GENE-THERAPY VECTORS FOR TREATING CARDIOMYOPATHY

[0001] The present invention relates to a gene therapy vector which is useful in the treatment or prevention of hypertrophic cardiomyopathy in a subject in need thereof. The gene therapy vector of the invention comprises a nucleic acid sequence encoding a cardiac sarcomeric protein and a cardiomyocyte-specific promoter which is operably linked to said nucleic acid sequence. The invention furthermore relates to a cell which comprises the gene therapy vector. Pharmaceutical compositions which comprise the gene therapy vector and/or a cell comprising said vector are also provided. In another aspect, the invention relates to a method for treating or preventing hypertrophic cardiomyopathy in a subject by introducing the gene therapy vector of the invention into a subject in need of treatment.

[0002] While considerable progress has been made in the prevention of heart diseases that are caused by environmental factors, such as nicotine, hypercholesterolemia or diabetes, and in the symptomatic treatment of heart conditions, there is still a need for methods that improve the treatment of inherited cardiomyopathies. Among the cardiomyopathies that are caused by genetic factors are hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and arrhythmogenic right ventricular cardiomyopathy (ARVC).

[0003] HCM is the most prevalent myocardial disease characterized by unexplained left ventricular hypertrophy in the absence of another cardiac or systemic disease that itself would be capable of producing the magnitude of hypertrophy evident in a given patient. HCM is associated with initially normal systolic, but impaired diastolic function (Elliott et al., 2008, Eur Heart J 29:270-276; Gersch et al., 2011, J Thorac Cardiovasc Surg 142:e153-203). HCM has a particularly high prevalence of about 1:500 in the general population (Maron et al., 1995, Circulation 92:785-789), and it is the leading cause of sudden cardiac death in younger people, particularly in athletes. Although HCM is a life-threatening disease, no curative treatment exists to date (Carrier et al., 2010, Cardiovasc Res 85:330-338; Schlossarek et al., 2011, J Mol Cell Cardiol 50:613-20).

[0004] HCM is an autosomal-dominant disease which is known to be caused by more than 1000 different mutations in at least 10 genes that encode components of the cardiac sarcomere, such as cardiac myosin binding protein C (MYBPC3), β-myosin heavy chain (MYH7), cardiac troponin T (TNNT2), cardiac troponin I (TNNI3), myosin ventricular essential light chain 1 (MYL3), myosin ventricular regulatory light chain 2 (MYL2), cardiac α actin (ACTC), α-tropomyosin (TPM1), titin (TTN), four-and-a-half LIM protein 1 (FHL1) (Richard et al., 2003, Circulation 107:2227-2232; Schlossarek et al., 2011, J Mol Cell Cardiol 50:613-20; Friedrich et al., 2012, Hum Mol Genet 21:3237-54). Most mutations are missense mutations which encode full-length mutant polypeptides. The most known exceptions are MYBPC3 and FHL1, which exhibit mainly frameshift mutations leading to C-terminal truncated proteins.

[0005] The most frequently mutated gene in HCM is MYBPC3 which encodes cardiac myosin binding protein C (cMyBP-C) (Bonne et al., 1995, Nature Genet 11:438-440; Watkins et al., N Engl J Med., 2011, 364:1643-56). cMyBP-C is a major component of the A-band of the sarcomere, where it interacts with myosin, actin and titin (Schlossarek et al., 2011, J Mol Cell Cardiol 50:613-20). In humans and mice cMyBP-C is exclusively detected in the heart (Fougerousse et al, 1998, Circ Res 82:130-133) and is involved in the regulation of cardiac contraction and relaxation (Pohlmann et al., 2007, Circ Res Circ Res 101, 928-38; Schlossarek et al., 2011, J Mol Cell Cardiol 50:613-20). About 70% of the mutations in the MYBPC3 gene result in a frameshift and produce C-terminal truncated proteins (Carrier et al., 1997, Circ Res 80:427-434). Truncated proteins are unstable and have never been detected in myocardial tissue of patients (Marston et al., 2009, Circ Res 105:219-222; van Dijk et al., 2009, Circulation 119:1473-1483; van Dijk et al., 2012, Circ Heart Fail 5:36-46).

[0006] Therefore, a reduced level of cMyBP-C protein is one argument that haploinsufficiency is a likely disease mechanism of HCM. An insufficient amount of full-length cMyBP-C could produce an imbalance in the stoichiometry of the thick filament components and alter sarcomeric structure and function. Haploinsufficiency is also involved in mouse and cat models of HCM that carry either missense or frameshift mutations (Meurs et al., 2005, Hum Mol Genet 14:3587-3593; Vignier et al., 2009, Circ Res 105:239-248). In addition, in both cats and mice, there is evidence for the presence of mutant cMyBP-C (full-length or truncated), even at low level. Therefore, a second likely disease mechanism is the generation of toxic polypeptide inducing a dominant-negative effect, most probably by competing with the wild-type (WT) gene product.

[0007] Current drug-based treatments of HCM are merely empiric, can alleviate the symptoms but do not treat the genetic cause underlying the disease. Clearly, a gene-based or RNA-based therapy would be the only curative treatment for HCM. Gene therapeutic approaches have successfully been tested in connection with non-genetic cardiac diseases (Jessup et al., 2011, Circulation 124:304-313).

[0008] US applications 2005/0276804 and 2007/0292438 disclose that cMyBP-C is associated with genetic cardiac disorders. However, U S 2005/0276804 suggests a reduction of retinol binding protein or retinoid to treat these disorders. US 2007/0292438 is limited to the disclosure of different mouse models having disruptions in various genes.

[0009] US applications 2004/0086876 and US 2002/0127548 disclose the diagnosis of mutations in the human MYBPC3 gene which are associated with HCM. Further, these applications suggest treating HCM by administration of a nucleic acid which encodes a non-mutated cMyBP-C to the patient.

[0010] Merkulov et al., 2012, Circ Heart Fail, 5:635-644 disclose the transfer of the murine Mybpc3 gene into the myocardium of cMyBP-C-deficient (cMyBP-C^−/−) mice. The authors assume that the absence of cMyBP-C results in dysfunction and hypertrophy. The gene transfer improved systolic and diastolic contractile function and led to reductions in left ventricular wall thickness in the cMyBP-C-deficient (cMyBP-C^−/−) mice.

[0011] Vignier et al., 2009, Circ Res 105:239-248 developed a Mybpc3-targeted knock-in (KI) mouse model carrying a G>A point mutation that results in different mutant mRNAs and proteins originating from abnormal gene transcription and splicing. It was shown that exogenous stress, such as adrenergic stress or aging, leads to a saturation and finally to an impairment of the ubiquitin-proteasome system (UPS) in the KI mice and potentially to a subsequent accumulation of the mutant cMyBP-C polypeptides.

[0012] The present inventors found that in subjects suffering from HCM due to a heteroallelic mutation acting in a dominant-negative fashion in a gene encoding a cardiac sarcomeric protein, the introduction of a gene transfer vector which provides the corresponding non-mutated gene not only restores normal levels of the sarcomeric protein, but also minimizes the deleterious effects of toxic mutant polypeptides that are otherwise generated through transcription of mutant allele(s).

[0013] A vector-induced expression of an exogenous wild-type (WT) gene under the control of a cardiomyocyte-specific promoter thus overcomes the dominant-negative effect of the mutant protein in a subject which carries a mutated MYBPC3 allele and is not toxic, because, surprisingly, expression of the normal allele via the gene therapy vector effectively reduces the expression of the endogenous mutant allele. This effect is considered to occur as a cardiac cell-autonomous phenomenon due to tight intracellular control of the homeostasis and turnover of sarcomeric proteins.

[0014] The invention relates to novel therapeutic approaches for treating or preventing HCM. It is known that mutations in a number of genes which encode cardiac sarcomeric proteins lead to a reduced level of functional full-length sarcomeric protein. This is due to frameshift mutations which produce truncated mutant polypeptides, which are normally degraded by the ubiquitin-proteasome system (UPS). However, under conditions of exogenous stress, the function of the UPS may be disturbed which results in the accumulation of the mutant polypeptides, which can thus be incorporated into the sarcomere and act as a poison peptide in a dominant-negative fashion on the wild-type cMyBP-C, contributing to the pathogenesis of HCM (Vignier et al., 2009, see above). Similar observations have been made for the four-and-a-half LIM protein 1 (Friedrich et al., 2012, Hum Mol Genet 21:3237-3254). A Mybpc3-targeted knock-out, which does not produce any mutant cMyBP-C polypeptides, did not show any impairment of the UPS under the same conditions (Schlossarek et al., 2012, Basic Res Cardiol 107:1-13; Schlossarek et al., 2012, J Muscle Res Cell Motil 33: 5-15.

[0015] It is shown herein that gene transfer of wild-type cDNA, which encodes a functional version of a cardiac sarcomeric protein (such as cMyBP-C), via a gene therapy vector into a subject which carries the mutation in the gene of said cardiac sarcomeric protein, not only restores the normal level of the protein in the myocardium (i.e. the muscle tissue of the heart which is constituted by cardiomyocytes), but also prevents the production of toxic mRNAs and/or toxic polypeptides that would otherwise result from expression of the mutated allele encoding the cardiac sarcomeric protein in the genome of said subject. It was also observed that the introduction of high amounts of the gene therapy vector is not associated with a high risk for the patient to be treated, since it was not found to result in excessive amounts of the exogenous wild-type (WT) protein within the cells. Unexpectedly, the expression of cardiac sarcomeric proteins appears to be stochiometrically tightly regulated in the cell which means that it is not possible to provide the exogenous protein in amounts that could be harmful to the patient. Accordingly, the invention provides a simple and safe method for the treatment of HCM in a subject.

