TW202421788A - Treatment of arrhythmogenic cardiomyopathy with aav gene therapy vectors - Google Patents

Abstract

Provided herein are gene therapy compositions and methods of treating loss of cardiomyocyte alignment due to reduced levels of functional plakophilin-2 protein in a subject having arrhythmogenic cardiomyopathy. Also provided herein are gene therapy vector components and methods to be used in gene therapy for improving cardiac expression of gene therapy products.

Description

AAV gene therapy vectors for the treatment of arrhythmogenic cardiomyopathy

Provided herein are recombinant adeno-associated virus (rAAV) gene therapy vectors and viral particles that can be used to treat and prevent arrhythmogenic cardiomyopathy by increasing the expression of plakophilin-2 (PKP2) and restoring skeletal function in ACM cardiomyocytes. Also provided herein are gene therapy vector components and methods for use in gene therapy that are used to improve gene therapy product expression and increase cellular uptake or delivery of therapeutic proteins provided by gene therapy vectors.

Although considerable progress has been made in preventing environmentally-induced heart disease, there is still a need for improved treatments for genetic cardiomyopathies, including arrhythmogenic cardiomyopathy (ACM), hypertrophic cardiomyopathy (HCM), and dilated cardiomyopathy (DCM).

Arrhythmogenic cardiomyopathy (ACM) was originally described as arrhythmogenic right ventricular cardiomyopathy (ARVC), but is now recognized to include both left-dominant and biventricular forms. (Asimaki et al., Prog. Pediatr. Cardiol. 37: 3-7 (2014). ACM is a hereditary heart disease that affects approximately 1:5000 people and accounts for 17% of sudden cardiac deaths in people younger than 35 years of age. At least 50% of cases of ACM are familial and are primarily inherited in an autosomal dominant manner, but recessive forms have been identified in the general population (Maron et al., Circulation, 92: 785-9 (1995) and are the leading cause of sudden cardiac death in young people, especially athletes.

ACM is characterized by leaky cardiomyocyte alignment due to abnormal sphenotypes, leading to poor electrical conduction, arrhythmic storms, myocardial fat replacement, and ultimately heart failure. (Sen-Chowdhry et al., J. Cardiovasc. Electrophysiol. 16: 937-35 (2005). Hereditary ACM is a genetic disease known to be caused by mutations and/or defects in genes encoding sphenotypes such as desmoplakin encoded by DSP, plakoglobin encoded by PKP2, plakoglobin encoded by JUP, desmocolin, and/or desmoglein.

Plafyn-2 and other cardiac basal proteins, including plakoglobin and plakoplakin 2, form the major cell adhesion complex that links basal calcineurin to plakoplakin and the interferon system, providing structural and functional integrity to neighboring cells that is important for cell rigidity and cell signaling. (van Tintelen et al., Circulation , 113(13): 1650-8 (2006); (Chidgey et al., Biochim. Biophys. Acta. Biomembranes, 1778: 572-87 (2008). Heterozygous PKP2 mutations are the most common mutations in AMC, with a prevalence of 1:7000 to 1:18,000.

Despite the availability of implantable cardioverter defibrillators and antiarrhythmic drugs, the disease burden of ACM remains high and no curative treatment exists to date (Carrier et al., Cardiovasc. Res . 85:330-338 (2010); Schlossarek et al., J. Mol. Cell. Cardiol. 50:613-20 (2011). Gene-based or RNA-based therapies will be the only curative treatment for ACM.

In one aspect of the present invention, the embodiments described herein relate to vector constructs, recombinant replication-deficient AAV particles, cells, and pharmaceutical compositions for delivering PKP2 to individuals with ACM or individuals with deficiencies of functional cardiac spondin proteins such as plakoflavan-2 (e.g., PKP2 mutations), spondin (e.g., DSP mutations), or plakoglobin (e.g., JUP mutations). The embodiments described herein also relate to the use of such AAV particles or such vector constructs to deliver a gene encoding PKP2 to the myocardium of such individuals. Without being bound by theory of the invention, we demonstrate herein that PKP2 gene therapy upregulates endogenous PKP2 cross-linking partners, which is expected to slow or halt the progression of myocardial adipose fibrosis associated with several genetic forms of ACM.

Gene therapy vectors are suitable for treating or preventing ACM in a mammalian individual, preferably a human individual, in need of treatment. In some embodiments, the individual in need of treatment is an individual carrying a mutation in at least one or two genes encoding PKP2. In other embodiments, the individual in need of treatment is an individual carrying a mutation in the DSP gene or the JUP gene. After administration to the individual to be treated, the vector provides for expression of the encoded PKP2 protein in the individual, preferably in the myocardium of the individual.

In one aspect, the embodiments described herein provide a vector construct comprising a nucleic acid sequence encoding a functional PKP2 protein. In one or more embodiments, the functional PKP2 protein comprises an amino acid sequence that is at least 90%, 95% or 98% identical to the amino acid sequence of SEQ ID NO: 2 (human PKP2 protein) or SEQ ID NO: 130, or SEQ ID NO: 131, or SEQ ID NO: 132. In some embodiments, the functional PKP2 protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2 or 130, or 131 or 132. In exemplary embodiments, the nucleic acid sequence encoding a functional PKP2 is a wild-type sequence (of which SEQ ID NO: 1 is an example or an allele variant), or is codon-optimized for human expression, or is a variant. Alternative codon optimized or variant human PKP2 coding sequences are shown in SEQ ID NOs: 69-73 and 129. In some embodiments, the nucleic acid sequence encoding a functional PKP2 is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1 or 129.

The protein to be expressed may also be a functional variant that exhibits a substantial amino acid sequence identity (i.e., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) to SEQ ID NO: 2 or 130 or 131 or 132. In this context, the term "functional variant" means that the variant of the PKP2 protein is able to perform the function of the naturally occurring PKP2 protein, such as providing structural and/or functional support to skeletons to restore normal electrical conduction in the heart, and optionally, is able to increase the expression of other skeletal proteins, such as skeletoplakin or plakoglobin.

Functional variants of PKP2 proteins may include proteins that differ from their naturally occurring counterparts, for example, by substitution, deletion or addition of one or more amino acids. For example, the variant protein of the human PKP2 protein of SEQ ID NO: 2 may have an amino acid sequence in which at least 2, 3, 4, 5, 6, 10 or more and/or at most 10, 20, 30 or more positions are substituted with another amino acid relative to SEQ ID NO: 2. As another example, the variant protein of the human PKP2 protein of SEQ ID NO: 2 may be a truncated version of a functional human PKP2, or may be SEQ ID NO: 130, or SEQ ID NO: 131, or SEQ ID NO: 132.

In one or more embodiments, the nucleic acid sequence encoding PKP2 is operably linked to one or more heterologous expression control elements. Preferably, the expression of the transgenic gene encoding PKP2 is controlled by at least one cardiomyocyte-specific expression control element. Therefore, in such embodiments, in the vector constructs described herein, the nucleic acid sequence encoding PKP2 is operably linked to a heterologous cardiomyocyte-specific transcriptional regulatory region. In some embodiments, in the vector constructs described herein, the expression control elements include one or more of the following: a promoter and/or enhancer; introns, if present; exons, if present; and a polyadenylation (polyA) signal. Such elements will be further described herein.

The cardiomyocyte-specific transcriptional regulatory region may comprise one or more cardiomyocyte-specific expression control elements, such as a cardiomyocyte-specific promoter. Preferably, the cardiomyocyte-specific promoter comprises at least one fragment or variant of the human cardiac calcified protein T (hTNNT2) promoter.

In some embodiments, the cardiomyocyte-specific promoter comprises a nucleic acid sequence that is at least or more than 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 85 (over the entire length of SEQ ID NO: 85). In some embodiments, the cardiomyocyte-specific promoter can be combined with an intron that enhances the expression of PKP2 protein, located 5' of the PKP2 coding sequence. For example, the vector construct and/or AAV particle comprises a cardiomyocyte-specific promoter, an intron that enhances the expression of PKP2 protein, and a nucleotide sequence encoding the PKP2 coding sequence in the 5' to 3' orientation. In some embodiments, the vector construct and/or AAV particle comprises (a) a cardiomyocyte-specific promoter comprising a nucleotide sequence at least 80% identical to any one of (i) SEQ ID NO: 85, (ii) SEQ ID NO: 86, (iii) SEQ ID NO: 87, (iv) SEQ ID NO: 88, (v) SEQ ID NO: 81, (vi) SEQ ID NO: 89, or (vii) SEQ ID NO: 82; and (b) an intron comprising a nucleotide sequence at least 60%, 70%, or 80% identical to SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID NO: 75.

In some embodiments, the intron sequence is located 5' of the PKP2 coding sequence. Preferably, the intron sequence is located within the nucleotide sequence encoding PKP2, such as between any exons, such as between exons 1 and 2. In some embodiments, the intron is located after position 261 of SEQ ID NO: 1 or SEQ ID NO: 129.

In some embodiments, the vector construct and/or the resulting AAV particle comprises a cardiomyocyte-specific promoter sequence that is a fragment or variant of the hTNNT2 promoter that is greater than 420 and less than 544 nucleotides in length and comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 85.

For example, the sequence of the cardiomyocyte-specific promoter comprises a nucleic acid sequence that is at least or more than 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of (i) SEQ ID NO: 85 or a fragment thereof, (ii) SEQ ID NO: 86 or a fragment thereof, (iii) SEQ ID NO: 87 or a fragment thereof, (iv) SEQ ID NO: 88 or a fragment thereof, (v) SEQ ID NO: 81 or a fragment thereof, (vi) SEQ ID NO: 89 or a fragment thereof, or (vii) SEQ ID NO: 82. In an exemplary embodiment, the sequence of the cardiomyocyte-specific promoter comprises a nucleotide sequence that is at least 96%, 97%, 98% or 99% identical to SEQ ID NO: 82. In some exemplary embodiments, the sequence of the hTNNT2 promoter comprises at least nucleotides 1-106 and 507-532 of SEQ ID NO: 81, or at least nucleotides 507-532 of SEQ ID NO: 81, or at least nucleotides 521-532 of SEQ ID NO: 81.

In some embodiments, the vector construct comprises one or more introns, which enhance the expression of PKP2 encoding nucleic acid, such as to detect increased expression in myocardium or heart. In some embodiments, the intron comprises a globin intron and/or a fragment or variant thereof, or a chimeric intron and/or a fragment or variant thereof. In one or more embodiments, the intron comprises a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% consistent with SEQ ID NO: 77. In one or more embodiments, the intron comprises a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% consistent with SEQ ID NO: 88. In one or more embodiments, the intron comprises a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 76.

In some embodiments, the vector construct may further comprise an exon sequence or a fragment thereof, preferably adjacent to an intron sequence, such as a globin intron adjacent to the 3' end of a fragment of beta globin exon 3 (SEQ ID NO: 76). The cardiomyocyte-specific transcriptional regulatory region may comprise a combination of intron and exon fragments, such as SEQ ID NO: 78.

In some embodiments, the vector construct comprises a polyadenylation signal, optionally a bovine growth hormone (bGH) polyadenylation signal (e.g., SEQ ID NO: 90, 91, 92, 93, 94, 95, or 96) or a fragment thereof, optionally a human growth hormone (hGH) polyadenylation signal (e.g., SEQ ID NO: 97) or a fragment thereof, optionally a SV40 polyadenylation signal (e.g., SEQ ID NO: 98) or a fragment thereof, optionally a Proudfoot synthetic polyadenylation signal (e.g., SEQ ID NO: 99) or a fragment thereof, or optionally a rabbit β-globulin polyadenylate (e.g., SEQ ID NO: 100). In some embodiments, the polyadenylation signal comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 90, for example, comprising SEQ ID NO: 91 or a fragment thereof, SEQ ID NO: 92 or a fragment thereof, SEQ ID NO: 93 or a fragment thereof, or SEQ ID NO: 94 or a fragment thereof. In exemplary embodiments, the polyadenylation signal is a fragment of SEQ ID NO: 97, having a length of about 100 to about 500 nucleotides, or a length of about 150 to about 400 nucleotides, or a length of about 200 to about 300 nucleotides, or a length of about 200 to about 250 nucleotides, comprising SEQ ID NO. 90.

In some embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98%, or 99% identical to any one of SEQ ID NOs: 3-68 or 126-128. In other embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98%, or 99% identical to the following nucleotide sequence: the nucleotide sequence is complementary to or negative (-) stranded with any one of SEQ ID NOs: 3-68 or 126-128.

In some embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98% or 99% identical to any one of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66 or 126. In other embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98% or 99% identical to any one of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66 or 126.

In some embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98% or 99% identical to any one of SEQ ID NO: 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67 or 127. In other embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98% or 99% identical to any one of SEQ ID NO: 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67 or 127.

In some embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98% or 99% identical to any one of SEQ ID NO: 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68 or 128. In other embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98% or 99% identical to any one of 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68 or 128.

Exemplary embodiments include the following: Construct A is 3691 bp in length (SEQ ID NO: 5) and comprises the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), hTNNT2 promoter (533 bp) (SEQ ID NO: 82), wild-type human PKP2 (hPKP2 or HuPKP2) (SEQ ID NO: 69) with a globin intron (131 bp) (SEQ ID NO: 77) between exons, bovine poly A (223 bp) (SEQ ID NO: 90), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct A optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complementary sequences) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complementary sequences), or fragments thereof. Construct B was 3691 bp in length (SEQ ID NO: 8) and comprised the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), hTNNT2 promoter (533 bp) (SEQ ID NO: 82), wild-type hPKP2 (SEQ ID NO: 69) with a globin intron (131 bp) (SEQ ID NO: 77) between exons, bovine poly A (223 bp) (SEQ ID NO: 90), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct B optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complementary sequences) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complementary sequences), or fragments thereof. Construct C was 3565 bp in length (SEQ ID NO: 11) and comprised the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), hTNNT2 promoter (533 bp) (SEQ ID NO: 82), wild-type hPKP2 (SEQ ID NO: 69) with a globin intron (131 bp) (SEQ ID NO: 77) between exons, bovine poly A (223 bp) (SEQ ID NO: 90), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct C optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complementary sequences) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complementary sequences), or fragments thereof. Construct D was 3565 bp in length (SEQ ID NO: 14) and comprised the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), hTNNT2 promoter (533 bp) (SEQ ID NO: 82), wild-type mouse (MsPKP2) (SEQ ID NO: 70) with a globin intron (131 bp) (SEQ ID NO: 77) between exons, bovine poly A (227 bp) (SEQ ID NO: 92), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct D optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complementary sequences) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complementary sequences), or fragments thereof. Construct E was 3651 bp in length (SEQ ID NO: 17) and comprised the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), cTNNT2 or cTNT promoter (421 bp) (SEQ ID NO: 83), globin intron (131 bp) (SEQ ID NO: 77), HBB exon 3 (SEQ ID NO: 76), wild-type hPKP2 (SEQ ID NO: 69), bovine poly A (227x bp) (SEQ ID NO: 93), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct E optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complementary sequences) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complementary sequences), or fragments thereof. Construct F was 4782 bp in length (SEQ ID NO: 20) and comprised the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), endogenous promoter (1417 bp) (SEQ ID NO: 84), globin intron (167 bp) (SEQ ID NO: 75), HBB exon 3 (SEQ ID NO: 76), wild-type hPKP2 with globin intron (131 bp) (SEQ ID NO: 77) between exons (SEQ ID NO: 69), bovine poly A (229 bp) (SEQ ID NO: 94), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct F optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complementary sequences) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complementary sequences), or fragments thereof. Construct G was 4651 bp in length (SEQ ID NO: 23) and comprised the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), endogenous promoter (1417 bp) (SEQ ID NO: 84), globin intron (131 bp) (SEQ ID NO: 77), HBB exon 3 (SEQ ID NO: 76), wild-type hPKP2 (SEQ ID NO: 69), bovine poly A (229 bp) (SEQ ID NO: 94), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct G optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complementary sequences) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complementary sequences), or fragments thereof. Construct H was 4596 bp in length (SEQ ID NO: 26) and comprised the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), endogenous promoter (1417 bp) (SEQ ID NO: 84), wild-type hPKP2 (SEQ ID NO: 69) with a globin intron (131 bp) (SEQ ID NO: 77) between exons, bovine poly A (229 bp) (SEQ ID NO: 94), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct H optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or its complementary sequence) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or its complementary sequence), or a fragment thereof. Construct I is 4465 bp in length (SEQ ID NO: 29) and comprises the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), endogenous promoter (1417 bp) (SEQ ID NO: 84), wild-type hPKP2 (SEQ ID NO: 69), bovine poly A (229 bp) (SEQ ID NO: 94), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct I optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complementary sequences) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complementary sequences), or fragments thereof. Construct J was 4596 bp in length (SEQ ID NO: 32) and comprised the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), endogenous promoter (1417 bp) (SEQ ID NO: 84), codon-optimized hPKP2 (SEQ ID NO: 71) with a globin intron (131 bp) (SEQ ID NO: 77) between exons, bovine poly A (229 bp) (SEQ ID NO: 94), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct J comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complements) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complements), or fragments thereof, as appropriate. Construct K is 4465 bp in length (SEQ ID NO: 35) and comprises the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), endogenous promoter (1417 bp) (SEQ ID NO: 84), codon-optimized hPKP2 (SEQ ID NO: 71), bovine poly A (229 bp) (SEQ ID NO: 94), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct K optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complementary sequences) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complementary sequences), or fragments thereof. Construct L was 4596 bp in length (SEQ ID NO: 38) and comprised the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), endogenous promoter (1417 bp) (SEQ ID NO: 84), codon-optimized hPKP2 (SEQ ID NO: 72) with a globin intron (131 bp) (SEQ ID NO: 77) between exons, bovine poly A (229 bp) (SEQ ID NO: 94), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct L optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or its complementary sequence) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or its complementary sequence), or a fragment thereof. Construct M is 4465 bp in length (SEQ ID NO: 41) and comprises the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), endogenous promoter (1417 bp) (SEQ ID NO: 84), codon-optimized hPKP2 (SEQ ID NO: 72), bovine poly A (229 bp) (SEQ ID NO: 94), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct M optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complementary sequences) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complementary sequences), or fragments thereof. Construct N was 4596 bp in length (SEQ ID NO: 44) and comprised the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), endogenous promoter (1417 bp) (SEQ ID NO: 84), codon-optimized hPKP2 (SEQ ID NO: 73) with a globin intron (131 bp) (SEQ ID NO: 77) between exons, bovine poly A (229 bp) (SEQ ID NO: 94), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct N optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or its complementary sequence) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or its complementary sequence), or a fragment thereof. Construct O is 4465 bp in length (SEQ ID NO: 47) and comprises the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), endogenous promoter (1417 bp) (SEQ ID NO: 84), codon-optimized hPKP2 (SEQ ID NO: 73), bovine poly A (229 bp) (SEQ ID NO: 94), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct O optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complementary sequences) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complementary sequences), or fragments thereof. Construct P was 3896 bp in length (SEQ ID NO: 50) and comprised the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), hTNNT2 promoter (533 bp) (SEQ ID NO: 82), globin intron (131 bp) (SEQ ID NO: 77), HBB exon 3 (SEQ ID NO: 76), wild-type hPKP2 with globin intron (131 bp) (SEQ ID NO: 77) between exons (SEQ ID NO: 69), bovine poly A (229 bp) (SEQ ID NO: 94), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct P optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complementary sequences) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complementary sequences), or fragments thereof. Construct Q was 3869 bp in length (SEQ ID NO: 53) and comprised the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), hTNNT2 promoter (533 bp) (SEQ ID NO: 82), globin intron (131 bp) (SEQ ID NO: 77), HBB exon 3 (SEQ ID NO: 76), codon-optimized hPKP2 (SEQ ID NO: 71) with globin intron (131 bp) (SEQ ID NO: 77) between exons, bovine poly A (229 bp) (SEQ ID NO: 94), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct Q optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complementary sequences) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complementary sequences), or fragments thereof. Construct R was 3765 bp in length (SEQ ID NO: 56) and comprised the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), hTNNT2 promoter (533 bp) (SEQ ID NO: 82), globin intron (131 bp) (SEQ ID NO: 77), HBB exon 3 (SEQ ID NO: 76), codon-optimized hPKP2 (SEQ ID NO: 71), bovine poly A (229 bp) (SEQ ID NO: 94), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct R optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complementary sequences) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complementary sequences), or fragments thereof. Construct S was 3896 bp in length (SEQ ID NO: 59) and comprised the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), hTNNT2 promoter (533 bp) (SEQ ID NO: 82), globin intron (131 bp) (SEQ ID NO: 77), HBB exon 3 (SEQ ID NO: 76), codon-optimized hPKP2 with globin intron (131 bp) (SEQ ID NO: 77) between exons (SEQ ID NO: 72), bovine poly A (229 bp) (SEQ ID NO: 94), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct S optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complementary sequences) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complementary sequences), or fragments thereof. Construct T was 3765 bp in length (SEQ ID NO: 62) and comprised the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), hTNNT2 promoter (533 bp) (SEQ ID NO: 82), globin intron (131 bp) (SEQ ID NO: 77), HBB exon 3 (SEQ ID NO: 76), codon-optimized hPKP2 (SEQ ID NO: 72), bovine poly A (229 bp) (SEQ ID NO: 94), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct T optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complementary sequences) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complementary sequences), or fragments thereof. Construct U was 3896 bp in length (SEQ ID NO: 65) and comprised the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), hTNNT2 promoter (533 bp) (SEQ ID NO: 82), globin intron (131 bp) (SEQ ID NO: 77), HBB exon 3 (SEQ ID NO: 76), codon-optimized hPKP2 with globin intron (131 bp) (SEQ ID NO: 77) between exons (SEQ ID NO: 73), bovine poly A (229 bp) (SEQ ID NO: 94), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct U optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or its complementary sequence) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or its complementary sequence), or a fragment thereof; and construct V is 3765 bp in length (SEQ ID NO: 68) and comprises the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), hTNNT2 promoter (533 bp) (SEQ ID NO: 81), globin intron (131 bp) (SEQ ID NO: 77), HBB exon 3 (SEQ ID NO: 76), codon-optimized hPKP2 (SEQ ID NO: 73), bovine polyA (229 bp) (SEQ ID NO: In other embodiments, construct V optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complementary sequences) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complementary sequences), or fragments thereof. Construct W was 3691 bp in length (SEQ ID NO: 128) and comprised the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), hTNNT2 promoter (533 bp) (SEQ ID NO: 82), wild-type hPKP2 with a globin intron (131 bp) (SEQ ID NO: 77) between exons (SEQ ID NO: 129), bovine polyA (223 bp) (SEQ ID NO: 90), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct W optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complementary sequences) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complementary sequences), or fragments thereof.

In any of the foregoing embodiments, the vector construct comprises at least one ITR sequence. Exemplary ITR sequences include but are not limited to SEQ ID NOs: 67-74, including any complementary sequences and/or combinations thereof.

In any of the foregoing embodiments, the length of the vector insert starting with one ITR and ending with the second ITR is between about 3.0 kb and about 5.5 kb. In one or more embodiments, the vector construct is an AAV vector genome, with a size of about 3.3 kb to about 5.0 kb, a size of about 3.5 kb to about 4.7 kb.

The vector construct is preferably a recombinant AAV vector construct. In some embodiments, the vector construct comprises (a) one or both of (i) AAV 5' inverted terminal repeat sequence (ITR) and (ii) AAV 3' ITR; (b) a promoter and/or enhancer, such as a cardiomyocyte-specific transcriptional regulatory region; and (c) a nucleic acid sequence encoding a functionally active human PKP2 protein. In some embodiments, the vector construct comprises (a) AAV 5' inverted terminal repeat (ITR) sequence; (b) a promoter and/or enhancer, such as a cardiomyocyte-specific transcriptional regulatory region; (c) a nucleic acid sequence encoding a functionally active human PKP2 protein; (d) an intron; (e) a polyadenylation signal; and (f) AAV 3' ITR. In some embodiments, the intron is downstream of the promoter and is located 5' of the PKP2 coding sequence, while in other embodiments, the intron is located between exons of the PKP2 coding sequence, such as between exon 1 and exon 2. In other embodiments, the vector construct comprises (a) an AAV 5' inverted terminal repeat (ITR) sequence; (b) a promoter and/or enhancer, such as a cardiomyocyte-specific transcriptional regulatory region; (c) a nucleic acid sequence encoding a functionally active human PKP2 protein; (d) introns; (e) and exons; (f) a polyadenylation signal; and (g) an AAV 3' ITR. The AAV 5' ITR and/or the AAV 3' ITR may be derived from a heterologous AAV pseudotype (which may or may not be modified as known in the art). In some embodiments, the 5' ITR and 3' ITR sequences are derived from AAV2 (e.g., SEQ ID NOs: 101-104 and 105-108, respectively).

In any of the aforementioned embodiments, the vector construct comprises a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to any one of SEQ ID NOs: 3-68 or 126-128 over the length of SEQ ID NOs: 3-68 or 126-128, respectively. In some embodiments, the vector construct is at least 95% identical to any one of SEQ ID NOs: 3-68 or 126-128 over the length of SEQ ID NOs: 3-68 or 126-128, respectively. In some embodiments, the vector construct is at least 96% identical to any one of SEQ ID NOs: 3-68 or 126-128 over the length of SEQ ID NOs: 3-68 or 126-128, respectively. In some embodiments, the vector construct is at least 97%, 98%, or 99% identical to any one of SEQ ID NOs: 3-68 or 126-128 over the length of SEQ ID NOs: 3-68 or 126-128, respectively. For example, such vectors preferably comprise flanking ITRs, a nucleic acid sequence encoding a functionally active human PKP2 protein coding sequence, a cardiomyocyte-specific regulatory region described herein, an expression enhancing intron, and a polyA signal.

In another aspect, a recombinant adeno-associated virus (rAAV) particle is provided herein, comprising an AAV capsid and a vector construct described in one or more of the embodiments herein. Any AAV capsid can be used, such as AAV1-13. In some embodiments, the recombinant AAV (rAAV) particle used to deliver the PKP2 encoding gene has a cardiotropism. In such embodiments, the rAAV comprises an AAV capsid having a cardiotropism or a muscle tropism, such as an AAV9-type capsid that is at least 85%, 90%, or 95% identical to SEQ ID NO: 109, or an AAV1-type, AAV6-type, or AAV7-type capsid that exhibits cardiotropism or, in some embodiments, exhibits muscle tropism, or a variant of any of these. In one or more embodiments, the AAV capsid is one that has reduced pre-existing humoral immunity compared to AAV9, e.g., when assessed by in vitro IVIG neutralization.

