Gene therapy vectors for treating heart disease

Abstract

The present disclosure provides methods and compositions useful for treating or preventing cardiac disease. In particular, the present disclosure provides a vector comprising a modified troponin T promoter operably linked to a therapeutic gene product for treating or preventing cardiac disease, such as cardiomyopathy. The gene product may be MYBPC3. The present disclosure also provides recombinant adeno-associated virus (rAAV) virions, rAAV viral genomes, and expression cassettes and pharmaceutical compositions thereof. The present disclosure further provides methods for treating diseases or disorders such as cardiac disease.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims priority to U.S. Provisional Application No. 63 / 047,633, filed on 2 July 2020, and U.S. Provisional Application No. 62 / 976,160, filed on 13 February 2020, the contents of which are incorporated herein by reference in their entirety.

[0002] Technical field This disclosure relates to compositions and methods for the treatment or prevention of cardiomyopathy in subjects. In particular, this disclosure relates to vectors comprising cardiac-specific promoter manipulability linked to therapeutic gene products for the treatment of cardiomyopathy.

[0003] Reference to sequence list This application was filed electronically via EFS-Web and includes an electronically submitted sequence listing in .txt format. The .txt file contains a sequence listing named “TENA_015_02WO_SeqList_ST25.txt” with a size of 824 kilobytes, created on February 9, 2021. The sequence listing contained in this .txt file is part of this specification and is incorporated herein by reference in its entirety. [Background technology]

[0004] background Gene therapy approaches for treating heart disease often utilize vectors configured to effectively transduce cardiac cells and express transgenes in a cardiac tissue-specific manner. Adeno-associated virus (AAV) vectors, cardiac-specific promoters, or a combination of both, can be used to deliver polynucleotides encoding gene products (e.g., therapeutic proteins) to cardiac tissue, thereby causing the tissue to express the gene product and treat heart disease. Cardiac-specific promoters include desmin (Des), alpha-myosin heavy chain (α-MHC), myosin light chain 2 (MLC-2), and cardiac troponin C (TNNC1 or cTnC) promoters, as well as the 600-base-pair cardiac troponin T (TNNT2) promoter. However, the delivery of polynucleotides encoding large proteins remains challenging, partly due to the packaging limitations of viral vectors.

[0005] Considering these challenges, there remains a need in this technology for improving gene therapy vectors for heart disease. [Overview of the project]

[0006] overview This disclosure relates, in general terms, to compositions and methods for the treatment or prevention of cardiomyopathy (e.g., cardiomyopathy). In a first aspect, this disclosure provides a vector comprising a promoter operably ligated to a polynucleotide encoding a therapeutic gene product for the treatment and prevention of cardiomyopathy, for example, a cardiac-specific promoter. The vector may be an adeno-associated virus (AAV) vector.

[0007] In some embodiments, the disclosure provides a cardiac troponin T promoter comprising a polynucleotide having 300 bp to 500 bp. In some embodiments, the polynucleotide comprises a sequence sharing at least 80%, at least 90%, or at least 100% identity with any one of SEQ ID NOs: 1 to 85. In some embodiments, the polynucleotide comprises a sequence sharing at least 80%, at least 90%, or at least 100% identity with SEQ ID NO: 1. In some embodiments, the polynucleotide comprises a sequence sharing at least 80%, at least 90%, or at least 100% identity with SEQ ID NO: 3. In some embodiments, the polynucleotide shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a genomic polynucleotide sequence containing the transcription start site of the troponin T gene upstream of it. In some embodiments, the polynucleotide shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a genomic polynucleotide sequence ranging from -450 bp to +1 bp relative to the transcription start site of the troponin T gene. In some embodiments, the polynucleotide shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a genomic polynucleotide sequence ranging from -350 bp to +1 bp relative to the transcription start site of the troponin T gene. In some embodiments, the polynucleotide shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a genomic polynucleotide sequence ranging from -250 bp to +1 bp relative to the transcription start site of the troponin T gene. In some embodiments, the polynucleotide shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a genomic polynucleotide sequence ranging from -450 bp to +50 bp relative to the transcription start site of the troponin T gene. In some embodiments, the polynucleotide shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a genomic polynucleotide sequence ranging from -350 bp to +50 bp relative to the transcription start site of the troponin T gene.In some embodiments, the polynucleotide shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a genomic polynucleotide sequence of -250 bp to +50 bp relative to the transcription start site of the troponin T gene. In some embodiments, the troponin T gene is the human troponin T gene.

[0008] In some embodiments, the promoter is a muscle-specific promoter. In some embodiments, the promoter is a cardiac cell-specific promoter. In some embodiments, the promoter is a cardiomyocyte-specific promoter. In some embodiments, the promoter has the same cell type specificity as the native troponin T promoter of about 600 bp. In some embodiments, the promoter described herein has the same cell type specificity as the reference promoter including SEQ ID NO: 1. In some embodiments, the promoter expresses at least about 10%, at least about 20%, and at least about 30% more operably ligated gene products than the native troponin T promoter. In some embodiments, the promoter described herein expresses at least about 10%, at least about 20%, and at least about 30% more operably ligated gene products than the reference promoter including SEQ ID NO: 1.