[0016] The combined effects of providing sufficient levels of normal cDNA resulting in adequate production of the cardiac sarcomeric protein and suppression of the toxic mRNAs/polypeptides result in an effective treatment of HCM. Without wishing to be bound by theory, it is assumed that the exogenous gene expression through the gene therapy vector reduces endogenous expression from the mutated allele by competing for sarcomeric-specific transcription factors.

[0017] In a first aspect, the invention therefore provides a gene therapy vector for expressing an exogenous nucleic acid sequence comprising:

(a) a nucleic acid sequence encoding a functional cardiac sarcomeric protein and,
(b) a cardiomyocyte-specific promoter which is operably linked to said nucleic acid sequence.

[0018] The gene therapy vector is suitable for use in treating or preventing HCM in a mammalian subject in need of treatment, preferably a human subject. The subject in need of treatment is one that carries a mutation in the corresponding gene encoding said cardiac sarcomeric protein which contributes to HCM. After administration into the subject to be treated, the vector provides for the expression of the encoded cardiac sarcomeric protein in the subject, preferably in the myocardium of said subject.

[0019] The cardiac sarcomeric protein to be expressed in the subject is known to be associated with HCM, i.e. mutations in the gene encoding the cardiac sarcomeric protein which lead to expression of a non-functional protein variant, e.g. a full-length or truncated variant or mutant, eventually cause HCM. To date mutations in at least ten different genes encoding cardiac sarcomeric proteins are known to cause HCM. The sarcomere is the basic unit of a muscle and is defined as the segment between two adjacent Z-discs. According to the invention, the sarcomeric protein to be expressed by the gene therapy vector is a cardiac sarcomeric protein which means that the protein naturally occurs in the sarcomere of the cardiac muscle.

[0020] The cardiac sarcomeric protein to be expressed in the subject may be a structural or regulatory protein which is present in the cardiac sarcomere. The protein is preferably selected from the group consisting of β-myosin heavy chain (encoded by the gene MYH7, RefSeqGene NG_007884.1), myosin ventricular essential light chain 1 (encoded by the gene MYL3, RefSeqGene NG_007555.2), myosin ventricular regulatory light chain 2 (encoded by the gene MYL2, RefSeqGene NG_007554.1), cardiac α actin (encoded by the gene ACTC1, RefSeqGene NG_007553.1), α-tropomyosin (encoded by the gene TPM1, RefSeqGene NG_007557.1), cardiac troponin T (encoded by the gene TNNT2, RefSeqGene NG_007556.1), cardiac troponin I (encoded by the gene TNNI3, RefSeqGene NG_007866.2), cardiac myosin binding protein C (encoded by the gene MYBPC3, RefSeqGene NG_007667.1), titin (encoded by the gene TTN, RefSeqGene NG_011618.1), and four-and-a-half LIM protein 1 (encoded by the gene FHL1, RefSeqGene NG_015895.1) (Richard et al., 2003, Circulation 107:2227-2232; Friedrich et al., 2012, Hum Mol Genet, 21:3237-54).

[0021] It is preferred that the cardiac sarcomeric protein to be expressed by the gene therapy vector of the invention is cardiac myosin-binding protein C (cMyBP-C). The protein can be derived from different species, such as mouse, cat, pig or monkey. In one preferred embodiment of the invention, the cMyBP-C protein to be expressed is a murine cMyBP-C, preferably a murine cMyBP-C having the amino acid sequence depicted in SEQ ID NO:4 (NCBI accession number: NP_032679.2) of the enclosed sequence listing or a sequence having at least 80% sequence identity thereto. The nucleotide sequence encoding the murine cMyBP-C of SEQ ID NO:4 is depicted in SEQ ID NO:3 (NCBI accession number: NM_008653.2).

[0022] The present invention particularly envisages the treatment of human patients suffering from HCM. Thus, in another preferred embodiment of the invention, the nucleic acid inserted in the vector which encodes a human cMyBP-C protein is of human origin. Preferably, the human cMyBP-C protein has the amino acid sequence depicted in SEQ ID NO:2 (NCBI accession number: NP_000247.2) or a sequence having at least 80% sequence identity thereto. The nucleotide sequence encoding the human cMyBP-C protein is shown in SEQ ID NO:1 (NM_000256.2).

[0023] The protein to be expressed may also be a functional variant of one of the above-mentioned proteins which exhibits a significant amino acid sequence identity compared to the original protein. Preferably, the amino acid identity amounts to at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%. Preferably, the amino acid identity of the variant is at least 70%, 80% or 90%. In this context, the term “functional variant” means that the variant of the sarcomeric protein is capable of fulfilling the function of the naturally occurring cardiac sarcomeric protein, e.g. providing structural/functional support.

[0024] Functional variants of a cardiac sarcomeric protein may include, for example, proteins which differ from their naturally occurring counterparts by one or more amino acid substitutions, deletions or additions. For example, a variant protein of the human cMyBP-C protein depicted in SEQ ID NO:2 may have an amino acid sequence with 2, 3, 4, 5, 6, or up to 10, 20, 30 or more positions which have been substituted by another amino acid relative to SEQ ID NO:2. For example, the functional variant may e.g. be selected from the group consisting of the naturally occurring Mybpc3 splice variant lacking exons 5 and 6, termed variant 4 (as shown in SEQ ID NO:28).

[0025] The amino acid substitutions can be conservative or non-conservative. It is preferred that the substitutions are conservative substitutions, i.e. a substitution of an amino acid residue by an amino acid of similar polarity, which 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 can be substituted with another hydrophobic residue, or a polar residue can be substituted with another polar residue having the same charge. Functionally homologous amino acids which may be used for a conservative substitution comprise, for example, non-polar amino acids such as glycine, valine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, and tryptophan. Examples of uncharged polar amino acids comprise serine, threonine, glutamine, asparagine, tyrosine and cysteine. Examples of charged polar (basic) amino acids comprise histidine, arginine and lysine. Examples of charged polar (acidic) amino acids comprise aspartic acid and glutamic acid.

[0026] Also considered as variants are proteins which 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 sarcomeric protein (i.e. as an insertion), or they may be added to one or both termini of the protein. Basically, insertions can take place at any position if the addition of amino acids does not impair the capability of the polypeptide to fulfill the function of the naturally occurring cardiac sarcomeric protein and/or rescue the haploinsufficiency in the treated subject. Moreover, variants of sarcomeric proteins also comprise proteins in which, compared to the original polypeptide, one or more amino acids are lacking. Such deletions may affect any amino acid position provided that it does not impair the ability to fulfill the normal function of the cardiac sarcomeric protein and/or rescue the haploinsufficiency.

[0027] Finally, variants of the cardiac sarcomeric proteins also refer to proteins which differ from the naturally occurring protein by structural modifications, such as modified amino acids. According to the invention, modified amino acids are amino acids which have been modified either by natural processes, such as processing or post-translational modifications, or by chemical modification processes known in the art. Typical amino acid modifications comprise phosphorylation, glycosylation, acetylation, 0-Linked N-acetylglucosamination, glutathionylation, acylation, branching, ADP ribosylation, crosslinking, disulfide bridge formation, formylation, hydroxylation, carboxylation, methylation, demethylation, amidation, cyclization and/or covalent or non-covalent bonding to phosphotidylinositol, flavine derivatives, lipoteichonic acids, fatty acids or lipids. Such modifications have been extensively described in the literature, e.g., in Proteins: Structure and Molecular Properties, T. Creighton, 2^nd edition, W. H. Freeman and Company, New York (1993). In a preferred embodiment of the invention, the nucleic acid sequence encodes a constitutively phosphorylated isoform of human cMyBP-C. It has been shown that these isoforms are particularly cardioprotective (Sadayappan et al. (2005), Circ Res 97:1156-1163; Sadayappan et al., 2006; Proc Natl Acad Sci USA 103:16918-16923).

[0028] The gene therapy vector is preferably for treating or preventing HCM in a subject in need thereof. The subject to be treated with the vectors can be a subject that has been diagnosed with HCM, a subject with an increased risk for developing HCM, or a subject predisposed to develop HCM. In a preferred aspect of the invention, the subject is a human subject diagnosed with HCM as a result of a mutation in at least one of the alleles of a gene encoding a cardiac sarcomeric protein which is known to be associated with HCM.

[0029] In another preferred aspect of the invention, the subject which is treated with the vector of the invention carries a gene mutation that impairs the function of said cardiac sarcomeric protein and, as a result of the mutation, produces one or more dysfunctional protein species which originate from the cardiac sarcomeric protein.

[0030] In another preferred aspect of the invention, the mutation in the cardiac sarcomeric protein causes haploinsufficiency in said subject. Haploinsufficiency designates a state of a diploid organism, which is characterized by one dysfunctional allele, wherein the remaining functional allele does not produce a sufficient level of the gene product to generate the wild-type phenotype.

[0031] The present invention is based on the surprising insight that the phenotype of HCM can be effectively ameliorated or eradicated by administration of a gene therapy vector which provides an intact, exogenous version of the wild-type gene which compensates for the mutated allele in the genome of the subject to be treated. It was unexpectedly found that an accumulation of toxic mRNA and/or polypeptides (both of which derive from the mutated allele) can be effectively prevented. Thus, in one embodiment, the described gene therapy vector is for use in a method of treating or preventing hypertrophic cardiomyopathy in a mammalian subject, wherein said subject produces one or more dysfunctional protein species which originate from the cardiac sarcomeric protein and, optionally, wherein administration of said therapy results in the reduction of one or more of the dysfunctional protein species. “Reduction” refers to a level of one or more of the dysfunctional protein species which is reduced by more than 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, compared to the level before administration. In a preferred embodiment, the level of one or more of the dysfunctional protein species is reduced by more than 20% compared to the level before administration.