In another aspect, provided herein is a method for producing AAV particles that can be used as gene delivery vectors, the method comprising the following steps: (1) providing a cell (e.g., a mammalian cell) with one or more nucleic acid constructs, which comprises (a) a vector construct described herein, the vector construct comprising a nucleic acid encoding PKP2 described herein, the nucleic acid flanked by two AAV ITR nucleotide sequences; (b) encoding one or more AAV (c) a nucleotide sequence encoding one or more AAV capsid proteins, which are operably linked to a promoter capable of driving the expression of the capsid proteins; and (d) optionally, genes encoding AAP and MAAP contained in VP2/3; (2) culturing the cells defined in (1) under conditions conducive to the expression of the Rep proteins and the capsid proteins; and optionally (3) recovering the AAV particles. In some embodiments, the cells are mammalian cells. In some embodiments, the mammalian cells are HEK293 cells. Also provided herein are rAAV particle populations produced by such methods.

In another aspect, provided herein is a pharmaceutical composition comprising a vector construct described herein or a rAAV particle or rAAV particle population described herein, and a sterile pharmaceutically acceptable diluent, excipient or carrier.

In another aspect, the present invention provides a method for delivering a PKP2 gene to a mammalian individual. Such methods include methods for expressing PKP2 in a mammalian individual, which comprises administering to the individual a composition comprising a vector construct described herein, a rAAV particle described herein, or a pharmaceutical composition described herein, thereby expressing the encoded PKP2 protein in the individual. Preferably, in such methods, the mammal is a human and the PKP2 protein is a functional human PKP2 protein described herein. Such methods include a method for expressing a PKP2 protein in the myocardial cells of a mammal, by administering an amount of a vector construct, a rAAV particle, or a pharmaceutical composition that effectively increases the amount of expression of the PKP2 protein in the myocardium of the mammal. Such methods also include a method of increasing the level of functional PKP2 protein in the heart tissue (e.g., myocardial cells) of a mammal by administering an amount of a vector construct, rAAV particle, or pharmaceutical composition effective to increase the level of functional PKP2 protein in the heart tissue (e.g., myocardial cells) of the mammal. Such methods also include a method of treating a functional wild-type PKP2 protein deficiency in a mammal by administering an amount of a vector construct, rAAV particle, or pharmaceutical composition effective to increase the level of functional PKP2 protein in the heart tissue (e.g., myocardial cells) of the mammal. In some embodiments, the amount of the vector construct, rAAV particle, or pharmaceutical composition is effective to increase the level of PKP2 protein in cardiac tissue (e.g., cardiomyocytes) by at least about 2-fold; and/or improve electrical conduction in cardiac tissue, improve cardiac contraction, and/or reduce the frequency of arrhythmias, in vitro in engineered heart tissue or in vivo in animal tissue.

Such methods also include a method of treating ACM in a mammal, or treating or preventing any symptom thereof, comprising administering a therapeutically effective amount of a vector construct, rAAV particle, or pharmaceutical composition. In one or more embodiments, such methods increase the amount of PKP2 expression in the heart by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% compared to the amount when not treated, or to the amount seen in healthy humans. For example, such methods reduce or eliminate the number of ICD shocks required over a period of, for example, 6 months, reduce or eliminate myocardial fibrosis or adipose fibrosis, reduce or eliminate ventricular wall thinning, reduce or eliminate dilated cardiomyopathy (DCM), reduce or eliminate sudden cardiac death, reduce or eliminate heart failure, arrhythmia, chest pain, shortness of breath, fatigue and/or dizziness, and/or reduce other symptoms of ACM disease. Such methods may also increase the expression of desmosome complex proteins, including nexin plakoglobin, desmoplakin, desmoglobin-2, nexin 43, and N-calcified mucin.

In any of the methods described herein, the rAAV particles are delivered in an aqueous suspension at a dose of about 1e12 to 6e14 vg/kg. In any of the methods described herein, administration of the vector construct, rAAV particles, or pharmaceutical composition may further comprise administration of prophylactic or therapeutic corticosteroid treatment, and/or may further comprise administration of a secondary therapy for treating ACM. In any of the methods herein, prior to administering AAV particles to a patient as described above, the prospective patient may be assessed for the presence of anti-AAV capsid antibodies or anti-AAV neutralizing antibodies that are capable of blocking cell transduction or otherwise reducing the overall efficiency of the treatment.

In another object of the present invention, the embodiments described herein relate to compositions comprising novel cardiomyocyte-specific promoter sequences, specifically novel hTNNT promoter sequences, and methods of using such compositions to deliver genes of interest encoding or expressing gene therapy products to individuals. Provided herein is a transgene comprising (a) a gene of interest capable of expressing a gene therapy product, (b) a cardiac-specific or more specifically cardiomyocyte-specific promoter operably linked to the gene of interest, (c) optionally an expression enhancing intron, and (d) optionally a polyadenylation signal. The compositions described herein include vector constructs (e.g., plasmids), donor constructs and viral vectors, or viral particles or cells, or pharmaceutical compositions comprising such transgenes. The compositions are suitable for expressing gene therapy products in an individual. Such methods include methods for delivering a gene of interest or delivering a gene therapy product to an individual in need, preferably to the individual's cardiac tissue and/or cardiac myocytes. Such methods also include delivering a gene therapy product (or expressing a gene therapy product) in an individual in need to treat a disease or condition that is improved by the gene therapy product, preferably a cardiac disease or condition or a cardiac genetic disease or condition. Compositions and methods include enhancing the expression of a gene of interest in the heart of such an individual, and more particularly, in cardiomyocytes. Gene therapy may include delivering a peptide, protein, or RNA. In some embodiments, the individual carries a mutation in at least one gene encoding a gene therapy product.

Gene therapy may include gene silencing, such as administration of a gene of interest expressing siRNA, shRNA, miRNA, or ribozyme. Gene therapy may also include delivery of a gene editing system, such as to inactivate a disease-causing gene by disruption or deletion, or to integrate a transgene from a donor construct. Targeted gene editing systems are known in the art and include zinc finger nucleases (ZFNs) with guide RNAs, transcription activator-like effector nucleases (TALENs), meganucleases, or clustered regulatory interspaced short tandem palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)-associated nucleases (e.g., Cas9, Cpf1/Cas12a, or variants thereof). In some embodiments, the transgene encodes a gene editing system of interest.

Gene therapy may include gene replacement, in which a functional copy of a gene of interest is administered, for example to supplement or replace the activity of a mutant gene. In such cases, the individual suffers from a genetic disease or genetic condition caused by the mutant endogenous gene of interest. Gene therapy may also include delivering a gene of interest that is not related to the genetic mutation, thereby producing a gene therapy product that prevents or treats the disease. In some cases, the individual does not have the mutant endogenous gene of interest, or has a mutant endogenous gene that is different from the gene of interest.

In some embodiments, when the gene therapy product has a natural source, the gene therapy product may be wild-type or may be a functional variant of a wild-type gene therapy product, comprising an amino acid sequence (in the case of a protein gene therapy product) or a nucleotide sequence (in the case of an RNA gene therapy product) that exhibits considerable sequence identity (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity) compared to the original wild-type sequence (amino acid or nucleotide sequence). Preferably, the sequence identity of the variant is at least 70%, 80% or 90%. In some embodiments, when the gene therapy product has a natural source, the gene of interest may be a wild-type gene or a codon-optimized gene, or a functional variant thereof.

According to this object of the present invention, the transgene comprises a gene of interest operably linked to a cardiomyocyte-specific promoter described herein. Examples of such cardiomyocyte-specific control elements include any one of SEQ ID NOs: 133-263, or a nucleic acid sequence at least about 90% identical thereto. For example, the cardiomyocyte-specific promoter provides at least about 2 times higher, at least about 3 times higher, at least about 4 times higher, at least about 5 times higher, at least about 6 times higher, at least about 7 times higher, at least about 8 times higher, at least about 9 times higher , at least about 10 times higher, at least about 11 times higher, at least about 12 times higher, at least about 13 times higher, at least about 14 times higher, at least about 15 times higher, at least about 16 times higher, at least about 17 times higher, at least about 18 times higher, at least about 19 times higher, at least about 20 times higher, at least about 21 times higher, at least about 22 times higher, at least about 23 times higher, at least about 24 times higher, at least about 25 times higher, at least about 26 times higher, at least about 27 times higher, at least about 28 times higher, at least about 29 times higher, at least about 30 times higher, at least about 31 times higher, at least about 32 times higher, at least about 33 times higher, at least about 34 times higher, at least about 35 times higher, at least about 36 times higher, at least about 37 times higher, at least about 38 times higher, at least about 39 times higher, at least about 40 times higher, at least about 41 times higher, at least about 15 times higher, at least about 16 times higher, at least about 17 times higher, at least about 18 times higher, at least about 19 times higher, at least about 20 times higher, at least about 21 times higher, at least about 22 times higher, at least about 23 times higher, at least about 24 times higher, at least about 25 times higher, at least about 26 times higher, at least about 27 times higher, at least about 28 times higher, at least about 29 times higher, at least about 30 times higher, at least about 31 times higher, at least about 32 times higher about 32 times higher, at least about 33 times higher, at least about 34 times higher, at least about 35 times higher, at least about 36 times higher, at least about 37 times higher, at least about 38 times higher, at least about 39 times higher, at least about 40 times higher, at least about 41 times higher, at least about 42 times higher, at least about 43 times higher, at least about 44 times higher, at least about 45 times higher, at least about 46 times higher, at least about 47 times higher, at least about 48 times higher, at least about 49 times higher, at least about 50 times higher, at least about 51 times higher, at least about 52 times higher, at least about 53 times higher, at least about 54 times higher, at least about 55 times higher, at least about 56 times higher, at least about 57 times higher, at least about 58 times higher, at least about 59 times higher, at least about 60 times higher, at least about 61 times higher, at least about 62 times higher, at least about 63 times higher, at least about 64 times higher, at least about 65 times higher, up to about 66 times higher at least about 66 times higher, at least about 67 times higher, at least about 68 times higher, at least about 69 times higher, at least about 70 times higher, at least about 71 times higher, at least about 72 times higher, at least about 73 times higher, at least about 74 times higher, at least about 75 times higher, at least about 76 times higher, at least about 77 times higher, at least about 78 times higher, at least about 79 times higher, at least about 80 times higher, at least about 81 times higher, at least about 82 times higher, at least about 83 times higher, at least about 84 times higher, at least about 85 times higher, at least about 86 times higher, at least about 87 times higher, at least about 88 times higher, at least about 89 times higher, at least about 90 times higher, at least about 91 times higher, at least about 92 times higher, at least about 93 times higher, at least about 94 times higher, at least about 95 times higher, at least about 96 times higher, at least about 97 times higher, at least about 98 times higher, at least about 99 times higher, At least about 100 times higher, at least about 200 times higher, at least about 300 times higher, at least about 400 times higher, at least about 500 times higher, at least about 600 times higher, at least about 700 times higher, at least about 800 times higher, at least about 900 times higher, at least about 1000 times higher, at least about 1500 times higher, at least about 2000 times higher, at least about 2500 times higher, at least about 3000 times higher, at least about 3500 times higher, at least about 4000 times higher, at least about 4500 times higher, at least about 5000 times higher, at least about 5500 times higher, at least about 6000 times higher, at least about 6500 times higher, at least about 7000 times higher, at least about 7500 times higher, at least about 8000 times higher, at least about 8500 times higher, at least about 9000 times higher, at least about 9500 times higher, or at least about 10000 times higher.

In some embodiments, the heart-specific or cardiomyocyte-specific transcriptional regulatory region comprises a cardiomyocyte-specific promoter sequence comprising a nucleic acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 133-263. The cardiomyocyte-specific promoter sequence preferably comprises nucleotides 1-12 at the 3' end of SEQ ID NO: 264. The heart-specific or cardiomyocyte-specific transcriptional regulatory region optionally comprises a fragment or variant of another promoter, and/or one or more enhancer sequences.

In some embodiments, the transgene also comprises an intron, which is optionally an intron that enhances the cardiac expression of the gene of interest when combined with a cardiac-specific promoter described herein. The intron may be close to the cardiac-specific promoter, or may be located 5' or 3' to the transcription start site of the gene of interest. In some embodiments, the intron is located within the gene of interest. In some embodiments, the intron is located 5' or 3' to the start of the coding sequence of the gene of interest. In some embodiments, the intron is located in the 5' half of the coding sequence of the gene of interest. In some embodiments, the intron is located within 1000 bp of the start of the coding sequence of the gene of interest. In some embodiments, introns are located between any naturally occurring exons of the nucleotide sequence encoding the gene therapy product (gene of interest). In some embodiments, the intron sequence is located between the exons of the gene of interest: between the first and second exons or between the second and third exons. In some embodiments, the transgenic gene may further include an exon sequence or a fragment thereof that is preferably adjacent to the intron sequence. In some embodiments, the transgenic gene may further include an exon sequence or a fragment thereof that is adjacent to the intron sequence at the 5' or 3' end of the intron to facilitate splicing and removal of the intron. The cardiomyocyte-specific transcriptional regulatory region may include a combination of introns and exon fragments. In some embodiments, the length of the intron is 250 bp or less. In one or more embodiments, the intron comprises a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 265-275 or SEQ ID NO: 77. In some embodiments, the intron comprises SEQ ID NOs: 74-75, SEQ ID NOs: 265-275, SEQ ID NO: 265, SEQ ID NOs: 266-269 or 272-274, SEQ ID NO: 271, SEQ ID NO: 275, or an expression-enhancing fragment or variant thereof. The transgene may also comprise a polyadenylation signal, optionally a bovine growth hormone (bGH) polyA signal or a fragment thereof, or a human growth hormone (hGH) polyA signal or a fragment thereof.

Other embodiments will be apparent to those skilled in the art upon reading this specification.

Cross-references to related patent applications

This application claims priority to U.S. Provisional Patent Application No. 63/376,713 filed on September 22, 2022, U.S. Provisional Patent Application No. 63/376,715 filed on September 22, 2022, and U.S. Provisional Patent Application No. 63/581,820 filed on September 11, 2023, each of which is incorporated herein by reference in its entirety. Sequence Listing Incorporated by Reference

This application includes a sequence listing in computer-readable form as an independent part of the disclosure (file name: PCT_SeqListing.xml; 628,569 bytes, created on September 12, 2023). The contents of the sequence listing xml file are incorporated herein by reference in their entirety.

Provided herein are nucleic acids or vector constructs encoding functionally active therapeutic PKP2 proteins, AAV vector genomes and replication-defective rAAV particles comprising such vector constructs, and pharmaceutical compositions comprising such vector constructs, vector genomes and AAV particles. The compositions and methods of the present invention can provide improved AAV virus yields and/or simplified purification and/or enhanced expression of PKP2 proteins in heart, especially myocardial cells (cardiomyocytes). Also provided herein are methods for making vector constructs, AAV vector genomes and replication-defective rAAV particles comprising such vector constructs. Further provided herein are methods for treating functional wild-type PKP2 deficiency (including ACM).

In another embodiment, methods of producing recombinant adeno-associated virus (AAV) particles comprising any of the AAV vector constructs provided herein are provided. The methods comprise the steps of culturing cells transfected with any of the AAV vector constructs provided herein (associated with various AAV cap and rep genes) and recovering recombinant therapeutic AAV particles from the transfected cells or the supernatant of the transfected cell cultures.

Cells suitable for the production of recombinant AAV provided herein are any cell type susceptible to bacillivirus infection, including insect cells such as High Five, Sf9, Se301, SeIZD2109, SeUCR1, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAm1, BM-N, Ha2302, Hz2E5, and Ao38. In another embodiment, mammalian cells such as HEK293, HeLa, CHO, NSO, SP2/0, PER.C6, Vero, RD, BHK, HT 1080, A549, Cos-7, ARPE-19, and MRC-5 can be used.

In another embodiment, the present invention provides an effective amount of a vector nucleic acid, a vector construct, or an AAV particle for use in the preparation of a drug for treating an individual suffering from ACM or a functional wild-type PKP2 protein deficiency. In one embodiment, the individual suffering from ACM is a human. In one embodiment, the drug is administered by intravenous (IV) administration. In another embodiment, the administration of the drug increases the level of functional PKP2 in myocardial cells, thereby improving ACM symptoms. In certain embodiments, the drug is also used for co-administration with preventive and/or therapeutic corticosteroids to prevent and/or treat any toxicity associated with the administration of AAV particles. Prophylactic or therapeutic corticosteroid treatment may comprise at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more mg/day of corticosteroid. In certain embodiments, the prophylactic or therapeutic corticosteroid may be administered for a continuous period of at least about 3, 4, 5, 6, 7, 8, 9, 10 weeks or longer.

In another embodiment, the methods of treating ACM provided herein optionally further comprise administering, e.g., concurrently administering, other therapies for treating ACM.

Also provided herein are novel cardiomyocyte-specific promoters, as well as transgenes, vector constructs, and donor constructs for integration into genomes, viral vectors, or viral particles comprising such novel promoters. Further provided herein are uses of such compositions for delivery of a gene of interest for use in treating a disease or condition, preferably a cardiac disease or condition, or a cardiac genetic disease or condition, in an individual in need of the gene of interest. The viral vector may be integrated into the genome of a host cell, resulting in stable gene expression, or may be an episomal, non-integrating virus. DNA from a donor construct comprising a gene of interest operably linked to a novel cardiomyocyte-specific promoter described herein can also be integrated into the genome via gene editing methods such as ZFN, TALEN, meganuclease, or CRISPR-Cas9. Definition:

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd Edition, J. Wiley & Sons (New York, N.Y. 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, N.Y. 1989). For the purposes of the present invention, the following terms are defined as follows.

As used herein, in the context of gene delivery, the term "vector" or "gene delivery vector" may refer to a particle that serves as a gene delivery vehicle and comprises a nucleic acid (i.e., a vector genome comprising any of the vector constructs described herein) packaged in, for example, an envelope or capsid. The gene delivery vector may be a viral gene delivery vector or a non-viral gene delivery vector. Alternatively, in some cases, the term "vector" may be used to refer to only the vector genome or the vector construct. Viral vectors suitable for use herein may be a small virus, an adenovirus, a retrovirus, a lentivirus, or a herpes simplex virus. The small virus may be an adeno-associated virus (AAV).

As used herein, the term "AAV" is a standard abbreviation for adeno-associated virus. Adeno-associated viruses are small single-stranded DNA viruses that grow only in cells where a co-infecting helper virus provides certain functions. There are multiple AAV serotypes that have been characterized. General information and reviews of AAV can be found, for example, in Carter, Handbook of Parvoviruses, Vol. 1, pp. 169-228 (1989); and Berns, Virology, pp. 1743-64, Raven Press, (New York) (1990); Gao et al., Meth. Mol. Biol . 807: 93-118 (2011); Ojala et al., Mol. Ther. 26(1): 304-19 (2018). However, it is fully expected that these same principles will apply to additional AAV serotypes, since it is well known that the various serotypes are very closely related structurally and functionally, even at the genetic level. (See, e.g., Blacklowe, 1988, pp. 165-174, Parvoviruses and Human Disease, JR Pattison, ed.; and Rose, Comprehensive Virology 3:1-61 (1974)). For example, all AAV serotypes apparently exhibit very similar replication properties mediated by homologous rep genes; and all carry three related capsid proteins. The degree of relatedness was further confirmed by heterologous duplex analysis revealing extensive cross-hybridization between serotypes along the length of the genome and the presence of similar self-adhesive segments at the ends corresponding to "inverted terminal repeats" (ITRs).

As used herein, "AAV vector construct" refers to a single-stranded or double-stranded nucleic acid having at least one of (i) an AAV 5' inverted terminal repeat (ITR) sequence and (ii) an AAV 3' ITR, flanked by a protein coding sequence (in one embodiment, a functional therapeutic protein coding sequence, such as a PKP2 protein coding sequence), which is operably linked to transcriptional regulatory elements (also referred to as "expression control elements") that are heterologous to the protein coding sequence and/or heterologous to the AAV viral genome, i.e., one or more promoters and/or enhancers and, optionally, a polyadenylation sequence and/or, optionally, one or more introns. Single-stranded AAV vectors refer to nucleic acids present in the genome of AAV viral particles and can be the sense strand or antisense strand of the nucleic acid sequences disclosed herein. The sizes of such single-stranded nucleic acids are provided in bases. Double-stranded AAV vectors refer to nucleic acids present in the DNA of plasmids (e.g., pUC19) or in the genome of double-stranded viruses (e.g., bacilli) used to express or transfer AAV vector nucleic acids. The sizes of such double-stranded nucleic acids are provided in base pairs (bp).

The AAV vector constructs provided herein in single-stranded form are less than about 5.5 kb in length, or less than 5.4 kb in length, or less than 5.3 kb in length, or less than 5.2 kb in length. The length of the AAV vector constructs in single-stranded form is also at least about 3.0 kb. Preferably, the length of the AAV vector construct is about 3.5 kb to about 4.0 kb. In some embodiments, the length of the AAV vector constructs provided herein in single-stranded form is less than about 7.0 kb, or less than 6.5 kb, or less than 6.4 kb, or less than 6.3 kb, or less than 6.2 kb, or less than 6.0 kb, or less than 5.8 kb, or less than 5.6 kb, or less than 5.5 kb, or less than 5.4 kb, or less than 5.3 kb, or less than 5.2 kb. The length of the AAV vector constructs in single-stranded form is also at least about 4.0 kb. Preferably, the length of the AAV vector constructs is also at least about 4.5 kb. In some embodiments, the length of an AAV vector construct provided herein in single-stranded form ranges from about 4.0 kb to about 5.8 kb.

Although AAV particles have been reported in the literature with AAV genomes > 5.0 kb, in many of these cases the 5' or 3' end of the encoded gene appears to be truncated (see Hirsch et al., Molec. Ther. 18: 6-8 (2010) and Ghosh et al., Biotech. Genet. Engin. Rev. 24: 165-78 (2007). However, overlapping homologous recombination has been shown to occur between nucleic acids with 5' and 3' truncations in AAV-infected cells, thereby generating an "intact" nucleic acid encoding a large protein, thereby reconstructing a functional full-length gene.

The oversized AAV vector is randomly truncated at the 5' end and lacks the 5' AAV ITR. Because AAV is a single-stranded DNA virus and packages either the sense strand or the antisense strand, the sense strand in the oversized AAV vector lacks the 5' AAV ITR and possible portions of the 5' end of the target protein coding gene, and the antisense strand in the oversized AAV vector lacks the 3' ITR and possible portions of the 3' end of the target protein coding gene. Functional transgenes are produced in cells infected with the oversized AAV vector by ligation of the sense and antisense truncated genomes within the target cell. Therefore, in certain embodiments, the AAV PKP2 vector and/or viral particle comprises at least one ITR.

As used herein, the term "inverted terminal repeat sequence (ITR)" refers to a region recognized in the art found at the 5' and 3' ends of the AAV genome that functions in a cis-mode as an origin of DNA replication and a packaging signal for the viral genome. The AAV ITR, together with the AAV rep coding region, enables efficient excision and recovery of the nucleotide sequence inserted between the two flanking ITRs and integration of the nucleotide sequence into the host cell genome. The sequences of certain AAV-related ITRs are disclosed in Yan et al., J. Virol. 79: 364-79 (2005), which is incorporated herein by reference in its entirety. The ITR sequences that can be used herein may be full-length, wild-type AAV ITRs or fragments thereof that retain functional capabilities, or may be sequence variants of full-length, wild-type AAV ITRs that are capable of functioning in a cis-mode as an origin of replication. The AAV ITRs that can be used in the recombinant AAV PKP2 vectors of the embodiments provided herein can be derived from any known AAV serotype, and in certain embodiments, from the AAV9 serotype.

The term "control sequence" refers to DNA sequences required for expression of an operably linked coding sequence in a particular host organism. Suitable control sequences for prokaryotes include, for example, a promoter, an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

"Transcriptional regulatory element" refers to the nucleotide sequence of a gene involved in the regulation of gene transcription, including a promoter, plus response elements, activators and enhancer sequences, which are used to bind transcription factors to help RNA polymerase bind and promote expression; and operator or silencer sequences, inhibitory proteins bind to these sequences to block RNA polymerase binding and prevent expression. The term "cardiac myocyte-specific transcriptional regulatory element" or "cardiac myocyte-specific expression control element" refers to a regulatory element or region that specifically produces better gene expression in cardiomyocytes, such as a promoter whose activity in heart cells is at least 2 times or at least 5 times higher than in any other non-cardiac cell type. In some embodiments, the cardiomyocyte-specific promoter provides at least 5-fold higher expression in cardiomyocytes than in skeletal muscle cells. In some embodiments, the cardiomyocyte-specific promoter 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 more active in cardiomyocytes than in non-cardiac cell types.

The heart-specific or myocardial cell-specific promoter is operably linked to a nucleic acid sequence encoding the PKP2 protein, which means that the promoter is combined with the encoding nucleic acid so that when integrated into the genome of a cell or present as an extragenomic nucleic acid construct in a cell, the encoding nucleic acid can be expressed in myocardial cells under the control of the promoter.

Transcriptional regulatory elements may include enhancer elements, introns, polyadenylation sequences or post-transcriptional regulatory elements, which are used to increase the expression of myosin binding protein. Examples include the SV40 early gene enhancer and the enhancer of the long terminal repeat sequence (LTR) of Rous Sarcoma Virus (Gorman et al. (1982) Proc. Natl. Acad. Sci. 79:6777). The vector may also include transcriptional termination sequences and polyadenylation sequences, which are used to improve the expression of human and/or non-human antigens. Suitable transcriptional terminators and polyadenylation signals can be derived, for example, from SV40 (Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual). Preferably, the bGH polyadenylation signal is used in the vector of the present invention. Any other elements known in the art that support expression efficiency or specificity may be added to the expression vector, such as the woodchuck hepatitis posttranscriptional regulatory element (wPRE). To increase cardiac or myocardial cell specificity, other elements may be introduced to inactivate gene expression in other tissues, such as sequences encoding miRNAs such as miR122 (Geisler et al., Gene Ther. 18: 199-209 (2011)).

As used herein, "introns" are broadly defined as nucleotide sequences that can be removed by RNA splicing. "RNA splicing" means the excision of introns from pre-mRNA to form mature mRNA. Introns can be located upstream, downstream or within the coding region of a gene. Insertion of introns into a nucleotide sequence can be achieved by any method known in the art. The only limitation on the location of the insertion of introns is to consider the packaging limitations of AAV viral particles (e.g., about 5 kb).

As used herein, the term "operably linked" is used to describe the connection between a regulatory element and a gene or its coding region. Typically, gene expression is under the control of one or more regulatory elements, such as but not limited to constitutive or inducible promoters, tissue-specific regulatory elements, and enhancers. A gene or coding region is said to be "operably linked to" or "operably linked to" a regulatory element or "operably associated with" a regulatory element, meaning that the gene or coding region is controlled or affected by the regulatory element. For example, if a promoter affects the transcription or expression of a coding sequence, then the promoter is operably linked to the coding sequence.

