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 a subject suffering from hypertrophic cardiomyopathy.

Description

Technical Field

[0001] Provided herein are recombinant adeno-associated virus (rAAV) gene therapy vectors and viral particles that can be used to treat and prevent hypertrophic cardiomyopathy by increasing the expression of cardiac myosin binding protein C (cMyBP-C).

[0002] Cross-Reference to Related Applications

[0003] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 376,712, filed on September 22, 2022, and U.S. Provisional Patent Application No. 63 / 519,967, filed on August 16, 2023, each of which is incorporated herein by reference in its entirety.

[0004] Incorporation of Sequence Listing

[0005] This patent application includes a sequence listing in electronic format (filename: PCT_SeqListing.xml; created on September 6, 2023; 763,166 bytes) and is incorporated herein by reference in its entirety. Background of the Invention

[0006] Although significant progress has been made in preventing heart diseases caused by environmental factors such as nicotine, hypercholesterolemia, or diabetes and in treating heart disorders symptomatically, there is still a need to improve the treatment of genetic cardiomyopathies. Cardiomyopathies caused by genetic factors are hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and arrhythmogenic right ventricular cardiomyopathy (ARVC).

[0007] Hypertrophic cardiomyopathy is the most common genetic heart disease and is characterized by unexplained left ventricular hypertrophy. Hypertrophic cardiomyopathy is associated with initially normal systolic function but impaired diastolic function (Elliott et al., Eur. Heart J. 29: 270-6 (2008); Gersch et al., J. Thorac. Cardiovasc. Surg. 142: el53-203 (2011)). The incidence of hypertrophic cardiomyopathy in the general population is particularly high, approximately 1:500 (Maron et al., Circulation, 92: 785-9 (1995)), and it is the leading cause of sudden cardiac death in young people, especially athletes. Although HCM is a life-threatening disease, there is no curative treatment to date (Carrier et al., Cardiovasc. Res. 85: 330-338 (2010); Schlossarek et al., J. Mol. Cell. Cardiol. 50: 613-20 (2011)).

[0008] Hypertrophic cardiomyopathy (HCM) is a genetic disorder known to be caused by over 1,000 different mutations in at least 10 genes encoding cardiac sarcomere components, such as cardiac myosin-binding protein C (cMyBP-C), β-myosin heavy chain (MYH7), cardiac troponin T (TNNT2), cardiac troponin I (TNNI3), myosin ventricular essential light chain 1 (MYL3), myosin ventricular regulatory light chain 2 (MYL2), cardiac α-actin (ACTC), α-tropomyosin (TPM1), titin (TTN), four and a half LIM domains 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., Hum. Mol. Genet. 21: 3237-54 (2012)). Many mutations are missense mutations encoding full-length mutant polypeptides, while other frameshift or splice-site mutations can lead to truncation (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 mainly exhibit frameshift mutations leading to C-terminal truncated proteins.

[0009] 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 only in the heart (Fougerousse et al., Circ. Res. 82: 130-3 (1998)) and is involved in the regulation of myocardial 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 the mutations in the MYBPC3 gene result in frameshifts and produce C-terminal truncated proteins (Carrier et al., Circ. Res. 80: 427-34(1997)). The truncated proteins are unstable and have 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)).

[0010] Current drug-based HCM treatments relieve symptoms but do not address the underlying genetic cause of the disease. Gene- or RNA-based therapies would be the only curative treatments for HCM. Gene therapy approaches have been successfully tested in combination with non-genetic heart diseases (Jessup et al., Circulation, 124: 304-13 (2011)). SUMMARY OF THE INVENTION

[0011] The embodiments described herein relate to a vector construct, a recombinant replication-defective AAV particle, a cell, and a pharmaceutical composition for delivering cardiac myosin binding protein C (cMyBP-C) to a subject having HCM or a subject lacking a functional cardiac sarcomeric protein such as cMyBP-C. The 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 a subject.

[0012] The gene therapy vector is suitable for treating or preventing HCM in mammalian subjects, preferably human subjects, in need thereof. In some embodiments, the subject in need of treatment is a subject carrying a mutation in at least one or two 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 myocardium of the subject.

[0013] In one aspect, the 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 that is 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 that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2. In an exemplary embodiment, the nucleic acid sequence encoding the functional cardiac myosin binding protein C is a wild-type sequence, where SEQ ID NO: 1, 42 and 43 are examples, or is codon-optimized, or is a variant. Alternative codon-optimized or variant human cardiac myosin binding protein C coding sequences are shown in SEQ ID NO: 44-46. In some embodiments, the coding sequence of cardiac myosin binding protein C (cMyBP-C) is codon-optimized for expression in humans. In some embodiments, the nucleic acid sequence encoding the functional cMyBP-C is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 1 or 42-46.

[0014] The protein to be expressed can also be a functional variant that exhibits significant amino acid sequence identity compared to SEQ ID NO: 2 (i.e., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%). In this context, the term "functional variant" means a variant of the cMyBP-C protein that is capable of performing the functions of the naturally occurring cMyBP-C protein, such as providing structural and / or functional support to the sarcomere to restore normal myocardial contractility, and optionally being capable of inhibiting the expression and / or reducing the level of the mutant cMyBP-C protein or other mutant sarcomeric proteins.

[0015] Functional variants of the cMyBP-C protein can include, for example, proteins that differ from their naturally occurring counterparts by one or more amino acid substitutions, deletions, or additions. For example, a variant protein of the human cMyBP-C protein of SEQ ID NO: 2 can have an amino acid sequence in which at least 2, 3, 4, 5, 6, 10 or more and / or at most 10, 20, 30 or more positions are substituted by another amino acid relative to SEQ ID NO: 2. As another example, a variant protein of the human cMyBP-C protein of SEQ ID NO: 2 can be a truncated form of the human cMyBP-C protein. For example, functional variants can be selected from the group consisting of naturally occurring MYBPC3 splice variants lacking exons 5 and 6, designated variant 4 (as shown in SEQ ID NO: 46).

[0016] In one or more embodiments, a nucleic acid sequence encoding cMyBP-C can be operably linked to one or more heterologous expression control elements. Preferably, the expression of the transgene encoding cMyBP-C 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; an optional intron; an optional exon; and a polyadenylation (polyA) signal. Such elements will be further described herein.

[0017] The cardiomyocyte-specific transcriptional regulatory region can contain one or more cardiomyocyte-specific expression control elements, such as a cardiomyocyte-specific promoter. Preferably, the cardiomyocyte-specific promoter contains at least one fragment or variant of the human cardiac troponin T (hTNNT2) promoter.

[0018] In some embodiments, the cardiomyocyte-specific promoter comprises a nucleic acid sequence that is at least or more than 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 47 (over the full length of SEQ ID NO: 47). In some embodiments, the cardiomyocyte-specific promoter can be combined with an intron located 5' of the cMyBP-C coding sequence, which enhances the expression of the cMyBP-C protein. For example, the vector construct and / or AAV particle comprises, in a 5' to 3' orientation, a cardiomyocyte-specific promoter, an intron that enhances the expression of the cMyBP-C protein, and a nucleotide sequence encoding the cMyBP-C coding sequence. In some embodiments, the vector construct and / or AAV particle comprises (a) a cardiomyocyte-specific promoter that comprises a nucleotide sequence that is 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' of the cMyBP-C coding sequence that comprises a nucleotide sequence that is 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 that is at least 60% identical to SEQ ID NO: 56 or SEQ ID NO: 58.

[0019] In other embodiments, the cardiomyocyte-specific promoter can be combined with an intron located within the cMyBP-C coding sequence, which enhances the expression of the cMyBP-C protein. In some embodiments, the intron sequence is located within the nucleotide sequence encoding cMyBP-C, for example, between any exons, such as between exon 2 and exon 3. In some embodiments, the intron is located at position 293 of any one of SEQ ID NO: 1 or 42 - 45.

[0020] In some embodiments, the vector construct and / or the resulting AAV particle comprises a cardiomyocyte-specific promoter sequence that is a fragment or variant of the hTNNT2 promoter, which is greater than 420 and less than 544 nucleotides in length and comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 47. In any of the embodiments described herein, the cardiomyocyte-specific promoter optionally does not include any one of SEQ ID NOs: 1 to 85 of U.S. Patent Publication No. 2021 / 0252165.

[0021] For example, a cardiomyocyte-specific promoter sequence comprises a nucleic acid sequence that is at least or more than 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of (i) SEQ ID NO: 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 one 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.

[0022] In some embodiments, the vector construct comprises one or more introns that enhance the expression of the cMyBP-C-encoding nucleic acid, such that, for example, elevated 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 that is 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 that is 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 that is 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' of the cMyBP-C-encoding sequence. In some embodiments, the intron is located within the nucleotide sequence encoding cMyBP-C, for example, between any exons, such as between exon 2 and exon 3. In an exemplary embodiment, the intron is inserted at nucleotide position 293 of the MYBPC3 wild-type cDNA sequence of SEQ ID NO: 1. In other exemplary embodiments, the intron is inserted at nucleotide position 293 of the MYBPC3 wild-type cDNA sequence of any one of SEQ ID NOs: 42-45.

[0023] In some embodiments, the vector construct may further comprise an exon sequence or a fragment thereof, preferably adjacent to an intron sequence, such as the globin intron adjacent to the 3' end of a fragment of the β-globin exon 3 (SEQ ID NO: 54). The cardiomyocyte-specific transcriptional regulatory region may comprise a combination of intron and exon fragments, such as SEQ ID NO: 55. In some exemplary embodiments, the cardiomyocyte-specific transcriptional regulatory region comprises SEQ ID NO: 56.

[0024] In some embodiments, the vector construct comprises a polyadenylation signal, optionally the bovine growth hormone (BGH) polyA signal (such as SEQ ID NO: 59, 60 or 61) or a fragment thereof, optionally the human growth hormone (hGH) polyA signal (such as SEQ ID NO: 62) or a fragment thereof, optionally the SV40 polyA signal (such as SEQ ID NO: 63) or a fragment thereof, optionally the Proudfoot synthetic polyA signal (such as SEQ ID NO: 65) or a fragment thereof, or optionally the rabbit β-globin polyA signal (such as SEQ ID NO: 66) or a fragment thereof. In some embodiments, the polyA signal comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 64. In some embodiments, the polyA signal comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 59, such as comprising 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, having a length of about 100 to about 500 nucleotides, or a length of about 150 to about 400 nucleotides, or a length of about 200 to about 300 nucleotides, or a length of about 200 to about 250 nucleotides, and comprising SEQ ID NO 59.

[0025] In some embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98% or 99% identical to any one of SEQ ID NOs: 3-41. 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 or is the negative (-) strand of any one of SEQ ID NOs: 3-41 or 92-169.

[0026] In some embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98%, or 99% identical to any one of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 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 or the negative (-) strand of any one of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, or 39.

[0027] In some embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98%, or 99% identical to any one of SEQ ID NO: 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 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 or the negative (-) strand of any one of SEQ ID NO: 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, or 40.

[0028] In some embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98%, or 99% identical to any one of SEQ ID NO: 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 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 or the negative (-) strand of any one of 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, or 41.

[0029] Exemplary embodiments include the following:

[0030] Construct C1 is 4950 bp in length (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), mini poly A (57bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71). In other embodiments, construct C1 optionally contains any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 67-70 (or its complementary sequence) and / or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 71-74 (or its complementary sequence), or a fragment thereof.

[0031] Construct C2 is 4801 bp in length (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), mini poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71). In other embodiments, construct C2 optionally contains any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 67-70 (or its complementary sequence) and / or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 71-74 (or its complementary sequence), or a fragment thereof.

[0032] Construct C3 is 4801 bp in length (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), mini poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71). In other embodiments, construct C3 optionally contains any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 67-70 (or its complementary sequence) and / or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 71-74 (or its complementary sequence), or a fragment thereof.

[0033] Construct C4 is 4950 bp in length (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), mini poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71). In other embodiments, construct C4 optionally contains any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 67-70 (or its complementary sequence) and / or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 71-74 (or its complementary sequence), or a fragment thereof.

[0034] Construct C5 is 4950 bp in length (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), micro poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71). In other embodiments, construct C5 optionally contains any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 67-70 (or its complementary sequence) and / or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 71-74 (or its complementary sequence), or a fragment thereof.

[0035] Construct A1 is 5074 bp in length (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 poly A (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71). In other embodiments, construct A1 optionally contains any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 67-70 (or its complementary sequence) and / or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 71-74 (or its complementary sequence), or a fragment thereof.

[0036] Construct A2 is 4939 bp in length (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH poly A (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71). In other embodiments, construct A2 optionally contains any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 67-70 (or its complementary sequence) and / or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 71-74 (or its complementary sequence), or a fragment thereof.

[0037] Construct A3 is 4939 bp in length (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH poly A (169 bp) (SEQ ID NO: 59), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71). In other embodiments, construct A3 optionally contains any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 67-70 (or its complementary sequence) and / or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 71-74 (or its complementary sequence), or a fragment thereof.

[0038] Construct A4 is 4939 bp in length (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH poly A (202 bp) (SEQ ID NO: 60), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71). In other embodiments, construct A4 optionally contains any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 67 - 70 (or its complementary sequence) and / or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 71 - 74 (or its complementary sequence), or a fragment thereof.

[0039] Construct A5 is 4871 bp in length (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 poly A (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71). In other embodiments, construct A5 optionally contains any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 67 - 70 (or its complementary sequence) and / or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 71 - 74 (or its complementary sequence), or a fragment thereof.

[0040] Construct A6 is 5002 bp in length (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH poly A (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71). In other embodiments, construct A6 optionally contains any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 67-70 (or its complementary sequence) and / or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 71-74 (or its complementary sequence), or a fragment thereof.

[0041] Construct A7 is 4781 bp in length (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), Kozac sequence, wild-type hMYBPC3 (SEQ ID NO: 42) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, mini poly A (57 bp) (SEQ ID NO: 64), 3'-UTR sequence, and 3' AAV2-ITR (130 bp) (SEQ ID NO: 71). In other embodiments, construct A7 optionally contains any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 67-70 (or its complementary sequence) and / or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 71-74 (or its complementary sequence), or a fragment thereof.

[0042] Construct A8 is 4,844 bp in length (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), Kozac sequence, wild-type hMYBPC3 (SEQ ID NO: 42) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, mini poly A (57 bp) (SEQ ID NO: 64), 3'-UTR sequence, and 3' AAV2-ITR (130 bp) (SEQ ID NO: 71). In other embodiments, construct A8 optionally contains any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 67-70 (or its complementary sequence) and / or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 71-74 (or its complementary sequence), or a fragment thereof.

[0043] In any of the foregoing embodiments, the vector construct contains at least one ITR sequence. Exemplary ITR sequences include, but are not limited to, SEQ ID NOs: 67-74, including any complementary sequences and / or combinations thereof.

[0044] In any of the foregoing embodiments, the length of the vector insert that begins with one ITR and ends with a second ITR is between about 4 kb and about 5.5 kb. In one or more embodiments, the vector construct is an AAV vector genome, sized between about 4 kb and about 5.4 kb, between about 4.5 kb and about 5.5 kb, or between about 4.8 kb and about 5.2 kb, or between about 4.5 kb and about 5 kb.

[0045] The vector construct is preferably a recombinant AAV vector construct. In some embodiments, the vector construct comprises (a) (i) an AAV 5' inverted terminal repeat (ITR) and / or (ii) an AAV 3' ITR; (b) a promoter and / or enhancer, such as a cardiomyocyte-specific transcriptional regulatory region; and (c) a nucleic acid sequence encoding a functionally active human 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, such as 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 5' to the cMyBP-C coding sequence, while in other embodiments, the intron is between exons of the cMyBP-C coding sequence, such as between exon 2 and exon 3. In other embodiments, the vector construct comprises (a) an AAV'5' inverted terminal repeat (ITR) sequence; (b) a promoter and / or enhancer, such as a cardiomyocyte-specific transcriptional regulatory region; (c) a nucleic acid sequence encoding a functionally active human 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 the AAV 3' ITR may be 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).

[0046] 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 one of SEQ ID NOs: 3-42 and 92-169 over the lengths 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 one of SEQ ID NOs: 3-42 and 92-169 over the lengths of SEQ ID NOs: 3-42 and 92-169, respectively. In a specific example, the vector construct comprises a nucleotide sequence that is at least 85% identical to any one of SEQ ID NO: 29, 32 or 41 or at least 95% identical to any one of SEQ ID NO: 35 or 38. In other examples, the vector construct comprises a nucleotide sequence that is at least 90% identical to any one of SEQ ID NO: 29, 32 or 41 or at least 98% identical to any one of SEQ ID NO: 35 or 38. By way of example, such vectors preferably comprise nucleic acid sequences flanked by ITRs, encoding a functional active human cMyBP-C protein coding sequence, a cardiomyocyte-specific regulatory region, an intron and a polyA signal.

[0047] In another aspect, the present disclosure provides a recombinant adeno-associated virus (rAAV) particle comprising an AAV capsid and a vector construct as described in one or more of the embodiments herein. Any AAV capsid can be used, such as AAV1-13. In some embodiments, the recombinant AAV (rAAV) particle for delivering the cMyBP-C encoding gene has cardiac tropism. In such embodiments, the rAAV comprises an AAV capsid with cardiac tropism, such as an AAV9 capsid that is at least 85%, 90% or 95% identical to SEQ ID NO: 75, or an AAV1, AAV6 or AAV7 capsid that exhibits cardiac tropism, or a variant of any of these. In one or more embodiments, the AAV capsid is a capsid with reduced pre-existing humoral immunity compared to AAV9, such as when evaluated by in vitro IVIG neutralization.

[0048] In another aspect, provided herein are methods for generating AAV particles that can be used as gene delivery vectors, the methods comprising the steps of: (1) providing to a cell (e.g., a mammalian cell) one or more nucleic acid constructs, the one or more nucleic acid constructs comprising (a) a vector construct as described herein, the vector construct comprising a nucleic acid encoding cMyBP-C as described herein, the nucleic acid 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 the expression of the one or more Rep proteins; (c) a nucleotide sequence encoding one or more AAV capsid proteins, operably linked to a promoter capable of driving the expression of the one or more capsid proteins; and (d) optionally, a gene encoding the AAP and MAAP contained in VP2 / 3; (2) culturing the cell as defined in (1) under conditions conducive to the expression of the Rep proteins and the capsid proteins; and optionally (3) recovering the AAV particles. In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is a HEK293 cell. Also provided herein is a population of rAAV particles produced by such methods.

[0049] In another aspect, provided herein are pharmaceutical compositions comprising a vector construct as described herein or an rAAV particle or population of rAAV particles as described herein, and a sterile pharmaceutically acceptable diluent, excipient, or carrier.

[0050] In another aspect, the present disclosure provides 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 mammalian is a human and the myosin-binding protein C is a functional human myosin-binding protein C as described herein. Such methods include a method for expressing myosin-binding protein C in a cardiomyocyte of a mammalian subject, the method comprising administering an amount of a vector construct, an rAAV particle, or a pharmaceutical composition effective to increase the expression level of myosin-binding protein C in the mammalian myocardium. Such methods further include a method for increasing the level of functional myosin-binding protein C in a heart tissue (e.g., cardiomyocyte) of a mammalian subject, the method comprising administering an amount of a vector construct, an rAAV particle, or a pharmaceutical composition effective to increase the level of functional myosin-binding protein C in the heart tissue (e.g., cardiomyocyte) of the mammalian subject. Such methods further include a method for treating a functional wild-type myosin-binding protein C deficiency in a mammalian subject, the method comprising administering an amount of a vector construct, an rAAV particle, or a pharmaceutical composition effective to increase the level of functional myosin-binding protein C in the heart tissue (e.g., cardiomyocyte) of the mammalian subject. In some embodiments, the amount of the vector construct, rAAV particle, or pharmaceutical composition is effective to increase the level of myosin-binding protein C in the heart tissue (e.g., cardiomyocyte) by at least about 2-fold; and / or restore the contractility, relative tension, calcium-activated tension, relaxation time of an engineered heart tissue in vitro or an animal tissue in vivo.