[0032] Preferably, the “level of one or more of the dysfunctional protein species” is the level of all dysfunctional species of the sarcomeric protein combined, i.e. the overall level of dysfunctional species of the sarcomeric protein.

[0033] HCM describes a deterioration of the heart muscle which results in a decreased integrity. This may lead to heart rhythm disorder and ultimately to heart failure. HCM is a genetically and clinically heterogeneous disease of the sarcomere characterized inter alia by a thickening of the muscular walls in the ventricular septum and left ventricle in the absence of another cardiac or systemic disease that itself would be capable of producing the magnitude of hypertrophy evident in a given patient. As a consequence of the wall thickening, the left ventricle outflow tract may be narrowed. Characteristics of HCM are myocyte hypertrophy, myocellular disarray, interstitial fibrosis, small vessel coronary disease, and/or left ventricular outflow obstruction. HCM is associated with initially normal systolic, but impaired diastolic function in majority of cases. Thus, in a preferred embodiment of the invention, the gene therapy vector is for treating or preventing HCM in a subject in need thereof, wherein HCM is characterized by a thickening of the muscular walls in the ventricular septum and/or left ventricle and diastolic dysfunction.

[0034] When expressing a cardiac sarcomeric protein, e.g. one of the proteins mentioned above, exogenously in the subject to which the therapeutic vector has been administered, it may turn out that it is not necessary to express the full-length protein to compensate for the dysfunctional mutant protein expressed from the mutated allele. Instead, it may be sufficient to express only a functional fragment of the full-length sarcomeric protein or its variants as defined above. Thus, the present invention also comprises the use of functional fragments of the cardiac sarcomeric protein or their variants for treating or preventing HCM in a subject in need thereof. As used herein, fragments of cardiac sarcomeric proteins of the invention are proteins which differ from the naturally occurring protein by the lack of one or several amino acids at the N-terminus and/or the C-terminus, wherein at least part of the ability to fulfill the normal function of the naturally occurring cardiac sarcomeric protein is retained.

[0035] The nucleic acid sequence encoding the cardiac sarcomeric protein is administered to the subject to be treated in the form of a gene therapy vector, i.e. a nucleic acid construct which comprises the coding sequence, including the translation and termination codons, next to other sequences required for providing expression of the exogenous nucleic acid such as promoters, kozak sequences, polyA signals and the like. Gene therapy vectors for expressing an exogenous nucleic acid sequence in a subject are well known in the art.

[0036] For example, the gene therapy vector may be part of a mammalian expression system. Useful mammalian expression systems and expression constructs have been described in the prior art. Also, several mammalian expression systems are distributed by different manufacturers and can be employed in the present invention, such as plasmid- or viral vector based systems, e.g. LENTI-Smart™ (InvivoGen), GenScript™ Expression vectors, pAdVAntage™ (Promega), ViraPower™ Lentiviral, Adenoviral Expression Systems (Invitrogen) and adeno-associated viral expression systems (Cell Biolabs).

[0037] The gene therapy vector of the invention can be, for example, a viral or non-viral expression vector which is suitable for introducing the exogenous nucleic acid into a cell for subsequent expression of the protein encoded by said nucleic acid. The vector should be specifically adapted to provide expression of the encoded sarcomeric protein in a cardiomyocyte. In a preferred embodiment the vector provides specific expression of the encoded sarcomeric protein in cardiomyocytes. The expression is “specific” when the expression is at least 2-fold higher than in other non-cardiac cell type or cardiac cell which is not a cardiomyocte.

[0038] The expression vector can be an episomal vector, i.e. one that is capable of self-replicating autonomously within the host cell, or an integrating vector, i.e. one which stably incorporates into the genome of the cell. The expression in the host cell can be constitutive or regulated (e.g. inducible). Preferably, the functional exogenous cardiac sarcomeric protein is located intracellularly, preferably in the sarcomere of the host cell.

[0039] A gene therapy vector of the invention will normally comprise a promoter which is functionally linked to the nucleic acid encoding the sarcomeric protein. The promoter sequence must be compact and ensure a strong expression. Preferably, the promoter provides for an expression of the sarcomeric protein in the myocardium of the patient that has been treated with the gene therapy vector. More preferably, the promoter provides for a specific expression of the sarcomeric protein in the myocardium of the patient. The expression is “specific” when the expression is at least 2-fold higher than in cells do not belong to the myocardium. It is further preferred that substantially no sarcomeric protein is expressed in cells that do not belong to the myocardium. “Substantially no” in this context means that less than 10%, less than 5%, less than 1% of the sarcomeric protein that is expressed from the vector is expressed in cells that do not belong to the myocardium.

[0040] Suitable promoters include, for example, the muscle creatine kinase (MCK), the cytomegalovirus enhancer+myosin light chain 2 promoter (CMV-MLC2, or CMV-MLC1.5, CMV-MLC260), the phosphoglycerate kinase (PGK), and the cardiac troponin T promoter (TNNT2), and any other sarcomere-specific promoters. Preferably, these promoters are derived from human genes.

[0041] In a particularly preferred embodiment, the gene therapy vector comprises a cardiac-specific promoter which is operably linked to the nucleic acid sequence encoding the cardiac sarcomeric protein. As used herein, a “cardiac-specific promoter” refers to a promoter whose activity in cardiac cells is at least 2-fold higher than in any other non-cardiac cell type. Preferably, a cardiac-specific promoter suitable for being used in the vector of the invention has an activity in cardiac cells which is 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 compared to its activity in a non-cardiac cell type.

[0042] In a further preferred embodiment, the gene therapy vector comprises a cardiomyocyte-specific promoter which is operably linked to the nucleic acid sequence encoding the cardiac sarcomeric protein. A “cardiomyocyte-specific promoter”, as used herein, specifies a promoter whose activity in cardiomyocytes is at least 2-fold higher than in any other non-cardiac cell type or cardiac cell which is not a cardiomyocte. Preferably, a cardiomyocte-specific promoter suitable for being used in the vector of the invention has an activity in cardiomyocytes which is 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 compared to its activity in a non-cardiac cell type or a cardiac cell type which is not a cardiomyocte.

[0043] Preferably, the cardiac-specific or cardiomyocyte-specific promoter is a human promoter. As can be seen from the enclosed examples, one promoter that has been proven useful for the vectors of the invention is a cardiac troponin T promoter (TNNT2), such as the human TNNT2 promoter set forth in SEQ ID NO:5. Accordingly, the cardiomyocyte-specific promoter of the invention preferably comprises the sequence of SEQ ID NO:5 or a functional equivalent sequence having at least 80%, more preferably 90%, 95%, 96%, 97%, 98%, 99%, sequence identity thereto. In a preferred embodiment, the gene therapy vector comprises a TNNT2 promoter operably linked to the nucleic acid sequence encoding the cardiac sarcomeric protein. In a further preferred embodiment, the gene therapy vector comprises the human TNNT2 promoter of SEQ ID NO:5 operably linked to the nucleic acid sequence encoding the cardiac sarcomeric protein. Other cardiac-specific promoters include the alpha myosin heavy chain promoter, the myosin light chain 2v promoter, the alpha myosin heavy chain promoter, the alpha-cardiac actin promoter, the alpha-tropomyosin promoter, the cardiac troponin C promoter, the cardiac troponin I promoter, the cardiac myosin-binding protein C promoter, and the sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) promoter (e.g. isoform 2 of this promoter (SERCA2)).

[0044] Cardiac muscle tissue is striated muscle tissue that has repeating sarcomeres. Thus, in a further embodiment the gene therapy vector comprises a striated muscle promoter, such as the desmin promoter.

[0045] The cardiac-specific or cardiomyocyte-specific promoter is operably linked to the nucleic acid sequence encoding the cardiac sarcomeric protein which means that the promoter is combined with the coding nucleic acid so as to enable the expression of said coding nucleic acid under the control of the promoter in cardiac myocytes cells when integrated into the genome of the cell or present as an extragenomic nucleic acid construct in the cell.

[0046] As an optional component, the gene therapy vector can include an enhancer element for increasing the expression level of the sarcomeric protein. Examples include the SV40 early gene enhancer and the enhancer of the long terminal repeat (LTR) of Rous Sarcoma Virus (Gorman et al. (1982) Proc. Natl. Acad. Sci. 79:6777). The vector also optionally comprises transcription termination sequences and polyadenylation sequences for improved expression of the human and/or non-human antigen(s). Suitable transcription terminator and polyadenylation signals can, for example, be derived from SV40 (Sambrook et al (1989), Molecular Cloning: A Laboratory Manual). Preferably, a SV40 polyadenylation signal comprising or consisting of the sequence of SEQ ID NO:6 is used in the vector of the invention. Any other element which is known in the art to support efficiency or specificity of expression may be added to the expression vector, such as the Woodchuck hepatitis post-transcriptional regulatory element (wPRE). To increase the cardiac specificity, other elements can be introduced to inactivate the expression of genes in other tissues, such as sequences encoding miRNAs such as miR122 (Geisler et al., 2011, Gene Therapy 18:199-209). To visualize the exogenous gene expression in the heart, other optional elements can be introduced such as tag sequences (myc, FLAG, HA, His, and the like), or fluorochromes such as GFP, YFP, RFP.

[0047] To further increase the gene expression level, a chimeric intron can be introduced into the gene therapy vector of the invention. A “chimeric intron” as used herein refers to an intron that comprises parts of at least two different introns which have been derived from two different genes. Particularly preferred chimeric introns for use in the gene therapy vector of the present invention comprise, e.g. intron sequences from the human beta globin gene and human immunoglobulin G (IgG). An exemplary intron is depicted in SEQ ID NO:7. Preferably, the chimeric intron is inserted immediately downstream from the promoter. It has e.g. been shown that insertion of the beta globin/Ig intron immediately downstream of the PGK promoter increases gene expression about 37-fold (Dominguez et al., 2011, Hum Mol Genet 20:681-693).

