. The vector of claim 19 or 20, wherein the promoter is a cardiac troponin T promoter.

[0095] [Appendix 23] 21. The vector of claim 19 or 20, wherein the promoter is a cardiac myosin light chain promoter, a cardiac myosin heavy chain promoter, or an elongation factor 1 alpha promoter-associated cardiac alpha actin enhancer.

[0096] [Appendix 24] 24. The vector of any one of claims 19 to 23, wherein the vector is an adenoviral vector.

[0097] [Appendix 25] 24. The vector of any one of claims 19 to 23, wherein the vector is an adeno-associated virus vector.

[0098] [Appendix 26] 26. The vector of any one of claims 19 to 25, wherein the sequence encoding connexin 43 is SEQ ID NO: 2.

Claims

1. A vector for use in a method for treating a structural cardiovascular disease or condition in a subject in need thereof, the method comprising administering to the subject the vector, the vector encoding a connexin 43 polypeptide sequence operably linked to a promoter that is active in myocardial tissue, wherein administration of an effective amount of the vector results in increased connexin 43 levels in at least a portion of the subject's heart.

2. The vector of claim 1 , wherein the subject has a mutation in a gene encoding a component of a plaque desmosome.

3. 3. The vector of claim 2, wherein the component is desmoplakin (DSP), plakoglobin (JUP), plakophilin 2 (PKP2), or desmoglein 2 (DSG2).

4. The vector according to claim 1 , wherein the disease or condition is characterized by a structural disorder of plaques.

5. The vector according to claim 1 , wherein the disease or condition is characterized by a deficiency of a plaque adhesion protein.

6. The disease or condition is Arrhythmogenic right ventricular cardiomyopathy, Right ventricular dysfunction, left ventricular dysfunction, fibrofatty replacement of the myocardium, Hypertrophic cardiomyopathy, or Electrical and physiological cardiac dysfunction in arrhythmogenic diseases, The vector according to any one of claims 1 to 5, wherein the vector is one or more of the following:

7. 7. The vector of claim 1, wherein the connexin 43 polypeptide sequence comprises the 382 amino acid sequence of P17302 (CXA_1HUMAN, UniProtKB) (SEQ ID NO: 1) or a functional fragment thereof.

8. The vector of claim 7, wherein the sequence encoding the connexin 43 polypeptide is SEQ ID NO:

2.

9. The vector according to any one of claims 1 to 8, wherein the vector is a viral vector.

10. The vector of claim 9 , wherein the viral vector is an adenoviral vector or an adeno-associated viral (AAV) vector.

11. The vector of claim 10 , wherein the AAV vector is from the AAV1, AAV2, or AAV9 serotype group.

12. 12. The vector of claim 1, wherein the promoter is a CMV immediate early enhancer / promoter, or the promoter is a cardiac specific promoter.

13. 13. The vector of claim 12, wherein the promoter is a troponin T promoter, or the promoter is a cardiac myosin light chain promoter, a cardiac myosin heavy chain promoter, or an elongation factor 1 alpha promoter-associated cardiac alpha actin enhancer.

14. The vector of any one of claims 1 to 13, wherein the vector is administered locally or systemically.

15. The vector of any one of claims 1 to 14, wherein the subject is a mammal.

16. The vector of claim 15 , wherein the subject is a human.

17. The effective amount is about 2×10 per kg of the subject's body weight. １１ From about 2 x 10 １４ 17. The vector of any one of claims 1 to 16, which is a viral genome.

18. 18. The vector of any one of claims 1 to 17, wherein said administration results in a reduction of electrical and / or physiological cardiac dysfunction.

19. 19. The vector of any one of claims 1 to 18, wherein said administration results in improved structural integrity of the heart.

20. 20. The vector of any one of claims 1 to 19, wherein said administration results in improved cardiac contractile function.

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