[0051] Such methods further include a method for treating HCM in a mammalian subject or treating or preventing any symptoms thereof, the method comprising administering a therapeutically effective amount of a vector construct, an rAAV particle, or a pharmaceutical composition. In one or more embodiments, such methods increase the cMyBP-C expression level in the heart by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% compared to the untreated level, or increase to the level seen in healthy humans. Such methods, for example, reduce heart size, reduce the cardiothoracic ratio, reduce the end-diastolic or end-systolic left ventricular diameter, reduce the anterior or posterior wall thickness, increase the ejection time, increase the aortic peak velocity or aortic flow time, and / or reduce disease symptoms. 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.

[0052] In any of the methods described herein, the rAAV particles are delivered in an aqueous suspension at a dose of about 1e12 to 6e14 vg / kg. In any of the methods described herein, the administration of the vector construct, rAAV particles, or pharmaceutical composition may further include the administration of prophylactic or therapeutic corticosteroid treatment, and / or may further include the administration of a second therapy for the treatment of HCM. In any of the methods herein, prior to administering the AAV particles to a patient as described above, it may be evaluated whether the prospective patient has anti-AAV capsid antibodies or anti-AAV neutralizing antibodies that can block cellular transduction or otherwise reduce the overall efficiency of the treatment.

[0053] Other embodiments will be apparent to those skilled in the art upon reading this specification. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Depicts tissues containing elements of AAV particles comprising the vectors designated as C1-C5 and A1-A6 herein.

[0055] Figure 2 Depicts the fold change in cMyBP-C protein detected by Western blot in whole cell lysates of engineered heart tissue of the vectors designated as C1-C5 and A1-A6 herein.

[0056] Figure 3 Depicts the normalized contractile force of cardiomyocytes in engineered heart tissue treated with AAV particles comprising the vectors designated as C2, C3, and A2-A6 herein.

[0057] Figures 4A - 4C Depicts the relaxation time of cardiomyocytes after contraction in engineered heart tissue treated with AAV particles comprising the vectors designated as C2, C3, and A1-A6 herein. Figure 4A Shows the relative percentage of delayed relaxation time. Figure 4B Shows the time to reach 20% relaxation, in seconds, Figure 4C Shows the time to reach 80% relaxation, in seconds, and Figure 4D Depicts the force % normalization of constructs A3 and A6 produced in HEK293 cells (Group 3) and insect cells (Group 4).

[0058] Figures 5A - 5C Respectively depicts the DNA copy number (vector genome), RNA copy number, and cMyBP-C protein (μg / g heart tissue) in mice administered AAV particles comprising the vectors designated as C1-C5 and A1-A6 herein.

[0059] Figure 6Depict the percentage of cardiomyocytes expressing human cMyBP-C in the heart tissue of mice administered with AAV particles comprising the vectors herein designated as C3, A5, and A6. Detailed Description

[0060] The present disclosure provides nucleic acids or vector constructs encoding a functionally active therapeutic cMyBP-C protein, AAV vector genomes comprising such vector constructs, and replication-deficient rAAV particles, and pharmaceutical compositions comprising such vector constructs, vector genomes, and AAV particles. The compositions and methods of the invention can provide increased AAV virus production and / or simplified purification and / or enhanced expression of the cMyBP-C protein in cells of the heart, particularly cardiomyocytes (cardiomyocytes). Also provided herein are methods of making vector constructs, AAV vector genomes comprising such vector constructs, and replication-deficient rAAV particles. Further provided herein are methods of treating functional wild-type cMyBP-C deficiency (including HCM).

[0061] In another embodiment, provided is a method of generating recombinant adeno-associated virus (AAV) particles comprising any one of the AAV vector constructs provided herein. The method comprises the steps of culturing cells transfected with any one of the AAV vector constructs provided herein (associated with various AAV cap and rep genes) and recovering recombinant therapeutic AAV particles from the supernatant of the transfected cells or transfected cell culture.

[0062] Cells that can be used for the production of recombinant AAV 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.

[0063] In another embodiment, the present disclosure provides for 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 functional wild-type cMyBP-C protein deficiency. In one embodiment, the subject suffering from HCM is human. In one embodiment, the medicament is administered by intravenous (IV) administration. In another embodiment, the administration of the medicament results in an increase in the level of functional cMyBP-C in cardiomyocytes, thereby improving HCM symptoms. In certain embodiments, the medicament is also used in co-administration with a prophylactic and / or therapeutic corticosteroid to prevent and / or treat any toxicity associated with the administration of the AAV particle. 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, the prophylactic or therapeutic corticosteroid may be administered for a continuous period of at least about 3, 4, 5, 6, 7, 8, 9, 10 weeks or longer.

[0064] In another embodiment, the hypertrophic cardiomyopathy therapy provided herein optionally further comprises administration, such as co-administration, of other therapies for treating HCM.

[0065] Definition:

[0066] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd ed., J. Wiley and Sons (New York, N.Y. 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, N.Y. 1989). For the purposes of this disclosure, the following terms are defined below.

[0067] As used herein, in the context of gene delivery, the term "vector" or "gene delivery vector" can refer to a particle that serves as a gene delivery vehicle and contains a nucleic acid (i.e., a vector genome containing any of the vector constructs described herein) packaged within, for example, an envelope or capsid. A gene delivery vector can be a viral gene delivery vector or a non-viral gene delivery vector. Alternatively, in some instances, 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. A parvovirus can be an adeno-associated virus (AAV).

[0068] 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 multiple characterized AAV serotypes. General information and reviews of AAV can be found, for example, in Carter, Handbook of Parvoviruses, Volume 1, pages 169 - 228 (1989); and Berns, Virology, pages 1743 - 64, Raven Press, (New York) (1990); Gao et al., Meth. Mol. Biol. 807: 93 - 118 (2011); Ojala et al., Mol. Ther. 26(1): 304 - 19(2018). However, it is fully anticipated that these same principles will apply to additional AAV serotypes, as it is well known that the various serotypes are extremely closely related structurally and functionally, even at the genetic level. (See, for example, Blacklowe, 1988, pages 165 - 174, Parvoviruses and Human Disease, edited by J. R. Pattison; and Rose, Comprehensive Virology 3:1 - 61 (1974)). For example, all AAV serotypes clearly exhibit extremely similar replication properties mediated by homologous rep genes; and all carry three related capsid proteins. The degree of relatedness is further determined by non-complementary duplex analysis, which reveals extensive cross-hybridization along the genome length between serotypes; and the presence of similar self-annealing segments at the termini corresponding to the "inverted terminal repeats" (ITRs).

[0069] As used herein, an "AAV vector construct" refers to a single-stranded or double-stranded nucleic acid that has (i) an AAV 5' inverted terminal repeat (ITR) sequence and (ii) at least one of the AAV 3' ITRs, flanking a protein-coding sequence (in one embodiment, a functional therapeutic protein-coding sequence, such as a cMyBP-C coding sequence), the protein-coding sequence being operably linked to a transcriptional regulatory element (also referred to as an "expression control element") that is heterologous to the protein-coding sequence and / or heterologous to the AAV viral genome, i.e., one or more promoters and / or enhancers and optionally a polyadenylation sequence and / or optionally one or more introns. A single-stranded AAV vector refers to a nucleic acid that is present in the genome of an AAV viral particle and can be the sense or antisense strand of the nucleic acid sequences disclosed herein. The size of such single-stranded nucleic acids is provided in bases. A double-stranded AAV vector refers to a nucleic acid for expressing or transferring an AAV vector nucleic acid that is present in the DNA of a plasmid (e.g., pUC19) or the genome of a double-stranded virus (e.g., baculovirus). The size of such double-stranded nucleic acids is provided in base pairs (bp).

[0070] The length of the AAV vector construct in single-stranded form provided herein is less than about 7.0 kb, or less than 6.5 kb, or less than 6.4 kb, or less than 6.3 kb, or less than 6.2 kb, or less than 6.0 kb, or less than 5.8 kb, or less than 5.6 kb, or less than 5.5 kb, or less than 5.4 kb, or less than 5.3 kb, or less than 5.2 kb. The length of the AAV vector construct in single-stranded form is also at least about 4.0 kb. Preferably, the length of the AAV vector construct is also at least about 4.5 kb. In some embodiments, the length of the AAV vector construct in single-stranded form provided herein ranges from about 4.0 kb to about 5.8 kb.

[0071] Although it has been reported in the literature that AAV particles have an AAV genome of > 5.0 kb, in many of these cases, the 5' or 3' end of the encoded gene appears to be truncated (see Hirsch et al., Molec. Ther. 18: 6-8 (2010) and Ghosh et al., Biotech. Genet. Engin. Rev. 24: 165-78 (2007)). However, it has been shown that overlapping homologous recombination occurs between nucleic acids with a truncated 5' end and nucleic acids with a truncated 3' end in cells infected with AAV, resulting in a "complete" nucleic acid encoding a large protein and thus reconstructing a functional full-length gene.

[0072] The oversized AAV vector is randomly truncated at the 5' end and lacks the 5' AAV ITR. Since AAV is a single-stranded DNA virus and packages either the sense or antisense strand, the sense strand in the oversized AAV vector lacks the 5' AAV ITR and possibly part of the 5' end of the target protein-encoding gene, and the antisense strand in the oversized AAV vector lacks the 3' ITR and possibly part of the 3' end of the target protein-encoding gene. The functional transgene is generated in cells infected with the oversized AAV vector by annealing of the sense and antisense truncated genomes within the target cell. Thus, in certain embodiments, the AAV cMyBP-C vector and / or viral particles comprise at least one ITR.

[0073] As used herein, the term "inverted terminal repeat (ITR)" refers to the region recognized in the art found at the 5' and 3' termini of the AAV genome that functions in cis as an origin of DNA replication and a packaging signal for the viral genome. The AAV ITR, together with the AAV rep coding region, enables efficient excision and rescue of the nucleotide sequence flanked by two ITRs and integration of the nucleotide sequence into the host cell genome. The sequences of certain AAV-related ITRs are disclosed in Yan et al., J. Virol. 79: 364-79 (2005), which is incorporated herein by reference in its entirety. The ITR sequences useful herein may be full-length, wild-type AAV ITRs or fragments thereof that retain functional capacity, or may be sequence variants of full-length, wild-type AAV ITRs that are capable of functioning in cis as an origin of replication. The AAV ITRs of the recombinant AAV cMyBP-C vectors useful in the embodiments provided herein may be derived from any known AAV serotype, and in certain embodiments, from AAV2 or AAV5 serotypes.

[0074] The term "control sequence" refers to the DNA sequences required for expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for prokaryotes include, for example, a promoter, an optional operator sequence, and a ribosome binding site. It is known that eukaryotic cells utilize promoters, polyadenylation signals, and enhancers.

[0075] "Transcription regulatory element" refers to the nucleotide sequence of a gene that participates in the regulation of gene transcription, including a promoter, plus response elements, activator elements, and enhancer sequences, which are used to bind transcription factors to assist RNA polymerase binding and promote expression; and operon or silencer sequences, where inhibitory proteins bind to the sequences to block RNA polymerase attachment and prevent expression. The terms "cardiomyocyte-specific transcription regulatory element" or "cardiomyocyte-specific expression control element" refer to regulatory elements or regions that specifically produce preferred gene expression in cardiomyocytes, such as a promoter that is at least 2-fold or at least 5-fold more active in cardiac cells than in any other non-cardiac cell type. In some embodiments, the cardiomyocyte-specific promoter provides at least 5-fold higher expression in cardiomyocytes than in skeletal muscle cells. In some embodiments, the cardiomyocyte-specific promoter is at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, or at least 50-fold more active in cardiomyocytes than in non-cardiac cell types.

[0076] A heart-specific or cardiomyocyte-specific promoter is operably linked to a nucleic acid sequence encoding the cMyBP-C protein, which means that the promoter is combined with the encoding nucleic acid so that when integrated into the genome of a cell or present as an episomal nucleic acid construct in a cell, the encoding nucleic acid can be expressed in cardiomyocytes under the control of the promoter.

[0077] The transcription regulatory element optionally includes enhancer elements, introns, polyadenylation sequences, or post-transcriptional regulatory elements for increasing the expression level of myosin binding protein. Examples include the enhancer of the SV40 early gene and the enhancer of the long terminal repeat (LTR) of Rous Sarcoma Virus (Gorman et al. (1982) Proc. Natl. Acad. Sci. 79:6777). The vector also optionally contains a transcription termination sequence and a polyadenylation sequence for improving the expression of human and / or non-human antigens. 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 element known in the art that supports the efficiency or specificity of expression can be added to the expression vector, such as the woodchuck hepatitis post-transcriptional regulatory element (wPRE). To increase heart or cardiomyocyte specificity, other elements can be introduced to inactivate gene expression in other tissues, such as a sequence encoding a miRNA (such as miR122) (Geisler et al., Gene Ther. 18: 199-209 (2011).

[0078] As used herein, an "intron" is broadly defined as a nucleotide sequence that can be removed by RNA splicing. "RNA splicing" means the excision of introns from pre-mRNA to form mature mRNA. Introns can be located upstream, downstream, or within the coding region of a gene. Insertion of an intron into a nucleotide sequence can be achieved by any method known in the art. The only limitation on the location of the inserted intron is consideration of the packaging limit of AAV viral particles (e.g., about 5 kb).

[0079] As used herein, the term "operably linked" is used to describe the linkage between a regulatory element and a gene or its coding region. Generally, gene expression is under the control of one or more regulatory elements, such as, but not limited to, constitutive or inducible promoters, tissue-specific regulatory elements, and enhancers. A gene or coding region is said to be "operably linked to" or "operatively linked to" or "operably associated with" a regulatory element, meaning that the gene or coding region is controlled or affected by the regulatory element. For example, if a promoter affects the transcription or expression of a coding sequence, it is operably linked to the coding sequence.

[0080] In certain embodiments, the recombinant AAV vector construct comprises (a) a nucleic acid comprising the 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).

[0081] In one embodiment, the vector construct comprises a nucleic acid encoding a functionally active cMyBP-C protein. The cMyBP-C coding sequence can be wild-type, codon-optimized, or a variant. To visualize the expression of a foreign gene in the heart, other optional elements can be introduced as part of the cMyBP-C coding sequence, such as tag sequences (myc, FLAG, HA, His, etc.) or fluorescent dyes such as GFP, YFP, RFP.

[0082] As used herein, wild-type cardiac myosin-binding protein C (MYBPC3 gene) has the following nucleic acid sequence SEQ ID NO: 1 (GenBank accession number NM_000256.2)

[0083]

[0084] As used herein, wild-type cardiac myosin binding protein C has the following amino acid sequence SEQ ID NO:2 (GenBank accession number NP_000247.1)

[0085]

[0086] As used in connection with the nucleic acid molecules of the present disclosure, the term "isolated" generally refers to a nucleic acid sequence that has been identified and separated from at least one contaminating nucleic acid normally associated with its natural source. An isolated nucleic acid can exist in a form or environment different from that in which it is found in nature. Thus, an isolated nucleic acid molecule is distinct from a nucleic acid molecule that exists in a natural cell.

[0087] As used herein, the term "variant" refers to a polynucleotide (or polypeptide) having a sequence that is substantially similar to a reference polynucleotide (or polypeptide). Procedures for introducing nucleotide and amino acid changes into 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 can have one or more nucleotide deletions, substitutions, or additions at the 5'-end, 3'-end, and / or 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 obtained synthetically, such as, for example, polynucleotides generated by site-directed mutagenesis. Generally, as determined by sequence alignment programs known to those of skill in the art, a variant of a polynucleotide (including, but not limited to, DNA) can 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 greater sequence identity to the reference polynucleotide. In the case of polypeptides, a variant can have one or more amino acid deletions, substitutions, or additions compared to the reference polypeptide. Sequence similarities and / or differences between a variant and a reference polypeptide can be detected using conventional techniques known in the art, such as Western blot. Generally, as determined by sequence alignment programs known to those of skill in the art, a variant of a polypeptide can 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 higher sequence identity to the reference polypeptide.

[0088] The amino acid substitutions can be conservative or non-conservative. Preferably, the substitutions are conservative substitutions, i.e., the amino acid residue is substituted with an amino acid having a similar polarity that serves as a functional equivalent. Preferably, the amino acid residue used as the substitute is selected from the same group of amino acids as the amino acid residue to be substituted. For example, a hydrophobic residue can be substituted with another hydrophobic residue, or a polar residue can be substituted with another polar residue having the same charge. Functionally homologous amino acids useful for conservative substitution include, for example, non-polar amino acids such as glycine, valine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, and tryptophan. Examples of uncharged polar amino acids include serine, threonine, glutamine, asparagine, tyrosine, and cysteine. Examples of charged polar (basic) amino acids include histidine, arginine, and lysine. Examples of charged polar (acidic) amino acids include aspartic acid and glutamic acid.

[0089] 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 can be present within the amino acid sequence of the native cMyBP-C protein (i.e., as an insertion), or they can be added to one or both termini of the protein. Such insertions, substitutions, or deletions can occur at any position, provided that they do not impair the ability of the polypeptide to perform the functions of the naturally occurring cMyBP-C protein and / or rescue haploinsufficiency in the treated subject. In addition, variants of the cMyBP-C protein also include proteins that lack one or more amino acids compared to the original polypeptide. Such deletions can affect any amino acid position, provided that they do not impair the ability to perform the normal functions of the cMyBP-C protein and / or rescue haploinsufficiency.

[0090] Finally, variants of the cardiac cMyBP-C protein also refer to proteins that differ from the naturally occurring protein by structural modifications, such as modified amino acids. Modified amino acids are amino acids that have been modified by natural processes, such as processing or post-translational modification, or by chemical modification methods known in the art. Typical amino acid modifications include phosphorylation, glycosylation, acetylation, O-linked N-acetylglucosaminylation, glutathionylation, acylation, branching, ADP-ribosylation, cross-linking, disulfide bridge formation, formylation, hydroxylation, carboxylation, methylation, demethylation, amidation, cyclization, and / or covalent or non-covalent binding to phosphatidylinositol, flavin derivatives, lipoteichoic acid, fatty acids, or lipids. Such modifications have been widely described in the literature, for example, in Proteins: Structure and Molecular Properties, T. Creighton, 2nd ed., W. H. Freeman and Company, New York (1993). In a preferred embodiment of the present invention, the nucleic acid sequence encodes a constitutively phosphorylated isoform of human cMyBP-C. These isoforms have been shown to be particularly cardioprotective (Sadayappan et al. (2005), Circ Res 97:1156-1163; Sadadayappan et al., 2006; Proc Natl Acad Sci U S A 103:16918-16923).

[0091] The terms "identity," "homology," and their grammatical variations mean that two or more of the entities mentioned are the same when they are "aligned" sequences. Thus, for example, when two polypeptide sequences are the same, they have the same amino acid sequence at least within the region or portion mentioned. When two polynucleotide sequences are the same, they have the same polynucleotide sequence at least within the region or portion mentioned. Identity can be within a defined region (region or domain) of the sequence. A "region" or "portion" of identity refers to a part of two or more of the entities mentioned that are the same. Thus, when two polypeptide or nucleic acid sequences are the same in one or more sequence regions or portions, they have identity in that portion. An "aligned" sequence refers to a plurality of polynucleotide or polypeptide (amino acid) sequences that, compared to a reference sequence, typically contain corrections for missing or additional bases or amino acids (gaps). "Substantially homologous" means that a molecule is structurally or functionally conserved such that it has or is predicted to have at least some of the structure or function of a reference molecule or a related / corresponding region or portion of the reference molecule with which it shares homology (e.g., biological function or activity).

