Treatment of cardiomyopathy with AAV gene therapy vectors

Abstract

Provided herein are gene therapy compositions and methods for treating reduced levels of functional cardiac myosin binding protein C in subjects with hypertrophic cardiomyopathy.

Description

[Technical Field]

[0001] Provided herein are recombinant adeno-associated virus (rAAV) gene therapy vectors and viral particles useful for the treatment and prevention of hypertrophic cardiomyopathy by increasing the expression of cardiac myosin-binding protein C (cMyBP-C).

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 376,712, filed September 22, 2022, and U.S. Provisional Patent Application No. 63 / 519,967, filed August 16, 2023, each of which is incorporated by reference in its entirety.

[0003] Sequence Listing Reference This patent application contains an electronic sequence listing (filename: PCT_SeqListing.xml; created September 6, 2023; 763,166 bytes), which is incorporated herein by reference in its entirety. [Background technology]

[0004] Although considerable progress has been made in preventing heart disease caused by environmental factors such as nicotine, hypercholesterolemia, or diabetes, and in treating the symptoms of heart disease, there remains a need for improved methods of treating genetic cardiomyopathies, including hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and arrhythmogenic right ventricular cardiomyopathy (ARVC).

[0005] Hypertrophic cardiomyopathy is the most common inherited heart disease and is characterized by left ventricular hypertrophy of unknown cause. Hypertrophic cardiomyopathy is associated with impaired diastolic function despite initially normal systolic function (Elliott et al., Eur. Heart J. 29:270-6 (2008); Gersch et al., J. Thorac. Cardiovasc. Surg. 142:153-203 (2011)). Hypertrophic cardiomyopathy has a particularly high prevalence in the general population, approximately 1:500 (Maron et al., Circulation 92:785-9 (1995)), and is the leading cause of sudden cardiac death in young people, especially athletes. HCM is a life-threatening disease, but there is currently no curative treatment (Carrier et al., Cardiovasc. Res. 85:330-338 (2010); Schlossarek et al., J. Mol. Cell. Cardiol. 50:613-20 (2011)).

[0006] Hereditary hypertrophic cardiomyopathy is a genetic disorder known to be caused by over 1000 different mutations in at least 10 genes encoding components of the myocardium, such as cardiac myosin-binding protein C (cMyBP-C), beta-myosin heavy chain (MYH7), cardiac troponin T (TNNT2), cardiac troponin I (TNNI3), myosin ventricular essential light chain 1 (MYL3), myosin ventricular regulatory light chain 2 (MYL2), cardiac actin (ACTC), α-tropomyosin (TPM1), titin (TTN), and four-and-a-half LIM protein 1 (FHL1) (Richard et al., Circulation, 107:2227-2232 (2003); Schlossarek et al., J. Mol. Cell Cardiol. 50:613-20 (2011); Friedrich et al., J. Mol. Cell Cardiol. 50:613-20 (2011)). (e.g., Marian et al., Hum. Mol. Genet. 21:3237-54 (2012)). While many mutations are missense mutations that encode full-length mutant polypeptides, other frameshift or splice site mutations can result in truncations (e.g., Marian et al., Circ. Res. 121:749-70 (2017); Walsh et al., Genet. Med. 19:192-203 (2017)). The most common truncated mutant polypeptides are MYBPC3 and FHL1, which primarily exhibit frameshift mutations, resulting in C-terminal truncated proteins.

[0007] The most frequently mutated gene in HCM is MYBPC3, which encodes cardiac myosin-binding protein C (cMyBP-C) (Bonne et al., Nat. Genet. 11:438-40 (1995); Watkins et al., N. Engl. J. Med. 364:1643-56 (2011)). cMyBP-C is a major component of the A-band of the sarcomere, where it interacts with myosin, actin, and titin (Schlossarek et al., J. Mol. Cell. Cardiol. 50:613-20 (2011)). In humans and mice, cMyBP-C is detected exclusively in the heart (Fougerousse et al., Circ. Res. 82:130-3 (1998)) and is involved in the regulation of cardiac contraction and relaxation (Pohlmann et al., Circ. Res. Circ. Res. 101:928-38 (2007); Schlossarek et al., J. Mol. Cell. Cardiol. 50:613-20 (2011)). Approximately 70% of mutations in the MYBPC3 gene result in a frameshift, resulting in the generation of a C-terminally truncated protein (Carrier et al., Circ. Res. 80:427-34 (1997)). The truncated protein is unstable and has never been detected in patient myocardial tissue (Marston et al., Circ. Res. 105:219-22 (2009); van Dijk et al., Circulation, 119:1473-83 (2009); van Dijk et al., Circ. Heart Fail. 5:36-46 (2012)).

[0008] Current drug-based treatments for HCM alleviate symptoms but do not treat the underlying genetic cause of the disease. Gene- or RNA-based therapy may be the only curative treatment for HCM. Gene therapy approaches have been successfully tested in the context of non-genetic heart disease (Jessup et al., Circulation, 124:304-13 (2011)). Summary of the Invention

[0009] Embodiments described herein relate to vector constructs, recombinant, replication-deficient AAV particles, cells, and pharmaceutical compositions for delivering cardiac myosin-binding protein C (cMyBP-C) to subjects with HCM or subjects with a deficiency of a functional cardiomyocyte protein, such as cMyBP-C. Embodiments described herein also relate to the use of such AAV particles or such vector constructs to deliver a gene encoding cMyBP-C to the myocardium of such subjects.

[0010] The gene therapy vector is suitable for use in treating or preventing HCM in a mammalian subject, preferably a human subject, in need of treatment. In some embodiments, the subject in need of treatment is a subject carrying a mutation in at least one or both of the genes encoding cMyBP-C. After administration to the subject to be treated, the vector provides expression of the encoded cardiac myosin binding protein in the subject, preferably in the subject's cardiac muscle.

[0011] In one aspect, embodiments described herein provide a vector construct comprising a nucleic acid sequence encoding a functional cMyBP-C protein. In one or more embodiments, the functional cMyBP-C protein comprises an amino acid sequence at least 90%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO:2 (human cardiac myosin binding protein C). In some embodiments, the functional cMyBP-C protein comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:2. In exemplary embodiments, the nucleic acid sequence encoding the functional cardiac myosin binding protein C is a wild-type sequence (examples include SEQ ID NOs:1, 42, and 43), or is codon-optimized or variant. Alternative codon-optimized or variant human cardiac myosin binding protein C coding sequences are set forth as SEQ ID NOs:44-46. The coding sequence for cardiac myosin binding protein C (cMyBP-C) is, in some embodiments, codon-optimized for expression in humans. In some embodiments, the nucleic acid sequence encoding functional cMyBP-C is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1 or 42-46.

[0012] The expressed protein may also be a functional variant that exhibits significant amino acid sequence identity (i.e., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) compared to SEQ ID NO: 2. In this context, the term "functional variant" means that the variant of the cMyBP-C protein is capable of performing the function of a naturally occurring cMyBP-C protein, e.g., providing structural and / or functional support to the sarcomere to restore normal cardiac muscle contractility, and optionally suppressing and / or reducing the levels of expression of a mutant cMyBP-C protein or other mutant sarcomere proteins.

[0013] Functional cMyBP-C protein variants can include proteins that differ from their naturally occurring counterparts, for example, by one or more amino acid substitutions, deletions, or additions. For example, a variant protein of the human cMyBP-C protein of SEQ ID NO: 2 can have an amino acid sequence in which at least 2, 3, 4, 5, 6, 10, or more positions, and / or up to 10, 20, 30, or more positions, relative to SEQ ID NO: 2, are substituted with different amino acids. As another example, a variant protein of the human cMyBP-C protein of SEQ ID NO: 2 can be a truncated version of the human cMyBP-C protein. For example, a functional variant can be selected from the group consisting of a naturally occurring MYBPC3 splice variant lacking exons 5 and 6, designated variant 4 (as set forth in SEQ ID NO: 46).

[0014] In one or more embodiments, the nucleic acid sequence encoding cMyBP-C is operably linked to one or more heterologous expression control elements. Preferably, expression of the cMyBP-C-encoding transgene is controlled by at least one cardiomyocyte-specific expression control element. Thus, in such embodiments, in the vector constructs described herein, the nucleic acid sequence encoding cMyBP-C 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, optionally an intron, optionally an exon, and a polyadenylation (polyA) signal. Such elements are further described herein.

[0015] 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 a fragment or variant of the human cardiac troponin T (hTNNT2) promoter.

[0016] In some embodiments, the cardiomyocyte-specific promoter comprises a nucleic acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical, or more than 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical (over the entire length of SEQ ID NO: 47) to SEQ ID NO: 47. In some embodiments, the cardiomyocyte-specific promoter may be combined with an intron that enhances expression of the cMyBP-C protein, located 5' to the cMyBP-C coding sequence. For example, the vector construct and / or AAV particle comprises, in the 5' to 3' direction, a nucleotide sequence encoding a cardiomyocyte-specific promoter, an intron that enhances expression of the cMyBP-C protein, and a cMyBP-C coding sequence. 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: 47, (ii) SEQ ID NO: 48, (iii) SEQ ID NO: 49, (iv) SEQ ID NO: 50, (v) SEQ ID NO: 51, or (vi) SEQ ID NO: 52; (b) an intron located 5' to the cMyBP-C coding sequence comprising a nucleotide sequence at least 60% identical to SEQ ID NO: 53; a nucleotide sequence encoding cMyBP-C; and optionally a polyadenylation signal sequence. Alternatively, the intron comprises a nucleotide sequence at least 60% identical to SEQ ID NO: 56 or SEQ ID NO: 58.

[0017] In other embodiments, the cardiomyocyte-specific promoter may be combined with an intron located within the cMyBP-C coding sequence that enhances expression of the cMyBP-C protein. In some embodiments, the intron sequence is located within the nucleotide sequence encoding cMyBP-C, e.g., between one of the exons, e.g., between exons 2 and 3. In some embodiments, the intron is located at position 293 of any one of SEQ ID NOs: 1 or 42-45.

[0018] In some embodiments, the vector construct and / or resulting AAV particles comprise a cardiomyocyte-specific promoter sequence that is more than 420 nucleotides in length and less than 544 nucleotides in length and is a fragment or variant of the hTNNT2 promoter, comprising a nucleic acid sequence at least 90% identical to SEQ ID NO: 47. In any of the embodiments described herein, the cardiomyocyte-specific promoter optionally excludes any one of SEQ ID NOs: 1-85 of U.S. Patent Publication No. 2021 / 0252165.

[0019] For example, the cardiomyocyte-specific promoter sequence comprises a nucleic acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, or 99%, or greater than 80%, 85%, 90%, 95%, 97%, 98%, or 99%, identical to any of: (i) SEQ ID NO: 47 or a fragment thereof, (ii) SEQ ID NO: 48 or a fragment thereof, (iii) SEQ ID NO: 49 or a fragment thereof, (iv) SEQ ID NO: 50 or a fragment thereof, (v) SEQ ID NO: 51 or a fragment thereof, or (vi) SEQ ID NO: 52 or a fragment thereof. In exemplary embodiments, the cardiomyocyte-specific promoter sequence comprises a nucleotide sequence at least 96%, 97%, 98%, or 99% identical to SEQ ID NO: 51. In some exemplary embodiments, the hTNNT promoter sequence comprises at least nucleotides 1-106 and 507-532 of SEQ ID NO: 51, or at least nucleotides 507-532 of SEQ ID NO: 51, or at least nucleotides 521-532 of SEQ ID NO: 51.

[0020] In some embodiments, the vector construct comprises one or more introns that enhance expression of the cMyBP-C-encoding nucleic acid, e.g., so that increased levels are detectable in the 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 at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 53. In one or more embodiments, the intron comprises a nucleotide sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 56. In one or more embodiments, the intron comprises a nucleotide sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 58. In some embodiments, the intron is inserted downstream of the promoter and 5' to the cMyBP-C coding sequence. In some embodiments, the intron is located within the nucleotide sequence encoding cMyBP-C, e.g., between one of the exons, e.g., between exons 2 and 3. In an exemplary embodiment, the intron is inserted at nucleotide position 293 of the wild-type cDNA sequence of MYBPC3 of SEQ ID NO: 1. In a further exemplary embodiment, the intron is inserted at nucleotide position 293 of the wild-type cDNA sequence of MYBPC3 of any one of SEQ ID NOs: 42-45.

[0021] In some embodiments, the vector construct may further comprise a globin intron adjacent to the 3' end of an exon sequence or fragment thereof, e.g., a fragment of beta globin exon 3 (SEQ ID NO: 54), preferably adjacent to an intron sequence. The cardiomyocyte-specific transcriptional regulatory region may comprise a combination of an intron and an exon fragment, e.g., SEQ ID NO: 55. In some exemplary embodiments, the cardiomyocyte-specific transcriptional regulatory region comprises SEQ ID NO: 56.

[0022] In some embodiments, the vector construct comprises a polyadenylation signal, optionally a bovine growth hormone (bGH) polyA signal (e.g., SEQ ID NO:59, 60, or 61) or a fragment thereof, optionally a human growth hormone (hGH) polyA signal (e.g., SEQ ID NO:62) or a fragment thereof, optionally an SV40 polyA signal (e.g., SEQ ID NO:63) or a fragment thereof, optionally a Proudfoot synthetic polyA signal (e.g., SEQ ID NO:65) or a fragment thereof, or optionally a rabbit beta-globin polyA signal (e.g., SEQ ID NO:66) or a fragment thereof. In some embodiments, the polyA signal comprises a nucleotide sequence at least 90% identical to SEQ ID NO:64. In some embodiments, the polyA signal comprises a nucleotide sequence at least 90% identical to SEQ ID NO:59, e.g., SEQ ID NO:60 or a fragment thereof, or SEQ ID NO:61 or a fragment thereof. In an exemplary embodiment, the polyA signal is a fragment of SEQ ID NO: 62, which contains SEQ ID NO: 59, and is about 100 to about 500 nucleotides in length, or about 150 to about 400 nucleotides in length, or about 200 to about 300 nucleotides in length, or about 200 to about 250 nucleotides in length.

[0023] In some embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98%, or 99% identical to any of SEQ ID NOs: 3-41. In other embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98%, or 99% identical to a nucleotide sequence that is complementary to, i.e., the minus (-) strand of, any of SEQ ID NOs: 3-41 or 92-169.

[0024] In some embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98%, or 99% identical to any of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, or 39. In other embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98%, or 99% identical to a nucleotide sequence that is complementary to, i.e., the minus (-) strand of, any of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, or 39.

[0025] In some embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98%, or 99% identical to any of SEQ ID NOs: 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, or 40. In other embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98%, or 99% identical to a nucleotide sequence that is complementary to, i.e., the minus (-) strand of, any of SEQ ID NOs: 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, or 40.

[0026] In some embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98%, or 99% identical to any of SEQ ID NOs: 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, or 41. In other embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98%, or 99% identical to a nucleotide sequence that is complementary to, i.e., the minus (-) strand of, any of SEQ ID NOs: 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, or 41.

[0027] Example embodiments include the following:

[0028] Construct C1 is 4950 bp long (SEQ ID NO:29) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO:67), hTNNT2 promoter (544 bp) (SEQ ID NO:52), chimeric intron (133 bp) (SEQ ID NO:58), codon-optimized hMYBPC3 (SEQ ID NO:44), minipolyA (57 bp) (SEQ ID NO:64), and 3' AAV2 ITR (130 bp) (SEQ ID NO:71). In a further embodiment, construct C1 optionally contains any of the 5' AAV2-ITR sequences of SEQ ID NOs:67-70 (or their complementary sequences) and / or any of the 3' AAV2-ITR sequences of SEQ ID NOs:71-74 (or their complementary sequences), or fragments thereof.

[0029] Construct C2 is 4801 bp long (SEQ ID NO: 32) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 67), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), codon-optimized hMYBPC3 (SEQ ID NO: 44), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71). In a further embodiment, construct C2 optionally contains any of the 5' AAV2-ITR sequences of SEQ ID NOs: 67-70 (or their complementary sequences) and / or any of the 3' AAV2-ITR sequences of SEQ ID NOs: 71-74 (or their complementary sequences), or fragments thereof.

[0030] Construct C3 is 4801 bp long (SEQ ID NO: 35) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 67), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), wild-type hMYBPC3 (SEQ ID NO: 43), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71). In a further embodiment, construct C3 optionally contains any of the 5' AAV2-ITR sequences of SEQ ID NOs: 67-70 (or their complementary sequences) and / or any of the 3' AAV2-ITR sequences of SEQ ID NOs: 71-74 (or their complementary sequences), or fragments thereof.

[0031] Construct C4 is 4950 bp long (SEQ ID NO:38) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO:67), hTNNT2 promoter (544 bp) (SEQ ID NO:52), chimeric intron (133 bp) (SEQ ID NO:58), wild-type hMYBPC3 (SEQ ID NO:43), minipolyA (57 bp) (SEQ ID NO:64), and 3' AAV2 ITR (130 bp) (SEQ ID NO:71). In a further embodiment, construct C4 optionally contains any of the 5' AAV2-ITR sequences of SEQ ID NOs:67-70 (or their complementary sequences) and / or any of the 3' AAV2-ITR sequences of SEQ ID NOs:71-74 (or their complementary sequences), or fragments thereof.

[0032] Construct C5 is 4950 bp long (SEQ ID NO:41) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO:67), hTNNT2 promoter (544 bp) (SEQ ID NO:52), chimeric intron (133 bp) (SEQ ID NO:58), CpG-free hMYBPC3 (SEQ ID NO:45), minipolyA (57 bp) (SEQ ID NO:64), and 3' AAV2 ITR (130 bp) (SEQ ID NO:71). In a further embodiment, construct C5 optionally contains any of the 5' AAV2-ITR sequences of SEQ ID NOs:67-70 (or their complementary sequences) and / or any of the 3' AAV2-ITR sequences of SEQ ID NOs:71-74 (or their complementary sequences), or fragments thereof.

[0033] Construct A1 is 5074 bp long (SEQ ID NO: 5) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 67), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), globin intron (131 bp) (SEQ ID NO: 53), HBB exon 3 (SEQ ID NO: 54), wild-type hMYBPC3 (SEQ ID NO: 42), bGH polyA (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71). In a further embodiment, construct A1 optionally contains any of the 5' AAV2-ITR sequences of SEQ ID NOs: 67-70 (or their complementary sequences) and / or any of the 3' AAV2-ITR sequences of SEQ ID NOs: 71-74 (or their complementary sequences), or fragments thereof.

[0034] Construct A2 is 4939 bp long (SEQ ID NO: 8) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 67), hTNNT2 promoter (469 bp) (SEQ ID NO: 49), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH polyA (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71). In a further embodiment, construct A2 optionally contains any of the 5' AAV2-ITR sequences of SEQ ID NOs: 67-70 (or their complementary sequences) and / or any of the 3' AAV2-ITR sequences of SEQ ID NOs: 71-74 (or their complementary sequences), or fragments thereof.

[0035] Construct A3 is 4939 bp long (SEQ ID NO: 11) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 67), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH polyA (169 bp) (SEQ ID NO: 59), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71). In a further embodiment, construct A3 optionally contains any of the 5' AAV2-ITR sequences of SEQ ID NOs: 67-70 (or their complementary sequences) and / or any of the 3' AAV2-ITR sequences of SEQ ID NOs: 71-74 (or their complementary sequences), or fragments thereof.

[0036] Construct A4 is 4939 bp long (SEQ ID NO: 14) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 67), hTNNT2 promoter (499 bp) (SEQ ID NO: 50), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH polyA (202 bp) (SEQ ID NO: 60), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71). In a further embodiment, construct A4 optionally contains any of the 5' AAV2-ITR sequences of SEQ ID NOs: 67-70 (or their complementary sequences) and / or any of the 3' AAV2-ITR sequences of SEQ ID NOs: 71-74 (or their complementary sequences), or fragments thereof.

[0037] Construct A5 is 4871 bp long (SEQ ID NO: 17) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 67), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), wild-type hMYBPC3 (SEQ ID NO: 42), bGH polyA (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71). In a further embodiment, construct A5 optionally contains any of the 5' AAV2-ITR sequences of SEQ ID NOs: 67-70 (or their complementary sequences) and / or any of the 3' AAV2-ITR sequences of SEQ ID NOs: 71-74 (or their complementary sequences), or fragments thereof.

[0038] Construct A6 is 5002 bp long (SEQ ID NO:20) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO:67), hTNNT2 promoter (532 bp) (SEQ ID NO:51), wild-type hMYBPC3 (SEQ ID NO:42) containing a globin intron (131 bp) (SEQ ID NO:53) between exons, bGH polyA (227 bp) (SEQ ID NO:61), and 3' AAV2 ITR (130 bp) (SEQ ID NO:71). In a further embodiment, construct A6 optionally contains any of the 5' AAV2-ITR sequences of SEQ ID NOs:67-70 (or their complementary sequences) and / or any of the 3' AAV2-ITR sequences of SEQ ID NOs:71-74 (or their complementary sequences), or fragments thereof.

[0039] Construct A7 is 4781 bp long (SEQ ID NO:23) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO:67), hTNNT2 promoter (469 bp) (SEQ ID NO:49), Kozak sequence, wild-type hMYBPC3 (SEQ ID NO:42) containing a globin intron (131 bp) (SEQ ID NO:53) between exons, minipolyA (57 bp) (SEQ ID NO:64), 3'-UTR sequence, and 3' AAV2-ITR (130 bp) (SEQ ID NO:71). In a further embodiment, construct A7 optionally contains any of the 5' AAV2-ITR sequences set forth in SEQ ID NOs:67-70 (or their complementary sequences) and / or any of the 3' AAV2-ITR sequences set forth in SEQ ID NOs:71-74 (or their complementary sequences), or fragments thereof.

[0040] Construct A8 is 4844 bp long (SEQ ID NO:26) and contains the following elements from 5' to 3': 5'AAV2-ITR (130 bp) (SEQ ID NO:67), hTNNT2 promoter (532 bp) (SEQ ID NO:51), Kozak sequence, wild-type hMYBPC3 (SEQ ID NO:42) containing a globin intron (131 bp) (SEQ ID NO:53) between exons, minipolyA (57 bp) (SEQ ID NO:64), 3'-UTR sequence, and 3'AAV2-ITR (130 bp) (SEQ ID NO:71). In a further embodiment, construct A8 optionally contains any of the 5'AAV2-ITR sequences set forth in SEQ ID NOs:67-70 (or their complementary sequences) and / or any of the 3'AAV2-ITR sequences set forth in SEQ ID NOs:71-74 (or their complementary sequences), or fragments thereof.

