Gene Therapy Compositions and Treatment of Arrhythmogenic Right Ventricular Cardiomyopathy

Abstract

Compositions and methods for treating or preventing cardiomyopathy in a human subject are disclosed. In some embodiments, the methods include delivering a therapeutic dose of a gene therapy vector to cardiomyocytes of the human subject, the gene therapy vector including a nucleic acid sequence encoding PKP2.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This invention claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 163,393, filed March 19, 2021, the disclosure of which is incorporated by reference in its entirety herein.

[0002] The present invention relates to gene therapy methods and pharmaceutical compositions for the treatment of cardiac diseases (eg, cardiac myopathies), in particular for the treatment of cardiomyopathies. [Background technology]

[0003] Despite pharmacological advances in the treatment of various cardiac diseases such as heart failure, mortality and morbidity remain unacceptably high. Moreover, certain therapeutic approaches are not suitable for many patients (e.g., patients with advanced heart failure conditions associated with other comorbidities). Alternative approaches such as gene therapy and cell therapy have attracted increasing attention because they may be uniquely tailored and effective in addressing the pathogenic mechanisms of the underlying causes of many cardiac diseases. Summary of the Invention

[0004] It is an object of the present invention to provide a method for delivering a therapeutic polynucleotide sequence to cardiomyocytes of a subject, such as a human subject.

[0005] A further object of certain embodiments of the present invention is to vectorize the polynucleotide sequence encoding the plakophilin-2 (PKP2) protein into a viral vector, such as an adeno-associated virus.

[0006] It is a further object of certain embodiments of the present invention to utilize gene therapy approaches to correct haploinsufficiency in PKP2 mutated cardiomyocytes.

[0007] It is a further object of certain embodiments of the present invention to increase expression of functional PKP2 protein in cells that are haploinsufficient for PKP2.

[0008] The above objects and others are met by the present invention, at least one aspect of which is directed to a method of treating or preventing cardiomyopathy in a subject (e.g., a human subject). The method includes, for example, delivering a therapeutic dose of a gene therapy vector to a cardiomyocyte of a subject, the gene therapy vector including a nucleic acid sequence encoding PKP2. In some embodiments, delivery of the gene therapy vector to a cardiomyocyte haploinsufficient for plakophilin-2 (PKP2) results in at least a 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, or 5-fold increase in desmosomal expression of PKP2 by the cardiomyocyte. In some embodiments, delivery of the gene therapy vector to a cardiomyocyte results in desmosomal expression of PKP2 that is at least 50% of the desmosomal expression by a non-haploinsufficient cardiomyocyte.

[0009] In at least one embodiment, the gene therapy vector comprises a viral vector. In at least one embodiment, the viral vector comprises one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, variants thereof, and combinations thereof. In at least one embodiment, the viral vector comprises AAV6 or AAV9. In at least one embodiment, the viral vector comprises AAV6.

[0010] In at least one embodiment, the nucleic acid sequence further encodes a cardiac-specific promoter.

[0011] In at least one embodiment, the therapeutic dose is effective to treat or prevent arrhythmogenic right ventricular cardiomyopathy (ARVC) by causing production of PKP2 or a functional variant thereof by the subject's cardiomyocytes.

[0012] In at least one embodiment, delivery of the therapeutic dose is intravenous.

[0013] In at least one embodiment, the subject is a human subject.

[0014] In another aspect, the gene therapy vector is adapted to express the nucleic acid sequence in the cardiomyocyte of a subject.In at least one embodiment, the nucleic acid sequence comprises a first sequence encoding PKP2 or its functional variant, and a second sequence comprising a cardiac-specific promoter.In at least one embodiment, the delivery of the gene therapy vector to the cardiomyocyte that is haploinsufficient for PKP2 results in at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, or 5-fold increase in the total desmosomal expression of PKP2 by the cardiomyocyte.In at least one embodiment, the delivery of the gene therapy vector to the cardiomyocyte that is haploinsufficient results in at least 50% of the total desmosomal expression by the cardiomyocyte that is non-haploinsufficient.

[0015] In at least one embodiment, the gene therapy vector comprises a viral vector. In at least one embodiment, the viral vector comprises one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, variants thereof, and combinations thereof. In at least one embodiment, the viral vector comprises AAV6 or AAV9.

[0016] In at least one embodiment, the cardiac specific promoter comprises TNNT2 or a functional sequence having at least 99%, 95%, 90%, 85%, 80%, 75%, or 70% similarity.

[0017] In at least one embodiment, the subject is a human subject.

[0018] In another aspect, the therapeutic formulation is formulated to treat or prevent cardiomyopathy in a subject.In at least one embodiment, the therapeutic formulation comprises a pharma- ceutically acceptable excipient or carrier and a viral vector comprising a nucleic acid sequence encoding PKP2 or a functional variant thereof.In at least one embodiment, the delivery of the therapeutic formulation to haploinsufficient cardiomyocytes for PKP2 results in at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, or 5-fold increase in total desmosomal expression of PKP2 by cardiomyocytes.In at least one embodiment, the delivery of the therapeutic formulation vector to haploinsufficient cardiomyocytes results in total desmosomal expression of PKP2 that is at least 50% of total desmosomal expression by non-haploinsufficient cardiomyocytes.

[0019] In at least one embodiment, the therapeutic formulation further comprises one or more additional viral vectors, each of which comprises a nucleic acid sequence encoding one or more non-PKP2 sarcomeric proteins or functional variants thereof. In at least one embodiment, the subject is a human subject.

[0020] In another aspect, a method of genetically modifying cardiomyocytes having a mutated PKP2 gene to express functional PKP2 or a functional variant thereof comprises transfecting or transducing cardiomyocytes with a nucleic acid sequence encoding functional PKP2, wherein the transfection or transduction results in at least a 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, or 5-fold increase in total desmosomal expression of functional PKP2 by the cardiomyocytes. In at least one embodiment, the transfection or transduction results in total desmosomal expression of functional PKP2 that is at least 50% of the total desmosomal expression by cardiomyocytes having a non-mutated PKP2 gene.

[0021] In at least one embodiment, the nucleic acid sequence is delivered by a viral vector comprising AAV6 or AAV9. In at least one embodiment, the viral vector comprises AAV6.

[0022] In at least one embodiment, the nucleic acid sequence further encodes a cardiac-specific promoter. In at least one embodiment, the cardiac-specific promoter comprises TNNT2 or a functional sequence having at least 99%, 95%, 90%, 85%, 80%, 75%, or 70% similarity.

[0023] In at least one embodiment, the PKP2 of any above method or formulation is PKP2 isoform 2a.

[0024] In at least one embodiment, the PKP2 of any of the above methods or formulations is PKP2 isoform 2b.

[0025] In another aspect, a therapeutic formulation for treating or preventing cardiomyopathy in a subject comprises a pharma- ceutically acceptable excipient or carrier, a first viral vector comprising a nucleic acid sequence encoding PKP2 isoform 2a or a functional variant thereof, and a second viral vector comprising a nucleic acid sequence encoding PKP2 isoform 2b or a functional variant thereof. In at least one embodiment, delivery of the therapeutic formulation to a cardiomyocyte haploinsufficient for PKP2 isoform 2a or isoform 2b results in at least a 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, or 5-fold increase in total desmosomal expression of PKP2 isoform 2a or isoform 2b by the cardiomyocyte. In at least one embodiment, delivery of the therapeutic formulation vector to a cardiomyocyte haploinsufficient results in at least a 50% total desmosomal expression of PKP2 isoform 2a or isoform 2b by the cardiomyocyte.

[0026] In another aspect, the isolated cell is transduced with any of the gene therapy vectors of the above embodiments.In at least one embodiment, the cell is a human cell.In at least one embodiment, the cell is a cardiac cell.In at least one embodiment, the cell is a cardiomyocyte derived from human induced pluripotent stem cells.

[0027] In another aspect, a method of upregulating one or more desmosomal proteins in cardiomyocytes having a mutated PKP2 gene comprises transfecting or transducing cardiomyocytes with a nucleic acid sequence encoding a functional PKP2 selected from PKP2 isoform 2a and PKP2 isoform 2b, wherein the transfection or transduction results in at least a 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, or 5-fold increase in total desmosomal expression of each of the one or more desmosomal proteins, wherein the one or more desmosomal proteins are selected from desmoplakin 1, desmoplakin 2, desmocollin 2, plakoglobin, desmoglein 2, and connexin 43.