[0048] The gene therapy vector can be constructed and cloned by standard methods known in the art, such as recombinant DNA technology or chemical synthesis. Standard cloning methods are described e.g. in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Lab Press.

[0049] In a particularly preferred aspect, the gene therapy vector is a viral expression vector. Viral vectors for use in the present invention typically comprise a viral genome in which a portion of the native sequence has been deleted in order to introduce a heterogeneous polynucleotide without destroying the infectivity of the virus. Due to the specific interaction between virus components and host cell receptors, viral vectors are highly suitable for efficient transfer of genes into target cells. Suitable viral vectors for facilitating gene transfer into a mammalian cell are well known in the art and can be derived from different types of viruses, for example, from a retrovirus, adenovirus, adeno-associated virus (AAV), orthomyxovirus, paramyxovirus, papovavirus, picornavirus, lentivirus, herpes simplex virus, vaccinia virus, pox virus or alphavirus. For an overview of the different viral vector systems, see Nienhuis et al., Hematology, Vol. 16: Viruses and Bone Marrow, N. S. Young (ed.), 353-414 (1993).

[0050] For example, retroviral vectors may be used. Retroviral vectors normally function by transducing and integrating the selected polynucleotide into the genome of the target cell. The retroviral vectors 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 Leukosis Virus can be used. The skilled person will be able to combine portions derived from different retroviruses, such as LTRs, tRNA binding sites, and packaging signals to provide a recombinant retroviral vector. These retroviral vectors are then normally used for producing transduction competent retroviral vector particles. For this purpose, the vectors are introduced into suitable packaging cell lines, such as those described in U.S. Pat. No. 5,591,624. Retrovirus vectors can also be constructed for site-specific integration into the DNA of the host cell by incorporating a chimeric integrase enzyme into the retroviral particle. See, for example, WO 96/37626.

[0051] According to the invention, it is particularly preferred that the gene therapy vector is an adeno-associated viral (AVV) vector, such as an AAV vector selected from the group consisting of serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 or chimeric AAV derived thereof, which will be even better suitable for high efficiency transduction in the tissue of interest (Wu et al., 2006, Mol Therapy 14:316-27; Bowles et al., 2012, Mol Therapy 20:443-455). Upon transfection, AAV elicits only a minor immune reaction (if any) in the host. Moreover, in contrast to other vector systems AAV vectors are also able to efficiently pass from the blood into terminally differentiated cardiomyocytes. In this respect the AAV system is superior e.g. to the use of lentivirus. Therefore, AAV is highly suited for gene therapy approaches. For transduction in mice, AAV serotype 6 and AAV serotype 9 are particularly suitable. For gene transfer into a human, AAV serotypes 1, 6, 8 and 9 are preferred. Thus, in a preferred embodiment of the invention, the gene therapy vector is an AAV serotype 6 vector. In a further preferred embodiment, the gene therapy vector is an AAV serotype 8 vector. Finally, it is most preferred that the gene therapy vector is an AAV serotype 9 vector. The AAV serotype 9 vector is particularly well suited for the induction of expression in cells of the myocardium/cardiomyocytes.

[0052] It was assumed in the prior art that the capacity of AAV for packaging a therapeutic gene is limited to approximately 4.9 kbp, while longer sequences lead to truncation of AAV particles (Wu et al., 2010, Mol Ther 18:80-86). However, it is demonstrated herein that packaging of an oversized DNA sequence of 5.4 kbp (including two inverted terminal repeats (ITRs), the FLAG-tagged Mybpc3 cDNA under the control of the human TNNT2 promoter, a chimeric intron and the SV40 polyadenylation signal) does not affect the production of the AAV serotype 6 or 9. Titers of 1-7×10¹² vector genomes per mL were achieved and the vectors induced marked expression of the FLAG-Mybpc3 gene in isolated mouse cardiac myocytes and in the mouse heart in vivo. Thus, in a preferred embodiment, the gene therapy vector comprises a polynucleotide sequence having a size of at least 4.0 kbp, at least 4.5 kbp, at least 5 kbp, at least 5.1 kbp, at least 5.2 kbp, at least 5.3 kbp, at least 5.4 kbp, at least 5.5 kbp or at least 5.6 kbp. In one embodiment, the gene therapy vector comprises a polynucleotide sequence having a size of at least 4.5 kbp. It is particularly preferred that the gene therapy vector comprises a polynucleotide sequence having a size of at least 5 kbp. In a further embodiment the gene therapy vector comprises a polynucleotide sequence having a size of at least 5.3 kbp.

[0053] Moreover, the gene therapy vectors of the invention preferably combine the advantages of a highly efficient and pharmaceutically acceptable transfection vector, such as AAV, with a cardiomyocyte-/myocardium-specific expression of the encoded cardiac sarcomeric protein, through a cardiac-specific promoter. Therefore, it is preferred that the gene therapy vector is an AAV vector that comprises a cardiac-specific promoter which is operably linked to the nucleic acid sequence encoding the cardiac sarcomeric protein. In a first preferred embodiment, the AAV vector is an AAV serotype 6. In a second preferred embodiment, the AAV vector is an AAV serotype 8. In a third preferred embodiment, the AAV vector is an AAV serotype 9. It is particularly preferred that the cardiac-specific promoter in any of these embodiments is the human TNNT2 of SEQ ID NO:5 or a functional equivalent sequence having at least 80%, more preferably 90%, 95%, 96%, 97%, 98%, 99%, sequence identity thereto. For example, the gene therapy vector is an AAV 9 vector that comprises a cardiac-specific promoter which is operably linked to the nucleic acid sequence encoding the cardiac sarcomeric protein, wherein the cardiac-specific promoter in any of these embodiments is the human TNNT2 of SEQ ID NO:5 or a functional equivalent sequence having at least 80%, more preferably 90%, 95%, 96%, 97%, 98%, 99%, sequence identity thereto.

[0054] As outlined above, the cardiac sarcomeric protein that is encoded by the gene therapy vector is preferably cardiac myosin-binding protein C (cMyBP-C). As shown in the below examples, a gene therapy vector combining the advantages of an AAV vector, a cardiomyocyte-specific promoter and expression of cardiac myosin-binding protein C is highly efficient in the treatment of patients suffering from HCM.

[0055] Thus, in a preferred embodiment, the gene therapy vector is an AAV serotype 9 vector that comprises the human TNNT2 promoter of SEQ ID NO:5 or a functionally equivalent sequence having at least 80%, more preferably 90%, 95%, 96%, 97%, 98%, 99%, sequence identity thereto, wherein the promoter is operably linked to a nucleic acid sequence encoding a cMyBP-C, preferably a murine cMyBP-C having the amino acid sequence depicted in SEQ ID NO:4 or a sequence having at least 80% sequence identity thereto, or the human cMyBP-C protein having the amino acid sequence depicted in SEQ ID NO:2 or a sequence having at least 80% sequence identity thereto. In a more preferred embodiment, the gene therapy vector is an AAV serotype 9 vector that comprises the human TNNT2 promoter of SEQ ID NO:5 or a functionally equivalent sequence having at least 80%, more preferably 90%, 95%, 96%, 97%, 98%, 99%, sequence identity thereto, wherein the promoter is operably linked to a nucleic acid sequence encoding the human cMyBP-C protein having the amino acid sequence depicted in SEQ ID NO:2 or a sequence having at least 80% sequence identity thereto.

[0056] Thus, in another embodiment, the gene therapy vector is an AAV serotype 6 vector that comprises the human TNNT2 promoter of SEQ ID NO:5 or a functionally equivalent sequence having at least 80%, more preferably 90%, 95%, 96%, 97%, 98%, 99%, sequence identity thereto, wherein the promoter is operably linked to a nucleic acid sequence encoding a cMyBP-C, preferably a murine cMyBP-C having the amino acid sequence depicted in SEQ ID NO:4 or a sequence having at least 80% sequence identity thereto, or the human cMyBP-C protein having the amino acid sequence depicted in SEQ ID NO:2 or a sequence having at least 80% sequence identity thereto. In a more preferred embodiment, the gene therapy vector is an AAV serotype 6 vector that comprises the human TNNT2 promoter of SEQ ID NO:5 or a functionally equivalent sequence having at least 80%, more preferably 90%, 95%, 96%, 97%, 98%, 99%, sequence identity thereto, wherein the promoter is operably linked to a nucleic acid sequence encoding the human cMyBP-C protein having the amino acid sequence depicted in SEQ ID NO:2 or a sequence having at least 80% sequence identity thereto.

[0057] Thus, in another embodiment, the gene therapy vector is an AAV serotype 8 vector that comprises the human TNNT2 promoter of SEQ ID NO:5 or a functionally equivalent sequence having at least 80%, more preferably 90%, 95%, 96%, 97%, 98%, 99%, sequence identity thereto, wherein the promoter is operably linked to a nucleic acid sequence encoding a cMyBP-C, preferably a murine cMyBP-C having the amino acid sequence depicted in SEQ ID NO:4 or a sequence having at least 80% sequence identity thereto, or the human cMyBP-C protein having the amino acid sequence depicted in SEQ ID NO:2 or a sequence having at least 80% sequence identity thereto. In a more preferred embodiment, the gene therapy vector is an AAV serotype 8 vector that comprises the human TNNT2 promoter of SEQ ID NO:5 or a functionally equivalent sequence having at least 80%, more preferably 90%, 95%, 96%, 97%, 98%, 99%, sequence identity thereto, wherein the promoter is operably linked to a nucleic acid sequence encoding the human cMyBP-C protein having the amino acid sequence depicted in SEQ ID NO:2 or a sequence having at least 80% sequence identity thereto.