[0009] In some embodiments, the Disclosure provides a vector comprising one of the promoters described herein operably ligated to a polynucleotide encoding a gene product. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adeno-associated viral vector (AAV). In some embodiments, the viral vector has a packaging limit of up to about 5.5 kb.

[0010] In some embodiments, the gene product is selected from the MYBPC3, KCNH2, TRPM4, DSG2, and ATP2A2 proteins. In some embodiments, the gene product is selected from the CACNA1C, DMD, DMPK, EPG5, EVC, EVC2, FBN1, NF1, SCN5A, SOS1, NPR1, ERBB4, VIP, and MYH7 proteins. In some embodiments, the gene product is Cas9, optionally selected from SpCas9, St1Cas9, and SaCas9.

[0011] In some embodiments, the vector described herein includes a polynucleotide encoding a second gene product. In some embodiments, the second gene product is a functional RNA, optionally a microRNA, or a guide RNA.

[0012] In some embodiments, the disclosure provides isolated cells containing any one of the promoters described herein. In some embodiments, the isolated cells are induced pluripotent stem cells or isolated cardiomyocytes.

[0013] In some embodiments, this disclosure provides a pharmaceutical composition comprising one of the vectors described herein.

[0014] In some embodiments, the Disclosure provides a cell therapy composition comprising any one of the isolated cells described herein.

[0015] In some embodiments, the disclosure provides a recombinant adeno-associated virus (AAV) vector genome comprising a MYBPC3 polynucleotide encoding the MYBPC3 protein, or a functional variant thereof, and a promoter, wherein the promoter is a 300 bp to 500 bp polynucleotide. In some embodiments, the polynucleotide comprises a sequence that shares at least 80%, at least 90%, or at least 100% identity with any one of SEQ ID NOs: 1 to 85. In some embodiments, the polynucleotide comprises a sequence that shares at least 80%, at least 90%, or at least 100% identity with any one of SEQ ID NOs: 1. In some embodiments, the polynucleotide comprises a sequence that shares at least 80%, at least 90%, or at least 100% identity with any one of SEQ ID NOs: 3. In some embodiments, the polynucleotide shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a genomic polynucleotide sequence containing the transcription start site of the troponin T gene upstream of it. In some embodiments, the polynucleotide shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a genomic polynucleotide sequence ranging from -450 bp to +1 bp relative to the transcription start site of the troponin T gene. In some embodiments, the polynucleotide shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a genomic polynucleotide sequence ranging from -350 bp to +1 bp relative to the transcription start site of the troponin T gene. In some embodiments, the polynucleotide shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a genomic polynucleotide sequence ranging from -250 bp to +1 bp relative to the transcription start site of the troponin T gene. In some embodiments, the polynucleotides share at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a genomic polynucleotide sequence of -450 bp to +50 bp relative to the transcription start site of the troponin T gene.In some embodiments, the polynucleotide shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a genomic polynucleotide sequence of -350 bp to +50 bp relative to the transcription start site of the troponin T gene. In some embodiments, the polynucleotide shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a genomic polynucleotide sequence of -250 bp to +50 bp relative to the transcription start site of the troponin T gene. In some embodiments, the troponin T gene is the human troponin T gene.

[0016] In some embodiments, the promoter is a muscle-specific promoter. In some embodiments, the promoter is a cardiac cell-specific promoter. In some embodiments, the promoter is a cardiomyocyte-specific promoter. In some embodiments, the promoter has the same cell type specificity as the approximately 600 bp native troponin T promoter. In some embodiments, the promoter has the same cell type specificity as the reference promoter containing SEQ ID NO: 1. In some embodiments, the promoter expresses at least approximately 10%, at least approximately 20%, and at least approximately 30% more operably ligated gene products than the native troponin T promoter. In some embodiments, the promoter expresses at least approximately 10%, at least approximately 20%, and at least approximately 30% more operably ligated gene products than the reference promoter containing SEQ ID NO: 1.

[0017] In some embodiments, the recombinant adeno-associated virus (AAV) vector genome described herein includes a MYBPC3 polynucleotide encoding the MYBPC3 protein. In some embodiments, the MYBPC3 polynucleotide contains at least about 3.5 kB. In some embodiments, the MYBPC3 polynucleotide contains about 3.8 kB. In some embodiments, MYBPC3 is full-length MYBPC3. In some embodiments, MYBPC3 is a shortened MYBPC3.

[0018] In some embodiments, the rAAV vector genome described herein expresses MYBPC3. In some embodiments, the rAAV vector genome expresses MYBPC3 at approximately the same level as a reference AAV vector containing a native troponin T promoter of about 600 bp. In some embodiments, the rAAV vector genome expresses MYBPC3 at a level at least about 10% higher than a reference AAV vector containing a native troponin T promoter of about 600 bp. In some embodiments, the rAAV vector genome expresses MYBPC3 at a level at least about 20% higher than a reference AAV vector containing a native troponin T promoter of about 600 bp.