[0028] In another aspect, a method of treating or preventing cardiomyopathy in a subject comprises delivering a therapeutic dose of a gene therapy vector to a cardiac muscle cell of the subject, the cardiac muscle cell being haploinsufficient for plakophilin-2 (PKP2), the gene therapy vector comprising a nucleic acid sequence encoding a non-dominant PKP2 isoform or a functional variant thereof, and delivery of the gene therapy vector to the cardiac muscle cell results in at least a 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, or 5-fold increase in total desmosomal expression of PKP2 by the cardiac muscle cell, the total desmosomal expression of PKP2 comprising expression of the dominant PKP2 isoform and the non-dominant PKP2 isoform. In at least one embodiment, the dominant PKP2 isoform is PKP2 isoform 2a and the non-dominant PKP2 isoform is PKP2 isoform 2b.

[0029] The above and other features of the present disclosure, its nature and various advantages will become apparent from a consideration of the following detailed description taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0030] [Figure 1] Shown are fluorescent microscopy images of PKP2 localized to desmosomal cell-cell junctions in wild-type 2D human induced pluripotent stem cell-derived cardiomyocytes ("hiPSC-CMs"). [Diagram 2] Fluorescence microscopy images confirming expression of PKP2 and its localization at desmosomal intercellular junctions following AAV9 transduction of control cardiomyocytes are shown. [Diagram 3] Western blots of PKP2 protein expression are shown, and haploinsufficiency of PKP2 mutant cell lines is evident by reduced expression of PKP2 compared to control cell lines. [Figure 4] Fluorescence microscopy images of the expression and precise localization of PKP2 isoform 2b following AAV9-mediated transduction of PKP2 mutant hiPSC-CMs are shown. [Diagram 5] PKP2 mutant hiPSC-CMs compared to two wild-type hiPSC-CM controls (Asi and Cau) are shown. [Figure 6] Decreased endogenous PKP2 expression compared to unrelated control cardiomyocytes, non-failing human heart (NFH) tissue, and PKP2 patient cardiomyocytes. [Figure 7] Figure 2 shows that PKP2 isoform 2a is the predominant PKP2 isoform in human tissues. [Figure 8] RNA levels following AAV9-mediated transduction of codon-optimized PKP2 isoform 2b compared to control and patient cells. [Figure 9A] PKP2 protein levels after transduction compared to control cells based on endogenous myosin-binding protein C levels are shown. [Figure 9B] PKP2 protein levels after transduction compared to control cells based on endogenous cardiac troponin T levels are shown. [Figure 10A] As a result of expression of exogenous PKP2 protein, there is upregulated expression of desmoplakin 1, desmoplakin 2, desmocollin 2, and plakoglobin. [Figure 10B] As a result of expression of exogenous PKP2 protein, upregulated expression of desmoglein 2 and connexin 43 is shown.

[0031] definition As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "drug" includes not only a single drug but also a mixture of two or more different drugs, reference to a "viral vector" includes not only a single viral vector but also a mixture of two or more different viral vectors, and so forth.

[0032] Also, as used herein, "about," when used in reference to a measured quantity, refers to normal variation in that measured quantity that would be expected by one of ordinary skill in the art when making the measurement and using a level of care commensurate with the purpose of the measurement and the precision of the measuring device. In certain embodiments, the term "about" includes the recited number ±10%, so that "about 10" would include 9-11.

[0033] Also, as used herein, "polynucleotide" has its normal and accustomed meaning in the art and includes any polymeric nucleic acid, such as a DNA molecule or an RNA molecule, as well as chemical derivatives known to those of skill in the art. Polynucleotides include those that code for therapeutic proteins, but also include sequences that can be used to reduce the expression of a target nucleic acid sequence using techniques known in the art (e.g., antisense, interference, or small interfering nucleic acids). Polynucleotides can also be used to initiate or increase the expression of a target nucleic acid sequence or the production of a target protein in cells of the cardiovascular system. Target nucleic acids and proteins include, but are not limited to, nucleic acids and proteins that are normally found in the target tissue, derivatives of such naturally occurring nucleic acids or proteins, naturally occurring nucleic acids or proteins that are not normally found in the target tissue, or synthetic nucleic acids or proteins. One or more polynucleotides can be used in combination and administered simultaneously and / or sequentially to increase and / or decrease one or more target nucleic acid sequences or proteins.

[0034] Also, as used herein, an "exogenous" nucleic acid or gene is one that is not naturally present in the vector utilized for nucleic acid transfer, e.g., not naturally found in a viral vector, although the term is not intended to exclude nucleic acids that encode proteins or polypeptides that are naturally present in the patient or host.

[0035] As used herein, "cardiac cells" also includes any cell of the heart involved in maintaining the structure or providing the function of the heart, such as cardiomyocytes, cells of the cardiovascular system, or cells present within the cardiac valves. Cardiac cells include cardiomyocytes (which have both normal and abnormal electrical properties), epithelial cells, endothelial cells, fibroblasts, cells of conductive tissue, cardiac pacemaker cells, and neurons.

[0036] Also, as used herein, the term "adeno-associated virus" or "AAV" encompasses all subtypes, serotypes, and pseudotypes, as well as naturally occurring and recombinant forms. Various AAV serotypes and strains are known in the art and are publicly available from sources such as ATCC and academic or commercial sources. Alternatively, sequences from AAV serotypes and strains that are publicly available and / or available from various databases can be synthesized using known techniques.

[0037] Also, as used herein, "serotype" refers to an AAV that is identified by and distinguished from other AAVs based on the reactivity of the capsid protein with a defined antiserum. There are at least 12 known serotypes of human AAV, including AAV1 to AAV12, although additional serotypes continue to be discovered, and the use of newly discovered serotypes is contemplated.

[0038] Also, as used herein, "pseudotyped" AAV refers to an AAV that contains capsid proteins of one serotype and a viral genome that contains 5' and 3' inverted terminal repeats (ITRs) of a different or heterologous serotype. A pseudotyped recombinant AAV (rAAV) would be expected to have the cell surface binding properties of the capsid serotype and genetic properties that match the ITR serotype. A pseudotyped rAAV can contain AAV capsid proteins, including VP1, VP2, and VP3 capsid proteins, and ITRs from any serotype of AAV, including any primate AAV serotype from AAV1 to AAV12, so long as the capsid proteins are of a heterologous serotype to the serotype(s) of the ITRs. In a pseudotyped rAAV, the 5' and 3' ITRs can be the same or heterologous. A pseudotyped rAAV is generated using standard techniques described in the art.

[0039] Also, as used herein, a "chimeric" rAAV vector includes AAV vectors that contain heterologous capsid proteins. That is, a rAAV vector may be chimeric with respect to its capsid proteins VP1, VP2, and VP3, such that VP1, VP2, and VP3 are not all of the same serotype of AAV. As used herein, a chimeric AAV includes AAVs in which the capsid proteins VP1, VP2, and VP3, including but not limited to capsid proteins from AAV1 and AAV2, are different in serotype, are a mixture of capsid proteins of other parvoviruses, or contain other viral proteins, or other proteins, such as proteins that target the delivery of AAV to a desired cell or tissue. As used herein, a chimeric rAAV also includes rAAVs that contain chimeric 5' and 3' ITRs.

[0040] Also, as used herein, "a pharma- ceutically acceptable excipient or carrier" refers to any inactive ingredient in a composition that is combined with an active agent in a formulation. Pharmaceutically acceptable excipients may include, but are not limited to, carbohydrates (such as glucose, sucrose, or dextran), antioxidants (such as ascorbic acid or glutathione), chelating agents, low molecular weight proteins, high molecular weight polymers, gel-forming agents, or other stabilizers and additives. Other examples of pharma- ceutically acceptable carriers include wetting agents, emulsifying agents, dispersing agents, or preservatives that are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. Examples of carriers, stabilizers, or adjuvants are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985).

[0041] Also, as used herein, "patient" refers to a subject, particularly humans (but will include non-humans), who is exhibiting clinical signs of a particular condition or symptoms indicating the need for treatment, is being prophylactically treated for a condition, or has been diagnosed with a condition to be treated.

[0042] Also, as used herein, "subject" encompasses the definition of the term "patient" and does not exclude otherwise healthy individuals.

[0043] Also, as used herein, "treatment of" and "treating" include the administration of a drug for the purpose of reducing the severity of or preventing a condition, e.g., heart disease.

[0044] As used herein, "prevention of" and "preventing" also include avoiding the onset of a condition, such as, for example, heart disease.

[0045] Also, as used herein, "condition" or "conditions" refers to those medical conditions, such as heart disease, that can be treated, alleviated, or prevented by administration of an effective amount of a drug to a subject.