[0092] "Percent(%) nucleic acid sequence identity or homology" is defined as the percentage of nucleotides in a candidate sequence that are identical to a reference sequence after aligning the corresponding sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity. The alignment for the purpose of determining the percent nucleic acid sequence identity can be accomplished in various ways within the skill in the art, e.g., using publicly available computer software such as ALIGN or Megalign (DNASTAR) software. One of ordinary skill in the art can determine the appropriate parameters for measuring the alignment, including any algorithms required to achieve the maximum alignment over the full length of the sequences being compared.

[0093] "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 the amino acid residues in the cMyBP-C polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity and without considering any conservative substitutions as part of the sequence identity. The alignment for the purpose of determining the percent amino acid sequence identity can be accomplished in various ways within the skill in the art, e.g., using publicly available computer software such as ALIGN or Megalign (DNASTAR) software. One of ordinary skill in the art can determine the appropriate parameters for measuring the alignment, including any algorithms required to achieve the maximum alignment over the full length of the sequences being compared.

[0094] "Codon optimization" or "codon optimized" refers to changes made in a nucleotide sequence to make it more likely to be expressed at a relatively high level compared to an unoptimized sequence. It does not change the amino acid encoded by each codon.

[0095] "AAV viral particle" or "AAV virion" 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. If the particle contains a heterologous polynucleotide (i.e., a polynucleotide that is not the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is commonly referred to as a "recombinant AAV vector particle" or simply an "AAV vector". The production of an AAV vector particle necessarily includes the production of an AAV vector genome, and thus the vector genome is contained within the AAV vector particle. It should be understood that reference to the polynucleotide AAV vector construct encapsulated within the vector particle and its replication refers to the AAV vector genome.

[0096] As used herein, "therapeutic AAV virus" refers to an AAV virion, AAV viral particle, AAV vector particle, or AAV virus that contains 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 that contains one or more polynucleotides (also referred to as transgenes) encoding a target protein, the polynucleotides flanked by at least one AAV inverted terminal repeat (ITR) and operably linked to one or more expression control elements. Such AAV vector constructs can replicate and package into infectious virus particles when present in a host cell that has been transfected with a vector encoding and expressing the rep and cap gene products. The term generally refers to recombinant AAV that is capable of infecting a cell such that the infected cell expresses (e.g., by transcription and / or by translation) a target element (e.g., nucleotide sequence, protein, etc.). To this extent, therapeutically effective rAAV particles can include AAV particles having capsids or vector genomes (vgs) with different characteristics. For example, therapeutically effective rAAV particles can have capsids carrying different post-translational modifications. In other instances, therapeutically effective AAV particles can contain vector genomes having different sizes / lengths, sense or antisense sequences, different sense / antisense, antisense / sense, sense / sense, antisense / antisense, etc. configurations, different numbers of ITRs (1, 2, 3, etc.), or truncated vector genomes. For example, overlapping homologous recombination occurs between a nucleic acid truncated at the 5' end and a nucleic acid truncated at the 3' end in a cell infected with rAAV, thereby generating a "complete" nucleic acid encoding a large protein and thus reconstructing a functional full-length gene. In other instances, complementary nucleic acid sequences truncated at the 5' end and truncated at the 3' end interact with each other, thereby forming a "complete" nucleic acid during second-strand synthesis. The "complete" nucleic acid encodes a large protein and thus reconstructs a functional full-length gene. Therapeutically effective rAAV particles are also referred to as re-capsids, full-capsids, or partial full-capsids. In contrast, a "non-therapeutic" AAV virus refers to an empty capsid, i.e., a capsid having a non-quantifiable or undetectable vector genome or a vector genome that cannot be recombined into a complete functional nucleic acid.

[0097] As used herein, "therapeutic protein" refers to a polypeptide having biological activity that replaces or compensates for the loss or reduction of endogenous protein activity. For example, a functional cMyBP-C protein is a therapeutic protein for HCM.

[0098] As used herein, "hypertrophic cardiomyopathy" refers to a genetic disease caused by mutations in genes encoding cardiac sarcomere components such as cardiac myosin binding protein C, and is characterized by symptoms such as heart failure, arrhythmia, chest pain, shortness of breath, fatigue, and dizziness, an increase in heart size, an increase in the cardiothoracic ratio, an increase in the end-diastolic left ventricular diameter, an increase in the end-systolic left ventricular diameter, an increase in the wall thickness of the ventricle (anterior or posterior or both), a decrease in ejection time, a decrease in aortic peak velocity, and / or a decrease in aortic blood flow time.

[0099] As used herein, "cardiac myosin binding protein C deficiency" or "functional wild-type cardiac myosin binding protein C deficiency" refers to a genetic disorder caused by a decrease in the level of functional cMyBP-C protein, which is attributed to the absence of the protein, reduced protein production, or production of a non-functional protein. This includes HCM.

[0100] As used herein, "effective in treating hypertrophic cardiomyopathy" or "hypertrophic cardiomyopathy therapy" refers to any therapeutic intervention in a subject with HCM that improves the characteristic deficiency of functional wild-type cMyBP-C, increases the level of cMyBP-C protein (e.g., in the myocardium), improves HCM symptoms, or reduces the frequency, duration, or severity of HCM symptoms.

[0101] As used herein, "hypertrophic cardiomyopathy gene therapy" refers to any therapeutic intervention in a subject with HCM that involves replacing, restoring, or increasing cMyBP-C by delivering one or more nucleic acid molecules to cells in the subject that express functional cMyBP. In certain embodiments, MYBPC3 gene therapy refers to gene therapy involving adeno-associated virus (AAV) particles that contain a vector construct expressing human cMyBP-C. In other embodiments, the gene therapy involves transfection of a plasmid expressing human cMyBP-C.

[0102] As used herein, "treat" or "treatment" refers to prophylactic or therapeutic treatment, which is treatment administered to a subject showing signs or symptoms of a pathology (i.e., HCM) with the aim of reducing or eliminating those signs or symptoms or improving their progression, severity, or duration. The signs or symptoms can be biochemical, cellular, histological, functional, subjective, or objective.

[0103] As used herein, "improve" refers to an action that reduces the severity, progression, or duration of the symptoms of a disease.

[0104] As used herein, "stably treating" or "stable treatment" refers to the use of a therapeutic vector construct, AAV particle, or cell administered to a subject, wherein the subject stably expresses a therapeutic protein expressed by the vector construct, AAV particle, or cell. Stably expressed therapeutic protein means that the protein is expressed for a clinically significant length of time. As used herein, "clinically significant length of time" means a length of time during which expression at a therapeutically effective level has a meaningful impact on the quality of life of the subject, such as demonstrated by a reduction in the signs or symptoms of the disease. In certain embodiments, the clinically significant length of time is at least six months, at least eight months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, at least ten years, or the lifetime of the subject.

[0105] As used herein, the term "effective amount" refers to an amount sufficient to achieve a beneficial or desired biological and / or clinical result.

[0106] As used herein, "subject" refers to an animal that is the object of treatment, observation, or experiment. "Animal" includes cold-blooded and warm-blooded vertebrates and invertebrates, such as fish, shellfish, reptiles, and particularly mammals. As used herein, the term "avian" includes, but is not limited to, chickens, ducks, geese, quails, turkeys, and pheasants. As used herein, "mammal" 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 pet animals. 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 particularly humans. In some embodiments, the mammal is a human, including an infant, child, or adolescent, such as a human up to 2, 2 - 4, 2 - 6, or 2 - 12 years of age.

[0107] Generally, a "pharmaceutically acceptable carrier" is a carrier that is non-toxic or not overly harmful to cells and is preferably sterile. Exemplary pharmaceutically acceptable carriers include sterile, pyrogen-free water and sterile, pyrogen-free saline or phosphate-buffered saline. Pharmaceutically acceptable carriers include physiologically acceptable carriers. The term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, etc., that are physiologically compatible.

[0108] Vector Constructs and AAV Vectors

[0109] The recombinant vector constructs of the present disclosure can be used as gene therapies themselves or can be used to generate rAAV particles by the methods described herein, which include providing the recombinant vector construct and the Rep and Cap genes to a suitable host cell. The vector constructs described herein contain a nucleic acid sequence encoding functional cMyBP-C. The recombinant vector construct can contain a nucleic acid encoding functional human cMyBP-C, which is operably linked to heterologous expression control elements such as a promoter and / or enhancer; an optional intron; and an optional polyadenylation (polyA) signal. The heterologous expression control element can be a heterologous cardiomyocyte-specific transcriptional regulatory region, such as those described herein.

[0110] When used to generate rAAV particles, the recombinant vector construct can contain (a) (i) an AAV 5' inverted terminal repeat (ITR) sequence and (ii) one or both of the AAV 3' ITRs, (b) a heterologous cardiomyocyte-specific transcriptional regulatory region, and (c) a nucleic acid encoding functional human cMyBP-C, optionally wherein the AAV ITR is an AAV2 ITR. Preferably, the nucleic acid encoding functional cMyBP-C is operably linked to a cardiomyocyte-specific expression control element. The vector construct can include additional expression control elements such as: a promoter and / or enhancer; an intron; an optional exon or fragment thereof; and a polyadenylation (polyA) signal. Such elements will be further described herein. In certain embodiments, the recombinant AAV vector construct contains a nucleic acid that includes (a) an AAV2 5' inverted terminal repeat sequence (ITR) (which can or cannot 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) an optional exon or fragment thereof, a polyadenylation sequence, and (f) an AAV2 3' ITR (which can or cannot be modified as known in the art).

[0111] Preferably, the rAAV particles further contain an AAV capsid with cardiac tropism, optionally an AAV9 capsid. Exemplary capsids with cardiac tropism include AAV1, 6, 7, and 9.

[0112] Other embodiments provided herein relate to vector constructs encoding functional cMyBP-C polypeptides, wherein the constructs contain one or more of the individual elements of the above constructs in one or more different orientations and combinations thereof. Another embodiment provided herein is the above construct in the opposite orientation.

[0113] The length of the single-stranded AAV vector construct provided herein ranges from about 4.5 kb to about 6.5 kb, or from about 4.5 kb to about 5.5 kb, or from about 4 kb to about 5.5 kb, or from about 4.8 kb to about 5.2 kb, or from 4.8 kb to 5.1 kb, or from about 4.9 kb to about 5.5 kb, or from about 4.8 kb to about 6.0 kb, or from about 5.0 kb to 6.2 kb, or from about 5.1 kb to about 6.3 kb, or from about 5.2 kb to about 6.4 kb, or from about 5.5 kb to about 6.5 kb, or from about 4.0 kb to about 5.0 kb, or from about 4 to about 4.5 kb, or from about 4.5 kb to about 5 kb.

[0114] When the AAV vector is produced from an overly large recombinant vector construct, it may lack a portion of the 5' or 3' end of the recombinant vector construct. Since AAV is a single-stranded DNA virus and packages either the sense or antisense strand, the sense strand in the overly large AAV vector lacks the 5' AAV ITR and a possible portion of the 5' end of the target protein-coding gene, and the antisense strand in the overly large AAV vector lacks the 3' ITR and a possible portion of the 3' end of the target protein-coding gene. The functional transgene is generated in cells infected with the overly large AAV vector by annealing the sense and antisense truncated genomes within the target cells. Thus, in certain embodiments, the rAAV particles of the present invention can comprise a recombinant vector construct that comprises at least one ITR and a majority of the nucleotide sequence encoding functional cMyBP-C, such as a fragment of SEQ ID NO: 1 or 42 - 45, which exceeds 50%, 60%, 70%, 80%, or 90% of the length of the nucleotide sequence. For example, the recombinant vector construct can comprise at least one ITR, a cardiomyocyte-specific transcriptional regulatory region, and a majority of the nucleotide sequence encoding functional cMyBP-C.

[0115] The production of the vector construct can be achieved using any suitable genetic engineering techniques well-known in the art, including but not limited to standard techniques of restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing, such as those described in Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, N.Y. (1989)).

[0116] The vector construct may incorporate sequences from the genomes of any known organism. The sequences may be incorporated in their native form or may be modified in any way to obtain the desired activity. For example, the sequences may contain insertions, deletions or substitutions.

[0117] When present in a host cell transfected with a polynucleotide encoding and expressing the rep and cap gene products, the AAV vector construct can be replicated and packaged into infectious AAV particles, preferably replication-defective AAV particles.

[0118] Transcription regulatory element or region

[0119] Promoters and enhancers.

[0120] In one or more embodiments, a nucleic acid sequence encoding cMyBP-C may be operably linked to one or more heterologous expression control elements. Preferably, the expression control element is a cardiomyocyte-specific expression control element. Examples of cardiomyocyte-specific control elements include, but are not limited to, the human cardiac troponin T (hTNNT2) promoter or fragments or variants thereof. Other promoters that are active in cardiomyocytes include fragments or variants of any of the following: muscle creatine kinase (MCK) promoter, cytomegalovirus enhancer + myosin light chain 2 promoter (CMV-MLC2, or CMV-MLC1.5, CMV-MLC260), phosphoglycerate kinase (PGK) promoter, sarcomere-specific promoter, α-myosin heavy chain promoter, myosin light chain 2v promoter, α-myosin heavy chain promoter, α-cardiac actin promoter, α-tropomyosin promoter, cardiac troponin C promoter, cardiac troponin I promoter, cardiac myosin binding protein C promoter and / or sarcoplasmic / endoplasmic reticulum Ca2+ ATPase (SERCA) promoter (e.g., isoform 2 of this promoter (SERCA2)) and / or striated muscle promoter, such as the intermedin promoter. Enhancers derived from cardiomyocyte-specific transcription factor binding sites are also contemplated.

[0121] Examples of fragments or variants of the hTNNT2 promoter include cardiomyocyte-specific promoter sequences that comprise a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 47. In some embodiments, the cardiomyocyte-specific transcriptional regulatory region comprises a cardiomyocyte-specific promoter sequence that comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 52. In some embodiments, the cardiomyocyte-specific transcriptional regulatory region comprises a cardiomyocyte-specific promoter sequence that comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 51. In some embodiments, the cardiomyocyte-specific transcriptional regulatory region comprises a cardiomyocyte-specific promoter sequence that comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 50. In some embodiments, the cardiomyocyte-specific transcriptional regulatory region comprises a cardiomyocyte-specific promoter sequence that comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 49. In some embodiments, the cardiomyocyte-specific transcriptional regulatory region comprises a cardiomyocyte-specific promoter sequence that comprises a nucleic acid sequence that is at least or more than 80%, 85%, 90%, 95%, 97%, 98% or 99% identical (over the length of the SEQ ID NO) to any one of SEQ ID Nos: 49-52. In any of the embodiments described herein, the cardiomyocyte-specific promoter optionally does not include any one of SEQ ID Nos: 1 to 85 of US Patent Publication No. 2021 / 0252165. In some embodiments, the cardiomyocyte-specific transcriptional regulatory region further comprises an intron that enhances the expression of the cMyBP-C protein, and optionally an exon or a fragment thereof 5' of the cMyBP-C coding sequence. By way of example, the vector construct and AAV particle comprise, in a 5' to 3' orientation, a cardiomyocyte-specific promoter that comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 47; an intron nucleotide sequence that is at least 70% identical to SEQ ID NO: 53; and a nucleotide sequence encoding cMyBP-C.

[0122] In other embodiments, the cardiomyocyte-specific promoter comprises (a) a nucleic acid sequence that is at least 80% identical to any one 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 that is 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 that is 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.

[0123] In some embodiments, the vector construct comprises (a) a nucleic acid sequence that is at least 90% identical to any one 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 that is 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 that is 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.

[0124] In some embodiments, the cardiomyocyte-specific transcriptional regulatory region may further comprise (in addition to fragments or variants of the hTNNT2 promoter and the globin intron) exon sequences or fragments thereof, for example, the 3' end of the globin intron is adjacent to a fragment of the β-globin exon 3 (SEQ ID NO: 54). The combination of the intron and exon fragments is, for example, SEQ ID NO: 55. In some exemplary embodiments, the cardiomyocyte-specific transcriptional regulatory region comprises SEQ ID NO: 56.

[0125] In some embodiments, the fragment or variant of the hTNNT2 promoter is greater than 420 and less than 544 nucleotides in length and comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 47. In any of the embodiments described herein, the cardiomyocyte-specific promoter optionally does not include any one of SEQ ID NOs: 1 to 85 of U.S. Patent Publication No. 2021 / 0252165.

[0126] In some embodiments, the cardiomyocyte-specific promoter sequence comprises a nucleic acid sequence that is at least or more than 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of (i) SEQ ID NO: 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 or more than 95%, 97%, 98% or 99% identical to any one 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.

[0127] In the vector constructs disclosed herein, various promoters may be operably linked to a nucleic acid comprising the coding region of the target protein human cardiac myosin binding protein C. In some embodiments, the promoter may drive the expression of the target protein in cells (such as target cells) infected with a virus from a viral vector. The promoter may be naturally occurring or non-naturally occurring. In some embodiments, the promoter is a synthetic promoter. In one embodiment, a synthetic promoter comprises a sequence that does not exist in nature and is designed to regulate the activity of an operably linked gene. In another embodiment, a synthetic promoter comprises a fragment of a natural promoter to form a new DNA sequence segment that does not exist in nature. Synthetic promoters generally comprise regulatory elements, promoters, enhancers, introns, splice donors and acceptors, which are designed to produce enhanced tissue-specific expression. Examples of promoters include, but are not limited to, viral promoters, plant promoters, and mammalian promoters. In another embodiment, the promoter is a cardiomyocyte-specific promoter.

[0128] 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 higher sequence identity to any one of SEQ ID No: 49 - 51. In some embodiments, the promoter is at least about or more than 95% identical to any one of SEQ ID No: 49 - 51.

[0129] In some embodiments, the promoter construct comprises one or more additional individual enhancer elements in one or more different orientations.

[0130] In some embodiments, the promoter is operably linked to a polynucleotide encoding one or more target proteins. In some embodiments, the promoter is operably linked to a polynucleotide encoding the cMyBP - C protein.

[0131] The size of the promoter can vary. Due to the limited packaging capacity of AAV, it is preferred to use a promoter with a smaller size that at the same time allows for high - level production of the target protein in the host cell. For example, in some embodiments, the promoter is at most about 1.5 kb, at most about 1.4 kb, at most about 1.35 kb, at most about 1.3 kb, at most about 1.25 kb, at most about 1.2 kb, at most about 1.15 kb, at most about 1.1 kb, at most about 1.05 kb, at most about 1 kb, at most about 800 base pairs, at most about 600 base pairs, at most about 400 base pairs, at most about 200 base pairs or at most about 100 base pairs.

[0132] Other regulatory elements.

[0133] A variety of 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 that further increase the expression level of the target protein in the host cell. In some embodiments, the regulatory elements can help to maintain the recombinant DNA molecule extrachromosomally in the host cell and / or improve vector efficacy (e.g., scaffold / matrix attachment region (S / MAR)). Such regulatory elements are well - known in the art.

[0134] The vector constructs disclosed herein may include regulatory elements such as transcription initiation regions and / or transcription termination regions. 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, mini-polyA, human growth hormone (hGH) poly(A), bovine growth hormone (BGH) poly(A), SV40 late poly(A), rabbit β-globin (rBG) poly(A), thymidine kinase (TK) poly(A) sequence, Proudfoot polyA, 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 mini-polyA (e.g., SEQ ID NO: 64), bGH polyA (e.g., any one of SEQ ID No: 59-61), hGH polyA (e.g., SEQ ID NO: 62), SV40 polyA (e.g., SEQ ID NO: 53), Proudfoot synthetic polyA (e.g., SEQ ID NO: 65), or rabbit β-globin polyA (e.g., SEQ ID NO: 66) sequence or a fragment thereof having a length of about 40 to 200 nucleotides.