[0019] In some embodiments, the Disclosure provides a recombinant adeno-associated virus (AAV) vector genome comprising, in 5' to 3' order, an expression cassette comprising a 5' segment containing a promoter, a polynucleotide encoding a gene product, a 3' segment containing a poly-A signal, and an expression cassette optionally flanked by one or both of a 5' inverted terminal repeat (ITR) and a 3'ITR, wherein the polynucleotide encoding the gene product comprises 3kb–11kb, 3kb–5kb, 3.5kb–4.5kb or 3.7kb–4kb, and a) the 5' segment and 3' segment both comprise up to 0.8kbp or up to 0.9kbp, b) the 5'ITR, 5' segment, 3' segment and 3'ITR each comprise up to 1.2kbp or up to 1.3kbp, and / or c) the vector genome comprises up to 4.7kbp, up to 4.8kbp, up to 4.9kbp or up to 5.0kbp. In some embodiments, the 5' segment includes a maximum of 500 bp or a maximum of 480 bp. In some embodiments, the 3' segment includes a maximum of 200 bp or a maximum of 150 bp.

[0020] In some embodiments, the rAAV vector genome contains a polynucleotide encoding a gene product ranging from 3.7 kbp to 3.9 kbp, optionally 3.8 kbp. In some embodiments, the gene product is MYBPC3 or a functional variant thereof. In some embodiments, the gene product is MYBPC3. In some embodiments, the polynucleotide encoding MYBPC3 shares at least 90% identity with SEQ ID NO: 86. In some embodiments, the polynucleotide encoding MYBPC3 shares at least 95% identity with SEQ ID NO: 86. In some embodiments, the polynucleotide encoding MYBPC3 is SEQ ID NO: 86. In some embodiments, MYBPC3 shares at least 90% identity with the peptide sequence of SEQ ID NO: 103. In some embodiments, MYBPC3 shares at least 95% identity with the peptide sequence of SEQ ID NO: 103. In some embodiments, MYBPC3 shares 100% identity with the peptide sequence of SEQ ID NO: 103.

[0021] In some embodiments, the rAAV vector genome includes a promoter, which is a polynucleotide having 300 bp to 500 bp. In some embodiments, the promoter includes a sequence that shares at least 80% identity with SEQ ID NOs. 1 to 85 or SEQ ID NOs. 91. In some embodiments, the promoter includes a sequence that shares at least 90% identity with SEQ ID NOs. 1 to 85 or SEQ ID NOs. 91. In some embodiments, the promoter includes a sequence that shares at least 100% identity with SEQ ID NOs. 1 to 85 or SEQ ID NOs. 91.

[0022] In some embodiments, the rAAV vector genome includes a polyA signal. In some embodiments, the polyA signal essentially consists of or includes a sequence that shares at least 90% identity with SEQ ID NO: 92. In some embodiments, the polyA signal essentially consists of or includes a sequence that shares at least 95% identity with SEQ ID NO: 92. In some embodiments, the polyA signal is SEQ ID NO: 92.

[0023] In some embodiments, the rAAV vector genome includes a 5' segment. In some embodiments, the 5' segment shares at least 80% identity with SEQ ID NO: 93. In some embodiments, the 5' segment shares at least 90% identity with SEQ ID NO: 93. In some embodiments, the 5' segment shares at least 95% identity with SEQ ID NO: 93. In some embodiments, the 5' segment is SEQ ID NO: 93.

[0024] In some embodiments, the rAAV vector genome includes a 3' segment. In some embodiments, the 3' segment shares at least 80% identity with SEQ ID NO: 94. In some embodiments, the 3' segment shares at least 90% identity with SEQ ID NO: 94. In some embodiments, the 3' segment shares at least 95% identity with SEQ ID NO: 94. In some embodiments, the 3' segment is SEQ ID NO: 94.

[0025] In some embodiments, the rAAV vector genome includes an expression cassette. In some embodiments, the expression cassette shares at least 80% identity with SEQ ID NO: 95. In some embodiments, the expression cassette shares at least 90% identity with SEQ ID NO: 95. In some embodiments, the expression cassette shares at least 95% identity with SEQ ID NO: 95. In some embodiments, the expression cassette is SEQ ID NO: 95.

[0026] In some embodiments, the rAAV genome includes an expression cassette adjacent to one or both of the 5' inverted terminal repeat (ITR) and the 3' ITR. In some embodiments, the 5' ITR includes a sequence that shares 95% identity with SEQ ID NO: 96. In some embodiments, the 3' ITR includes a sequence that shares at least 95% identity with SEQ ID NO: 97.

[0027] In some embodiments, this disclosure provides recombinant AAV (rAAV) virions. In some embodiments, the rAAV virion comprises one of the rAAV vector genomes and AAV capsid proteins described herein.

[0028] In some embodiments, the Disclosure provides a method for expressing the MYBPC3 protein in cells, comprising transducing the cells with either an rAAV virion described herein or an rAAV vector genome described herein. In some embodiments, the cells express the MYBPC3 protein - / - The cell is a cell. In some embodiments, the cell contains an inactivating mutation in one or both copies of the endogenous MYBPC3 gene.

[0029] In certain embodiments, the Disclosure provides a method for treating and / or preventing cardiomyopathy in subjects in need, comprising administering either an rAAV virion described herein or an rAAV vector genome described herein to a subject who is or is at risk of developing cardiomyopathy.

[0030] In certain embodiments, the Disclosure provides a method for expressing the MYBPC3 protein in the heart of a subject requiring it, and includes administering either an rAAV virion or an rAAV vector genome as described herein to a subject who is selectively suffering from or at risk of cardiomyopathy.

[0031] In some embodiments, administration of the AAV vector induces specific expression of MYBPC3 in the subject's heart. In some embodiments, administration of the AAV vector induces low or undetectable expression of MYBPC3 in the subject's skeletal tissue, brain, and / or liver, and optionally, the subject has or is at risk of cardiomyopathy.