[0041] 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 complements and / or combinations thereof).

[0042] In any of the foregoing embodiments, the length of the vector insert, beginning with one ITR and ending with the second ITR, is about 4 kb to about 5.5 kb in size. In one or more embodiments, the vector construct is an AAV vector genome about 4 kb to about 5.4 kb in size, about 4.5 kb to about 5.5 kb in size, or about 4.8 kb to about 5.2 kb in size, or about 4.5 kb to about 5 kb in size.

[0043] The vector construct is preferably a recombinant AAV vector construct. In some embodiments, the vector construct comprises (a) one or both of (i) an AAV 5' inverted terminal repeat (ITR) and (ii) an AAV 3' ITR, (b) a promoter and / or enhancer, e.g., a cardiomyocyte-specific transcriptional regulatory region, and (c) a nucleic acid sequence encoding a functionally active human cMyBP-C protein. In some embodiments, the vector construct comprises (a) an AAV 5' inverted terminal repeat (ITR) sequence, (b) a promoter and / or enhancer, e.g., a cardiomyocyte-specific transcriptional regulatory region, (c) a nucleic acid sequence encoding a functionally active human cMyBP-C protein, (d) an intron, (e) a polyadenylation signal, and (f) an AAV 3' ITR. In some embodiments, the intron is downstream of the promoter and located 5' to the cMyBP-C coding sequence, while in other embodiments, the intron is located between exons of the cMyBP-C coding sequence, e.g., between exon 2 and exon 3. In further embodiments, the vector construct comprises (a) an AAV 5' inverted terminal repeat (ITR) sequence, (b) a promoter and / or enhancer, e.g., a cardiomyocyte-specific transcriptional regulatory region, (c) a nucleic acid sequence encoding a functionally active human cMyBP-C protein, (d) an intron, (e) and an exon, (f) a polyadenylation signal, and (g) an AAV 3' ITR. The AAV 5' ITR and / or AAV 3' ITR can 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: 67-70 and 71-74, respectively).

[0044] In any of the foregoing 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 of SEQ ID NOs: 3-42 and 92-169 over the entire length of SEQ ID NOs: 3-42 and 92-169, respectively. In some embodiments, the vector construct is at least 97%, 98%, or 99% identical to any of SEQ ID NOs: 3-42 and 92-169 over the entire length of SEQ ID NOs: 3-42 and 92-169, respectively. In particular examples, the vector construct comprises a nucleotide sequence at least 85% identical to any of SEQ ID NOs: 29, 32, or 41, or at least 95% identical to any of SEQ ID NOs: 35 or 38. In other examples, the vector construct comprises a nucleotide sequence at least 90% identical to any of SEQ ID NOs: 29, 32, or 41, or at least 98% identical to any of SEQ ID NOs: 35 or 38. Such vectors preferably comprise, for example, flanking ITRs, a nucleic acid sequence encoding a functionally active human cMyBP-C protein coding sequence, a cardiomyocyte-specific regulatory region, an intron, and a polyA signal.

[0045] In another aspect, provided herein is a recombinant adeno-associated virus (rAAV) particle comprising an AAV capsid and a vector construct described in one or more of the embodiments herein. Any AAV capsid, for example, AAV1-13, may be used. In some embodiments, the recombinant AAV (rAAV) particle used to deliver a cMyBP-C-encoding gene has cardiotropy. In such embodiments, the rAAV comprises an AAV capsid with cardiotropy, for example, an AAV9 capsid at least 85%, 90%, or 95% identical to SEQ ID NO: 75, or an AAV1, AAV6, or AAV7 capsid that exhibits cardiotropy, or a variant of any of these. In one or more embodiments, the AAV capsid is a capsid that reduces pre-existing humoral immunity compared to AAV9, for example, as assessed by in vitro IVIG neutralization.

[0046] In another aspect, provided herein is a method for producing AAV particles useful as gene delivery vectors, the method comprising: (1) providing a cell (e.g., a mammalian cell) with one or more nucleic acid constructs comprising: (a) a vector construct described herein, comprising a nucleic acid encoding cMyBP-C described herein flanked by two AAV ITR nucleotide sequences; (b) a nucleotide sequence encoding one or more AAV Rep proteins operably linked to a promoter capable of driving expression of the Rep proteins; (c) a nucleotide sequence encoding one or more AAV capsid proteins operably linked to a promoter capable of driving expression of the capsid proteins; and (d) optionally, genes encoding AAP and MAAP contained in VP2 / 3; (2) culturing the cell defined in (1) under conditions promoting expression of the Rep and capsid proteins; and, optionally, (3) recovering the AAV particles. In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is a HEK293 cell. Also provided herein are populations of rAAV particles produced by such methods.

[0047] In yet another aspect, provided herein is a pharmaceutical composition comprising a vector construct described herein, or an rAAV particle or population of rAAV particles described herein, and a sterile, pharmaceutically acceptable diluent, excipient, or carrier.

[0048] In a further aspect, provided herein are methods for delivering the MYBPC3 gene to a mammalian subject. Such methods include methods for expressing myosin binding protein C in a mammalian subject, the methods comprising administering to the subject a composition comprising a vector construct described herein, an rAAV particle described herein, or a pharmaceutical composition described herein, thereby expressing the encoded myosin binding protein in the subject. Preferably, in such methods, the mammal is a human, and the myosin binding protein C is a functional human myosin binding protein C described herein. Such methods include methods for expressing myosin binding protein C in cells of the mammalian cardiac muscle by administering an amount of a vector construct, rAAV particle, or pharmaceutical composition effective to increase the level of expression of myosin binding protein C in the mammalian cardiac muscle. Such methods also include methods of increasing the level of functional myosin binding protein C in cardiac tissue (e.g., cardiomyocytes) of a mammal by administering a vector construct, rAAV particle, or pharmaceutical composition in an amount effective to increase the level of functional myosin binding protein C in cardiac tissue (e.g., cardiomyocytes) of the mammal. Such methods also include methods of treating a deficiency of functional wild-type myosin binding protein C in a mammal by administering a vector construct, rAAV particle, or pharmaceutical composition in an amount effective to increase the level of functional myosin binding protein C in cardiac tissue (e.g., cardiomyocytes) of the mammal. In some embodiments, the amount of vector construct, rAAV particle, or pharmaceutical composition is effective to increase the level of myosin binding protein C in cardiac tissue (e.g., cardiomyocytes) by at least about two-fold and / or is effective to restore contractile force, relative tension, calcium-activated tension, relaxation time in engineered cardiac tissue in vitro or in animal tissue in vivo.

[0049] Such methods also include methods for treating HCM in a mammal, or treating or preventing any symptoms 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 level of cMyBP-C 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 levels without treatment, or to levels seen in healthy humans. Such methods may, for example, reduce heart size, reduce cardiothoracic ratio, reduce end-diastolic or end-systolic left ventricular diameter, reduce anterior or posterior wall thickness, increase ejection time, increase aortic peak flow velocity or aortic flow time, 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, arrhythmia, chest pain, shortness of breath, fatigue, and dizziness.

[0050] In any of the methods described herein, the rAAV particles are delivered in an aqueous suspension at a dose of approximately 1e12-6e14 vg / kg. In any of the methods described herein, administration of the vector construct, rAAV particles, or pharmaceutical composition may further include administration of a prophylactic or therapeutic corticosteroid treatment and / or may further include administration of a second treatment for treating HCM. In any of the methods described herein, prior to administration of AAV particles to a patient as described above, the prospective patient may be evaluated for the presence of anti-AAV capsid antibodies or anti-AAV neutralizing antibodies that may block cellular transduction or otherwise reduce the overall efficacy of treatment.

[0051] From reading this specification, other embodiments will be apparent to those skilled in the art. [Brief explanation of the drawings]

[0052] [Figure 1] Illustrated is the organization of elements of an AAV particle containing vectors designated herein as C1-C5 and A1-A6. [Figure 2] Illustrated is the fold change in cMyBP-C protein detected by Western blot in total protein lysates of engineered cardiac tissue for vectors designated herein as C1-C5 and A1-A6. [Figure 3] 1 illustrates the normalized contractile force of myocytes in engineered cardiac tissue treated with AAV particles containing vectors designated herein as C2, C3, and A2-A6. [Figure 4A] 1 illustrates the relaxation time after contraction of myocytes in engineered cardiac tissue treated with AAV particles containing vectors designated herein as C2, C3, and A1-A6. The relative percentage of late relaxation time is shown. [Figure 4B] 1 illustrates the relaxation time after contraction of myocytes in engineered cardiac tissue treated with AAV particles containing vectors designated herein as C2, C3, and A1-A6. Time to 20% relaxation is shown in seconds. [Figure 4C] 1 illustrates the relaxation time after contraction of myocytes in engineered cardiac tissue treated with AAV particles containing vectors designated herein as C2, C3, and A1-A6. Time to 80% relaxation is shown in seconds. [Figure 4D] Normalized % force for constructs A3 and A6 produced in HEK293 cells (group 3) and insect cells (group 4) is shown. [Figure 5A] The DNA copy numbers (vector genomes) in mice administered AAV particles containing vectors designated herein as C1 to C5 and A1 to A6, respectively, are shown. [Figure 5B] 1 illustrates the RNA copy numbers in mice administered AAV particles containing vectors designated herein as C1 to C5 and A1 to A6, respectively. [Figure 5C] Illustrated is cMyBP-C protein (ug / gram of heart tissue) in mice administered AAV particles containing vectors designated herein as C1-C5 and A1-A6, respectively. [Figure 6]1 depicts the percentage of cardiomyocytes in cardiac tissue expressing human cMyBP-C from mice administered AAV particles containing vectors designated herein as C3, A5, and A6. DETAILED DESCRIPTION OF THE INVENTION

[0053] Provided herein are nucleic acids or vector constructs encoding functionally active therapeutic cMyBP-C protein, AAV vector genomes and replication-deficient rAAV particles containing such vector constructs, and pharmaceutical compositions containing such vector constructs, vector genomes, and AAV particles. The compositions and methods of the present invention may provide improved AAV virus production yields and / or simplified purification, and / or enhanced expression of cMyBP-C protein in the heart, particularly in cardiac muscle cells (cardiomyocytes). Also provided herein are methods for producing vector constructs, AAV vector genomes, and replication-deficient rAAV particles containing such vector constructs. Further provided herein are methods for treating deficiencies of functional wild-type cMyBP-C, including HCM.

[0054] In another embodiment, a method is provided for producing recombinant adeno-associated virus (AAV) particles comprising any of the AAV vector constructs provided herein, the method comprising 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 culture.

[0055] Cells useful for recombinant AAV production provided herein are any cell type susceptible to baculovirus 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.

[0056] In another embodiment, provided herein is the use of an effective amount of a vector nucleic acid, vector construct, or AAV particle for the preparation of a medicament for treating a subject suffering from HCM or a deficiency of functional wild-type cMyBP-C protein. In one embodiment, the subject suffering from HCM is a human. In one embodiment, the medicament is administered intravenously (IV). In another embodiment, administration of the medicament results in an increase in the level of functional cMyBP-C in cardiac muscle cells to ameliorate the symptoms of HCM. In certain embodiments, the medicament is also suitable for co-administration with prophylactic and / or therapeutic corticosteroids for the prevention and / or treatment of any toxicity associated with administration of AAV particles. Prophylactic or therapeutic corticosteroid treatment may include at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more mg / day of corticosteroid. In certain embodiments, prophylactic or therapeutic corticosteroids may be administered for a duration of at least about 3, 4, 5, 6, 7, 8, 9, 10 weeks, or longer.

[0057] In another embodiment, the hypertrophic cardiomyopathy treatment provided herein optionally further comprises the administration, eg, co-administration, of other therapeutic agents used to treat HCM.

[0058] definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. See, e.g., Singleton et al., Dictionary of Microbiology and Molecular Biology 2 nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). For purposes of this disclosure, the following terms are defined below.

[0059] As used herein, in the context of gene delivery, the term "vector" or "gene delivery vector" can refer to a particle that functions as a gene delivery vehicle and contains, for example, a nucleic acid packaged within an envelope or capsid (i.e., a vector genome including any of the vector constructs described herein). The gene delivery vector can be a viral gene delivery vector or a non-viral gene delivery vector. Alternatively, in some contexts, the term "vector" can be used to refer only to the vector genome or vector construct. Viral vectors suitable for use herein can be parvoviruses, adenoviruses, retroviruses, lentiviruses, or herpes simplex viruses. The parvovirus can be an adenovirus-associated virus (AAV).

[0060] As used herein, the term "AAV" is the standard abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells in which certain functions are provided by a co-infecting helper virus. There are many characterized serotypes of AAV. 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 well known that the various serotypes are fairly closely related structurally and functionally, even at the genetic level, and it is fully expected that these same principles will be applicable to additional AAV serotypes (see, e.g., Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, J.R.P.Tattison, ed., and Rose, Comprehensive Virology 3:1-61 (1974)). For example, all AAV serotypes appear to exhibit very similar replication properties mediated by homologous rep genes, and all possess three related capsid proteins. The extent of relatedness is further suggested by heteroduplex analysis, which reveals extensive cross-hybridization between serotypes along the entire length of the genome, and the presence of similar self-annealing segments at the ends corresponding to "inverted terminal repeats" (ITRs).

[0061] As used herein, "AAV vector construct" refers to a nucleic acid that is either single-stranded or double-stranded and has at least one of (i) an AAV 5' inverted terminal repeat (ITR) sequence and (ii) an AAV 3' ITR, and optionally a polyadenylation sequence and / or optionally one or more introns, flanking a protein-coding sequence (in one embodiment, a functional therapeutic protein-coding sequence, e.g., a cMyBP-C coding sequence) operably linked to transcriptional regulatory elements (also known as "expression control elements") heterologous to the protein-coding sequence and / or heterologous to the AAV viral genome. A single-stranded AAV vector refers to a nucleic acid present in the genome of an AAV viral particle and can be either the sense strand or the antisense strand of a nucleic acid sequence disclosed herein. The size of such single-stranded nucleic acids is determined in base units. A double-stranded AAV vector refers to the DNA of a plasmid, such as pUC19, or the nucleic acid present in the genome of a double-stranded virus, such as a baculovirus, used to express or transfer the AAV vector nucleic acid. The size of such double-stranded nucleic acids is measured in base pairs (bp).

[0062] The single-stranded form of the AAV vector constructs provided herein is less than about 7.0 kb in length, or less than 6.5 kb in length, or less than 6.4 kb in length, or less than 6.3 kb in length, or less than 6.2 kb in length, or less than 6.0 kb in length, or less than 5.8 kb in length, or less than 5.6 kb in length, or less than 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 single-stranded form of the AAV vector constructs also is at least about 4.0 kb in length. Preferably, the AAV vector constructs are also at least about 4.5 kb in length. In some embodiments, the single-stranded form of the AAV vector constructs provided herein is in the range of about 4.0 kb to about 5.8 kb in length.

[0063] Although AAV particles with AAV genomes >5.0 kb have been reported in the literature, 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, it has been shown that overlapping homologous recombination can occur between nucleic acids with 5' and 3' truncations in AAV-infected cells, resulting in the generation of "intact" nucleic acids encoding large proteins, thereby reconstituting functional full-length genes.

[0064] 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 of the oversized AAV vector lacks the 5' AAV ITR and, in some cases, a portion of the 5' end of the target protein-encoding gene, and the antisense strand of the oversized AAV vector lacks the 3' ITR and, in some cases, a portion of the 3' end of the target protein-encoding gene. A functional transgene is produced in the oversized AAV vector-infected cell by annealing the sense and antisense truncated genomes in the target cell. Thus, in certain embodiments, the AAV cMyBP-C vector and / or viral particle comprises at least one ITR.

[0065] As used herein, the term "inverted terminal repeat (ITR)" refers to art-recognized regions found at the 5' and 3' ends of the AAV genome that function in cis as origins of DNA replication and as packaging signals for the viral genome. Together with the AAV rep-coding region, the AAV ITRs enable efficient excision and rescue from nucleotide sequences inserted between the two flanking ITRs, and integration of such nucleotide sequences into the host cell genome. The sequences of certain AAV-related ITRs are disclosed by Yan et al., J. Virol. 79:364-79 (2005), which is incorporated herein by reference in its entirety. ITR sequences useful herein may be full-length wild-type AAV ITRs or fragments thereof that retain functional capability, or may be sequence variants of full-length wild-type AAV ITRs that can act in cis as origins of replication. The AAV ITRs useful in the recombinant AAV cMyBP-C vectors of the embodiments provided herein may be derived from any known AAV serotype, and in certain embodiments, may be derived from the AAV2 or AAV5 serotype.

[0066] The term "control sequence" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences that are suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0067] "Transcriptional regulatory element" refers to the nucleotide sequence of a gene involved in regulating gene transcription, including a promoter, a positive response element, activator and enhancer sequences for binding of transcription factors that assist RNA polymerase binding and promote expression, and operator or silencer sequences to which repressor proteins bind to prevent RNA polymerase binding and block expression. The term "cardiomyocyte-specific transcriptional regulatory element" or "cardiomyocyte-specific expression control element" refers to a regulatory element or region that results in preferred gene expression specifically in cardiomyocytes, for example, a promoter whose activity in cardiac cells is at least two-fold or at least five-fold higher than in any other non-cardiac cell type. In some embodiments, a cardiomyocyte-specific promoter provides at least five-fold higher expression in cardiomyocytes than in skeletal muscle cells. In some embodiments, a cardiomyocyte-specific promoter has at least five-fold, at least ten-fold, at least fifteen-fold, at least twenty-fold, at least twenty-fold, or at least fifty-fold higher activity in cardiomyocytes compared to its activity in non-cardiac cell types.

[0068] A cardiac-specific or cardiomyocyte-specific promoter is operably linked to a nucleic acid sequence encoding a cMyBP-C protein, meaning that the promoter is combined with the encoding nucleic acid so as to allow expression of the encoding nucleic acid under the control of the promoter in cardiomyocytes when integrated into the genome of a cell or present in a cell as an extragenomic nucleic acid construct.

[0069] The transcriptional regulatory elements optionally include enhancer elements, introns, polyadenylation sequences, or post-transcriptional regulatory elements for increasing the expression level of myosin-binding proteins. Examples include the SV40 early gene enhancer and the Rous sarcoma virus long terminal repeat (LTR) enhancer (Gorman et al. (1982) Proc. Natl. Acad. Sci. 79:6777). The vector also optionally includes transcription termination and polyadenylation sequences for improving the expression of human and / or non-human antigen(s). Suitable transcription 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 vectors of the present invention. Any other elements known in the art to support expression efficiency or specificity, such as the woodchuck hepatitis posttranscriptional regulatory element (wPRE), may be added to the expression vector. To increase cardiac or cardiac myocyte specificity, other elements, such as sequences encoding miRNAs such as miR122, can be introduced to inactivate gene expression in other tissues (Geisler et al., Gene Ther. 18:199-209 (2011)).

[0070] As used herein, "intron" is broadly defined as a sequence of nucleotides that can be removed by RNA splicing. "RNA splicing" refers to the excision of an intron from a pre-mRNA to form a mature mRNA. An intron can be located upstream, downstream, or within the coding region of a gene. The insertion of an intron into a nucleotide sequence can be accomplished by any method known in the art. The only restriction on where an intron is inserted is to consider the packaging limit (e.g., about 5 kb) of AAV viral particles.

[0071] 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 placed under the control of one or more regulatory elements, including, 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" or "operably associated with" a regulatory element, meaning that the gene or coding region is controlled or influenced by the regulatory element. For example, a promoter is operably linked to a coding sequence if the promoter causes transcription or expression of the coding sequence.

[0072] In certain embodiments, the recombinant AAV vector construct comprises (a) a nucleic acid comprising an AAV2 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 cMyBP-C protein coding region; (d) optionally, one or more introns; (e) a polyadenylation sequence; and (f) an AAV2 3' ITR, which may or may not be modified as known in the art.

[0073] In one embodiment, the vector construct comprises a nucleic acid encoding a functionally active cMyBP-C protein. The cMyBP-C coding sequence may be wild-type, codon-optimized, or variant. To visualize the expression of the exogenous gene in the heart, other optional elements, such as a tag sequence (e.g., myc, FLAG, HA, His), or a fluorescent dye such as GFP, YFP, or RFP, may be introduced as part of the cMyBP-C coding sequence.

[0074] As used herein, wild-type cardiac myosin binding protein C (MYBPC3 gene) has the following nucleic acid sequence, SEQ ID NO: 1 (GenBank Accession No. NM_000256.2).

[0075] As used herein, wild-type cardiac myosin binding protein C has the following amino acid sequence, SEQ ID NO: 2 (GenBank Accession No. NP_000247.1).

[0076] The term "isolated" when used in connection with the nucleic acid molecules of the present disclosure typically refers to a nucleic acid sequence that is identified and separated from at least one contaminating nucleic acid that is normally associated in its natural source.An isolated nucleic acid may exist in a form or environment that is different from the form or environment that it is found in nature.Therefore, an isolated nucleic acid molecule is distinguished from the nucleic acid molecule when it exists in natural cells.

[0077] As used herein, the term "variant" refers to a polynucleotide (or polypeptide) having a sequence 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 of skill in the art (see, e.g., Sambrook et al. (1989)). In the case of polynucleotides, a variant may have one or more nucleotide deletions, substitutions, or additions at the 5' end, the 3' end, and / or at one or more internal sites compared to the reference polynucleotide. Sequence similarities and / or differences between a variant and a reference polynucleotide can be detected using conventional techniques known in the art, such as polymerase chain reaction (PCR) and hybridization techniques. Variant polynucleotides also include polynucleotides of synthetic origin, such as, for example, polynucleotides generated 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, as determined by sequence alignment programs known to those skilled in the art. In the case of polypeptides, variants may have one or more amino acid deletions, substitutions, or additions compared to the reference polypeptide. Sequence similarities and / or differences between variants and reference polypeptides can be detected using conventional techniques known in the art, such as Western blots. Generally, a variant 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 as determined by sequence alignment programs known to those of skill in the art.

[0078] Amino acid substitutions can be conservative or non-conservative. Substitutions are preferably conservative, i.e., replacing an amino acid residue with an amino acid of similar polarity that acts as a functional equivalent. Preferably, the amino acid residue used as a substitute is selected from the same group of amino acids as the amino acid residue being replaced. For example, a hydrophobic residue can be replaced with another hydrophobic residue, or a polar residue can be replaced with another polar residue with the same charge. Functionally related amino acids that can be used for conservative substitutions include nonpolar amino acids such as glycine, valine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, and tryptophan. Examples of uncharged polar amino acids include serine, threonine, glutamine, asparagine, tyrosine, and cysteine. Examples of charged polar (basic) amino acids include histidine, arginine, and lysine. Examples of charged polar (acidic) amino acids include aspartic acid and glutamic acid.