[0135] In some embodiments, the vector construct comprises a polyadenylation signal, optionally a bovine growth hormone (BGH) polyA signal (e.g., SEQ ID No: 59-61) or a human growth hormone (hGH) polyA signal (e.g., SEQ ID NO: 62) or a fragment thereof.

[0136] The length of the polyA signal may be about 150 to about 250 nucleotides, about 160 to about 240 nucleotides, about 170 to about 230 nucleotides, about 180 to about 220 nucleotides, or about 200 to about 210 nucleotides.

[0137] In some embodiments, the vector construct may include additional transcription and translation initiation sequences and / or additional transcription and translation termination sequences that are known in the art.

[0138] The target protein and the nucleic acid encoding the target protein.

[0139] As used herein, "target protein" is any functional cMyBP-C protein, including naturally occurring and non-naturally occurring variants thereof. In some embodiments, polynucleotides encoding one or more target cMy-BP-C proteins can be inserted into the viral vectors disclosed herein, wherein the polynucleotides are operably linked to a promoter. In some cases, the promoter can drive the expression of one or more target proteins in a host cell (e.g., human myocardium).

[0140] In one or more embodiments, the functional cMyBP-C comprises an amino acid sequence that is at least 90%, 95% or 98% identical to SEQ ID NO: 2 (human cardiac myosin binding protein C). The present disclosure also provides an isolated nucleic acid molecule encoding such a functional wild-type cMY-BP-C protein. 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% homology or at least 98% homology to the nucleotide sequence of SEQ ID NO: 1, or at least 100, 200, 300, 400 or 500 consecutive nucleotides of SEQ ID NO: 1 or 42-43. In an exemplary embodiment, the nucleotide sequence encoding the functional cardiac myosin binding protein C is codon-optimized or is a variant and can be at least 85%, 90%, 95%, 97%, 98% or 99% identical to any one of SEQ ID Nos: 44-46.

[0141] In certain embodiments, the nucleic acid molecule has at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homology or at least 98% homology to the nucleotide sequence of SEQ ID No: 1 or 42-46, or at least 100, 200, 300, 400 or 500 consecutive nucleotides of SEQ ID NO: 1 or 42-46.

[0142] In an exemplary embodiment, the nucleic acid sequence encoding functional cMyBP-C is a wild-type MYBPC3 sequence, of which SEQ ID NO: 1 is an example, or is codon-optimized, or is a variant. The vector constructs described herein may comprise a nucleotide sequence that is different 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. In accordance with this aspect, the nucleotide sequence may comprise 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, provided that 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 an exemplary embodiment, the nucleotide sequence may comprise a portion having at least 90% homology to at least 100, 200, 300, 400 or 500 contiguous bases of SEQ ID NO: 1, provided that the nucleotide sequence encodes a functional human cMyBP-C protein that is at least 90% identical to SEQ ID NO: 2. In an exemplary embodiment, the nucleotide sequence has substantial homology to the nucleotide sequence of SEQ ID NO: 1 or 42-46 and encodes functional cMyBP-C. The term substantial homology may be further defined with reference to the percentage of homology (%), for example at least 80%, 85%, 90% or 95% homology. This is discussed in further detail elsewhere herein.

[0143] In exemplary embodiments, the nucleotide sequence of the target gene is codon-optimized, preferably codon-optimized for more efficient expression in humans, or in a target organ, tissue, and / or cell of a human. The target organ, tissue, or cell includes heart tissue and / or cardiomyocytes. The adaptation of the nucleotide sequence encoding the gene therapy product to the codon usage of human cells can be represented by the codon adaptation index (CAI). The codon adaptation index is defined herein as a measure of the relative adaptation of the codon usage of a gene to the codon usage of highly expressed human genes. The relative adaptation (w) of each codon is the ratio of the usage of each codon to the usage of the most abundant codon for the same amino acid. The CAI is defined as the geometric mean of these relative adaptation values. Non-synonymous codons and stop codons (depending on the genetic code) are not included. CAI values range from 0 to 1, with higher values indicating a higher proportion of the most abundant codons (see Sharp and Li, 1987, Nucleic Acids Research 15: 1281-1295; also see: Kim et al., Gene. 1997, 199:293-301; zur Megede et al., Journal of Virology, 2000, 74: 2628-2635). In certain embodiments, the CAI of the target gene is at least 0.75, 0.80, 0.85, 0.90, 0.95, or 0.99.

[0144] 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, Volume 7, Article number: 285 (2006) or using the Operon / Eurofins Genomics codon optimization software or other codon optimization tools, such as Grote et al., “Jcat: a novel tool to adapt codon usage of a target gene to its potential expression host,” Nucleic Acids Res. 33:W526-31 (2005).

[0145] Alternatively or as an alternative to codon optimization, the nucleotide sequence of the target gene can be adjusted to reduce the CpG dinucleotide content and optionally remove any additional ORFs in the sense and antisense directions. The CpG dinucleotide content has been shown to activate TLR9 in dendritic cells, leading to potential immune activation and CTL responses. Reducing the CpG content can reduce liver inflammation and ALT. In some embodiments, the CpG dinucleotide content of the nucleotide sequence of the target gene is less than 25, less than 20, less than 15, or less than 10. In another embodiment, the GC content of the nucleotide sequence of the target gene is less than 65%, less than 60%, or less than 55%.

[0146] Generally, codon optimization or CpG reduction does not change the amino acid encoded by each codon. It only changes the nucleotide sequence to make it more likely to be expressed at a relatively high level compared to the non-optimized sequence.

[0147] As described herein, the nucleotide sequence encoding the cMyBP-C protein can be modified to improve the protein expression efficiency. The methods that can be used to improve the transcription and / or translation of the genes herein are not particularly limited. For example, the nucleotide sequence can be modified to better reflect the host codon usage to increase the gene expression (such as protein production) in the host (such as a mammal). As another non-limiting example of the modification, one or more of the splice donors and / or splice acceptors in the nucleotide sequence of the target protein are modified to reduce the likelihood of aberrant splicing. As another non-limiting example of the modification, one or more introns can be inserted within or near the nucleotide sequence of the target protein to optimize AAV vector packaging and enhance expression.

[0148] 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 that contains a sequence that varies relative to any of the wild-type amino acids, the protein should still be a functional protein. Those skilled in the art should understand that a small number of changes can be made to some of the amino acids of the protein without adversely affecting the function of the protein.

[0149] In certain embodiments, when the nucleic acid molecule is expressed in a suitable system (such as a host cell), it produces a functional cMyBP-C protein and at a relatively high level. Since the produced cMyBP-C is functional, it will have at least a portion of the same conformation as the wild-type cMyBP-C. In certain embodiments, the functional cMyBP-C protein produced as described herein effectively treats subjects suffering from wild-type cMyBP-C protein deficiency and / or HCM.

[0150] One of ordinary skill in the art is fully capable of generating the nucleic acid molecules provided herein. This can be done, for example, using chemical synthesis of a given sequence. In addition, suitable methods for determining whether the nucleic acids described herein express a functional protein will be apparent to one of ordinary skill in the art. By way of example, one suitable in vitro method involves inserting the nucleic acid into a vector such as an AAV vector, transducing a host cell such as 293T or HeLa cells with the vector, and assaying for cMyBP-C. Alternatively, a suitable in vivo method involves transducing a vector containing the nucleic acid into an HCM mouse and assaying for functional cMyBP-C.

[0151] Intron

[0152] In some embodiments, the vector contains one or more introns. Introns can aid in the processing of RNA transcripts in mammalian host cells, increase the expression of the target protein, and / or optimize the packaging of AAV particles into the vector. Non-limiting examples of such introns are the human β-globin intron, the human immunoglobulin G (IgG) intron, or the native cMyBP-C intron. In some embodiments, the intron is a synthetic intron.

[0153] In some embodiments, the vector construct and / or AAV particle contains a cardiomyocyte-specific promoter and one or more additional heterologous expression control elements, such as an intron that enhances the expression of the cMyBP-C protein. By way of example, the vector construct and / or AAV particle contains any of the cardiomyocyte-specific promoters described above, and optionally an intron nucleotide sequence located 5' of the nucleotide sequence encoding cMyBP-C. In other examples, the vector construct and / or AAV particle contains 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 exons). In some embodiments, the intron sequence is located between exons 2 and 3. In some embodiments, the intron sequence is located at position 293 corresponding to SEQ ID NO: 1 or positions 42 - 46 of the nucleic acid encoding cMyBP-C.

[0154] In one or more embodiments, the intron comprises a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 53, and the intron can be about 50 to about 150 nucleotides in length, or about 100 to about 135 nucleotides in length. In an exemplary embodiment, the intron comprises SEQ ID NO: 53 or a fragment thereof, the fragment being about 50 - 150 nucleotides, 75 - 145 nucleotides, 100 - 135 nucleotides, or 120 - 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 can comprise a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 53.

[0155] In one or more embodiments, the intron comprises a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 58, and the intron can be about 50 to about 150 nucleotides in length, or about 100 to about 135 nucleotides in length. In an exemplary embodiment, the intron comprises SEQ ID NO: 58 or a fragment thereof, the fragment being about 50 - 150 nucleotides, 75 - 145 nucleotides, 100 - 135 nucleotides, or 120 - 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 can comprise a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 58.

[0156] Other exemplary introns comprise a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs: 53 - 58.

[0157] 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 an intron sequence. In one exemplary embodiment, the vector construct comprises a globin intron adjacent to an exon, the exon comprising a nucleotide sequence that is at least 80% or 85% or 90% or 95% identical to SEQ ID NO: 54. In another exemplary embodiment, the vector construct comprises a globin intron adjacent to an exon sequence, the exon sequence comprising a nucleotide sequence that is at least 80% or 85% or 90% or 95% identical to SEQ ID NO: 53. In one exemplary embodiment, the vector construct comprises a globin intron adjacent to an HbB exon sequence, the exon sequence comprising a nucleotide sequence that is at least 80% or 85% or 90% identical to SEQ ID NO: 54.

[0158] The position and size of the intron in the vector can vary. In some embodiments, the intron is located between the promoter and the sequence encoding the target protein. In some embodiments, the intron is located downstream of the sequence encoding the target protein. In some embodiments, the intron is located within the promoter. In some embodiments, the intron includes enhancer elements. In some embodiments, the intron is located within the sequence encoding the target protein, preferably between the exons of the sequence encoding the target protein. In some embodiments, the intron may comprise all or a portion of a naturally occurring intron within the sequence encoding the target protein. 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.

[0159] Inclusion of an intron element can enhance expression compared to expression in the absence of the intron element (see, e.g., Kurachi et al., J. Biol. Chem. 270(10): 5276-81 (1995)). AAV vectors generally accept DNA inserts having a defined size range, which generally ranges from about 4 kb to about 5.4 kb, or slightly more. However, there is no minimum size for extremely efficient packaging and packaging of a smaller vector genome. Introns and intron fragments meet this requirement while also enhancing expression. Thus, the present disclosure is not limited to including the cMyBP-C intron sequence in an AAV vector and includes other introns or other DNA sequences in place of portions of the cMyBP-C intron. Additionally, other 5' and 3' nucleic acid untranslated regions can be used in place of those recited for human cMyBP-C.

[0160] In some embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98%, or 99% identical to any one of SEQ ID NOs: 3-41 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 or is the negative (-) strand of any one of SEQ ID NOs: 3-41 or 92-169.

[0161] In some embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98%, or 99% identical to any one of SEQ ID NOs: 3, 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 or is the negative (-) strand of any one of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, or 39.

[0162] In some embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98%, or 99% identical to any one of SEQ ID NOs: 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 or is the negative (-) strand of any one of SEQ ID NOs: 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, or 40.

[0163] In some embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98%, or 99% identical to any one of SEQ ID NOs: 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 or is the negative (-) strand of any one of SEQ ID NOs: 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, or 41.

[0164] Exemplary embodiments include the following: Construct C1 has a length of 4950 bp (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), micro poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0165] In some embodiments, construct C1 has a length of 4980 bp (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), micro poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 72).

[0166] In some embodiments, construct C1 has a length of 4950 bp (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), micro poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0167] In some embodiments, construct C1 is 4980 bp in length (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), mini poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 74).

[0168] In some embodiments, construct C1 is 4950 bp in length (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), mini poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0169] In some embodiments, construct C1 is 4980 bp in length (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), mini poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 74).

[0170] In some embodiments, construct C1 has a length of 4950 bp (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), mini poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 69).

[0171] In some embodiments, construct C1 has a length of 4980 bp (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), mini poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 72).

[0172] In some embodiments, construct C1 has a length of 4640 bp (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 poly A (57 bp) (SEQ ID NO: 64). In other embodiments, construct C1 optionally contains any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 67-70 (or its complementary sequence) and / or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 71-74 (or its complementary sequence), or a fragment thereof.

[0173] Construct C2 is 4801 bp in length (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), micro poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0174] In some embodiments, construct C2 is 4831 bp in length (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), micro poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 72).

[0175] In some embodiments, construct C2 is 4801 bp in length (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), micro poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0176] In some embodiments, construct C2 is 4831 bp in length (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), micro poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 74).

[0177] In some embodiments, construct C2 has a length of 4801 bp (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), mini poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0178] In some embodiments, construct C2 has a length of 4831 bp (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), mini poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 74).

[0179] In some embodiments, construct C2 has a length of 4801 bp (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), mini poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0180] In some embodiments, construct C2 has a length of 4831 bp (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), mini poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 72).

[0181] In some embodiments, construct C2 has a length of 4491 bp (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 a micro poly A (57 bp) (SEQ ID NO: 64). In other embodiments, construct C2 optionally contains any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 67-70 (or its complementary sequence) and / or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 71-74 (or its complementary sequence), or a fragment thereof.

[0182] Construct C3 has a length of 4801 bp (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), a micro poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0183] In some embodiments, construct C3 has a length of 4831 bp (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), a micro poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 72).

[0184] In some embodiments, construct C3 is 4801 bp in length (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), mini poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0185] In some embodiments, construct C3 is 4831 bp in length (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), mini poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 74).

[0186] In some embodiments, construct C3 is 4801 bp in length (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), mini poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0187] In some embodiments, construct C3 is 4831 bp in length (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), mini poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 74).

[0188] In some embodiments, construct C3 is 4801 bp in length (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), mini poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0189] In some embodiments, construct C3 is 4831 bp in length (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), mini poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 72).

[0190] In some embodiments, construct C3 is 4491 bp in length (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 mini poly A (57 bp) (SEQ ID NO: 64). In other embodiments, construct C3 optionally contains any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 67-70 (or its complementary sequence) and / or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 71-74 (or its complementary sequence), or a fragment thereof.

[0191] Construct C4 is 4950 bp in length (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), micro poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0192] In some embodiments, construct C4 is 4980 bp in length (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), micro poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 72).

[0193] In some embodiments, construct C4 is 4950 bp in length (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), micro poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0194] In some embodiments, construct C4 is 4980 bp in length (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), micro poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 74).

[0195] In some embodiments, construct C4 is 4950 bp in length (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), micro poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0196] In some embodiments, construct C4 is 4980 bp in length (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), micro poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 74).

[0197] In some embodiments, construct C4 has a length of 4950 bp (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), micro poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0198] In some embodiments, construct C4 has a length of 4980 bp (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), micro poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 72).

[0199] In some embodiments, construct C4 has a length of 4640 bp (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 micro poly A (57 bp) (SEQ ID NO: 64). In other embodiments, construct C4 optionally contains any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 67 - 70 (or its complementary sequence) and / or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 71 - 74 (or its complementary sequence), or a fragment thereof.

[0200] Construct C5 is 4950 bp in length (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), micro poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0201] In some embodiments, construct C5 is 4980 bp in length (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), micro poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 72).

[0202] In some embodiments, construct C5 is 4950 bp in length (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), micro poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0203] In some embodiments, construct C5 is 4980 bp in length (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), mini poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 74).

[0204] In some embodiments, construct C5 is 4950 bp in length (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), mini poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0205] In some embodiments, construct C5 is 4980 bp in length (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), mini poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 74).

[0206] In some embodiments, construct C5 is 4950 bp in length (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), micro poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0207] In some embodiments, construct C5 is 4980 bp in length (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), micro poly A (57 bp) (SEQ ID NO: 64), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 72).

[0208] In some embodiments, construct C5 is 4806 bp in length (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 micro poly A (57 bp) (SEQ ID NO: 64). In other embodiments, construct C5 optionally contains any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 67 - 70 (or its complementary sequence) and / or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 71 - 74 (or its complementary sequence), or a fragment thereof.

[0209] Construct A1 is 5074 bp in length (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 poly A (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0210] In some embodiments, construct A1 is 5104 bp in length (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 poly A (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 72);

[0211] In some embodiments, construct A1 is 5074 bp in length (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 poly A (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73);

[0212] In some embodiments, construct A1 is 5104 bp in length (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 poly A (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 74);

[0213] In some embodiments, construct A1 is 5074 bp in length (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 poly A (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73);

[0214] In some embodiments, construct A1 is 5104 bp in length (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 poly A (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 74);

[0215] In some embodiments, construct A1 has a length of 5074 bp (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 poly A (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71);

[0216] In some embodiments, construct A1 has a length of 5104 bp (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 poly A (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 72).

[0217] In some embodiments, construct A1 has a length of 4786 bp (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 poly A (227 bp) (SEQ ID NO: 61). In other embodiments, construct A1 optionally contains any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 67-70 (or its complementary sequence) and / or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 71-74 (or its complementary sequence), or a fragment thereof.

[0218] Construct A2 is 4939 bp in length (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH poly A (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0219] In some embodiments, construct A2 is 4969 bp in length (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH poly A (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 72).

[0220] In some embodiments, construct A2 is 4939 bp in length (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH poly A (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0221] In some embodiments, construct A2 is 4969 bp in length (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH poly A (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 74).

[0222] In some embodiments, construct A2 is 4939 bp in length (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH poly A (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0223] In some embodiments, construct A2 is 4969 bp in length (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH poly A (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 74).

[0224] In some embodiments, construct A2 is 4939 bp in length (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH poly A (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0225] In some embodiments, construct A2 is 4969 bp in length (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH poly A (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 72).

[0226] In some embodiments, construct A2 is 4663 bp in length (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, and bGH poly A (227 bp) (SEQ ID NO: 61). In other embodiments, construct A2 optionally contains any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 67 - 70 (or its complementary sequence) and / or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 71 - 74 (or its complementary sequence), or a fragment thereof.

[0227] Construct A3 is 4939 bp in length (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH poly A (169 bp) (SEQ ID NO: 59), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0228] In some embodiments, construct A3 is 4969 bp in length (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH poly A (169 bp) (SEQ ID NO: 59), and 3' AAV2 ITR (145) (SEQ ID NO: 72).

[0229] In some embodiments, construct A3 is 4939 bp in length (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH poly A (169 bp) (SEQ ID NO: 59), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0230] In some embodiments, construct A3 has a length of 4969 bp (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH poly A (169 bp) (SEQ ID NO: 59), and 3' AAV2 ITR (145) (SEQ ID NO: 74).

[0231] In some embodiments, construct A3 has a length of 4939 bp (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH poly A (169 bp) (SEQ ID NO: 59), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0232] In some embodiments, construct A3 has a length of 4969 bp (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH poly A (169 bp) (SEQ ID NO: 59), and 3' AAV2 ITR (145) (SEQ ID NO: 74).

[0233] In some embodiments, construct A3 has a length of 4939 bp (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH poly A (169 bp) (SEQ ID NO: 59), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0234] In some embodiments, construct A3 has a length of 4969 bp (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH poly A (169 bp) (SEQ ID NO: 59), and 3' AAV2 ITR (145) (SEQ ID NO: 72).