[0032] In certain embodiments, the Disclosure provides a method for treating and / or preventing cardiomyopathy caused by MYBPC3 mutations in subjects in need thereof, comprising administering either an rAAV virion described herein or an rAAV vector genome described herein, optionally, to subjects who have cardiomyopathy or are at risk of developing cardiomyopathy.

[0033] In certain embodiments, the Disclosure provides a method for increasing MYBPC3 activity and / or increasing cardiac function in the heart of a subject requiring such action, comprising administering either one of the rAAV virions described herein or one of the rAAV vector genomes described herein, optionally, the subject having cardiomyopathy or being at risk of cardiomyopathy.

[0034] In some embodiments, the methods described herein treat cardiomyopathy. In some embodiments, the methods described herein prevent cardiomyopathy. In some embodiments, the cardiomyopathy is hypertrophic cardiomyopathy.

[0035] In some embodiments, the methods described herein include intravenous administration to either the rAAV virion described herein or the rAAV vector genome described herein. In some embodiments, the methods described herein include intracardiac administration to either the rAAV virion described herein or the rAAV vector genome described herein. In some embodiments, the methods described herein include direct administration to either the rAAV virion described herein or the rAAV vector genome described herein. In some embodiments, the methods described herein involve approximately 10 per kg 11 ~about 10 14 This includes administering a dose of rAAV virion or a dose of viral genome per kg.

[0036] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is an adult.

[0037] In some embodiments, the pharmaceutical compositions described herein are intended for use as agents in the therapeutic or prophylactic treatment of cardiomyopathy, for example. [Brief explanation of the drawing]

[0038] [Figure 1A] Figure 1A shows a map of insertion sequences in the AAV vector genome adapted for large cargo, illustrating the deletion or cleavage of two cis-regulatory elements.

[0039] [Figure 1B] Figure 1B shows flow cytometry analysis of human cardiac fibroblasts (n=2) with an AAV packaging construct and an MOI of 160,000, 2 days after infection.

[0040] [Figure 1C] Figure 1C shows a map of insertion sequences in the AAV vector genome adapted for the large cargo, illustrating deletions or cleavage of two cis-regulatory elements, intron deletions, and partial deletions of the 3'–5'ITR sequence.

[0041] [Figure 2A] Figure 2A shows schematic diagrams of the original and modified versions of the viral genome, including the cardiac-specific troponin (TNNT2) promoter and the myosin-binding protein C (MYBPC3) transgene.

[0042] [Figure 2B] Figure 2B shows the detection of MYBPC3 protein by immunofluorescence in MYBPC3- / - iPSC-derived cardiomyocytes transduced with an AAV6 package construct encoding MYBPC3 driven by a heart-specific TNNT2 promoter.

[0043] [Figure 3A] Figure 3A shows the detection of MYBPC3 protein by Western blotting in MYBPC3- / - iPSC-derived cardiomyocytes transduced with AAV6 package constructs encoding human MYBPC3 driven by cardiac-specific TNNT2 promoters of varying sizes (400–600 bp). GAPDH was used as a loading control.

[0044] [Figure 3B] Figure 3B shows the detection of MYBPC3 protein by Western blotting in MYBPC3- / - iPSC-derived cardiomyocytes transduced with AAV6 package constructs encoding human MYBPC3 driven by human cardiac TNNT2 promoters of varying sizes (400 or 600 bp). Kozak sequences were not used as a negative control, and GAPDH was used as a loading control.

[0045] [Figure 3C] Figure 3C shows the detection of MYBPC3 protein by Western blotting in MYBPC3- / -iPSC-derived cardiomyocytes transfected with AAV6 plasmids encoding human MYBPC3 driven by human cardiac TNNT2 promoters of varying sizes (400 or 600 bp). GAPDH was used as a loading control.

[0046] [Figure 4A] Figure 4A shows a dot blot map of the introns of the Mybpc3 gene and MYBPC3 protein expression in the founder mouse of the KO strain.

[0047] [Figure 4B] Figure 4B shows bar graphs of littermates of 2-week-old wild-type (WT) or KO (Mybpc3- / -) mice. WT (n=11), Mybpc3+ / - (n=7), and Mybpc3- / - (n=13).

[0048] [Figure 4C]Figure 4C shows a bar graph of ejection fraction (%) measured by echocardiography in 2-week-old wild-type (WT), heterozygous KO (Mybpc3+ / -), or homozygous KO (Mybpc3- / -) mice.

[0049] [Figure 4D] Figure 4D shows a bar graph of shortening rates measured by echocardiography in 2-week-old wild-type (WT), heterozygous KO (Mybpc3+ / -), or homozygous KO (Mybpc3- / -) mice.

[0050] [Figure 4E] Figure 4E shows a bar graph of left ventricular (LV) mass normalized by body weight (BW) in 2-week-old wild-type (WT), heterozygous KO (Mybpc3+ / -), or homozygous KO (Mybpc3- / -) mice.

[0051] [Figure 4F] Figure 4F shows a bar graph of left ventricular diameter (LVID) during cardiac contraction, normalized by body weight, for 2-week-old wild-type (WT), heterozygous KO (Mybpc3+ / -), or homozygous KO (Mybpc3- / -) mice.