[0079] Also considered variants are proteins that differ from their naturally occurring counterparts by the addition, substitution, or deletion of one or more (e.g., 2, 3, 4, 5, 10, or 15) additional amino acids. The additional amino acids may be present within the amino acid sequence of the original cMyBP-C protein (i.e., as insertions), or they may be added to one or both termini of the protein. Such insertions, substitutions, or deletions may occur at any position, so long as they do not impair the ability of the polypeptide to perform the function of the naturally occurring cMyBP-C protein and / or to rescue haploinsufficiency in the treated subject. Furthermore, cMyBP-C protein variants also include proteins lacking one or more amino acids compared to the original polypeptide. Such deletions may affect any amino acid position, so long as they do not impair the ability of the cMyBP-C protein to perform the normal function and / or rescue haploinsufficiency.

[0080] Finally, a variant of cardiac cMyBP-C protein also refers to a protein that differs from the naturally occurring protein in terms of structural modifications, such as modified amino acids. Modified amino acids are amino acids that have been modified either by natural processes, such as processing or post-translational modification, or by chemical modification processes known in the art. 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, amidation, cyclization, and / or covalent or non-covalent attachment to phosphatidylinositol, flavin derivatives, lipoteichoic acid, fatty acids, or lipids. Such modifications are described in the literature, e.g., in Proteins: Structure and Molecular Properties, T. Creighton, 2002. nd This is extensively described in the "Cell Signaling Genetics and Neuroscience: A Novel Approach to Cardiovascular Disease," WH 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 cMyBP-C. These 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).

[0081] The terms "identity," "homology," and their grammatical variations mean that two or more referent entities are the same when they are "aligned" sequences. Thus, by way of example, if two polypeptide sequences are identical, they have the same amino acid sequence, at least within a region or portion of the sequence. If two polynucleotide sequences are identical, they have the same polynucleotide sequence, at least within a region or portion of the sequence. Identity can be over a defined area (region or domain) of a sequence. An "area" or "region" of identity refers to a portion of two or more referent entities that is the same. Thus, if two polypeptide or nucleic acid sequences are identical over one or more sequence regions or regions, they share identity within that region. An "aligned" sequence refers to multiple polynucleotide or polypeptide (amino acid) sequences, often including corrections for missing or additional bases or amino acids (gaps) compared to a reference sequence. "Substantial homology" means that the molecule is structurally or functionally conserved such that it has, or is predicted to have, at least a partial structure or function (e.g., biological function or activity) of one or more of the structures or functions of the reference molecule, or related / corresponding regions or portions of the reference molecule with which it shares homology.

[0082] "Nucleic acid sequence identity or homology percentage (%)" is defined as the percentage of nucleotides in a candidate sequence that are identical to a reference sequence after aligning the corresponding sequences and introducing gaps, if necessary, to obtain the maximum sequence identity percentage. Alignment for determining nucleic acid sequence identity percentage can be achieved in a variety of ways within the skill of those skilled in the art, for example, using publicly available computer software such as ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithms required to obtain maximum alignment over the entire length of the sequences being compared.

[0083] "Percent (%) amino acid sequence identity or homology," with respect to the cMyBP-C amino acid sequences identified herein, is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the cMyBP-C polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without taking into account any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of one of ordinary 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 algorithms needed to achieve maximum alignment over the full length of the sequences being compared.

[0084] "Codon optimization" or "codon optimized" refers to changes made in a nucleotide sequence that make it more likely to be expressed at relatively high levels compared to a sequence that is not codon optimized, which does not change the amino acid that each codon encodes.

[0085] An "AAV virion" or "AAV virus particle" or "AAV vector particle" or "AAV virus" refers to a viral particle composed of at least one AAV capsid protein and an encapsulated AAV vector construct as described herein. When the particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is typically referred to as a "recombinant AAV vector particle" or simply an "AAV vector." Production of AAV vector particles necessarily includes production of an AAV vector genome, as such vector genome is contained within the AAV vector particle. Reference to a polynucleotide AAV vector construct encapsulated within a vector particle, and its replication, is understood to refer to the AAV vector genome.

[0086] As used herein, "therapeutic AAV virus" refers to an AAV virion, AAV viral particle, AAV vector particle, or AAV virus comprising a heterologous polynucleotide encoding a therapeutic protein, such as cMyBP-C, as described herein. As used herein, "AAV vector construct" or "AAV vector genome" refers to a vector construct comprising one or more polynucleotides encoding a protein of interest (also known as a transgene), flanked by at least one AAV terminal repeat (ITR) and operably linked to one or more expression control elements. Such AAV vector constructs, when present in a host cell transfected with a vector encoding and expressing the rep and cap gene products, can be replicated and packaged into infectious viral particles. This term generally refers to a recombinant AAV capable of infecting cells such that the infected cells express (e.g., by transcription and / or translation) an element of interest (e.g., a nucleotide sequence, a protein, etc.). To this extent, therapeutically effective rAAV particles can include AAV particles having capsid or vector genomes (vg) with different properties. For example, therapeutically effective rAAV particles may have capsids with different post-translational modifications. In other examples, therapeutically effective AAV particles may contain various sizes / lengths, plus or minus strand sequences, different flip / flop ITR configurations (flip / flop, flop / flip, flip / flip, flop / flop, etc.), different numbers of ITRs (1, 2, 3, etc.), or truncated vector genomes. For example, in rAAV-infected cells, overlapping homologous recombination occurs between nucleic acids with 5'- and 3'-truncations, resulting in the generation of a "complete" nucleic acid encoding a large protein, thereby reconstituting a functional full-length gene. In other examples, complementary nucleic acid sequences with 5'- and 3'-truncations interact with each other, thereby forming a "complete" nucleic acid during second-strand synthesis. The "complete" nucleic acid encodes a large protein, thereby reconstituting a functional full-length gene.Therapeutically effective rAAV particles are also referred to as heavy capsids, full capsids, or partially filled capsids. Conversely, "therapeutically ineffective" AAV viruses refer to empty capsids, i.e., capsids with unquantifiable or undetectable vector genomes or vector genomes that are unable to recombine into complete, functional nucleic acids.

[0087] As used herein, a "therapeutic protein" refers to a polypeptide having a biological activity that replaces or compensates for the loss or reduction of activity of an endogenous protein. For example, a functional cMyBP-C protein is a therapeutic protein for HCM.

[0088] As used herein, "hypertrophic cardiomyopathy" refers to a genetic disease caused by mutations in genes encoding components of the myocardium, such as cardiac myosin-binding protein C, and characterized by symptoms such as heart failure, arrhythmias, chest pain, shortness of breath, fatigue, and dizziness, increased heart size, increased cardiothoracic ratio, increased left ventricular end-diastolic diameter, increased left ventricular end-systolic diameter, increased ventricular wall thickness (anterior wall thickness or posterior wall thickness, or both), decreased ejection time, decreased peak aortic flow velocity, and / or decreased aortic flow time.

[0089] As used herein, "cardiac myosin binding protein C deficiency" or "deficiency of functional wild-type cardiac myosin binding protein C" refers to an inherited condition caused by reduced levels of functional cMyBP-C protein due to the absence of the protein, reduced production of the protein, or production of a non-functional protein, including HCM.

[0090] As used herein, "therapeutically effective against hypertrophic cardiomyopathy" or "hypertrophic cardiomyopathy treatment" refers to any therapeutic intervention in a subject with HCM that ameliorates the characteristic defect of functional wild-type cMyBP-C, e.g., increases cMyBP-C protein levels in the myocardium, ameliorates symptoms of HCM, or reduces the frequency, duration, or severity of symptoms of HCM.

[0091] As used herein, "gene therapy for hypertrophic cardiomyopathy" refers to any therapeutic intervention in a subject with HCM that involves the replenishment, restoration, or increase of cMyBP-C via the delivery of one or more nucleic acid molecules to the subject's cells that express functional cMyBP. In certain embodiments, MYBPC3 gene therapy refers to gene therapy involving adeno-associated virus (AAV) particles containing a vector construct that expresses human cMyBP-C. In other embodiments, gene therapy involves transfecting a plasmid that expresses human cMyBP-C.

[0092] As used herein, "treating" or "treatment" refers to prophylactic or therapeutic treatment administered to a subject who exhibits signs or symptoms of pathology (i.e., HCM) with the intent of diminishing or eliminating those signs or symptoms, or ameliorating their progression, severity, or duration. Signs or symptoms may be biochemical, cellular, histological, functional, subjective, or objective.

[0093] As used herein, "ameliorate" refers to the act of reducing the severity, progression, or duration of symptoms of a disease.

[0094] As used herein, "stable treating" or "stable treatment" refers to the use of a therapeutic vector construct, AAV particle, or cell administered to a subject such that the subject stably expresses the therapeutic protein expressed by the vector construct, AAV particle, or cell. A stably expressed therapeutic protein means that the protein is expressed for a clinically significant length of time. As used herein, a "clinically significant length of time" means expression at a therapeutically effective level for a length of time that has a meaningful impact on the subject's quality of life, as indicated, for example, by a reduction in signs or symptoms of disease. In certain embodiments, a clinically significant length of time is expression for at least 6 months, at least 8 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, at least 10 years, or for the lifetime of the subject.

[0095] As used herein, the term "effective amount" refers to an amount sufficient to effect beneficial or desired biological and / or clinical results.

[0096] As used herein, "subject" refers to an animal that is the object of treatment, observation, or experiment. "Animal" includes cold- and warm-blooded vertebrates and invertebrates, such as fish, crustaceans, reptiles, and especially mammals. The term "avian" as used herein includes, but is not limited to, chickens, ducks, geese, quail, turkeys, and pheasants. "Mammal" as used herein refers to an individual belonging to the class Mammalia, and includes, but is not limited to, humans, domestic and farm animals, zoo animals, sport animals, and pets. Non-limiting examples of mammals include mice; rats; rabbits; guinea pigs; dogs; cats; sheep; goats; cows; horses; primates such as monkeys, chimpanzees, and apes, and especially humans. In some embodiments, the mammal is a human, including an infant, child, or young human, e.g., a human up to the age of 2, 2-4, 2-6, or 2-12 years.

[0097] Generally, a "pharmaceutically acceptable carrier" is one that is not toxic or excessively 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 and absorption delaying agents, and the like that are physiologically compatible.

[0098] Vector constructs and AAV vectors The recombinant vector constructs of the present disclosure may be used by themselves as gene therapy or may be used to produce rAAV particles by the methods described herein, which involve providing the recombinant vector construct together with Rep and Cap genes to a suitable host cell. The vector constructs described herein include a nucleic acid sequence encoding functional cMyBP-C. The recombinant vector construct may also include heterologous expression control elements, such as a nucleic acid encoding functional human cMyBP-C operably linked to a promoter and / or enhancer, optionally an intron, and optionally a polyadenylation (polyA) signal. The heterologous expression control element may be, for example, a heterologous cardiomyocyte-specific transcriptional regulatory region described herein.

[0099] When used to produce rAAV particles, the recombinant vector construct may include (a) one or both of (i) an AAV 5' inverted terminal repeat (ITR) sequence and (ii) an AAV 3' ITR, (b) a heterologous cardiomyocyte-specific transcriptional regulatory region, and (c) a nucleic acid encoding functional human cMyBP-C, optionally the AAV ITR is an AAV2 ITR. Preferably, the nucleic acid encoding functional cMyBP-C is operably linked to cardiomyocyte-specific expression control elements. The vector construct may also 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 nucleic acid comprising: (a) an AAV2 5' inverted terminal repeat (ITR), which may or may not be modified as known in the art; (b) a cardiomyocyte-specific transcriptional regulatory region, a functional MYBPC3 protein coding region; (c) one or more introns, including fragments of longer introns; (d) optionally, an exon or fragment thereof, a polyadenylation sequence; and (f) an AAV2 3' ITR, which may or may not be modified as known in the art.

[0100] Preferably, the rAAV particles also include an AAV capsid with cardiotropy, optionally an AAV type 9 capsid. Exemplary capsids with cardiotropy include AAV1, 6, 7, and 9.

[0101] Other embodiments provided herein relate to vector constructs encoding functional cMyBP-C polypeptides, which constructs comprise one or more of the individual elements and combinations thereof of the above-described constructs in one or more different orientation(s). Further embodiments provided herein relate to the above-described constructs in the opposite orientation.

[0102] The AAV vector constructs provided herein in single-stranded form are in the range of about 4.5 kb to about 6.5 kb in length, or about 4.5 kb to about 5.5 kb in length, or about 4 kb to about 5.5 kb in length, or about 4.8 kb to about 5.2 kb in length, or in the range of 4.8 kb to 5.1 kb in length, or about 4.9 kb to about 5.5 kb in length, or about 4.8 kb to about 6.0 kb in length, or about 5.0 kb to 6.2 kb in length, or about 5.1 kb to about 6.3 kb in length, or about 5.2 kb to about 6.4 kb in length, or about 5.5 kb to about 6.5 kb in length, or in the range of about 4.0 kb to about 5.0 kb in length, or in the range of about 4 to about 4.5 kb in length, or in the range of about 4.5 kb to about 5 kb in length.

[0103] When AAV vectors are produced from oversized recombinant vector constructs, they may lack portions of the 5' or 3' ends of the recombinant vector construct. Because AAV is a single-stranded DNA virus and packages either the sense or antisense strand, the sense strand of an oversized AAV vector lacks the 5' AAV ITR and, optionally, a portion of the 5' end of the target protein-encoding gene, and the antisense strand of an oversized AAV vector lacks the 3' ITR and, optionally, a portion of the 3' end of the target protein-encoding gene. A functional transgene is produced in oversized AAV vector-infected cells by annealing of the sense and antisense truncated genomes within the target cell. Thus, in certain embodiments, rAAV particles of the present invention may comprise a recombinant vector construct comprising at least one ITR and a substantial portion of the nucleotide sequence encoding functional cMyBP-C, e.g., a fragment of SEQ ID NO: 1 or SEQ ID NO: 42-45, that is greater than 50%, 60%, 70%, 80%, or 90% of the length of the nucleotide sequence. For example, the recombinant vector construct may include at least one ITR, a cardiomyocyte-specific transcriptional regulatory region, and a substantial portion of a nucleotide sequence encoding functional cMyBP-C.

[0104] Generation of vector constructs can be accomplished using any suitable genetic engineering techniques known in the art, including, but not limited to, standard techniques of restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing, for example, as described in Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY (1989)).

[0105] The vector construct can incorporate sequences derived from the genome of any known organism. The sequences can be incorporated in their native form or can be modified in any way to obtain the desired activity. For example, the sequences can contain insertions, deletions, or substitutions.

[0106] The AAV vector construct, when present in a host cell transfected with a polynucleotide encoding and expressing the rep and cap gene products, can be replicated and packaged into infectious AAV particles, preferably replication-deficient AAV particles.

[0107] Transcriptional Regulatory Elements or Regions Promoters and enhancers.

[0108] In one or more embodiments, the nucleic acid sequence encoding cMyBP-C is operably linked to one or more heterologous expression control elements. Preferably, the expression control elements are cardiomyocyte-specific expression control elements. Examples of cardiomyocyte-specific control elements include, but are not limited to, the human cardiac troponin 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 plus myosin light chain 2 promoter (CMV-MLC2, or CMV-MLC1.5, CMV-MLC260), phosphoglycerate kinase (PGK) promoter, sarcomere-specific promoter, alpha myosin heavy chain promoter, myosin light chain 2v promoter, alpha myosin heavy chain promoter, alpha-cardiac promoter, alpha-tropomyosin promoter, cardiac troponin C promoter, cardiac troponin I promoter, cardiac myosin binding protein C promoter, and / or sarco / endoplasmic reticulum Ca2+" ATPase (SERCA) promoter (e.g., isoform 2 of this promoter (SERCA2)), and / or striated muscle promoters, such as the desmin promoter. Enhancers derived from cardiomyocyte-specific transcription factor binding sites are also contemplated.

[0109] Examples of fragments or variants of the hTNNT2 promoter include a cardiomyocyte-specific promoter sequence comprising a nucleic acid sequence at least 90% identical to SEQ ID NO: 47. In some embodiments, the cardiomyocyte-specific transcriptional regulatory region comprises a cardiomyocyte-specific promoter sequence comprising a nucleic acid sequence at least 80% identical to SEQ ID NO: 52. In some embodiments, the cardiomyocyte-specific transcriptional regulatory region comprises a cardiomyocyte-specific promoter sequence comprising a nucleic acid sequence at least 80% identical to SEQ ID NO: 51. In some embodiments, the cardiomyocyte-specific transcriptional regulatory region comprises a cardiomyocyte-specific promoter sequence comprising a nucleic acid sequence at least 80% identical to SEQ ID NO: 50. In some embodiments, the cardiomyocyte-specific transcriptional regulatory region comprises a cardiomyocyte-specific promoter sequence comprising a nucleic acid sequence at least 80% identical to SEQ ID NO: 49. In some embodiments, the cardiomyocyte-specific transcriptional regulatory region comprises a cardiomyocyte-specific promoter sequence comprising a nucleic acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, or 99%, or more than 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical (over the entire length of the SEQ ID NO: 49-52) to any one of SEQ ID NOs: 49-52. In any of the embodiments described herein, the cardiomyocyte-specific promoter optionally excludes any one of SEQ ID NOs: 1-85 of U.S. Patent Publication No. 2021 / 0252165. In some embodiments, the cardiomyocyte-specific transcriptional regulatory region also comprises an intron, and optionally an exon or fragment thereof, 5' to the cMyBP-C coding sequence that enhances expression of the cMyBP-C protein. For example, the vector construct and AAV particle comprise, in the 5' to 3' direction, a cardiomyocyte-specific promoter comprising a nucleotide sequence at least 80% identical to SEQ ID NO: 47, an intron nucleotide sequence at least 70% identical to SEQ ID NO: 53, and a nucleotide sequence encoding cMyBP-C.

[0110] In other embodiments, the cardiomyocyte-specific promoter comprises (a) a nucleic acid sequence at least 80% identical to any of (i) SEQ ID NO: 49 or a fragment thereof, (ii) SEQ ID NO: 50 or a fragment thereof, or (iii) SEQ ID NO: 51 or a fragment thereof, and (b) an intron nucleotide sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 53. 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: 58. Other exemplary introns are SEQ ID NOs: 53-58.

[0111] In some embodiments, the vector construct comprises (a) a nucleic acid sequence at least 90% identical to any of (i) SEQ ID NO: 49 or a fragment thereof, (ii) SEQ ID NO: 50 or a fragment thereof, or (iii) SEQ ID NO: 51 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: 53. 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: 58. Other exemplary introns are SEQ ID NOs: 53-58.

[0112] In some embodiments, the cardiomyocyte-specific transcriptional regulatory region may further comprise (in addition to the hTNNT2 promoter and a fragment or variant of the globin intron) an exon sequence or fragment thereof, e.g., a globin intron adjacent to the 3' end of a fragment of beta globin exon 3 (SEQ ID NO: 54). An example of a combination of the intron and exon fragment is SEQ ID NO: 55. In some exemplary embodiments, the cardiomyocyte-specific transcriptional regulatory region comprises SEQ ID NO: 56.

[0113] In some embodiments, the fragment or variant of the hTNNT2 promoter is more than 420 nucleotides in length and less than 544 nucleotides in length and comprises a nucleic acid sequence at least 90% identical to SEQ ID NO: 47. In any of the embodiments described herein, the cardiomyocyte-specific promoter optionally excludes any one of SEQ ID NOs: 1-85 of U.S. Patent Publication No. 2021 / 0252165.

[0114] In some embodiments, the cardiomyocyte-specific promoter sequence comprises a nucleic acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99%, or more than 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to any of (i) SEQ ID NO: 49 or a fragment thereof, (ii) SEQ ID NO: 50 or a fragment thereof, (iii) SEQ ID NO: 51 or a fragment thereof, or (iv) SEQ ID NO: 52 or a fragment thereof. For example, the cardiomyocyte-specific promoter sequence comprises a nucleic acid sequence that is at least 95%, 97%, 98%, or 99%, or more than 95%, 97%, 98%, or 99% identical to any of (i) SEQ ID NO: 49 or a fragment thereof, (ii) SEQ ID NO: 50 or a fragment thereof, or (iii) SEQ ID NO: 51 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: 51. In some exemplary embodiments, the sequence of the hTNNT promoter comprises at least nucleotides 1-106 and 507-532 of SEQ ID NO:51, or at least nucleotides 507-532 of SEQ ID NO:51, or at least nucleotides 521-532 of SEQ ID NO:51.

[0115] Various promoters can be operably linked to a nucleic acid comprising the coding region of the protein of interest, human cardiac myosin binding protein C, in the vector constructs disclosed herein. In some embodiments, the promoter is capable of driving expression of the protein of interest in cells infected with the virus derived from the viral vector, such as target cells. The promoter can be naturally occurring or non-naturally occurring. In some embodiments, the promoter is a synthetic promoter. In one embodiment, a synthetic promoter comprises a non-naturally occurring sequence designed to regulate the activity of an operably linked gene. In another embodiment, a synthetic promoter comprises a fragment of a natural promoter, thereby forming a new stretch of DNA sequence not found in nature. Synthetic promoters are typically composed of regulatory elements designed to confer enhanced tissue-specific expression: promoter, enhancer, intron, splice donor, and acceptor. 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.

[0116] In some embodiments, the promoter comprises the human cardiac troponin T (hTNNT2) promoter. A portion of the hTNNT2 promoter can 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 of SEQ ID NOs: 49-51. In some embodiments, the promoter is at least about 95%, or greater than 95%, identical to any of SEQ ID NOs: 49-51.

[0117] In some embodiments, the promoter construct comprises one or more of the additional individual enhancer elements in one or more different orientation(s).

[0118] In some embodiments, the promoter is operably linked to a polynucleotide encoding one or more proteins of interest, hi some embodiments, the promoter is operably linked to a polynucleotide encoding a cMyBP-C protein.

[0119] The size of the promoter can vary.Because the packaging capacity of AAV is limited, it is preferable to use a promoter that is small in size but at the same time allows high-level production of target protein(s) in host cells.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.

[0120] Other regulatory elements.

[0121] Various additional regulatory elements can be used in the vector construct, such as enhancers, polyadenylation signals, ribosome binding sequences, and / or consensus splice acceptor or splice donor sites to further increase the expression level of the protein of interest in the host cell. In some embodiments, the regulatory elements can facilitate extrachromosomal maintenance of the recombinant DNA molecule in the host cell and / or improve the efficiency of the vector (e.g., scaffold / matrix attachment region (S / MAR)). Such regulatory elements are well known in the art.