[0235] In some embodiments, construct A3 has a length of 4663 bp (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, and bGH poly A (169 bp) (SEQ ID NO: 59). In other embodiments, construct A3 optionally contains any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 67 - 70 (or its complementary sequence) and / or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 71 - 74 (or its complementary sequence), or a fragment thereof.

[0236] Construct A4 is 4939 bp in length (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH poly A (202 bp) (SEQ ID NO: 60), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0237] In some embodiments, construct A4 is 4969 bp in length (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH poly A (202 bp) (SEQ ID NO: 60), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 72).

[0238] In some embodiments, construct A4 is 4939 bp in length (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH poly A (202 bp) (SEQ ID NO: 60), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0239] In some embodiments, construct A4 has a length of 4969 bp (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH poly A (202 bp) (SEQ ID NO: 60), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 74).

[0240] In some embodiments, construct A4 has a length of 4939 bp (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH poly A (202 bp) (SEQ ID NO: 60), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0241] In some embodiments, construct A4 has a length of 4969 bp (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH poly A (202 bp) (SEQ ID NO: 60), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 74).

[0242] In some embodiments, construct A4 has a length of 4939 bp (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH poly A (202 bp) (SEQ ID NO: 60), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0243] In some embodiments, construct A4 has a length of 4969 bp (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH poly A (202 bp) (SEQ ID NO: 60), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 72).

[0244] In some embodiments, construct A4 has a length of 4663 bp (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, and bGH poly A (202 bp) (SEQ ID NO: 60). In other embodiments, construct A4 optionally contains any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 67 - 70 (or its complementary sequence) and / or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 71 - 74 (or its complementary sequence), or a fragment thereof.

[0245] Construct A5 is 4871 bp in length (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 poly A (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0246] In some embodiments, construct A5 is 4901 bp in length (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 poly A (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 71).

[0247] In some embodiments, construct A5 is 4871 bp in length (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 poly A (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0248] In some embodiments, construct A5 is 4901 bp in length (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 poly A (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 74).

[0249] In some embodiments, construct A5 is 4871 bp in length (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 poly A (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0250] In some embodiments, construct A5 is 4901 bp in length (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 poly A (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 74).

[0251] In some embodiments, construct A5 is 4871 bp in length (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 poly A (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0252] In some embodiments, construct A5 is 4901 bp in length (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 poly A (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 72).

[0253] In some embodiments, construct A5 has a length of 4595 bp (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 poly A (227 bp) (SEQ ID NO: 61). In other embodiments, construct A5 optionally contains any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 67-70 (or its complementary sequence) and / or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 71-74 (or its complementary sequence), or a fragment thereof.

[0254] Construct A6 has a length of 5002 bp (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH poly A (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0255] In some embodiments, construct A6 has a length of 5032 bp (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH poly A (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 72).

[0256] In some embodiments, construct A6 is 5002 bp in length (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH poly A (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0257] In some embodiments, construct A6 is 5032 bp in length (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH poly A (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 74).

[0258] In some embodiments, construct A6 is 5002 bp in length (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH poly A (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 73).

[0259] In some embodiments, construct A6 is 5032 bp in length (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH poly A (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 74).

[0260] In some embodiments, construct A6 is 5002 bp in length (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH poly A (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (130 bp) (SEQ ID NO: 71).

[0261] In some embodiments, construct A6 is 5032 bp in length (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, bGH poly A (227 bp) (SEQ ID NO: 61), and 3' AAV2 ITR (145 bp) (SEQ ID NO: 72).

[0262] In some embodiments, construct A6 has a length of 4726 bp (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) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, and bGH poly A (227 bp) (SEQ ID NO: 61). In other embodiments, construct A6 optionally contains any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 67-70 (or its complementary sequence) and / or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 71-74 (or its complementary sequence), or a fragment thereof.

[0263] Construct A7 has a length of 4781 bp (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), Kozac sequence, wild-type hMYBPC3 (SEQ ID NO: 42) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, mini poly A (57 bp) (SEQ ID NO: 64), 3'-UTR sequence, and 3' AAV2-ITR (130 bp) (SEQ ID NO: 71).

[0264] In some embodiments, construct A7 has a length of 4811 bp (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), Kozac sequence, wild-type hMYBPC3 (SEQ ID NO: 42) with a globin intron (131 bp) (SEQ ID NO: 53), mini poly A (57 bp) (SEQ ID NO: 64), 3'-UTR sequence, and 3' AAV2-ITR (145 bp) (SEQ ID NO: 71).

[0265] In some embodiments, construct A7 is 4781 bp in length (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), Kozac sequence, wild-type hMYBPC3 (SEQ ID NO: 42) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, micro poly A (57 bp) (SEQ ID NO: 64), 3'-UTR sequence, and 3' AAV2-ITR (130 bp) (SEQ ID NO: 73).

[0266] In some embodiments, construct A7 is 4811 bp in length (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), Kozac sequence, wild-type hMYBPC3 (SEQ ID NO: 42) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, micro poly A (57 bp) (SEQ ID NO: 64), 3'-UTR sequence, and 3' AAV2-ITR (145 bp) (SEQ ID NO: 74).

[0267] In some embodiments, construct A7 is 4781 bp in length (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), Kozac sequence, wild-type hMYBPC3 (SEQ ID NO: 42) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, micro poly A (57 bp) (SEQ ID NO: 64), 3'-UTR sequence, and 3' AAV2-ITR (130 bp) (SEQ ID NO: 73).

[0268] In some embodiments, construct A7 has a length of 4811 bp (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), Kozac sequence, wild-type hMYBPC3 (SEQ ID NO: 42) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, mini poly A (57 bp) (SEQ ID NO: 64), 3'-UTR sequence, and 3' AAV2-ITR (145 bp) (SEQ ID NO: 74).

[0269] In some embodiments, construct A7 has a length of 4781 bp (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), Kozac sequence, wild-type hMYBPC3 (SEQ ID NO: 42) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, mini poly A (57 bp) (SEQ ID NO: 64), 3'-UTR sequence, and 3' AAV2-ITR (130 bp) (SEQ ID NO: 71).

[0270] In some embodiments, construct A7 has a length of 4811 bp (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), Kozac sequence, wild-type hMYBPC3 (SEQ ID NO: 42) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, mini poly A (57 bp) (SEQ ID NO: 64), 3'-UTR sequence, and 3' AAV2-ITR (145 bp) (SEQ ID NO: 72).

[0271] In some embodiments, construct A7 is 4505 bp in length (SEQ ID NO: 21) and contains the following elements 5' to 3': hTNNT2 promoter (469 bp) (SEQ ID NO: 49), Kozac sequence, wild-type hMYBPC3 (SEQ ID NO: 42) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, micro poly A (57 bp) (SEQ ID NO: 64), and 3'-UTR sequence. In other embodiments, construct A7 optionally contains any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 67-70 (or its complementary sequence) and / or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 71-74 (or its complementary sequence), or a fragment thereof.

[0272] Construct A8 is 4844 bp in length (SEQ ID NO: 26) and contains the following elements 5' to 3': 5' AAV2-ITR (130 bp) (SEQ ID NO: 67), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), Kozac sequence, wild-type hMYBPC3 (SEQ ID NO: 42) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, micro poly A (57 bp) (SEQ ID NO: 64), 3'-UTR sequence, and 3' AAV2-ITR (130 bp) (SEQ ID NO: 71).

[0273] In some embodiments, construct A8 is 4874 bp in length (SEQ ID NO: 25) and contains the following elements 5' to 3': 5' AAV2-ITR (145 bp) (SEQ ID NO: 67), hTNNT2 promoter (532 bp) (SEQ ID NO: 51), Kozac sequence, wild-type hMYBPC3 (SEQ ID NO: 42) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, micro poly A (57 bp) (SEQ ID NO: 64), 3'-UTR sequence, and 3' AAV2-ITR (145 bp) (SEQ ID NO: 71).

[0274] In some embodiments, construct A8 is 4844 bp in length (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), Kozac sequence, wild-type hMYBPC3 (SEQ ID NO: 42) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, micro poly A (57 bp) (SEQ ID NO: 64), 3'-UTR sequence, and 3' AAV2-ITR (130 bp) (SEQ ID NO: 73).

[0275] In some embodiments, construct A8 is 4874 bp in length (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), Kozac sequence, wild-type hMYBPC3 (SEQ ID NO: 42) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, micro poly A (57 bp) (SEQ ID NO: 64), 3'-UTR sequence, and 3' AAV2-ITR (145 bp) (SEQ ID NO: 72).

[0276] In some embodiments, construct A8 is 4844 bp in length (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), Kozac sequence, wild-type hMYBPC3 (SEQ ID NO: 42) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, micro poly A (57 bp) (SEQ ID NO: 64), 3'-UTR sequence, and 3' AAV2-ITR (130 bp) (SEQ ID NO: 73).

[0277] In some embodiments, construct A8 has a length of 4874 bp (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), Kozac sequence, wild-type hMYBPC3 (SEQ ID NO: 42) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, micro poly A (57 bp) (SEQ ID NO: 64), 3'-UTR sequence, and 3' AAV2-ITR (145 bp) (SEQ ID NO: 73).

[0278] In some embodiments, construct A8 has a length of 4844 bp (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), Kozac sequence, wild-type hMYBPC3 (SEQ ID NO: 42) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, micro poly A (57 bp) (SEQ ID NO: 64), 3'-UTR sequence, and 3' AAV2-ITR (130 bp) (SEQ ID NO: 71).

[0279] In some embodiments, construct A8 has a length of 4874 bp (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), Kozac sequence, wild-type hMYBPC3 (SEQ ID NO: 42) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, micro poly A (57 bp) (SEQ ID NO: 64), 3'-UTR sequence, and 3' AAV2-ITR (145 bp) (SEQ ID NO: 72).

[0280] In some embodiments, construct A8 is 4568 bp in length (SEQ ID NO: 24) and contains the following elements from 5' to 3': hTNNT2 promoter (532 bp) (SEQ ID NO: 51), Kozac sequence, wild-type hMYBPC3 (SEQ ID NO: 42) with a globin intron (131 bp) (SEQ ID NO: 53) between exons, micro poly A (57 bp) (SEQ ID NO: 64), and 3'-UTR sequence. In other embodiments, construct A8 optionally contains any one of the 5' AAV2-ITR sequences of SEQ ID NOs: 67 - 70 (or its complementary sequence) and / or any one of the 3' AAV2-ITR sequences of SEQ ID NOs: 71 - 74 (or its complementary sequence), or a fragment thereof.

[0281] In any of the foregoing embodiments, the vector construct contains at least one ITR sequence. Exemplary ITR sequences include, but are not limited to, SEQ ID No: 67 - 74, including any complementary sequence and / or combinations thereof.

[0282] Modified forms of polynucleotides and polypeptides can be prepared using a variety of standard cloning, recombinant DNA techniques, cell expression known to those skilled in the art, or in vitro translation and chemical synthesis techniques (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition).

[0283] Gene delivery methods.

[0284] Also provided is a method of using a vector construct or AAV particle as described herein to deliver a gene encoding a target protein. In one embodiment, the gene delivery vector can be a viral gene delivery vector, such as a viral particle, or a non-viral gene delivery vector, such as a vector construct or nucleic acid encoding the target protein. Viral vectors include lentiviral vectors, adenoviral vectors, herpesviral vectors. Preferably, it is a recombinant adeno-associated virus (rAAV) vector. Alternatively, a non-viral system can be used, including using naked DNA (with or without chromatin attachment regions) or conjugated DNA, which is introduced into cells by various transfection methods such as lipids or electroporation.

[0285] Non-limiting examples of vector constructs as described herein include any one of SEQ ID No: 3 - 41 or 92 - 169.

[0286] 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 higher sequence identity to any one of SEQ ID No: 3-41 or 92-169 (over the full length of SEQ ID No: 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 one of SEQ ID No: 3-41 or 92-169. Preferably, the AAV vector genome of the vector construct or 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 higher sequence identity to any one of SEQ ID No: 3-41 or 92-169. Even more preferably, the nucleotide sequence of the vector construct is at least 97% or 98% or 99% or higher identical to any one of SEQ ID No: 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 or is the negative (-) strand of any one of SEQ ID No: 3-41 or 92-169.

[0287] In some embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98% or 99% identical to any one of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36 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 or is the negative (-) strand of any one of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36 or 39.

[0288] In some embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98% or 99% identical to any one of SEQ ID NO: 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37 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 or is the negative (-) strand of any one of SEQ ID NO: 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37 or 40.

[0289] In some embodiments, the vector construct comprises a nucleotide sequence that is at least 97%, 98% or 99% identical to any one of SEQ ID NO: 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38 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 or is the negative (-) strand of any one of 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38 or 41.

[0290] The present disclosure can be used in veterinary and medical applications. Suitable subjects for the gene delivery methods described herein include avians and mammals, where mammals are preferred and humans are most preferred. Human subjects include neonates, infants, adolescents and adults.

[0291] Non-viral gene delivery.

[0292] Non-viral gene delivery can be carried out using naked DNA, which is the simplest non-viral transfection method. For example, it may be possible to administer the vector constructs provided herein using naked plasmid DNA. Alternatively, the vector constructs can be delivered using methods involving: electroporation; sonoporation; or using a "gene gun", which uses, for example, high-pressure gas or an inverted.22 caliber gun (Helios® gene gun system (BIO-RAD)) to shoot DNA-coated gold particles into cells; microinjection; laser; hyperthermia; ultrasound; hydrodynamic gene transfer; magnetofection; chemical transfection (e.g., calcium phosphate, DEAE-dextran); liposomes; lipid complexes; dendrimers; lipid nanoparticles or inorganic nanoparticles, all of which are known in the art.

[0293] To improve the delivery of the vector construct into cells, it may be necessary to protect it from damage and facilitate its entry into cells. For this purpose, lipid complexes and polymeric complexes that are able to protect nucleic acids from unwanted degradation during transfection can be used.

[0294] The vector construct can be coated with lipids in organized structures such as micelles or liposomes. When the organized structure is complexed with DNA, it is called a lipid complex. Anionic and neutral lipids can be used to construct the lipid complexes of synthetic vectors. In one embodiment, cationic lipids can be used to condense negatively charged DNA molecules due to their positive charge to facilitate encapsulation of DNA into liposomes. If necessary, co-lipids (usually electro-neutral lipids such as DOPE) may be added to the cationic lipids to form lipid complexes (Dabkowska et al., J. R. Soc. Interface. 9(68): 548-61(2012).

[0295] In certain embodiments, complexes of polymers and DNA, termed polyplexes, can be used as delivery vehicle constructs. Most polyplexes consist of cationic polymers and their formation is governed by ionic interactions. Polyplexes generally do not release their DNA payloads into the cytoplasm. Thus, co-transfection with an endosomolytic agent such as inactivated adenovirus may be required (to lyse the endosomes generated during endocytosis, i.e., the process by which the polyplexes enter the cell) (Akinc et al., J. Gene Medic. 7(5): 657-63).

[0296] In certain embodiments, hybrid methods can be used to deliver vehicle constructs that combine two or more technologies. One example is a virinos; which combines liposomes with inactivated HIV or influenza virus. In another embodiment, other methods involve mixing other viral vectors with cationic lipids or hybridizing viruses and can be used to deliver nucleic acids (Khan, Firdos Alam, Biotechnology Fundamentals, CRC Press, November 18, 2015, p. 395).

[0297] In certain embodiments, dendrimers can be used to deliver vehicle constructs, particularly cationic dendrimers, i.e., dendrimers with a positive surface charge. In the presence of genetic material such as DNA or RNA, charge complementarity causes a transient association of the nucleic acid with the cationic dendrimer. After reaching the destination, the dendrimer-nucleic acid complex is then imported into the cell via endocytosis (Amiji, Mansoor M., ed., Polymeric Gene Delivery: Principles and Applications, CRC Press, September 29, 2004, p. 142).

[0298] Viral particles.

[0299] In one embodiment, suitable viral gene delivery vehicles such as viral particles can be used to deliver nucleic acids. In certain embodiments, viral gene delivery vehicles 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 adeno-associated virus (AAV).

[0300] Accordingly, the present disclosure provides virus particles (comprising the vector constructs provided herein) that serve as gene delivery vectors, which are based on parvoviruses, particularly dependoviruses, such as infectious human or simian AAV, and their components (e.g., parvovirus genomes), for introducing and / or expressing cMyBP-C protein in mammalian cells. Thus, as used herein, the term “parvovirus” encompasses dependoviruses, such as any type of AAV.

[0301] Viruses of the family Parvoviridae are small DNA animal viruses. The family Parvoviridae can be divided into two subfamilies: Parvovirinae, which infect vertebrates; and Densovirinae, which infect insects. Members of the subfamily Parvovirinae are referred to herein as parvoviruses and include the genus Dependovirus. As can be inferred from their genus name, members of the genus Dependovirus are unique in that they generally require co-infection with a helper virus such as adenovirus or herpesvirus for productive infection in cell culture. The genus Dependovirus includes AAVs that typically infect humans (e.g., serotypes 1, 2, 3A, 3B, 4, 5, and 6), primates (e.g., serotypes 1 and 4), and related viruses that infect other warm-blooded animals other than birds and reptiles (e.g., bovine, canine, equine, murine, rat, and ovine adeno-associated viruses). More information regarding parvoviruses and other members of the family Parvoviridae is described in Kenneth I. Berns, “Parvoviridae: The Viruses and Their Replication,” Fields Virology Chapter 69 (3rd ed. 1996). For convenience, the present disclosure is further illustrated and described herein by reference to AAV. However, it should be understood that the present disclosure is not limited to AAV, but may equally apply to other parvoviruses.

[0302] Production of AAV particles requires the AAV “rep” and “cap” genes, which are genes encoding replication and capsid proteins, respectively. The AAV rep and cap genes have been found in all AAV serotypes examined to date and are described herein and in the references cited. In wild-type AAV, the rep and cap genes are typically found adjacent to each other in the viral genome (i.e., they are “coupled” together in an adjacent or overlapping transcriptional unit), and they are generally conserved among AAV serotypes. The AAV rep and cap genes are also referred to independently and collectively as the “AAV packaging genes.” As used herein, the AAV cap gene encodes the Cap protein, which is capable of packaging an AAV vector in the presence of rep and adenoviral helper functions and is capable of binding to target cell receptors. In some embodiments, the AAV cap gene encodes a capsid protein having an amino acid sequence derived from a particular AAV serotype.

[0303] The AAV sequences used to generate AAV can be derived from the genomes of any AAV serotype. Generally, AAV serotypes have genomic sequences that are significantly homologous at the amino acid and nucleic acid levels, provide a set of similar genetic functions, produce virions that are physically and functionally substantially equivalent, and replicate and assemble by virtually the same mechanisms. Discussions of the genomic sequences and genomic similarities of AAV serotypes. (See, for example, 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)).

[0304] The genomic organization of all known AAV serotypes is extremely similar. The genome of AAV is a linear, single-stranded DNA molecule less than about 5,000 nucleotides (nt) in length. Inverted terminal repeats (ITRs) flank the unique coding nucleotide sequences of the non-structural replication (Rep) proteins and the structural (VP) proteins. The VP proteins form the capsid. The assembly-activating protein (AAP) rapidly chaperones capsid assembly and prevents degradation of free capsid proteins (Grosse et al., J. Virol. 91(20): e01198-17 (2017). The terminal 145 nt are self-complementary and are organized such that an energetically stable intramolecular double helix that forms a T-shaped hairpin can form. These hairpin structures serve as the origin of viral DNA replication and act as primers for the cellular DNA polymerase complex. The Rep gene encodes the Rep proteins Rep78, Rep68, Rep52, and Rep40. Rep78 and Rep68 are transcribed by the p5 promoter, and Rep 52 and Rep40 are transcribed by the p19 promoter. The cap gene encodes the VP proteins VP1, VP2, and VP3. The cap gene is transcribed by the p40 promoter. The ITRs employed in the vectors of the embodiments of the present invention can correspond to the same serotype as the associated cap gene, or can be different. In one embodiment, the ITRs employed herein correspond to the AAV2 serotype, and the cap gene corresponds to the AAV5 serotype.