[0052] [Figure 4G] Figure 4G shows a bar graph of left ventricular diameter (LVID) during diastole, normalized by body weight, for 2-week-old wild-type (WT), heterozygous KO (Mybpc3+ / -), or homozygous KO (Mybpc3- / -) mice.

[0053] [Figure 5] Figure 5 shows the detection of MYBPC3 mRNA by qRT-PCR in cardiac, skeletal, brain, and liver tissues isolated from mice injected postorbitally with E12 GC AAV9 package constructs encoding human MYBPC3 driven by human cardiac TNNT2 promoters of various sizes (400 or 600 bp).

[0054] [Figure 6A] Figure 6A shows a bar graph illustrating the absolute quantification of vector genome per microgram of genomic DNA in the heart and liver of adult mice four weeks after intravenous administration of an AAV9 vector containing a 400 bp modified TNNT2 promoter cassette.

[0055] [Figure 6B] Figure 6B shows a bar graph illustrating the doubling of transgene RNA in the vehicle in the heart and liver of adult mice four weeks after intravenous administration of an AAV9 vector containing a 400 bp modified TNNT2 promoter cassette.

[0056] [Figure 7] Figure 7 shows a Western blot of MYBPC3 protein expression in 2-week-old homozygous Mybpc3- / - mice injected postorbitally with the test vector 1E14 vg·kg-1 encoding Mybpc3 or the vehicle, HBSS.

[0057] [Figure 8] Figure 8 shows a bar graph illustrating MYBPC3 expression in 2-week-old homozygous Mybpc3- / - mice injected postorbitally with test vectors 3E13 vg·kg-1 and 1E14 vg·kg-1, which encode Mybpc3 or the vehicle, HBSS.

[0058] [Figure 9A] Figure 9A shows bar graphs of left ventricular mass (LVM / BM), normalized to body weight, 6 weeks after post-orbital injection of test vectors 1E13 vg·kg-1, 3E13 vg·kg-1, and 1E14 vg·kg-1 encoding Mybpc3 or vehicle, HBSS, into 2-week-old homozygous Mybpc3- / - mice.

[0059] [Figure 9B]Figure 9B shows a bar graph representing the change in LV diameter between systole and diastolic position, expressed as a percentage, at 6 weeks after post-orbital injection of test vectors 1E13 vg·kg-1, 3E13 vg·kg-1, and 1E14 vg·kg-1 encoding Mybpc3 or the vehicle, HBSS, into 2-week-old homozygous Mybpc3- / - mice.

[0060] [Figure 9C] Figure 9C shows a bar graph of ejection rates 6 weeks after post-orbital injection of test vectors 1E13 vg·kg-1, 3E13 vg·kg-1, and 1E14 vg·kg-1 encoding Mybpc3 or the vehicle, HBSS, into 2-week-old homozygous Mybpc3- / - mice.

[0061] [Figure 9D] Figure 9D shows a bar graph of body-normalized left ventricular mass (LVM / BM) 31 weeks after post-orbital injection of test vectors 1E13 vg·kg-1, 3E13 vg·kg-1, and 1E14 vg·kg-1 encoding Mybpc3 or vehicle, HBSS, into 2-week-old homozygous Mybpc3- / - mice.

[0062] [Figure 9E] Figure 9E shows a bar graph representing the change in LV diameter between systole and diastolic position, expressed as a percentage, at 31 weeks after post-orbital injection of test vectors 1E13 vg·kg-1, 3E13 vg·kg-1, and 1E14 vg·kg-1 encoding Mybpc3 or the vehicle HBSS into 2-week-old homozygous Mybpc3- / - mice.

[0063] [Figure 9F] Figure 9F shows a bar graph of ejection rates 31 weeks after post-orbital injection of test vectors 1E13 vg·kg-1, 3E13 vg·kg-1, and 1E14 vg·kg-1 encoding Mybpc3 or the vehicle, HBSS, into 2-week-old homozygous Mybpc3- / - mice.

[0064] [Figure 10A] Figure 10A shows the AAV9 vector encoding Mybpc3 in association with a 5.4 kbp or 4.7 kbp expression cassette.

[0065] [Figure 10B] Figure 10B shows a bar graph indicating the ejection rate 18 weeks after post-orbital injection of the 3E13 vg·kg-1 or 1E14 vg·kg-1 AAV9 vector encoding Mybpc3 into 3-month-old homozygous Mybpc3- / - mice, or after injection of a vehicle control or HBSS, in association with a 5.4 kbp or 4.7 kbp cassette.

[0066] [Figure 10C] Figure 10C shows a bar graph illustrating the advancement of ejection fraction after post-orbital injection of AAV9 vectors encoding Mybpc3, 3E13 vg·kg-1 or 1E14 vg·kg-1, into 3-month-old homozygous Mybpc3- / - mice, or after injection of a vehicle control or HBSS, in association with a 5.4 kbp or 4.7 kbp cassette.

[0067] [Figure 10D] Figure 10D shows bar graphs normalized to body weight left ventricular mass (LVM / BM) 18 weeks after post-orbital injection of 3E13 vg·kg-1 or 1E14 vg·kg-1 AAV9 vectors encoding Mybpc3 into 3-month-old homozygous Mybpc3- / - mice, or after injection of vehicle control, HBSS, in association with 5.4 kbp or 4.7 kbp cassettes.