[0058] Recombinant viral vectors can be generated according to standard techniques. For example, recombinant adenoviral or adeno-associated viral vectors can be propagated in human 293 cells (which provide E1A and E1B functions in trans) to titers in the range of 10⁷-10¹³ viral particles/mL. Prior to their in vivo application viral vectors may be desalted by gel filtration methods, such as sepharose columns, and purified by subsequent filtering. Purification reduces potential deleterious effects in the subject to which the vectors are administered. The administered virus is substantially free of wild-type and replication-competent virus. The purity of the virus can be proven by suitable methods, such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining. This is applicable for both AAV and adenoviral vectors.

[0059] As described in the below examples, transduction of the gene therapy vectors of the invention into the subject to be treated can be achieved by systemic application, e.g., by intravenous, intraarterial or intraperitoneal delivery of a vector in analogy to what has been shown in animal models (Katz et al., 2012, Gene Ther 19:659-669. In a preferred embodiment, the gene therapy vectors are for use in the described method of treating or preventing hypertrophic cardiomyopathy, wherein the gene therapy vector is administered systemically.

[0060] Alternatively, the gene therapy vectors of the invention can be delivered by direct administration to the heart tissue, e.g. by intracoronary administration. In a preferred embodiment, the gene therapy vectors are administered as a single dose by antegrade epicardial coronary artery infusion over a 10-minute period in a cardiac catheterization laboratory after angiography (percutaneous intracoronary delivery without vessel balloon occlusion) with the use of standard 5F or 6F guide or diagnostic catheters (Jaski et al., 2009, J Card Fail 15:171-181).

[0061] In another preferred embodiment, tissue transduction of the myocardium is achieved by catheter-mediated intramyocardial delivery (Gao et al., 2011, Hum Gene Ther 22:979-84). Importantly, this latter form of delivery can also be used to transfer vector-free cDNA coupled or not to transduction-enhancing carriers into myocardium. Cell-derived exosomes or microparticles with cardiac tropism can also be used to transport the vectors of the invention (Lee et al., 2012, Hum Mol Genet 21:R125-134). A suitable dose of AAV for humans would be in the range of about 1×10¹⁰ to 1×10¹⁴ virus particles, and in particular about 1×10¹².

[0062] Apart from viral vectors, non-viral expression constructs may also be used for introducing a gene encoding a functional cardiac sarcomeric protein or a functioning variant or fragment thereof into a cell or a human subject. Non-viral expression vectors which permit the in vivo expression of protein in the target cell include, for example, vectors such as pBK-CMV, pcDNA3.1, and pZeoSV (Invitrogen, Stratagene). Suitable methods for the transfer of non-viral vectors into target cells are, for example, the lipofection method, the calcium-phosphate co-precipitation method, the DEAE-dextran method and direct DNA introduction methods using micro-glass tubes and the like. Prior to the introduction of the vector, the cardiac muscle cells may be treated with a permeabilization agent, such as phosphatidylcholine, streptolysins, sodium caprate, decanoylcarnitine, tartaric acid, lysolecithin, Triton X-100, and the like.

[0063] Alternatively, isolated cells that have been removed from a subject, for example, by a biopsy procedure, may be transfected with the vector in an ex vivo procedure. The cells can then be re-implanted into or otherwise administered to a subject, preferably into the subject from whom they were obtained. In another aspect, the invention thus relates to an isolated cell, such as a cardiomyocyte or a stem cell, which has been transduced with the gene therapy vector of the invention. After transduction of the vector, the cell expresses the cardiac sarcomeric protein that was encoded by the vector. The cell preferably is a cardiac cell, such as a cardiomyocyte, or a cardiomyocyte derived from induced pluripotent stem cell (iPSC). The cell may also be a stem cell, preferably an embryonal or pluripotent adult stem cell, more preferably an endogenous cardiac stem cells (eCSCs) or an iPSC derived from fibroblasts (Okita et al., 2007, Nature 448:313-7; Yu et al., 207, Science 318:1917-20; Maekawa et al., 2011, Nature 474: 225-229), from keratinocytes (Aasen et al., 2008, Nat Biotech 11:1276-1284; Aasen & Belmonte, 2010, Nat Protocol 5:371-382) or from blood cells (Staerk et al., 2010, Stem Cell Stem 7: 20-24; Seki et al., 2012, Nat Protocol 7:718-728).

[0064] It is furthermore preferred that the cell is a human cell. The likelihood of rejection of transplanted cells is reduced when the subject from whom the cell is explanted is genetically similar to the subject to whom the cell is administered. Therefore, the cell of the invention is preferably an autologous cell that is transduced with the gene therapy vector of the invention ex vivo. After transduction of the autologous cell, the cell is reintroduced into the subject by appropriate administration means, such as transplantation or infusion.

[0065] The cell is preferably for use in a method of treating or preventing HCM in a subject, wherein mutations in the gene encoding said cardiac sarcomeric protein are associated with HCM and the subject carries a gene mutation that impairs the function of said cardiac sarcomeric protein.

[0066] The invention further relates to a pharmaceutical composition comprising the gene therapy vector of the invention. In a preferred embodiment, the composition is for use in a method of treating or preventing HCM in a subject having a dysfunctional cardiac sarcomeric protein.

[0067] Methods for the preparation of pharmaceutical compositions that contain gene therapy vectors are well known by those working in the field of pharmaceutics. Typically, such compositions are prepared either as liquid solutions or suspensions. The pharmaceutical composition of the invention can include commonly used pharmaceutically acceptable excipients, such as diluents and carriers. In particular, the composition comprises a pharmaceutically acceptable carrier, e.g., water, saline, Ringer's Solutions, or dextrose solution. Further examples of suitable carriers are described in standard textbooks, for example, in “Remington's Pharmaceutical Sciences”, Mack Pub. Co., New Jersey (1991). In addition to the carrier, the composition may also contain emulsifying agents, pH buffering agents, stabilizers, dyes and the like.

[0068] The pharmaceutical composition will comprise a therapeutically effective gene dose. A therapeutically effective gene dose is one that is capable of preventing or treating cardiomyopathy in a subject, without being toxic to the subject. Prevention or treatment of cardiomyopathy can be assessed as a change in a phenotypic characteristic associated with cardiomyopathy, such change being effective to prevent or treat cardiomyopathy. Phenotypic characteristics associated with cardiomyopathy are for example left ventricular (LV) hypertrophy, reduced fractional shortening, interstitial fibrosis as well as diastolic and systolic dysfunction. A therapeutically effective gene dose typically elicits a positive change in the phenotype of HCM, i.e. a change that approximates the phenotype of the subject suffering from HCM to the phenotype of a healthy subject which does not carry a HCM gene mutation. Thus, a therapeutically effective gene dose is typically one that, when administered in a physiologically tolerable composition, is sufficient to improve or prevent the pathogenic heart phenotype in the treated subject.

[0069] In yet another aspect, the invention relates to methods for treating or preventing HCM in a subject by introducing a gene therapy vector for expressing an exogenous nucleic acid sequence in a subject, said vector comprising:

• [0070] (a) a nucleic acid sequence encoding a cardiac sarcomeric protein as defined elsewhere herein, and
• [0071] (b) a cardiomyocyte-specific promoter which is operably linked to said nucleic acid sequence, wherein mutations in the gene encoding said cardiac sarcomeric protein are associated with HCM and said subject carries a gene mutation that impairs the function of said cardiac sarcomeric protein.

[0079] For both ex vivo and in vivo studies, a knock-in mouse carrying a G>A transition in the Mybpc3 gene (Mybpc3-targeted knock-in; KI) has been developed by gene targeting using the Cre/lox system (Vignier et al., 2009, Circ Res 105:239-248). Briefly, a 8105 bp-fragment containing the 5′ part of mouse Mybpc3 gene, which covers 1747 bp upstream of exon 1 up to exon 15, was obtained by long-range PCR or cloning from a FIX II genomic library derived from a 129/Svj mouse strain, and then cloned into the pBluescript® II KS+ vector (Stratagene). The G>A transition on the last nucleotide of exon 6 was obtained by site-directed mutagenesis (Stratagene) on a 258 bp PCR fragment, which was then cloned into the Eco47RI/Nsi I sites.

[0080] The phenotype of KI mice appeared normal and they were viable for up to two years (Vignier et al., 2009, Circ Res, 105:239-248). Echocardiography was performed on wild-type (WT) and homozygous KI mice using the Vevo 2100 System (VisualSonics, Toronto, Canada). Mice were anesthetized with isofluorane (1-2%) and fixed to a warming platform in a supine position. B-mode images were obtained using a MS400 transducer for adult mice and a MS550 transducer for neonatal mice. Images were obtained in a parasternal short and long axis view and dimensions of the left ventricle were measured in a short axis view in diastole and systole. KI mice exhibited left ventricular hypertrophy, reduced fractional shortening and interstitial fibrosis compared to WT mice at 3-4 months after birth (Vignier et al., 2009, Circ Res, 105:239-248).

[0081] The G>A transition resulted in three different mutant mRNAs (see FIG. 1A). Mutant 1 (missense) contains the G>A transition and produces an E264K mutant protein of about 150 kDa. Mutant 2 (nonsense) is a result from the skipping of exon 6, which leads to a frameshift and a premature termination codon (PTC) in exon 9. The expected protein is 32 kDa. Mutant 3 also results from the skipping of exon 6 and a partial retention of intron 8, which restores the reading frame. In this case, a 147 kDa-mutant protein is produced. None of these mutants encodes a functional protein.