[0305] It is known that AAV VP proteins determine the tropism of AAV virions. The VP protein coding sequences are significantly less conserved compared to the Rep proteins and genes in different AAV serotypes. The ability of Rep and ITR sequences to cross-complement corresponding sequences of other serotypes allows for the generation of pseudotyped AAV particles that contain the capsid proteins of one serotype (e.g., AAV1, 5, or 8) and the Rep and / or ITR sequences of another AAV serotype (e.g., AAV2). Such pseudotyped rAAV particles are part of the present disclosure.

[0306] The AAV particles (and the encoding AAV vector genomes) described herein can include any of the capsid proteins described in WO-2018 / 022608 or WO-2019 / 222136, the disclosures of which patents regarding human and simian AAV capsids and their properties, such as transduction efficiency, tissue tropism, glycan binding, and resistance to IVIG neutralization, are incorporated herein by reference in their entirety, including but not limited to any of the capsids in the sequence listings and their variants, such as those having chimeric swapped variable regions and / or glycan binding sequences and / or GH loops.

[0307] In one embodiment, the AAV ITR sequences for use in the context of the present disclosure are derived from AAV1, AAV2, AAV4, and / or AAV6. Similarly, in one embodiment, the Rep (e.g., Rep78 and Rep52) coding sequences are derived from AAV1, AAV2, AAV4, and / or AAV6. However, the sequences encoding the VP1, VP2, and VP3 capsid proteins for use in the context of the present disclosure can be obtained from any serotype, such as from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12, or from simian AAV, including any of the capsid proteins described in WO 2018 / 022608 or PCT / US19 / 32097, or newly developed AAV-like particles obtained, for example, by capsid shuffling techniques and AAV capsid libraries, or any capsid that is at least 90% identical to any of SEQ ID Nos: 75-91.

[0308] For example, the amino acid sequences of various capsids are disclosed. See, for example

[0309] AAVRh.1 / hu.14 / AAV9 AAS99264.1 (SEQ ID NO: 75)

[0310] AAVRh.8 SEQ97 of U.S. Patent Publication 2013 / 0045186 (SEQ ID NO: 76)

[0311] AAVRh.10 SEQ81 (SEQ ID NO: 77) of U.S. Patent Publication 2013 / 0045186

[0312] AAVRh.74 SEQ 1 (SEQ ID NO: 78) of International Patent Publication WO 2013 / 123503

[0313] AAV1 AAB_95452.1 (SEQ ID NO: 79)

[0314] AAV2 YP_680426.1 (SEQ ID NO: 80)

[0315] AAV3 NP_043941.1 (SEQ ID NO: 81)

[0316] AAV3B AAB95452.1 (SEQ ID NO: 82)

[0317] AAV4 NP_044927.1 (SEQ ID NO: 83)

[0318] AAV5 YP_068409.1 (SEQ ID NO: 84)

[0319] AAV6 AAB95450.1 (SEQ ID NO: 85)

[0320] AAV7 YP_077178.1 (SEQ ID NO: 86)

[0321] AAV8 YP_077180.1 (SEQ ID NO: 87)

[0322] AAV10 AAT46337.1 (SEQ ID NO: 88)

[0323] AAV11 AAT46339.1 (SEQ ID NO: 89)

[0324] AAV12 ABI16639.1 (SEQ ID NO: 90)

[0325] AAV13 ABZ10812.1 (SEQ ID NO: 91)

[0326] Modified “AAV” sequences can also be used in the context of the present disclosure, for example, for generating AAV gene therapy vectors. Such modified sequences, for example, sequences having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or higher nucleotide and / or amino acid sequence identity with AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9 ITR, Rep or VP (e.g., sequences having about 75%-99% nucleotide sequence identity), can be used to replace wild-type AAV ITR, Rep or VP sequences.

[0327] In some embodiments, a nucleic acid sequence encoding an AAV capsid protein is operably linked to an expression control sequence for expression in a particular cell type, such as Sf9 or HEK cells. The embodiments can be practiced using techniques known to those of skill in the art for expressing foreign genes in insect host cells or mammalian host cells. Methods for molecular engineering and expressing polypeptides in insect cells are described, for example, in Summers and Smith (1986) A Manual of Methods for Baculovirus Vectors and Insect Culture Procedures, Texas Agricultural Experimental Station Bull. No. 7555, College Station, Tex.; Luckow (1991) In Prokop et al., Cloning and Expression of Heterologous Genes in Insect Cells with Baculovirus Vectors' Recombinant DNA Technology and Applications, 97-152; King, L. A. and R. D. Possee (1992) The baculovirus expression system, Chapman and Hall, United Kingdom; O'Reilly, D. R., L. K. Miller, V. A. Luckow (1992) Baculovirus Expression Vectors: A Laboratory Manual, New York; W.H. Freeman and Richardson, C. D. (1995) Baculovirus Expression Protocols, Methods in Molecular Biology, Vol. 39; U.S. Patent No. 4,745,051; US-2003148506; and WO-03 / 074714, all of which are incorporated herein by reference in their entirety. Promoters particularly suitable for transcribing nucleotide sequences encoding AAV capsid proteins are, for example, the polyhedron promoter. However, other promoters known to be active in insect cells, such as the p10, p35, or IE-1 promoters, are also known in the art, and other promoters described in the above references are also contemplated.

[0328] The use of insect cells for the expression of heterologous proteins, as well as 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, have been well demonstrated. (See, e.g., METHODS IN MOLECULAR BIOLOGY, edited by Richard, Humana Press, N J (1995); O'Reilly et al., BACULOVIRUS EXPRESSION VECTORS, A LABORATORY MANUAL, Oxford Univ. Press (1994); Samulski et al., J. Virol. 63: 3822-8 (1989); Kajigaya et al., Proc. Nat'l. Acad. Sci. USA, 88: 4646-50 (1991); Ruffing et al., J. Virol. 66: 6922-30 (1992); Kirnbauer et al., Virol. 219: 37-44 (1996); Zhao et al., Virol. 272: 382-93 (2000); and U.S. Patent No. 6,204,059). In some embodiments, the nucleic acid construct encoding an AAV protein (e.g., an AAV rep or cap protein) in insect cells 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 compatible with insect cells. The vector can integrate into the insect cell genome, but the vector need not be permanently present in the insect cell and also includes transient episomal vectors. The vector can be introduced by any known means, e.g., by chemically treating the cells, electroporation, or infection. In some embodiments, the vector is a baculovirus, a viral vector, or a plasmid. In one embodiment, the vector is a baculovirus, i.e., the construct is a baculovirus vector. Baculovirus vectors and methods of using them are described in the references cited above regarding the molecular engineering of insect cells.

[0329] Method for producing recombinant AAV particles

[0330] The present disclosure provides materials and methods for generating 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 target proteins, wherein the promoter and the restriction site are 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 containing the coding region of the target protein inserted at the restriction site and operably linked to the promoter. Those skilled in the art will appreciate that any of the AAV vector constructs disclosed in this application can be used in methods for generating recombinant AAV particles.

[0331] In some embodiments, the helper functions for AAV production are provided by one or more helper plasmids or helper viruses comprising adenovirus or baculovirus helper genes. Non-limiting examples of adenovirus or baculovirus helper genes include, but are not limited to, E1A, E1B, E2A, E4, and VA, which can provide helper functions for AAV packaging.

[0332] Helper viruses for AAV are known in the art and include, for example, viruses from the families Adenoviridae and Herpes viridae. Examples of helper viruses for AAV include, but are not limited to, the SadV-13 helper virus and SadV-13-like helper viruses described in U.S. Publication No. 20110201088, the disclosure of which is incorporated herein by reference, and the helper vector pHELP (Applied Viromics). Those skilled in the art will appreciate that any helper virus or helper plasmid for AAV that can provide sufficient helper functions for AAV can be used herein.

[0333] In some embodiments, the AAV cap gene is present in a plasmid. The plasmid may also 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 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.

[0334] In some embodiments, insect or mammalian cells can be transfected with a helper plasmid or helper virus, a vector construct encoding the AAV cap gene, and a plasmid; and the recombinant AAV virus can be collected at different time points after co-transfection. For example, the recombinant AAV virus can be collected at about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 96 hours, about 120 hours after co-transfection, or at a time between any two of these time points.

[0335] Recombinant AAV particles can also be produced using any conventional method known in the art suitable for generating infectious recombinant AAV. In some cases, recombinant AAV can be produced by using insect or mammalian cells that stably express some of the components required to produce AAV particles. For example, a plasmid (or plasmids) containing the AAV rep and cap genes and a selectable marker (such as the neomycin resistance gene) can be integrated into the genome of the cells. Then, the insect or mammalian cells can be co-infected with a helper virus (such as an adenovirus or baculovirus that provides helper functions) and a viral vector construct containing 5' and 3' AAV ITRs (and, if necessary, a nucleotide sequence encoding a heterologous protein). 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, instead of a plasmid, an adenovirus or baculovirus can be used to introduce the rep and cap genes into the packaging cells. As yet another non-limiting example, a viral vector construct containing 5' and 3' AAV LTRs and the rep-cap genes can be stably integrated into the DNA of the production cells, and the helper functions can be provided by wild-type adenovirus to produce recombinant AAV.

[0336] In one aspect, the present disclosure provides a method for producing AAV particles that can be used as gene delivery vectors, the method comprising the steps of:

[0337] (a) Providing one or more nucleic acid constructs to cells that permit AAV replication (such as insect cells or mammalian cells), the nucleic acid constructs comprising:

[0338] (i) A nucleic acid molecule provided herein (such as a recombinant vector construct) flanked by at least one AAV inverted terminal repeat nucleotide sequence;

[0339] (ii) A nucleotide sequence encoding one or more AAV Rep proteins operably linked to a promoter capable of driving the expression of the one or more Rep proteins in the cells;

[0340] (iii) A nucleotide sequence encoding one or more AAV capsid proteins operably linked to a promoter capable of driving the expression of the one or more capsid proteins in the cells;

[0341] (iv) And optionally AAP and MAAP contained in VP2 / 3 mRNA

[0342] (b) Culturing the cells defined in (a) under conditions favorable for the expression of Rep proteins and capsid proteins; and

[0343] Optionally, (c) recovering the AAV gene delivery vector, and

[0344] Optionally, (d) purifying the AAV particles. By way of example, the recombinant vector construct of (i) comprises (1) at least one AAV ITR, (2) a heterologous cardiomyocyte-specific transcriptional regulatory region as described herein, and (3) a nucleic acid encoding functional cMyBP-C. Preferably, the recombinant vector construct of (i) comprises 5' and 3' AAV ITRs.

[0345] Generally, the methods provided herein for generating AAV gene delivery vectors include: providing to cells that permit AAV replication (a) a nucleotide sequence encoding a template for generating a vector genome, such as the vector constructs of the present disclosure (as described in detail herein); (b) a nucleotide sequence sufficient to replicate the template to generate a vector genome (such as the first expression cassette defined above); (c) a nucleotide sequence sufficient to package the vector genome into an AAV capsid under conditions sufficient to replicate the vector genome and package it into the AAV capsid (such as the second expression cassette defined above), thereby generating AAV particles containing a vector genome encapsulated within an AAV capsid in the cells.

[0346] 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)) are two of the most commonly used methods for generating AAV vectors.

[0347] Viral particles containing the vector constructs described herein can be produced using any cell type, such as mammalian and invertebrate cell types, that permit the production of AAV or a biological product and can be maintained in culture.

[0348] There are multiple methods for generating AAV viral particles: for example, but not limited to, transfection using a vector and AAV helper sequences in combination with co-infection with one of AAV helper viruses (such as adenovirus, herpesvirus, or vaccinia virus), or transfection with a recombinant AAV vector, an AAV helper vector, and an accessory function vector. Methods for manufacturing AAV viral particles are described in, for example, U.S. Patent Nos. US6204059, US5756283, US6258595, US6261551, US6270996, US6281010, US6365394, US6475769, US6482634, US6485966, US6943019, US6953690, US7022519, US7238526, US7291498, and US7491508, US5064764, US6194191, US6566118, US8137948; or International Publications WO1996039530, WO1998010088, WO1999014354, WO1999015685, WO1999047691, WO2000055342, WO2000075353, WO2001023597, WO2015191508, WO2019217513, WO2018022608, WO2019222136, WO2020232044, WO2019222132; Methods In Molecular Biology, edited by Richard, Humana Press, NJ (1995); O'Reilly et al., Baculovirus Expression Vectors, A Laboratory Manual, Oxford Univ. Press (1994); Samulski et al., J. Vir. 63:3822-8 (1989); Kajigaya et al., Proc. Nat''l. Acad. Sci. USA 88: 4646-50 (1991); Ruffing et al., J. Vir. 66:6922-30 (1992); Kimbauer et al., Vir., 219:37-44 (1996); Zhao et al., Vir. 272:382-93 (2000); the contents of each of which are incorporated herein by reference in their entirety.For a detailed description of methods for generating AAV viral particles, see, for example, U.S. Patent Nos. 6,001,650, 6,004,797, and 9,504,762, each incorporated herein by reference in its entirety. In one embodiment, a triple transfection method (see, for example, U.S. Patent No. 6,001,650, which is incorporated herein by reference in its entirety) is used to generate AAV viral particles. This method does not require the use of infectious helper virus, thus enabling the generation of AAV viral particles in the absence of any detectable helper virus. This is achieved by using three vectors for generating AAV viral particles, namely, an AAV helper function vector, an accessory function vector, and an AAV viral particle expression vector. However, those skilled in the art will appreciate that the nucleic acid sequences encoded by these vectors can be provided in various combinations on two or more vectors. In other embodiments, the host cell can be transfected with a helper plasmid or helper virus, a viral construct, and a plasmid encoding the AAV cap gene; and the AAV viral particles can be collected at different time points after co-transfection.

[0349] For example, wild-type AAV and a helper virus can be used to provide the replication functions necessary for generating AAV viral particles (see, for example, U.S. Patent No. 5,139,941, which is incorporated herein by reference in its entirety). Alternatively, a combination of a plasmid containing helper function genes and an infection with one of the well-known helper viruses can be used as a source of replication functions (see, for example, U.S. Patent Nos. 5,622,856 and 5,139,941, both incorporated herein by reference in their entirety). Similarly, a plasmid containing accessory function genes can be used in combination with a wild-type AAV infection to provide the necessary replication functions. Those skilled in the art can also employ other methods described herein and / or well-known in the art to generate AAV viral particles.

[0350] The term "vector" shall be understood to mean any genetic element, such as a plasmid, phage, transposon, cosmid, bacmid, minicircle plasmid (e.g., a plasmid without bacterial elements), Doggybone DNA (e.g., a minimal closed linear construct), chromosome, virus, virion (e.g., baculovirus), etc., which is capable of replicating and transferring gene sequences between cells when combined with appropriate control elements. As used herein, a "mammalian cell-compatible vector" or "vector" refers to a nucleic acid molecule capable of productively transforming or transfecting a mammal or mammalian cells. As used herein, an "insect cell-compatible vector" or "vector" refers to a nucleic acid molecule capable of productively transforming or transfecting an insect or insect cells. Exemplary biological vectors include plasmids, linear nucleic acid molecules, and recombinant viruses. Any vector can be used as long as it is compatible with insect cells. The vector can be integrated into the insect cell genome, but the vector does not need to permanently exist in the insect cells and also includes transient episomal vectors. The vector can be introduced by any known means, such as by chemically treating the cells, electroporation, or infection. The vectors and methods of using them are described in the references on molecular engineering of cells cited above.

[0351] The vector for the cell to produce 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 target proteins, wherein the promoter and the restriction site are downstream of the 5' AAV ITR and upstream of the 3' AAV ITR. The vector may also contain a post-transcriptional regulatory element, which is downstream of the restriction site and upstream of the 3' AAV ITR. The viral construct may also contain a polynucleotide inserted at the restriction site and operably linked to the promoter, wherein the polynucleotide contains the coding region of the target protein. In some embodiments, the viral construct also includes a promoter and a restriction site downstream of the promoter to allow insertion of a polynucleotide encoding one or more target proteins, wherein the promoter and the restriction site are downstream of the 5' AAV ITR and upstream of the 3' AAV ITR. In some embodiments, the viral construct also includes a post-transcriptional regulatory element, which is downstream of the restriction site and upstream of the 3' AAV ITR. In some embodiments, the viral construct also includes a polynucleotide inserted at the restriction site and operably linked to the promoter, wherein the polynucleotide includes the coding region of the target protein. Those skilled in the art should understand that any of the AAV vectors disclosed in the present application can be used as a viral construct in the method to produce rAAV virions.

[0352] The term "AAV helper" refers to an AAV-derived coding sequence that can be expressed to provide an AAV gene product that in turn acts in trans to permit productive AAV replication. Thus, AAV helper functions include both the major AAV open reading frames (ORFs) rep and cap. Rep expression products have been shown to have a number of functions, including in particular: recognition, binding, and cleavage of the AAV DNA replication origin; DNA helicase activity; and regulation of transcription from AAV (or other heterologous) promoters. The capsid (Cap) expression products provide the essential packaging functions. AAV helper functions are used herein to complement trans-acting AAV functions missing from the AAV vector genome.

[0353] For production, cells having AAV helper functions produce recombinant capsid proteins sufficient to form capsids. This includes at least the VP1 and VP3 proteins, but more typically all three of the VP1, VP2, and VP3 proteins as found in native AAV. The sequence of the capsid proteins determines the serotype of the AAV virions produced by the host cell. Capsids useful in the present invention include capsids derived from a variety of AAV serotypes, including 1, 2, 3, 3B, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or mixed serotypes (see for example the disclosure of unnatural mixed serotypes in U.S. Patent No. 8,318,480). The capsid proteins can also be variants of the native VP1, VP2, and VP3, including mutant, chimeric, or shuffled proteins. The capsid proteins can be the capsid proteins of rh.10 or other subtypes within the various branches of AAV; the various branches and subtypes are disclosed in for example U.S. Patent No. 7,906,111. Due to the wide availability of constructs and extensive characterization, the illustrative AAV vectors disclosed below are all derived from serotype 2. The construction and use of AAV vectors and AAV proteins of different serotypes are discussed in Chao et al., Mol. Ther. 2:619-623, 2000; Davidson et al., PNAS 97:3428-3432, 2000; Xiao et al., J. Virol. 72:2224-2232, 1998; Halbert et al., J. Virol. 74:1524-1532, 2000; Halbert et al., J. Virol. 75:6615-6624, 2001; and Auricchio et al., Hum. Molec. Genet. 10:3075-3081, 2001.

[0354] In various embodiments, a nucleotide sequence encoding a VP protein is operably linked to a suitable expression control sequence. In various embodiments, a nucleotide sequence encoding a Rep protein is operably linked to a suitable expression control sequence, such as a eukaryotic promoter. By way of example, the nucleotide sequence is operably linked to a eukaryotic promoter such as the SV40 promoter, CMV promoter, RSV promoter, UBC promoter, EF1A promoter, PGK promoter, dihydrofolate reductase promoter, β-actin promoter, TRE (Tet, Tet-On, Tet-Off) promoter, Cumate control 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 is 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.