In certain embodiments, the recombinant AAV vector construct comprises (a) a nucleic acid comprising the AAV9 5' inverted terminal repeat (ITR) (which may or may not be modified as known in the art), (b) a cardiomyocyte-specific transcriptional regulatory region, (c) a functional PKP2 protein coding region, (d) optionally one or more introns, an I polyadenylation sequence, and (f) the AAV9 3' ITR (which may or may not be modified as known in the art).

In one embodiment, the vector construct comprises a nucleic acid encoding a functionally active PKP2 protein. The PKP2 coding sequence may be wild type, codon optimized or a variant. In order to visualize the expression of the exogenous gene in the heart, other optional elements may be introduced as part of the PKP2 coding sequence, such as a tag sequence (myc, FLAG, HA, His and the like) or a fluorescent dye, such as GFP, YFP, RFP.

As used herein, the wild-type PKP2 protein (PKP2 gene) has the following nucleic acid sequence SEQ ID NO: 1 (Genbank accession number BC094762.1) atggcagcccccggcgccccagctgagtacggctacatccggaccgtcctgggccagcagatcctgggacaactggacagctccagcctggcgctgccctccgaggccaagctgaagctggcggggagcagcggccgcggcggccagacagtcaagagcctgcggatccaggagcaggtgcagcagaccctcgcccggaagggccgcagctccgtgggcaacggtaagtactagcagctacaatccagctaccattctgcttttattttatggttgggataaggctggattattctgagtccaagctaggcccttttgctaatcatgttc atacctcttatcttcctcccacaggaaatcttcaccgaaccagcagtgttcctgagtatgtctacaacctacacttggttgaaaatgattttgttggaggccgttcccctgttcctaaaacctatgacatgctaaaggctggcacaactgccacttatgaaggtcgctggggaagaggaacagcacagtacagctcccagaagtccgtggaagaaaggtccttgaggcatcctctgaggagactggagatttctcctgacagcagcccggagagggctcactacacgcacagcgattaccagtacagccagagaagccaggctgggcacac cctgcaccaccaagaaagcaggcgggccgccctcctagtgccaccgagatatgctcgttccgagatcgtgggggtcagccgtgctggcaccacaagcaggcagcgccactttgacacataccacagacagtaccagcatggctctgttagcgacaccgtttttgacagcatccctgccaacccggccctgctcacgtaccccaggccagggaccagccgcagcatgggcaacctcttggagaaggagaactacctgacggcagggctcactgtcgggcaggtcaggccgctggtgcccctgcagcccgtcactcagaacagggcttccagg tcctcctggcatcagagctccttccacagcacccgcacgctgagggaagctgggcccagtgtcgccgtggattccagcgggaggagagcgcacttgactgtcggccaggcggccgcagggggaagtgggaatctgctcactgagagaagcactttcactgactcccagctggggaatgcagacatggagatgactctggagcgagcagtgagtatgctcgaggcagaccacatgctgccatccaggatttctgctgcagctactttcatacagcacgagtgcttccagaaatctgaagctcggaagagggttaaccagcttcgtggcatcc tcaagcttctgcagctcctaaaagttcagaatgaagacgttcagcgagctgtgtgtgtggggccttgagaaacttagtatttgaagacaatgacaacaaattggaggtggctgaactaaatggggtacctcggctgctccaggtgctgaagcaaaccagagacttggagactaaaaaacaaataacaggtttgctgtggaatttgtcatctaatgacaaactcaagaatctcatgataacagaagcattgcttacgctgacggagaatatcatcatccccttttctgggtggcctgaaggagactacccaaaagcaaatggtttgctcgattt tgacatattctacaacgtcactggatgcctaagaaacatgagttctgctggcgctgatgggagaaaagcgatgagaagatgtgacggactcattgactcactggtccattatgtcagaggaaccattgcagattaccagccagatgacaaggccacggagaattgtgtgtgcattcttcataacctctcctaccagctggaggcagagctcccagagaaatattcccagaatatctatattcaaaaccggaatatccagactgacaacaaaagtattggatgtttttggcagtcgaagcaggaaagtaaaagagcaataccaggacgtg ccgatgccggaggaaaagagcaaccccaagggcgtggagtggctgtggcattccattgttataaggatgtatctgtccttgatcgccaaaagtgtccgcaactacacacaagaagcatccttaggagctctgcagaacctcacggccggaagtggaccaatgccgacatcagtggctcagacagttgtccagaaggaaagtggcctgcagcacacccgaaagatgctgcatgttggtgacccaagtgtgaaaaagacagccatctcgctgctgaggaatctgtcccggaatctttctctgcagaatgaaattgccaaagaaactctccctg atttggtttccatcattcctgacacagtcccgagtactgaccttctcattgaaactacagcctctgcctgttacacattgaacaacataatccaaaacagttaccagaatgcacgcgaccttctaaacaccgggggcatccagaaaattatggccattagtgcaggcgatgcctatgcctccaacaaagcaagtaaagctgcttccgtccttctgtattctctgtgggcacacacggaactgcatcatgcctacaagaaggctcagtttaagaagacagattttgtcaacagccggactgccaaagcctaccactcccttaaagactga.

As used herein, the wild-type PKP2 protein has the following amino acid sequence SEQ ID NO: 2 (Genbank accession number AAH94762.1) MAAPGAPAEYGYIRTVLGQQILGQLDSSSLALPSEAKLKLAGSSGRGGQTVKSLRIQEQVQQTLARKGRSSVGNGNLHRTSSVPEYVYNLHLVENDFVGGRSPVPKTYDMLKAGTTATYEGRWGRGTAQYSSQKSVEERSLRHPLRRLEISPDSSPERAHYTHSDYQYSQRSQAGHTLHHQESRRAALLVPPRYARSEIVGVSRAGTTS RQRHFDTYHRQYQHGSVSDTVFDSIPANPALLTYPRPGTSRSMGNLLEKENYLTAGLTVGQVRPLVPLQPVTQNRASRSSWHQSSFHSTRTLREAGPSVAVDSSGRRAHLTVGQAAAGGSGNLLTERSTFTDSQLGNADMEMTLERAVSMLEADHMPPSRISAAATFIQHECFQKSEARKRVNQLRGILKLLQLLKVQNEDVQRAVCGAL RNLVFEDNDNKLEVAELNGVPRLLQVLKQTRDLETKKQITGLLWNLSSNDKLKNLMITEALLTLTENIIIPFSGWPEGDYPKANGLLDFDIFYNVTGCLRNMSSAGADGRKAMRRCDGLIDSLVHYVRGTIADYQPDDKATENCVCILHNLSYQLEAELPEKYSQNIYIQNRNIQTDNNKSIGCFGSRSRKVKEQYQDVPMPEEKSNPKG VEWLWHSIVIRMYLSLIAKSVRNYTQEASLGALQNLTAGSGPMPTSVAQTVVQKESGLQHTRKMLHVGDPSVKKTAISLLRNLSRNLSLQNEIAKETLPDLVSIIPDTVPSTDLLIETTASACYTLNNIIQNSYQNARDLLNTGGIQKIMAISAGDAYASNKASKAASVLLYSLWAHTELHHAYKKAQFKKTDFVNSRTAKAYHSLKD.

The term "isolated" when used in relation to the nucleic acid molecules of the present invention generally refers to a nucleic acid sequence that has been identified and separated from at least one contaminating nucleic acid normally associated with its natural source. An isolated nucleic acid may exist in a form or environment different from that found in nature. Thus, an isolated nucleic acid molecule is distinct from a nucleic acid molecule that exists in a natural cell.

As used herein, the term "variant" refers to a polynucleotide (or polypeptide) having a sequence that is substantially similar to a reference polynucleotide (or polypeptide). Procedures for introducing nucleotide and amino acid changes in polynucleotides, proteins or polypeptides are known to those skilled in the art (see, e.g., Sambrook et al. (1989)). In the case of polynucleotides, a variant may have a deletion, substitution, addition of one or more nucleotides at the 5' end, the 3' end and/or at one or more internal sites relative to a reference polynucleotide. Sequence similarity and/or differences between a variant and a reference polynucleotide may be detected using techniques known in the art, such as using polymerase chain reaction (PCR) and hybridization techniques. Variant polynucleotides also include synthetically derived polynucleotides, such as those induced by using site-directed mutagenesis. Generally, variants of polynucleotides (including but not limited to DNA) may have at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to a reference polynucleotide when determined by sequence alignment programs known to those skilled in the art. In the case of polypeptides, variants may have one or more deletions, substitutions, additions of amino acids relative to a reference polypeptide. Sequence similarities and/or differences between variants and reference polypeptides may be detected using techniques known in the art, such as using Western blot. Generally, variants of a polypeptide may have at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to a reference polypeptide when measured by sequence alignment programs known to those skilled in the art.

Amino acid substitutions can be conservative or non-conservative. Preferably, the substitution is a conservative substitution, that is, the amino acid residue is substituted by an amino acid with similar polarity that serves as a functional equivalent. Preferably, the amino acid residue used as a substitute is selected from the same amino acid group as the amino acid residue to be substituted. For example, a hydrophobic residue can be substituted by another hydrophobic residue, or a polar residue can be substituted by another polar residue with the same charge. Functional homologous 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 glutamine.

Proteins that differ from their naturally occurring counterparts by adding, replacing or deleting one or more (e.g., 2, 3, 4, 5, 10 or 15) additional amino acids are also considered variants. Additional amino acids may be present in the amino acid sequence of the original PKP2 protein (i.e., as an insertion), or they may be added to one or both ends of the protein. Such insertions, substitutions or deletions may occur at any position, as long as they do not impair the ability of the polypeptide to perform the function of the naturally occurring PKP2 protein and/or repair haploinsufficiency of the treated individual. In addition, variants of the PKP2 protein also include proteins that lack one or more amino acids compared to the original polypeptide. Such deletions may affect any amino acid position, with the proviso that it does not impair the ability to perform the normal function of the PKP2 protein and/or repair haploinsufficiency.

Finally, variants of PKP2 proteins also refer to proteins that differ from naturally occurring proteins by structural modifications, such as modified amino acids. Modified amino acids are amino acids that have been modified by natural processes, such as processing or post-translational modification, or by chemical modification methods known in the art. Typical 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, acylation, cyclization and/or covalent or non-covalent bonding to phosphatidylinositol, flavin derivatives, lipoteichoic acid, fatty acids or lipids. Such modifications have been extensively described in the literature, for example in Proteins: Structure and Molecular Properties, T. Creighton, 2nd edition, W. H. Freeman and Company, New York (1993). In a preferred embodiment of the present invention, the nucleic acid sequence encodes a constitutively phosphorylated isoform of human PKP2. Such isoforms have been shown to be particularly cardioprotective (Sadayappan et al. (2005), Circ Res 97:1156-1163; Sadayappan et al., 2006; Proc Natl Acad Sci USA 103:16918-16923).

The terms "identity", "homology" and grammatical variations thereof mean that two or more of the referenced entities are identical when they are "aligned" sequences. Thus, for example, when two polypeptide sequences are identical, they have the same amino acid sequence at least in the referenced region or portion. When two polynucleotide sequences are identical, they have the same polynucleotide sequence at least in the referenced region or portion. Identity can be within a defined region (region or domain) of the sequence. A "region" or "region" of identity refers to portions of two or more of the referenced entities that are identical. Thus, when two polypeptide or nucleic acid sequences are identical in one or more sequence regions or regions, they share identity in that region. An "aligned" sequence refers to multiple polynucleotide or polypeptide (amino acid) sequences compared to a reference sequence, typically containing corrections for missing or additional bases or amino acids (gaps). "Substantial homology" means that the molecule is conserved structurally or functionally such that it has or is predicted to have at least a portion of one or more structures or functions (e.g., biological functions or activities) of a reference molecule or a related/corresponding region or portion of a reference molecule with which it shares homology.

"Percent nucleic acid sequence identity or homology (%)" is defined as the percentage of nucleotides in a candidate sequence that are identical to a reference sequence, after aligning the respective sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for the purpose of determining percent nucleic acid sequence identity can be achieved by various means within the skill in the art, for example, using publicly available computer software such as ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm needed to achieve maximum alignment over the full length of the sequences being compared.

"Amino acid sequence identity or homology percentage (%)" with respect to the PKP2 amino acid sequences identified herein is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the PKP2 polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum sequence identity percentage and without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining the percentage of amino acid sequence identity can be achieved by various means within the skill of the art, such as using publicly available computer software such as ALIGN or Megalign (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

"Codon optimization" or "codon optimized" refers to changes made in a nucleotide sequence in order to make it more likely to be expressed at a relatively higher level than a non-codon optimized sequence. It does not change the amino acids encoded by each codon.

"AAV virus particle" or "AAV virus particle" or "AAV vector particle" or "AAV virus" refers to a virus particle composed of at least one AAV capsid protein described herein and an encapsulated AAV vector construct. If the particle contains a heterologous polynucleotide (i.e., a polynucleotide that is not the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is usually referred to as a "recombinant AAV vector particle" or simply "AAV vector". The production of AAV vector particles necessarily includes the production of the AAV vector genome, so the vector genome is contained in the AAV vector particle. It should be understood that references to the polynucleotide AAV vector construct encapsulated in the vector particle and its replication refer to the AAV vector genome.

As used herein, "therapeutic AAV virus" refers to an AAV viral particle, AAV viral particle, AAV vector particle, or AAV virus that includes a heterologous polynucleotide encoding a therapeutic protein (such as PKP2 described herein). As used herein, "AAV vector construct" or "AAV vector genome" refers to a vector construct that includes one or more genes of interest, such as one or more polynucleotides encoding a protein of interest (also called a transgene), which is flanked by at least one AAV terminal repeat sequence (ITR) and is operably linked to one or more expression control elements. Such AAV vector constructs can replicate and be packaged into infectious viral particles when present in host cells transfected with vectors that have encoded and expressed the rep and cap gene products.

As used herein, "therapeutic protein" refers to a polypeptide having a biological activity that replaces or compensates for the loss or reduction of endogenous protein activity. For example, a functional PKP2 protein is a therapeutic protein for ACM.

As used herein, "arrhythmogenic cardiomyopathy" refers to a genetic disease caused by mutations in genes encoding cardiac skeletal components such as the plaquenil protein, characterized by symptoms such as arrhythmias, tissue damage, fibrofatty scarring, thinning of the ventricular walls, dilated cardiomyopathy (DCM), heart failure, palpitations, chest pain, shortness of breath, fatigue and dizziness, syncope, sudden cardiac death, and swelling of the feet, ankles, legs, or abdomen.

As used herein, "genetic disease or condition" refers to a genetic disease or condition caused by a mutation in an endogenous gene of interest ("mutated endogenous gene of interest") of an individual, which in turn produces a defective endogenous gene therapy product (protein or RNA) that is not functional or has reduced or abnormal activity. As used herein, "gene therapy product deficiency" refers to a genetic condition caused by a reduction in the level of an endogenous functional gene therapy product, which is attributed to the absence of an endogenous gene therapy product, reduced production of an endogenous gene therapy product, or the absence of a mutant defective endogenous gene therapy product that has reduced or abnormal activity. Gene therapy product deficiency includes genetic diseases and/or conditions.

As used herein, "therapeutically effective" or "gene therapy" refers to any therapeutic intervention for an individual suffering from a disease or disorder that would benefit from administration of a gene therapy product (e.g., where the therapeutic invention improves symptoms of the disease or disorder). For example, the therapy improves endogenous functional gene therapy product deficiency, increases the level of functional gene therapy product in an individual (e.g., in cardiac tissue and/or cardiac myocytes), and/or improves symptoms of the disease or disorder, including reducing the frequency, duration, or severity of symptoms of the disease or disorder.

As used herein, "plaquephrine protein-2 (PKP2) protein deficiency" or "functional wild-type plaquephrine protein-2 (PKP2) protein deficiency" refers to a genetic condition caused by reduced levels of functional PKP2 protein, which is due to the absence of protein, reduced protein production, or the production of non-functional protein. This condition includes ACM and ARCV.

As used herein, "effective for treatment of ACM" or "ACM treatment" refers to any therapeutic intervention in an individual with ACM that improves the characteristic deficiency of functional wild-type PKP2, increases the level of PKP2 protein, such as in the myocardium, improves ACM symptoms, or reduces the frequency, duration, or severity of ACM symptoms.

As used herein, "arrhythmogenic cardiomyopathy gene therapy" refers to any therapeutic intervention for an individual with ACM that involves replacing or restoring or increasing PKP2 by delivering one or more nucleic acid molecules to the individual's cells expressing functional PKP2. In certain embodiments, PKP2 gene therapy refers to gene therapy involving adeno-associated virus (AAV) particles comprising a vector construct expressing human PKP2. In other embodiments, gene therapy involves transfection of plasmids expressing human PKP2.

As used herein, "treat" or "treatment" refers to preventive or therapeutic treatment, which is treatment administered to an individual exhibiting signs or symptoms of a pathology (i.e., ACM) with the goal of reducing or eliminating those signs or symptoms or ameliorating their progression, severity, or duration. Such signs or symptoms may be biochemical, cellular, histological, functional, subjective, or objective.

As used herein, "amelioration" refers to the effect of reducing the severity, progression, or duration of symptoms of a disease.

As used herein, "stable treatment" refers to the use of a therapeutic vector construct, AAV particle or cell administered to an individual, wherein the individual stably expresses the therapeutic protein expressed by the vector construct, AAV particle or cell. Stably expressed therapeutic protein means that the protein is expressed for a clinically significant length of time. As used herein, "clinically significant length of time" means the length of time that a therapeutically effective amount is expressed to have a meaningful effect on the individual's quality of life, for example, as evidenced by a reduction in signs or symptoms of a disease. In certain embodiments, the clinically significant length of time is manifested for at least six months, at least eight months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, at least ten years, or the lifetime of the individual.

As used herein, the term "effective amount" refers to an amount sufficient to achieve beneficial or desired biological and/or clinical results.

As used herein, "subject" refers to an animal that is the subject of treatment, observation, or experiment. "Animals" include cold-blooded and warm-blooded vertebrates and invertebrates, such as fish, shellfish, reptiles, and especially mammals. As used herein, the term "avian" includes, but is not limited to, chickens, ducks, geese, quails, turkeys, and pheasants. As used herein, "mammals" refers to individuals belonging to the class Mammalia, and include, but are not limited to, humans, domestic and farm animals, zoo animals, sports animals, and pet animals. Non-limiting examples of mammals include mice; rats; rabbits; guinea pigs; dogs; cats; sheep; goats; cattle; horses; primates, such as monkeys, chimpanzees, and apes, and especially humans. In some embodiments, the mammal is a human, including an infant, child, or adolescent, such as a human up to 2, 2-4, 2-6, or 2-12 years of age.

In general, a "pharmaceutically acceptable carrier" is a carrier that is not toxic or overly harmful to cells and is preferably sterile. Exemplary pharmaceutically acceptable carriers include sterile, pyrogen-free water and sterile, pyrogen-free saline or phosphate-buffered saline. Pharmaceutically acceptable carriers include physiologically acceptable carriers. The term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents, and the like that are physiologically compatible. Vector Constructs and AAV Vectors

The recombinant vector constructs of the present invention can be used as gene therapy themselves, or can be used to produce rAAV particles by the method described herein, which comprises providing the recombinant vector construct and the Rep and Cap genes to a suitable host cell. The vector constructs described herein comprise a nucleic acid sequence encoding a functional PKP2 protein. The recombinant vector constructs may comprise a nucleic acid encoding a functional human PKP2, which is operably linked to a heterologous expression control element, such as a promoter and/or enhancer; an intron, if present; and a polyadenylation (polyA) signal, if present. The heterologous expression control element may be a heterologous cardiomyocyte-specific transcriptional regulatory region, such as described herein.

When used to generate rAAV particles, the recombinant vector construct may comprise (a) one or both of (i) AAV 5' inverted terminal repeat (ITR) sequences and (ii) AAV 3' ITRs, (b) a heterologous cardiomyocyte-specific transcriptional regulatory region, and (c) a nucleic acid encoding functional human PKP2, optionally wherein the AAV ITR is an AAV9 ITR. Preferably, the nucleic acid encoding functional PKP2 is operably linked to a cardiomyocyte-specific expression control element. The vector construct may include additional expression control elements, such as: a promoter and/or enhancer; an intron; optionally an exon or fragment thereof; and a polyadenylation (polyA) signal. Such elements are further described herein. In certain embodiments, the recombinant AAV vector construct comprises (a) a nucleic acid comprising the AAV9 5' inverted terminal repeat sequence (ITR) (which may or may not be modified as known in the art), (b) a cardiomyocyte-specific transcriptional regulatory region, a functional PKP2 protein coding region, (c) one or more introns including fragments of longer introns, (d) optionally, exons or fragments thereof, (e) a polyadenylation sequence, and (f) the AAV9 3' ITR (which may or may not be modified as known in the art).

Preferably, the rAAV particle also comprises an AAV capsid with cardiotropism, optionally an AAV9-type capsid. Exemplary capsids with cardiotropism include AAV1, 6, 7, and 9.

Other embodiments provided herein are directed to vector constructs encoding functional PKP2 polypeptides, wherein the constructs comprise one or more of the individual elements of the constructs described above in one or more different orientations and combinations thereof. Another embodiment provided herein is directed to the constructs described above in the opposite orientation.

The size of the AAV vector constructs provided herein in single strand form ranges from about 3.0 kb to about 5.5 kb. In one or more embodiments, the vector construct is an AAV vector genome of about 3.3 kb to about 5.0 kb, about 3.5 kb to about 4.8 kb, or about 3.6 kb to about 4.6 kb, or about 3.7 kb to about 4.5 kb.

When an AAV vector is produced from an oversized recombinant vector construct, it may lack a portion of the 5' or 3' end of the recombinant vector construct. Because AAV is a single-stranded DNA virus and packages either the sense or antisense strand, the sense strand in the oversized AAV vector lacks the 5' AAV ITR and possibly the 5' end of the target protein-coding gene, and the antisense strand in the oversized AAV vector lacks the 3' ITR and possibly the 3' end of the target protein-coding gene. Functional transgenes are produced in cells infected with oversized AAV vectors by ligation of the sense and antisense truncated genomes within the target cell. Thus, in certain embodiments, the rAAV particles of the present invention may comprise a recombinant vector construct comprising at least one ITR and a substantial portion of a nucleotide sequence encoding a functional PKP2, such as a fragment of SEQ ID NO: 1 or 69-73 or 129 that exceeds 50%, 60%, 70%, 80% or 90% of the length of the nucleotide sequence. For example, a recombinant vector construct may comprise at least one ITR, a cardiomyocyte-specific transcriptional regulatory region, and a substantial portion of a nucleotide sequence encoding a functional PKP2.

The production of vector constructs can be achieved using any suitable genetic engineering technique well known in the art, including but not limited to standard techniques of restriction endonuclease digestion, ligation, transformation, plasmid purification and DNA sequencing, such as those described in Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, N.Y. (1989)).

The vector construct may incorporate sequences from the genome of any known organism. The sequence may be incorporated in its native form or may be modified in any way to obtain the desired activity. For example, the sequence may include insertions, deletions, or substitutions.

When present in host cells transfected with polynucleotides encoding and expressing the rep and cap gene products, the AAV vector construct can be replicated and packaged into infectious AAV particles, preferably replication-defective AAV particles .

In one or more embodiments, the nucleic acid sequence encoding PKP2 is operably linked to one or more heterologous expression control elements. Preferably, the expression control element is a cardiomyocyte-specific expression control element. Examples of cardiomyocyte-specific control elements include, but are not limited to, the human cardiac necrosis factor T (hTNNT2) promoter or a fragment or variant thereof. Other promoters active in cardiomyocytes include fragments or variants of any of the following: muscle creatine kinase (MCK) promoter, cytomegalovirus enhancer + myosin light chain 2 promoter (CMV-MLC2 or CMV-MLC1.5, CMV-MLC260), phosphoglycerate kinase (PGK) promoter, crosslinked plasmid Specific promoter, α-myosin heavy chain promoter, myosin light chain 2v promoter, α-myosin heavy chain promoter, α-actin promoter, α-actin promoter, cardiac myosin C promoter, cardiac myosin I promoter, cardiac myosin binding protein C promoter and/or sarcoplasmic reticulum/endoplasmic reticulum Ca2+'' adenosine triphosphatase (SERCA) promoter (e.g., isoform 2 (SERCA2) of this promoter) and/or striated muscle promoter, such as desmin promoter. Enhancers derived from myocardial cell-specific transcription factor binding sites are also contemplated.

Examples of fragments or variants of the hTNNT2 promoter include a cardiomyocyte-specific promoter sequence comprising a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 85. In some embodiments, the cardiomyocyte-specific transcriptional regulatory region comprises a cardiomyocyte-specific promoter sequence comprising a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 89. In some embodiments, the cardiomyocyte-specific transcriptional regulatory region comprises a cardiomyocyte-specific promoter sequence comprising a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 81. In some embodiments, the cardiomyocyte-specific transcriptional regulatory region comprises a cardiomyocyte-specific promoter sequence comprising a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 88. In some embodiments, the cardiomyocyte-specific transcriptional regulatory region comprises a cardiomyocyte-specific promoter sequence comprising a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 87. In some embodiments, the cardiomyocyte-specific transcriptional regulatory region comprises a cardiomyocyte-specific promoter sequence comprising a nucleic acid sequence that is at least or more than 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 81-89 (in the length of the SEQ ID NO). In some embodiments, the cardiomyocyte-specific transcriptional regulatory region also comprises an intron that enhances the expression of the PKP2 protein, and, if appropriate, an exon or a fragment thereof 5' of the PKP2 coding sequence. For example, the vector construct and AAV particle comprise a cardiomyocyte-specific promoter in the 5' to 3' orientation comprising a nucleotide sequence at least 80% identical to SEQ ID NO: 85; an intronic nucleotide sequence at least 70% identical to SEQ ID NO: 77; and a nucleotide sequence encoding PKP2.

In other embodiments, the cardiomyocyte-specific promoter comprises (a) a nucleic acid sequence at least 80% identical to any one of (i) SEQ ID NO: 87 or a fragment thereof, (ii) SEQ ID NO: 88 or a fragment thereof, (iii) SEQ ID NO: 81 or (iv) SEQ ID NO: 82 or a fragment thereof; and (b) an intronic nucleotide sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 77. In alternative embodiments, the intron comprises a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 75. Other exemplary introns are SEQ ID NOs: 74-80 or 265-275.

In some embodiments, the vector construct comprises (a) a nucleic acid sequence at least 90% identical to any of (i) SEQ ID NO: 87 or a fragment thereof, (ii) SEQ ID NO: 88 or a fragment thereof, or (iii) SEQ ID NO: 82 or a fragment thereof; and (b) an intron comprising a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 77. In alternative embodiments, the intron comprises a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 75. Other exemplary introns are SEQ ID NOs: 74-80 or 265-275.