[0068] [Figure 11A] Figure 11A is a bar graph showing GFP expression in cardiac tissue of adult mice with or without the GFP-coded cassette after systemic delivery of the AAV9 capsid variant CR9-10 (p < 0.05, one-way ANOVA, Dunnett's multiple comparison test).

[0069] [Figure 11B] Figure 11B shows a bar graph illustrating the ejection fraction when AAV9 vectors encoding 1E13 vg·kg-1 and 3E13 vg·kg-1 Mybpc3, CR9-10 vectors encoding Mybpc3, or vehicle control HBSS were injected postorbitally into 2-week-old Mybpc3- / - mice.

[0070] [Figure 11C] Figure 11C shows bar graphs comparing ejection fraction (ΔEF) to baseline before administration when AAV9 vectors encoding the 1E13 vg·kg-1 and 3E13 vg·kg-1 expression cassette Mybpc3 genes, CR9-10 vectors encoding the Mybpc3 expression cassette gene, or vehicle control HBSS were injected postorbitally into 2-week-old Mybpc3- / - mice.

[0071] [Figure 12A] Figure 12A is a bar graph showing GFP expression in the left ventricle of non-human primates one month after intravenous delivery of an AAV vector at a dose of 1E13 vg·kg-1 encoding GFP packaged in one of 14 different capsid proteins.

[0072] [Figure 12B] Figure 12B is a bar graph showing GFP expression in the liver of non-human primates one month after intravenous delivery of an AAV vector at a dose of 1E13 vg·kg-1 encoding GFP, packaged in one of 14 different capsid proteins.

[0073] [Figure 12C] Figure 12C is a bar graph showing the ratio of GFP expression in the left ventricle:liver of non-human primates one month after intravenous delivery of an AAV vector at a dose of 1E13 vg·kg-1 encoding GFP packaged in one of 14 different capsid proteins.

[0074] [Figure 13A] Figure 13A is a plot showing the advancement of ejection fraction when AAV9 encoding the mouse Mybpc3 gene (mMybpc3) (1E14 vg·kg-1), AAV9 encoding the human MYBPC3 gene (hMYBPC3) (1E14 vg·kg-1), or the vehicle HBSS was injected postorbitally into 2-week-old homozygous Mybpc3- / - mice.

[0075] [Figure 13B] Figure 13B is a plot showing the advancement of left ventricular mass (LVM / BW), normalized to body weight, when AAV9 encoding the mouse Mybpc3 gene (mMybpc3) (1E14 vg·kg-1), AAV9 encoding the human MYBPC3 gene (hMYBPC3) (1E14 vg·kg-1), or the vehicle HBSS was injected postorbitally into 2-week-old homozygous Mybpc3- / - mice.

[0076] [Figure 13C] Figure 13C is a bar graph showing the diastolic left ventricular posterior wall thickness (LVPW;d) when AAV9 encoding the mouse Mybpc3 gene (mMybpc3) (1E14 vg·kg-1), AAV9 encoding the human MYBPC3 gene (hMYBPC3) (1E14 vg·kg-1), or the vehicle HBSS was injected postorbitally into 2-week-old homozygous Mybpc3- / - mice.

[0077] [Figure 14A] Figure 14A shows a bar graph illustrating the ejection fraction when test vectors 1E13 vg·kg-1, 1E14 vg·kg-1, and 3E14 vg·kg-1, which encode the human MYBPC3 gene or vehicle, or HBSS, were injected postorbitally into 2-week-old homozygous Mybpc3- / - mice.

[0078] [Figure 14B]Figure 14B shows bar graphs comparing ejection fractions (ΔEF) to baseline before administration when test vectors 1E13 vg·kg-1, 1E14 vg·kg-1, and 3E14 vg·kg-1 encoding the human MYBPC3 gene or vehicle, HBSS, were injected postorbitally into 2-week-old homozygous Mybpc3- / - mice.

[0079] [Figure 14C] Figure 14C shows bar graphs of left ventricular mass (LVM / BW), normalized to body weight, when test vectors 1E13 vg·kg-1, 1E14 vg·kg-1, and 3E14 vg·kg-1 encoding human MYBPC3 or vehicle, HBSS, were injected postorbitally into 2-week-old homozygous Mybpc3- / - mice. [Modes for carrying out the invention]

[0080] Detailed explanation This disclosure provides compositions and methods for gene therapy using cardiac cells and / or large genes. The disclosed polynucleotides and vectors may be used to treat or prevent diseases (e.g., cardiomyopathy, a type of heart disease). This disclosure provides cardiac-specific promoters, expression cassettes, recombinant adeno-associated virus (rAAV) viral genomes, rAAV virions, pharmaceutical compositions, and methods of use. The expression cassettes and rAAV viral genomes may include a cardiac-specific promoter operably ligated to a polynucleotide encoding a gene product. The gene product may be a therapeutic gene product, such a therapeutic gene product used to treat and / or prevent cardiomyopathy, a type of heart disease. This disclosure further provides rAAV viral genomes and expression cassettes engineered to deliver and express large gene products. In some embodiments, the vector genome includes a MYBPC3 polypeptide, or a functional variant thereof, and a polynucleotide encoding a promoter. In some embodiments, the promoter is a cardiac troponin T promoter (i.e., a TNNT2 promoter). In some embodiments, the rAAV vector genome comprises a gene product, e.g., a polynucleotide encoding MYBPC3, and an expression cassette comprising a promoter, e.g., a TNNT2 promoter, adjacent to one or more inverted terminal repeat polynucleotide sequences. In some embodiments, the rAAV virion comprises a polynucleotide comprising the rAAV vector genome described herein and an AAV capsid protein. This disclosure also provides pharmaceutical compositions comprising the vector genome, rAAV vector genome, and rAAV virion described herein. Furthermore, methods for treating and / or preventing cardiomyopathy in a subject are provided, comprising administering the rAAV virion or vector genome described herein.