[0082] RNA or protein was extracted from ventricular tissue of homozygous KI and WT mice. Total RNA was isolated from ventricular tissue (30 mg) using the SV Total RNA Isolation System Kit (Promega) according to the manufacturer's instructions. RNA concentration, purity and quality were determined using the NanoDrop® ND-1000 spectrophotometer (Thermo Scientific). Reverse transcription (RT) was performed from 150-200 ng RNA using oligo-dT primers (SuperScript®-III kit, Life Technologies). Quantitative polymerase chain reaction (qPCR) was performed using primers #1 (forward: 5′-GGA TTA CAA GGA TGA CGA CGA-3′; SEQ ID NO:9) and #2 (reverse: 5′-TCC AGA GTC CCA GCA TCT TC-3′; SEQ ID NO:10) and SYBR green. The level of total Mybpc3 mRNA was 80% lower in homozygous KI mice than in wild WT mice (FIG. 1B).

[0083] Crude protein extract was obtained from about 15 mg of ventricular tissue homogenized in 5% SDS, 50 mM Tris-HCl, pH 7.5, 250 mM sucrose, 75 mM urea, 1 mM DTT at 4° C. and centrifuged at 13000 rpm for 2 min. The supernatant was collected and its concentration was determined using the BCA Protein Assay Kit (Pierce). Proteins were loaded on 10%-acrylamide/bisacrylamide (29:1) gels and electrotransferred on a 0.45 μm pore size nitrocellulose membrane (Invitrogen). Membranes were stained with a polyclonal antibody directed against cMyBP-C(C0-C1 1:1,000). The secondary antibody was coupled to HRP (Sigma). Signal was revealed with SuperSignal® West Pico chemiluminescent substrate (Pierce) and acquired with a ChemiImager™ 5500 (Alpha Innotech). Quantification of the signal was done using the NIH Image 1.63 software. Homozygous Mybpc3-targeted knock-in mice expressed only low levels of mutant proteins (FIG. 1C).

[0084] The results show that the presence of low levels of cMyBP-C proteins (=haploinsufficiency), including mutant polypeptides (=poison-polypeptides) results in left ventricular hypertrophy and dysfunction, which are hallmarks of HCM.

[0085] The vector PGG2-hTNNT2-WT-Mybpc3 was constructed by first amplifying the full-length FLAG-tagged mouse Mybpc3 cDNA (GenBank accession number NM_008653.2) including exons 1-34 by RT-PCR from mouse ventricular RNA using the forward primer #3 (5′-TTC GAC CTC GAG ATG GAT TAC AAG GAT GAC GAC GAT AAG CCT GGT GTG ACT GTT CTC AA-3′; SEQ ID NO:11) containing the XhoI restriction site and the FLAG sequence and reverse primer #4 (5′-TTC GAC GGA TCC CTG GTC ACT GAG GAA CTC G-3′; SEQ ID NO:12) containing BamHI restriction site. The human cardiac troponin T (hTNNT2) 5′ region from base −502 to +42 (GenBank accession number NG_007556.1; SEQ ID NO:5) was originally amplified from a human cDNA library by PCR using forward primer #5 (5′-AAA AAA ACG CGT CTC AGT CCA TTA GGA GCC AGT AGC-3′; SEQ ID NO:13) and reverse primer #6 (5′-CCC CCC CAA GCT TCT GCC GAC AGA TCC TGG AGG CG-3′; SEQ ID NO:14) enabling cloning with MluI/HindIII restriction enzymes in a plasmid containing a renilla luciferase reporter gene (pdsTNNT2(−502+42)-Rluc) and the chimeric (□-globin/Ig) intron, which has been shown to increase gene expression (Dominguez et al., 2011, Hum Mol Genet 20, 681-93). For generation of the pGG2-hTNNT2-WT-Mybpc3 plasmid the hTNNT2 promoter and the chimeric intron (SEQ ID NO:7; FIG. 2) were excised with the restriction enzymes EcoRI and NheI and ligated into the PGG2 plasmid vector containing the SV polyA signal (SEQ ID NO:6). Together the vector has a size of 9027 bp (FIG. 2), including 5.4 kbp of insert between two ITRs (FIG. 2), which exceeds the packaging capacity of adeno-associated virus (AAV; Wu et al., 2010, Mol Ther 18:80-86).

[0086] AAV6 and AAV9 pseudotyped vectors were produced with the two (AAV6; Muller et al., 2006, Cardiovasc Res 70:70-78) or the three (AAV9; Kaya et al., 2011, Cardiovasc Res 91:116-123) plasmids transfection method. AAV6 pseudotyped vectors were generated by co-transfection of HEK293T cells with the pGG2-hTNNT2-WT-Mybpc3 transfer plasmid and the AAV packaging plasmid pDP6rs, which provides the AAV2 rep and AAV6 cap genes and adenoviral helper functions (Grimm et al., 2003, Mol Ther, 7:839-850). AAV9 pseudotyped vectors were generated by triple-transfection of pGG2-hTNNT2-WT-Mybpc3 transfer plasmid with p5E18-VD2-9 and pDGdeltaVP encoding adenoviral helper functions (Kaya et al., 2011, Cardiovasc Res 91:116-23). Generation of recombinant AAV6 and AAV9 particles was carried out as described previously (Grieger et al., 2006, Nat Protoc 1:1412-1428), with some modifications. Plasmids were transfected into 293T HEK cells in cell stacks or in plates with a diameter of 15 cm using polyethylenimine (PEI) as described before (Hauswirth et al., 2000, Methods Enzymol 316:743-761). The HEK293T-AAV cells were cultivated in DMEM, High Glucose supplemented with 10% (v/v) heat-inactivated fetal calf serum, 0.1 mM MEM non-essential amino acids, 2 mM L-glutamine, 100 UI/ml penicillin and 100 μg/ml streptomycin. Tissue culture reagents were obtained from Life technologies. Cells were harvested after 72 h, washed three times with phosphate-buffered saline (PBS). After three freeze-thaw cycles, benzonase (Merck; 250 U/ml) was added for 1 h at 37° C. Cell debris was pelleted and vector-containing lysates were purified using iodixanol step gradients (Hauswirth et al., 2000, Methods Enzymol 316:743-761).

[0087] The genomic titers of DNase-resistant AAV particles were determined by qPCR using the SYBR Green qPCR Master MIX 2 (Fermentas) and an ABI PRISM® 7900HT cycler (Applied Biosystem) as reported before (Veldwijk et al., 2002, Mol Ther 6:272-278). Vectors were quantified using primers #7 (forward: 5′-CTC AGT CCA TTA GGA GCC AGT-3′; SEQ ID NO:15) and #8 (reverse: 5′-AAG GCA ACC TCC AAG ACA CT-3; SEQ ID NO:16) specific for TNNT2 promoter sequence. Real-time PCR was performed in a total volume of 10 μl with 0.3 μM for each primer. The pdsAAV-TNNT2-eGFP plasmid was used as a copy number standard. A standard curve for quantification was generated by serial dilutions of the respective plasmid DNA. The cycling conditions were as follows: 50° C./2 min, 95° C./10 min, followed by 35 cycles of 95° C./15 sec and 60° C./60 sec. Calculations were done using the SDS 2.4 software (Applied Biosystem).

[0088] Neonatal mouse cardiac myocytes were isolated from neonatal mouse hearts as previously described (Vignier et al., 2009, Circ Res 105:239-248). Cardiac myocytes were immediately transduced with AAV6-FLAG-Mybpc3 under the control of hTNNT2 at a multiplicity of infection (MOI) of 3000 for 30 min at 37° C. in suspension prior to plating (4.4×10⁵ cells/well). Cardiac myocytes were kept in culture for 7 days at 37° C. and 10% CO₂ prior to harvesting.

[0089] HEK293 cells were plated at a density of 2×10⁵ cells in 12-well dishes in DMEM (10% FCS, 1% penicillin-streptomycin) and incubated at 37° C. with 7% CO₂ until the recommended confluence of 50-70% was reached. The transient transfection of FLAG-Mybpc3 plasmid into adherent HEK293 cells was performed using the TurboFect transfection reagent (Fermentas) according to the manufacturer's protocol.

[0090] Total RNA was isolated from cultured cardiac myocytes using the SV Total RNA Isolation System Kit (Promega) according to the manufacturer's instructions. RNA concentration, purity and quality were determined using the NanoDrop® ND-1000 spectrophotometer (Thermo Scientific). RT was performed from 150-200 ng RNA using oligo-dT primers (SuperScript®-III kit, Life Technologies). As a control for genomic contamination a reaction without RT was performed. Touch-down PCR amplifications (65° C.-60° C.) were performed using AmpliTaq® Gold Polymerase (Applied Biosystems) in a total volume of 20 μl for 35 cycles with different primer pairs: FLAG-Mybpc3 mRNA was amplified using forward primer #9 (5′-GGA TTA CAA GGA TGA CGA CGA-3′; SEQ ID NO:17) and reverse primer #10 (5′-TCC AGA GTC CCA GCA TCT TC-3′; SEQ ID NO:18); total Mybpc3 mRNA was amplified with forward primer #11 (5′-CCT GGT GTG ACT GTT CTC AA-3′; SEQ ID NO:19) and reverse primer #12 (5′-TCC AGA GTC CCA GCA TCT TC-3′; SEQ ID NO:20); Myh6 mRNA (encoding α-myosin heavy chain) was amplified with forward primer #13 (5′-CTC AAG CTC ATG GCT ACA CTC TTC TC-3′; SEQ ID NO:21) and reverse primer #14 (5′-AGA GCA GAC ACT GTT TGG AAG GA-3′; SEQ ID NO:22). PCR products were visualized on 1.5% agarose gels (FIG. 3A). In untransduced cells (Untr.), only mutant mRNAs were detected (total Mybpc3 panel). In contrast, after AAV6-FLAG-Mybpc3 gene transfer in KI cardiac myocytes, FLAG-Mybpc3 mRNA was detected (FLAG-Mybpc3 panel) and was associated with a reduced level of mutant mRNAs (total Mybpc3 panel). The level of Myh6 did not differ between the groups (Myh6 panel). Quantitative PCR using forward primer #15 (5′-GAT GCG AGC CCT GAT GAC-3′; SEQ ID NO:23) and reverse primer #16 (5′-GAC TTG AGA CAC TTT CTT CC-3′; SEQ ID NO:24) and SYBR green demonstrated further that the level of total Mybpc3 mRNA in KI cardiac myocytes transduced with AAV6-FLAG-Mybpc3 reached the level found in WT cardiac myocytes (FIG. 3B). Moreover, quantitative PCR using specific hydrolysing Taqman probes were performed to determine the level of the different mutant mRNAs: Mutant-1 was revealed with probe #1 (5′-VIC-CTC ACT GTC CAT AAG G-MGB-3′; SEQ ID NO:25), mutants 2+3 with probe #2 (5′-FAM-CCA GCA AGA GGC CA-MGB-3′; SEQ ID NO:26) and mutant 3 with probe #3 (5′-FAM-TCG GAG AAC CAG CCC CTG CTA GCT C-TAMRA-3′; SEQ ID NO:27). This shows that mutant-1 and mutant-3 mRNA are completely absent, whereas levels of mutant-2 mRNA are markedly reduced in KI cardiac myocytes from KI transduced with AAV6-FLAG-Mybpc3 (FIG. 3C).