[0122] The vector constructs disclosed herein may include regulatory elements such as a transcription initiation region and / or a transcription termination region. Examples of transcription termination regions include, but are not limited to, polyadenylation signal sequences. Examples of polyadenylation signal sequences include, but are not limited to, minipolyA, human growth hormone (hGH) poly(A), bovine growth hormone (bGH) poly(A), SV40 late poly(A), rabbit beta-globin (rBG) poly(A), thymidine kinase (TK) poly(A) sequence, Proudfoot poly(A), and any variants thereof. In some embodiments, the transcription termination region is located downstream of the post-transcriptional regulatory element. In some embodiments, the transcription termination region is a polyadenylation signal sequence. In some embodiments, the transcription termination region is a minipoly A (e.g., SEQ ID NO: 64), bGH poly A (e.g., any of SEQ ID NOs: 59-61), hGH poly A (e.g., SEQ ID NO: 62), SV40 poly A (e.g., SEQ ID NO: 53), Proudfoot synthetic poly A (e.g., SEQ ID NO: 65), or rabbit beta-globin poly A (e.g., SEQ ID NO: 66) sequence, or a fragment thereof of about 40 to 200 nucleotides in length.

[0123] In some embodiments, the vector construct includes a polyadenylation signal, optionally a bovine growth hormone (bGH) polyA signal (e.g., SEQ ID NOs: 59-61) or a human growth hormone (hGH) polyA signal (e.g., SEQ ID NO: 62) or a fragment thereof.

[0124] 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.

[0125] In some embodiments, the vector construct can include additional transcription and translation initiation sequences, and / or additional transcription and translation terminator sequences, as known in the art. A protein of interest and a nucleic acid encoding the protein of interest.

[0126] As used herein, a "protein of interest" is any functional cMyBP-C protein (including naturally occurring and non-naturally occurring variants thereof). In some embodiments, a polynucleotide encoding one or more cMy-BP-C proteins of interest can be inserted into a viral vector disclosed herein, where the polynucleotide is operably linked to a promoter. In some cases, the promoter is capable of driving expression of the protein(s) of interest in a host cell (e.g., human cardiac muscle).

[0127] In one or more embodiments, functional cMyBP-C comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO:2 (human cardiac myosin binding protein C). The present disclosure also provides isolated nucleic acid molecules encoding such functional wild-type cMY-BP-C proteins. The nucleotide sequence can be homologous to the wild-type nucleotide sequence of SEQ ID NO:1. In certain embodiments, the nucleic acid molecule has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the nucleotide sequence of SEQ ID NO:1, or at least 100, 200, 300, 400, or 500 contiguous nucleotides of SEQ ID NOs:1 or 42-43. In exemplary embodiments, the nucleotide sequence encoding functional cardiac myosin binding protein C is codon-optimized or a variant and can be at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any of SEQ ID NOs:44-46.

[0128] In certain embodiments, the nucleic acid molecule has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% homology to the nucleotide sequence of SEQ ID NO: 1 or 42-46, or at least 100, 200, 300, 400, or 500 consecutive nucleotides of SEQ ID NO: 1 or 42-46.

[0129] In exemplary embodiments, the nucleic acid sequence encoding functional cMyBP-C is the wild-type MYBPC3 sequence (SEQ ID NO: 1 is an example), or is codon-optimized or a variant. The vector constructs described herein may include a nucleotide sequence that differs from the wild-type nucleotide sequence but still encodes a functional cMyBP-C amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 2. According to this aspect, the nucleotide sequence may include a portion having at least 80%, 85%, 90%, or 95% homology to at least 100 contiguous bases of SEQ ID NO: 1 or 42-46, so long as the nucleotide sequence encodes a functional human cMyBP-C protein that is at least 90%, 95%, or 98% identical to SEQ ID NO: 2. In exemplary embodiments, the nucleotide sequence may include a portion having at least 90% homology for at least 100, 200, 300, 400, or 500 contiguous bases of SEQ ID NO: 1, so long as the nucleotide sequence encodes a functional human cMyBP-C protein that is at least 90% identical to SEQ ID NO: 2. In exemplary embodiments, the nucleotide sequence has substantial homology to the nucleotide sequence of SEQ ID NO: 1 or 42-46 and encodes a functional cMyBP-C. The term substantial homology may be further defined with reference to percent (%) homology, e.g., at least 80%, 85%, 90%, or 95% homology. This is discussed in more detail elsewhere herein.

[0130] In an exemplary embodiment, the nucleotide sequence of a gene of interest is codon-optimized, preferably for more efficient expression in humans or in human target organs, tissues, and / or cells. Target organs, tissues, or cells include cardiac tissue and / or cardiomyocytes. The adaptability of a nucleotide sequence encoding a 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 a gene's codon usage relative to the codon usage of highly expressed human genes. The relative adaptability (w) of each codon is the ratio of the usage frequency of each codon to the usage frequency of the most abundant codon for the same amino acid. The CAI is defined as the geometric mean of these relative adaptability values. Nonsynonymous codons and stop codons (depending on the genetic code) are excluded. 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 gene of interest has a CAI of at least 0.75, 0.80, 0.85, 0.90, 0.95, or 0.99.

[0131] 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 Operon / Eurofins Genomics codon optimization software or other codon optimization tools (e.g., 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)).

[0132] In addition to or as an alternative to codon optimization, the nucleotide sequence of the gene of interest can be adjusted to reduce CpG dinucleotide content and optionally remove any extra ORFs in the sense and antisense directions. CpG dinucleotide content has been shown to activate TLR9 in dendritic cells, resulting in potential immune activation and CTL responses. Reducing the CpG content can reduce liver inflammation and ALT. In some embodiments, the nucleotide sequence of the gene of interest has a CpG dinucleotide content of less than 25, less than 20, less than 15, or less than 10. In other embodiments, the nucleotide sequence of the gene of interest has a GC content of less than 65%, less than 60%, or less than 55%.

[0133] Generally, codon optimization or CpG reduction does not change the amino acid that each codon encodes, it simply changes the nucleotide sequence, resulting in a higher likelihood of being expressed at a relatively higher level compared to a non-optimized sequence.

[0134] As described herein, the nucleotide sequence encoding the cMyBP-C protein can be modified to improve the expression efficiency of the protein. 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 codon usage of the host (e.g., a mammal) to increase gene expression (e.g., protein production) in the host. As another non-limiting example of modification, one or more splice donors and / or splice acceptors in the nucleotide sequence of the protein of interest can be modified to reduce the possibility of irrelevant splicing. As another non-limiting example of modification, one or more introns can be inserted within or adjacent to the nucleotide sequence of the protein of interest to optimize AAV vector packaging and enhance expression.

[0135] The nucleic acid molecule encodes a functional cMyBP-C protein that is at least 90% identical to the wild-type amino acid sequence of SEQ ID NO: 2. If the nucleic acid encodes a protein containing a sequence with a change to any of the wild-type amino acids, the protein will still be a functional protein. One skilled in the art will understand that minor changes can be made to some of the amino acids of a protein without adversely affecting the function of the protein.

[0136] In certain embodiments, the nucleic acid molecule, when expressed in a suitable system (e.g., a host cell), produces relatively high levels of functional cMyBP-C protein. Because the MyBP-C produced is functional, it will have a conformation that is the same as at least a portion of wild-type cMyBP-C. In certain embodiments, the functional cMyBP-C protein produced as described herein effectively treats subjects suffering from a deficiency of wild-type cMyBP-C protein and / or HCM.

[0137] Producing the nucleic acid molecules provided herein is well within the capabilities of those skilled in the art. This can be done, for example, using chemical synthesis of a given sequence. Furthermore, suitable methods for determining whether the nucleic acids described herein express functional proteins will be apparent to those skilled in the art. For example, one suitable in vitro method involves inserting the nucleic acid into a vector, such as an AAV vector, transducing the vector into host cells, such as 293T or HeLa cells, and assaying for cMyBP-C. Alternatively, a suitable in vivo method involves transducing a vector containing the nucleic acid into HCM mice and assaying for functional cMyBP-C.

[0138] Introns In some embodiments, the vector contains one or more introns. Introns may facilitate processing of the RNA transcript in mammalian host cells, increase expression of the protein of interest, and / or optimize packaging of the vector into AAV particles. Non-limiting examples of such introns are the human beta globin intron, human immunoglobulin G (IgG) intron, or the naturally occurring cMyBP-C intron. In some embodiments, the intron is a synthetic intron.

[0139] 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 enhance expression of the cMyBP-C protein. 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' to the nucleotide sequence encoding cMyBP-C. In a further example, 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 cMyBP-C, e.g., between any of the exons. In some embodiments, the intron sequence is located between exons 2 and 3. In some embodiments, the intron sequence is located at a position within the nucleic acid encoding cMyBP-C corresponding to position 293 of SEQ ID NO: 1 or 42-46.

[0140] In one or more embodiments, the intron comprises a nucleotide sequence at least 60%, 65%, 70%, 75%, 80%, or 85%, or 90%, or 95% identical to SEQ ID NO: 53, 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: 53 or a fragment thereof that is about 50 to 150 nucleotides, 75 to 145 nucleotides, 100 to 135 nucleotides, or 120 to 135 nucleotides of SEQ ID NO: 53, or a variant of the fragment that 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: 53.

[0141] In one or more embodiments, the intron comprises a nucleotide sequence at least 60%, 65%, 70%, 75%, 80%, or 85%, or 90%, or 95% identical to SEQ ID NO: 58, 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: 58 or a fragment thereof that is about 50 to 150 nucleotides, 75 to 145 nucleotides, 100 to 135 nucleotides, or 120 to 135 nucleotides of SEQ ID NO: 58, or a variant of the fragment that 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: 58.

[0142] Other exemplary introns comprise a nucleotide sequence at least 60%, 65%, 70%, 75%, 80%, or 85%, or 90%, or 95% identical to any one of SEQ ID NOs: 53-58.

[0143] 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 an exon comprising a nucleotide sequence at least 80%, or 85%, or 90%, or 95% identical to SEQ ID NO: 54. In a further exemplary embodiment, the vector construct comprises a globin intron adjacent to an exon sequence comprising a nucleotide sequence at least 80%, or 85%, or 90%, or 95% identical to SEQ ID NO: 53. In an exemplary embodiment, the vector construct comprises a globin intron adjacent to an HbB exon sequence comprising a nucleotide sequence at least 80%, or 85%, or 90% identical to SEQ ID NO: 54.

[0144] The location and size of the intron within the vector can vary. 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 comprises 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 comprise all or a portion of a naturally occurring intron within the sequence encoding the protein of interest. In some embodiments, the intron is a globin intron. In some embodiments, the intron is a chimeric intron and comprises a fragment of a human IgG intron.

[0145] Incorporation of intron elements can enhance expression compared to expression in the absence of intron elements (see, e.g., Kurachi et al., J. Biol. Chem. 270(10):5276-81 (1995)). AAV vectors typically accept DNA inserts within a defined size range, generally from about 4 kb to about 5.4 kb, or slightly larger. However, there is no minimum size for packaging, and small vector genomes are packaged very efficiently. Introns and intron fragments meet this requirement while also enhancing expression. Thus, the present disclosure is not limited to incorporation of the cMyBP-C intron sequence within AAV vectors, but includes other introns or other DNA sequences in place of portions of the cMyBP-C intron. Furthermore, other 5' and 3' untranslated regions of nucleic acids may be used in place of those recited for human cMyBP-C.

[0146] In some embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98%, or 99% identical to any of SEQ ID NOs: 3-41 or 92-169. In other embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98%, or 99% identical to a nucleotide sequence that is complementary to, i.e., the minus (-) strand of, any of SEQ ID NOs: 3-41 or 92-169.

[0147] In some embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98%, or 99% identical to any of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, or 39. In other embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98%, or 99% identical to a nucleotide sequence that is complementary to, i.e., the minus (-) strand of, any of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, or 39.

[0148] In some embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98%, or 99% identical to any of SEQ ID NOs: 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, or 40. In other embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98%, or 99% identical to a nucleotide sequence that is complementary to, i.e., the minus (-) strand of, any of SEQ ID NOs: 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, or 40.

[0149] In some embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98%, or 99% identical to any of SEQ ID NOs: 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, or 41. In other embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98%, or 99% identical to a nucleotide sequence that is complementary to, i.e., the minus (-) strand of, any of SEQ ID NOs: 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, or 41.

[0150] Exemplary embodiments include the following: Construct C1 is 4950 bp long (SEQ ID NO: 29) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 67), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), chimeric intron (133 bp) (SEQ ID NO: 58), codon-optimized hMYBPC3 (SEQ ID NO: 44), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0151] In some embodiments, construct C1 is 4980 bp long (SEQ ID NO: 28) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 68), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), chimeric intron (133 bp) (SEQ ID NO: 58), codon-optimized hMYBPC3 (SEQ ID NO: 44), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 72).

[0152] In some embodiments, construct C1 is 4950 bp long (SEQ ID NO: 92) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 69), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), chimeric intron (133 bp) (SEQ ID NO: 58), codon-optimized hMYBPC3 (SEQ ID NO: 44), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0153] In some embodiments, construct C1 is 4980 bp long (SEQ ID NO: 93) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 70), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), chimeric intron (133 bp) (SEQ ID NO: 58), codon-optimized hMYBPC3 (SEQ ID NO: 44), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 74).

[0154] In some embodiments, construct C1 is 4950 bp long (SEQ ID NO: 94) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 67), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), chimeric intron (133 bp) (SEQ ID NO: 58), codon-optimized hMYBPC3 (SEQ ID NO: 44), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0155] In some embodiments, construct C1 is 4980 bp long (SEQ ID NO: 95) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 68), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), chimeric intron (133 bp) (SEQ ID NO: 58), codon-optimized hMYBPC3 (SEQ ID NO: 44), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 74).

[0156] In some embodiments, construct C1 is 4950 bp long (SEQ ID NO: 96) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 69), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), chimeric intron (133 bp) (SEQ ID NO: 58), codon-optimized hMYBPC3 (SEQ ID NO: 44), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 69).

[0157] In some embodiments, construct C1 is 4980 bp long (SEQ ID NO: 97) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 70), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), chimeric intron (133 bp) (SEQ ID NO: 58), codon-optimized hMYBPC3 (SEQ ID NO: 44), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 72).

[0158] In some embodiments, construct C1 is 4640 bp long (SEQ ID NO:27) and contains the following elements from 5' to 3': hTNNT2 promoter (544 bp) (SEQ ID NO:52), chimeric intron (133 bp) (SEQ ID NO:58), codon-optimized hMYBPC3 (SEQ ID NO:44), and mini-polyA (57 bp) (SEQ ID NO:64). In further embodiments, construct C1 optionally contains any of the 5' AAV2-ITR sequences of SEQ ID NOs:67-70 (or their complements) and / or any of the 3' AAV2-ITR sequences of SEQ ID NOs:71-74 (or their complements), or fragments thereof.

[0159] Construct C2 is 4801 bp long (SEQ ID NO: 32) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 67), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), codon-optimized hMYBPC3 (SEQ ID NO: 44), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0160] In some embodiments, construct C2 is 4831 bp long (SEQ ID NO: 31) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 68), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), codon-optimized hMYBPC3 (SEQ ID NO: 44), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 72).

[0161] In some embodiments, construct C2 is 4801 bp long (SEQ ID NO: 98) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 69), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), codon-optimized hMYBPC3 (SEQ ID NO: 44), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0162] In some embodiments, construct C2 is 4831 bp long (SEQ ID NO: 99) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 70), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), codon-optimized hMYBPC3 (SEQ ID NO: 44), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 74).

[0163] In some embodiments, construct C2 is 4801 bp long (SEQ ID NO: 100) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 67), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), codon-optimized hMYBPC3 (SEQ ID NO: 44), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0164] In some embodiments, construct C2 is 4831 bp long (SEQ ID NO: 101) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 68), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), codon-optimized hMYBPC3 (SEQ ID NO: 44), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 74).

[0165] In some embodiments, construct C2 is 4801 bp long (SEQ ID NO: 102) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 69), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), codon-optimized hMYBPC3 (SEQ ID NO: 44), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0166] In some embodiments, construct C2 is 4831 bp long (SEQ ID NO: 103) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 70), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), codon-optimized hMYBPC3 (SEQ ID NO: 44), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 72).

[0167] In some embodiments, construct C2 is 4491 bp long (SEQ ID NO: 30) and contains the following elements from 5' to 3': hTNNT2 promoter (544 bp) (SEQ ID NO: 52), codon-optimized hMYBPC3 (SEQ ID NO: 44), and minipolyA (57 bp) (SEQ ID NO: 64). In further embodiments, construct C2 optionally contains any of the 5' AAV2-ITR sequences of SEQ ID NOs: 67-70 (or their complements) and / or any of the 3' AAV2-ITR sequences of SEQ ID NOs: 71-74 (or their complements), or fragments thereof.

[0168] Construct C3 is 4801 bp long (SEQ ID NO: 35) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 67), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), wild-type hMYBPC3 (SEQ ID NO: 43), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0169] In some embodiments, construct C3 is 4831 bp long (SEQ ID NO: 34) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 68), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), wild-type hMYBPC3 (SEQ ID NO: 43), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 72).

[0170] In some embodiments, construct C3 is 4801 bp long (SEQ ID NO: 104) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 69), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), wild-type hMYBPC3 (SEQ ID NO: 43), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0171] In some embodiments, construct C3 is 4831 bp long (SEQ ID NO: 105) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 70), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), wild-type hMYBPC3 (SEQ ID NO: 43), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 74).

[0172] In some embodiments, construct C3 is 4801 bp long (SEQ ID NO: 106) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 67), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), wild-type hMYBPC3 (SEQ ID NO: 43), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0173] In some embodiments, construct C3 is 4831 bp long (SEQ ID NO: 107) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 68), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), wild-type hMYBPC3 (SEQ ID NO: 43), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 74).

[0174] In some embodiments, construct C3 is 4801 bp long (SEQ ID NO: 108) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 69), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), wild-type hMYBPC3 (SEQ ID NO: 43), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0175] In some embodiments, construct C3 is 4831 bp long (SEQ ID NO: 109) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 70), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), wild-type hMYBPC3 (SEQ ID NO: 43), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 72).

[0176] In some embodiments, construct C3 is 4491 bp long (SEQ ID NO: 33) and contains the following elements from 5' to 3': hTNNT2 promoter (544 bp) (SEQ ID NO: 52), wild-type hMYBPC3 (SEQ ID NO: 43), and minipolyA (57 bp) (SEQ ID NO: 64). In further embodiments, construct C3 optionally contains any of the 5' AAV2-ITR sequences of SEQ ID NOs: 67-70 (or their complements) and / or any of the 3' AAV2-ITR sequences of SEQ ID NOs: 71-74 (or their complements), or fragments thereof.

[0177] Construct C4 is 4950 bp long (SEQ ID NO: 38) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 67), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), chimeric intron (133 bp) (SEQ ID NO: 58), wild-type hMYBPC3 (SEQ ID NO: 43), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0178] In some embodiments, construct C4 is 4980 bp long (SEQ ID NO: 37) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 68), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), chimeric intron (133 bp) (SEQ ID NO: 58), wild-type hMYBPC3 (SEQ ID NO: 43), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 72).

[0179] In some embodiments, construct C4 is 4950 bp long (SEQ ID NO: 110) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 69), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), chimeric intron (133 bp) (SEQ ID NO: 58), wild-type hMYBPC3 (SEQ ID NO: 43), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0180] In some embodiments, construct C4 is 4980 bp long (SEQ ID NO: 111) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 70), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), chimeric intron (133 bp) (SEQ ID NO: 58), wild-type hMYBPC3 (SEQ ID NO: 43), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 74).

[0181] In some embodiments, construct C4 is 4950 bp long (SEQ ID NO: 112) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 67), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), chimeric intron (133 bp) (SEQ ID NO: 58), wild-type hMYBPC3 (SEQ ID NO: 43), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0182] In some embodiments, construct C4 is 4980 bp long (SEQ ID NO: 113) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 68), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), chimeric intron (133 bp) (SEQ ID NO: 58), wild-type hMYBPC3 (SEQ ID NO: 43), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 74).

[0183] In some embodiments, construct C4 is 4950 bp long (SEQ ID NO: 114) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 69), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), chimeric intron (133 bp) (SEQ ID NO: 58), wild-type hMYBPC3 (SEQ ID NO: 43), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0184] In some embodiments, construct C4 is 4980 bp long (SEQ ID NO: 115) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 70), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), chimeric intron (133 bp) (SEQ ID NO: 58), wild-type hMYBPC3 (SEQ ID NO: 43), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 72).

[0185] In some embodiments, construct C4 is 4640 bp long (SEQ ID NO: 36) and contains the following elements from 5' to 3': hTNNT2 promoter (544 bp) (SEQ ID NO: 52), chimeric intron (133 bp) (SEQ ID NO: 58), wild-type hMYBPC3 (SEQ ID NO: 43), and minipolyA (57 bp) (SEQ ID NO: 64). In further embodiments, construct C4 optionally contains any of the 5' AAV2-ITR sequences of SEQ ID NOs: 67-70 (or their complements) and / or any of the 3' AAV2-ITR sequences of SEQ ID NOs: 71-74 (or their complements), or fragments thereof.

[0186] Construct C5 is 4950 bp long (SEQ ID NO: 41) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 67), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), chimeric intron (133 bp) (SEQ ID NO: 58), CpG-free hMYBPC3 (SEQ ID NO: 45), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0187] In some embodiments, construct C5 is 4980 bp long (SEQ ID NO: 40) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 68), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), chimeric intron (133 bp) (SEQ ID NO: 58), CpG-free hMYBPC3 (SEQ ID NO: 45), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 72).

[0188] In some embodiments, construct C5 is 4950 bp long (SEQ ID NO: 116) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 69), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), chimeric intron (133 bp) (SEQ ID NO: 58), CpG-free hMYBPC3 (SEQ ID NO: 45), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0189] In some embodiments, construct C5 is 4980 bp long (SEQ ID NO: 117) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 70), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), chimeric intron (133 bp) (SEQ ID NO: 58), CpG-free hMYBPC3 (SEQ ID NO: 45), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 74).

[0190] In some embodiments, construct C5 is 4950 bp long (SEQ ID NO: 118) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 67), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), chimeric intron (133 bp) (SEQ ID NO: 58), CpG-free hMYBPC3 (SEQ ID NO: 45), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0191] In some embodiments, construct C5 is 4980 bp long (SEQ ID NO: 119) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 68), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), chimeric intron (133 bp) (SEQ ID NO: 58), CpG-free hMYBPC3 (SEQ ID NO: 45), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 74).