[0046] Also, as used herein, an "effective amount" refers to an amount of a drug that is sufficient to produce a beneficial or desired effect at a level that is readily detectable by a method commonly used to detect such an effect. In some embodiments, such an effect produces a change of at least 10% from the basal level value when the drug is not administered. In other embodiments, the change is at least 20%, 50%, 80%, or even higher from the basal level. As explained below, the effective amount of a drug may vary from subject to subject depending on the subject's age, general condition, the severity of the condition being treated, the particular drug being administered, and the like. The appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art by reference to the relevant texts and literature and / or by using routine experimentation.

[0047] Also, as used herein, "active agent" refers to any substance intended to produce a therapeutic, prophylactic, or other intended effect, regardless of whether it has been approved by a government agency for that purpose.

[0048] The recitation of ranges of values ​​herein is intended to serve merely as a shorthand method of individually referring to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated herein as if it were individually recited herein. All methods described herein may be performed in any suitable order, unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended merely to facilitate understanding of certain materials and methods, and does not limit the scope of the claims. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosed materials and methods. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a major myocardial disorder and the leading cause of sudden cardiac death (SCD) in young individuals. Arrhythmogenic right ventricular cardiomyopathy is characterized by myocardial degeneration and fibrofatty replacement of the myocardium, which may be present in the right and / or left ventricle and ultimately lead to progressive heart failure. The clinical cardiac phenotype is characterized by the presence of typical electrocardiographic abnormalities, an increased burden of ventricular arrhythmias, and extensive myocardial scarring on cardiac magnetic resonance imaging.

[0050] ARVC is familial in approximately 50% of cases and is usually inherited as an autosomal dominant trait. Approximately 30% of patients of Caucasian descent have dominant mutations in the PKP2 gene. The majority of mutations result in abnormal or truncated proteins as a result of insertion / deletion, nonsense, or splice site mutations, causing haploinsufficiency.

[0051] ARVC is thought to be a disease of the desmosomals, electron-dense structures that provide mechanical adhesion between cardiac myocytes. PKP2 is one of several genes that form part of the desmosomal protein complex, in which mutations have been identified that lead to ARVC. Absence of the PKP2 protein, due to haploinsufficiency, destabilizes the desmosomal protein complex, with mechanical and signaling consequences.

[0052] Mechanical components are highlighted in vitro by abnormal gene expression patterns, including downregulation of several extracellular matrix genes, such as various collagens, and strong upregulation of other profibrotic markers, such as fibrillogenic collagens, fibronectin, and TIMP1, due to the lack of PKP2 protein under mechanical stress conditions. In preclinical and clinical settings, this is reflected by the aggravation of ARVC by exercise in PKP2 mouse models and by the adverse effects of exercise on the phenotype in humans, e.g., athletes. At the signaling level, the lack of plakophilin causes the translocation of plakoglobin to the nucleus, which leads to a decrease in classical Wnt / b-catenin signaling and an increase in the expression of fibrogenic and adipogenic genes.

[0053] The two major forms of PKP2 include PKP2 isoform 2a (SEQ ID NO: 3) and PKP2 isoform 2b (SEQ ID NO: 5). The protein coding portion of the PKP2 gene for PKP2 isoform 2a is contained in a 2764 bp cDNA sequence (GenBank: BC126199.1; SEQ ID NO: 1), which can be vectorized into AAV according to the present invention. As used herein, "PKP2" or "PKP2 protein" should be construed to encompass isoforms of PKP2, including PKP2 isoform 2a and PKP2 isoform 2b, unless otherwise stated or suggested by the context.

[0054] Certain embodiments can correct haploinsufficiency of PKP2 protein by replacing it with a normal allele via AAV9-TNNT2-PKP2 mediated gene transfer. In certain embodiments, the compositions and methods of the present invention may be capable of, for example, (1) precisely localizing PKP2 protein to desmosomes and (2) correcting haploinsufficiency in PKP2 mutated human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs), thereby correcting the desmosomal protein complex. Certain embodiments are also expected to result in complete or near complete PKP2 deficiency in iPSC-CMs carrying two pathogenic mutations in trans. Non-limiting exemplary embodiments for testing delivery of PKP2 polynucleotides to cardiomyocytes include: (1) vectorizing PKP2 into AAV9 and / or AAV6 using the TNNT2 promoter; generating iPSC-CMs with PKP2 mutations (either one mutation or two mutations in trans); transducing 2D PKP2 mutant cardiomyocyte cultures (with one or two mutations) with AAV6-PKP2 or AAV9-PKP2 in vitro and testing subcellular localization to desmosomes; testing molecular and physiological data including cell size, contractility, and transcriptome analysis.

[0055] Although many embodiments are described herein with respect to PKP2 protein, it should be understood that the expression of additional proteins (e.g., sarcomeric proteins) is envisioned.In addition to PKP2, exemplary proteins may include, without limitation, one or more of SERCA2, MYBPC3, MYH7, MYL3, MYL2, ACTC1, TPM1, TNNT2, TNNI3, TTN, FHL1, ALPK3, dystrophin, FKRP, their mutants, or combinations thereof.The one or more proteins used may also be functional mutants of the proteins mentioned herein, and may show significant amino acid sequence identity compared to the original protein. For example, the amino acid identity may reach at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%. In this context, the term "functional variant" means that a variant of a protein can partially or completely exert the function of the corresponding naturally occurring protein. A functional variant of a protein may include a protein that differs from its naturally occurring counterpart by, for example, the substitution, deletion, or addition of one or more amino acids. The amino acid substitutions may be conservative or non-conservative. It is preferred that the substitutions are conservative substitutions, i.e., replacement of an amino acid residue with an amino acid of similar polarity that acts as a functional equivalent. Preferably, the amino acid residue used as a substitute is selected from the same group of amino acids as the amino acid residue to be substituted. For example, a hydrophobic residue may be replaced with another hydrophobic residue, a polar residue may be replaced with another polar residue having the same charge, etc. Functionally compatible amino acids that can be used for conservative substitutions include, for example, non-polar amino acids such as glycine, valine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, and tryptophan.Examples of uncharged polar amino acids include serine, threonine, glutamine, asparagine, tyrosine and cysteine. Examples of charged polar (basic) amino acids include histidine, arginine and lysine. Examples of charged polar (acidic) amino acids include aspartic acid and glutamic acid.

[0056] Also considered as variants are proteins that differ from their naturally occurring counterparts by one or more (e.g., 2, 3, 4, 5, 10, or 15) additional amino acids. These additional amino acids may be present within the amino acid sequence of the original protein (i.e., as an insertion) or may be added to one or both termini of the protein. Essentially, insertions can be made at any position, provided that the addition of the amino acids does not inhibit the function of the polypeptide to perform the function of the naturally occurring protein in the subject to be treated. Furthermore, variants of proteins also include proteins that, compared to the original polypeptide, lack one or more amino acids. Such deletions may affect any amino acid position, provided that they do not impair the ability of the protein to perform its normal function.

[0057] Finally, a variant of a cardiac sarcomere protein (e.g., PKP2) also refers to a protein that differs from the naturally occurring protein by structural modifications, such as modified amino acids. Modified amino acids are amino acids that have been modified either by natural processes, such as processing or post-translational modification, or by chemical modification processes known in the art. Exemplary amino acid modifications include phosphorylation, glycosylation, acetylation, O-linked N-acetylglucosaminylation, glutathionylation, acylation, branching, ADP-ribosylation, cross-linking, disulfide bridge formation, formylation, hydroxylation, carboxylation, methylation, demethylation, amidation, cyclization, and / or covalent or non-covalent binding with phosphatidylinositol, flavin derivatives, lipoteichoic acid, fatty acids, or lipids.

[0058] Therapeutic polynucleotide sequences encoding a target protein may be administered to the subject to be treated in the form of a gene therapy vector, i.e., a nucleic acid construct that contains a coding sequence including translation and termination codons, next to other sequences required to provide expression of the exogenous nucleic acid, such as a promoter, a Kozak sequence, a polyA signal, etc.

[0059] For example, the gene therapy vector may be part of a mammalian expression system. Useful mammalian expression systems and expression constructs are commercially available. Also, several mammalian expression systems, such as plasmid or viral vector-based systems, e.g., LENTI-Smart™ (InvivoGen), GenScript™ Expression Vector, pAdVAntage™ (Promega), ViraPower™ Lentiviral, Adenoviral Expression Systems (Invitrogen), and Adeno-associated Virus Expression System (Cell Biolabs), are sold by different manufacturers and can be used in the present invention.