[0091] Crude proteins from transduced cultured cardiac myocytes or transfected HEK293 cells were extracted in lysis buffer (30 mM Tris base pH 8.8, 5 mM EDTA, 30 mM NaF, 3% SDS, 10% glycerol) and protein concentration was determined by Bradford protein assay (BioRad). Total proteins (cardiac myocytes 30 μg/lane, HEK293 2.5 μg/lane) were separated on 10% SDS-polyacrylamide (29:1) mini-gels (BioRad) and transferred on PVDF membranes by electroblotting. Membranes were stained overnight with the primary antibody directed against the MyBP-C motif of cMyBP-C (1:1,000). After incubation with anti-rabbit (1:6,000, Sigma) peroxidase-conjugated secondary antibodies, proteins were visualized using Super Signal® West Dura detection reagent (Thermo Scientific) and signals were detected with the ChemiGenius² Bio Imaging System. Western blot analysis shows a specific cMyBP-C band in all lanes. Furthermore, the cMyBP-C levels in AAV6-FLAG-Mybpc3-transduced KI cardiac myocytes reached the levels found in untransduced WT cardiac myocytes (FIG. 3D).

[0092] Immunofluorescence analysis was performed in order to examine the localization of the transgenic FLAG-tagged cMyBP-C protein (FIG. 4D). KI cardiac myocytes transduced with AAV6-TNNT2-FLAG-WT-Mybpc3 (MOI 3,000) were analyzed by confocal microscopy after fixation of the cells and staining with antibodies directed against the FLAG epitope and total full-length cMyBP-C protein. Immunofluorescence of transduced cardiac myocytes using the anti-cMyBP-C antibody showed the classic striation pattern of total cMyBP-C protein located in doublets in the A-band of the sarcomere (FIG. 4D, cMyBP-C). Furthermore, FLAG-positive signal (FIG. 4D; FLAG) colocalized with cMyBP-C protein striation, confirming the correct sarcomeric incorporation of the transgenic FLAG-tagged cMyBP-C protein.

[0093] These data demonstrate that Mybpc3 gene transfer in KI cardiac myocytes rescues cMyBP-C haploinsufficiency and at the same time prevents transcription of mutant alleles and accumulation of toxic mutant cMyBP-C proteins.

[0094] Hearts derived from wild-type (WT) and Mybpc3-targeted knock-in (KI) neonatal mice were taken (postnatal day 0-1) for cell isolation using a trypsin/collagenase overnight digestion (Laugwitz et al., 2005, Nature 433:647-653; Moretti et al., 2006, Cell 127:1151-65). To generate engineered heart tissue (EHT), a reconstitution mix was prepared on ice as follows (final concentration): Unpurified 6.8×10⁶ cells/ml, 5 mg/ml bovine fibrinogen (stock solution: 200 mg/ml plus aprotinin, 0.5 μg/mg fibrinogen in NaCl 0.9%, Sigma F4753), 100 μl/ml Matrigel (BD Bioscience 356235). 2×DMEM was added to match the volumes of fibrinogen and thrombin stock (100 U/ml, Sigma Aldrich T7513) to ensure isotonic conditions. Casting molds were prepared as previously described (Hansen et al., 2010, Circ Res 107:35-44).

[0095] AAV6-FLAG-Mybpc3 or AAV6-FLAG-GFP, or a control without virus was added directly into the EHT master mix before casting, at a MOI of 1000 or 3000. The volume of 2×DMEM was adapted to the volume of virus to maintain isotonic conditions. For each EHT a 97-μl-reconstitution mix was mixed briefly with 3 μl thrombin and pipetted into the agarose slot. For fibrinogen polymerization, the constructs were placed in a 37° C., 7% CO₂ humidified cell culture incubator for 2 h. The racks were transferred to 24-well plates filled with culture medium. EHTs were kept in a 37° C., 7% CO₂ humidified cell culture incubator. Cell culture medium was changed after 48 h and consisted of DMEM (Biochrom F0415), 10% horse serum (Gibco 26050), 2% chick embryo extract, 1% Penicillin/Streptomycin (Gibco 15140), insulin (10 μg/ml, Sigma-Aldrich I9278) and aprotinin (33 μg/ml, Sigma Aldrich A1153). On day 5 of the EHT culture, cytosine β-D-arabinofuranoside (25 μg/ml, Sigma-Aldrich C1768) was added to the culture medium for 48 h. Spontaneous contractile activity of EHTs was monitored from day 7 to day 19 via video-optical recording (Hansen, et al., 2010, Circ Res 107, 35-44). Contraction graphs were automatically recorded and evaluated. The CTMV software (Pforzheim, Germany) was used to measure spontaneous contractions of murine EHTs as recently published (Hansen et al., 2010, Circ Res 107:35-44; Stöhr et al., 2013, J Mol Cell Cardiol 63:189-98). For this purpose, the 24-well plate was placed in a cell incubator unit with control of CO₂, humidity and temperature, and a glass roof for monitoring purposes. A Basler camera (Type A 602f-2) was placed above the cell culture unit in a PC-controlled manner. During measuring time the distance between the ends of the muscle strip was recorded during contractions. The force was calculated according to a recently published equation (Vandenburgh et al., 2008, Muscle Nerve, 37:438-47) based on post geometry, elastic modulus of Sylgard 184 (Dow Corning) and delta of post distance (post deflection). Squares in recorded contraction graphs indicated the identified peaks, which were taken for frequency, average force, contraction and relaxation times (T1, T2, respectively) calculation. T1 and T2 were determined at 10% of peak maximum. At the end of the experiments, EHTs were removed from posts, and total RNA was extracted.

[0096] FLAG-Mybpc3 mRNA was amplified as described in the Example 3 and detected only in transduced EHTs, and its level increased with increasing MOI (FIG. 4A). In addition, PCR amplification of all types of Mybpc3 mRNAs (Total Mybpc3, as described in Example 3) revealed that (FIG. 4B): i) the different mutant mRNAs were detected at a similar level in both untransduced KI-EHT and in EHT transduced with AAV6-GFP; ii) FLAG-Mybpc3 gene transfer in KI EHTs lead to a single type of mRNA. The level of this mRNA did not differ from the level detected in WT-EHT. This shows that gene transfer of FLAG-Mybpc3 repaired the mRNA haploinsufficiency and reduced the content of mutant mRNAs in EHT derived from KI neonatal cardiac cells.

[0097] Spontaneous contractile activity of EHTs was monitored from day 7 to day 19 of culture via video optical recording (FIG. 4C). In all groups, maximum force was reached at 14 days. The developed force is higher in KI (about 65 μN) than in WT EHTs (about 40 μN, data not shown), indicating hypercontractility. The developed force was significantly lower after Mybpc3 gene transfer in KI EHT than in other groups (FIG. 4C), reaching levels previously found in WT EHTs.

[0098] Together, these data show that gene transfer of FLAG-Mybpc3 in EHT derived from KI neonatal cardiac cells rescues both the molecular phenotype (no haploinsufficiency and no mutant mRNAs) and the function (absence of hypercontractility).

[0099] All experimental in vivo studies were in accordance with the guidelines for the care and use of laboratory animals published by the NIH (Publication No. 85-23, revised 1985) as well as the German Law for the Protection of Animals and accepted by the Ministry of Science and Public Health of the City State of Hamburg, Germany (Nr. 69/10).

[0100] AAV9-FLAG-Mybpc3 (5×10¹² vector genomes (vg)) or PBS as a control were administered in 3-day-old mice via temporal vein injection using a 30-G needle (Sands and Barker, 1999, Lab Anim Sci, 49:328-330) as described previously (Dominguez et al., 2011, Hum Mol Genet 20:681-693). All mice recovered quickly from the injection. The cardiac phenotype was evaluated every week from 3 weeks of age by echocardiography (see details in Example 1). The mice were sacrificed at 7 weeks of age and different organs were extracted. RNA and proteins were extracted. FLAG-Mybpc3 and total Mybpc3 mRNAs in ventricles, liver and skeletal muscle were evaluated by RT-PCR as described in Example 3 (FIG. 5A).

[0101] The level of FLAG-Mybpc3 mRNA was much higher in the ventricles than in other organs (FIG. 5A, upper FLAG-Mybpc3 panel). In the ventricles, the different mutant Mybpc3 mRNAs were amplified in the control KI mice (FIG. 5A, lower total Mybpc3 panel), whereas a major unique band was detected in wild-type control mouse and in the KI mouse transduced with AAV9-FLAG-Mybpc3 (FIG. 5A, total Mybpc3 panel). A band was also detected in liver and skeletal muscle after FLAG-Mybpc3 gene transfer, although at a lower level than in the ventricles.