[0192] In some embodiments, construct C5 is 4950 bp long (SEQ ID NO: 120) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 69), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), chimeric intron (133 bp) (SEQ ID NO: 58), CpG-free hMYBPC3 (SEQ ID NO: 45), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0193] In some embodiments, construct C5 is 4980 bp long (SEQ ID NO: 121) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 70), hTNNT2 promoter (544 bp) (SEQ ID NO: 52), chimeric intron (133 bp) (SEQ ID NO: 58), CpG-free hMYBPC3 (SEQ ID NO: 45), minipolyA (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 72).

[0194] In some embodiments, construct C5 is 4806 bp long (SEQ ID NO:39) and contains the following elements from 5' to 3': hTNNT2 promoter (544 bp) (SEQ ID NO:52), chimeric intron (133 bp) (SEQ ID NO:58), CpG-free hMYBPC3 (SEQ ID NO:45), and mini-polyA (57 bp) (SEQ ID NO:64). In further embodiments, construct C5 optionally contains any of the 5' AAV2-ITR sequences of SEQ ID NOs:67-70 (or their complements) and / or any of the 3' AAV2-ITR sequences of SEQ ID NOs:71-74 (or their complements), or fragments thereof.

[0195] Construct A1 is 5074 bp long (SEQ ID NO: 5) and contains the following elements from 5' to 3': 5'AAV2-ITR (130 bp) (SEQ ID NO: 67), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), globin intron (131 bp) (SEQ ID NO: 53), HBB exon 3 (SEQ ID NO: 54); wild-type hMYBPC3 (SEQ ID NO: 42), bGH polyA (227 bp) (SEQ ID NO: 61), and 3'AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0196] In some embodiments, construct A1 is 5104 bp long (SEQ ID NO: 4) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 68), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), globin intron (131 bp) (SEQ ID NO: 53), HBB exon 3 (SEQ ID NO: 54); wild-type hMYBPC3 (SEQ ID NO: 42), bGH polyA (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 72);

[0197] In some embodiments, construct A1 is 5074 bp long (SEQ ID NO: 122) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 69), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), globin intron (131 bp) (SEQ ID NO: 53), HBB exon 3 (SEQ ID NO: 54); wild-type hMYBPC3 (SEQ ID NO: 42), bGH polyA (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73);

[0198] In some embodiments, construct A1 is 5104 bp long (SEQ ID NO: 123) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 70), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), globin intron (131 bp) (SEQ ID NO: 53), HBB exon 3 (SEQ ID NO: 54); wild-type hMYBPC3 (SEQ ID NO: 42), bGH polyA (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 74);

[0199] In some embodiments, construct A1 is 5074 bp long (SEQ ID NO: 124) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 67), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), globin intron (131 bp) (SEQ ID NO: 53), HBB exon 3 (SEQ ID NO: 54); wild-type hMYBPC3 (SEQ ID NO: 42), bGH polyA (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73);

[0200] In some embodiments, construct A1 is 5104 bp long (SEQ ID NO: 125) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 68), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), globin intron (131 bp) (SEQ ID NO: 53), HBB exon 3 (SEQ ID NO: 54); wild-type hMYBPC3 (SEQ ID NO: 42), bGH polyA (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 74);

[0201] In some embodiments, construct A1 is 5074 bp long (SEQ ID NO: 126) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 69), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), globin intron (131 bp) (SEQ ID NO: 53), HBB exon 3 (SEQ ID NO: 54); wild-type hMYBPC3 (SEQ ID NO: 42), bGH polyA (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71);

[0202] In some embodiments, construct A1 is 5104 bp long (SEQ ID NO: 127) and contains the following elements from 5' to 3': 5'AAV2-ITR (145 bp) (SEQ ID NO: 70), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), globin intron (131 bp) (SEQ ID NO: 53), HBB exon 3 (SEQ ID NO: 54); wild-type hMYBPC3 (SEQ ID NO: 42), bGH polyA (227 bp) (SEQ ID NO: 61), and 3'AAV2 ITR (145 bp) (SEQ ID NO: 72).

[0203] In some embodiments, construct A1 is 4786 bp long (SEQ ID NO:3) and contains the following elements from 5' to 3': hTNNT2 promoter (532 bp) (SEQ ID NO:51), globin intron (131 bp) (SEQ ID NO:53), HBB exon 3 (SEQ ID NO:54); wild-type hMYBPC3 (SEQ ID NO:42), and bGH polyA (227 bp) (SEQ ID NO:61). In further embodiments, construct A1 optionally contains any of the 5' AAV2-ITR sequences of SEQ ID NOs:67-70 (or their complementary sequences) and / or any of the 3' AAV2-ITR sequences of SEQ ID NOs:71-74 (or their complementary sequences), or fragments thereof.

[0204] Construct A2 is 4939 bp long (SEQ ID NO: 8) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 67), hTNNT2 promoter (469 bp) (SEQ ID NO: 49), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH polyA (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0205] In some embodiments, construct A2 is 4969 bp long (SEQ ID NO: 7) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 68), hTNNT2 promoter (469 bp) (SEQ ID NO: 49), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH polyA (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 72).

[0206] In some embodiments, construct A2 is 4939 bp long (SEQ ID NO: 128) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 69), hTNNT2 promoter (469 bp) (SEQ ID NO: 49), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH polyA (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0207] In some embodiments, construct A2 is 4969 bp long (SEQ ID NO: 129) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 70), hTNNT2 promoter (469 bp) (SEQ ID NO: 49), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH polyA (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 74).

[0208] In some embodiments, construct A2 is 4939 bp long (SEQ ID NO: 130) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 67), hTNNT2 promoter (469 bp) (SEQ ID NO: 49), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH polyA (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0209] In some embodiments, construct A2 is 4969 bp long (SEQ ID NO: 131) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 68), hTNNT2 promoter (469 bp) (SEQ ID NO: 49), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH polyA (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 74).

[0210] In some embodiments, construct A2 is 4939 bp long (SEQ ID NO: 132) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 69), hTNNT2 promoter (469 bp) (SEQ ID NO: 49), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH polyA (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0211] In some embodiments, construct A2 is 4969 bp long (SEQ ID NO: 133) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 70), hTNNT2 promoter (469 bp) (SEQ ID NO: 49), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH polyA (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 72).

[0212] In some embodiments, construct A2 is 4663 bp long (SEQ ID NO: 6) and contains the following elements from 5' to 3': hTNNT2 promoter (469 bp) (SEQ ID NO: 49), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, and bGH polyA (227 bp) (SEQ ID NO: 61). In further embodiments, construct A2 optionally contains any of the 5' AAV2-ITR sequences of SEQ ID NOs: 67-70 (or their complementary sequences) and / or any of the 3' AAV2-ITR sequences of SEQ ID NOs: 71-74 (or their complementary sequences), or fragments thereof.

[0213] Construct A3 is 4939 bp long (SEQ ID NO: 11) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 67), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH polyA (169 bp) (SEQ ID NO: 59), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0214] In some embodiments, construct A3 is 4969 bp long (SEQ ID NO: 10) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 68), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH polyA (169 bp) (SEQ ID NO: 59), and 3' AAV2 ITR (145) (SEQ ID NO: 72).

[0215] In some embodiments, construct A3 is 4939 bp long (SEQ ID NO: 134) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 69), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH polyA (169 bp) (SEQ ID NO: 59), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0216] In some embodiments, construct A3 is 4969 bp long (SEQ ID NO: 135) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 70), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH polyA (169 bp) (SEQ ID NO: 59), and 3' AAV2 ITR (145) (SEQ ID NO: 74).

[0217] In some embodiments, construct A3 is 4939 bp long (SEQ ID NO: 136) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 67), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH polyA (169 bp) (SEQ ID NO: 59), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0218] In some embodiments, construct A3 is 4969 bp long (SEQ ID NO: 137) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 68), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH polyA (169 bp) (SEQ ID NO: 59), and 3' AAV2 ITR (145) (SEQ ID NO: 74).

[0219] In some embodiments, construct A3 is 4939 bp long (SEQ ID NO: 138) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 69), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH polyA (169 bp) (SEQ ID NO: 59), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0220] In some embodiments, construct A3 is 4969 bp long (SEQ ID NO: 139) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 70), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH polyA (169 bp) (SEQ ID NO: 59), and 3' AAV2 ITR (145) (SEQ ID NO: 72).

[0221] In some embodiments, construct A3 is 4663 bp long (SEQ ID NO: 9) and contains the following elements from 5' to 3': hTNNT2 promoter (532 bp) (SEQ ID NO: 51), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, and bGH polyA (169 bp) (SEQ ID NO: 59). In further embodiments, construct A3 optionally contains any of the 5' AAV2-ITR sequences of SEQ ID NOs: 67-70 (or their complements) and / or any of the 3' AAV2-ITR sequences of SEQ ID NOs: 71-74 (or their complements), or fragments thereof.

[0222] Construct A4 is 4939 bp long (SEQ ID NO: 14) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 67), hTNNT2 promoter (499 bp) (SEQ ID NO: 50), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH polyA (202 bp) (SEQ ID NO: 60), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0223] In some embodiments, construct A4 is 4969 bp long (SEQ ID NO: 13) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 68), hTNNT2 promoter (499 bp) (SEQ ID NO: 50), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH polyA (202 bp) (SEQ ID NO: 60), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 72).

[0224] In some embodiments, construct A4 is 4939 bp long (SEQ ID NO: 140) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 69), hTNNT2 promoter (499 bp) (SEQ ID NO: 50), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH polyA (202 bp) (SEQ ID NO: 60), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0225] In some embodiments, construct A4 is 4969 bp long (SEQ ID NO: 141) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 70), hTNNT2 promoter (499 bp) (SEQ ID NO: 50), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH polyA (202 bp) (SEQ ID NO: 60), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 74).

[0226] In some embodiments, construct A4 is 4939 bp long (SEQ ID NO: 142) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 67), hTNNT2 promoter (499 bp) (SEQ ID NO: 50), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH polyA (202 bp) (SEQ ID NO: 60), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0227] In some embodiments, construct A4 is 4969 bp long (SEQ ID NO: 143) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 68), hTNNT2 promoter (499 bp) (SEQ ID NO: 50), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH polyA (202 bp) (SEQ ID NO: 60), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 74).

[0228] In some embodiments, construct A4 is 4939 bp long (SEQ ID NO: 144) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 69), hTNNT2 promoter (499 bp) (SEQ ID NO: 50), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH polyA (202 bp) (SEQ ID NO: 60), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0229] In some embodiments, construct A4 is 4969 bp long (SEQ ID NO: 145) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 70), hTNNT2 promoter (499 bp) (SEQ ID NO: 50), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH polyA (202 bp) (SEQ ID NO: 60), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 72).

[0230] In some embodiments, construct A4 is 4663 bp long (SEQ ID NO: 12) and contains the following elements from 5' to 3': hTNNT2 promoter (499 bp) (SEQ ID NO: 50), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, and bGH polyA (202 bp) (SEQ ID NO: 60). In further embodiments, construct A4 optionally contains any of the 5' AAV2-ITR sequences of SEQ ID NOs: 67-70 (or their complements) and / or any of the 3' AAV2-ITR sequences of SEQ ID NOs: 71-74 (or their complements), or fragments thereof.

[0231] Construct A5 is 4871 bp long (SEQ ID NO: 17) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 67), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), wild-type hMYBPC3 (SEQ ID NO: 42), bGH polyA (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0232] In some embodiments, construct A5 is 4901 bp long (SEQ ID NO: 16) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 67), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), wild-type hMYBPC3 (SEQ ID NO: 42), bGH polyA (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 71).

[0233] In some embodiments, construct A5 is 4871 bp long (SEQ ID NO: 146) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 69), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), wild-type hMYBPC3 (SEQ ID NO: 42), bGH polyA (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0234] In some embodiments, construct A5 is 4901 bp long (SEQ ID NO: 147) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 70), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), wild-type hMYBPC3 (SEQ ID NO: 42), bGH polyA (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 74).

[0235] In some embodiments, construct A5 is 4871 bp long (SEQ ID NO: 148) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 67), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), wild-type hMYBPC3 (SEQ ID NO: 42), bGH polyA (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0236] In some embodiments, construct A5 is 4901 bp long (SEQ ID NO: 149) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 68), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), wild-type hMYBPC3 (SEQ ID NO: 42), bGH polyA (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 74).

[0237] In some embodiments, construct A5 is 4871 bp long (SEQ ID NO: 150) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 69), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), wild-type hMYBPC3 (SEQ ID NO: 42), bGH polyA (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0238] In some embodiments, construct A5 is 4901 bp long (SEQ ID NO: 151) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 70), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), wild-type hMYBPC3 (SEQ ID NO: 42), bGH polyA (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 72).

[0239] In some embodiments, construct A5 is 4595 bp long (SEQ ID NO: 15) and contains the following elements from 5' to 3': hTNNT2 promoter (532 bp) (SEQ ID NO: 51), wild-type hMYBPC3 (SEQ ID NO: 42), and bGH polyA (227 bp) (SEQ ID NO: 61). In further embodiments, construct A5 optionally contains any of the 5' AAV2-ITR sequences of SEQ ID NOs: 67-70 (or their complements) and / or any of the 3' AAV2-ITR sequences of SEQ ID NOs: 71-74 (or their complements), or fragments thereof.

[0240] Construct A6 is 5002 bp long (SEQ ID NO: 20) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 67), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH polyA (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0241] In some embodiments, construct A6 is 5032 bp long (SEQ ID NO: 19) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 68), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH polyA (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 72).

[0242] In some embodiments, construct A6 is 5002 bp long (SEQ ID NO: 152) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 69), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH polyA (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0243] In some embodiments, construct A6 is 5032 bp long (SEQ ID NO: 153) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 70), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH polyA (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 74).

[0244] In some embodiments, construct A6 is 5002 bp long (SEQ ID NO: 154) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 67), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH polyA (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0245] In some embodiments, construct A6 is 5032 bp long (SEQ ID NO: 155) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 68), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH polyA (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 74).

[0246] In some embodiments, construct A6 is 5002 bp long (SEQ ID NO: 156) and contains the following elements from 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 69), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH polyA (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0247] In some embodiments, construct A6 is 5032 bp long (SEQ ID NO: 157) and contains the following elements from 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 70), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH polyA (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 72).

[0248] In some embodiments, construct A6 is 4726 bp long (SEQ ID NO: 18) and contains the following elements from 5' to 3': hTNNT2 promoter (532 bp) (SEQ ID NO: 51), wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, and bGH polyA (227 bp) (SEQ ID NO: 61). In further embodiments, construct A6 optionally contains any of the 5' AAV2-ITR sequences of SEQ ID NOs: 67-70 (or their complements) and / or any of the 3' AAV2-ITR sequences of SEQ ID NOs: 71-74 (or their complements), or fragments thereof.

[0249] Construct A7 is 4781 bp long (SEQ ID NO: 23) and contains the following elements from 5' to 3': 5'AAV2-ITR (130 bp) (SEQ ID NO: 67), hTNNT2 promoter (469 bp) (SEQ ID NO: 49), Kozak sequence, wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, minipolyA (57 bp) (SEQ ID NO: 64), 3'-UTR sequence, and 3'AAV2-ITR (130 bp) (SEQ ID NO: 71).

[0250] In some embodiments, construct A7 is 4811 bp long (SEQ ID NO: 22) and contains the following elements from 5' to 3': 5'AAV2-ITR (145 bp) (SEQ ID NO: 67), hTNNT2 promoter (469 bp) (SEQ ID NO: 49), Kozak sequence, wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, mini-polyA (57 bp) (SEQ ID NO: 64), 3'-UTR sequence, and 3'AAV2-ITR (145 bp) (SEQ ID NO: 71).

[0251] In some embodiments, construct A7 is 4781 bp long (SEQ ID NO: 158) and contains the following elements from 5' to 3': 5'AAV2-ITR (130 bp) (SEQ ID NO: 69), hTNNT2 promoter (469 bp) (SEQ ID NO: 49), Kozak sequence, wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, mini-polyA (57 bp) (SEQ ID NO: 64), 3'-UTR sequence, and 3'AAV2-ITR (130 bp) (SEQ ID NO: 73).

[0252] In some embodiments, construct A7 is 4811 bp long (SEQ ID NO: 159) and contains the following elements from 5' to 3': 5'AAV2-ITR (145 bp) (SEQ ID NO: 70), hTNNT2 promoter (469 bp) (SEQ ID NO: 49), Kozak sequence, wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, mini-polyA (57 bp) (SEQ ID NO: 64), 3'-UTR sequence, and 3'AAV2-ITR (145 bp) (SEQ ID NO: 74).

[0253] In some embodiments, construct A7 is 4781 bp long (SEQ ID NO: 160) and contains the following elements from 5' to 3': 5'AAV2-ITR (130 bp) (SEQ ID NO: 67), hTNNT2 promoter (469 bp) (SEQ ID NO: 49), Kozak sequence, wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, mini-polyA (57 bp) (SEQ ID NO: 64), 3'-UTR sequence, and 3'AAV2-ITR (130 bp) (SEQ ID NO: 73).

[0254] In some embodiments, construct A7 is 4811 bp long (SEQ ID NO: 161) and contains the following elements from 5' to 3': 5'AAV2-ITR (145 bp) (SEQ ID NO: 68), hTNNT2 promoter (469 bp) (SEQ ID NO: 49), Kozak sequence, wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, mini-polyA (57 bp) (SEQ ID NO: 64), 3'-UTR sequence, and 3'AAV2-ITR (145 bp) (SEQ ID NO: 74).

[0255] In some embodiments, construct A7 is 4781 bp long (SEQ ID NO: 162) and contains the following elements from 5' to 3': 5'AAV2-ITR (130 bp) (SEQ ID NO: 69), hTNNT2 promoter (469 bp) (SEQ ID NO: 49), Kozak sequence, wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, mini-polyA (57 bp) (SEQ ID NO: 64), 3'-UTR sequence, and 3'AAV2-ITR (130 bp) (SEQ ID NO: 71).

[0256] In some embodiments, construct A7 is 4811 bp long (SEQ ID NO: 163) and contains the following elements from 5' to 3': 5'AAV2-ITR (145 bp) (SEQ ID NO: 70), hTNNT2 promoter (469 bp) (SEQ ID NO: 49), Kozak sequence, wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, mini-polyA (57 bp) (SEQ ID NO: 64), 3'-UTR sequence, and 3'AAV2-ITR (145 bp) (SEQ ID NO: 72).

[0257] In some embodiments, construct A7 is 4505 bp long (SEQ ID NO:21) and contains the following elements from 5' to 3': hTNNT2 promoter (469 bp) (SEQ ID NO:49), Kozak sequence, wild-type hMYBPC3 (SEQ ID NO:42) containing a globin intron (131 bp) (SEQ ID NO:53) between exons, mini-polyA (57 bp) (SEQ ID NO:64), and 3'-UTR sequence. In further embodiments, construct A7 optionally contains any of the 5'AAV2-ITR sequences of SEQ ID NOs:67-70 (or their complementary sequences) and / or any of the 3'AAV2-ITR sequences of SEQ ID NOs:71-74 (or their complementary sequences), or fragments thereof.

[0258] Construct A8 is 4844 bp long (SEQ ID NO: 26) and contains the following elements from 5' to 3': 5'AAV2-ITR (130 bp) (SEQ ID NO: 67), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), Kozak sequence, wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, minipolyA (57 bp) (SEQ ID NO: 64), 3'-UTR sequence and 3'AAV2-ITR (130 bp) (SEQ ID NO: 71).

[0259] In some embodiments, construct A8 is 4874 bp long (SEQ ID NO: 25) and contains the following elements from 5' to 3': 5'AAV2-ITR (145 bp) (SEQ ID NO: 67), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), Kozak sequence, wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, mini-polyA (57 bp) (SEQ ID NO: 64), 3'-UTR sequence, and 3'AAV2-ITR (145 bp) (SEQ ID NO: 71).

[0260] In some embodiments, construct A8 is 4844 bp long (SEQ ID NO: 164) and contains the following elements from 5' to 3': 5'AAV2-ITR (130 bp) (SEQ ID NO: 69), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), Kozak sequence, wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, mini-polyA (57 bp) (SEQ ID NO: 64), 3'-UTR sequence, and 3'AAV2-ITR (130 bp) (SEQ ID NO: 73).

[0261] In some embodiments, construct A8 is 4874 bp long (SEQ ID NO: 165) and contains the following elements from 5' to 3': 5'AAV2-ITR (145 bp) (SEQ ID NO: 70), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), Kozak sequence, wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, mini-polyA (57 bp) (SEQ ID NO: 64), 3'-UTR sequence, and 3'AAV2-ITR (145 bp) (SEQ ID NO: 72).

[0262] In some embodiments, construct A8 is 4844 bp long (SEQ ID NO: 166) and contains the following elements from 5' to 3': 5'AAV2-ITR (130 bp) (SEQ ID NO: 67), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), Kozak sequence, wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, mini-polyA (57 bp) (SEQ ID NO: 64), 3'-UTR sequence, and 3'AAV2-ITR (130 bp) (SEQ ID NO: 73).

[0263] In some embodiments, construct A8 is 4874 bp long (SEQ ID NO: 167) and contains the following elements from 5' to 3': 5'AAV2-ITR (145 bp) (SEQ ID NO: 68), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), Kozak sequence, wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, mini-polyA (57 bp) (SEQ ID NO: 64), 3'-UTR sequence, and 3'AAV2-ITR (145 bp) (SEQ ID NO: 73).

[0264] In some embodiments, construct A8 is 4844 bp long (SEQ ID NO: 168) and contains the following elements from 5' to 3': 5'AAV2-ITR (130 bp) (SEQ ID NO: 69), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), Kozak sequence, wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, mini-polyA (57 bp) (SEQ ID NO: 64), 3'-UTR sequence, and 3'AAV2-ITR (130 bp) (SEQ ID NO: 71).

[0265] In some embodiments, construct A8 is 4874 bp long (SEQ ID NO: 169) and contains the following elements from 5' to 3': 5'AAV2-ITR (145 bp) (SEQ ID NO: 70), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), Kozak sequence, wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, mini-polyA (57 bp) (SEQ ID NO: 64), 3'-UTR sequence, and 3'AAV2-ITR (145 bp) (SEQ ID NO: 72).