[0060] The gene therapy vector for expressing the exogenous therapeutic polynucleotide sequence of the present invention may be a viral or non-viral expression vector suitable for introducing the exogenous therapeutic polynucleotide sequence into cells, for example, for the subsequent expression of the protein encoded by the nucleic acid. The expression vector may be an episomal vector, i.e., a vector capable of autonomously replicating itself in a host cell, or an integrative vector, i.e., a vector that is stably integrated into the genome of a cell. Expression in a host cell may be constitutive or regulated (e.g., inducible).

[0061] In certain embodiments, the gene therapy vector is a viral expression vector. The viral vector for use in the present invention may comprise a viral genome with a deletion of a portion of the native sequence in order to introduce a heterologous polynucleotide without destroying the infectivity of the virus. Due to the specific interaction between the viral components and the host cell receptor, the viral vector is highly suitable for efficient introduction of genes into target cells. The viral vector suitable for facilitating gene transfer into mammalian cells can be derived from different types of viruses, such as, for example, from AAV, adenovirus, retrovirus, herpes simplex virus, bovine papilloma virus, lentivirus, vaccinia virus, polyoma virus, Sendai virus, orthomyxovirus, paramyxovirus, papovavirus, picornavirus, poxvirus, alphavirus, or any other viral shuttle suitable for gene therapy, their mutants, and their combinations.

[0062] "Adenoviral expression vector" or "adenovirus" is intended to include those constructs that contain sufficient adenoviral sequences to (a) support packaging of a therapeutic polynucleotide sequence construct and / or (b) ultimately express a tissue- and / or cell-specific construct that has been cloned therein. In one embodiment of the present invention, the expression vector comprises a genetically engineered form of adenovirus. Knowledge of the genetic organization of adenovirus, a 36 kilobase (kb) linear double-stranded DNA virus, allows for the replacement of large pieces of adenoviral DNA with foreign sequences up to 7 kb.

[0063] Adenovirus growth and manipulation is known to those of skill in the art and exhibits a broad host range in vitro and in vivo. This group of viruses can be produced in high titers, e.g., 10 per mL. 9 ~10 11Adenoviruses can be acquired in plaque-forming units of 10 ...

[0064] Retroviruses (also called "retroviral vectors") may be chosen as gene delivery vectors due to their ability to integrate genes into the host genome, introduce large amounts of foreign genetic material, infect a wide range of species and cell types, and be packaged in specialized cell lines.

[0065] The retroviral genome contains three genes, gag, pol, and env, which code for the capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence upstream from the gag gene contains a signal for packaging of the genome into virions. Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genes. These contain strong promoter and enhancer sequences and are also required for integration into the host cell genome.

[0066] To construct a retroviral vector, a nucleic acid encoding a gene of interest is inserted into the viral genome in place of a specific viral sequence to generate a replication-defective virus. To produce virions, a packaging cell line is constructed that contains the gag, pol, and / or env genes, but does not contain the LTR and / or packaging components. When a recombinant plasmid containing a cDNA is introduced into this cell line (e.g., by calcium phosphate acid precipitation) along with retroviral LTRs and packaging sequences, the packaging sequences allow packaging of the RNA transcripts of the recombinant plasmid into viral particles that are then secreted into the medium. The medium containing the recombinant retrovirus is then collected, optionally condensed, and used for gene transfer. Retroviral vectors are capable of infecting a wide variety of cell types. However, integration and stable expression require the division of the host cell.

[0067] Retroviruses can be derived from any of the subfamilies. For example, vectors from murine sarcoma virus, bovine leukemia virus, Rous sarcoma virus, murine leukemia virus, mink cell focus-inducing virus, reticuloendotheliosis virus, or avian leukemia virus can be used. Those skilled in the art can combine parts from different retroviruses, such as LTR, tRNA binding site, and packaging signal, to provide recombinant retroviruses. These retroviruses are then usually used to produce transduction-competent retroviral vector particles. For this purpose, the vector is introduced into a suitable packaging cell line. Retroviruses can also be constructed for site-specific integration into the DNA of host cells by incorporating a chimeric integrase enzyme into the retroviral particle.

[0068] Herpes simplex virus (HSV) is of great interest for the treatment of nervous system diseases because of its neurotropism. Furthermore, the ability of HSV to establish latent infection in non-dividing neuronal cells without integrating into the host cell chromosome or otherwise altering the host cell metabolism, as well as the presence of promoters that are active during latency, make HSV an attractive vector. And although much attention has been focused on the neurotropic applications of HSV, the vector has a broad host range and can therefore be used in other tissues.

[0069] Another factor that makes HSV an attractive vector is the size and organization of its genome. Because HSV is large, the integration of multiple genes or expression cassettes is less problematic than with other smaller viral systems. In addition, the ability to use different viral regulatory sequences with different performance (time, strength, etc.) allows for greater control of expression than with other systems. Another advantage is that the virus has relatively few spliced ​​messages, making genetic manipulation easier.

[0070] HSV is also relatively easy to manipulate and can be grown to high titers. Thus, delivery is less of a problem, both in terms of the amount needed to achieve a sufficient multiplicity of infection (MOI) and in terms of reducing the need for repeated dosing. Non-virulent mutants of HSV have been developed and are readily available for use in the context of gene therapy.

[0071] Lentiviruses are complex retroviruses that contain, in addition to the common retroviral genes gag, pol, and env, other genes with regulatory or structural functions. The increased complexity allows the virus to regulate its life cycle, such as during latent infection. Examples of lentiviruses include human immunodeficiency viruses (HIV-1, HIV-2) or simian immunodeficiency viruses (SIV). Lentiviral vectors have been generated by multiple attenuation of HIV toxic genes, e.g., genes env, vif, vpr, vpu, and nef are deleted, making the vector biologically safe.

[0072] Lentiviral vectors are plasmid-based or virus-based, and are constructed to carry the necessary sequences for the selection and introduction of nucleic acid into host cells in order to incorporate foreign nucleic acid. The gag, pol and env genes of interest are also known in the art. Thus, the relevant genes are cloned into the selected vector and then used to transform the target cells of interest.

[0073] Vaccinia virus vectors have been widely used because they are easy to construct, can produce relatively high levels of expression, have a wide host range, and have a large DNA carrying capacity. Vaccinia contains a linear double-stranded DNA genome of approximately 186 kb that shows a pronounced "AT" preference. Approximately 10.5 kb of inverted terminal repeats flank the genome. The majority of essential genes appear to map within a central region that is most highly conserved among poxviruses. There are an estimated 150-200 open reading frames in vaccinia virus. Although both strands are encoded, extensive overlap of the reading frames is not common.

[0074] At least 25 kb can be inserted into the vaccinia virus genome. Prototypical vaccinia vectors contain a transgene inserted into the viral thymidine kinase gene via homologous recombination. Vectors are selected on the basis of the tk phenotype. Inclusion of the non-translated leader sequence from encephalomyocarditis virus results in expression levels higher than those of conventional vectors, with the transgene accumulating to more than 10% of the protein in infected cells at 24 hours.

[0075] Empty capsids of papovaviruses, such as mouse polyomavirus, have attracted attention as potential vectors for gene transfer. The use of empty polyoma was first reported when polyoma DNA and purified empty capsids were incubated in a cell-free system. The DNA of the new particles was protected from the action of pancreatic DNase. The reconstituted particles were used to introduce transforming polyoma DNA fragments into rat FIII cells. The empty capsids and reconstituted particles consist of all three of the polyoma capsid antigens VP1, VP2, and VP3.

[0076] AAV is a parvovirus belonging to the Dependovirus genus. They are small, non-enveloped, single-stranded DNA viruses that require a helper virus to replicate. Superinfection with a helper virus (such as adenovirus, herpesvirus, or vaccinia virus) is required to form a functionally complete AAV virion. In vitro, in the absence of superinfection with a helper virus, AAV establishes a latent state in which the viral genome exists in episomal form, but no infectious virions are produced. Subsequent infection with a helper virus "rescues" the genome, allowing it to replicate and be packaged into viral capsids, thereby reconstituting infectious virions. Recent data indicate that in vivo, both wild-type and recombinant AAV exist primarily as large episomal concatemers. In one embodiment, the gene therapy vector used herein is an AAV vector. The AAV vector may be a purified, replication-incompetent, pseudotyped rAAV particle.