In some embodiments, the cardiomyocyte-specific transcriptional regulatory region may further comprise (in addition to the hTNNT2 promoter and the fragment or variant of the globin intron) an exon sequence or a fragment thereof, such as the 3' end of the fragment of the globin intron and the beta globin exon 3 (SEQ ID NO: 76). The combination of the intron and the exon fragment is, for example, SEQ ID NO: 78. In some exemplary embodiments, the cardiomyocyte-specific transcriptional regulatory region comprises SEQ ID NO: 79.

In some embodiments, the fragment or variant of the hTNNT2 promoter is greater than 420 and less than 544 nucleotides in length and comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 85.

In some embodiments, the cardiomyocyte-specific promoter sequence comprises a nucleic acid sequence that is at least or more than 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of (i) SEQ ID NO: 87 or a fragment thereof, (ii) SEQ ID NO: 88 or a fragment thereof, (iii) SEQ ID NO: 81 or a fragment thereof, (iv) SEQ ID NO: 89 or a fragment thereof, or (v) SEQ ID NO: 82. For example, the cardiomyocyte-specific promoter sequence comprises a nucleic acid sequence that is at least or more than 95%, 97%, 98% or 99% identical to any one of (i) SEQ ID NO: 87 or a fragment thereof, (ii) SEQ ID NO: 88 or a fragment thereof, (iii) SEQ ID NO: 81 or a fragment thereof, or (iv) SEQ ID NO: 82 or a fragment thereof. In an exemplary embodiment, the sequence of the cardiomyocyte-specific promoter comprises a nucleotide sequence that is at least 96%, 97%, 98% or 99% identical to SEQ ID NO: 81. In some exemplary embodiments, the sequence of the hTNNT promoter comprises at least nucleotides 1-106 and 507-532 of SEQ ID NO: 81, or at least nucleotides 507-532 of SEQ ID NO: 81, or at least nucleotides 521-532 of SEQ ID NO: 81.

Various promoters can be operably linked to nucleic acids comprising the coding region of the protein of interest, human PKP2, in the vector construct disclosed herein. In some embodiments, the promoter can drive the protein of interest to be expressed in cells (such as target cells) infected by viruses derived from viral vectors. Promoters can be naturally occurring or non-naturally occurring. In some embodiments, the promoter is a synthetic promoter. In one embodiment, the synthetic promoter comprises a sequence that does not exist in nature and is designed to regulate the activity of an operably linked gene. In another embodiment, the synthetic promoter comprises a fragment of a natural promoter to form a new DNA sequence segment that does not exist in nature. Synthetic promoters typically include regulatory elements, promoters, enhancers, introns, splice donors, and receptors, which are designed to produce enhanced tissue-specific expression. Examples of promoters include, but are not limited to, viral promoters, plant promoters, and mammalian promoters. In another embodiment, the promoter is a cardiomyocyte-specific promoter.

In some embodiments, the promoter comprises the human cardiac tyrosin T (hTNNT2) promoter. The portion of the hTNNT2 promoter may comprise a nucleic acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to any one of SEQ ID NOs: 87-89. In some embodiments, the promoter is at least about or more than 95% identical to any one of SEQ ID NOs: 87-89. Promoter

According to the second object of the present invention, a tissue-specific promoter is provided herein, which can be operably linked to any gene of interest, meaning that the promoter is combined with the gene of interest so that the gene of interest is expressed in cardiac tissue and/or myocardial cells under the control of the promoter when integrated into the genome of the cell or present in the cell as an exogenous nucleic acid construct. The present invention further contemplates the use of the hTNNT2 promoter with any gene of interest for expression in myocardial cells.

The transgene or vector construct described herein comprises a nucleotide sequence encoding or expressing a functional gene therapy product (i.e., a gene of interest). The transgene or vector construct may comprise a gene of interest operably linked to a cardiac-specific promoter described herein, and optionally other expression control elements that together form a transcriptional regulatory region; optionally an expression enhancing intron; and optionally a polyadenylation (polyA) signal.

A nucleic acid comprising a coding region for a protein of interest (a nucleic acid sequence encoding or expressing a gene therapy product) is operably linked to a heterologous transcriptional regulatory region comprising a cardiomyocyte-specific promoter, optionally a promoter derived from the cardiac calcification protein T (TNNT2) promoter. In such embodiments, the transgene comprises a gene of interest as described herein operably linked to a cardiomyocyte-specific promoter.

Examples of such cardiomyocyte-specific promoters include any one of SEQ ID NOs: 133-263, or a nucleic acid sequence at least about 80% identical thereto. The cardiomyocyte-specific promoter is expressed at least about 5 times or at least about 10 times higher in vitro or in vivo in cardiac tissue and/or cardiomyocytes than in another non-cardiac cell type.

In some embodiments, the cardiac specific promoter sequence comprises a nucleic acid sequence that is at least about 85% identical to any one of SEQ ID NOs: 133-263. In some embodiments, the cardiac specific promoter sequence comprises a nucleic acid sequence that is at least about 90% identical to any one of SEQ ID NOs: 133-263. In some embodiments, the cardiac specific promoter sequence comprises a nucleic acid sequence that is at least about 95% identical to any one of SEQ ID NOs: 133-263. In some embodiments, the cardiac specific promoter sequence comprises a nucleic acid sequence that is at least about 98% identical to any one of SEQ ID NOs: 133-263. In some embodiments, the cardiac specific promoter sequence comprises a nucleic acid sequence that is at least about 99% identical to at least one of SEQ ID NOs: 133-263. Such a cardiac-specific promoter preferably comprises nucleotides 1-12 of SEQ ID NO: 264 at the 3' end.

In some embodiments, the length of the promoter is at least about 300 bp to about 600 bp, or at least about 400 bp to about 600 bp, or at least about 300 bp to about 500 bp, or at least about 400 bp to about 450 bp. In any of the embodiments described herein, the cardiac-specific promoter does not consist of any of SEQ ID NOs: 1 to 85 of U.S. Patent Publication No. 2021/0252165. The cardiac-specific transcriptional regulatory region optionally comprises a fragment or variant of another promoter, and/or one or more enhancer sequences.

In some embodiments, the activity of a cardiomyocyte-specific promoter in cardiac tissue and/or cardiomyocytes is at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, or at least about 50-fold greater than its activity in non-cardiac cell types.

In some embodiments, any of the activation substructures described herein include one or more additional individual reinforcing subelements in one or more different orientations.

In some embodiments, the promoter is operably linked to a polynucleotide encoding one or more proteins or RNAs of interest. In some embodiments, the promoter is operably linked to a polynucleotide encoding a gene of interest.

In some embodiments, the promoter is operably linked to a polynucleotide encoding one or more proteins of interest. In some embodiments, the promoter is operably linked to a polynucleotide encoding a PKP2 protein.

The size of the promoter can vary. Due to the limited packaging capacity of AAV, it is preferred to use a promoter that is smaller in size but at the same time allows for the production of a protein of interest in large quantities in the host cell. For example, in some embodiments, the promoter is at most about 1.5 kb, at most about 1.4 kb, at most about 1.35 kb, at most about 1.3 kb, at most about 1.25 kb, at most about 1.2 kb, at most about 1.15 kb, at most about 1.1 kb, at most about 1.05 kb, at most about 1 kb, at most about 800 base pairs, at most about 600 base pairs, at most about 400 base pairs, at most about 200 base pairs, or at most about 100 base pairs. Other Regulatory Elements .

Various additional regulatory elements can be used in the vector constructs, such as enhancers, polyadenylation signals, ribosome binding sequences, and/or common splice acceptor or splice donor sites that further increase the expression of the protein of interest in the host cell. In some embodiments, the regulatory elements can help maintain the recombinant DNA molecule outside the chromosome of the host cell and/or improve vector efficiency (e.g., backbone/matrix attachment region (S/MAR)). Such regulatory elements are well known in the art.

The vector constructs disclosed herein may include regulatory elements, such as a transcriptional initiation region and/or a transcriptional termination region. Examples of transcriptional termination regions include, but are not limited to, polyadenylation signal sequences. Examples of polyadenylation signal sequences include, but are not limited to, mini-poly(A), human growth hormone (hGH) poly(A), bovine growth hormone (bGH) poly(A), SV40 late poly(A), rabbit β-globulin (rBG) poly(A), thymidine kinase (TK) poly(A) sequence, Proudfoot polyA, and any variants thereof. In some embodiments, the transcriptional termination region is located downstream of the post-transcriptional regulatory element. In some embodiments, the transcriptional termination region is a polyadenylation signal sequence. In some embodiments, the transcriptional termination region is a bGH polyA (e.g., any one of SEQ ID NOs: 90-96), hGH polyA (e.g., SEQ ID NO: 97), SV40 polyA (e.g., SEQ ID NO: 98), Proudfoot synthetic polyA (e.g., SEQ ID NO: 99), or rabbit β-globin polyA (e.g., SEQ ID NO: 100) sequence or a fragment thereof of about 50 to 75 nucleotides in length.

In some embodiments, the vector construct comprises a polyadenylation signal, optionally a bovine growth hormone (bGH) polyA signal (e.g., SEQ ID NOs: 90-96) or a human growth hormone (hGH) polyA signal, or a fragment thereof.

The polyA signal can be about 150 to about 250 nucleotides in length, about 160 to about 240 nucleotides in length, about 170 to about 230 nucleotides in length, about 180 to about 220 nucleotides in length, or about 200 to about 210 nucleotides in length.

In some embodiments, the vector construct may include additional transcriptional and translational initiation sequences and/or additional transcriptional and translational termination sequences, which are known in the art. Proteins of interest and nucleic acids encoding proteins of interest .

As used herein, "protein of interest" is any functional PKP2 protein, including naturally occurring and non-naturally occurring variants thereof. In some embodiments, polynucleotides encoding one or more PKP2 proteins of interest can be inserted into a viral vector disclosed herein, wherein the polynucleotide is operably linked to a promoter. In some cases, the promoter can drive the expression of the protein of interest in host cells (e.g., human myocardium).

In one or more embodiments, the functional PKP2 protein comprises an amino acid sequence that is at least 90%, 95% or 98% identical to SEQ ID NO: 2 (human plaquenol protein 2) or SEQ ID NO: 130, or SEQ ID NO: 131, or SEQ ID NO: 132. The present invention also provides an isolated nucleic acid molecule encoding such a functional wild-type PKP2 protein. The nucleotide sequence may be homologous to the wild-type nucleotide sequence of SEQ ID NO: 1 or 129. In certain embodiments, the nucleic acid molecule has at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homology or at least 98% homology to the nucleotide sequence of SEQ ID NO: 1 or 129, or at least 100, 200, 300, 400 or 500 consecutive nucleotides of SEQ ID NO: 1 or 69-73 or 129. In exemplary embodiments, the nucleotide sequence encoding a functional PKP2 is codon optimized or a variant and may be at least 85%, 90%, 95%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 71-73.

In certain embodiments, the nucleic acid molecule has at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homology or at least 98% homology to the nucleotide sequence of SEQ ID NO: 1 or 69-73 or 129, or at least 100, 200, 300, 400 or 500 consecutive nucleotides of SEQ ID NO: 1 or 69-73 or 129.

In exemplary embodiments, the nucleic acid sequence encoding a functional PKP2 is a wild-type PKP2 sequence, wherein SEQ ID NO: 1 is an example, or SEQ ID NO: 129 is another example, or is codon-optimized, or is a variant. The vector constructs described herein may include a nucleotide sequence that is different from the wild-type nucleotide sequence but still encodes a functional PKP2 amino acid sequence that is at least 90%, 95% or 98% identical to SEQ ID NO: 2 or SEQ ID NO: 130, SEQ ID NO: 131 or SEQ ID NO: 132. According to this aspect, the nucleotide sequence may include a portion having at least 80%, 85%, 90% or 95% homology to at least 100 consecutive bases of SEQ ID NO: 1 or 69-73 or 129, as long as the nucleotide sequence encodes a functional human PKP2 protein that is at least 90%, 95% or 98% identical to SEQ ID NO: 2 or 130 or 131 or 132. In exemplary embodiments, the nucleotide sequence may include a portion having at least 90% homology to at least 100, 200, 300, 400 or 500 consecutive bases of SEQ ID NO: 1 or 129, as long as the nucleotide sequence encodes a functional human PKP2 protein that is at least 90% identical to SEQ ID NO: 2 or 130 or 131 or 132. In exemplary embodiments, the nucleotide sequence has substantial homology to the nucleotide sequence of SEQ ID NO: 1 or 69-73 or 129 and encodes a functional PKP2. The term substantial homology can be further defined with reference to a homology percentage (%), such as at least 80%, 85%, 90% or 95% homology. This is further discussed in detail elsewhere herein.

In an exemplary embodiment, the nucleotide sequence of the gene of interest is codon optimized, preferably codon optimized so as to be more efficiently expressed in humans, or more efficiently expressed in target organs, target tissues and/or target cells of humans. Target organs, tissues or cells include cardiac tissues and/or cardiac myocytes. The adaptability of the nucleotide sequence encoding the gene therapy product to the codon usage of human cells can be expressed as a codon adaptability index (CAI). The codon adaptability index is defined herein as a measure of the relative adaptability of the codon usage of a gene to the codon usage of highly expressed human genes. The relative adaptability (w) of each codon is the ratio of the usage of each codon to the usage of the most abundant codon of the same amino acid. CAI is defined as the geometric average of these relative adaptability values. Non-synonymous codons and stop codons (depending on the genetic code) are not included. CAI values range from 0 to 1, with higher values indicating a higher proportion of the most abundant codons (see Sharp and Li, 1987, Nucleic Acids Research 15: 1281-1295; see also: Kim et al., Gene. 1997, 199: 293-301; zur Megede et al., Journal of Virology, 2000, 74: 2628-2635). In certain embodiments, the CAI of the gene of interest is at least 0.75, 0.80, 0.85, 0.90, 0.95 or 0.99.

Codon optimization can be performed, for example, using the DNA2.0 codon optimization algorithm, see Villalobos et al., "Gene Designer: a synthetic biology tool for constructing artificial DNA segments," BMC Bioinformatics, Vol. 7, Article No. 285 (2006) or using Operon/Eurofins Genomics codon optimization software or other codon optimization tools, such as Grote et al., "Jcat: a novel tool to adapt codon usage of a target gene to its potential expression host", Nucleic Acids Res. 33:W526-31 (2005).

In addition, or as an alternative to codon optimization, the nucleotide sequence of the gene of interest can be adjusted to reduce the CpG dinucleotide content and remove any additional ORFs in the sense and antisense directions as appropriate. CpG dinucleotide content has been shown to activate TLR9 in dendritic cells, leading to potential immune activation and CTL response. Reducing CpG content can reduce liver inflammation and ALT. In some embodiments, the CpG dinucleotide content of the nucleotide sequence of the gene of interest is less than 25, less than 20, less than 15 or less than 10. In another embodiment, the GC content of the nucleotide sequence of the gene of interest is less than 65%, less than 60% or less than 55%.

In general, codon optimization or CpG reduction does not change the amino acids encoded by each codon. It simply changes the nucleotide sequence so that it is more likely to be expressed in relatively high amounts compared to a non-optimized sequence.

As described herein, the nucleotide sequence encoding the PKP2 protein can be modified to increase the expression efficiency of the protein. The methods that can be used to improve the transcription and/or translation of the genes herein are not particularly limited. For example, the nucleotide sequence can be modified to better reflect the host codon usage to increase the gene expression (e.g., protein production) of the host (e.g., mammal). As another non-limiting example of modification, one or more of the splicing donors and/or splicing acceptors in the nucleotide sequence of the protein of interest are modified to reduce the possibility of foreign splicing. As another non-limiting example of modification, one or more introns can be inserted into or near the nucleotide sequence of the protein of interest to optimize AAV vector packaging and enhance expression.

The nucleic acid molecule encodes a functional PKP2 protein that is at least 90% identical to the wild-type amino acid sequence of SEQ ID NO: 2, or at least 90% identical to SEQ ID NO: 130, or at least 90% identical to SEQ ID NO: 131, or at least 90% identical to SEQ ID NO: 132. If the nucleic acid encodes a protein having a sequence that is altered relative to any of the wild-type amino acids, the protein should still be a functional protein. The skilled person will appreciate that minor changes may be made to some of the amino acids of a protein without adversely affecting the function of the protein.

In certain embodiments, the nucleic acid molecules, when expressed in a suitable system (e.g., a host cell), produce a functional PKP2 protein at a relatively high level. Because the PKP2 produced is functional, it will have a conformation identical to at least a portion of wild-type PKP2. In certain embodiments, the functional PKP2 protein produced as described herein is effective in treating individuals suffering from wild-type PKP2 protein deficiency and/or ACM.

In other embodiments, the nucleic acid molecules, when expressed in a suitable system (e.g., a host cell), increase the expression of various endogenous bridge and GAP proteins, such as nexin plakoglobin, desmoplakin, desmoglobin-2, nexin 43, and N-calcineurin. In other embodiments, functional PKP2 proteins produced as described herein and increasing the expression of various endogenous bridge and GAP proteins are effective in treating individuals with wild-type PKP2 protein deficiency and/or ACM.

Those skilled in the art are well within the ability to generate the nucleic acid molecules provided herein. This can be done, for example, using chemical synthesis of a given sequence. In addition, suitable methods for determining whether the nucleic acids described herein express functional proteins will be apparent to those skilled in the art. For example, a suitable in vitro method involves inserting the nucleic acid into a vector (such as an AAV vector), transducing host cells with the vector, and analyzing exogenous PKP2 expression. Alternatively, a suitable in vivo method involves transducing a vector containing the nucleic acid into mice using an ACM disease model and analyzing reduced ACM symptoms. Introns

In some embodiments, the vector comprises one or more introns. Introns can aid in the processing of RNA transcripts in mammalian host cells, increase the expression of a protein of interest, and/or optimize vector packaging in AAV particles. Non-limiting examples of such introns are human beta globulin introns, human immunoglobulin G (IgG) introns, or native PKP2 introns. In some embodiments, the introns are synthetic introns.

In some embodiments, the vector construct and/or AAV particle comprises a cardiomyocyte-specific promoter and one or more additional heterologous expression control elements, such as an intron that enhances PKP2 protein expression. For example, the vector construct and/or AAV particle comprises any of the cardiomyocyte-specific promoters described above, and optionally an intron nucleotide sequence located 5' of the nucleotide sequence encoding PKP2. In other examples, the vector construct and/or AAV particle comprises any of the cardiomyocyte-specific promoters described above, and optionally an intron nucleotide sequence located within the nucleotide sequence encoding PKP2, such as between any exons. In some embodiments, the intron sequence is located between exons 1 and 2. In some embodiments, the intron sequence is located within the nucleic acid encoding PKP2 at a position between positions 261-262 of SEQ ID NO: 1 or 129.

In one or more embodiments, the intron comprises a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 77, and the intron may be about 50 to about 150 nucleotides in length, or about 100 to about 135 nucleotides in length. In exemplary embodiments, the intron comprises SEQ ID NO: 77 or a fragment thereof, which is about 50-150 nucleotides, 75-145 nucleotides, 100-135 nucleotides, or 120-135 nucleotides of SEQ ID NO: 77, or a variant of the fragment, which is at least 80%, 85%, 90%, or 95% identical to the fragment. In some embodiments, the intron may comprise a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 77.

In one or more embodiments, the intron comprises a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 77, and the intron may be about 50 to about 150 nucleotides in length, or about 100 to about 135 nucleotides in length. In exemplary embodiments, the intron comprises SEQ ID NO: 74 or a fragment thereof, which is about 50-150 nucleotides, 75-145 nucleotides, 100-135 nucleotides, or 120-135 nucleotides of SEQ ID NO: 74, or a variant of the fragment, which is at least 80%, 85%, 90%, or 95% identical to the fragment. In some embodiments, the intron may comprise a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 74.

Other exemplary introns comprise a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80% or 85% or 90% or 95% identical to any one of SEQ ID NOs: 74-80 or 265-275.

In some embodiments, the vector construct may further comprise an exon sequence or a fragment thereof; preferably adjacent to the 5' or 3' end of the intron sequence. In an exemplary embodiment, the vector construct comprises a globin intron adjacent to the exon, the exon comprising a nucleotide sequence at least 80% or 85% or 90% or 95% identical to SEQ ID NO: 76. In another exemplary embodiment, the vector construct comprises a globin intron adjacent to the exon sequence, comprising a nucleotide sequence at least 80% or 85% or 90% or 95% identical to SEQ ID NO: 77. In an exemplary embodiment, the vector construct comprises a globin intron adjacent to the HbB exon sequence, the exon sequence comprising a nucleotide sequence at least 80% or 85% or 90% identical to SEQ ID NO: 77.

The position and size of introns in the vector may be different. In some embodiments, the intron is located between the promoter and the sequence encoding the protein of interest. In some embodiments, the intron is located downstream of the sequence encoding the protein of interest. In some embodiments, the intron is located within the promoter. In some embodiments, the intron includes an enhancer element. In some embodiments, the intron is located within the sequence encoding the protein of interest, preferably between exons of the sequence encoding the protein of interest. In some embodiments, the intron may be included in all or part of a naturally occurring intron within the sequence encoding the protein of interest. In some embodiments, the intron is a globulin intron. In some embodiments, the intron is a chimeric intron and comprises a fragment of a human IgG intron.

Inclusion of intronic elements can enhance expression compared to expression in the absence of intronic elements (see, e.g., Kurachi et al., J. Biol. Chem . 270(10): 5276-81 (1995). AAV vectors generally accept DNA inserts of a limited size range, generally from about 3.5 kb to about 5.4 kb, or slightly more. However, there is no minimum size for extremely efficient packaging and packaging of genomes in smaller vectors. Introns and intronic fragments satisfy this requirement while also enhancing expression. Thus, the present invention is not limited to the inclusion of PKP2 intronic sequences in AAV vectors, and includes other introns or other DNA sequences in place of portions of PKP2 introns. Furthermore, other 5' and 3' nucleic acid non-translated regions can be used in place of those described for human PKP2.

In some embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98%, or 99% identical to any one of SEQ ID NOs: 3-68 or 126-128. In other embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98%, or 99% identical to the following nucleotide sequence: the nucleotide sequence is complementary to or negative (-) stranded with any one of SEQ ID NOs: 3-68 or 126-128.

In some embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98% or 99% identical to any one of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66 or 126. In other embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98% or 99% identical to any one of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66 or 126.

In some embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98% or 99% identical to any one of SEQ ID NO: 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67 or 127. In other embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98% or 99% identical to any one of SEQ ID NO: 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67 or 127.

In some embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98% or 99% identical to any one of SEQ ID NO: 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68 or 128. In other embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98% or 99% identical to any one of 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68 or 128.