[0266] In some embodiments, construct A8 is 4568 bp long (SEQ ID NO: 24) and contains the following elements from 5' to 3': hTNNT2 promoter (532 bp) (SEQ ID NO: 51), Kozak sequence, wild-type hMYBPC3 (SEQ ID NO: 42) containing a globin intron (131 bp) (SEQ ID NO: 53) between exons, mini-polyA (57 bp) (SEQ ID NO: 64), and 3'-UTR sequence. In further embodiments, construct A8 optionally contains any of the 5' AAV2-ITR sequences of SEQ ID NOs: 67-70 (or their complementary sequences) and / or any of the 3' AAV2-ITR sequences of SEQ ID NOs: 71-74 (or their complementary sequences), or fragments thereof.

[0267] 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 complements and / or combinations thereof).

[0268] Polynucleotides and polypeptides, including modified forms, can be produced using a variety of standard cloning, recombinant DNA techniques known to those skilled in the art, via cellular expression or in vitro translation, and chemical synthesis techniques (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2002). nd edition).

[0269] Methods of gene delivery.

[0270] Also provided is a method for delivering a gene encoding a protein of interest using the vector construct or AAV particle described herein.In one embodiment, the gene delivery vector may 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 herpesvirus vectors.Preferably, it is a recombinant adeno-associated virus (rAAV) vector.Alternatively, non-viral systems may be used, including the use of naked DNA (with or without chromatin-binding regions) or conjugated DNA, which is introduced into cells by various transfection methods such as lipid or electroporation.

[0271] Non-limiting examples of vector constructs described herein include any of SEQ ID NOs: 3-41 or 92-169.

[0272] 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 of SEQ ID NOs: 3-41 or 92-169 (over the entire length of SEQ ID NOs: 3-41 or 92-169, respectively). In some embodiments, the vector construct comprises a nucleotide sequence having at least about 85% sequence identity to any of SEQ ID NOs: 3-41 or 92-169. Preferably, the vector construct or AAV vector genome of an AAV particle comprises a nucleotide sequence having at least about 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5% or more sequence identity to any of SEQ ID NOs: 3-41 or 92-169. Even more preferably, the nucleotide sequence of the vector construct is at least 97%, or 98%, or 99% or more identical to any of SEQ ID NOs: 3-41 or 92-169. In other embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98%, or 99% identical to a nucleotide sequence that is complementary to, i.e., the minus (-) strand of, any of SEQ ID NOs: 3-41 or 92-169.

[0273] In some embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98%, or 99% identical to any of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, or 39. In other embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98%, or 99% identical to a nucleotide sequence that is complementary to, i.e., the minus (-) strand of, any of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, or 39.

[0274] In some embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98%, or 99% identical to any of SEQ ID NOs: 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, or 40. In other embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98%, or 99% identical to a nucleotide sequence that is complementary to, i.e., the minus (-) strand of, any of SEQ ID NOs: 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, or 40.

[0275] In some embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98%, or 99% identical to any of SEQ ID NOs: 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, or 41. In other embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98%, or 99% identical to a nucleotide sequence that is complementary to, i.e., the minus (-) strand of, any of SEQ ID NOs: 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, or 41.

[0276] The present disclosure is useful for both veterinary and medical applications. Suitable subjects for the gene delivery methods described herein include both birds and mammals, with mammals being preferred and humans being most preferred. Human subjects include neonates, infants, adolescents, and adults.

[0277] Non-viral gene delivery.

[0278] Non-viral gene delivery can be carried out using naked DNA, which is the simplest method of non-viral transfection.For example, it may be possible to administer the vector constructs provided herein using naked plasmid DNA.Alternatively, the vector constructs can be delivered using electroporation, sonoporation, or the use of a "gene gun" that shoots DNA-coated gold particles into cells using, for example, high pressure gas or an inverted .22 caliber gun (Helios® Gene Gun System (BIO-RAD)), microinjection, laser, high temperature, ultrasound, hydrodynamic gene transfer, magnetic transfection, chemical transfection (e.g., calcium phosphate, DEAE-dextran), liposomes, lipoplexes, dendrimers, lipid nanoparticles, or inorganic nanoparticles, all of which are known in the art.

[0279] To improve the delivery of a vector construct into a cell, it may be necessary to protect it from damage and facilitate its entry into the cell. For this purpose, lipoplexes and polyplexes may be used, which have the ability to protect nucleic acids from unwanted degradation during the transfection process.

[0280] Vector constructs may be coated with lipids in organized structures such as micelles or liposomes. When the organized structures are complexed with DNA, they are called lipoplexes. Anionic and neutral lipids may be used to construct lipoplexes for synthetic vectors. In one embodiment, cationic lipids, due to their positive charge, may be used to condense negatively charged DNA molecules to facilitate encapsulation of DNA into liposomes. It may be necessary to add a helper lipid (usually an electrically neutral lipid such as DOPE) to the cationic lipid to form lipoplexes (Dabkowska et al., JR Soc. Interface. 9(68):548-61(2012)).

[0281] In certain embodiments, vector constructs may be delivered using polymer-DNA complexes called polyplexes. Most polyplexes are composed of cationic polymers, and their formation is regulated by ionic interactions. Polyplexes typically cannot release their DNA into the cytoplasm. Therefore, co-transfection with an endosomolytic agent, such as an inactivated adenovirus, may be required to dissolve endosomes generated during endocytosis, the process by which polyplexes enter cells (Akinc et al., J. Gene Medic. 7(5):657-63).

[0282] In certain embodiments, vector constructs may be delivered using hybrid methods that combine two or more techniques. Virosomes are one example, and they combine liposomes with inactivated HIV or influenza viruses. In other embodiments, other methods involve mixing other viral vectors with cationic lipids or hybridizing viruses, which may be used to deliver nucleic acids (Khan, Firdos Alam, Biotechnology Fundamentals, CRC Press, November 18, 2015, p. 395).

[0283] In certain embodiments, dendrimers, particularly cationic dendrimers, i.e., those with a positive surface charge, may be used to deliver vector constructs. When in the presence of genetic material such as DNA or RNA, charge complementarity results in a transient association between the nucleic acid and the cationic dendrimer. Upon reaching its destination, the dendrimer-nucleic acid complex is then transported into the cell via endocytosis (Amiji, Mansoor M. ed., Polymeric Gene Delivery: Principles and Applications, CRC Press, September 29, 2004, p. 142).

[0284] Virus particles.

[0285] In one embodiment, nucleic acid can be delivered using a suitable viral gene delivery vector, such as a viral particle.In certain embodiments, the viral gene delivery vector suitable for use herein can be parvovirus, adenovirus, retrovirus, gamma-retrovirus, lentivirus, herpes simplex virus, vaccinia virus, measles virus, vesicular stomatitis virus, poliovirus or reovirus.The parvovirus can be adenovirus-associated virus (AAV).

[0286] Thus, the present disclosure provides viral particles for use as gene delivery vectors (including the vector constructs provided herein) based on animal parvoviruses, particularly infectious human or simian AAV-like dependoviruses, and components thereof (e.g., animal parvovirus genomes), for the introduction and / or expression of cMyBP-C protein in mammalian cells. Thus, as used herein, the term "parvovirus" encompasses any type of AAV-like dependovirus.

[0287] Viruses of the Parvoviridae family are small DNA animal viruses. The Parvoviridae family is divided into two subfamilies: the Parvovirinae, which infect vertebrates, and the Densovirinae, which infect insects. Members of the Parvovirinae subfamily are referred to herein as parvoviruses and include the genus Dependovirus. As can be inferred from their genus name, Dependovirus members are unique in that they typically require co-infection with a helper virus, such as an adenovirus or a herpesvirus, for productive infection in cell culture. The Dependovirus genus includes AAVs that typically infect humans (e.g., serotypes 1, 2, 3A, 3B, 4, 5, and 6), AAVs that infect primates (e.g., serotypes 1 and 4), and related viruses that infect other warm-blooded animals in addition to birds and reptiles (e.g., bovine, canine, equine, murine, rat, and ovine adeno-associated viruses). Further information about parvoviruses and other members of the Parvoviridae family is provided in Kenneth I. Berns, "Parvoviridae: The Viruses and Their Replication," Chapter 69 in Fields Virology (3d Ed. 1996). For convenience, the present disclosure will be further illustrated and described herein with reference to AAV. However, it should be understood that the present disclosure is not limited to AAV and may be equally applicable to other parvoviruses.

[0288] AAV particle production requires the AAV "rep" and "cap" genes, which encode replication and encapsidation proteins, respectively. The AAV rep and cap genes are found in all AAV serotypes examined to date and are described herein and in the cited references. 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 as adjacent or overlapping transcription units), and they are generally conserved among AAV serotypes. The AAV rep and cap genes are also individually and collectively referred to as "AAV packaging genes." As used herein, the AAV cap gene encodes a Cap protein that is capable of packaging an AAV vector in the presence of rep and adenovirus helper functions and of binding to a target cell receptor. In some embodiments, the AAV cap gene encodes a capsid protein having an amino acid sequence derived from a particular AAV serotype.

[0289] AAV sequences used in AAV production can be derived from the genome of any AAV serotype. Generally, AAV serotypes have genomic sequences with significant homology at the amino acid and nucleic acid levels, exhibit similar sets of gene functions, produce virions that are essentially physically and functionally equivalent, and replicate and assemble by virtually identical mechanisms. For a discussion of the genomic sequences and genomic similarities of AAV serotypes, see, e.g., GenBank Accession number U89790, GenBank Accession number J01901, GenBank Accession number AF043303, GenBank Accession number 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)).

[0290] The genomic organization of all known AAV serotypes is very similar. The AAV genome is a linear, single-stranded DNA molecule less than approximately 5,000 nucleotides (nt) in length. Inverted terminal repeats (ITRs) flank the unique coding nucleotide sequences for the nonstructural replication (Rep) and structural (VP) proteins. The VP proteins form the capsid. Assembly-activating proteins (AAPs) rapidly attend capsid assembly and prevent degradation of free capsid proteins (Grosse et al., J. Virol. 91(20):e01198-17(2017)). The terminal 145 nt are self-complementary and organized, allowing the formation of energetically stable intramolecular duplexes that form T-shaped hairpins. These hairpin structures serve as origins of viral DNA replication and as primers for the cellular DNA polymerase complex. The Rep genes encode the Rep proteins Rep78, Rep68, Rep52, and Rep40. Rep78 and Rep68 are transcribed from the p5 promoter, and Rep52 and Rep40 are transcribed from the p19 promoter. The cap gene encodes the VP proteins VP1, VP2, and VP3. The cap gene is transcribed from the p40 promoter. The ITRs used in the vectors of this embodiment 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 AAV2 serotype and the cap gene corresponds to the AAV5 serotype.

[0291] AAV VP proteins are known to determine the cell tropism of AAV virions. VP protein coding sequences are significantly less conserved among different AAV serotypes than Rep proteins and genes. The ability of Rep and ITR sequences to cross-complement corresponding sequences of other serotypes allows for the production of pseudotyped AAV particles containing capsid proteins from one serotype (e.g., AAV1, 5, or 8) and Rep and / or ITR sequences from another AAV serotype (e.g., AAV2). Such pseudotyped rAAV particles are part of the present disclosure.

[0292] The AAV particles (and encoding AAV vector genomes) described herein may comprise any of the capsid proteins described in WO-2018 / 022608 or WO-2019 / 222136 (which are incorporated by reference in their entireties for their disclosure of human and simian AAV capsids and their properties such as transduction efficiency, tissue tropism, glycan binding, and resistance to neutralization by IVIG), including, but not limited to, any of the capsids in the Sequence Listing, and variants thereof, e.g., with chimeric swapped variable regions and / or glycan binding sequences and / or GH loops.

[0293] In one embodiment, AAV ITR sequences for use in the context of the present disclosure are derived from AAV1, AAV2, AAV4, and / or AAV6. Similarly, Rep (e.g., Rep78 and Rep52) coding sequences are, in one embodiment, derived from AAV1, AAV2, AAV4, and / or AAV6. However, sequences encoding the VP1, VP2, and VP3 capsid proteins for use in the context of the present disclosure may be obtained from any serotype, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12, or from simian AAV, including the capsid proteins described in any of WO2018 / 022608 or PCT / US19 / 32097, or newly developed AAV-like particles obtained, for example, by capsid shuffling techniques and AAV capsid libraries, or any capsid at least 90% identical to any of SEQ ID NOs: 75-91.

[0294] For example, the amino acid sequences of various capsids have been published. AAVRh.1 / hu.14 / AAV9 AAS99264.1 (SEQ ID NO: 75) AAVRh.8 sequence 97 (SEQ ID NO: 76) of U.S. Patent Publication 2013 / 0045186 AAVRh.10 sequence 81 (SEQ ID NO: 77) of U.S. Patent Publication 2013 / 0045186 AAVRh.74 Sequence 1 (SEQ ID NO: 78) of International Patent Publication WO2013 / 123503 AAV1 AAB_95452.1 (SEQ ID NO: 79) AAV2 YP_680426.1 (SEQ ID NO: 80) AAV3 NP_043941.1 (SEQ ID NO: 81) AAV3B AAB95452.1 (SEQ ID NO: 82) AAV4 NP_044927.1 (SEQ ID NO: 83) AAV5 YP_068409.1 (SEQ ID NO: 84) AAV6 AAB95450.1 (SEQ ID NO: 85) AAV7 YP_077178.1 (SEQ ID NO: 86) AAV8 YP_077180.1 (SEQ ID NO: 87) AAV10 AAT46337.1 (SEQ ID NO: 88) AAV11 AAT46339.1 (SEQ ID NO: 89) AAV12 ABI16639.1 (SEQ ID NO: 90) See AAV13 ABZ10812.1 (sequence number 91).

[0295] Modified "AAV" sequences can also be used in the context of the present disclosure, e.g., for the production of AAV gene therapy vectors. Such modified sequences, e.g., 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 an ITR, Rep, or VP of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 (e.g., sequences having about 75-99% nucleotide sequence identity), can be used in place of the wild-type AAV ITR, Rep, or VP sequences.

[0296] In some embodiments, the nucleic acid sequences encoding the AAV capsid proteins are operably linked to expression control sequences for expression in a particular cell type, such as Sf9 or HEK cells. Techniques known to those of skill in the art for expressing foreign genes in insect or mammalian host cells can be used to practice this embodiment. Techniques for molecular engineering and expression of 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 Bulletin No. 7555, College Station, Texas; Luckow (1991) In Prokop et al., Cloning and Expression of Heterologous Genes in Insect Cells with Baculovirus Vectors' Recombinant DNA Technology and Applications, pp. 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 entireties. A particularly suitable promoter for transcription of nucleotide sequences encoding AAV capsid proteins is, for example, the polyhedron promoter.However, other promoters active in insect cells are known in the art, for example, the p10, p35 or IE-1 promoters and further promoters described in the above references are also contemplated.

[0297] The use of insect cells for the expression of heterologous proteins is well documented, as are methods for introducing nucleic acids, such as vectors, e.g., insect cell-compatible vectors, into such cells and methods for maintaining such cells in culture (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. (See, for example, Zhao 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, "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 insect cell-compatible. While the vector can be integrated into the insect cell genome, the presence of the vector in the insect cell need not be permanent; transient episomal vectors are also included. The vector can be introduced by any known means, such as by chemical treatment, electroporation, or infection of the cells. In some embodiments, the vector is a baculovirus, viral vector, or plasmid. In one embodiment, the vector is a baculovirus, ie, the construct is a baculovirus vector.Baculovirus vectors and methods for their use are described in the references cited above regarding molecular engineering of insect cells.

[0298] Methods for producing recombinant AAV particles The present disclosure 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 the protein of interest. Those skilled in the art will understand that any one of the AAV vector constructs disclosed herein can be used in methods for producing recombinant AAV particles.

[0299] In some embodiments, helper functions for AAV production are provided by one or more helper plasmids or viruses containing adenovirus or baculovirus helper genes, non-limiting examples of which include, but are not limited to, E1A, E1B, E2A, E4, and VA, which can provide helper functions for AAV packaging.

[0300] AAV helper viruses are known in the art and include, for example, viruses from the Adenoviridae and Herpesviridae families. Examples of AAV helper viruses 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 understand that any AAV helper virus or helper plasmid that can provide sufficient helper function for AAV can be used herein.

[0301] In some embodiments, the AAV cap gene is present on a 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 and / or rep genes from any of the AAV serotypes described herein (including, but not limited to, AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, and any variants thereof) can be used to produce 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 a variant thereof.

[0302] In some embodiments, insect or mammalian cells can be transfected with a helper plasmid or helper virus, a vector construct, and a plasmid encoding the AAV cap gene, and the recombinant AAV virus can be harvested at various time points after co-transfection, for example, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 96 hours, about 120 hours, or any time between these two time points after co-transfection.

[0303] Recombinant AAV particles can also be produced using any conventional method known in the art that is suitable for producing infectious recombinant AAV. In some cases, recombinant AAV can be produced by using insect or mammalian cells that stably express some of the components necessary for AAV particle production. For example, a plasmid (or multiple plasmids) containing 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. The insect or mammalian cells can then be co-infected with a viral vector construct containing a helper virus (e.g., an adenovirus or baculovirus that provides helper functions) and the 5' and 3' AAV ITRs (and, if desired, nucleotide sequences encoding heterologous proteins). The advantage of this method is that the cells are selectable and suitable for large-scale production of recombinant AAV particles. As another non-limiting example, the rep and cap genes can be introduced into packaging cells using adenovirus or baculovirus rather than a plasmid. As yet another non-limiting example, a viral vector construct containing both the 5' and 3' AAV ITRs and the rep-cap gene can be stably integrated into the DNA of a producer cell, with helper functions provided by wild-type adenovirus, to produce recombinant AAV.

[0304] In one aspect, provided herein is a method for the production of AAV particles useful as gene delivery vectors, the method comprising: (a) into cells permissive for AAV replication (e.g., insect cells or mammalian cells), (i) a nucleic acid molecule (e.g., a recombinant vector construct) provided herein flanked by at least one AAV inverted terminal repeat nucleotide sequence; (ii) a nucleotide sequence encoding one or more AAV Rep proteins operably linked to a promoter capable of driving expression of the Rep protein(s) in a cell; (iii) a nucleotide sequence encoding one or more AAV capsid proteins operably linked to a promoter capable of driving expression of the capsid protein(s) in the cell; (iv) and optionally providing one or more nucleic acid constructs comprising the AAP and MAAP contained in the VP2 / 3 mRNA; (b) culturing the cells defined in (a) under conditions that promote expression of Rep and capsid proteins; 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) a heterologous cardiomyocyte-specific transcriptional regulatory region described herein, and (3) a nucleic acid encoding functional cMyBP-C. Preferably, the recombinant vector construct of (i) comprises both the 5' AAV ITR and the 3' AAV ITR.

[0305] Typically, then, the methods provided herein for producing an AAV gene delivery vector involve providing a cell permissive for AAV replication with (a) a nucleotide sequence encoding a template for producing a vector genome, e.g., a vector construct of the present disclosure (as detailed herein), (b) a nucleotide sequence sufficient for replication of the template for producing the vector genome (a first expression cassette, as defined above), and (c) a nucleotide sequence sufficient for packaging the vector genome into an AAV capsid (a second expression cassette, as defined above), under conditions sufficient for replication and packaging of the vector genome into an AAV capsid, such that AAV particles comprising the vector genome encapsulated within the AAV capsid are produced in the cell.

[0306] Transient transfection of adherent HEK293 cells (Chahal et al., J. Virol. Meth. 196:163-73 (2014)) and transfection of Sf9 cells using the baculovirus expression vector system (BEVS) (Mietzsch et al., Hum. Gene Ther. 25:212-22 (2014)) (Mietzsch et al., Hum. Gene Ther. 25:212-22 (2014)) are two of the most commonly used methods for producing AAV vectors.

[0307] Viral particles containing the vector constructs described herein may be produced using any cell type that allows for the production of AAV or biological products and that can be maintained in culture, including mammalian and invertebrate cell types.

[0308] There are several methods for generating AAV viral particles, including, but not limited to, transfection using a vector and AAV helper sequences, along with co-infection with one of the AAV helper viruses (e.g., adenovirus, herpesvirus, or vaccinia virus), or transfection with a recombinant AAV vector, an AAV helper vector, and an accessory function vector.Methods for producing AAV viral particles are described, for example, in U.S. Pat. Nos. US6,204,059, US5,756,283, US6,258,595, US6,261,551, US6,270,996, US6,281,010, US6,365,394, US6,475,769, US6,482,634, US6,485,966, US6,943,019, US6,953,690, US7,022,519, US7,238,526, US7,291,498, and US7,491,508, US5,064,764, US6,194,191, No. US6566118, No. US8137948, or International Publication Nos. WO1996039530, WO1998010088, WO1999014354, WO1999015685, WO1999047691, WO2000055342, WO 2000075353, WO2001023597, WO2015191508, WO2019217513, WO2 018022608, WO2019222136, WO2020232044, WO2019222132, Methods In Molecular Biology, ed. Richard, Humana Press, NJ (1995), O'Reilly et al., Baculovirus Expression Vectors, A Laboratory Manual, Oxford University 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, for example, 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, for example, U.S. Patent No. 6,001,650, incorporated herein by reference in its entirety) is used to produce AAV viral particles. This method does not require the use of an infectious helper virus and allows AAV viral particles to be produced without the presence of any detectable helper virus. This is accomplished by using three vectors for AAV viral particle production: an AAV helper function vector, an accessory function vector, and an AAV viral particle expression vector. However, those skilled in the art will understand that the nucleic acid sequences encoded by these vectors can be provided in two or more vectors in various combinations. In other embodiments, host cells can be transfected with a helper plasmid or helper virus, a viral construct, and a plasmid encoding the AAV cap gene, and AAV viral particles can be harvested at various time points after co-transfection.

[0309] For example, wild-type AAV and a helper virus may be used to provide the replication functions necessary to produce AAV viral particles (see, e.g., U.S. Pat. No. 5,139,941, incorporated herein by reference in its entirety). Alternatively, a plasmid containing helper function genes in combination with infection with one of the well-known helper viruses may be used as a source of replication functions (see, e.g., U.S. Pat. Nos. 5,622,856 and 5,139,941, both of which are incorporated herein by reference in their entirety). Similarly, a plasmid containing accessory function genes may be used in combination with infection with wild-type AAV to provide the necessary replication functions. Other approaches described herein and / or known in the art can also be used by those skilled in the art to produce AAV viral particles.