[0077] AAV is not associated with any known human disease, is generally not considered pathogenic, and does not appear to alter the physiological properties of host cells upon integration. AAV can infect a wide range of host cells, including non-dividing cells, and can infect cells of different species. In contrast to some vectors that are rapidly cleared or inactivated by both cellular and humoral responses, AAV vectors have been shown to induce sustained transgene expression in various tissues in vivo. The persistence of recombinant AAV-mediated transgenes in non-dividing cells in vivo may be due to the lack of native AAV viral genes and the vector's ability to form ITR-associated episomal concatemers.

[0078] AAV is an attractive vector system for use in cell transduction according to the invention because it has a high frequency of persistence as episomal concatemers, is capable of infecting non-dividing cells, including cardiomyocytes, and is therefore useful, for example, for delivery of genes to mammalian cells in tissue culture and in vivo.

[0079] Typically, rAAV is produced by co-transfecting a plasmid containing the gene of interest flanked by two AAV terminal repeats, such as pIM45, and / or an expression plasmid containing a wild-type AAV coding sequence without the terminal repeats. Cells are also infected and / or transfected with adenovirus and / or a plasmid carrying the adenovirus genes required for AAV helper function. Stocks of rAAV produced in such a way are contaminated with adenovirus and must be physically separated from the rAAV particles (e.g., by cesium chloride density centrifugation or column chromatography). Alternatively, adenovirus containing the AAV coding region and / or a cell line containing some or all of the AAV coding region and / or adenovirus helper genes could be used. Cell lines with rAAV DNA as an integrated provirus can also be used.

[0080] Multiple serotypes of AAV exist in nature, with at least 12 serotypes (AAV1-AAV12). Despite high homology, different serotypes have tropism for different tissues. Upon transfection, AAV induces only minor (if any) immune responses in the host. Therefore, AAV is well suited for gene therapy approaches.

[0081] The present disclosure may be directed to a drug comprising an AAV vector, which in some embodiments is one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, ANC AAV, their chimeric AAV derivatives, their mutants, and combinations thereof, which are more suitable for high efficiency transduction in the tissue of interest. In certain embodiments, the gene therapy vector is an AAV serotype 1 vector. In certain embodiments, the gene therapy vector is an AAV serotype 2 vector. In certain embodiments, the gene therapy vector is an AAV serotype 3 vector. In certain embodiments, the gene therapy vector is an AAV serotype 4 vector. In certain embodiments, the gene therapy vector is an AAV serotype 5 vector. In certain embodiments, the gene therapy vector is an AAV serotype 6 vector. In certain embodiments, the gene therapy vector is an AAV serotype 7 vector. In certain embodiments, the gene therapy vector is an AAV serotype 8 vector. In certain embodiments, the gene therapy vector is an AAV serotype 9 vector. In certain embodiments, the gene therapy vector is an AAV serotype 10 vector. In certain embodiments, the gene therapy vector is an AAV serotype 11 vector. In certain embodiments, the gene therapy vector is an AAV serotype 12 vector.

[0082] The appropriate dose of AAV for humans is approximately 1 × 10 per kilogram of body weight. 8 Vector genome (vg / kg) ~ approx. 3 × 10 14 vg / kg, approx. 1×10 8 vg / kg, approx. 1×10 9 vg / kg, approx. 1×1010 vg / kg, approx. 1×10 11 vg / kg, approx. 1×10 12 vg / kg, approx. 1×10 13 vg / kg, or approximately 1 × 10 14 The total viral load, or DRP, can be in the range of about 5×10 15 vg / kg, 4 × 10 15 vg / kg, 3 × 10 15 vg / kg, 2×10 15 vg / kg, 1×10 15 vg / kg, 9×10 14 vg / kg, 8×10 14 vg / kg, 7×10 14 vg / kg, 6×10 14 vg / kg, 5×10 14 vg / kg, 4 × 10 14 vg / kg, 3 × 10 14 vg / kg, 2×10 14 vg / kg, 1×10 14 vg / kg, 9×10 13 vg / kg, 8×10 13 vg / kg, 7×10 13 vg / kg, 6×10 13 vg / kg, 5×10 13 vg / kg, 4 × 10 13 vg / kg, 3 × 10 13 vg / kg, 2×10 13 vg / kg, 1×10 13 vg / kg, 9×10 12 vg / kg, 8×10 12 vg / kg, 7×10 12 vg / kg, 6×10 12 vg / kg, 5×10 12 vg / kg, 4 × 10 12 vg / kg, 3 × 10 12 vg / kg, 2×10 12 vg / kg, 1×10 12 vg / kg, 9×10 11 vg / kg, 8×10 11 vg / kg, 7×10 11 vg / kg, 6×10 11 vg / kg, 5×10 11 vg / kg, 4 × 1011 vg / kg, 3 × 10 11 vg / kg, 2×10 11 vg / kg, 1×10 11 vg / kg, 9×10 10 vg / kg, 8×10 10 vg / kg, 7×10 10 vg / kg, 6×10 10 vg / kg, 5×10 10 vg / kg, 4 × 10 10 vg / kg, 3 × 10 10 vg / kg, 2×10 10 vg / kg, 1×10 10 vg / kg, 9×10 9 vg / kg, 8×10 9 vg / kg, 7×10 9 vg / kg, 6×10 9 vg / kg, 5×10 9 vg / kg, 4 × 10 9 vg / kg, 3 × 10 9 vg / kg, 2×10 9 vg / kg, 1×10 9 vg / kg, 9×10 8 vg / kg, 8×10 8 vg / kg, 7×10 8 vg / kg, 6×10 8 vg / kg, 5×10 8 vg / kg, 4 × 10 8 vg / kg, 3 × 10 8 vg / kg, 2×10 8 vg / kg, or 1 × 10 8 vg / kg, about these, at least these, at least about these, less than these, or less than these, or within a range defined by any two of these values. The dosages listed above are in vg / kg cardiac tissue.

[0083] Apart from viral vectors, non-viral expression constructs can also be used to introduce the gene encoding target protein or its functional variant or fragment into patient's cells.The non-viral expression vectors that allow in vivo expression of protein in target cells include, for example, plasmid, modified RNA, mRNA, cDNA, antisense oligomer, DNA lipid complex, nanoparticle, exosome, any other non-viral shuttle suitable for gene therapy, their variants, and their combinations.

[0084] Apart from viral and non-viral vectors, nuclease systems may be used in conjunction with vectors and / or electroporation systems to enter and introduce into the patient's cells a gene encoding a target protein or a functional variant or fragment thereof. Exemplary nuclease systems may include, but are not limited to, clustered regularly interspaced short palindromic repeats (CRISPR), DNA cleavage enzymes (e.g., Cas9), meganucleases, TALENs, zinc finger nucleases, any other nuclease system suitable for gene therapy, their variants, and their combinations. For example, in one embodiment, one viral vector (e.g., AAV) may be used for a nuclease (e.g., CRISPR), and another viral vector (e.g., AAV) may be used for a DNA cleavage enzyme (e.g., Cas9) to introduce both (nuclease and DNA cleavage enzyme) into the target cell.

[0085] Other vector delivery systems that can be used to deliver therapeutic polynucleotide sequences encoding therapeutic genes into cells are receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Due to the cell type-specific distribution of various receptors, delivery can be highly specific. Receptor-mediated gene targeting vehicles can contain two components: a ligand specific for a cell receptor, and a DNA-binding agent.

[0086] Suitable methods for the introduction of non-viral vectors into target cells include, for example, lipofection, calcium phosphate coprecipitation, DEAE-dextran, and direct DNA introduction using microglass tubes, ultrasound, electroporation, etc. Prior to the introduction of the vector, cardiomyocytes can be treated with permeabilizing agents such as phosphatidylcholine, streptolysin, sodium caprate, decanoylcarnitine, tartaric acid, lysolecithin, Triton X-100, etc. Exosomes can also be used to introduce naked DNA or DNA wrapped in the capsid of AAV.

[0087] The gene therapy vector of the present invention may include a promoter operably linked to a nucleic acid sequence encoding a target protein. The promoter sequence must be compact and ensure strong expression. Preferably, the promoter provides expression of the target protein in the myocardium of a patient who has been treated with the gene therapy vector. In some embodiments, the gene therapy vector includes a cardiac-specific promoter operably linked to a nucleic acid sequence encoding the target protein. As used herein, "cardiac-specific promoter" refers to a promoter whose activity in cardiac cells is at least two-fold higher than in any other non-cardiac cell type. Preferably, cardiac-specific promoters suitable for use in the vectors of the present invention have at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, or at least 50-fold higher activity in cardiac cells compared to activity in non-cardiac cell types.