Exemplary embodiments include the following: Construct A is 3691 bp in length (SEQ ID NO: 5) and comprises the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), hTNNT2 promoter (533 bp) (SEQ ID NO: 82), wild-type hPKP2 with a globin intron (131 bp) (SEQ ID NO: 77) between exons (SEQ ID NO: 69), bovine poly A (223 bp) (SEQ ID NO: 90), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct A optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complementary sequences) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complementary sequences), or fragments thereof. Construct B was 3691 bp in length (SEQ ID NO: 8) and comprised the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), hTNNT2 promoter (533 bp) (SEQ ID NO: 82), wild-type hPKP2 (SEQ ID NO: 69) with a globin intron (131 bp) (SEQ ID NO: 77) between exons, bovine poly A (223 bp) (SEQ ID NO: 90), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct B optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complementary sequences) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complementary sequences), or fragments thereof. Construct C was 3565 bp in length (SEQ ID NO: 11) and comprised the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), hTNNT2 promoter (533 bp) (SEQ ID NO: 82), wild-type hPKP2 (SEQ ID NO: 69) with a globin intron (131 bp) (SEQ ID NO: 77) between exons, bovine poly A (223 bp) (SEQ ID NO: 90), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct C optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complementary sequences) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complementary sequences), or fragments thereof. Construct D was 3565 bp in length (SEQ ID NO: 14) and comprised the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), hTNNT2 promoter (533 bp) (SEQ ID NO: 82), wild-type MsPKP2 (SEQ ID NO: 70) with a globin intron (131 bp) (SEQ ID NO: 77) between exons, bovine poly A (227 bp) (SEQ ID NO: 92), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct D optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complementary sequences) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complementary sequences), or fragments thereof. Construct E was 3651 bp in length (SEQ ID NO: 17) and comprised the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), cTNNT2 promoter (421 bp) (SEQ ID NO: 83), globin intron (131 bp) (SEQ ID NO: 77), HBB exon 3 (SEQ ID NO: 76), wild-type hPKP2 (SEQ ID NO: 69), bovine poly A (227x bp) (SEQ ID NO: 93), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct E optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complementary sequences) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complementary sequences), or fragments thereof. Construct F was 4782 bp in length (SEQ ID NO: 20) and comprised the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), endogenous promoter (1417 bp) (SEQ ID NO: 84), globin intron (167 bp) (SEQ ID NO: 75), HBB exon 3 (SEQ ID NO: 76), wild-type hPKP2 with globin intron (131 bp) (SEQ ID NO: 77) between exons (SEQ ID NO: 69), bovine poly A (229 bp) (SEQ ID NO: 94), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct F optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complementary sequences) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complementary sequences), or fragments thereof. Construct G was 4651 bp in length (SEQ ID NO: 23) and comprised the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), endogenous promoter (1417 bp) (SEQ ID NO: 84), globin intron (131 bp) (SEQ ID NO: 77), HBB exon 3 (SEQ ID NO: 76), wild-type hPKP2 (SEQ ID NO: 69), bovine poly A (229 bp) (SEQ ID NO: 94), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct G optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complementary sequences) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complementary sequences), or fragments thereof. Construct H was 4596 bp in length (SEQ ID NO: 26) and comprised the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), endogenous promoter (1417 bp) (SEQ ID NO: 84), wild-type hPKP2 (SEQ ID NO: 69) with a globin intron (131 bp) (SEQ ID NO: 77) between exons, bovine poly A (229 bp) (SEQ ID NO: 94), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct H optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or its complementary sequence) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or its complementary sequence), or a fragment thereof. Construct I is 4465 bp in length (SEQ ID NO: 29) and comprises the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), endogenous promoter (1417 bp) (SEQ ID NO: 84), wild-type hPKP2 (SEQ ID NO: 69), bovine poly A (229 bp) (SEQ ID NO: 94), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct I optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complementary sequences) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complementary sequences), or fragments thereof. Construct J was 4596 bp in length (SEQ ID NO: 32) and comprised the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), endogenous promoter (1417 bp) (SEQ ID NO: 84), codon-optimized hPKP2 (SEQ ID NO: 71) with a globin intron (131 bp) (SEQ ID NO: 77) between exons, bovine poly A (229 bp) (SEQ ID NO: 94), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct J comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complements) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complements), or fragments thereof, as appropriate. Construct K is 4465 bp in length (SEQ ID NO: 35) and comprises the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), endogenous promoter (1417 bp) (SEQ ID NO: 84), codon-optimized hPKP2 (SEQ ID NO: 71), bovine poly A (229 bp) (SEQ ID NO: 94), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct K optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complementary sequences) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complementary sequences), or fragments thereof. Construct L was 4596 bp in length (SEQ ID NO: 38) and comprised the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), endogenous promoter (1417 bp) (SEQ ID NO: 84), codon-optimized hPKP2 (SEQ ID NO: 72) with a globin intron (131 bp) (SEQ ID NO: 77) between exons, bovine poly A (229 bp) (SEQ ID NO: 94), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct L optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or its complementary sequence) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or its complementary sequence), or a fragment thereof. Construct M is 4465 bp in length (SEQ ID NO: 41) and comprises the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), endogenous promoter (1417 bp) (SEQ ID NO: 84), codon-optimized hPKP2 (SEQ ID NO: 72), bovine poly A (229 bp) (SEQ ID NO: 94), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct M optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complementary sequences) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complementary sequences), or fragments thereof. Construct N was 4596 bp in length (SEQ ID NO: 44) and comprised the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), endogenous promoter (1417 bp) (SEQ ID NO: 84), codon-optimized hPKP2 (SEQ ID NO: 73) with a globin intron (131 bp) (SEQ ID NO: 77) between exons, bovine poly A (229 bp) (SEQ ID NO: 94), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct N optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or its complementary sequence) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or its complementary sequence), or a fragment thereof. Construct O is 4465 bp in length (SEQ ID NO: 47) and comprises the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), endogenous promoter (1417 bp) (SEQ ID NO: 84), codon-optimized hPKP2 (SEQ ID NO: 73), bovine poly A (229 bp) (SEQ ID NO: 94), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct O optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complementary sequences) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complementary sequences), or fragments thereof. Construct P was 3896 bp in length (SEQ ID NO: 50) and comprised the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), hTNNT2 promoter (533 bp) (SEQ ID NO: 82), globin intron (131 bp) (SEQ ID NO: 77), HBB exon 3 (SEQ ID NO: 76), wild-type hPKP2 with globin intron (131 bp) (SEQ ID NO: 77) between exons (SEQ ID NO: 69), bovine poly A (229 bp) (SEQ ID NO: 94), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct P optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complementary sequences) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complementary sequences), or fragments thereof. Construct Q was 3869 bp in length (SEQ ID NO: 53) and comprised the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), hTNNT2 promoter (533 bp) (SEQ ID NO: 82), globin intron (131 bp) (SEQ ID NO: 77), HBB exon 3 (SEQ ID NO: 76), codon-optimized hPKP2 (SEQ ID NO: 71) with globin intron (131 bp) (SEQ ID NO: 77) between exons, bovine poly A (229 bp) (SEQ ID NO: 94), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct Q optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complementary sequences) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complementary sequences), or fragments thereof. Construct R was 3765 bp in length (SEQ ID NO: 56) and comprised the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), hTNNT2 promoter (533 bp) (SEQ ID NO: 82), globin intron (131 bp) (SEQ ID NO: 77), HBB exon 3 (SEQ ID NO: 76), codon-optimized hPKP2 (SEQ ID NO: 71), bovine poly A (229 bp) (SEQ ID NO: 94), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct R optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complementary sequences) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complementary sequences), or fragments thereof. Construct S was 3896 bp in length (SEQ ID NO: 59) and comprised the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), hTNNT2 promoter (533 bp) (SEQ ID NO: 82), globin intron (131 bp) (SEQ ID NO: 77), HBB exon 3 (SEQ ID NO: 76), codon-optimized hPKP2 with globin intron (131 bp) (SEQ ID NO: 77) between exons (SEQ ID NO: 72), bovine poly A (229 bp) (SEQ ID NO: 94), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct S optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complementary sequences) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complementary sequences), or fragments thereof. Construct T was 3765 bp in length (SEQ ID NO: 62) and comprised the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), hTNNT2 promoter (533 bp) (SEQ ID NO: 82), globin intron (131 bp) (SEQ ID NO: 77), HBB exon 3 (SEQ ID NO: 76), codon-optimized hPKP2 (SEQ ID NO: 72), bovine poly A (229 bp) (SEQ ID NO: 94), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct T optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complementary sequences) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complementary sequences), or fragments thereof. Construct U was 3896 bp in length (SEQ ID NO: 65) and comprised the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), hTNNT2 promoter (533 bp) (SEQ ID NO: 82), globin intron (131 bp) (SEQ ID NO: 77), HBB exon 3 (SEQ ID NO: 76), codon-optimized hPKP2 with globin intron (131 bp) (SEQ ID NO: 77) between exons (SEQ ID NO: 73), bovine poly A (229 bp) (SEQ ID NO: 94), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct U optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or its complementary sequence) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or its complementary sequence), or a fragment thereof; and construct V is 3765 bp in length (SEQ ID NO: 68) and comprises the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), hTNNT2 promoter (533 bp) (SEQ ID NO: 81), globin intron (131 bp) (SEQ ID NO: 77), HBB exon 3 (SEQ ID NO: 76), codon-optimized hPKP2 (SEQ ID NO: 73), bovine poly A (229 bp) (SEQ ID NO: In other embodiments, construct V optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complementary sequences) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complementary sequences), or fragments thereof. Construct W was 3691 bp in length (SEQ ID NO: 128) and comprised the following elements from 5' to 3': 5' AAV2-ITR (SEQ ID NO: 101), hTNNT2 promoter (533 bp) (SEQ ID NO: 82), wild-type hPKP2 with a globin intron (131 bp) (SEQ ID NO: 77) between exons (SEQ ID NO: 129), bovine poly A (223 bp) (SEQ ID NO: 90), and 3' AAV2 ITR (SEQ ID NO: 105). In other embodiments, construct W optionally comprises any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 101-104 (or their complementary sequences) and/or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 105-108 (or their complementary sequences), or fragments thereof.

In any of the foregoing embodiments, the vector construct comprises at least one ITR sequence. Exemplary ITR sequences include but are not limited to SEQ ID NOs: 67-74, including any complementary sequences and/or combinations thereof.

Polynucleotides and polypeptides, including modified forms, can be prepared using a variety of standard cloning, recombinant DNA techniques, by cell expression or in vitro translation and chemical synthesis techniques known to those skilled in the art (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed.). Gene Delivery Methods .

Also provided is a method of delivering a gene of interest using a transgene, vector construct, donor construct, or viral vector or viral particle described herein, or a pharmaceutical composition described herein. Also provided is a method of delivering a gene encoding a protein of interest using a vector construct or AAV particle described herein. In one embodiment, the gene delivery vector can be a viral gene delivery vector, such as a viral particle, or a non-viral gene delivery vector, such as a vector construct or nucleic acid encoding a protein of interest. Viral vectors include lentiviral vectors, adenoviral vectors, and herpes virus vectors. Preferably, it is a recombinant adeno-associated virus (rAAV) vector. Alternatively, non-viral systems can be used, including the use of naked DNA (with or without chromatin attachment regions) or conjugated DNA, which is introduced into cells by various transfection methods such as lipid or electroporation.

Non-limiting examples of vector constructs described herein include any one of SEQ ID NOs: 3-68 or 126-128.

In some embodiments, the vector construct or AAV vector genome comprises a nucleotide sequence having at least about 80%, 85%, 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to any one of SEQ ID NOs: 3-68 or 126-128 (over the entire length of SEQ ID NOs: 3-68 or 126-128, respectively). In some embodiments, the vector construct comprises a nucleotide sequence having at least about 95% sequence identity to any one of SEQ ID NOs: 3-68 or 126-128. Preferably, the vector construct of the AAV particle or the AAV vector genome comprises a nucleotide sequence having at least about 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5% or higher sequence identity to any one of SEQ ID NOs: 3-68 or 126-128. Even more preferably, the nucleotide sequence of the vector construct is at least 97% or 98% or 99% or higher identical to any one of SEQ ID NOs: 3-68 or 126-128. In other embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98% or 99% identical to the following nucleotide sequence: the nucleotide sequence is complementary or negative (-) stranded to any one of SEQ ID NOs: 3-68 or 126-128.

In some embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98% or 99% identical to any one of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66 or 126. In other embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98% or 99% identical to any one of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66 or 126.

In some embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98% or 99% identical to any one of SEQ ID NO: 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67 or 127. In other embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98% or 99% identical to any one of SEQ ID NO: 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67 or 127.

In some embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98% or 99% identical to any one of SEQ ID NO: 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68 or 128. In other embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98% or 99% identical to any one of 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68 or 128.

The invention may be used in veterinary and medical applications. Subjects suitable for the gene delivery methods described herein include avians and mammals, with mammals being preferred and humans being most preferred. Human subjects include newborns, infants, adolescents, and adults. Donor Construct Delivery .

In some embodiments, DNA can be integrated from a donor construct containing a gene of interest and introns described herein flanked by homology arms. DNA from a donor construct can also be integrated into the genome via targeted gene editing methods with nucleases such as ZFNs, TALENs, meganucleases, or CRISPR-Cas9. The targeted nuclease cleaves the target site in the gene, and donor constructs containing homology arms that are complementary to the region flanking the target site facilitate homology directed repair, which results in integration of sequences between the homology arms. The donor construct or donor template can be, for example, a single-stranded donor oligonucleotide (ssODN), double-stranded DNA (e.g., a PCR product), a minicircle, or a virus (rAAV or lentivirus). Non-viral gene delivery .

Non-viral gene delivery can be carried out using naked DNA, which is the simplest non-viral transfection method. For example, it may be possible to administer the vector constructs provided herein using naked plasmid DNA. Alternatively, the vector construct can be delivered using methods involving: electroporation; sonoporation; biolistic or using a "gene gun" which uses, for example, high pressure gas or an inverted .22 caliber gun (Helios® Gene Gun System (BIO-RAD)) to shoot DNA-coated gold particles into cells; microinjection; laser; hyperthermia; ultrasound; hydrodynamic gene transfer; magnetofection; chemical transfection (e.g., calcium phosphate, DEAE-polydextrose); liposomes; lipoplexes; dendrimers; lipid nanoparticles or inorganic nanoparticles, all of which are known in the art.

In order to improve the delivery of a vector construct into cells, it may be necessary to protect it from damage and facilitate its entry into the cell. For this purpose, lipid complexes and polymer complexes can be used that are able to protect the nucleic acid from unwanted degradation during the transfection process.

The vector construct can be coated with lipids in an organized structure such as micelles or liposomes. When the organized structure is complexed with DNA, it is called a lipoplex. Anionic and neutral lipids can be used to construct lipoplexes of synthetic vectors. In one embodiment, cationic lipids can be used to condense negatively charged DNA molecules due to their positive charge to promote DNA encapsulation into liposomes. If necessary, auxiliary lipids (usually neutral lipids such as DOPE) are added to the cationic lipids to form lipoplexes (Dabkowska et al., JR Soc. Interface. 9(68): 548-61 (2012).

In certain embodiments, complexes of polymers and DNA, called polyplexes, can be used to deliver vector constructs. Most polyplexes consist of cationic polymers, and their production is regulated by ionic interactions. Polyplexes are generally unable to release their DNA cargo into the cytoplasm. Therefore, co-transfection with an endosomolytic agent (to dissolve endosomes generated during endocytosis, the process by which polyplexes enter cells) such as an inactivated adenovirus may be required (Akinc et al., J. Gene Medic. 7 (5): 657-63).

In certain embodiments, hybrid approaches can be used to deliver vector constructs that combine two or more technologies. Virosomes are one example; they combine liposomes with inactivated HIV or influenza viruses. In another embodiment, other approaches involve mixing other viral vectors with cationic lipids or hybridizing viruses and can be used to deliver nucleic acids (Khan, Firdos Alam, Biotechnology Fundamentals, CRC Press, November 18, 2015, page 395).

In certain embodiments, dendrimers can be used to deliver vector constructs, particularly cationic dendrimers, i.e., dendrimers with positive surface charges. When in the presence of genetic material such as DNA or RNA, charge complementarity causes a temporary association of nucleic acid with the cationic dendrimer. Upon reaching the destination, the dendrimer-nucleic acid complex is then introduced into the cell via endocytosis (Amiji, Mansoor M., ed., Polymeric Gene Delivery: Principles and Applications, CRC Press, September 29, 2004, p. 142). Viral particles .

In one embodiment, a suitable viral gene delivery vector, such as a viral particle, can be used to deliver nucleic acid. In certain embodiments, the viral gene delivery vector suitable for use herein can be a small virus, an adenovirus, a retrovirus, a gamma-retrovirus, a lentivirus, herpes simplex virus, poxvirus, measles virus, vesicular stomatitis virus, poliovirus, or Riovirus. The small virus can be an adenovirus-associated virus (AAV).

Therefore, the present invention provides viral particles (including vector constructs provided herein) for use as gene delivery vectors, which are based on animal miniviruses, particularly dependoviruses, such as infectious human or simian AAV, and components thereof (e.g., animal minivirus genomes), for introduction and/or expression of PKP2 protein in mammalian cells. Therefore, as used herein, the term "minivirus" encompasses dependoviruses, such as any type of AAV.

Viruses of the family Parvoviridae are small DNA animal viruses. The family Parvovirinae can be divided into two subfamilies: the Parvovirinae, which infect vertebrates, and the Denuvirinae, which infect insects. Members of the Parvovirinae are referred to herein as miniviruses and include the genus Dependent virus. As can be inferred from their genus name, Dependent virus members are unique in that they typically require co-infection with a helper virus, such as adenovirus or herpes virus, for productive infection in cell culture. The Dependent virus genus includes AAVs that typically infect humans (e.g., serotypes 1, 2, 3A, 3B, 4, 5, and 6), primates (e.g., serotypes 1 and 4), and related viruses that infect other warm-blooded animals except birds and reptiles (e.g., bovine, canine, equine, mouse, rat, and ovine adenovirus-related viruses). More information about parvoviruses and other members of the Parvoviridae family is described in Kenneth I. Berns, "Parvoviridae: The Viruses and Their Replication," Fields Virology, Chapter 69 (3rd ed. 1996). For convenience, the present invention is further exemplified and described herein by reference to AAV. However, it should be understood that the present invention is not limited to AAV, but is equally applicable to other parvoviruses.

The production of AAV particles requires the AAV "rep" and "cap" genes, which are genes encoding replication and encapsidation proteins, respectively. AAV rep and cap genes have been found in all AAV serotypes examined to date and are described herein and in the references cited. In wild-type AAV, the rep and cap genes are generally found adjacent to each other in the viral genome (i.e., they are "coupled" together in adjacent or overlapping transcription units) and are generally conserved among AAV serotypes. The AAV rep and cap genes are also independently and collectively referred to as "AAV packaging genes." The AAV cap gene used herein encodes the Cap protein, which is capable of packaging AAV vectors in the presence of rep and adeno-helper functions and is capable of binding to target cellular receptors. In some embodiments, the AAV cap gene encodes a capsid protein having an amino acid sequence derived from a particular AAV serotype.

AAV sequences used to generate AAV can be derived from the genome of any AAV serotype. In general, AAV serotypes have genomic sequences that are significantly homologous at the amino acid and nucleic acid levels, provide a similar set of genetic functions, produce physically and functionally essentially equivalent viral particles, and replicate and assemble by virtually identical mechanisms. Discussion of genomic sequences and genomic similarities among AAV serotypes. (See, e.g., GenBank Accession No. U89790; GenBank Accession No. J01901; GenBank Accession No. AF043303; GenBank Accession No. AF085716; Chiorini et al., J. Virol. 71: 6823-33 (1997); Srivastava et al., J. Virol. 45: 555-64 (1983); Chiorini et al., J. Virol. 73: 1309-19 (1999); Rutledge et al., J. Virol. 72: 309-19 (1998); and Wu et al., J. Virol. 74: 8635-47 (2000)).

The genome organization of all known AAV serotypes is very similar. The AAV genome is a linear, single-stranded DNA molecule of less than approximately 5,000 nucleotides (nt) in length. Inverted terminal repeats (ITRs) flank unique nucleotide sequences encoding nonstructural replicative (Rep) and structural (VP) proteins. The VP proteins form the capsid. The assembly activating protein (AAP) rapidly chaperones capsid assembly and protects free capsid proteins from degradation (Grosse et al., J. Virol . 91(20): e01198-17 (2017). The terminal 145 nt are self-complementary and organized to enable the formation of an energetically stable intramolecular double helix that forms a T-shaped hairpin. These hairpin structures serve as the starting point for viral DNA replication, acting as primers for the cellular DNA polymerase complex. The Rep gene encodes the Rep proteins Rep78, Rep68, Rep52, and Rep40. Rep78 and Rep68 are transcribed from the p5 promoter, and Rep 52 and Rep40 are transcribed from the p19 promoter. The cap gene encodes VP proteins VP1, VP2 and VP3. The cap gene is transcribed from the p40 promoter. The ITRs used in the vectors of the embodiments of the present invention may correspond to the same serotype as the associated cap gene, or may be different. In one embodiment, the ITRs used herein correspond to the AAV9 serotype, and the cap gene corresponds to the AAV9 serotype.

It is known that the AAV VP protein determines the cellular tropism of the AAV virion. The conservation of the VP protein coding sequence is significantly lower than that of the Rep protein and gene among different AAV serotypes. The ability of the Rep and ITR sequences to cross-complement the corresponding sequences of other serotypes allows the generation of pseudo-patterned AAV particles comprising capsid proteins of one serotype (e.g., AAV1, 5, 8, or 9) and Rep and/or ITR sequences of another AAV serotype (e.g., AAV2). Such pseudo-patterned rAAV particles are part of the present invention.

The AAV particles described herein (and the encoding AAV vector genome) may comprise any of the capsid proteins described in WO-2018/022608 or WO-2019/222136, the disclosures of which regarding human and simian AAV capsids and their properties, such as transduction efficiency, tissue tropism, glycan binding, and resistance to IVIG neutralization, are incorporated herein by reference in their entirety, including but not limited to any of the capsids in the sequence listing and variants thereof, such as those having chimeric exchange variable regions and/or glycan binding sequences and/or GH loops.

In one embodiment, the AAV ITR sequences used in the context of the present invention are derived from AAV1, AAV2, AAV4, AAV6 and/or AAV9. Similarly, in one embodiment, the Rep (e.g., Rep78 and Rep52) coding sequences are derived from AAV1, AAV2, AAV4, AAV6 and/or AAV9. However, the sequences encoding the VP1, VP2 and VP3 capsid proteins for use in the context of the present invention may be obtained from any serotype, such as from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or AAV12, or from simian AAV, including any of the capsid proteins described in WO 2018/022608 or PCT/US19/AAV32097, or from newly developed AAV-like particles obtained by, for example, capsid shuffling techniques and AAV capsid libraries, or any capsid that is at least 90% identical to any one of SEQ ID NOs: 109-125.

For example, the amino acid sequences of various capsids are disclosed. See, for example AAVRh.1 / hu.14 / AAV9 AAS99264.1 (SEQ ID NO: 109) AAVRh.8 SEQ97 (SEQ ID NO: 110) in U.S. Patent Publication 2013/0045186 AAVRh.10 SEQ81 (SEQ ID NO: 111) in U.S. Patent Publication 2013/0045186 AAVRh.74 SEQ 1 (SEQ ID NO: 112) in International Patent Publication WO 2013/123503 AAV1 AAB_95452.1 (SEQ ID NO: 113) AAV2 YP_680426.1 (SEQ ID NO: 114) AAV3 NP_043941.1 (SEQ ID NO: 115) AAV3B AAB95452.1 (SEQ ID NO: 116) AAV4 NP_044927.1 (SEQ ID NO: 117) AAV5 YP_068409.1 (SEQ ID NO: 118) AAV6 AAB95450.1 (SEQ ID NO: 119) AAV7 YP_077178.1 (SEQ ID NO: 120) AAV8 YP_077180.1 (SEQ ID NO: 121) AAV10 AAT46337.1 (SEQ ID NO: 122) AAV11 AAT46339.1 (SEQ ID NO: 123) AAV12 ABI16639.1 (SEQ ID NO: 124) AAV13 ABZ10812.1 (SEQ ID NO: 125)

Modified "AAV" sequences can also be used in the context of the present invention, for example, for making AAV gene therapy vectors. Such modified sequences, for example, sequences having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or more nucleotide and/or amino acid sequence identity to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9 ITR, Rep or VP (e.g., sequences having about 75-99% nucleotide sequence identity), can be used to replace wild-type AAV ITR, Rep or VP sequences.

In some embodiments, the nucleic acid sequence encoding the AAV capsid protein is operably linked to an expression control sequence for expression in a specific cell type, such as Sf9 or HEK cells. The embodiments can be practiced using techniques known to those skilled in the art for expressing foreign genes in insect host cells or mammalian host cells. Methods for molecular engineering and expressing polypeptides in insect cells are described, for example, in Summers and Smith (1986) A Manual of Methods for Baculovirus Vectors and Insect Culture Procedures, Texas Agricultural Experimental Station Bull. No. 7555, College Station, Tex.; Luckow (1991) In Prokop et al., Cloning and Expression of Heterologous Genes in Insect Cells with Baculovirus Vectors' Recombinant DNA Technology and Applications, 97-152; King, L. A. and R. D. Possee (1992) The baculovirus expression system, Chapman and Hall, United Kingdom; O'Reilly, D. R., L. K. Miller, V. A. Luckow (1992) Baculovirus Expression Vectors: A Laboratory Manual, New York; W. H. Freeman and Richardson, C. D. (1995) Baculovirus Expression Protocols, Methods in Molecular Biology, Vol. 39; U.S. Patent No. 4,745,051; US-2003148506; and WO-03/074714, all of which are incorporated herein by reference in their entirety. A promoter particularly suitable for transcribing a nucleotide sequence encoding an AAV capsid protein is, for example, the polyhedron promoter. However, other promoters active in insect cells are known in the art, such as the p10, p35 or IE-1 promoters, and the other promoters described in the above references are also encompassed.

The use of insect cells for the expression of heterologous proteins and methods for introducing nucleic acids (e.g., vectors, e.g., insect cell-compatible vectors) into such cells and methods for maintaining such cells in culture are well established. (See, e.g., METHODS IN MOLECULAR BIOLOGY, ed. Richard, Humana Press, NJ (1995); O'Reilly et al., BACULOVIRUS EXPRESSION VECTORS, A LABORATORY MANUAL, Oxford Univ. Press (1994); Samulski et al., J. Virol. 63: 3822-8 (1989); Kajigaya et al., Proc. Nat'l . Acad. Sci. USA , 88: 4646-50 (1991); Ruffing et al., J. Virol. 66: 6922-30 (1992); Kirnbauer et al., Virol. 219: 37-44 (1996); Zhao et al., Virol . 272: 382-93 (2000); and U.S. Patent No. 6,204,059). In some embodiments, the nucleic acid construct encoding an AAV protein (e.g., an AAV rep or cap protein) in an insect cell is an insect cell-compatible vector. As used herein, an "insect cell-compatible vector" or "vector" refers to a nucleic acid molecule capable of productive transformation or transfection of an insect or insect cell. Exemplary biological vectors include plasmids, linear nucleic acid molecules, and recombinant viruses. Any vector may be used as long as it is insect cell-compatible. The vector may be integrated into the insect cell genome, but the vector need not be permanently present in the insect cell, and transient episomal vectors are also included. The vector can be introduced by any known means, such as by chemical treatment of cells, electroporation or infection. In some embodiments, the vector is a bacilli, a viral vector or a plasmid. In one embodiment, the vector is a bacilli, that is, the construct is a bacilli vector. Bacilli vectors and methods of using them are described in the references cited above regarding molecular engineering of insect cells. Methods of Producing Recombinant AAV Particles

The present invention provides materials and methods for producing recombinant AAV particles in insect or mammalian cells comprising any of the vector constructs described herein. In some embodiments, the vector construct further comprises a promoter and a restriction site downstream of the promoter to allow insertion of a polynucleotide encoding one or more proteins of interest, wherein the promoter and restriction site are located downstream of the 5' AAV ITR and upstream of the 3' AAV ITR. In some embodiments, the vector construct further comprises a post-transcriptional regulatory element downstream of the restriction site and upstream of the 3' AAV ITR. In some embodiments, the vector construct further comprises a polynucleotide inserted into the restriction site and operably linked to the promoter, wherein the polynucleotide comprises a coding region for a protein of interest. Those skilled in the art will appreciate that any of the AAV vector constructs disclosed in this application can be used in a method for producing recombinant AAV particles.

In some embodiments, the helper functions for producing AAV are provided by one or more helper plasmids or helper viruses comprising adenoviral or bacilli helper genes. Non-limiting examples of adenoviral or bacilli helper genes include, but are not limited to, E1A, E1B, E2A, E4, and VA, which can provide helper functions for AAV packaging.

Helper viruses for AAV are known in the art and include, for example, viruses from the Adenoviridae and Herpes viridae families. Examples of helper viruses for AAV include, but are not limited to, the SAdV-13 helper virus and SAdV-13-like helper virus described in U.S. Publication No. 20110201088 (the disclosure of which is incorporated herein by reference), and the helper vector pHELP (Applied Viromics). Those skilled in the art will appreciate that any helper virus or helper plasmid of AAV that can provide sufficient helper function to AAV can be used herein.

In some embodiments, the AAV cap gene is present in a plasmid. The plasmid may further comprise an AAV rep gene, which may or may not correspond to the same serotype as the cap gene. Cap genes and/or rep genes from any AAV serotype described herein (including but not limited to AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, and any variants thereof) may be used to generate recombinant AAV. In some embodiments, the AAV cap gene encodes a capsid from serotype 1, serotype 2, serotype 4, serotype 5, serotype 6, serotype 7, serotype 8, serotype 9, serotype 10, serotype 11, serotype 12, serotype 13, or variants thereof.

In some embodiments, insect or mammalian cells can be transfected with a helper plasmid or helper virus, a vector construct encoding an AAV cap gene, and a plasmid; and the recombinant AAV virus can be collected at different time points after co-transfection. For example, the recombinant AAV virus can be collected at about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 96 hours, about 120 hours, or a time between any of these two time points after co-transfection.