[0081] Other embodiments, features, and advantages of the present invention are evident from and encompassed by the following detailed description and claims. I. Definition

[0082] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless otherwise explicitly indicated by the content.

[0083] When used herein, unless otherwise suggested, the terms “and / or” are used in this disclosure to mean either “and” or “or.”

[0084] Throughout this specification, unless otherwise required by context, the term “contains,” or variations thereof such as “contains,” will be understood to imply the inclusion of a specified element or integer or group of elements or integers, but not the exclusion of other elements or integers or groups of elements or integers.

[0085] Where used in this application, the terms “about” and “approximately” are used as equivalents. Any figures used in this application, with or without “about” or “approximately,” are intended to encompass any normal variation recognized by a person skilled in the art. In certain embodiments, unless otherwise stated or otherwise evident from the context, the terms “approximately” or “about” refer to values ​​in the range of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or the smaller of these (except where such figures may exceed 100%).

[0086] The terms “polynucleotide” and “nucleic acid,” as used interchangeably herein, refer to polymeric forms of nucleotides exceeding approximately 100 nucleotides, either ribonucleotides or deoxyribonucleotides. Therefore, these terms include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, unnatural, or derivatized nucleotide bases. “Oligonilocyte” generally refers to polynucleotides of approximately 5 to approximately 100 nucleotides in single-stranded or double-stranded DNA. However, for the purposes of this disclosure, there is no upper limit to the length of oligonucleotides. Oligonucleotides are also known as “oligomers” or “oligos” and may be isolated from genes or chemically synthesized by methods known in the art. The terms “polynucleotide” and “nucleic acid” should be understood to include single-stranded (e.g., sense or antisense) and double-stranded polynucleotides, where applicable to the embodiments described.

[0087] As used herein, the term “promoter” refers to a polynucleotide sequence having a recognition site to which RNA polymerase binds, enabling RNA polymerase to initiate transcription of the polynucleotide sequence “downstream” of the promoter in a host or target cell. Similarly, a “promoter” is operably ligated to or operablely ligated to a polynucleotide sequence in which RNA polymerase initiates transcription of the polynucleotide at the transcription state site in a host or target cell where the promoter is active. Promoters acting in mammalian cells generally include an AT-rich region located approximately 25–30 bases upstream of the site where transcription is initiated, and / or another sequence, the CNCAAT region, found 70–80 bases upstream from the start of transcription, where N may be any nucleotide.

[0088] The terms “upstream” and “upstream end” refer to a portion of a polynucleotide that is 5' to TSS on the sense strand (or coding strand) and 3' to TSS on the antisense strand, with reference to the transcription start site (TSS). The terms “downstream” and “downstream end” refer to a portion of a polynucleotide that is 3' to TSS on the sense strand (or coding strand) and 5' to TSS on the antisense strand, with reference to the TSS. Therefore, a deletion from the upstream end of a promoter is a deletion of one or more base pairs in the non-transcription region of a polynucleotide from 5' to TSS on the sense strand (or from 3' to TSS on the antisense strand). A deletion from the downstream end of a promoter is a deletion of one or more base pairs in the transcription region of a polynucleotide from 5' to TSS on the sense strand (or from 3' to TSS on the antisense strand).

[0089] As used herein, the term “transgene” refers to a nucleic acid sequence that codes for a protein or RNA (e.g., a therapeutic protein) that is partially or completely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or homologous to an endogenous gene in the transgenic animal or cell into which it is introduced, but is designed or inserted into the animal’s genome in such a way that it alters the genome of the cell into which it is inserted (e.g., inserted at a different location than the native gene, or resulting in knockout). A transgene may include one or more transcriptional regulatory sequences and any other nucleic acids, such as introns, which may be necessary for the optimal expression of the selected nucleic acid.

[0090] The term "sequence identity" refers to the ratio of bases or amino acids between two polynucleotide or polypeptide sequences that are identical and occupy the same relative positions. Thus, one polynucleotide or polypeptide sequence has a certain degree of sequence identity compared to another polynucleotide or polypeptide sequence. For sequence comparison, typically, one sequence acts as a reference sequence, against which the test sequence is compared. The term "reference sequence" refers to the molecule against which the test sequence is compared.

[0091] Methods for sequence alignment for comparing and determining sequence identity percentages are well known in the art. Optimal alignment of sequences for comparison can be achieved, for example, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), by manual alignment and visual inspection (see, e.g., Brent et al., Current Protocols in Molecular Biology (2003)), by use of algorithms known in the art, including the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977), and Altschul et al. This was performed by al., J.Mol.Biol.215:403-410 (1990). The software for performing BLAST analysis is publicly available through the National Biotechnology Information Center.