[0088] A cardiac-specific promoter may be a selected human promoter or a promoter comprising a functionally equivalent sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a selected human promoter. An exemplary non-limiting promoter that may be used is the cardiac troponin T promoter (TNNT2). Other non-limiting examples of promoters include α-myosin heavy chain promoter, myosin light chain 2v promoter, α-myosin heavy chain promoter, α-cardiac actin promoter, α-tropomyosin promoter, cardiac troponin C promoter, cardiac troponin I promoter, cardiac myosin binding protein C promoter, and sarco / endoplasmic reticulum Ca promoter. 2+ -ATPase (SERCA) promoter (e.g., isoform 2 of this promoter (SERCA2)).

[0089] Vectors useful in the present invention may have various transduction efficiencies. As a result, viral or non-viral vectors transduce, equal to, or at least about, about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% or more of the cells in the target vascular area. Multiple vectors (viral or non-viral, or combinations thereof) can be used simultaneously or sequentially. This can be used to introduce multiple polynucleotides and / or target multiple cell types. When multiple vectors or multiple drugs are used, multiple transduction / transfection efficiencies can occur.

[0090] The pharmaceutical composition containing the gene therapy vector can be prepared as either a liquid solution or a suspension. The pharmaceutical composition of the present invention may contain commonly used pharma- ceutical acceptable excipients, such as diluents and carriers. In particular, the composition contains a pharma- ceutical acceptable carrier, such as water, saline, Ringer's solution, or dextrose solution. In addition to carriers, the pharmaceutical composition may also contain emulsifiers, pH buffers, stabilizers, dyes, and the like.

[0091] In certain embodiments, the pharmaceutical composition comprises a therapeutic gene dose, which is a dose that is not toxic to the subject and can prevent or treat the subject's cardiomyopathy.Prevention or treatment of cardiomyopathy may be evaluated as a change in phenotypic characteristics associated with cardiomyopathy, and such change is effective for preventing or treating cardiomyopathy.Thus, a therapeutic gene dose is usually a dose that is sufficient to improve or prevent the pathogenic cardiac phenotype of the subject being treated when administered in a physiologically acceptable composition.

[0092] In certain embodiments, gene therapy vectors may be transduced into subjects by several different methods, including intravenous, intraarterial, or intraperitoneal delivery. In some embodiments, gene therapy vectors may be directly administered to cardiac tissue, for example, by intracoronary administration. In some embodiments, myocardial tissue transduction may be achieved by intramyocardial delivery via a catheter, which can be used to introduce vector-free cDNA, with or without a transduction-facilitating carrier, into the myocardium.

[0093] In certain embodiments, the medicament will comprise a therapeutic gene dose, which is a dose that is non-toxic to the patient and capable of preventing or treating a particular cardiac condition in the patient.

[0094] Cardiac conditions that may be treated by the methods disclosed herein include, but are not limited to, genetically determined heart disease (e.g., genetically determined cardiomyopathies), arrhythmic heart disease, heart failure, ischemia, arrhythmias, myocardial infarction, congestive heart failure, transplant rejection, abnormal cardiac contractility, non-ischemic cardiomyopathy, mitral regurgitation, aortic stenosis or regurgitation, abnormal Ca 2+ These may include one or more of metabolism, congenital heart disease, primary or secondary cardiac tumors, and combinations thereof. EXAMPLES

[0095] The following examples are provided to aid in the understanding of the present disclosure and should not be construed as specifically limiting the embodiments described and claimed herein. Such variations of the embodiments, including the substitution of all currently known or later developed equivalents that would be within the skill of a person skilled in the art, and changes in formulations or minor changes in experimental design, should be construed as being included within the scope of the embodiments incorporated herein.

[0096] Example 1 (Prophetic) In an illustrative example of an in vitro system, the PKP2 isoform 2a cDNA sequence (2764 bp cDNA, GenBank: BC126199.1; SEQ ID NO: 1) is cloned under the cardiac-specific TNNT2 promoter (SEQ ID NO: 6) using the AAV2 internal terminal repeat (ITR): ITR-TNNT2-PKP2cDNA-ITR. The nucleic acid sequence encoding PKP2 may be a codon-optimized version of the PKP2 gene encoding the PKP2 isoform 2a protein (SEQ ID NO: 2). As another illustrative example, the nucleic acid sequence encoding PKP2 may be a codon-optimized version of the PKP2 gene encoding the PKP2 isoform 2b protein (SEQ ID NO: 4).

[0097] The construct can be vectorized into AAV, such as AAV6 and AAV9. A Flag-tagged construct (Flag-PKP2) can be made so that anti-Flag can identify the protein after transfection and distinguish it from endogenous protein. SEQ ID NO: 7 is an exemplary construct sequence for expressing PKP2 isoform 2b, for example. The expression of PKP2 in vitro can be observed by immunofluorescence microscopy using PKP2 primary antibody, which reveals the localization of PKP2 in the cell membrane and dense plaques.

[0098] It is anticipated that one or more new introns can be incorporated into the gene therapy vectors described herein to further increase gene expression levels. For example, a "chimeric intron" may be utilized, which refers to an intron that contains parts of at least two different introns from two different genes, such as an intron sequence from the human beta globin gene and human immunoglobulin G. In some embodiments, the new intron may be inserted immediately downstream of the promoter. In some embodiments, the new intron may be located in different locations in the PKP2 cDNA sequence, such as after exon 1 and before exon 2.

[0099] AAV6-TNNT2-PKP2 is used to transfect iPSC-CMs in 2D cell cultures, including normal cardiomyocytes, cardiomyocytes with one heterozygous PKP2 mutation (from an ARVC patient), and cardiomyocytes with two PKP2 mutations in trans.

[0100] Following successful transfection and characterization of PKP2 RNA and protein levels, comparisons of normal CM versus PKP2-deficient and PKP2-corrected CM are performed for a number of measurements, including cell size and correction of genes known to be altered in expression in PKP2 deficiency (MYL2, SCN5A (whose protein product is NaV1.5), GJA1, and TTN).

[0101] It is anticipated that similar approaches will be adaptable for ex vivo treatment in human 3D culture models as well as in vivo treatment in PKP2 mutant mouse models.

[0102] Expression of (Flag-)PKP2 protein would allow it to reach its correct subcellular location (desmosomes), and transfection would correct PKP2 haploinsufficient or completely deficient cells at the RNA and protein levels. In completely PKP2 deficient cells, PKP2 transfection would also be able to restore desmosomal protein complexes in desmosomes, in particular plakoglobin, which is reduced when PKP2 is reduced.

[0103] It is further expected that gene therapy vectors for expressing PKP2 isoform 2a, PKP2 isoform 2b, or both, can be delivered to cardiac tissue of a human subject. For example, the gene therapy vectors can be formulated into a therapeutic formulation that includes one or more gene therapy vectors and a pharma- ceutically acceptable excipient or carrier. The formulation can be transduced into a human subject by several different methods, including intravenous, intraarterial, or intraperitoneal delivery. The gene therapy vectors can be administered directly to cardiac tissue, for example, by intracoronary administration. The gene therapy vectors can also be delivered by intramyocardial delivery via a catheter.

[0104] It is further anticipated that the gene therapy vector can be administered locally to the subject's cardiac tissue, for example, by forming a closed circuit by isolating the subject's coronary circulation from the subject's systemic circulation, and perfusing a fluid (e.g., a formulation containing the gene therapy vector) into the isolated coronary circulation of the subject. The perfusion may be performed in the patient's unarrested beating heart. The closed circuit may be formed, for example, with a first drug delivery catheter placed in the patient's right coronary artery, a second drug delivery catheter placed in the patient's left main coronary artery, a drug retrieval catheter placed in the coronary sinus, an extracorporeal membrane oxygenator inserted between the coronary artery, the coronary venous system, and the venous and arterial branches. Such local delivery may be performed as described in International Application No. PCT / IB2020 / 000692, filed August 26, 2020, the disclosure of which is incorporated herein by reference in its entirety.

[0105] Example 2: Desmosomal PKP2 expression in hiPSC-derived cardiomyocytes Proteins of the desmosomal complex were expressed in human induced pluripotent stem cell-derived (hiPSC-derived) normal cardiomyocytes in two-dimensional (2D) cell culture and located in the forming intracellular structures of desmosomes. Figure 1 shows a fluorescent microscopy image of PKP2 localized to the desmosomal cell-cell junctions in wild-type 2D hiPSC cardiomyocytes.

[0106] Control hiPSC cardiomyocytes were transduced with AAV9-TNNT2-PKP2b, the same vector described in Example 1, which further contains a FLAG tag. Figure 2 shows fluorescent microscopy images confirming that the FLAG tag signal is expressed and precisely localized to the desmosomal cell-cell junctions of wild-type cardiomyocytes.