Recombinant AAV particles may also be produced using any known method known in the art suitable for producing infectious recombinant AAV. In some cases, recombinant AAV may be produced by using insect or mammalian cells that stably express some of the components required for the production of AAV particles. For example, a plasmid (or plasmids) containing the AAV rep and cap genes and a selectable marker (such as a neomycin resistance gene) may be integrated into the genome of the cell. The insect or mammalian cell may then be co-infected with a helper virus (e.g., an adenovirus or bacilli that provides helper functions) and a viral vector construct containing the 5' and 3' AAV ITRs (and, if desired, nucleotide sequences encoding heterologous proteins). The advantage of this method is that these cells are selectable and suitable for large-scale production of recombinant AAV particles. As another non-limiting example, adenovirus or bacilli can be used instead of plasmids to introduce rep and cap genes into packaging cells. As yet another non-limiting example, a viral vector construct containing 5' and 3' AAV LTRs and rep-cap genes can be stably integrated into the DNA of production cells, and helper functions can be provided by wild-type adenovirus to produce recombinant AAV.

In one embodiment, the present invention provides a method for producing AAV particles that can be used as gene delivery vectors, the method comprising the following steps: (a) providing one or more nucleic acid constructs to cells that allow AAV replication (e.g., insect cells or mammalian cells), which contain: (i) a nucleic acid molecule provided herein (e.g., a recombinant vector construct), which is flanked by at least one AAV reverse terminal repeat nucleotide sequence; (ii) a nucleotide sequence encoding one or more AAV Rep proteins, which is operably linked to a promoter capable of driving the expression of the one or more Rep proteins in the cell; (iii) a nucleotide sequence encoding one or more AAV capsid proteins, which is operably linked to a promoter capable of driving the expression of the one or more capsid proteins in the cell; (iv) and optionally AAP and MAAP contained in VP2/3 mRNA (b) culturing the cells defined in (a) under conditions conducive to the expression of Rep protein and capsid protein; and optionally (c) recovering the AAV gene delivery vector, and optionally (d) purifying the AAV particles. For example, the recombinant vector construct of (i) comprises (1) at least one AAV ITR, (2) the heterologous cardiomyocyte-specific transcriptional regulatory region described herein, and (3) a nucleic acid encoding functional PKP2. Preferably, the recombinant vector construct of (i) comprises 5' and 3' AAV ITRs.

Typically, then, the methods provided herein for producing an AAV gene delivery vector comprise: providing to cells permissive for AAV replication (a) a nucleotide sequence encoding a template for producing a vector genome, such as a vector construct of the present invention (as described in detail herein); (b) a nucleotide sequence sufficient to replicate the template to produce the vector genome (the first expression cassette defined above); and (c) a nucleotide sequence sufficient to package the vector genome into an AAV capsid under conditions sufficient to replicate the vector genome and package it into an AAV capsid (the second expression cassette defined above), thereby producing AAV particles comprising the vector genome encapsulated in the AAV capsid in the cells.

Transient transfection of adherent HEK293 cells (Chahal et al., J. Virol. Meth . 196: 163-73 (2014)) and transfection of Sf9 cells using the bacilliform viral expression vector system (BEVS) (Mietzsch et al., Hum. Gene Ther . 25: 212-22 (2014)) are the two most commonly used methods for producing AAV vectors.

Viral particles comprising the vector constructs described herein can be produced using any cell type, such as mammalian and invertebrate cell types that allow production of AAV or biologics and can be maintained in culture.

There are a variety of methods for producing AAV viral particles: for example, but not limited to, transfection using a vector and AAV helper sequences combined with co-infection with one of the AAV helper viruses (e.g., adenovirus, herpes virus, or vaccinia virus), or transfection with a recombinant AAV vector, an AAV helper vector, and an accessory function vector. Methods for making AAV viral particles are described, for example, in U.S. Patent Nos. 6204059, 5756283, 6258595, 6261551, 6270996, 6281010, 6365394, 6475769, 6482634, 6485966, 6943019, 6953690, 7022519, 7238526, 7291498, 7491508, 5064764, 6194191, 65 66118, US8137948; or International Publication Nos. WO1996039530, WO1998010088, WO1999014354, WO1999015685, WO1999047691, WO2000055342, WO2000075353, WO2001023597, WO2015191508, WO2019217513, WO2018022608, WO2019222136, WO2020232044, WO2019222132; Methods In Molecular Biology, ed. Richard, Humana Press, NJ (1995); O'Reilly et al., Baculovirus Expression Vectors, A Laboratory Manual, Oxford Univ. Press (1994); Samulski et al., J. Vir. 63:3822-8 (1989); Kajigaya et al., Proc. Nat'l. Acad. Sci. USA 88: 4646-50 (1991); Ruffing et al., J. Vir. 66:6922-30 (1992); Kimbauer et al., Vir., 219:37-44 (1996); Zhao et al., Vir. 272:382-93 (2000); the contents of each of which are incorporated herein by reference in their entirety. For detailed descriptions of methods for producing AAV viral particles, see, e.g., U.S. Patent Nos. 6,001,650, 6,004,797, and 9,504,762, each of which is incorporated herein by reference in its entirety. In one embodiment, a triple transfection method (see, e.g., U.S. Patent No. 6,001,650, which is incorporated herein by reference in its entirety) is used to produce AAV viral particles. This method does not require the use of infectious helper viruses, thereby enabling the production of AAV viral particles in the absence of any detectable helper viruses. This is achieved by using three vectors for producing AAV viral particles, namely, an AAV helper function vector, an accessory function vector, and an AAV viral particle expression vector. However, it will be appreciated by those skilled in the art that the nucleic acid sequences encoded by such vectors can be provided on two or more vectors in various combinations. In other embodiments, host cells can be transfected with a helper plasmid or a helper virus, a viral construct, and a plasmid encoding an AAV cap gene; and AAV viral particles can be collected at different time points after co-transfection.

For example, wild-type AAV and helper viruses can be used to provide the replication functions necessary for producing AAV viral particles (see, e.g., U.S. Patent No. 5,139,941, which is incorporated herein by reference in its entirety). Alternatively, a plasmid containing a helper function gene in combination with infection with one of the well-known helper viruses can be used as a source of replication function (see, e.g., U.S. Patent No. 5,622,856 and U.S. Patent No. 5,139,941, both of which are incorporated herein by reference in their entirety). Similarly, a plasmid containing an accessory function gene can be used in combination with infection with wild-type AAV to provide the necessary replication functions. Other methods described herein and/or known in the art can also be used by those skilled in the art to produce AAV viral particles.

The term "vector" should be understood to refer to any genetic element, such as a plasmid, a phage, a transposon, a cosmid, a bacilli plasmid, a miniplasmid (e.g., a plasmid without bacterial elements), a doggybone DNA (e.g., a minimal closed linear construct), a chromosome, a virus, a virion (e.g., a bacilli virus), etc., which is capable of replication and transfer of genetic sequences between cells when combined with appropriate control elements. As used herein, a "mammalian cell-compatible vector" or "vector" refers to a nucleic acid molecule capable of productive transformation or transfection of a mammal or a mammalian cell. As used herein, an "insect cell-compatible vector" or "vector" refers to a nucleic acid molecule capable of productive transformation or transfection of an insect or an insect cell. Exemplary biological vectors include plasmids, linear nucleic acid molecules, and recombinant viruses. Any vector may be used as long as it is compatible with insect cells. The vector may be integrated into the insect cell genome, but the vector need not be permanently present in the insect cell, and also includes transient free vectors. The vector may be introduced by any known means, such as by chemical treatment of cells, electroporation, or infection. The vector and its use are described in the references cited above for molecular engineering of cells.

The vector for the cell production of rAAV vector genome may contain a promoter and a restriction site downstream of the promoter to allow insertion of polynucleotides encoding one or more proteins of interest, wherein the promoter and restriction site are located downstream of the 5' AAV ITR and upstream of the 3' AAV ITR. The vector may also contain a post-transcriptional regulatory element downstream of the restriction site and upstream of the 3' AAV ITR. The viral construct may further include a polynucleotide inserted into the restriction site and operably linked to the promoter, wherein the polynucleotide comprises a coding region for a protein of interest. In some embodiments, the viral construct further includes a promoter and a restriction site downstream of the promoter to allow insertion of polynucleotides encoding one or more proteins of interest, wherein the promoter and restriction site are located downstream of the 5' AAV ITR and upstream of the 3' AAV ITR. In some embodiments, the viral construct further comprises a post-transcriptional regulatory element downstream of the restriction site and upstream of the 3' AAV ITR. In some embodiments, the viral construct further comprises a polynucleotide inserted into the restriction site and operably linked to a promoter, wherein the polynucleotide comprises a coding region for a protein of interest. Those skilled in the art will appreciate that any of the AAV vectors disclosed in this application can be used as a viral construct in a method to produce rAAV virions.

The term "AAV helper" refers to AAV-derived coding sequences that can be expressed to provide AAV gene products that in turn act in trans for productive AAV replication. Thus, AAV helper functions include both the major AAV open reading frames (ORFs) rep and cap. The Rep expression product has been shown to have many functions, including, among others: recognition, binding, and cleavage of the AAV origin of DNA replication; DNA helicase activity; and regulation of transcription from AAV (or other heterologous) promoters. The capsid (Cap) expression product supplies the essential packaging function. AAV helper functions are used herein to complement trans-acting AAV functions that are missing in the AAV vector genome.

For production, cells with AAV helper function produce recombinant capsid proteins sufficient to form capsids. This includes at least VP1 and VP3 proteins, but more typically, all three of the VP1, VP2, and VP3 proteins found in native AAV. The sequence of the capsid protein determines the serotype of the AAV virions produced by the host cell. Capsids that can be used in the present invention include capsids derived from a variety of AAV serotypes, including 1, 2, 3, 3B, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or mixed serotypes (see, e.g., U.S. Patent No. 8,318,480 for disclosure of non-natural mixed serotypes). The capsid protein can also be a variant of natural VP1, VP2, and VP3, including mutant, chimeric, or reorganized proteins. The capsid protein can be rh.10 or other subtypes within the various clades of AAV; various clades and subtypes are disclosed, for example, in U.S. Patent No. 7,906,111. Due to the wide availability and extensive characterization of the constructs, the illustrative AAV vectors disclosed below are all derived from serotype 2. The construction and use of AAV vectors and AAV proteins of different serotypes are discussed in Chao et al., Mol. Ther. 2:619-623, 2000; Davidson et al., PNAS 97:3428-3432, 2000; Xiao et al., J. Virol. 72:2224-2232, 1998; Halbert et al., J. Virol. 74:1524-1532, 2000; Halbert et al., J. Virol. 75:6615-6624, 2001; and Auricchio et al., Hum. Molec. Genet. 10:3075-3081, 2001.

In various embodiments, the nucleotide sequence encoding the VP protein is operably linked to a suitable expression control sequence. In various embodiments, the nucleotide sequence encoding the Rep protein is operably linked to a suitable expression control sequence, such as a eukaryotic promoter. For example, the nucleotide sequence is operably linked to a eukaryotic promoter, such as SV40 promoter, CMV promoter, RSV promoter, UBC promoter, EF1A promoter, PGK promoter, dihydrofolate reductase promoter, b-actin promoter, TRE (Tet, Tet-On, Tet-Off) promoter, Cumate control system (CuR/CuO) (see US2004/0205834), temperature-induced HSP70 promoter, p5 promoter, p10 promoter, p19 promoter and p40 promoter. In another example, the nucleotide sequence is operably linked to a bacillivirus promoter, such as a polyhedrin (Polh) promoter, a ΔIE1 promoter, a p5 promoter, a p10 promoter, a p19 promoter, a p40 promoter, a metallothionein promoter, a 39K promoter, a p6.9 promoter, and an orf46 promoter.

For production, cells with AAV helper function will produce Rep proteins to promote the production of rAAV. It has been found that when at least one large Rep protein (Rep78 or Rep68) and at least one small Rep protein (Rep52 and Rep40) are expressed in the cell, infectious particles can be produced. In a specific embodiment, all four of Rep 78, Rep68, Rep52 and Rep 40 are expressed. Alternatively, Rep78 and Rep52, Rep78 and Rep40, Rep 68 and Rep52, or Rep68 and Rep40 are expressed. The following examples demonstrate the use of Rep78/Rep52 combinations. Rep proteins can be derived from AAV-2 or other serotypes. In various embodiments, the nucleotide sequence encoding the Rep protein can be operably linked to a suitable expression control sequence. In various embodiments, the nucleotide sequence encoding the Rep protein is operably linked to a suitable expression control sequence, such as a eukaryotic promoter. For example, the nucleotide sequence is operably linked to a eukaryotic promoter, such as SV40 promoter, CMV promoter, RSV promoter, UBC promoter, EF1A promoter, PGK promoter, dihydrofolate reductase promoter, b-actin promoter, TRE (Tet, Tet-On, Tet-Off) promoter, Cumate control system (CuR/CuO) (see US2004/0205834), and temperature-induced HSP70 promoter, p5 promoter, p10 promoter, p19 promoter, and p40 promoter. In other examples, the nucleotide sequence is operably linked to a bacillivirus promoter, such as the polyhedrin (Polh) promoter, ΔIE1 promoter, p5 promoter, p10 promoter, p19 promoter, p40 promoter, metallothionein promoter, 39K promoter, p6.9 promoter, and orf46 promoter.

In some embodiments, the AAV cap gene is present in a plasmid or a bacilliform virus plasmid. The plasmid may further include an AAV rep gene, which may or may not correspond to the same serotype as the cap gene. The cap gene and/or the rep gene are from any AAV serotype.

Cells with AAV helper function can also produce assembly activation protein (AAP), which helps assemble the capsid. In various embodiments, the nucleotide sequence encoding AAP is operably linked to a suitable expression control sequence. For example, the nucleotide sequence is operably linked to a eukaryotic promoter. In other examples, the nucleotide sequence is operably linked to a bacillary viral promoter, such as the polyhedrin (Polh) promoter, the ΔIE1 promoter, the p5 promoter, the p10 promoter, the p19 promoter, the p40 promoter, the metallothionein promoter, the 39K promoter, the p6.9 promoter, and the orf46 promoter.

The term "non-AAV helper functions" refers to non-AAV derived viral and/or cellular functions that AAV replication depends on. Thus, the term encompasses proteins and RNAs required for AAV replication, including those involved in AAV gene transcriptional activation, stage-specific AAV mRNA splicing, AAV DNA replication, Cap expression product synthesis, and AAV capsid assembly. Virus-based accessory functions may be derived from any of the known helper viruses, such as adenovirus, herpes virus (except herpes simplex virus type 1), and vaccinia virus.

The term "non-AAV helper function vector" generally refers to a nucleic acid molecule that includes a nucleotide sequence that provides an accessory function. The accessory function vector can be transfected into a suitable host cell, wherein the vector is then capable of supporting the production of AAV virus particles in the host cell. The term expressly excludes infectious viral particles that occur in nature, such as adenovirus, herpes virus or vaccinia virus particles. Thus, the accessory function vector may be in the form of a plasmid, phage, transposon or cosmid. In particular, it has been demonstrated that the accessory helper function does not require the complete complement of an adenoviral gene. For example, adenovirus mutants that are incapable of DNA replication and subsequent gene synthesis have been shown to permit AAV replication. Ito et al., (1970) J. Gen. Virol. 9:243; Ishibashi et al., (1971) Virology 45:317. Similarly, mutants within the E2B and E3 regions have been shown to support AAV replication, suggesting that the E2B and E3 regions may not be involved in providing accessory functions. Carter et al., (1983) Virology 126:505. However, adenoviruses defective in the E1 region or deleted in the E4 region are unable to support AAV replication. Therefore, the E1A and E4 regions may be required directly or indirectly for AAV replication. Laughlin et al., (1982). J. Virol. 41:868; Janik et al., (1981) Proc. Natl. Acad. Sci. USA 78:1925; Carter et al., (1983) Virology 126:505. Other characterized Ad mutants include: E1B (Laughlin et al. (1982), supra; Janik et al. (1981), supra; Ostrove et al., (1980) Virology 104:502); E2A (Handa et al., (1975) J. Gen. Virol. 29:239; Strauss et al., (1976) J. Virol. 17:140; Myers et al., (1980) J. Virol. 35:665; Jay et al., (1981) Proc. Natl. Acad. Sci. USA 78:2927; Myers et al., (1981) J. Biol. Chem. 256:567); E2B (Carter, Adeno-Associated Virus Helper Functions, in I CRC Handbook of Parvoviruses (P. Tijssen, ed., (1990)); E3 (Carter et al. (1983), supra); and E4 (Carter et al. (1983), supra; Carter (1995)). Although studies of accessory functions provided by adenoviruses with mutations in the E1B coding region have produced conflicting results, Samulski et al., (1988) J. Virol. 62:206-210 recently reported that E1B55k, but not E1B19k, is required for AAV virion production. In addition, International Publication No. WO 97/17458 and Matshushita et al., (1998) Gene Therapy 5:938-945 describe accessory function vectors encoding various Ad genes. Particularly preferred accessory function vectors include adenovirus VA RNA coding region, adenovirus E4 ORF6 coding region, adenovirus E2A 72 kD coding region, adenovirus E1A coding region, and adenovirus E1B region lacking the complete E1B55k coding region. Such vectors are described in International Publication No. WO 01/83797.

In another embodiment, the methods provided herein are performed using any mammalian cell type that allows AAV replication or bioproduct production and can be maintained in culture. In one embodiment, the mammalian cells used can be HEK293, HeLa, CHO, NSO, SP2/0, PER.C6, Vero, RD, BHK, HT 1080, A549, Cos-7, ARPE-19 and MRC-5 cells.

The use of insect cells for the expression of heterologous proteins and methods for introducing nucleic acids (e.g., vectors, e.g., insect cell-compatible vectors) into such cells and methods for maintaining such cells in culture are well established. (See, e.g., METHODS IN MOLECULAR BIOLOGY, Richard, ed., Humana Press, N J (1995); O'Reilly et al., BACULOVIRUS EXPRESSION VECTORS, A LABORATORY MANUAL, Oxford Univ. Press (1994); Samulski et al., J. Vir. (1989) Vol. 63, pp. 3822-3828; Kajigaya et al., Proc. Nat'l. Acad. Sci. USA (1991) Vol. 88, pp. 4646-4650; Ruffing et al., J. Vir. (1992) Vol. 66, pp. 6922-6930; Kirnbauer et al., Vir. (1996) Vol. 219, pp. 37-44; Zhao et al., Vir. (2000) Vol. 272, pp. 37-44.) 382-393; and U.S. Patent No. 6,204,059). In some embodiments, the nucleic acid construct encoding AAV in an insect cell is an insect cell-compatible vector. "Expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector includes sufficient cis-acting elements for expression; other expression elements may be supplied by a host cell or an in vitro expression system. Expression vectors include all expression vectors known in the art, such as muscisomes, plasmids (e.g., plasmids contained in naked plasmids or liposomes), artificial chromosomes, and viruses incorporating recombinant polynucleotides. As used herein, "insect cell compatible vector" or "vector" refers to a nucleic acid molecule capable of productive transformation or transfection of an insect or insect cell. Exemplary biological vectors include plasmids, linear nucleic acid molecules, and recombinant viruses. Any vector can be used as long as it is compatible with insect cells. The vector can be integrated into the insect cell genome, but the vector does not need to be permanently present in the insect cell, and short-lived free-type vectors are also included. The vector can be introduced by any known means, such as by chemical treatment of cells, electroporation, or infection. In some embodiments, the vector is a bacillivirus, a viral vector, or a plasmid. In a more preferred embodiment, the vector is a bacillivirus, that is, the construct is a bacillivirus vector. Bacitraviral vectors and methods of their use are described in the references cited above regarding molecular engineering of insect cells.

For example, the insect cell strain used may be from Spodoptera frugiperda, such as SF9, SF21, SF900+; fruit fly cell strain; mosquito cell strain, such as Aedes albopictus derived cell strain; silkworm cell strain, such as Bombyx mori cell strain; Trichoplusia ni cell strain, such as High Five cell strain; or Lepidoptera cell strain, such as Ascalapha odorata cell strain. In one embodiment, the insect cell is a cell from an insect species susceptible to bacillivirus infection, including High Five, Sf9, Se301, SeIZD2109, SeUCR1, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAm1, BM-N, Ha2302, Hz2E5, and Ao38.

Baculoviruses are enveloped DNA viruses of arthropods, two members of which are well-known expression vectors for producing recombinant proteins in cell culture. Baculoviruses have a circular double-stranded genome (80-200 kbp) that can be engineered to allow delivery of large genomic content to specific cells. The viruses used as vectors are generally Autographa californica polycapsid nuclear polyhedrosis virus (AcMNPV) or house moth nuclear polyhedrosis virus (BmNPV) (Kato et al., Appl. Microbiol. Biotechnol . 85(3): 459-70 (2010).

Baculoviruses are commonly used to infect insect cells to express recombinant proteins. Specifically, heterologous gene expression in insects can be achieved as described, for example, in U.S. Patent No. 4,745,051; EP 127,839; EP 155,476; Vlak et al., J. Gen. Virol. 68: 765-76 (1988); Miller et al., Ann. Rev. Microbiol. 42: 177-9 (1988); Carbonell et al., Gene , 73(2): 409-18 (1998); Maeda et al., Nature , 315: 592-4 (1985); Lebacq-Veheyden et al., Molec. Cell. Biol . 8(8): 3129-35 (1988); Smith et al., PNAS , 82: 8404-8 (1985); and Miyajima et al., Gene , 58: 273-81 (1987). Many bacilli and variants useful for protein production and corresponding permissive insect host cells are described in Luckow et al., Nat. Biotechnol . 6: 47-55 (1988); Maeda et al., Nature , 315: 592-4 (1985); and McKenna et al., J. Invert. Pathol. 71(1): 82-90 (1998).

Baculovirus shuttle vectors or bacilli are used to produce bacilli. Baculovirus plasmids are propagated as large plasmids in bacteria such as E. coli. When transfected into insect cells, bacilli produce bacilli. In another embodiment, the methods provided herein are performed with any mammalian cell type that allows AAV replication or bioproduct production and can be maintained in culture. In one embodiment, the mammalian cells used can be HEK293, HeLa, CHO, NSO, SP2/0, PER.C6, Vero, RD, BHK, HT 1080, A549, Cos-7, ARPE-19, and MRC-5 cells.

rAAV particles can also be produced using the methods disclosed in the various embodiments. In some cases, rAAV particles can be produced by using insect or mammalian cells that stably express some of the components required for producing AAV particles. For example, a plasmid (or plasmids) comprising the AAV rep and cap genes and a selectable marker (such as a neomycin resistance gene) can be integrated into the genome of the cell. In another example, a plasmid (or plasmids) comprising a selectable marker (such as a neomycin resistance gene) can be integrated into the genome of the cell. The insect, fungal or mammalian cells can then be co-infected with a helper virus (e.g., an adenovirus or bacilli that provide helper functions) and a viral vector comprising the 5' and 3' AAV ITRs (and, if necessary, nucleotide sequences encoding heterologous proteins). The advantage of this approach is that the cells are selectable and suitable for large-scale production of rAAV. As another non-limiting example, host regulatory genes, rep genes and cap genes can be introduced into packaging cells using adenovirus or bacilli instead of plasmids.

In one embodiment, after the transfected cells are expanded in suspension cell culture through a series of increasingly larger culture platforms, the suspension of transfected cells is purified through a multi-step process to remove process impurities, including recombinant bacilli and host cells, and to enrich for viral particles comprising a recombinant small virus (rAAV) vector construct. In another embodiment, the methods provided herein may include a step of affinity purification of the rAAV vector construct using an anti-AAV antibody (in one embodiment, an immobilized antibody). In another embodiment, the anti-AAV antibody is a monoclonal antibody. One type of antibody used herein is a single-chain camel antibody or a fragment thereof, such as that obtained from camel or llama (see, e.g., Muyldermans, Biotechnol . 74: 277-302 (2001). Antibodies used for affinity purification of rAAV are antibodies that specifically bind to an antigenic determinant on an AAV capsid protein, whereby in one embodiment, the antigenic determinant is an antigenic determinant present on the capsid protein of more than one AAV serotype. For example, the antibody may be generated or selected based on specific binding to an AAV5 capsid, but may also specifically bind to an AAV1, AAV2, AAV3, AAV6, AAV8 or AAV9 capsid.

The methods provided herein for producing rAAV particles produce a population of rAAV particles. In some embodiments, the population is enriched for particles containing full-length or nearly full-length vector genomes by reducing the number of empty capsids.

The rAAV particle populations produced by the methods provided herein are, for example, used for administration in any of the therapeutic methods described herein. Host Organisms and / or Cells

In another embodiment, a host cell comprising the vector described above is provided. In one embodiment, the vector construct is capable of replicating or expressing the nucleic acid molecules provided herein in the host cell. In some embodiments, an ACM therapeutic agent is provided herein, which is a host cell comprising a vector construct comprising a nucleic acid encoding PKP2 for use in ACM cell therapy. The cell may be autologous or allogeneic to the individual.

As used herein, the term "host" refers to an organism and/or cell carrying a nucleic acid molecule or vector construct of the present invention, as well as an organism and/or cell suitable for expressing a recombinant gene or protein. The present invention is not intended to be limited to any particular type of cell or organism. In fact, it is contemplated that any suitable organism and/or cell may be used as a host herein. The host cell may be in the form of a single cell, a group of similar or different cells, such as a culture (such as a liquid culture or a culture on a solid substrate), an organism, or a portion thereof. In one embodiment, the host cell allows the expression of the nucleic acid molecules provided herein. Thus, the host cell may be, for example, a bacterial, yeast, insect or mammalian cell or a human cell.

In another embodiment, a method for delivering the nucleic acids provided herein to a wide range of cells (including dividing and non-dividing cells) is provided. The present invention can be used to deliver the nucleic acids provided herein to cells in vitro, for example, to produce polypeptides encoded by such nucleic acid molecules in vitro or for ex vivo gene therapy.

The nucleic acid molecules, vector constructs, cells and methods/uses of the present invention can also be used in methods for delivering the nucleic acids provided herein to a host, typically a host suffering from ACM. Pharmaceutical Formulations

In one embodiment, a pharmaceutical composition is provided, which comprises a nucleic acid or vector provided herein and a pharmaceutically acceptable diluent, excipient or carrier. The pharmaceutical composition may comprise a transgene, vector construct, donor construct, viral vector or viral particle described herein, such as a rAAV particle or a population of rAAV particles described herein. The pharmaceutical composition may further comprise a second therapeutic agent or adjuvant, etc. Preferably, if parenteral administration is intended, the composition is sterile. Preferably, the composition is free of infectious viruses and toxins. Preferably, the composition is stable for a suitable period of time under storage conditions.

"Pharmaceutically acceptable" means a material that is not biologically or otherwise undesirable, that is, the material can be administered to an individual without causing any undesirable biological effects. Thus, such pharmaceutical compositions can be used, for example, for in vitro cell transfection or for direct administration of viral particles or cells to an individual.