[0310] The term "vector" is understood to refer to any genetic element, such as a plasmid, phage, transposon, cosmid, bacmid, miniplasmid (e.g., a plasmid lacking bacterial elements), doggybone DNA (e.g., a minimal closed linear construct), chromosome, virus, virion (e.g., baculovirus), etc., that is capable of replication and transfer of genetic sequences between cells when associated with the 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 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 insect cell. Exemplary biological vectors include plasmids, linear nucleic acid molecules, and recombinant viruses. Any vector can be used as long as it is insect cell-compatible. While the vector can be integrated into the insect cell genome, the presence of the vector in the insect cell need not be permanent; transient episomal vectors are also included. The vector can be introduced by any known means, for example, by chemical treatment, electroporation, or infection of the cells. Vectors and methods for their use are described in the references cited above regarding molecular engineering of cells.

[0311] The vector from which the cell produces the rAAV vector genome may contain a promoter and a restriction site downstream of the promoter to allow insertion of a polynucleotide encoding one or more proteins of interest, where 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 post-transcriptional regulatory elements downstream of the restriction site and upstream of the 3' AAV ITR. The viral construct further comprises a polynucleotide inserted into the restriction site and operably linked to the promoter, where the polynucleotide may further comprise a coding region for a protein of interest. In some embodiments, the viral 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, where 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 post-transcriptional regulatory elements 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. One skilled in the art will understand that any one of the AAV vectors disclosed herein can be used in the method as a viral construct for producing AAV virions.

[0312] The term "AAV helper" refers to coding sequences from AAV that can be expressed to produce AAV gene products that then function 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, inter alia, recognition, binding, and nicking of the AAV DNA replication origin, DNA helicase activity, and regulation of transcription from AAV (or other heterologous) promoters. The capsid (Cap) expression product supplies the necessary packaging function. AAV helper functions are used herein to complement in trans AAV functions missing from the AAV vector genome.

[0313] For production, cells with AAV helper functions produce sufficient recombinant capsid proteins to form capsids, including at least the 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 proteins determines the serotype of the AAV virions produced by the host cells. Capsids useful in the present invention include those derived from several 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 its disclosure of non-naturally occurring mixed serotypes). Capsid proteins can also be variants of native VP1, VP2, and VP3, including mutated, chimeric, or shuffled proteins. The capsid protein can be of AAV rh.10 or other subtypes within the various clades of AAV, the various clades and subtypes being disclosed, for example, in U.S. Patent No. 7,906,111. Due to the wide range of construct availability and extensive characterization, the exemplary AAV vectors disclosed below are 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.

[0314] In various embodiments, the nucleotide sequence encoding the VP protein can be operably linked to a suitable expression control sequence. In various embodiments, the nucleotide sequence encoding the Rep protein can be operably linked to a suitable expression control sequence, such as a eukaryotic promoter. For example, the nucleotide sequence can be operably linked to a eukaryotic promoter such as the 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 regulatory system (CuR / CuO) (see US2004 / 0205834), temperature-inducible HSP70 promoter, p5 promoter, p10 promoter, p19 promoter, and p40 promoter. In another example, the nucleotide sequence may be operably linked to a baculovirus 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.

[0315] Regarding production, cells with AAV helper functions produce Rep proteins to facilitate rAAV production. It has been found that infectious particles can be produced when at least one large Rep protein (Rep78 or Rep68) and at least one small Rep protein (Rep52 and Rep40) are expressed in cells. In certain embodiments, all four of Rep78, Rep68, Rep52, and Rep40 are expressed. Alternatively, Rep78 and Rep52, Rep78 and Rep40, Rep68 and Rep52, or Rep68 and Rep40 are expressed. The examples below demonstrate the use of the Rep78 / Rep52 combination. Rep proteins can be derived from AAV-2 or other serotypes. In various embodiments, the nucleotide sequence encoding the Rep proteins can be operably linked to suitable expression control sequences. In various embodiments, the nucleotide sequence encoding the Rep protein can be operably linked to a suitable expression control sequence, such as a eukaryotic promoter. For example, the nucleotide sequence can be operably linked to a eukaryotic promoter such as the 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 regulatory system (CuR / CuO) (see US 2004 / 0205834), and temperature-inducible HSP70 promoter, p5 promoter, p10 promoter, p19 promoter, and p40 promoter. In another example, the nucleotide sequence can be operably linked to a baculovirus 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.

[0316] In some embodiments, the AAV cap gene is present on the plasmid or bacmid. The plasmid may further contain an AAV rep gene, which may or may not correspond to the same serotype as the cap gene. The cap gene and / or rep gene from any AAV serotype.

[0317] Cells with AAV helper functions can also produce assembly-activating proteins (AAPs), which help capsids assemble. In various embodiments, the nucleotide sequence encoding the AAP can be operably linked to a suitable expression control sequence. For example, the nucleotide sequence can be operably linked to a eukaryotic promoter. In another example, the nucleotide sequence can be operably linked to a baculovirus 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.

[0318] The term "non-AAV helper functions" refers to non-AAV-derived viral and / or cellular functions on which AAV depends for its replication. Thus, this term captures proteins and RNAs required for AAV replication, including those involved in activation of AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, Cap expression product synthesis, and AAV capsid assembly. Viral-based helper functions can be derived from any of the known helper viruses, such as adenovirus, herpesvirus (other than herpes simplex virus type 1), and vaccinia virus.

[0319] The term "non-AAV helper function vector" generally refers to a nucleic acid molecule containing a nucleotide sequence that provides an accessory function. The accessory function vector can be transfected into a suitable host cell, and the vector can then support AAV virion production in the host cell. Infectious virus particles, such as adenovirus, herpesvirus, or vaccinia virus particles, are explicitly excluded from this term because they exist in nature. Therefore, accessory function vectors can be in the form of plasmids, phages, transposons, or cosmids. In particular, it has been demonstrated that a complete complement of adenovirus genes is not required for accessory helper function. For example, it has been shown that adenovirus mutants incapable of DNA replication and the synthesis of late genes are permissive for AAV replication. (Ito et al., (1970) J. Gen. Virol. 9:243; Ishibashi et al., (1971) Virology 45:317). Similarly, mutations in the E2B and E3 regions have been shown to support AAV replication, indicating that the E2B and E3 regions are probably not 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 cannot support AAV replication. Therefore, the E1A and E4 regions are likely required, either 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) (see above), Janik et al. (1981) (see above), 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 These include E1B (Parvoviruses (P. Tijssen ed., 1990)), E3 (Carter et al. (1983) (see above)), and E4 (Carter et al. (1983) (see above), Carter (1995)). Studies of accessory functions provided by adenoviruses with mutations in the E1B coding region have yielded conflicting results, although Samulski et al. (1988) J. Virol. 62:206-210 recently reported that E1B55k is required for AAV virion production, whereas E1B19k is not. Furthermore, International Publication WO 97 / 17458 and Matshushita et al. (1998) Gene Therapy 5:938-945 describe accessory function vectors encoding various Ad genes. A particularly preferred accessory function vector comprises an adenovirus VA RNA coding region, an adenovirus E4 ORF6 coding region, an adenovirus E2A 72 kD coding region, an adenovirus E1A coding region, and an adenovirus E1B region lacking an intact E1B55k coding region. Such vectors are described in International Publication No. WO 01 / 83797.

[0320] In another embodiment, the methods provided herein are practiced using any mammalian cell type that allows for AAV replication or biological product 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.

[0321] The use of insect cells for the expression of heterologous proteins is well documented, as are methods for introducing nucleic acids, such as vectors, e.g., insect cell-compatible vectors, into such cells and methods for maintaining such cells in culture (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. 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. (1991) vol. 88, pp. 4646-4650; Ruffing et al., J. Vir. (1991) vol. 88, pp. 4646-4650). (See, e.g., Kirnbauer 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. 382-393; and U.S. Patent No. 6,204,059.) In some embodiments, the nucleic acid construct encoding AAV in insect cells is an insect cell-compatible vector. An "expression vector" refers to a vector containing a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), artificial chromosomes, and viruses that incorporate 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 insect cell-compatible.The vector can be integrated into the insect cell genome, but the presence of the vector in the insect cell need not be permanent; transient episomal vectors are also included. The vector can be introduced by any known means, for example, by chemical treatment, electroporation, or infection of the cells. In some embodiments, the vector is a baculovirus, a viral vector, or a plasmid. In a more preferred embodiment, the vector is a baculovirus, i.e., the construct is a baculoviral vector. Baculoviral vectors and methods for their use are described in the references cited above regarding molecular engineering of insect cells.

[0322] For example, the insect cell line used can be a Spodoptera frugiperda derived cell line, e.g., SF9, SF21, SF900+, a Drosophila cell line, a mosquito cell line, e.g., an Aedes albopictus derived cell line, a silkworm cell line, e.g., a Bombyx mori cell line, a Trichoplusia ni cell line, e.g., High Five cells, or a Lepidoptera cell line, e.g., an Ascalapha odorata cell line. In one embodiment, the insect cell is a cell from an insect species susceptible to baculovirus 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.

[0323] Baculoviruses are enveloped arthropod DNA viruses, two of which are well-known expression vectors for producing recombinant proteins in cell culture. Baculoviruses have circular, double-stranded genomes (80-200 kbp) that can be engineered to allow delivery of large genome contents to specific cells. Viruses used as vectors are generally Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) or Bombyx mori nuclear polyhedrosis virus (BmNPV) (Kato et al., Appl. Microbiol. Biotechnol. 85(3):459-70 (2010)).

[0324] Baculoviruses are commonly used to infect insect cells for the expression of recombinant proteins. In particular, the expression of heterologous genes in insects has been described, for example, in U.S. Pat. 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. et al., Gene, 58:273-81 (1987). Numerous baculovirus strains and variants and corresponding permissive insect host cells that can be used for protein production 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).

[0325] Baculovirus shuttle vectors, or bacmids, are used to generate baculovirus. Bacmids propagate as large plasmids in bacteria, such as Escherichia coli. When transfected into insect cells, bacmids generate baculovirus. In another embodiment, the methods provided herein are performed using any mammalian cell type that allows AAV replication or biological product 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.

[0326] rAAV particles can also be produced using the methods disclosed in various embodiments. In some cases, rAAV particles can be produced by using insect or mammalian cells that stably express some of the components necessary for rAAV particle production. For example, a plasmid (or multiple plasmids) containing 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 multiple plasmids) containing a selectable marker such as a neomycin resistance gene can be integrated into the genome of the cell. Insect, fungal, or mammalian cells can then be co-infected with a helper virus (e.g., an adenovirus or baculovirus providing 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 the cells are selectable and suitable for large-scale production of rAAV. As another non-limiting example, adenovirus or baculovirus, rather than plasmids, can be used to introduce host regulatory genes, rep genes, and cap genes into packaging cells.

[0327] In one embodiment, following propagation of transfected cells in suspension cell culture through a series of increasingly larger culture platforms, the suspension of transfected cells is purified via a multi-step process to remove process impurities, including recombinant baculovirus and host cells, and to enrich for virions containing the recombinant parvovirus (rAAV) vector construct. In another embodiment, the methods provided herein may include an affinity purification step 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 antibody for use herein is a single-chain camelid antibody or fragment thereof, such as those obtainable from camels or llamas (see, e.g., Muyldermans, Biotechnol. 74:277-302 (2001)). Antibodies for affinity purification of rAAV are antibodies that specifically bind to an epitope on an AAV capsid protein, such that in one embodiment, the epitope is an epitope present on the capsid protein of more than one AAV serotype. For example, an antibody may be produced or selected based on specific binding to an AAV5 capsid, but at the same time, it may also specifically bind to an AAV1, AAV2, AAV3, AAV6, AAV8, or AAV9 capsid.

[0328] The methods provided herein for producing rAAV particles produce a population of AAV particles, which in some embodiments are enriched for particles containing full-length or near-full-length vector genomes by reducing the number of empty capsids.

[0329] The populations of rAAV particles produced by the methods provided herein are used, for example, for administration in any of the therapeutic methods described herein.

[0330] Host organisms and / or cells In further embodiments, a host cell containing the above-described vector is provided. In one embodiment, the vector construct is capable of replicating in the host cell or expressing the nucleic acid molecule provided herein. In some embodiments, provided herein is a therapeutic agent for HCM, which is a host cell containing a vector construct containing a nucleic acid encoding cMyBP-C for use in HCM cell therapy. The cells can be autologous or allogeneic to the subject.

[0331] As used herein, the term "host" refers to organisms and / or cells that harbor a nucleic acid molecule or vector construct of the present disclosure, as well as organisms and / or cells that are suitable for use in expressing a recombinant gene or protein. It is not intended that the present disclosure be limited to any particular type of cell or organism. Indeed, any suitable organism and / or cell is contemplated as being useful herein as a host. Host cells may be in the form of a single cell, a population of similar or different cells, e.g., a culture (such as a liquid culture or a culture on a solid substrate), an organism, or a portion thereof. In one embodiment, host cells are capable of permitting expression of the nucleic acid molecules provided herein. Thus, host cells may be, for example, bacterial, yeast, insect, or mammalian cells, or human cells.

[0332] In another embodiment, means are provided for delivering the nucleic acids provided herein into a wide range of cells, including dividing and non-dividing cells. Using the present disclosure, the nucleic acids provided herein may be delivered to cells in vitro, for example, to produce polypeptides encoded by such nucleic acid molecules for in vitro or ex vivo gene therapy.

[0333] The nucleic acid molecules, vector constructs, cells and methods / uses of the present disclosure are further useful in methods of delivering the nucleic acids provided herein into a host, typically a host suffering from HCM.

[0334] Pharmaceutical preparations In one embodiment, a pharmaceutical composition is provided comprising the nucleic acid or vector provided herein and a pharmaceutically acceptable diluent, excipient, or carrier. The pharmaceutical composition may further comprise a second therapeutic agent or an adjuvant. Preferably, the composition is sterile when intended for parenteral administration. Preferably, the composition is free of infectious viruses and toxins. Preferably, the composition is stable for a suitable period under storage conditions.

[0335] "Pharmaceutically acceptable" means a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject without causing any undesired biological effects. Thus, such pharmaceutical compositions may be used, for example, in transfection of cells ex vivo or in administering viral particles or cells directly to a subject.

[0336] 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 aqueous dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or injectable dispersions. The use of such media and agents for pharmaceutical active substances is well known in the art. Unless any conventional media or agent is incompatible with the active compound, its use in the pharmaceutical compositions provided herein is contemplated.

[0337] Another embodiment provided herein is a pharmaceutical composition (i.e., formulation) of AAV particles useful for administration to a subject suffering from a genetic disorder to deliver a gene encoding a protein of interest.In certain embodiments, the pharmaceutical formulation provided herein is a liquid formulation comprising recombinant AAV particles comprising any of the vector constructs disclosed herein.The concentration of recombinant AAV virions in the formulation may vary.

[0338] In other embodiments, the pharmaceutical formulations of AAV particles provided herein comprise one or more sterile, pharmaceutically acceptable excipients to provide the formulation with advantageous properties for storage and / or administration to a subject for treatment of a genetic disorder.

[0339] In certain embodiments, the formulation comprising the recombinant AAV particles further comprises one or more buffering agents.

[0340] In another embodiment, the recombinant AAV particle formulations provided herein may include one or more isotonicity agents, such as sodium chloride. Other buffers and isotonicity agents known in the art are suitable and may be routinely employed for use in the formulations provided herein.

[0341] 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, dextran, lactose, trehalose, and povidone (PVP K24).

[0342] In yet another embodiment, the recombinant AAV particle formulations provided herein may contain 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, etc., and combinations thereof.

[0343] The recombinant AAV particle formulations provided herein are typically sterile, stable, and capable of being stored for extended periods of time without unacceptable changes in quality, potency, or purity.

[0344] In some embodiments, isotonic agents, such as sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride, are included in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, such as monostearate salts and gelatin. In certain embodiments, the nucleic acid or vector constructs provided herein may be administered in sustained- or controlled-release formulations, including implants and microencapsulated delivery systems, in compositions containing, for example, slow-release polymers or other carriers that protect the compound against rapid release. For example, biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, and polylactic acid-polyglycolic acid copolymers (PLG) may be used.

[0345] In certain embodiments, pharmaceutical compositions comprising the vector constructs or AAV particles provided herein can be useful for transferring 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, the method comprising contacting a nucleic acid, vector construct, or pharmaceutical composition described herein under conditions such that the nucleic acid or vector provided herein enters the cell. The cell can be an in vitro, ex vivo, or in vivo cell.

[0346] Treatment method The vector construct or AAV particle described herein is administered to a mammalian subject in a dose effective to deliver the MYBPC3 gene to the subject's heart. The subject is preferably a human, including a young subject. A young subject can be, for example, between 0 and 2 years old, 2 and 6 years old, 2 and 10 years old, 2 and 12 years old, 2 and 15 years old, 2 and 18 years old, 12 and 18 years old, or 0 and 18 years old.

[0347] Such methods include a method of expressing cMyBP-C in the heart of a mammalian subject, the method comprising administering to the subject an effective amount of a composition comprising a vector construct described herein, an rAAV particle described herein, or a pharmaceutical composition described herein, thereby expressing cMyBP-C in the subject's cardiac tissue (e.g., myocardium or cardiomyocytes).

[0348] Such methods also include methods of treating a deficiency of functional wild-type myosin binding protein C in a mammalian subject by administering an amount of a vector construct, rAAV particle, or pharmaceutical composition effective to increase the level of functional myosin binding protein C in cardiac tissue (e.g., cardiomyocytes). In one or more embodiments, such methods increase the level of expression of cMyBP-C 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 level without treatment, or to levels seen in healthy humans. In some embodiments, the amount of vector construct, rAAV particle, or pharmaceutical composition is effective to increase the level of myosin binding protein C in cardiac tissue (e.g., cardiomyocytes) by at least about two-fold and / or is effective to restore contractile force, relative tension, calcium-activated tension, and relaxation time in engineered cardiac tissue in vitro or in animal tissue in vivo.

[0349] Such methods also include methods for treating HCM in a mammal, or treating or preventing any of its symptoms, comprising administering a therapeutically effective amount of a vector construct, rAAV particles, or pharmaceutical composition. The mammal may have a mutation in one or both alleles of the cMyBP-C gene. Such methods may, for example, reduce heart size, reduce cardiothoracic ratio, reduce end-diastolic or end-systolic left ventricular diameter, reduce anterior or posterior wall thickness, increase ejection time, increase aortic peak flow velocity or aortic flow time, 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, arrhythmia, chest pain, shortness of breath, fatigue, and dizziness.

[0350] In any of the methods described herein, the rAAV particles are delivered in an aqueous suspension at a dose of about 1e12 to about 6e14 vg / kg.

[0351] 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 treatment, and / or may further comprise administration of a second therapeutic agent for treating HCM, including, but not limited to, beta-blockers, calcium channel blockers, antiarrhythmic agents, and small molecule inhibitors of cardiac myosin.

[0352] In any of the methods herein, prior to administration of AAV particles to a patient as described above, the prospective patient may be evaluated for the presence of anti-AAV capsid antibodies or anti-AAV neutralizing antibodies that may block cellular transduction or otherwise reduce the overall efficacy of the treatment.

[0353] Detection of anti-AAV antibodies To maximize the likelihood of successful cardiac transduction by systemic AAV-mediated therapeutic gene transfer, prior to administration of AAV particles in a therapeutic regimen to a human patient as described above, the prospective patient may be evaluated for the presence of anti-AAV capsid antibodies or anti-AAV neutralizing antibodies that may block cellular transduction or otherwise reduce the overall efficiency of the therapeutic regimen. Such antibodies may be present in the serum of the prospective patient and may target AAV capsids of any serotype. In one embodiment, the serotype targeted by the pre-existing antibodies is AAV5.

[0354] Methods for detecting pre-existing AAV immunity are well known and routinely used in the art, including cell-based in vitro transduction inhibition (TI) assays, in vivo (e.g., in mice) TI assays, and ELISA-based detection of total anti-capsid antibodies (tAbs) (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 may use host cells pre-transfected with an AAV-inducible reporter vector. The reporter vector may contain an inducible reporter gene, such as GFP, whose expression is induced upon transduction of host cells by the AAV virus. Anti-AAV capsid antibodies, which are present in human serum and can prevent / reduce host cell transduction, will thereby reduce the overall expression of the reporter gene in the system. Therefore, such assays may be used to detect the presence of anti-AAV capsid antibodies in human serum that are capable of blocking / reducing cell transduction by therapeutic AAV particles.

[0355] An assay for detecting anti-AAV capsid antibodies may use a solid-phase-bound AAV capsid as a "capture agent" by passing human serum over it, allowing anti-capsid antibodies present in the serum to bind to the solid-phase-bound capsid "capture agent." After washing to remove nonspecific binding, a "detection agent" may be used to detect the anti-capsid antibodies bound to the capture agent. The detection agent may be an antibody, an AAV capsid, etc., and may be detectably labeled to aid in the detection and quantification of bound anti-capsid antibodies. In one embodiment, the detection agent is labeled with ruthenium or a ruthenium complex, which may be detected using electrochemiluminescence techniques and instruments.

[0356] The same techniques described above can be used to assess and detect the generation of anti-AAV capsid immune responses in patients previously treated with a therapeutic AAV virus of interest. Thus, these techniques can be used not only to assess the presence of anti-AAV capsid antibodies before treatment with a therapeutic AAV virus, but also to assess and measure the induction of an immune response to the administered therapeutic AAV virus after administration. Thus, a method is contemplated herein that combines a technique for detecting anti-AAV capsid antibodies in human serum with the administration of a therapeutic AAV virus for the treatment of HCM, where the technique for detecting anti-AAV capsid antibodies in human serum can be performed either before or after administration of the therapeutic AAV virus.

[0357] Other aspects and advantages of the present disclosure will be understood by consideration of the following illustrative examples. [Example]

[0358] Example 1: Production of AAV particles Figure 1 shows the organization of elements of vector constructs designated A1 to A8 and C1 to C5 (SEQ ID NOS: 3 to 41 or 92 to 169, respectively), which contain nucleic acids encoding human cMyBP-C. AAV particles containing the AAV9 capsid and vector constructs of SEQ ID NOS: 3 to 26 were produced in HEK293 cells and Sf9 cells. Vectors for AAV production were generated. For example, for AAV production in HEK293 cells, plasmids were generated. These plasmids contain nucleotide sequences that provide the AAV vector genome, encode Rep and capsid proteins, and provide non-helper AAV functions. These plasmids were transfected into HEK293 cells using a transfection reagent. After culturing HEK293 cells for the specified times following transfection, the produced rAAV particles were isolated from the culture, purified, and titered. For AAV production in Sf9 cells, bacmids were generated. These bacmids contain nucleotide sequences that provide the AAV vector genome and encode the Rep and capsid proteins. The bacmids were transfected into naive Sf9 cells using a transfection reagent. After culturing the transfected Sf9 cells for a predetermined time, recombinant baculovirus (rBV) was isolated, purified, and titered. To produce rAAV, separate naive cultures of Sf9 cells were infected with rBV at a predetermined multiplicity of infection (MOI). The Sf9 cells were cultured for a predetermined time post-infection. After the predetermined time, the produced rAAV particles were isolated from the culture, purified, and titered.