[0107] Next, we characterized PKP2-mutated hiPSC-derived cardiomyocytes and compared them with various wild-type cell lines of Asian (Asi) and Caucasian (Cau) origin as controls to demonstrate haploinsufficiency at the cellular level. Figure 3 shows a Western blot of PKP2 protein expression, and haploinsufficiency of PKP2-mutated cell lines is evident by the reduced expression of PKP2 compared to control cell lines (quantification relative to cardiac troponin T).

[0108] As shown in the fluorescence microscopy images in Figure 4, AAV9-mediated transduction of a FLAG-tagged PKP2 isoform 2b transgene containing the TNNT2 promoter into PKP2-mutated hiPSC-derived cardiomyocytes was shown to result in the expression and precise localization of PKP2 isoform 2b.

[0109] In the experiments described herein, the hiPSC-derived cardiomyocytes used were found to express only PKP2 isoform 2a, indicating that this is a less mature and developmentally regulated isoform. In contrast, in mature human cardiac tissue, the full-length PKP2b isoform 2b predominates. At the total protein level, Western blot was used to confirm that transduction with AAV9-TNNT2-PKP2b-FLAG corrected the haploinsufficient state to full PKP2 protein expression in PKP2 mutant hiPSC cardiomyocytes. Figure 5 shows PKP2 mutant hiPSC-CMs ("PKP2") compared to two wild-type hiPSC-CM controls (Asi and Cau). MYBPC3 and cTnT were used as reference proteins to calculate the relative amount of PKP2 expressed. PKP2 mutant hi-IPSC-CMs showed greatly reduced PKP2 protein expression compared to the two control cell lines. PKP2 expression was quantitatively corrected after transduction with AAV9-PKP2. Note that transduced cells displayed a doublet of PKP2, indicative of PKP2 isoform 2b produced from the AAV-mediated transgene and PKP2 isoform 2a naturally expressed in hiPSC-CMs. De novo expression of PKP2 isoform 2b was well tolerated and did not lead to obvious functional changes in PKP2 mutant or wild-type control cardiomyocytes.

[0110] Example 3: Reduction of endogenous PKP2 expression FIG. 6 shows reduced endogenous PKP2 expression compared to unrelated control cardiomyocytes, non-failing human heart (NFH) tissue, and PKP2 patient cardiomyocytes (haploinsufficient for PKP2). As shown, PKP2 patient cardiomyocytes express less PKP2 compared to normal control cells and non-failing human heart (NFH) cells. Endogenous PKP2 content was unchanged when treated with neuraminidase (utilized during transduction with AAV9 in cell culture) without transduction ("PKP2 Pat NT NA"). Transduction was performed with a codon-optimized PKP2 isoform 2b vector ("PKP2 pat TD 2b opt") using primers selected not to bind to the wild-type PKP2 sequence, resulting in no change after transduction.

[0111] Figure 7 shows that PKP2 isoform 2a is the predominant PKP2 isoform in human tissues (irrelevant control, non-failing human heart). It remains unchanged under neuraminidase and after transduction of codon-optimized PKP2b (using unbound primers as above). The full-length isoform of PKP2 isoform 2b was not detected in NFH cells, control cells, or PKP2 patient cardiomyocytes. PKP2 isoform 2a was present as approximately half of the total PKP2.

[0112] Example 4: Restoration of total PKP2 levels Figures 8A and 8B show RNA levels after AAV9-mediated transduction of codon-optimized PKP2 isoform 2b ("TD") compared to the average of NFH cells and two unrelated control cells ("wt"), patient cells, and treated with ("NT NA"). Figures 9A and 9B show total protein levels after transduction, comparing healthy control CM levels and PKP2 patient CM levels (without transduction) with PKP2 levels in transduced patient cells. In Figure 9A, total PKP2 protein levels are measured relative to endogenous myosin-binding protein C (MYBPC3) levels, and in Figure 9B, PKP2 protein levels are measured relative to endogenous cardiac troponin T (cTnT) levels. As shown in Figures 8-9, transduction restores total PKP2 levels in PKP2 patient CM. This is achieved using exogenous expression of PKP2 isoform 2b, even though PKP2 isoform 2a is the predominant isoform, as shown in FIG.

[0113] Example 5: Recovery of other proteins in the desmosomal protein complex 10A and 10B show expression of various proteins of the desmosomal protein complex, including desmoplakin 1, desmoplakin 2, desmocollin 2, plakoglobin, desmoglein 2, and connexin 43, in untreated patient CM compared to patient CM following AAV9-mediated transduction of codon-optimized PKP2 isoform 2b. Without wishing to be bound by theory, it is believed that expression of exogenous PKP2 results in upregulation of various desmosomal proteins compared to cells haploinsufficient for PKP2.

[0114] In the above description, many specific details are set forth, such as specific materials, dimensions, process parameters, etc., to provide a thorough understanding of the invention. Particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments. The word "example" or "exemplary" is used herein to mean serving as an example, instance, or illustration. Any aspect or design described herein as "example" or "exemplary" should not necessarily be construed as preferred or advantageous over other aspects or designs. Rather, the use of the word "example" or "exemplary" is merely intended to present a concept in a concrete manner. As used in this application, "or" is intended to mean an inclusive "or" rather than an exclusive "or." That is, unless otherwise specified or clear from the context, "X includes A or B" is intended to mean all natural inclusive permutations. That is, if X includes A, if X includes B, or if X includes both A and B, then "X includes A or B" is satisfied under any of the above illustrative examples. References throughout this specification to "an embodiment," "a particular embodiment," or "one embodiment" mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment. Thus, the appearances of the phrases "one embodiment," "a particular embodiment," or "one embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment.

[0115] The invention has been described with reference to specific exemplary embodiments thereof. The specification and drawings are accordingly to be regarded in an illustrative rather than a restrictive sense. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art and are intended to fall within the scope of the appended claims.

[0116] SEQ ID NO:1 below is a cDNA copy of the mRNA sequence (GenBank: BC126199.1) containing the protein coding sequence of PKP2 isoform 2a.

[0117] SEQ ID NO:2 below is the codon-optimized DNA sequence (5' to 3') encoding PKP2 isoform 2a.

[0118] SEQ ID NO:3 below is the amino acid sequence of PKP2 isoform 2a. MAAPGAPAEYGYIRTVLGQQILGQLDSSSLALPSEAKLKLAGSSGRGGQTVKSLRIQEQVQQTLARKGRSSVGNGNLHRTSSVPEYVYNLHLVENDFVGGRSPV PKTYDMLKAGTTATYEGRWGRGTAQYSSQKSVEERSLRHPLRRLEISPDSSPERAHYTHSDYQYSQRSQAGHTLHHQESRRAALLVPPRYARSEIVGVSRAGTTS RQRHFDTYHRQYQHGSVSDTVFDSIPANPALLTYPRPGTSRSMGNLLEKENYLTAGLTVGQVRPLVPLQPVTQNRASRSSWHQSSFHSTRTLREAGPSVAVDSSGRRAHLTVGQAAAGGSGNLLTERSTFTDSQLGNADMEMTLERAVSMLEADHMLPSRISAAATFIQHECFQKSEARKRVNQLRGILKLLQLLKVQNEDVQRAVCGA LRNLVFEDNDNKLEVAELNGVPRLLQVLKQTRDLETKKQITGLLWNLSSNDKLKNLMITEALLTLTENIIIPFSGWPEGDYPKANGLLDFDIFYNVTGCLRNMS SAGADGRKAMRRCDGLIDSLVHYVRGTIADYQPDDKATENCVCILHNLSYQLEAELPEKYSQNIYIQNRNIQTDNNKSIGCFGSRSRKVKEQYQDVPMPEEKSNP KGVEWLWHSIVIRMYLSLIAKSVRNYTQEASLGALQNLTAGSGPMPTSVAQTVVQKESGLQHTRKMLHVGDPSVKKTAISLLRNLSRNLSLQNEIAKETLPDLVS IIPDTVPSTDLLIETTASACYTLNNIIQNSYQNARDLLNTGGIQKIMAISAGDAYASNKASKAASVLLYSLWAHTELHHAYKKAQFKKTDFVNSRTAKAYHSLKD

[0119] SEQ ID NO:4 below is the codon-optimized DNA sequence (5' to 3') encoding PKP2 isoform 2b.