[0355] For production, cells with AAV helper functions produce Rep proteins to facilitate the production of rAAV. 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 the cells. In one particular embodiment, 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 following examples demonstrate the use of the Rep78 / Rep52 combination. The Rep proteins can be derived from AAV-2 or other serotypes. In various embodiments, the nucleotide sequence encoding the Rep protein is operably linked to a suitable expression control sequence. In various embodiments, the nucleotide sequence encoding the Rep protein is operably linked to a suitable expression control sequence, such as a eukaryotic promoter. For example, the nucleotide sequence is operably linked to a eukaryotic promoter such as the SV40 promoter, CMV promoter, RSV promoter, UBC promoter, EF1A promoter, PGK promoter, dihydrofolate reductase promoter, β-actin promoter, TRE (Tet, Tet-On, Tet-Off) promoter, Cumate control system (CuR / CuO) (see US2004 / 0205834), and temperature-inducible HSP70 promoter, p5 promoter, p10 promoter, p19 promoter, and p40 promoter. In other examples, the nucleotide sequence is 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.

[0356] In some embodiments, the AAV cap gene is present in a plasmid or bacmid. The plasmid may further include the AAV rep gene, which may or may not correspond to the same serotype as the cap gene. The cap gene and / or the rep gene are from any AAV serotype.

[0357] Cells with AAV helper functions can also produce assembly-activating protein (AAP), which aids in capsid assembly. In various embodiments, the nucleotide sequence encoding AAP is operably linked to a suitable expression control sequence. For example, the nucleotide sequence is operably linked to a eukaryotic promoter. In other examples, the nucleotide sequence is operably linked to a 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.

[0358] The term "non-AAV helper function" refers to non-AAV-derived viral and / or cellular functions upon which AAV replication depends. Thus, the term encompasses the proteins and RNAs required for AAV replication, including those involved in AAV gene transcriptional activation, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of Cap expression products, and AAV capsid assembly. Viral-based accessory functions can be derived from any of the known helper viruses, such as adenovirus, herpesviruses (except herpes simplex virus type 1), and vaccinia virus.

[0359] The term "non-AAV helper function vector" generally refers to a nucleic acid molecule comprising a nucleotide sequence that provides an accessory function. The accessory function vector can be transfected into a suitable host cell, where the vector is subsequently capable of supporting the production of AAV viral particles in the host cell. The term specifically excludes infectious viral particles that exist in nature, such as adenovirus, herpesvirus, or vaccinia virus particles. Thus, the accessory function vector can be in the form of a plasmid, phage, transposon, or cosmid. In particular, it has been demonstrated that a complete complement of adenovirus genes is not required for the accessory helper function. For example, adenovirus mutants that are unable to perform DNA replication and late gene synthesis have been shown to permit AAV replication. Ito et al., (1970) J. Gen. Virol. 9:243; Ishibashi et al., (1971) Virology 45:317. Similarly, mutants within the E2B and E3 regions have been shown to support AAV replication, indicating that the E2B and E3 regions may not be involved in providing the accessory function. Carter et al., (1983) Virology 126:505. However, adenoviruses that are defective in the E1 region or deleted in the E4 region cannot support AAV replication. Thus, the E1A and E4 regions may be directly or indirectly required for AAV replication. Laughlin et al., (1982). J. Virol. 41:868; Janik et al.,(1981) Proc. Natl. Acad. Sci. USA 78:1925; Carter et al., (1983) Virology 126:505.Other characterized Ad mutants include: E1B (Laughlin et al. (1982), supra; Janik et al. (1981), supra; Ostrove et al., (1980) Virology 104:502); E2A (Handa et al., (1975) J. Gen. Virol. 29:239; Strauss et al., (1976) J. Virol. 17:140; Myers et al., (1980) J. Virol. 35:665; Jay et al., (1981) Proc. Natl. Acad. Sci. USA 78:2927; Myers et al.,(1981) J. Biol. Chem. 256:567); E2B (Carter, Adeno-Associated Virus Helper Functions, in I CRC Handbook of Parvoviruses (P. Tijssen ed., 1990)); E3 (Carter et al. (1983), supra); and E4 (Carter et al. (1983), supra; Carter (1995)). Although studies of the helper functions provided by adenoviruses with mutations in the E1B coding region have yielded conflicting results, Samulski et al., (1988) J. Virol. 62:206-210 recently reported that E1B55k is required for AAV virion production, while E1B19k is not. Additionally, International Publication WO 97 / 17458 and Matshushita et al., (1998) Gene Therapy 5:938-945 describe helper function vectors encoding various Ad genes. Particularly preferred helper function vectors contain the adenovirus VA RNA coding region, the adenovirus E4 ORF6 coding region, the adenovirus E2A 72 kD coding region, the adenovirus E1A coding region, and an adenovirus E1B region lacking the intact E1B55k coding region. Such vectors are described in International Publication No. WO 01 / 83797.

[0360] In another embodiment, the methods provided herein are performed using any mammalian cell type that permits AAV replication or bioproduct production and can be maintained in culture. In one embodiment, the mammalian cells used can be HEK293, HeLa, CHO, NSO, SP2 / 0, PER.C6, Vero, RD, BHK, HT 1080, A549, Cos-7, ARPE-19, and MRC-5 cells.

[0361] The use of insect cells for the expression of heterologous proteins, and methods for introducing nucleic acids (such as vectors, e.g., insect cell-compatible vectors) into such cells and for maintaining such cells in culture have been well demonstrated. (See, e.g., METHODS IN MOLECULAR BIOLOGY, edited by Richard, Humana Press, N J (1995); O'Reilly et al., BACULOVIRUS EXPRESSION VECTORS, A LABORATORY MANUAL, Oxford Univ. Press (1994); Samulski et al., J. Vir. (1989) Vol. 63, pp. 3822-3828; Kajigaya et al., Proc. Nat'l. Acad. Sci. USA (1991) Vol. 88, pp. 4646-4650; Ruffing et al., J. Vir. (1992) Vol. 66, pp. 6922-6930; Kirnbauer et al., Vir. (1996) Vol. 219, pp. 37-44; Zhao et al., Vir. (2000) Vol. 272, pp. 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 comprising a recombinant polynucleotide that includes an expression control sequence operably linked to a nucleotide sequence to be expressed. Expression vectors include sufficient cis-acting elements for expression; other expression elements may be supplied by the host cell or an in vitro expression system. Expression vectors include all expression vectors known in the art, such as cosmids, plasmids (e.g., naked plasmids or plasmids contained in liposomes), artificial chromosomes, and viruses incorporating recombinant polynucleotides. As used herein, an "insect cell-compatible vector" or "vector" refers to a nucleic acid molecule capable of effecting productive transformation or transfection of an insect or insect cell. Exemplary biological vectors include plasmids, linear nucleic acid molecules, and recombinant viruses. Any vector may be used so long as it is compatible with insect cells. The vector may integrate into the insect cell genome, but the vector need not be permanently present in the insect cell and also includes transient episomal vectors. The vector may be introduced by any known means, such as by chemically treating the cells, electroporation, or infection. In some embodiments, the vector is a baculovirus, viral vector, or plasmid. In a more preferred embodiment, the vector is a baculovirus, i.e., the construct is a baculovirus vector. Baculovirus vectors and methods for their use are described in the references cited above regarding the molecular engineering of insect cells.)

[0362] For example, the insect cell line used can be from Spodoptera frugiperda, such as SF9, SF21, SF900+; Drosophila cell line; mosquito cell line, e.g., Aedes albopictus-derived cell line; silkworm cell line, e.g., Bombyx mori cell line; Trichoplusia ni cell line, such as High Five cell; or Lepidoptera cell line, such as Ascalapha odorata cell line. In one embodiment, the insect cells are cells from an insect species susceptible to baculovirus infection, including HighFive, Sf9, Se301, SeIZD2109, SeUCR1, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAm1, BM-N, Ha2302, Hz2E5, and Ao38.

[0363] Baculoviruses are enveloped DNA viruses of arthropods, and two of its members are well-known expression vectors for producing recombinant proteins in cell culture. Baculoviruses have a circular double-stranded genome (80 - 200 kbp), which can be engineered to allow delivery of large genomic inclusions to specific cells. The virus used as a vector is generally Autographa californica multiple nucleopolyhedrovirus (AcMNPV) or Bombyx mori nucleopolyhedrovirus (BmNPV) (Kato et al., Appl. Microbiol. Biotechnol. 85(3): 459 - 70 (2010).

[0364] Baculoviruses are commonly used to infect insect cells for the expression of recombinant proteins. Specifically, heterologous gene expression in insects can be achieved as described, for example, in U.S. Patent No. 4,745,051; EP 127,839; EP 155,476; Vlak et al., J. Gen. Virol. 68: 765-76 (1988); Miller et al., Ann. Rev. Microbiol. 42:177-9 (1988); Carbonell et al., Gene, 73(2): 409-18 (1998); Maeda et al., Nature, 315: 592-4 (1985); Lebacq-Veheyden et al., Molec. Cell. Biol. 8(8): 3129-35 (1988); Smith et al., PNAS, 82: 8404-8 (1985); and Miyajima et al., Gene, 58: 273-81 (1987). Many baculovirus strains and variants and corresponding permissive insect host cells useful 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).

[0365] Baculovirus shuttle vectors or bacmids are used to generate baculoviruses. Bacmids are propagated as large plasmids in bacteria such as Escherichia coli. When transfected into insect cells, bacmids generate baculoviruses. In another embodiment, the methods provided herein are performed with any mammalian cell type that permits AAV replication or bioproduct production and can be maintained in culture. In one embodiment, the mammalian cells used can be HEK293, HeLa, CHO, NSO, SP2 / 0, PER.C6, Vero, RD, BHK, HT 1080, A549, Cos-7, ARPE-19, and MRC-5 cells.

[0366] 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 required for the production of AAV particles. For example, a plasmid (or plasmids) comprising the AAV rep and cap genes and a selectable marker (such as the neomycin resistance gene) can be integrated into the genome of the cells. In another example, a plasmid (or plasmids) comprising a selectable marker (such as the neomycin resistance gene) can be integrated into the genome of the cells. Subsequently, the insect, fungal, or mammalian cells can be co-infected with a helper virus (such as an adenovirus or baculovirus that provides helper functions) and a viral vector comprising 5' and 3' AAV ITRs (and, if necessary, a nucleotide sequence encoding a heterologous protein). The advantage of this method is that the cells are selectable and suitable for large-scale production of rAAV. As another non-limiting example, a host regulatory gene, rep gene, and cap gene can be introduced into the packaging cells using an adenovirus or baculovirus instead of a plasmid.

[0367] In one embodiment, after the transfected cells are amplified in suspension cell culture via a series of increasingly large culture platforms, the suspension of the transfected cells is purified via a multi-step process to remove process impurities, including recombinant baculovirus and host cells, and to enrich the viral particles containing the recombinant parvovirus (rAAV) vector construct. In another embodiment, the methods provided herein can include the step of affinity purifying 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 used herein is a single-chain camelid antibody or a fragment thereof, which can be obtained, for example, from camels or llamas (see, e.g., Muyldermans, Biotechnol. 74: 277-302 (2001)). The antibody used for the affinity purification of rAAV is an antibody that specifically binds to an epitope on the AAV capsid protein, whereby in one embodiment, the epitope is an epitope present on the capsid proteins of more than one AAV serotype. For example, the antibody can be generated or selected based on specific binding to the AAV5 capsid, but it can also specifically bind to the AAV1, AAV2, AAV3, AAV6, AAV8, or AAV9 capsid.

[0368] The methods provided herein for producing rAAV particles produce a population of rAAV particles. In some embodiments, the population is enriched for particles containing a full-length or near-full-length vector genome by a step that reduces the number of empty capsids.

[0369] The population of rAAV particles produced by the methods provided herein is, for example, used for administration in any of the therapeutic methods described herein.

[0370] Host organism and / or cell

[0371] In another embodiment, a host cell comprising the above-described vector is provided. In one embodiment, the vector construct is capable of replicating or expressing the nucleic acid molecules provided herein in the host cell. In some embodiments, the present disclosure provides an HCM therapeutic agent, which is a host cell comprising a vector construct, the vector construct comprising a nucleic acid encoding cMyBP-C, for HCM cell therapy. The cells can be autologous or allogeneic to the subject.

[0372] As used herein, the term "host" refers to an organism and / or cell that harbors a nucleic acid molecule or vector construct of the present disclosure, and an organism and / or cell suitable for expressing a recombinant gene or protein. The present disclosure is not intended to be limited to any particular type of cell or organism. Indeed, any suitable organism and / or cell can be used as a host herein. The host cell can be in the form of a single cell, a population of similar or different cells, such as in a culture (such as a liquid culture or a culture on a solid substrate), an organism, or a part thereof. In one embodiment, the host cell allows expression of the nucleic acid molecules provided herein. Thus, the host cell can be, for example, a bacterial, yeast, insect, or mammalian cell or a human cell.

[0373] In another embodiment, a means for delivering the nucleic acids provided herein to a wide range of cells, including dividing and non-dividing cells, is provided. The present disclosure can be used to deliver the nucleic acids provided herein to cells in vitro, such as to produce polypeptides encoded by such nucleic acid molecules in vitro or for ex vivo gene therapy.

[0374] The nucleic acid molecules, vector constructs, cells, and methods / uses of the present disclosure can additionally be used in methods for delivering the nucleic acids provided herein to a host, typically a host suffering from HCM.

[0375] Pharmaceutical preparation

[0376] In one embodiment, a pharmaceutical composition is provided that comprises a nucleic acid or vector provided herein and a pharmaceutically acceptable diluent, excipient, or carrier. The pharmaceutical composition can also comprise a second therapeutic agent or adjuvant, etc. Preferably, if parenteral administration is intended, the composition is sterile. Preferably, the composition is free of infectious virus and toxins. Preferably, the composition is stable for a suitable period of time under storage conditions.

[0377] "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 undesirable biological effects. Thus, such pharmaceutical compositions can be used, for example, for ex vivo cell transfection or for direct administration of viral particles or cells to a subject.

[0378] The carrier may be suitable for parenteral administration, which includes intravenous, intraperitoneal, or intramuscular administration. Alternatively, the carrier may be suitable for sublingual or oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the temporary preparation of sterile injectable solutions or dispersions. The use of such media and reagents for active pharmaceutical substances is well known in the art. Unless any conventional medium or reagent is incompatible with the active compound, it is contemplated for use in the pharmaceutical compositions provided herein.

[0379] In other embodiments, provided herein are pharmaceutical compositions (i.e., formulations) of AAV particles that can be used to administer to a subject suffering from a genetic disorder to deliver a gene encoding a target protein. In certain embodiments, the pharmaceutical formulations provided herein are liquid formulations comprising recombinant AAV particles containing any one of the vector constructs disclosed herein. The concentration of recombinant AAV virions in the formulation can vary.

[0380] In other embodiments, the AAV particle pharmaceutical formulations provided herein comprise one or more pharmaceutically acceptable excipients to provide a formulation having properties conducive to storage and / or administration to a subject for treating a genetic disorder.

[0381] In certain aspects, the formulation comprising recombinant AAV particles further comprises one or more buffering agents.

[0382] In another embodiment, the recombinant AAV particle formulations provided herein may comprise one or more tonicity agents, such as sodium chloride. Other buffering agents and tonicity agents known in the art are suitable and can be routinely employed in the formulations provided herein.

[0383] In another embodiment, the recombinant AAV particle formulations provided herein may comprise one or more bulking agents. Exemplary bulking agents include, but are not limited to, mannitol, sucrose, dextran, lactose, trehalose, and polyvinylpyrrolidone (PVP K24).

[0384] In yet another embodiment, the recombinant AAV particle formulations provided herein may comprise one or more surfactants, which may be nonionic surfactants. Exemplary surfactants include ionic surfactants, nonionic surfactants, and combinations thereof. By way of example, the surfactant may be, but is not limited to, TWEEN 80 (also known as polysorbate 80, or its chemical name polyoxyethylene sorbitan monooleate), sodium dodecyl sulfate, sodium stearate, ammonium lauryl sulfate, TRITON AG 98 (Rhone-Poulenc), poloxamer 407, poloxamer 188, and the like, and combinations thereof.

[0385] The recombinant AAV particle preparations provided herein are generally sterile and stable and can be stored long - term without unacceptable changes in quality, potency, or purity.

[0386] In some embodiments, the composition includes an isotonic agent such as a sugar, a polyol (such as mannitol, sorbitol), or sodium chloride. Prolonged absorption of the injectable composition can be achieved by including a delayed - absorption agent such as monostearate and gelatin in the composition. In certain embodiments, the nucleic acid or vector construct provided herein can be administered in a timed or controlled - release formulation, such as in a composition that includes a slow - release polymer or other carriers (including implants and microencapsulation delivery systems) that protect the compound from rapid release. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, and polylactic - polyglycolic acid copolymers (PLG) can be used, for example.

[0387] In certain embodiments, a pharmaceutical composition comprising the vector construct or AAV particle provided herein can be used to transfer genetic material into cells. Such transfer can be carried out 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 as described herein under conditions that allow the nucleic acid or vector provided herein to enter the cell. The cell can be an in - vitro, ex - vivo, or in - vivo cell.

[0388] Therapeutic method

[0389] The vector construct or AAV particle described herein is administered to a subject in a dose effective to deliver the MYBPC3 gene to the heart of a mammalian subject. The subject is preferably human, including pediatric subjects. The age range of pediatric subjects can be, for example, 0 - 2, 2 - 6, 2 - 10, 2 - 12, 2 - 15, 2 - 18, 12 - 18, or 0 - 18 years.

[0390] Such methods include methods for expressing cMyBP - C in the heart of a mammalian subject, the method comprising administering to the subject an effective amount of a composition comprising the vector construct described herein, the rAAV particle described herein, or the pharmaceutical composition described herein, thereby expressing cMyBP - C in the heart tissue (such as myocardium or cardiomyocytes) of the subject.

[0391] Such methods also include a method of treating a mammalian subject for a deficiency of functional wild-type myosin binding protein C, the method being effected 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 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 the untreated level, or increase it to the level seen in healthy humans. In some embodiments, the amount of the vector construct, rAAV particle, or pharmaceutical composition can be effective to increase the level of myosin binding protein C in cardiac tissue (e.g., cardiomyocytes) by at least about 2-fold; and / or restore the contractility, relative tension, calcium-activated tension, relaxation time of engineered heart tissue in vitro or animal tissue in vivo.

[0392] Such methods also include a method of treating HCM in a mammal or treating or preventing any symptoms thereof, the method comprising administering a therapeutically effective amount of a vector construct, rAAV particle, or pharmaceutical composition. In such methods, the mammal can have a mutation in one or both alleles of the cMyBP-C gene. Such methods, for example, reduce heart size, reduce the cardiothoracic ratio, reduce the end-diastolic or end-systolic left ventricular diameter, reduce the anterior or posterior wall thickness, increase the ejection time, increase the aortic peak velocity or aortic blood flow time, and / or reduce disease symptoms. 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.

[0393] 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.

[0394] In any of the methods described herein, the administration of the vector construct, rAAV particle, or pharmaceutical composition can also include the administration of prophylactic or therapeutic corticosteroid treatment, and / or can further include the administration of a second therapeutic agent for treating HCM, including but not limited to beta blockers, calcium channel blockers, antiarrhythmic drugs, and small molecule inhibitors of cardiac myosin.

[0395] In any of the methods herein, prior to administering the AAV particles to a patient as described above, it can be evaluated whether the prospective patient has anti-AAV capsid antibodies or anti-AAV neutralizing antibodies that can block cell transduction or otherwise reduce the overall efficiency of the treatment.

[0396] Detection of anti - AAV antibody

[0397] To maximize the likelihood of successful cardiac transduction using systemic AAV-mediated therapeutic gene transfer, prior to administering AAV particles to a human patient in a treatment regimen as described above, it may be evaluated whether the prospective patient has anti-AAV capsid antibodies or anti-AAV neutralizing antibodies that can block cellular transduction or otherwise reduce the overall efficiency of the treatment regimen. Such antibodies may be present in the serum of the prospective patient and may be directed against the capsid of any serotype of AAV. In one embodiment, the serotype against which pre-existing antibodies are directed is AAV5.