The carrier may be suitable for parenteral administration, including intravenous, intraperitoneal or intramuscular administration. Alternatively, the carrier may be suitable for sublingual or oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and reagents for pharmaceutically active substances is well known in the art. Unless any known media or reagents are incompatible with the active compound, they are considered for use in the pharmaceutical compositions provided herein.

In other embodiments, provided herein are pharmaceutical compositions (i.e., formulations) of AAV particles suitable for administration to individuals suffering from genetic diseases to deliver a gene of interest, such as a gene encoding a protein of interest. In certain embodiments, provided herein are pharmaceutical formulations that are liquid formulations comprising recombinant AAV particles comprising any of the vector constructs disclosed herein. The concentration of recombinant AAV virions in the formulation can vary.

In other embodiments, the AAV particle pharmaceutical formulations provided herein comprise one or more pharmaceutically acceptable excipients to provide the formulation with properties that facilitate storage and/or administration to a subject for treating a genetic disorder.

In certain aspects, the formulation comprising recombinant AAV particles further comprises one or more buffers.

In another embodiment, the recombinant AAV particle formulations provided herein may include one or more isotonic agents, such as sodium chloride. Other buffers and isotonic agents known in the art are suitable and can be routinely employed in the formulations provided herein.

In another embodiment, the recombinant AAV particle formulations provided herein may include one or more bulking agents. Exemplary bulking agents include, but are not limited to, mannitol, sucrose, polydextrose, lactose, trehalose, and povidone (PVP K24).

In yet another embodiment, the recombinant AAV particle formulations provided herein may include one or more surfactants, which may be non-ionic surfactants. Exemplary surfactants include ionic surfactants, non-ionic surfactants, and combinations thereof. For example, the surfactant may be, but is not limited to, TWEEN 80 (also known as polysorbate 80, or its chemical name polyoxyethylene sorbitan monooleate), sodium dodecyl sulfate, sodium stearate, ammonium lauryl sulfate, TRITON AG 98 (Rhone-Poulenc), poloxamer 407, poloxamer 188, and the like, and combinations thereof.

The recombinant AAV particle formulations provided herein are generally sterile and stable and can be stored for extended periods of time without unacceptable changes in quality, potency, or purity.

In some embodiments, isotonic agents such as sugars, polyols (such as mannitol, sorbitol) or sodium chloride are included in the composition. Extended absorption of injectable compositions can be achieved by including delayed absorption agents such as monostearate and gelatin in the composition. In certain embodiments, the nucleic acids or carrier constructs provided herein can be administered in time or controlled release formulations, such as in compositions including slow release polymers or other carriers (including implants and microencapsulation delivery systems) that protect the compound from rapid release. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic acid, polyglycolic acid copolymers (PLG) can be used, for example.

In certain embodiments, pharmaceutical compositions comprising a vector construct or AAV particle provided herein can be used to transfer genetic material into cells. Such transfer can be performed in vitro, ex vivo, or in vivo. Thus, one embodiment provides a method for delivering a nucleotide sequence to a cell, comprising contacting a nucleic acid, vector construct, or pharmaceutical composition described herein under conditions that allow the nucleic acid or vector provided herein to enter the cell. The cell can be a cell in vitro, ex vivo, or in vivo. Treatment Methods

The vector constructs or AAV particles described herein are administered to a subject in an amount effective to deliver the PKP2 gene to the heart of a mammalian subject. The subject is preferably a human, including a juvenile subject. The age range of the juvenile subject can be, for example, 0-2, 2-6, 2-10, 2-12, 2-15, 2-18, 12-18 or 0-18 years old.

Such methods include methods of expressing PKP2 in the heart of a mammalian subject, comprising administering to the subject an effective amount of a composition comprising a vector construct described herein, a rAAV particle described herein, or a pharmaceutical composition described herein, thereby expressing PKP2 in the subject's heart tissue (e.g., myocardium or myocardial cells).

Such methods also include a method of treating functional wild-type PKP2 deficiency in a mammalian subject by administering an amount of a vector construct, rAAV particle, or pharmaceutical composition effective to increase the level of functional PKP2 in cardiac tissue (e.g., cardiac myocytes). In one or more embodiments, such methods increase the amount of PKP2 expression in the heart by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% compared to the untreated amount, or to the amount seen in a healthy human. In some embodiments, the amount of the vector construct, rAAV particle, or pharmaceutical composition is effective to increase the level of PKP2 in cardiac tissue (e.g., cardiac myocytes) by at least about 2-fold; and/or improve the electrical conductivity of the myocardium. In some aspects, the amount is effective to reduce or eliminate the number of ICD shocks required per month, myocardial fibrosis or fat fibrosis, ventricular wall thinning, dilated cardiomyopathy (DCM), sudden cardiac death, reduce heart failure episodes, arrhythmias, chest pain, shortness of breath, fatigue and/or dizziness, and/or reduce other symptoms of ACM disease.

Such methods also include a method of treating ACM in a mammal, or treating or preventing any symptom thereof, comprising administering a therapeutically effective amount of a vector construct, rAAV particle, or pharmaceutical composition. In such methods, the mammal may have a mutation in one or both alleles of the PKP2 gene. For example, such methods reduce arrhythmias, reduce tissue damage, reduce fibrofatty scarring, reduce ventricular wall thinning, and/or reduce symptoms of the disease. In one or more embodiments, such methods reduce the frequency or severity of symptoms such as heart failure, arrhythmias, chest pain, shortness of breath, fatigue, and dizziness.

In any of the methods described herein, rAAV particles are delivered in an aqueous suspension at a dose of about 1e12 to about 6e14 vg/kg.

In any of the methods described herein, administration of the vector construct, rAAV particle, or pharmaceutical composition may further comprise administration of a prophylactic or therapeutic corticosteroid therapy, and/or may further comprise administration of a second therapeutic agent for the treatment of ACM, including but not limited to angiotensin converting enzyme (ACE) inhibitors or angiotensin II receptor blockers, beta blockers, calcium channel blockers, and/or antiarrhythmic drugs. In some cases, symptom-specific therapies are also administered, such as blood deviscosities to prevent clotting and diuretics to reduce swelling.

According to the second object of the present invention, when delivering a gene of interest for treating a cardiac disorder or condition, or a cardiac genetic disease or condition, the treatment method can, for example, restore contractile force, relative tension, calcium activated tension, and/or relaxation time in an engineered cardiac tissue in vitro or in mammalian tissue in vivo. For example, such methods reduce heart size, reduce heart-to-chest ratio, reduce end-diastolic or end-systolic left ventricular diameter, reduce anterior or posterior wall thickness, increase or normalize ejection time, increase aortic peak flow velocity or aortic blood flow time, and/or reduce symptoms of a cardiac disease or condition in an individual. In one or more embodiments, such methods reduce the frequency or severity of symptoms of a cardiac disease or disorder in a subject.

In any of the methods herein, prior to administering AAV particles to a patient as described above, the patient may be assessed for the presence of anti-AAV capsid antibodies or anti-AAV neutralizing antibodies that could block cell transduction or otherwise reduce the overall efficiency of treatment. Detection of anti -AAV antibodies

In order to maximize the likelihood of successful cardiac transduction by systemic AAV-mediated therapeutic gene transfer, prior to administering AAV particles to a human patient in a treatment regimen as described above, the prospective patient may be assessed for the presence of anti-AAV capsid antibodies or anti-AAV neutralizing antibodies that could block cell transduction or otherwise reduce the overall efficiency of the treatment regimen. Such antibodies may be present in the serum of the prospective patient and may be directed against any serotype of AAV capsid. In one embodiment, the serotype against which the pre-existing antibodies are directed is AAV9.

Methods for detecting pre-existing AAV immunity are well known and routinely used in this technology, and include cell-based in vitro transduction inhibition (TI) assays, in vivo (e.g., in mice) TI assays, and ELISA-based total anti-capsid antibody (TAb) detection (see, e.g., Masat et al., Discov. Med., Vol. 15, pp. 379-389; and Boutin et al., (2010) Hum. Gene Ther., Vol. 21, pp. 704-712). TI assays can employ host cells into which an AAV inducible reporter vector has been previously introduced. The reporter vector can comprise an inducible reporter gene, such as GFP, etc. After the host cell is transduced with the AAV virus, its expression is induced. Anti-AAV capsid antibodies present in human serum that are able to prevent/reduce host cell transduction will thereby reduce the overall expression of the reporter gene in the system. Therefore, this type of assay can be used to detect the presence of anti-AAV capsid antibodies in human serum that are able to prevent/reduce cell transduction by therapeutic AAV particles.

Assays for detecting anti-AAV capsid antibodies can employ solid-phase bound AAV capsids as a "capture agent" over which human serum is passed, thereby allowing anti-capsid antibodies present in the serum to bind to the solid-phase bound capsid "capture agent." After washing to remove non-specific binding, the presence of anti-capsid antibodies bound to the capture agent can be detected using a "detector." The detector can be an antibody, AAV capsid, or the like, and can be detectably labeled to aid in the detection and quantification of bound anti-capsid antibodies. In one embodiment, the detector is labeled with ruthenium or a ruthenium complex, which can be detected using electrochemical luminescence techniques and equipment.

The same methods described above can be used to assess and detect the generation of anti-AAV capsid immune responses in patients who are previously treated with the therapeutic AAV virus of interest. Thus, these techniques can be used not only to assess the presence of anti-AAV capsid antibodies prior to treatment with the therapeutic AAV virus, but also to assess and measure the induction of immune responses to the administered therapeutic AAV virus after administration. Thus, the present invention contemplates combining techniques for detecting anti-AAV capsid antibodies in human serum with methods for administering therapeutic AAV viruses to treat ACM, wherein the techniques for detecting anti-AAV capsid antibodies in human serum can be performed before or after administration of the therapeutic AAV virus.

Other aspects and advantages of the present invention will be understood after considering the following illustrative examples. Examples Example 1 : Production of AAV Particles

AAV particles comprising AAV9 capsid and vector constructs of SEQ ID NO: 3-68 or 126-128 are produced in HEK293 cells. Vectors for AAV production are produced. For example, when AAV is produced using HEK293 cells, plasmids are produced. These plasmids have nucleotide sequences that provide the AAV vector genome, encode Rep and capsid proteins, and provide non-adjuvant AAV functions. These plasmids are transfected into HEK293 cells using a transfection reagent. After culturing the HEK293 for a predetermined time after transfection, the rAAV particles produced are isolated from the culture, purified, and titrated.

To produce AAV in Sf9 cells, rod-shaped viral plasmids are produced. These rod-shaped viral plasmids have nucleotide sequences that provide the AAV vector genome and encode Rep and capsid proteins. The rod-shaped viral plasmids are transfected into untreated Sf9 cells using a transfection reagent. After the transfected Sf9 cells are cultured for a predetermined time, the recombinant rod-shaped virus (rBV) is isolated, purified and titrated. To produce rAAV, another untreated culture of Sf9 cells is infected with rBV at a predetermined infection multiplicity (MOI). The Sf9 cells are cultured for a predetermined time after infection. After the predetermined time, the produced rAAV particles are isolated from the culture, purified and titrated. Example 2 : Evaluation of the effect of AAV particles on PKP2 expression .

PKP2 expression was observed in healthy human donor iPS cardiomyocytes (iCM2) and HK1 mouse cardiomyocytes. On day 2 of culture, human UNK-CM2 cells were transduced with 0.2, 1, or 5e6 vg/CM of AAV9-hTNNT2-MsPKP2a encoding mouse wild-type praegyrin 2 or AAV9-hTNNT2-HuPKP2a encoding human wild-type praegyrin 2 constructs. The cells were then electrically paced for three weeks to allow for maturation. At 3 weeks post-transduction, no changes in beating rate or contractility were observed by Cardio xCELLigence or nuclear high-content imaging. PKP2 expression was measured by Western blotting and liquid chromatography mass spectrometry (LCMS). Dose-dependent expression of HuPKP2 and MsPKP2 mRNA and protein was observed in healthy human donor iPS-CM2 cardiomyocytes. For all doses, the AAV9-hTNNT2-HuPKP2 construct resulted in higher PKP2 expression compared to the AAV9-hTNNT2-MsPKP2a construct (Figure 1A and Figure 1B). There were no safety signals, and the ability to overnormalize PKP2 protein levels was limited, and no displacement of endogenous mitochondrial binding partners was found. These results support the conclusion that the AAV-HuPKP2 construct results in higher PKP2 expression in iPS cardiomyocytes. In addition, a dose-dependent increase in endogenous DSP and JUP proteins was observed (Figure 2), thus increasing the potential of PKP2 GT to expand into the DSP and JUP gene segments of ACM. Example 3 : Evaluation of the effect of AAV particles in vivo

An 8-week single-dose performance study was performed in RAG2-/- mice to evaluate the effects of AAV particles in vivo. RAG2-/- mice were dosed with AAV9-hTNNT2-HuPKP2 or AAV9-hTNNT2-MsPKP2 particles at 6e13 vg/kg, and heart tissue was harvested at 8 weeks. Both AAV9-hTNNT2-HuPKP2 and AAV9-hTNNT2-MsPKP2 constructs resulted in increased PKP2 protein levels in RAG2-/- heart tissue, and no obvious safety signals were observed. Vector-derived PKP2 mRNA and PKP2 protein were detected in the myocardium. Both constructs had limited ability to over-normalize PKP2 protein. The AAV9-hTNNT2-HuPKP2 construct showed higher PKP2 protein expression than AAV9-hTNNT2-MsPKP2 (Figure 3). Although both MsPKP2 and HuPKP2 constructs increased endogenous levels of the desmosomal DSP protein (desmotoplakin) and JUP protein (plakoglobin) after transcription, no increase in DSP or JUP mRNA was detected. See Figure 4. These results suggest that PKP2 replacement therapy itself has a positive effect on the deficiency of these two proteins.

AAV9-hTNNT2-HuPKP2 expression in the hearts of RAS2-/- mice also promoted differential expression of genes involved in fatty acid metabolism. Specifically, when the negative regulator Arid5b was upregulated, the expression of Bckhda, Kif2, and Sik1 was downregulated. These results suggest that PKP2 replacement therapy itself has a positive effect on fatty acid metabolism. Example 4 : Evaluation of the effect of AAV particles in cardiomyocytes of patients with ACM disease

R79X PKP2 HET ACM patient iPSC-CMs (Khloris Biosciences ACM cell line 410 (PKP2 p.Arg79Stop; c.235 C＞T)) or gene-edited R79X PKP2 HET iPSC co-cultures were used for experiments. The beating waveform of ACM R79X PKP2 Het (line 410) was measured by cardio-xCELLingence and compared with healthy controls (line 274). The beating amplitude at day 3 post-transduction showed an increase of approximately 0.03 in ACM line 410 relative to WT line 274 (P=0.0243). Irregular waveforms (arrhythmias) were observed in several wells, but WT wells did not exhibit arrhythmias.

ACM R79X PKP2 (strain 410) were transduced with 0.15E6 vg/CM, 0.3E6 vg/CM, 0.6E6 vg/CM, 1.25E6 vg/CM, or 2.5E6 vg/CM AAV9-hPKP2. Beat amplitude, a surrogate for contractility, was then measured at day 6 post-transduction (Figure 5). Beat amplitude has been shown to be restored by AAV9-hPKP2 in a dose-dependent manner, and iPSC cardiomyocytes exhibit increased contractility. PKP2 expression was then measured at day 11 post-transduction (Figure 6), demonstrating comparable expression for all doses evaluated relative to healthy controls.

These results indicate that the HuPKP2 construct rescues or normalizes the contractile defects of ACM patient iPSC-CM heterozygous for the R79X PKP2 mutation in a dose-dependent manner without over-normalization. In addition, the HuPKP2 construct also prevents fibrosis in a co-culture model of gene-edited hiPSCs with the same R79X PKP2 heterozygous mutation. Example 5 : Evaluation of the effect of AAV particles in vivo in a murine disease model

The constructs described above were tested in a PKP2 mouse model. One model involves administration of flecainide to heterozygous PKP2+/- mice to expose arrhythmias, for example, at a dose of 30 mg/kg of flecainide. In this model, arrhythmias are monitored by measuring ventricular early contraction/triggering activity; 60% of PKP2+/- mice exhibit arrhythmias compared to only 20% of WT sibling counterparts.

Alternatively, isoproterenol was used to expose ACM in PKP2 heterozygous mice by inducing tachycardia. Administration of isoproterenol to PKP2 +/- mice resulted in arrhythmias attributed to poor electrical conductivity (ECG), leading to tissue damage, fibrofatty scarring, ventricular wall thinning/dilated cardiomyopathy, and ultimately heart failure.

Administration of AAV9-hTNNT2-HuPKP2 or AAV9-hTNNT2-MsPKP2 particles in this mouse model resulted in a decrease in arrhythmic frequency, improved electrical conductivity, improved cardiac contractility, reduced cardiac fibrosis, or reduced ventricular wall thinning. Example 6 : Evaluation of AAV9-MsPKP2 Administration in the PKP2- Mouse Model of Arrhythmogenic Cardiomyopathy

A conditional PKP2 knockout (PKP2-cKO) mouse model was generated by crossing PKP2fl/fl with alphaMHC-Cre-ERT2 as described in Cerrone et al., Nature Communications 8, 106 (2017). This mouse model shows rapid disease progression from right ventricular predominant arrhythmogenic cardiomyopathy to end-stage biventricular cardiomyopathy failure and death within approximately 50 days.

Conditional PKP2 knockout mice were administered rAAV9 particles (AAV9-hTNNT2-MsPKP2, AAV9-MsPKP2, e.g., one of SEQ ID NOs: 12-14 ) generated using a vector construct described herein (comprising an hTNNT2 promoter sequence operably linked to a wild-type murine PKP2a (MsPKP2) gene) and monitored for 8 weeks. AAV9-hTNNT2-MsPKP2 was administered to mice 28 days prior to disease induction. On day 0, Tamoxifen (CAS#: 10540-29-1) was administered to induce disease. Different conditional PKP2 knockout mice were administered vehicle control 28 days prior to ACM disease induction via tamoxifen administration, and these mice were administered tamoxifen on day 0. Healthy mice of the same background (Cre -/-) were used as controls.

MsPKP2 mRNA expression was evaluated in cardiac tissue of conditional PKP2 knockout mice receiving vehicle control or AAV9-MsPKP2 and healthy control mice of the same background (Cre -/-). Heart tissue was extracted 28 days after arrhythmogenic cardiomyopathy disease was induced by tamoxifen administration. AAV9-hTNNT2-MsPKP2 administration resulted in a significant increase in MsPKP2 mRNA levels in conditional PKP2 knockout mice receiving AAV9-MsPKP2 compared to control mice, ranging from approximately 130% copies per nanogram of RNA. The mRNA has been shown to be evenly distributed throughout cardiac tissue and is detected in the majority of cardiomyocytes (e.g., 60%-74% or 94% of cardiomyocytes are positive for MsPKP2 mRNA).

Cardiac tissues of conditional PKP2 knockout mice receiving vehicle control or AAV9-MsPKP2 and healthy control mice of the same background (Cre -/-) were evaluated for plasminogen activator 2 protein expression. Heart tissues were extracted 28 days after arrhythmogenic cardiomyopathy disease was induced by tamoxifen administration. Plasminogen activator 2 protein expression was measured using rapid-in-situ-hybridisation (RISH), dual in situ hybridization (DISH), immunohistochemistry (IHC), liquid chromatography mass spectrometry (LCMS), or trichrome staining. Heart tissues of conditional PKP2 knockout mice that received vehicle control exhibited statistically significantly reduced levels of Bf2 protein relative to control mice (FIG. 7). Heart tissues of conditional PKP2 knockout mice that received AAV9-MsPKP2 exhibited statistically significantly increased levels of Bf2 protein relative to vehicle control conditional PKP2 knockout mice, which had levels substantially similar to control mice (FIG. 7). Of particular note, AAV9-hTNNT2-MsPKP2 administration increased Bf2 protein expression to approximately 120% protein compared to control mice.

AAV9-hTNNT2-MsPKP2 was able to prevent arrhythmias and fibrosis at all doses tested, as determined by electrocardiogram (ECG) 21 days after disease induction. Mice were sacrificed 28 days after disease induction. It was also observed that the expression of plaquenil protein 2 in the liver of conditional PKP2 knockout mice dosed with AAV9-hTNNT2-MsPKP2 was comparable to that of control mice. The data highlight that the cardiomyocyte-specific promoter of the vector constructs tested was shown to effectively restrict the expression of most of the PKP2 transgene to the heart of conditional PKP2 knockout mice.

Cardiac tissue fibrosis was assessed in conditional PKP2 knockout mice receiving vehicle control or AAV9-MsPKP2 and healthy control mice of the same background (Cre -/-). Heart tissue from the right and left ventricles was extracted 28 days after induction of arrhythmogenic cardiomyopathy disease by tamoxifen administration. Heart tissue from both ventricles of conditional PKP2 knockout mice receiving vehicle control showed statistically significant increased fibrosis, as evidenced by increased collagen content in heart tissue (Figure 8). Heart tissue from conditional PKP2 knockout mice receiving AAV9-MsPKP2 exhibited statistically significant reductions in fibrosis of both ventricles relative to vehicle-controlled conditional PKP2 knockout mice, which had substantially similar fibrosis to control mice ( FIG. 8 ).

The hearts of conditional PKP2 knockout mice that received vehicle control or AAV9-MsPKP2 and healthy control mice of the same background (Cre -/-) were evaluated for the number of ectopic beats. Ectopic beats were measured by cardiac ultrasound after administration of isoproterenol (CAS#: 7683-59-2). Analysis was also performed 21 days after induction of arrhythmogenic cardiomyopathy disease by administration of tamoxifen. Hearts of conditional PKP2 knockout mice that received vehicle control exhibited a statistically significant increase in the number of ectopic beats, indicative of cardiac arrhythmias (Figure 9). Hearts of conditional PKP2 knockout mice that received AAV9-MsPKP2 exhibited a statistically significant reduction in the number of ectopic beats relative to vehicle control conditional PKP2 knockout mice, which had levels comparable to control mice ( FIG. 9 ).

Thus, administration of AAV9-MsPKP2 to conditional PKP2 knockout mice unexpectedly prevented the induction of arrhythmogenic cardiomyopathy disease, as evidenced by a reduction in fibrosis and the number of ectopic beats, compared to vehicle control conditional PKP2 knockout mice following tamoxifen administration.

The effect of AAV9-MsPKP2 administration on the expression of other proteins that are critical for functional bridge formation was also analyzed. Figures 10A to 10E depict the expression of different endogenous bridge and GAP proteins in heart tissue of conditional PKP2 knockout mice that had received vehicle control or AAV9-MsPKP2 and healthy control mice of the same background (Cre -/-). The expression of these proteins was measured using RISH, DISH, IHC, LCMS or trichrome staining. Heart tissue was extracted 28 days after induction of arrhythmogenic cardiomyopathy disease by tamoxifen administration. Conditional PKP2 knockout mice receiving vehicle control showed significantly reduced expression of nexin plakoglobin, desmoplakin, desmoplakin-2, nexin 43, and N-calcineurin (Figures 10A-10E). Reduced expression of these endogenous proteins indicates reduced levels of or damage to the spondylomeric complex in the heart, which disrupts cell-cell connectivity and causes mechanical and electrical dysfunction, leading to arrhythmogenic cardiomyopathy. Conditional PKP2 knockout mice that received AAV9-MsPKP2 unexpectedly displayed a significant increase in the expression of plakoglobin (FIG. 10A), desmoplakin (FIG. 10B), desmoplakin-2 (FIG. 10C), nexin 43 (FIG. 10D), and N-calcein (FIG. 10E) relative to vehicle control conditional PKP2 knockout mice. It should be further noted that the expression of each of these proteins was comparable to control mice. This data highlights the role of AAV9-MsPKP2 administration in unexpectedly preventing the induction of arrhythmogenic cardiomyopathy disease.

AAV9-hTNNT2-MsPKP2 administration also normalized proteins involved in glycosphingolipid metabolism, lipogenesis, and high-density lipoprotein (HDL) clearance in this mouse model of the disease. The levels of CTSA, ApoE, and acid cermidase were normalized compared to wild-type mice. The increased levels of fatty acid binding proteins (i.e., fatty acid binding protein 5 (FABP-5), fatty acid binding protein, adipocytes, and ApoA I) in PKP2-cKP heart tissue were also normalized after administration of AAV9-hTNNT2-MsPKP2. In addition, AAV9-hTNNT2-MsPKP2 administration normalized saposins (i.e., prosaposin, saposin B, and saposin D).

Overall, it was shown that AAV9-hTNNT2-MsPKP2 was evenly distributed throughout the PKP2-cKO hearts, where it increased PKP2 protein levels and prevented the arrhythmias and fibrosis that cause ACM. Example 7 : Evaluation of AAV9-HuPKP2 Administration in PKP2-cKO for ACM

Conditional PKP2 knockout mice are administered rAAV9 particles (AAV9-hTNNT2-hPKP2, AAV9-hPKP2, AAV9-hTNNT2-HuPKP2, AAV9-HuPKP2, e.g., one of SEQ ID NOs: 3-11, 126-128 ) produced using a vector construct described herein comprising an hTNNT2 promoter sequence operably linked to a wild-type or functional human PKP2a (HuPKP2 or hPKP2) gene, and the mice are monitored for an extended period of time after induction of arrhythmogenic cardiomyopathy by tamoxifen administration. Conditional PKP2 knockout mice are administered AAV9-HuPKP2 28 days prior to tamoxifen administration, and tamoxifen is administered on day 0. Different conditional PKP2 knockout mice were administered vehicle controls 28 days prior to ACM disease induction via tamoxifen administration, and these mice were administered tamoxifen on day 0. Healthy wild-type mice were used as controls.

Hearts from conditional PKP2 knockout mice receiving vehicle control or AAV9-HuPKP2 and healthy wild-type mice were evaluated for right ventricular (RV) size/area. RV size/area was measured by cardiac ultrasound and occurred on days -28, -7, 28, 63, and 137 after induction of ACM by tamoxifen administration. Conditional PKP2 knockout mice receiving vehicle control exhibited significantly larger RV size/area relative to wild-type mice by 28 days after induction of disease, which was symptomatic or ACM (Figure 11). Conditional PKP2 knockout mice that received AAV9-HuPKP2 exhibited RV size/area substantially similar to that of wild-type mice until 137 days after induction of arrhythmogenic cardiomyopathy disease by tamoxifen administration (Figure 11). Thus, AAV9-HuPKP2 administration provides long-term protection against RV enlargement.