[0120] SEQ ID NO:5 below is the amino acid sequence of PKP2 isoform 2b. MAAPGAPAEYGYIRTVLGQQILGQLDSSSLALPSEAKLKLAGSSGRGGQTVKSLRIQEQVQQTLARKGRSSVGNGNLHRTSSVPEYVYNLHLVENDFVGGRSPVPKTYDM LKAGTTATYEGRWGRGTAQYSSQKSVEERSLRHPLRRLEISPDSSPERAHYTHSDYQYSQRSQAGHTLHHQESRRAALLVPPRYARSEIVGVSRAGTTSRQRHFDTYHRQ YQHGSVSDTVFDSIPANPALLTYPRPGTSRSMGNLLEKENYLTAGLTVGQVRPLVPLQPVTQNRASRSSWHQSSFHSTRTLREAGPSVAVDSSGRRAHLTVGQAAAGGSGNLLTERSTFTDSQLGNADMEMTLERAVSMLEADHMLPSRISAAATFIQHECFQKSEARKRVNQLRGILKLLQLLKVQNEDVQRAVCGALRNLVFEDNDNKLEVAELNGVP RLLQVLKQTRDLETKKQITDHTVNLRSRNGWPGAVAHACNPSTLGGQGGRITRSGVRDQPDQHGLLWNLSSNDKLKNLMITEALLTLTENIIIPFSGWPEGDYPKANGLL DFDIFYNVTGCLRNMSSAGADGRKAMRRCDGLIDSLVHYVRGTIADYQPDDKATENCVCILHNLSYQLEAELPEKYSQNIYIQNRNIQTDNNKSIGCFGSRSRKVKEQYQ DVPMPEEKSNPKGVEWLWHSIVIRMYLSLIAKSVRNYTQEASLGALQNLTAGSGPMPTSVAQTVVQKESGLQHTRKMLHVGDPSVKKTAISLLRNLSRNLSLQNEIAKET LPDLVSIIPDTVPSTDLLIETTASACYTLNNIIQNSYQNARDLLNTGGIQKIMAISAGDAYASNKASKAASVLLYSLWAHTELHHAYKKAQFKKTDFVNSRTAKAYHSLKD

[0121] SEQ ID NO:6 below is the nucleic acid sequence (5' to 3') encoding the TNNT2 promoter. GTCATGGAGAAGACCCACCTTGCAGATGTCCTCACTGGGGCTGGCAGAGCCGGCAACCTGCCTAAGGCTGCTCAGTCCATTAGGAGCCAGTAGCCTGGAAGATGTCTTTACCCCCAGCATCAGTTCAAGTGGAGCAGCACATAACTCTTG CCCTCTGCCTTCCAAGATTCTGGTGCTGAGACTTATGGAGTGTCTTGGAGGTTGCCTTCTGCCCCCAACCCTGCTCCCAGCTGGCCTCCCAGGCCTGGGTTGCTGGCCTCTGCTTTATCAGGATTCTCAAGAGGGACAGCTGGTTTAT GTTGCATGACTGTTCCCTGCATATCTGCTCTGGTTTTAAATAGCTTATCTGAGCAGCTGGAGGACCACATGGGCTTATATGGCGTGGGGTACATGTTCCTGTAGCCTTGTCCCTGGCACCTGCCAAAATAGCAGCCAACACCCCCCACCC CCACCGCCATCCCCCTGCCCCACCCGTCCCCTGTCGCACATTCCTCCCTCCGCAGGGCTGGCTCACCAGGCCCAGCCCACATGCCTGCTTAAAGCCCTCTCCATCCTCTGCCTCACCCAGTCCCCGCTGAGACTGAGCAGACGCCTCCA

[0122] SEQ ID NO:7 below is an exemplary vector construct for expressing PKP2 isoform 2b in cardiomyocytes.

Claims

1. 1. A gene therapy vector adapted for expressing a nucleic acid sequence in a cardiac myocyte of a subject, the nucleic acid sequence comprising: A first sequence encoding PKP2 or a functional variant thereof; a second sequence comprising a cardiac specific promoter; Including, The gene therapy vector, wherein delivery of the gene therapy vector to a cardiomyocyte haploinsufficient for PKP2 results in at least a 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, or 5-fold increase in total desmosomal expression of PKP2 by the cardiomyocyte.

2. 1. A gene therapy vector adapted for expressing a nucleic acid sequence in a cardiac myocyte of a subject, the nucleic acid sequence comprising: A first sequence encoding PKP2 or a functional variant thereof; a second sequence comprising a cardiac specific promoter; Including, The gene therapy vector, wherein delivery of said gene therapy vector to haploinsufficient cardiomyocytes results in total desmosomal expression of said PKP2 that is at least 50% of the total desmosomal expression by non-haploinsufficient cardiomyocytes.

3. The gene therapy vector of claim 1 or 2, wherein the gene therapy vector comprises a viral vector.

4. 4. The gene therapy vector of claim 3, wherein the viral vector comprises one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, mutants thereof, and combinations thereof.

5. The gene therapy vector of claim 3 , wherein the viral vector comprises AAV6 or AAV9.

6. The gene therapy vector of any one of claims 1 to 5, wherein the cardiac-specific promoter comprises TNNT2 or a functional sequence having at least 99%, 95%, 90%, 85%, 80%, 75%, or 70% similarity.

7. The gene therapy vector of any one of claims 1 to 5, wherein the subject is a human subject.

8. A therapeutic formulation for treating or preventing cardiomyopathy in a subject, the therapeutic formulation comprising: A pharma- ceutically acceptable excipient or carrier; A viral vector comprising a nucleic acid sequence encoding PKP2 or a functional variant thereof. Including, The therapeutic preparation, wherein delivery of the therapeutic preparation to cardiomyocytes haploinsufficient for PKP2 results in at least a 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, or 5-fold increase in total desmosomal expression of PKP2 by the cardiomyocytes.

9. A therapeutic formulation for treating or preventing cardiomyopathy in a subject, the therapeutic formulation comprising: A pharma- ceutically acceptable excipient or carrier; A viral vector comprising a nucleic acid sequence encoding PKP2 or a functional variant thereof. Including, The therapeutic formulation, wherein delivery of said therapeutic formulation vector to haploinsufficient cardiomyocytes results in total desmosomal expression of said PKP2 that is at least 50% of the total desmosomal expression by non-haploinsufficient cardiomyocytes.

10. 10. The therapeutic formulation of claim 8 or 9, further comprising one or more additional viral vectors, each comprising a nucleic acid sequence encoding one or more non-PKP2 sarcomeric proteins or functional variants thereof.

11. The therapeutic formulation according to any one of claims 8 to 10, wherein the subject is a human subject.

12. The gene therapy vector or therapeutic preparation according to any one of claims 1 to 11, wherein the PKP2 is PKP2 isoform 2a.

13. The gene therapy vector or therapeutic preparation according to any one of claims 1 to 11, wherein the PKP2 is PKP2 isoform 2b.

14. A therapeutic formulation for treating or preventing cardiomyopathy in a subject, the therapeutic formulation comprising: A pharma- ceutically acceptable excipient or carrier; A first viral vector comprising a nucleic acid sequence encoding PKP2 isoform 2a or a functional variant thereof; a second viral vector comprising a nucleic acid sequence encoding PKP2 isoform 2b or a functional variant thereof; Including, The therapeutic preparation, wherein delivery of the therapeutic preparation to cardiomyocytes that are haploinsufficient for PKP2 isoform 2a or isoform 2b results in at least a 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, or 5-fold increase in total desmosomal expression of PKP2 isoform 2a or isoform 2b by the cardiomyocytes.

15. A therapeutic formulation for treating or preventing cardiomyopathy in a subject, the therapeutic formulation comprising: A pharma- ceutically acceptable excipient or carrier; A first viral vector comprising a nucleic acid sequence encoding PKP2 isoform 2a or a functional variant thereof; a second viral vector comprising a nucleic acid sequence encoding PKP2 isoform 2b or a functional variant thereof; Including, The therapeutic formulation, wherein delivery of said therapeutic formulation vector to haploinsufficient cardiomyocytes results in total desmosomal expression of PKP2 isoform 2a or isoform 2b that is at least 50% of the total desmosomal expression by non-haploinsufficient cardiomyocytes.

16. An isolated cell transduced with a gene therapy vector according to any one of claims 1 to 7.

17. The isolated cell of claim 16 , wherein the cell is a human cell.

18. 17. The isolated cell of claim 16, wherein the cell is a cardiac cell.

19. 17. The isolated cell of claim 16, wherein the cell is a cardiomyocyte derived from a human induced pluripotent stem cell.

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