[0398] Methods for detecting pre-existing AAV immunity are well-known and routinely used in the art and include cell-based in vitro transduction inhibition (TI) assays, in vivo (e.g., in mice) TI assays, and ELISA-based total anti-capsid antibody (tAb) detection (see, e.g., Masat et al., Discov. Med., Vol. 15, pp. 379-389; and Boutin et al., (2010) Hum. Gene Ther., Vol. 21, pp. 704-712). TI assays may employ host cells that have been pre-introduced with an AAV-inducible reporter vector. The reporter vector may contain an inducible reporter gene, such as GFP, etc. After the host cells are transduced with the AAV virus, its expression is induced. Anti-AAV capsid antibodies present in human serum that can prevent / reduce host cell transduction will thus reduce the overall expression of the reporter gene in the system. Thus, such assays can be used to detect the presence of anti-AAV capsid antibodies in human serum that can prevent / reduce cellular transduction by therapeutic AAV particles.

[0399] Assays for detecting anti-AAV capsid antibodies may employ a solid-phase bound AAV capsid as a "capture agent" across which human serum is passed, thereby allowing anti-capsid antibodies present in the serum to bind to the solid-phase bound capsid "capture agent". After washing to remove non-specific binding, a "detection agent" may be used to detect the presence of anti-capsid antibodies bound to the capture agent. The detection agent may be an antibody, an AAV capsid, or the like, and may be detectably labeled to aid in the detection and quantification of the bound anti-capsid antibodies. In one embodiment, the detection agent is labeled with ruthenium or a ruthenium complex that can be detected using electrochemiluminescence technology and equipment.

[0400] The same methods described above can be used to evaluate and detect the generation of an anti-AAV capsid immune response in patients pre-treated with a target therapeutic AAV virus. Thus, prior to treatment with a therapeutic AAV virus, these techniques can be used not only to evaluate the presence of anti-AAV capsid antibodies, but also to evaluate and measure the induction of an immune response against the administered therapeutic AAV virus after administration. Accordingly, a method of combining techniques for detecting anti-AAV capsid antibodies in human serum and administering a therapeutic AAV virus to treat HCM is contemplated herein, wherein the techniques for detecting anti-AAV capsid antibodies in human serum can be performed before or after administration of the therapeutic AAV virus.

[0401] Other aspects and advantages of the present disclosure will be understood after considering the following illustrative embodiments.

[0402] Example

[0403] Example 1: Generation of AAV Particles

[0404] Figure 1 The organization of elements of vector constructs, designated A1-A8 and C1-C5 SEQ ID NO: 3-41 or 92-169, respectively, that contain nucleic acids encoding human cMyBP-C is shown. AAV particles containing the AAV9 capsid and vector constructs of SEQ ID NO: 3-26 are generated in HEK293 cells and Sf9 cells. Vectors for AAV production are generated. For example, when generating AAV in HEK293 cells, plasmids are generated. These plasmids have nucleotide sequences that provide the AAV vector genome, encode Rep and capsid proteins, and provide non-helper AAV functions. These plasmids are transfected into HEK293 cells using a transfection reagent. After culturing the HEK293 for a predetermined time after transfection, the generated rAAV particles are isolated from the culture, purified, and titrated. To generate AAV in Sf9 cells, bacmids are generated. These bacmids have nucleotide sequences that provide the AAV vector genome and encode Rep and capsid proteins. The bacmids are transfected into untreated Sf9 cells using a transfection reagent. After culturing the transfected Sf9 cells for a predetermined time, recombinant baculovirus (rBV) is isolated, purified, and titrated. To produce rAAV, another untreated culture of Sf9 cells is infected with rBV at a predetermined multiplicity of infection (MOI). The Sf9 cells are cultured for a predetermined time after infection. After the predetermined time, the generated rAAV particles are isolated from the culture, purified, and titrated.

[0405] Example 2: Evaluation of the Effect of AAV Particles on Engineered Heart Tissue.

[0406] Analyze the effects of AAV9 particles prepared using the MYBPC3 vector construct described herein on human iPSC-derived cardiomyocytes in 2D and 3D formats. In the 2D format, the exogenous protein and RNA content are measured, as well as the inhibition of mutant MYBPC3 transcripts (see Example 5 below). In the 3D format, the effects on contractile function are also measured, including beat frequency, contractility, and kinetics.

[0407] Two hiPSC lines were created by CRISPR / Cas9 genome editing using patient-specific human induced pluripotent stem cell (hiPSC) lines carrying heterozygous MYBPC3 truncating mutations: 1) cpHet carrying an additional homozygous MYBPC3 truncating mutation, which results in the complete absence of the MYBPC3 protein; and 2) an isogenic control carrying two wild-type alleles, resulting in normal levels of the 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.

[0408] In 2D and engineered heart tissue (EHT) formats, AAV9 particles containing the vector construct described herein were tested by contacting human cardiomyocytes (CMs) derived from the cpHet hiPSC line and the isogenic control hiPSC line.

[0409] hiPSC cardiomyocytes (hiPSC-CMs) are prepared by passaging cpHet or isogenic control hiPSC cells, dissociating the hiPSC cells, and enhancing cardiac differentiation over 14 days by using different media. The differentiated cardiomyocytes are cultured in 2D monolayers or in the form of 3D engineered heart tissue (EHT). EHTs are prepared by embedding 1 million hiPSC-CMs in a fibrin matrix, generally as described by 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.

[0410] Briefly, an EHT well with a spacer is placed in a 24-well plate filled with 2% agarose (1.6 ml / well). When the agarose is solid, the spacer is removed and a polydimethylsiloxane scaffold with a pair of struts is placed in each mold. For each EHT casting, approximately 1 million hiPSC-CMs are used in a master medium containing horse serum, Y-27632 (Biorbyt, orb154626), fibrinogen, L-glutamine, DMEM, penicillin / streptomycin, and thrombin. The hiPSC-CMs and the casting medium are placed in the agarose well. After 1.5 hours, the EHT solidifies around the polydimethylsiloxane scaffold and is transferred to a culture plate filled with medium. Subsequently, the EHTs are maintained at 37 °C, 7% CO2, 40% O2, and 98% relative humidity.

[0411] For transduction with AAV, HiPSC-CMs in 2D are re-plated and cultured as adherent cells, 220K cells per 24-well plate or 20K cells per 96-well plate. Approximately 4 days after re-plating, 2D cpHet hiPSC-CMs are transduced by adding AAV particles (at a multiplicity of infection (MOI) of 300K) to the medium. Approximately 2 weeks after generation, cpHet EHTs are transduced by adding AAV particles (MOI of 300K) to the tissue culture medium.

[0412] For 2D and 3D EHT formats, human cMyBP-C protein expression was detected 7 or 14 days after transduction, respectively. Briefly, 2D or EHT hiPSC-CMs were harvested to extract proteins for further analysis. Custom antibodies against hMYBPC3 were used to measure exogenous protein levels using whole protein lysates by Western blotting. α-Actinin and / or cTnT protein levels were used as references. MYBPC3 was not detected in untransduced cpHet hiPSC-CMs. After transduction with AAV9 particles, exogenous MYBPC3 protein was detected, and protein levels were depicted as fold increases compared to isogenic controls. The results are shown in Figure 2 , and showed at least 2.5-fold increases in expression with A2, A3, A4, A6, where A6 achieved the highest increase, and at least 0.1 - 1.0-fold increases in expression with C1, C2, C4, and C5, where C3 achieved the highest increase. Additional AAV9 particles containing the vector constructs described herein, produced in HEK293 cells or Sf9-derived cells, were tested, and Western blot analysis showed even higher 5 - 12-fold changes compared to isogenic controls.

[0413] 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 evaluate contractile function, engineered heart tissue was stimulated to contract and relax. hiPSC-CMs in the EHT format began to exhibit intrinsic spontaneous contractility 1 - 2 weeks after generation. For the spontaneous beating frequency as a functional endpoint, the intrinsic spontaneous contraction frequency was analyzed without further stimulation.

[0414] Spontaneous beating activity was evaluated in low glucose DMEM medium with 1.8 mM calcium and measured 1 week after transduction. Higher spontaneous beating frequencies were consistently observed in cpHet EHTs compared to isogenic control EHTs. The goal of gene therapy with AAV9 particles containing the vector constructs described herein was to reduce this abnormally high frequency. At one and two weeks after transduction, the frequency over time was analyzed in complete medium and serum-free DMEM. Constructs A2, A3, A4, A5, and A6 restored the abnormal phenotype to a normal phenotype. Specifically, constructs A3, A4, and A6 showed the best and most consistent decrease in beating rate (BPM). In contrast, construct A5 showed only a minor effect on the beating rate.

[0415] Pacing was performed using electrical stimulation to evaluate systolic function at a fixed beat frequency. 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, short-term electrical pacing (1 Hz, 2.5 V) was used in a medium containing 1.8 mM calcium to evaluate endpoints at a fixed frequency, with baseline force as a confirmatory QC endpoint and systolic kinetics as a functional endpoint (relative time to delayed relaxation, RT 20% and RT 80% ). The transduction protocol was optimized to provide stable force development by adjusting the time point, transduction medium, and transduction chamber, with no significant difference between untransduced (NT) cpHets and vector-transduced EHTs.

[0416] After activation, the contractile force of cardiomyocytes in engineered heart tissue was measured. The contractile force and kinetics of EHTs were monitored in an EHT test system that combines semi-automatic video optical analysis with pattern recognition software. The force (mN) development over time (seconds) was calculated from the increment in the distance (column deflection) of a polydimethylsiloxane column with a known Sylgard 184 (2.6 kPa) elastic modulus. To depict the normalized mean peak, the baseline of each EHT was set to 0% and the peak force was set to 100%. Untransduced (NT) cpHet EHTs were compared and normalized with transduced cpHets and syngeneic control EHTs. The results are shown in Figure 3 .

[0417] The relaxation time and rate from peak sarcomere contraction to re-elongation were measured. The recorded contractions were identified by peak criteria. Based on the identified contractions, values for frequency, mean force, fractional shortening, contraction time (time to peak), and relaxation time (RT) were calculated. The relaxation time percentage represents the percentage of time required to return from the maximum column deflection to the baseline. For example, RT20% represents the time (seconds) from the maximum value (100%) to 80% column deflection. Based on the absolute RT, the relative delayed RT (fraction or %) = (RT80% - RT50%) / RT80%. The relative percentage of delayed relaxation time is shown in Figure 4A . The relaxation times (seconds) for re-elongation of 20% or 80% are shown in Figure 4B and 4CAmong them. Compared with the isogenic control, cpHet showed consistently higher scores for relative delay RT (RT80% - RT50% / RT80%). The goal of gene therapy is to normalize the contractile kinetics, including by reducing the abnormally high relative delay RT.

[0418] Evaluate the effects of AAV9 particles containing various MYBPC3 vector constructs produced in HEK293 cells, Sf9, and Sf9-derived cells on contractile function. One week after transduction, cpHet EHT transduced with AAV9-MYBPC3 was compared with time-matched isogenic control EHT. Most of the tested vector constructs, such as A2, A3, A4, A5, A6, and C3, restored the abnormally high relative delay RT to a phenotype closer to normal. AAV9 particles produced in HEK293 cells had a better effect on contractile function than those produced in Sf9 cells. A3, A4, A5, and A6 had the greatest effect and completely restored the abnormally high relative delay RT.

[0419] The absolute relaxation time was also evaluated. Compared with the isogenic control, cpHet EHT showed significantly shorter absolute relaxation times at 20% and 80% (RT 20% and RT 80% ). The tested vector constructs, such as 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 showed the most obvious recovery of both (RT 20% and RT 80% ) compared with untransduced cpHet EHT cardiomyocytes. Construct A6 had a statistically significant effect on RT 20% and RT 80% . EHT transduced with vector A6 showed significantly longer RT 20% and longer RT 80% .

[0420] In addition, the normalized mean contraction peak of AAV-transduced cpHet EHT was analyzed and compared with the normalized mean contraction peaks of cpHet and isogenic control EHT. Some effects were visible for all tested vectors, and constructs such as A2, A3, A4, A5, A6, and C3 always had the greatest effect. AAV9 particles containing vector construct A6 produced in HEK293 cells completely normalized the contractile kinetics. Transduction of cpHet EHT with AAV9 particles containing vector constructs A3 and A6 produced in HEK293-derived cells improved the relaxation kinetics to be close to that of the isogenic control. As Figure 4DAs shown, the normalized force % of A3- and A6-transduced cells (Group 3) generated in HEK293 cells was significantly greater than that of A3- and A6-transduced cells (Group 4) generated in insect cells. Overall, compared with AAV9 particles produced in insect cells, AAV9 particles produced in mammalian cells exhibited excellent activity in improving contractile function and contractile kinetics.

[0421] Example 3: Evaluation of the in vivo effect of AAV particles

[0422] AAV particles prepared as in Example 1 were administered to mice (n = 10) at a dose of 2e14 vg / kg, and heart tissues were collected at 8 weeks.

[0423] The number of vector genomes encoding human cMyBP-C was evaluated by ddPCR. The results (vector genomes per diploid gene number) are shown in Figure 5A . All tested AAV particles provided efficient delivery of at least one gene copy encoding human cMyBP-C per cell.

[0424] The number of human cMyBP-C mRNA transcripts was evaluated by ddPCR. The results (mRNA transcripts per RPLP0 ribosomal protein transcript) are shown in Figure 5B . All tested AAV particles provided efficient translation into mRNA, with A2 and A4 providing the highest levels.

[0425] The amount of cMyBP-C protein was determined by liquid chromatography / mass spectrometry (LC / MS). The results (μg / g heart tissue and percentage of human cMyBP-C in total cMyBP-C protein in murine hearts) are shown in Figure 5C .

[0426] Heart tissues were stained with antibodies specific for human cMyBP-C and ASG (α-sarcoglycan, a muscle cell membrane marker). Intact cell nuclei were also stained with DAPI. cMyBP-C protein was widely detected in most cardiomyocytes, and 77% and 65% of cardiomyocytes were positive for cMyBP-C in preparations from mice administered A5, A6, and C3, respectively. See Figure 6 .

[0427] Heart tissues were also stained with antibodies specific for human cMyBP-C and actin present in the sarcomere. cMyBP-C protein was observed to localize to the sarcomere.

[0428] Results showed that the A5 and A6 vector constructs provided efficient delivery of human cMyBP-C protein to mice administered with AAV particles containing these vector constructs, and the human cMyBP-C protein was effectively incorporated into the sarcomeres of most cardiomyocytes. Integration of functional human cMyBP-C protein was expected to improve contractility and alleviate hypertrophic cardiomyopathy and its related symptoms.

[0429] Example 4: Further evaluation of the in vivo effects of AAV particles

[0430] Various doses of rAAV particles containing the vector constructs described herein were administered to MYBPC3 KO mice to evaluate the correction of the HCM phenotype, including hypertrophy and cardiac dysfunction. Echocardiography will be used to monitor the functional correction of these mice throughout the study, and at the end of the study, heart tissue will be used to evaluate the transduction and expression of the vector constructs.

[0431] Example 5: Evaluation of mutant MYBPC3 mRNA levels

[0432] 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.

[0433] Semi-quantitative RT-PCR was performed to monitor the effect on mutant MYBPC3 mRNA. RT-PCR with primers around the MYBPC3 c.2308G>A mutation site showed only one band at 912 bp in the isogenic control CMs, corresponding to wild-type mRNA, and two additional mRNA bands in untransduced CpHet CMs, which were larger in size compared to the wild-type band (i.e., 912 bp). The accumulation of these two abnormal MYBPC3 mRNAs produced by the endogenous mutant gene was prevented by transduction with AAV9-MYBPC3 vector constructs as described herein (e.g., A1, A2, A3, A4, A5, A6). C1, C4, and C5 had the least effect on the mutant mRNA bands.

[0434] Cardiovascular safety and toxicology studies will be conducted in relevant models. Dose-response studies will be performed to determine the transduction / protein expression % in MYBPC3 - / - mice to inform dose selection.

[0435] The embodiments described herein are only intended to be exemplary, and those skilled in the art will recognize, or will be able to determine using only routine experimentation, many equivalents of specific constructs, materials, and procedures. All such equivalents are considered to be within the scope of this disclosure.

[0436] All patents, patent applications, and publications mentioned herein are hereby incorporated by reference in their entirety. The 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 will be better understood from the appended claims.

Claims

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

2. The vector construct according to claim 1, wherein the nucleic acid encoding the functional cMyBP-C amino acid sequence is at least 85% identical to SEQ ID NO: 1 or any one of SEQ ID NOs: 42-45, or a complementary sequence thereof.

3. The vector construct according to any one of claims 1-2, wherein the nucleic acid has a sequence that is at least 97%, 98% or 99% identical to the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NOs: 42-45, or a complementary sequence thereof.

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

5. The vector construct according to 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 vector construct according to any one of claims 1-5, wherein the cardiomyocyte-specific transcriptional regulatory region comprises (a) a cardiomyocyte-specific promoter comprising a nucleotide sequence that is at least 80% identical to any one of SEQ ID NOs: 47-52, or a fragment or complementary sequence thereof, and (b) an intron comprising a nucleotide sequence that is 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 the functional human cMyBP-C.

7. The vector construct according to claim 6, which comprises a fragment of an exon, optionally an HbB exon.

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

9. The vector construct according to claim 7, which comprises the nucleotide sequence of SEQ ID NO: 56, or a complementary sequence thereof.

10. The vector construct according to any one of claims 1-3, which further comprises an intron.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

38. The rAAV particle according to claim 36, wherein the AAV capsid is an AAV serotype 9 capsid.

39. A method for producing the rAAV particle according to any one of claims 36 - 38, the method comprising the steps of: (a) providing a mammalian cell comprising one or more nucleic acid constructs, the one or more nucleic acid constructs comprising (i) the vector construct according to any one of claims 1 - 35, (ii) a nucleotide sequence encoding one or more AAV Rep proteins operably linked to a promoter, and (iii) a nucleotide sequence encoding one or more AAV capsid proteins operably linked to a promoter, (b) culturing the mammalian cell under conditions conducive to the expression of the Rep protein and the capsid protein, and (c) recovering the rAAV particle.

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

41. A method for producing rAAV particles, the method comprising the steps of: (a) providing to a cell permissive for AAV replication one or more nucleic acid constructs comprising: (i) A recombinant vector construct comprising (1) at least one AAV ITR, (2) a heterologous cardiomyocyte-specific transcriptional regulatory region, and (3) a nucleic acid encoding a functional human cardiac myosin binding protein C, (ii) A nucleotide sequence encoding one or more AAV Rep proteins, the nucleotide sequence being operably linked to a promoter capable of driving the expression of the one or more Rep proteins in the cell; and (iii) A nucleotide sequence encoding one or more AAV capsid proteins, the nucleotide sequence being operably linked to a promoter capable of driving the expression of the one or more capsid proteins in the cell; (b) Culturing the cells under conditions permitting the expression of the Rep proteins and the capsid proteins; and optionally (c) recovering the AAV particles.

42. The method of claim 41, wherein the cells are insect cells.

43. The method of claim 41, wherein the cells are mammalian cells.

44. The method of any one of claims 41-43, wherein the cells have the recombinant vector construct of any one of claims 1-35.

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

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

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

48. A method of treating hypertrophic cardiomyopathy, the method comprising administering to a patient with hypertrophic cardiomyopathy a therapeutically effective amount of the vector construct of any one of claims 1-35 or the rAAV particles of any one of claims 36-38 or the population of rAAV particles of claim 45 or the pharmaceutical composition of claim 46.

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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