[0359] Example 2: Evaluation of the effects of AAV particles in engineered cardiac tissue. AAV9 particles prepared using the MYBPC3 vector constructs described herein were analyzed in 2D and 3D formats for their effects on human iPSC-derived cardiomyocytes. In the 2D format, exogenous protein and RNA content, as well as suppression of mutant MYBPC3 transcripts, were measured (see Example 5 below). In the 3D format, effects on contractile function, including beating frequency, contractile force, and kinetics, were also measured.

[0360] Using a patient-specific human induced pluripotent stem cell (hiPSC) line carrying a heterozygous MYBPC3 truncating mutation, two hiPSC lines were generated by CRISPR / Cas9 genome editing: 1) cpHet, carrying an additional homozygous MYBPC3 truncating mutation resulting in the complete absence of MYBPC3 protein, and 2) an isogenic control carrying two wild-type alleles resulting in normal levels of MYBPC3 protein. See Warnecke et al., Generation of bi-allelic MYBPC3 truncating mutant and isogenic control from an iPSC line of a patient with hypertrophic cardiomyopathy. Stem Cell Res. 55(2021):102489.

[0361] AAV9 particles containing the vector constructs described herein were tested in contact with human cardiomyocytes (CMs) derived from the cpHet hiPSC line and an isogenic control hiPSC line in 2D and engineered cardiac tissue (EHT) formats.

[0362] HiPSC-CMs were prepared by passaging cpHet or isogenic control hiPSC cells, dissociating the hiPSC cells, and enhancing cardiac differentiation using different media over a 14-day period. Differentiated cardiomyocytes were cultured in 2D monolayers or as 3D engineered heart tissue (EHT). EHT was prepared by embedding one million hiPSC-CMs in a fibrin matrix, as generally described in Breckwoldt K, et al., Differentiation of cardiomyocytes and generation of human engineered heart tissue. Nat Protoc 12, 1177-1197 (2017) and Hansen et al., Development of a drug screening platform based on engineered heart tissue. Circ. Res. 107.1 (2010):35-44.

[0363] Briefly, EHT slots with spacers were placed into a 24-well plate filled with 2% agarose (1.6 ml / well). Once the agarose solidified, the spacers were removed, and a silicone rack with a pair of posts was placed into each mold. Approximately 1 million hiPSC-CMs were used to cast each EHT in master medium containing horse serum, Y-27632 (Biorbyt, orb154626), fibrinogen, L-glutamine, DMEM, penicillin / streptomycin, and thrombin. The hiPSC-CMs and casting medium were then placed into the agarose slots. After 1.5 hours, the EHT solidified around the silicone rack, and was then transferred to a culture plate filled with medium. The EHTs were then maintained at 37°C, 7% CO2, 40% O2, and 98% relative humidity.

[0364] For AAV transduction, 2D HiPSC-CMs were replated and cultured as adherent cells at 220K cells per 24-well plate or 20K cells per 96-well plate. Approximately 4 days after replated, 2D cpHet hiPSC-CMs were transduced by adding AAV particles to the culture medium at a multiplicity of infection (MOI) of 300K. Approximately 2 weeks after generation, cpHet EHTs were transduced by adding AAV particles to the tissue culture medium at a multiplicity of infection (MOI) of 300K.

[0365] Human cMyBP-C protein expression was detected 7 or 14 days after transduction for 2D and 3D EHT formats, respectively. Briefly, 2D or EHT hiPSC-CMs were harvested and protein extracted for further analysis. Total protein lysates were used to measure exogenous protein levels by Western blot using a custom-made antibody against hMYBPC3. Alpha-actinin and / or cTnT protein levels were used as references. MYBPC3 was not detected in non-transduced cpHet hiPSC-CMs. After transduction with AAV9 particles, exogenous MYBPC3 protein was detected, and protein levels were expressed as fold-increase over isogenic controls. The results are shown in Figure 2, which shows that A2, A3, A4, and A6 achieved at least a 2.5-fold increase in expression, with A6 achieving the highest increase, and C1, C2, C4, and C5 achieved at least a 0.1-1.0-fold increase in expression, with C3 achieving the highest increase. Testing of additional AAV9 particles containing the vector constructs described herein, produced in HEK293 cells or Sf9-derived cells, resulted in even higher 5-12-fold changes compared to isogenic controls, as analyzed by Western blot.

[0366] In the 3D EHT format, the effects on spontaneous beating frequency and contractile function were evaluated after administration of the AAV9-MYBPC3 vector construct described herein. To assess contractile function, engineered cardiac tissue was stimulated to contract and relax. hiPSC-CMs in the EHT format exhibit intrinsic spontaneous contractility starting 1-2 weeks after generation. For spontaneous beating frequency as a functional endpoint, intrinsic spontaneous contractile frequency was analyzed without further stimulation.

[0367] Spontaneous beating activity was assessed in low-glucose DMEM medium containing 1.8 mM calcium and measured one week after transduction. Higher spontaneous beating frequencies were consistently observed in cpHet EHT compared with isogenic control EHT. The goal of gene therapy with AAV9 particles containing the vector constructs described herein was to reduce this abnormally high frequency. Frequency was analyzed over time in complete medium and one and two weeks after transduction in serum-free DMEM. Constructs A2, A3, A4, A5, and A6 resulted in reversion of the abnormal phenotype to a normal phenotype. Specifically, constructs A3, A4, and A6 showed the best and most consistent reduction in beating rate (BPM). In contrast, construct A5 showed only a slight effect on beating rate.

[0368] Contractile function was assessed at a fixed beat frequency by pacing using electrical stimulation. See Hirt et al., Functional improvement and maturation of rat and human engineered heart tissue by chronic electrical stimulation. J. Molec. Cell. Cardiol. 74 (2014): 151-161. One or two weeks after transduction, endpoints were assessed at a fixed frequency using short-term electrical pacing (1 Hz, 2.5 V) in medium containing 1.8 mM calcium, with baseline force as a confirmatory QC endpoint and contractile kinetics (relative late relaxation time, RT) as a functional endpoint. 20% and RT 80%The transduction procedure was optimized to provide stable force generation with no significant difference between non-transduced (NT) cpHet and vector-transduced EHT by adjusting the time points, transduction medium, and transduction chambers.

[0369] After activation, the contractile force of myocytes in the engineered cardiac tissue was measured. EHT contractile force and kinetics were monitored using an EHT testing system that combines semi-automated video optical analysis with shape recognition software. The force (mN) development over time (seconds) was calculated by the delta distance (post deflection) of a silicone post with a known elastic modulus (2.6 kPa) of Sylgard 184. To show the normalized average peak, the baseline was set to 0% and the peak force to 100% for each EHT. Non-transduced (NT) cpHet EHTs were compared and normalized to transduced cpHets and isogenic control EHTs. The results are shown in Figure 3.

[0370] Relaxation time and velocity were measured from peak sarcomere contraction to re-extension. Recorded contractions were identified by the peak criterion. Based on the identified contractions, values ​​for frequency, mean force, fractional shortening, contraction (time to peak), and relaxation time (RT) were calculated. The relaxation time percentage represents the time required from maximum post-deflection to baseline in percentage units; for example, RT20% represents the time (seconds) from maximum (100%) to 80% post-deflection. From the absolute RT, the relative late RT (percentage or %) was calculated as RT80%-RT50% / RT80%. The relative percentage of late relaxation time is shown in Figure 4A. The relaxation time (seconds) to 20% or 80% re-extension is shown in Figures 4B and 4C, respectively. cpHet exhibits a consistently higher relative late RT (RT80%-RT50% / RT80%) compared to isogenic controls. The goal of gene therapy is to normalize contractile kinetics, including by reducing abnormally high relative late RT.

[0371] AAV9 particles containing various MYBPC3 vector constructs produced in HEK293, Sf9, and Sf9-derived cells were evaluated for their effects on contractile function. cpHet EHT transduced with AAV9-MYBPC3 were compared with time-matched isogenic control EHT one week after transduction. Most of the vector constructs tested, such as A2, A3, A4, A5, A6, and C3, reversed the abnormally high relative late RT to near-normal phenotype. The effects of AAV9 particles produced in HEK293 cells on contractile function were superior to those produced in Sf9 cells. A3, A4, A5, and A6 had the greatest effect, completely reversing the abnormally high relative late RT.

[0372] Absolute relaxation time was also assessed. cpHet EHT was measured at 20% and 80% (RT 20% and RT 80% ) showed significantly shorter absolute relaxation times compared to isogenic controls. The vector constructs tested, e.g., A2, A3, A4, A5, A6, and C3, significantly prolonged the abnormally short RT. Treatment of cpHet EHT cardiomyocytes with AAV particles produced in HEK293 cells significantly prolonged the (RT 20% and RT 80% ) showed the most obvious recovery compared to non-transduced cpHet EHT cardiomyocytes. 20% and RT 80% Vector A6-transduced EHTs had a statistically significant effect on both RT and RT. 20% and longer RT 80% Both were shown.

[0373] Furthermore, the normalized mean contraction peaks of AAV-transduced cpHet EHT were analyzed and compared with those of cpHet and isogenic control EHT. All test vectors showed some effect, with A2, A3, A4, A5, A6, and C3 consistently showing the greatest effect. AAV9 particles containing vector construct A6 produced in HEK293 cells resulted in a complete normalization of contraction kinetics. cpHet EHT transduced with AAV9 particles containing vector constructs A3 and A6 produced in HEK293-derived cells improved relaxation kinetics toward the isogenic control. As shown in Figure 4D, the normalized force % of cells transduced with A3 and A6 produced in HEK293 cells (Group 3) was significantly greater than the normalized force % of cells transduced with A3 and A6 produced in insect cells (Group 4). Overall, AAV9 particles produced in mammalian cells showed greater activity in improving contractile function and contractile kinetics compared to AAV9 particles produced in insect cells.

[0374] Example 3: Evaluation of the effects of AAV particles in vivo Mice (n=10) were administered AAV particles prepared as in Example 1 at a dose of 2e14vg / kg and cardiac tissue was collected at 8 weeks.

[0375] The number of vector genomes encoding human cMyBP-C was assessed by ddPCR. The results (vector genomes per diploid gene number) are shown in Figure 5A. All of the AAV particles tested resulted in efficient delivery of at least one copy of the gene encoding human cMyBP-C per cell.

[0376] The number of human cMyBP-C mRNA transcripts was assessed by ddPCR. The results (mRNA transcripts per RPLP0 ribosomal protein transcript) are shown in Figure 5B. All of the AAV particles tested resulted in efficient translation into mRNA, with A2 and A4 producing the highest levels.

[0377] The amount of cMyBP-C protein was determined by liquid chromatography / mass spectrometry (LC / MS). The results (µg of human cMyBP-C in mouse hearts / gram of heart tissue and the percentage of human cMyBP-C in total cMyBP-C protein) are shown in Figure 5C.

[0378] Cardiac tissue was stained with antibodies specific for human cMyBP-C and ASG (α-sarcoglycan, a sarcolemmal marker). Intact cell nuclei were also stained with DAPI. cMyBP-C protein was widely detected throughout the majority of cardiomyocytes, with 77% and 65% of cardiomyocytes positive for cMyBP-C in preparations from mice administered A5, A6, and C3, respectively (see Figure 6).

[0379] Cardiac tissue was also stained with antibodies specific for human cMyBP-C and actin present in the sarcomere, and human cMyBP-C protein was observed to be localized to the sarcomere.

[0380] These results indicate that the A5 and A6 vector constructs effectively delivered human cMyBP-C protein to mice administered AAV particles containing these vector constructs, and that the human cMyBP-C protein was effectively incorporated into the majority of cardiomyocyte sarcomeres. The incorporation of functional human cMyBP-C protein is expected to improve contractility and reduce hypertrophic cardiomyopathy and its associated symptoms.

[0381] Example 4: Further evaluation of the effects of AAV particles in vivo To evaluate the correction of HCM phenotypes, including hypertrophy and cardiac dysfunction, various doses of rAAV particles containing the vector constructs described herein will be administered to MYBPC3 KO mice. Functional correction in these mice will be monitored throughout the study using echocardiography, and upon completion of the study, cardiac tissue will be used to evaluate transduction and expression of the vector constructs.

[0382] Example 5: Assessment of mutant MYBPC3 mRNA levels The effect of AAV9 particles containing the vector constructs described herein on mutant MYBPC3 mRNA levels was also evaluated in 2D hiPSC-CMs prepared as described in Example 2.

[0383] Semiquantitative RT-PCR was performed to monitor the effect on mutant MYBPC3 mRNA. RT-PCR using primers surrounding the MYBPC3 c.2308G>A mutation site revealed only one band at 912 bp corresponding to wild-type mRNA in isogenic control CM, and two additional mRNA bands of larger size compared to the wild-type band (i.e., 912 bp) in untransduced CpHet CM. The accumulation of these two aberrant MYBPC3 mRNAs due to the endogenous mutant gene was prevented by transduction with the AAV9-MYBPC3 vector constructs described herein, e.g., A1, A2, A3, A4, A5, and A6. C1, C4, and C5 had the least effect on the mutant mRNA band.

[0384] Cardiovascular safety and toxicology studies will be conducted in relevant models. A dose-response study will be performed to determine the % transduction / protein expression in MYBPC3- / - mice to inform dose selection.

[0385] The embodiments described herein are intended to be merely illustrative, and those skilled in the art will recognize, or 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 disclosure.

[0386] All patents, patent applications, and publications mentioned herein are hereby incorporated by reference in their entirety. Citation or identification of any reference in this application is not an admission that such reference is available as prior art to this application. The full scope of the present disclosure is better understood with reference to the appended claims.

Claims

1. (a) a nucleic acid encoding a functional human cardiac myosin binding protein C (cMyBP-C) comprising an amino acid sequence at least 95% identical to SEQ ID NO:2, or its complementary sequence; (b) a heterologous cardiomyocyte-specific transcriptional regulatory region comprising a fragment or variant of the hTNNT2 promoter, to which the nucleic acid is operably linked; (c) a polyadenylation signal, and (d) a recombinant vector construct comprising one or both of the 5' and 3' AAV inverted terminal repeat (ITR) sequences.

2. 2. The vector construct of claim 1, wherein the nucleic acid encoding a functional cMyBP-C amino acid sequence is at least 85% identical to any one of SEQ ID NOs: 1 or 42-45 or a complementary sequence thereof.

3. 3. The vector construct of claim 1, 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 or 42-45, or a complementary sequence thereof.

4. 2. The vector construct of claim 1, optionally comprising introns and / or exons or fragments or variants thereof.

5. 5. The vector construct of claim 4, wherein the intron comprises a nucleotide sequence that is at least 60% identical to SEQ ID NO: 53 or 58 or a complementary sequence thereof.

6. The cardiomyocyte-specific transcriptional regulatory region is (a) a cardiomyocyte-specific promoter comprising a nucleotide sequence at least 80% identical to any one of SEQ ID NOs: 47-52, or a fragment or complement thereof; and (b) an intron comprising a nucleotide sequence at least 60% identical to any one of SEQ ID NOs: 53, 56 or 58, or a complementary sequence thereof, wherein the intron is located 5' to the nucleic acid encoding functional human cMyBP-C.

7. 7. The vector construct of claim 6, comprising an exon, optionally a fragment of an HbB exon.

8. 8. The vector construct of claim 7, wherein the exon comprises the nucleotide sequence of SEQ ID NO: 54 or its complementary sequence.

9. 8. The vector construct of claim 7, comprising the nucleotide sequence of SEQ ID NO: 56 or its complementary sequence.

10. The vector construct of any one of claims 1 to 3, further comprising an intron.

11. 11. The vector construct of claim 10, wherein the intron comprises a nucleotide sequence at least 60% identical to SEQ ID NO: 53 or SEQ ID NO: 58, or a complementary sequence thereof.

12. The vector construct of claim 10, wherein the intron is located within the nucleic acid encoding functional human cMyBP-C.

13. 11. The vector construct of claim 10, wherein the intron is located between two exons of the nucleic acid encoding functional human MyBP-C.

14. 11. The vector construct of claim 10, wherein the intron is located between exon 2 and exon 3 of the nucleic acid encoding functional human cMyBP-C.

15. The vector construct of claim 10, wherein the intron is located at position 293 of SEQ ID NO: 1 or 42 to 45.

16. The vector construct of any one of claims 1 to 15, wherein the cardiomyocyte-specific transcriptional regulatory region comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 49 or its complementary sequence.

17. The vector construct of any one of claims 1 to 15, wherein the cardiomyocyte-specific transcriptional regulatory region comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 50 or its complementary sequence.

18. The vector construct of any one of claims 1 to 15, wherein the cardiomyocyte-specific transcriptional regulatory region comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 51 or its complementary sequence.

19. 2. The vector construct of claim 1, wherein the cardiomyocyte-specific transcriptional regulatory region comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 49 or its complementary sequence.

20. 2. The vector construct of claim 1, wherein the cardiomyocyte-specific transcriptional regulatory region comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 50 or its complementary sequence.

21. 2. The vector construct of claim 1, wherein the cardiomyocyte-specific transcriptional regulatory region comprises a nucleotide sequence that is more than 95% identical to SEQ ID NO: 51 or its complementary sequence.

22. 2. The vector construct of claim 1, wherein the cardiomyocyte-specific transcriptional regulatory region comprises a nucleotide sequence that is more than 95% identical to SEQ ID NO: 52 or its complementary sequence.

23. 2. The vector construct of claim 1, wherein the cardiomyocyte-specific transcriptional regulatory region comprises a nucleotide sequence that is at least 97% identical to SEQ ID NO: 49 or its complementary sequence.

24. 2. The vector construct of claim 1, wherein the cardiomyocyte-specific transcriptional regulatory region comprises a nucleotide sequence that is at least 97% identical to SEQ ID NO: 50 or its complementary sequence.

25. 2. The vector construct of claim 1, wherein the cardiomyocyte-specific transcriptional regulatory region comprises a nucleotide sequence that is at least 97% identical to SEQ ID NO: 51 or its complementary sequence.

26. 2. The vector construct of claim 1, wherein the cardiomyocyte-specific transcriptional regulatory region comprises a nucleotide sequence that is at least 97% identical to SEQ ID NO: 52 or its complementary sequence.

27. The vector construct of any one of claims 1 to 26, wherein the polyadenylation signal is a mini-polyadenylation signal, a growth hormone polyadenylation signal, an SV40 polyadenylation signal, or a fragment thereof.

28. 28. The vector construct of claim 27, wherein the polyadenylation signal comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 64 or its complementary sequence.

29. 28. The vector construct of claim 27, wherein the polyadenylation signal is a bovine growth hormone polyadenylation signal or a fragment thereof.

30. 30. The vector construct of claim 29, wherein the polyadenylation signal comprises a nucleotide sequence at least 90% identical to any of SEQ ID NOs: 59-61 or their complementary sequences.

31. 28. The vector construct of claim 27, wherein the polyadenylation signal is a human growth hormone polyadenylation signal or a fragment thereof.

32. 32. The vector construct of claim 31 , wherein the polyadenylation signal comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 62 or any fragment or complementary sequence thereof.

33. 33. The vector construct of any one of claims 1 to 32, wherein the rAAV vector construct is about 4 to about 5.5 kb in size.

34. The vector construct of any one of claims 1 to 32, wherein the AAV 5' ITR and / or the AAV 3' ITR is derived from AAV2.

35. 33. The vector construct of any one of claims 1 to 32, comprising a nucleotide sequence that is at least 97%, 98% or 99% identical to any of SEQ ID NOs: 3 to 41 or 92 to 169 or a complementary sequence thereof.

36. A rAAV particle comprising the vector construct of any one of claims 1 to 35 and an AAV capsid.

37. The rAAV particle of claim 36, wherein the AAV capsid has cardiac tropism.

38. The rAAV particle of claim 36, wherein the AAV capsid is an AAV type 9 capsid.

39. A method for producing rAAV particles according to any one of claims 36 to 38, comprising: (a) (i) a vector construct according to 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) providing a mammalian cell containing one or more nucleic acid constructs comprising a nucleotide sequence encoding one or more AAV capsid proteins operably linked to a promoter; (b) culturing the mammalian cells under conditions that promote expression of the Rep and the capsid protein; and (c) recovering the rAAV particles.

40. 40. The method of claim 39, wherein the mammalian cell is a HEK293 cell.

41. 1. A method for producing rAAV particles, comprising: (a) Into cells permissive for AAV replication, (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 cardiac myosin binding protein C; (ii) a nucleotide sequence encoding one or more AAV Rep protein(s) operably linked to a promoter capable of driving expression of said Rep protein(s) in said cell; and (iii) providing one or more nucleic acid constructs comprising a nucleotide sequence encoding one or more AAV capsid protein(s) operably linked to a promoter capable of driving expression of said capsid protein(s) in said cell; (b) culturing the cells under conditions permissive for expression of the Rep and the capsid proteins, and optionally (c) recovering the AAV particles.

42. 42. The method of claim 41, wherein the cell is an insect cell.

43. 42. The method of claim 41, wherein the cell is a mammalian cell.

44. The method of any of claims 41 to 43, wherein the cell is provided with a recombinant vector construct according to any of claims 1 to 35.

45. 45. A population of rAAV particles produced by the method of any one of claims 39 to 44, optionally enriched for particles comprising full-length or near-full-length vector genomes by reducing the number of empty capsids.

46. A pharmaceutical composition comprising a vector construct according to any one of claims 1 to 35, or an rAAV particle according to any one of claims 36 to 38, or a population of rAAV particles according to claim 45, in an aqueous suspension comprising a sterile, pharmaceutically acceptable excipient.

47. 46. ​​A method for delivering a human cardiac myosin binding protein C coding sequence, comprising administering to a patient having hypertrophic cardiomyopathy a vector construct described in any one of claims 1 to 35, or an rAAV particle described in any one of claims 36 to 38, or a population of rAAV particles described in claim 45, or a pharmaceutical composition described in claim 46.

48. A method for treating hypertrophic cardiomyopathy, comprising administering to a patient having hypertrophic cardiomyopathy a therapeutically effective amount of a vector construct described in any one of claims 1 to 35, or an rAAV particle described in any one of claims 36 to 38, or a population of rAAV particles described in claim 45, or a pharmaceutical composition described in claim 46.

49. 49. The method of claim 47 or 48, wherein the patient exhibits a mutation in one or both cMyBP-C alleles.

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