Left ventricular ejection fraction (LVEF) was assessed in hearts of conditional PKP2 knockout mice receiving vehicle control or AAV9-HuPKP2 and healthy wild-type mice. LVEF was measured by cardiac ultrasound and occurred on days -28, -7, 28, 63, and 137 after induction of ACM by tamoxifen administration. Conditional PKP2 knockout mice receiving vehicle control exhibited significantly impaired LVEF by 28 days after induction of disease, which was symptomatic or ACM (Figure 12). Conditional PKP2 knockout mice that received AAV9-HuPKP2 exhibited a LVEF substantially similar to that of healthy wild-type mice until 137 days after induction of arrhythmogenic cardiomyopathy disease by tamoxifen administration ( FIG. 12 ). Thus, AAV9-HuPKP2 administration preserves LVEF long term. Thus, administration of AAV9-HuPKP2 to conditional PKP2 knockout mice unexpectedly prevented the induction of arrhythmogenic cardiomyopathy disease after tamoxifen administration compared to vehicle-controlled conditional PKP2 knockout mice, as demonstrated by preventing increases in RV enlargement (measured by RV size/area) and maintaining a LVEF similar to that of wild-type mice. AAV9-HuPKP2 administration also increased survival in this mouse model, as supported by the survival curves shown in FIG. 13 . All conditional PKP2 knockout mice receiving vehicle control died of ACM disease approximately 53 days after tamoxifen administration induced the disease (Figure 13). On the other hand, conditional PKP2 knockout mice and wild-type mice receiving AAV9-HuPKP2 all survived (Figure 13). Therefore, AAV9-HuPKP2 administration was able to prevent ACM-related mortality during the observed period.

Studies were also performed to evaluate human PKP2 protein expression and fibrillation in the hearts of conditional PKP2 knockout mice and healthy wild-type mice that received vehicle control or AAV9-HuPKP2. Human PKP2 protein expression and fibrillation were measured by RISH/DISH/IHC at different predetermined time points. Example 8 : Evaluation of AAV9-HuPKP2 or AAV9-MsPKP2 Administration in the PKP2-cKO Mouse Model of Arrhythmogenic Cardiomyopathy

Conditional PKP2 knockout mice are administered rAAV9 particles produced using a vector construct described herein (AAV9-hTNNT2-hPKP2, AAV9-hPKP2, AAV9-hTNNT2-HuPKP2, AAV9-HuPKP2, e.g., one of SEQ ID NOs: 3-11, 126-128) or a wild-type murine PKP2a (MsPKP2) gene (AAV9-hTNNT2-MsPKP2, AAV9-MsPKP2, e.g., one of SEQ ID NOs: 12-14), the vector construct comprising a wild-type or functional human PKP2a operably linked to The hTNNT2 promoter sequence of the (HuPKP2 or hPKP2) gene was expressed in the mice, and the mice were monitored for an extended period of time after the arrhythmogenic cardiomyopathy disease was induced by tamoxifen administration. AAV9-HuPKP2 and AAV9-MsPKP2 were administered to the conditional PKP2 knockout mice 7 days after tamoxifen administration or on day 0. Tamoxifen was administered on day -7. Vehicle controls were administered to the different conditional PKP2 knockout mice 7 days after tamoxifen administration or on day 0, and tamoxifen was administered on day -7. Healthy wild-type mice were used as controls.

The number of ectopic beats in the hearts of conditional PKP2 knockout mice and wild-type mice that received vehicle control, AAV9-MsPKP2 or AAV9-HuPKP2. Ectopic beats were measured by cardiac ultrasound after isoproterenol administration. Analysis was also performed 21 days and 56 days after induction of arrhythmogenic cardiomyopathy by tamoxifen administration. At 21 days after induction, the hearts of conditional PKP2 knockout mice that received vehicle control showed a statistically significant increase in the number of ectopic beats, which is indicative of cardiac arrhythmia (Figure 14A). At 21 and 56 days after induction, hearts of conditional PKP2 knockout mice that received AAV9-MsPKP2 or AAV9-HuPKP2 exhibited a statistically significant reduction in the number of ectopic beats at day 21 relative to vehicle-controlled conditional PKP2 knockout mice, whose levels were comparable to those of wild-type mice that received vehicle controls ( FIGS. 14A and 14B ).

Thus, administration of AAV9-MsPKP2 and AAV9-HuPKP2 to conditional PKP2 knockout mice unexpectedly intervened to prevent the induction of arrhythmogenic cardiomyopathy, as evidenced by a decrease in the number of ectopic waves, compared to vehicle-controlled conditional PKP2 knockout mice after tamoxifen administration. This is further supported by the survival curves shown in FIG. 15 . All conditional PKP2 knockout mice receiving vehicle control died of arrhythmogenic cardiomyopathy disease approximately 53 days after induction of the disease by tamoxifen administration ( FIG. 15 ). On the other hand, all conditional PKP2 knockout mice receiving AAV9-HuPKP2 survived ( FIG. 15 ). Most conditional PKP2 knockout mice and wild-type mice receiving AAV9-MsPKP2 survived ( FIG. 15 ). Thus, AAV9-PKP2 administration was able to correct the defect attributable to the failure to express functional PKP2 protein and prevent mortality associated with arrhythmogenic cardiomyopathy.

Studies were also performed to evaluate the expression of Pf2 and fibrillation in the hearts of conditional PKP2 knockout mice that received vehicle control, AAV9-HuPKP2, AAV9-MSPKP2, and healthy wild-type mice. Pf2 expression and fibrillation were measured by RISH, DISH, IHC, LCMS, or trichrome staining of heart tissue extracted at different predetermined times after induction.

A vector construct is generated, which includes a hTNNT2 promoter comprising nucleotides 1-12 of SEQ ID NO: 264, such as a promoter having a sequence of any one of SEQ ID NO: 133-263. The vector construct comprises the elements described herein (5' and 3' ITRs, a transcriptional regulatory region comprising a cardiac-specific promoter, a gene of interest, and an expression enhancing intron, if present). The rAAV vector construct is temporarily transfected into a variety of different cell types, and the relative level of the gene of interest or the relative expression of the gene therapy product is detected by various methods known in the art. For example, the number of vector genomes comprising the gene of interest and the number of transcripts of the gene of interest are assessed by qPCR or ddPCR. The amount of gene therapy products was measured by liquid chromatography/mass spectrometry (LC/MS) or Western blot.

The results showed that higher expression was achieved in cardiac tissues and/or cardiomyocytes compared to non-cardiac tissues or cell types, and the effect was superior to that of the hTNNT2 promoter that did not contain nucleotides 1-12 of SEQ ID NO: 264.

Expression analysis of different promoters was performed using an intravital imaging system (IVIS) to determine luciferase expression in mice transduced with rAAV vectors having a vector genome having a nucleotide sequence encoding luciferase and operably linked to different promoters. The promoters evaluated included RSV LTR/promoter (RSV), murine cytokeratin 8 promoter (mCK8), hTNNT2 promoter, chicken TNNT2 promoter (cTNNT2), human creatine kinase M promoter (hCKM), and human desmin promoter (hDES). rAAVs containing different promoters operably linked to a nucleotide sequence encoding luciferase were generated. Different rAAVs were administered to a first group of mice. After a predetermined period of time, luciferin was administered to the mice and the location of luciferase expression was identified using a whole mouse IVIS. A second group of mice was also transduced with a different rAAV. After a predetermined period of time, the organs of the mice were harvested. Luciferase expression was measured using a luciferase reporter assay using luciferin. Luciferase expression was calculated as photons/second. Table 1 is a graph showing expression in the heart, gastrocnemius (e.g., skeletal muscle), and diaphragm. The values were normalized to mCK8 expression. The hTNNT2 promoter is cardiac-specific, with a signal that is 1.71 times that of mCK8, and very low signal in skeletal muscle and diaphragm. Table 1 Starter Luciferase expression (ratio to mCK8) Heart Gastrocnemius muscle Diaphragm RSV 0.13 0.12 0.5 mCK* 1.00 1.00 1.00 hTNNT2 1.71 0.00 0.05 TNT2 0.19 0.01 0.19 HkDJ 0.04 0.11 0.09 oeLh 0.61 0.94 0.47

The embodiments described herein are intended to be illustrative only, and those skilled in the art will recognize, or will be able to ascertain using no more than routine experimentation, numerous equivalents to the specific structures, materials, and procedures. All such equivalents are considered to be within the scope of the present invention.

All patents, patent applications, and publications mentioned herein are incorporated herein by reference in their entirety. The citation or identification of any reference in this application is not an admission that the reference is available as prior art for this application. The full scope of the invention will be better understood from the attached patent claims.

Figures 1A and 1B depict the dose-dependent expression of human PKP2 (HuPKP2) and mouse (MsPKP2) constructs in healthy human donor iPS-CM2 cardiomyocytes, where all HuPKP2s expressed higher PKP2 than MsPKP2.

FIG2 shows the protein expression levels of PKP2, desmoplaxin (DSP) and juxtaplaxin (JUP) in human iPSC-CM2 cardiomyocytes.

FIG. 3 depicts PKP2 protein expression in heart tissue of RAG2-/- mice transduced with HuPKP2 or MsPKP2.

FIG. 4 shows the protein expression levels of DSP and JUP proteins in the heart tissues of RAG2-/- mice transduced with HuPKP2 or MsPKP2.

FIG. 5 depicts the dose-dependent beat amplitude (surrogate of contraction) of ACM R79X PKP2 (strain 410) transduced with 0.15E6 vg/CM, 0.3E6 vg/CM, 0.6E6 vg/CM, 1.25E6 vg/CM, or 2.5E6 vg/CM AAV9-hPKP2.

Figure 6 depicts PKP2 expression in ACM R79X PKP2 (strain 410) transduced with 0.15E6 vg/CM, 0.3E6 vg/CM, 0.6E6 vg/CM, 1.25E6 vg/CM, or 2.5E6 vg/CM AAV9-hPKP2 relative to healthy controls assessed.

FIG. 7 depicts the expression of PKP2 protein/Pfizer 2 in heart tissues of conditional PKP2 knockout mice that had received vehicle control or AAV9-MsPKP2 and healthy control mice of the same background (Cre −/−).

FIG. 8 depicts fibrosis in heart tissue of conditional PKP2 knockout mice that had received vehicle control or AAV9-MsPKP2 and healthy control mice of the same background (Cre −/−).

FIG. 9 depicts ventricular ectopic beats in hearts of conditional PKP2 knockout mice that had received vehicle control or AAV9-MsPKP2 and healthy control mice of the same background (Cre −/−).

Figures 10A to 10E depict the expression of different desmosome proteins in heart tissues of conditional PKP2 knockout mice that have received vehicle control or AAV9-MsPKP2 and healthy control mice (Cre -/-) of the same background. Figure 10A shows the expression of nexin plakoglobin. Figure 10B shows the expression of desmoplakoglobin. Figure 10C shows the expression of desmoplakin-2. Figure 10D shows the expression of connexin 43. Figure 10E shows the expression of N-calcified mucin.

Figure 11 depicts right ventricular size/area of hearts from conditional PKP2 knockout mice and healthy wild-type mice receiving vehicle control or AAV9-HuPKP2.

Figure 12 depicts left ventricular ejection fraction of hearts from conditional PKP2 knockout mice and healthy wild-type mice receiving vehicle control or AAV9-HuPKP2.

FIG. 13 depicts the survival curves of wild-type mice and conditional PKP2 knockout mice receiving AAV9-HuPKP2 or vehicle control.

Figures 14A and 14B depict ventricular ectopic beats in the hearts of healthy wild-type mice and conditional PKP2 knockout mice that received vehicle control, AAV9-MsPKP2, or AAV9-HuPKP2.

FIG. 15 depicts the survival curves of wild-type mice and conditional PKP2 knockout mice receiving vehicle control, AAV9-MsPKP2, or AAV9-HuPKP2.

TW202421788A_112136195_SEQL.xml

Claims (76)

A recombinant vector construct comprising: (a) a nucleic acid encoding a functional human plakophilin-2 (PKP2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 2 or 130; (b) a heterologous cardiomyocyte-specific transcriptional regulatory region comprising at least nucleotides 521-532 of SEQ ID NO: 82; (c) an intron at least 85% identical to SEQ ID NO: 77; (d) a polyadenylation signal, and (d) one or both of the 5' and 3' AAV inverted terminal repeat (ITR) sequences. The vector construct of any one of claims 1 to 2, wherein the nucleic acid has a sequence that is at least 97%, 98% or 99% identical to the nucleic acid sequence of SEQ ID NO: 1, 69-73 or 129. The vector construct of any one of claims 1 to 2, wherein the cardiomyocyte-specific transcriptional regulatory region comprises SEQ ID NO: 82. The vector construct of any one of claims 1 to 3, wherein the intron comprises the nucleotide sequence of SEQ ID NO: 77. The vector construct of claim 4, wherein the intron is located within the nucleic acid encoding functional human PKP2. The vector construct of claim 4, wherein the intron is located between two exons of the nucleic acid encoding functional human PKP2. The vector construct of claim 4, wherein the intron is located between exon 1 and exon 2 of the nucleic acid encoding functional human PKP2. The vector construct of claim 4, wherein the intron is located after position 261 of SEQ ID NO: 1 or 129. The vector construct of any one of claims 1 to 8, wherein the polyadenylation signal is a mini-polyadenylation signal, a growth hormone polyadenylation signal, an SV40 polyadenylation signal or a fragment thereof. The vector construct of claim 9, wherein the polyadenylation signal comprises a nucleotide sequence that is at least 90% identical to: any one of SEQ ID NOs: 90-96; or any one of SEQ ID NOs: 97-100, or a fragment thereof. The vector construct of any one of claims 1 to 10, wherein the rAAV vector construct has a size of about 3.5 to about 4.0 kb. The vector construct of any one of claims 1 to 11, wherein the AAV 5' ITR and/or AAV 3' ITR are from AAV9. The vector construct of any one of claims 1 to 11, comprising a nucleotide sequence that is at least 97%, 98% or 99% identical to any one of SEQ ID NOs: 3-68 or 126-128. A rAAV particle comprising the vector construct of any one of claims 1 to 13 and an AAV capsid. The rAAV particle of claim 14, wherein the AAV capsid is cardiotropic. The rAAV particle of claim 14, wherein the AAV capsid is an AAV9 type capsid. A method for producing rAAV particles as claimed in any one of claims 14 to 16, comprising the following steps: (a) providing a mammalian cell comprising one or more nucleic acid constructs, wherein the one or more nucleic acid constructs comprise (i) a vector construct as claimed in any one of claims 1 to 35, (ii) a nucleotide sequence encoding one or more AAV Rep proteins operably linked to a promoter, and (iii) a nucleotide sequence encoding one or more AAV capsid proteins operably linked to a promoter, (b) culturing the mammalian cell under conditions conducive to the expression of the Rep proteins and capsid proteins, and (c) recovering the rAAV particles. The method of claim 17, wherein the mammalian cell is a HEK293 cell. A method for producing rAAV particles, comprising the following steps: (a) providing one or more nucleic acid constructs to cells that are permissive for AAV replication, the one or more nucleic acid constructs comprising: (i) a recombinant vector construct comprising (1) at least one AAV ITR, (2) a heterologous cardiomyocyte-specific transcriptional regulatory region, and (3) a nucleic acid encoding a functional human PKP2 protein, (ii) a nucleotide sequence encoding one or more AAV Rep proteins, which is operably linked to a promoter capable of driving the expression of the one or more Rep proteins in the cell; and (iii) a nucleotide sequence encoding one or more AAV capsid proteins, which is operably linked to a promoter capable of driving the expression of the one or more capsid proteins in the cell; (b) culturing the cell under conditions that allow the expression of the Rep proteins and the capsid proteins; and, if appropriate, (c) recovering the AAV particles. The method of claim 19, wherein the cell is an insect cell. The method of claim 19, wherein the cell is a mammalian cell. The method of any one of claims 19 to 21, wherein the cell has a recombinant vector construct of any one of claims 1 to 13. A population of rAAV particles produced by the method of any one of claims 17 to 22, optionally enriched for particles containing full-length or nearly full-length vector genomes by a step of reducing the number of empty capsids. A pharmaceutical composition comprising a vector construct as described in any one of claims 1 to 13, or a rAAV particle as described in any one of claims 14 to 16, or a rAAV particle population as described in claim 23 in the form of an aqueous suspension, and a sterile pharmaceutically acceptable formulation. A method for delivering a human PKP2 coding sequence, comprising administering a vector construct as described in any one of claims 1 to 13, or a rAAV particle as described in any one of claims 14 to 16, or a rAAV particle population as described in claim 23, or a pharmaceutical composition as described in claim 24 to a patient suffering from arrhythmogenic cardiomyopathy. A method for treating arrhythmogenic cardiomyopathy, comprising administering to a patient suffering from arrhythmogenic cardiomyopathy a therapeutically effective amount of a vector construct as described in any one of claims 1 to 13, or an rAAV particle as described in any one of claims 14 to 16, or an rAAV particle population as described in claim 23, or a pharmaceutical composition as described in claim 24. The method of claim 25 or 26, wherein the patient exhibits a mutation in one or both PKP2 alleles. A method for restoring PKP2 protein expression in cardiac tissue of a subject suffering from arrhythmogenic cardiomyopathy, comprising administering to the subject the vector construct of claim 1. A method of reducing cardiac damage or inflammation in a subject suffering from arrhythmogenic cardiomyopathy comprises administering to the subject the vector construct of claim 1. A method for normalizing cardiomyocytes and/or cardiac function, comprising administering the vector construct of claim 1 to a subject in need thereof. A method for reducing premature ventricular contractions in a subject suffering from arrhythmogenic cardiomyopathy comprises administering to the subject the vector construct of claim 1. A transgene comprising: (a)     a gene of interest capable of expressing a gene therapy product, (b)     a cardiac-specific promoter operably linked to the gene of interest and comprising a nucleic acid sequence at least 80% identical to any one of SEQ ID NOs: 133-263, wherein the cardiac-specific promoter comprises SEQ ID NO: 264, (c)     optionally a cardiac expression enhancing intron, and (d)     optionally a polyadenylation signal. The transgene of claim 32, wherein the heart-specific promoter comprises a nucleic acid sequence that is at least 90% identical to any one of SEQ ID NOs: 133-263. The transgene of any one of claims 32 to 33, comprising a polyadenylation signal. The transgene of any one of claims 32 to 34, comprising a cardiac expression enhancing intron. The transgene of claim 35, wherein the cardiac expression enhancing intron is located within the coding sequence of the gene of interest. The transgene of claim 36, wherein the cardiac expression enhancing intron is located between naturally occurring exons of the gene of interest, between the first and second exons or the second and third exons of the gene of interest, as the case may be. The transgene of any one of claims 32 to 37, wherein the cardiac-specific promoter provides at least about 5-fold higher expression in cardiomyocytes than in another cell type. The transgene of any one of claims 32 to 38, wherein the intron comprises any one of SEQ ID NOs: 270 or 265-275, or an expression-enhancing fragment thereof. The transgene of any one of claims 32 to 39, wherein the polyadenylation signal is a growth hormone polyadenylation signal or a fragment thereof. The transgene of any one of claims 32 to 39, wherein the polyadenylation signal is a bovine growth hormone polyadenylation signal, a human growth hormone polyadenylation signal or a fragment thereof. A vector construct comprising the transgene of any one of claims 32 to 41. A plasmid comprising the transgene of any one of claims 32 to 41. A donor construct comprising the transgene of any one of claims 32 to 41. A viral vector or viral particle comprising the transgene of any one of claims 32 to 41. A cell comprising the transgene of any one of claims 32 to 41. A recombinant vector construct comprising: (a) one or both of the 5' and 3' AAV inverted terminal repeat (ITR) sequences, (b) a gene of interest capable of expressing a gene therapy product, (c) a cardiac-specific promoter operably linked to the gene of interest and comprising a nucleic acid sequence at least 80% identical to any one of SEQ ID NOs: 233-263, wherein the cardiac-specific promoter comprises SEQ ID NO: 264, (d) an expression enhancing intron, if present, and (e) a polyadenylation signal, if present. The vector construct of claim 47, comprising an expression enhancing intron. The vector construct of claim 48, comprising a polyadenylation signal. The vector construct of claim 49, further comprising a fragment of an exon adjacent to the intron. The vector construct of any one of claims 47 to 50, wherein the rAAV vector construct has a size ranging from 4 kb to about 5.5 kb. A rAAV particle comprising the vector construct of any one of claims 47 to 51 and an AAV capsid, optionally a capsid having cardiac-specific tropism. A method for producing rAAV particles as claimed in claim 52, comprising the following steps: (a) providing a mammalian cell comprising one or more nucleic acid constructs, wherein the one or more nucleic acid constructs comprise (i) a vector construct as claimed in any one of claims 47 to 51, (ii) a nucleotide sequence encoding one or more AAV Rep proteins operably linked to a promoter, and (iii) a nucleotide sequence encoding one or more AAV capsid proteins operably linked to a promoter, (b) culturing the mammalian cell under conditions conducive to the expression of the Rep proteins and capsid proteins, and, if appropriate, (c) recovering the rAAV particles. The method of claim 53, wherein the mammalian cell is a HEK293 cell. A method for producing rAAV particles, comprising the following steps: (a) providing one or more nucleic acid constructs to cells permissive for AAV replication, the one or more nucleic acid constructs comprising: (i) a vector construct as described in any one of claims 47 to 51, (ii) a nucleotide sequence encoding one or more AAV Rep proteins, which is operably linked to a promoter capable of driving the expression of the one or more Rep proteins in the cell; and (iii) a nucleotide sequence encoding one or more AAV capsid proteins, which is operably linked to a promoter capable of driving the expression of the one or more capsid proteins in the cell; (b) culturing the cells under conditions that allow expression of the Rep proteins and the capsid proteins; and optionally (c) recovering the AAV particles. The method of claim 55, wherein the cell is an insect cell, optionally High Five, Sf9, Se301, SeIZD2109, SeUCR1, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAm1, BM-N, Ha2302, Hz2E5, or Ao38. The method of claim 55, wherein the cell is a mammalian cell, optionally HEK293, HeLa, CHO, NSO, SP2/0, PER.C6, Vero, RD, BHK, HT 1080, A549, Cos-7, ARPE-19 or MRC-5. A population of rAAV particles produced by the method of any one of claims 53 to 57, optionally enriched for particles containing full-length or nearly full-length vector genomes by a step of reducing the number of empty capsids. A pharmaceutical composition comprising the vector construct of any one of claims 47 to 51, or the rAAV particles of claim 52, or the rAAV particle population of claim 58 in the form of an aqueous suspension and a sterile pharmaceutically acceptable formulation. A method for delivering a gene of interest to cardiac tissue and/or cardiac myocytes of an individual in need thereof using any of the vector constructs, plasmids, viral vectors, viral particles or cells of any of claims 42 to 51, or the AAV particles of claim 52, or the rAAV particle population of claim 58, or the pharmaceutical composition of claim 59. A method for expressing a gene of interest in cardiac tissue and/or cardiac myocytes of an individual in need thereof using any of the vector constructs, plasmids, viral vectors, viral particles or cells of any of claims 42 to 51, or the AAV particles of claim 52, or the rAAV particle population of claim 58, or the pharmaceutical composition of claim 59. A method for expressing a gene therapy product in cardiac tissue and/or myocardial cells of an individual in need thereof using any of the vector constructs, plasmids, viral vectors, viral particles or cells of any one of claims 42 to 51, or the AAV particles of claim 52, or the rAAV particle population of claim 58, or the pharmaceutical composition of claim 59, for treating a disease or condition improved by the gene therapy product. The method of any one of claims 60 to 62, wherein the transgene comprises providing enhanced expression of the gene of interest in cardiac tissue and/or cardiac myocytes of such individual. A method as in any one of claims 60 to 62, wherein the method results in an increase in the level of the gene of interest or an increase in the level of the gene therapy product in the cardiac tissue and/or cardiac myocytes of such an individual. The method of any one of claims 60 to 62, wherein the individual suffers from a genetic disease or genetic disorder caused by a mutant endogenous gene of interest, wherein a functional copy of the gene of interest is administered to supplement or replace the activity of the mutant endogenous gene of interest. The method of claim 65, wherein the method increases the level of the functional gene therapy product in the cardiac tissue and/or cardiac myocytes of the individual by at least about 2-fold compared to the level in the untreated subject. A method as in any one of claims 60 to 62, wherein the individual suffers from a deficiency of a gene therapy product, and the method improves the frequency of deficiency of the gene therapy product and/or the severity of symptoms associated therewith. The method of any one of claims 60 to 62, wherein the individual suffers from a deficiency of the gene therapy product and the method increases the level of the functional gene therapy product in the individual to at least about 50% of the level seen in healthy humans. The method of any one of claims 60 to 68, wherein the individual suffers from coronary artery disease, cardiovascular disease, ischemic heart disease, angina, myocardial infarction, heart failure, congestive heart failure, hypertensive heart disease, inflammatory heart disease, cardiomyopathy, dilated cardiomyopathy, systolic cardiomyopathy, diastolic cardiomyopathy, hypertrophic cardiomyopathy, valvular heart disease, atherosclerosis, cardiac hypertrophy, cardiac fibrosis, cardiac inflammation, arrhythmia, atrial fibrillation, ventricular fibrillation, neointimal hyperplasia, or pulmonary hypertension. The method of any one of claims 60 to 69, wherein the method results in at least about 2-fold higher in vitro expression in cardiac tissue and/or cardiac myocytes compared to non-cardiac tissue or cells. The method of any one of claims 60 to 70, further comprising administering a second therapeutic agent. The method of any one of claims 60 to 70, wherein rAAV particles or a population of rAAV particles are administered to the individual, and the method comprises administering a prophylactic and/or therapeutic corticosteroid. The method of any one of claims 60 to 72, wherein the subject is a human subject, preferably a subject suffering from a cardiac disease or disorder. A vector construct, plasmid, viral vector, viral particle or cell as in any one of claims 42 to 51, or an AAV particle as in claim 52, or a rAAV particle population as in claim 58, or a pharmaceutical composition as in claim 59, or a method as in any one of claims 60 to 72, wherein the gene of interest expresses siRNA, shRNA, miRNA or ribozyme. A vector construct, plasmid, viral vector, viral particle or cell as in any one of claims 42 to 51, or an AAV particle as in claim 52, or a rAAV particle population as in claim 58, or a pharmaceutical composition as in claim 59, or a method as in any one of claims 60 to 72, wherein the gene of interest expresses a peptide or protein. A vector construct, plasmid, viral vector, viral particle or cell as in any one of claims 42 to 51, or an AAV particle as in claim 52, or a rAAV particle population as in claim 58, or a pharmaceutical composition as in claim 59, or a method as in any one of claims 60 to 72, wherein the component of the gene editing system expressing the gene of interest is, as the case may be, a zinc finger nuclease (ZFN) with a guide RNA, a transcription activator-like effector nuclease (TALEN), a meganuclease, or a clustered regulatory interspaced short tandem palindromic repeat (CRISPR)/CRISPR-associated protein (Cas)-associated nuclease.