[0102] Western blot analyses was performed as described in Example 3 using an antibody directed against cMyBP-C and revealed that the cMyBP-C protein level after AAV9-FLAG-Mybpc3 gene transfer is higher than in PBS- or AAV9-GFP-injected KI mice and reached the level found in WT mouse (FIG. 5B). cMyBP-C protein was not detected in the liver and skeletal muscle after Mybpc3 gene transfer (FIG. 5B), due to the cardiac-specificity of the vector.

[0103] In order to examine the localization of the exogenously expressed FLAG-tagged cMyBP-C protein, immunofluorescence analysis was performed on ventricular cryosections of the KI mouse injected with AAV9-FLAG-Mybpc3 for 7 weeks using antibodies directed against FLAG epitope and total cMyBP-C protein. The staining showed the classic striation pattern of the cMyBP-C protein located in doublets in the A-band of the sarcomere (FIG. 5C; cMyBP-C), which entirely co-stained with the FLAG signal (FIG. 5C; FLAG). Nuclei were stained with DRAQ5™. Taken together, the overexpressed cMyBP-C protein was properly incorporated within the sarcomere and the majority of FLAG-positive-striated cardiomyocytes were co-stained with total cMyBP-C protein, suggesting that exogenous cMyBP-C protein replaced the endogenous mutant ones.

[0104] Echocardiographic analyses were performed as described in Example 1 above. Fractional area shortening (FAS) and left ventricular mass-to-body weight (LVM/BW) ratio were examined in wild-type (WT), PBS-injected knock-in (KI−) mice and KI mice injected with AAV9-FLAG-Mybpc3 (KI+) at 3, 5, 6 and 7 weeks of age. Evaluation of the cardiac function by echocardiography showed a rescue of the fractional area shortening (FAS) and a reduction of the left ventricular mass-to-body weight (LVM/BW) ratio after FLAG-Mybpc3 gene transfer (FIG. 5D).

[0105] Together, these data showed that a single administration of AAV9-FLAG-Mybpc3 in neonatal KI mice rescues the molecular phenotype (no cMyBP-C haploinsufficiency and no mutant polypeptides) and the functional phenotype (no left ventricular hypertrophy and dysfunction).

[0106] Induced pluripotent stem cells (iPSC) were generated by reprogramming of fibroblasts expanded from a skin biopsy of a human control individual. Cardiac myocyte differentiation was adapted from a protocol from the group of Gordon Keller (Yang L et al., 2008, Nature 22:524-8).

[0107] After differentiation, human cardiac myocytes were plated at a density of 2×10⁵ cells/well in a 12-well plate for RNA and protein analysis, or 2.5×10⁴ cells/chamber in a four chamber dish (35-mm diameter) for immunofluorescence analysis. Cardiac myocytes were transduced for 8 days with a myc-tagged MYBPC3 adenovirus encoding human myc-cMyBP-C (DNA sequence: SEQ ID NO:29 followed by SEQ ID NO:1) at different MOI.

[0108] Construction of the myc-tagged human MYBPC3 plasmid was described previously (Flavigny J et al., 1999, J Mol Biol 294, 443-456; Sarikas et al., 2005, Cardiovasc Res 66:33-44). Briefly, an ATG plus 30-nucleotide sequence (SEQ ID NO:29) encoding the myc epitope (SEQ ID NO:30) was inserted behind the CMV promoter (SEQ ID NO:31) and before the human MYBPC3 cDNA (SEQ ID NO:1). The insert encodes a myc-tagged human cMyBP-C(SEQ ID NO:32). Recombinant adenovirus were generated by cloning the insert (myc-tagged human MYBPC3 cDNA) into the shuttle vector pAdTrack-CMV and subsequent cotransformation of this plasmid with pAdEasy-1 into Escherichia coli as described previously (He T et al., 1998 Proc Natl Acad Sci USA 95, 2509-2514). Expression of cMyBP-C is driven by the constitutively active CMV promoter (SEQ ID NO:31).

[0109] The evaluation of the transcription of the different MYBPC3 mRNAs (exogenous myc-MYBPC3 and total MYBPC3) in human cardiac myocytes was performed by RT-PCR as described before (FIG. 6A). Exogenous MYBPC3 mRNA was amplified with specific primers (Forward primer in the myc sequence, 5′-GCA AAA GCT TAT TAG CGA GGA A-3′ (SEQ ID NO:33) and reverse primer in exon 2, 5′-CAG GCC GTA CTT GTT GCT G-3′ (SEQ ID NO:34)), and total MYBPC3 mRNA with primers that recognized both exogenous and endogenous MYBPC3 (Forward primer in exon 1, 5′-GGG GAA GAA GCC AGT CTC AG-3′ (SEQ ID NO:35) and reverse primer in exon 2, 5′-CAG GCC GTA CTT GTT GCT G-3′ (SEQ ID NO:34)). The level of Myc-MYBPC3 mRNA increased with increasing virus dose (FIG. 6A, left panel). No Myc-MYBPC3 mRNA was detected in non-transduced cells (NT) and in negative controls lacking reverse transcriptase (RT).

[0110] Western blot analysis was performed as described before using antibodies directed either against the CO-C1 domains of cMyBP-C (FIG. 6B, total cMyBP-C, kindly given by collaborator) or against the myc tag (FIG. 6B, exogenous myc-cMyBP-C (rabbit polyclonal Sigma; catalog # C3956). The positive control (+) was a sample of murine cardiac myocytes transduced with the same virus. The level of total cMyBP-C was slightly increased after adenoviral gene transfer, whereas the myc-tagged cMyBP-C protein was absent in non-transduced (NT) sample and its level increased with increasing MOI (FIG. 6B).

[0111] Localization of exogenous myc-cMyBP-C in human cardiac myocytes derived from iPSC was analysed by immunofluorescence. Human cardiac myocytes were stained with anti-cMyBP-C antibody (FIG. 6C, cMyBP-C), which showed expected sarcomeric striations. Exogenous cMyBP-C was stained with the anti-myc antibody (FIG. 6C, myc). The anti-myc antibody binds to the myc tag, which is located at the N-terminus of the protein. It was observed that exogenous myc-tagged cMyBP-C was correctly incorporated into the sarcomere of human iPSC-derived cardiac myocytes as a doublet in the A band.

[0112] These data showed for the first time expression of exogenous human myc-MYBPC3 in human cardiac myocytes derived from iPSC. Expression of exogenous human myc-MYBPC3 resulted in a stable human cMyBP-C protein, which is incorporated into the sarcomere. Thus, overexpression of MYBPC3 cDNA may be used for gene therapy in human hypertrophic cardiomyopathy.

[0113] Different doses of adeno-associated virus serotype 9 (AAV9)-Mybpc3 (1×10¹¹, 3×10¹¹, 1×10¹² and 3×10¹² vector genomes (vg)/mouse) or PBS were administered into the temporal vein of 1-day-old Mybpc3-targeted knock-in (KI) mice, before the appearance of the cardiac disease phenotype. After 34 weeks, mice were subjected to in vivo hemodynamics and tissue analysis. WT mice were used as controls.

[0114] Analyses of systolic (=dP/dtmax) and diastolic (=dP/dtmin) function and determination of the heart weight to body weight ratio (HW/BW) were performed in 34-week-old WT, KI treated with PBS and KI mice treated with the highest dose of 3×10¹² vg (FIG. 7.1 A). Compared to WT, the slight reduction in systolic function, the marked reduction in diastolic function and the marked increase in HW/BW ratio were prevented by Mybpc3 gene therapy in KI mice.

[0115] Further, RT-PCR was performed for evaluation of the mRNA levels of exogenous FLAG-tagged Mybpc3 (FIG. 7.1 B, upper panel) and total Mybpc3 (lower panel). RNA was extracted from ventricular tissues and pooled in each group (n=5-10/group). The size of the PCR-amplified bands is shown on the left side of FIG. 7.1 B. This shows that the expression of Mybpc3 (both exogenous alone and total) increased in a AAV9-Mybpc3 dose-dependent manner. Importantly and conversely, the expression of mutant mRNAs (as represented by the amplicons for mutant-1, mutant-2 and mutant-3) decreased in a AAV9-Mybpc3 dose-dependent manner.

[0116] Moreover, total Mybpc3 mRNA level was determined by RT-qPCR performed in 34-week-old WT, KI treated with PBS and KI mice treated with the highest dose of 3×10¹² vg (FIG. 7.1 C). Compared to WT, the marked reduction in Mybpc3 mRNA level was fully prevented by Mybpc3 gene therapy in KI mice.

[0117] Western blot analysis was performed for evaluation of the protein levels of exogenous FLAG-tagged cMyBP-C (FIG. 7.2 D, upper panels) and total cMyBP-C (lower panels). Ventricular protein extracts from each group were pooled for the analysis. Blots were stained with antibodies directed against the FLAG epitope or total cMyBP-C (upper part in each condition). An antibody directed against GAPDH was used as loading control (lower parts in each condition). As for the Mybpc3 mRNA, this shows that the protein level of cMyBP-C (both exogenous alone and total) increased in a AAV9-Mybpc3 dose-dependent manner.

[0118] cMyBP-C protein level was quantified, normalized to GAPDH and related to WT (FIG. 7.2 E). Compared to WT, the marked reduction in cMyBP-C protein level was significantly prevented by Mybpc3 gene therapy in KI mice.

[0119] Taken together, these data showed that long-term Mybpc3 gene therapy not only restored the level of Mybpc3 WT in KI mice but also prevented the transcription of mutant Mybpc3 mRNAs. Both partially significantly prevented the development of left ventricular hypertrophy and diastolic dysfunction, which are the key features of hypertrophic cardiomyopathy.