[0092] In some embodiments, the determination of the sequence identity ratio may be performed after local alignment. Such alignments are well known in the art, and for example, the service EMBOSS Matcher identifies local similarities between two sequences using an algorithm based on the LALIGN application, version 2.0u4. In one example, identity between two nucleic acid sequences may be calculated using the service Matcher (EMBOSS) set to default parameters such as matrix (DNAfull), gap open (16), gap extended (4), and substitute match (1).

[0093] An "expression cassette" or "expression construct" refers to a DNA polynucleotide sequence that is operably ligated to a promoter. "Operablely ligated" or "operable ligated" means that the components described in this way are juxtaposed in a relationship that allows them to function in the manner intended. For example, if a promoter affects the transcription or expression of a polynucleotide sequence, the promoter is operably ligated to the polynucleotide sequence.

[0094] As used herein, the term “delivery” is used interchangeably with “transduction,” but refers to the process by which an exogenous nucleic acid molecule is delivered into a cell so that it is located within the cell. Nucleic acid delivery is a different process from nucleic acid expression.

[0095] The term "modified" refers to a substance or compound (e.g., cell, polynucleotide sequence, and / or polypeptide sequence) that has been altered or modified compared to the corresponding unmodified substance or compound.

[0096] The term “sample” refers to a biological composition (e.g., a portion of a cell or tissue) subjected to analysis and / or genetic modification. In some embodiments, the sample is a “primary sample” obtained directly from the subject, and in some embodiments, it is the result of processing the primary sample to remove specific components and / or isolate or purify specific target components.

[0097] The term “gene” or “recombinant gene” refers to a nucleic acid containing an open reading frame that codes for a polypeptide, which includes both exon sequences and (optionally) intron sequences.

[0098] The term "transfection" refers to the uptake of exogenous DNA by a cell. When exogenous DNA is introduced into the cell membrane, the cell is "transfected." Several transfection techniques are generally known in this art. For example, Graham et al., Virology 52:456 (1973); Sambrook et al., Molecular Cloning: A Laboratory Manual (1989); Davis et al., Basic Methods in Molecular Biology (1986); Chu et al., Gene 13:197 (see 1981). These techniques can be used to introduce one or more exogenous DNA portions, such as nucleotide embedding vectors and other nucleic acid molecules, into suitable host cells. The term encompasses both chemical and electrical transfection procedures.

[0099] The term "expression" refers to the process by which nucleic acids are translated into peptides or transcribed into RNA, which can be, for example, peptides, polypeptides, or proteins. If the nucleic acid originates from genomic DNA, expression may involve splicing of mRNA, provided that a suitable eukaryotic host cell or organism is selected. For heterologous nucleic acids to be expressed in a host cell, they must first be delivered into the cell, then temporarily within the cell, and finally within the nucleus.

[0100] The term "gene therapy" involves the transfer of heterologous DNA to mammalian, particularly human, cells that have a disorder or condition for which treatment or diagnosis is sought. The DNA is introduced into selected target cells so that the heterologous DNA is expressed and the therapeutic product encoded by it is produced. Alternatively, the heterologous DNA may in some way mediate the expression of the DNA encoding the therapeutic product, or it may encode a product such as a peptide or RNA, and in some way directly or indirectly mediate the expression of the therapeutic product. Gene therapy can also be used to deliver nucleic acids encoding a gene product to replace a defective gene, or to supplement gene products produced by the mammal or the cells into which it is introduced. The introduced nucleic acid may encode a therapeutic gene product that is not normally produced in the mammalian host, or not produced in therapeutically effective amounts or for a therapeutically effective duration. The heterologous DNA encoding the therapeutic product may be modified before being introduced into the cells of the affected host to enhance the product or its expression, or otherwise alter it.

[0101] As used herein, “heterogeneous” polynucleotides or nucleic acids refer to polynucleotides or portions of polynucleotides derived from sources other than the host organism, or, in the case of viral vectors, from naturally occurring non-recombinant viruses. Examples of heterogeneous DNA include, but are not limited to, DNA encoding traceable marker proteins such as drugs that confer resistance, DNA encoding therapeutic agents such as anticancer drugs, enzymes and hormones, and DNA encoding other types of proteins such as antibodies.

[0102] The term "wild-type" refers to a protein, a part thereof, a protein sequence, or a naturally occurring polynucleotide sequence that codes for a protein or a part thereof, which are normally present in vivo in normal or healthy subjects.

[0103] The term "variant" refers to a protein or nucleic acid that has one or more genetic alterations (e.g., insertions, deletions, substitutions, etc.) that restore all or substantially all of the function of a reference protein or nucleic acid. For example, a variant of a therapeutic protein retains the same or substantially the same activity and / or provides the same or substantially the same therapeutic benefit to the target that needs it. A variant of a promoter sequence retains the same or substantially the same ability to initiate transcription at the same or substantially the same level as the reference promoter and retains the same or substantially the same cell type specificity. In certain embodiments, the polynucleotide variant has at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence reference. In certain embodiments, the protein variant has at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence reference.

[0104] The term "subject" includes animals, such as mammals. In some embodiments, mammals are primates. In some embodiments, mammals are humans. In some embodiments, subjects are livestock such as cattle, sheep, goats, pigs, or domestic animals such as dogs and cats. In some embodiments (for example, particularly in the research context), subjects are rodents (e.g., mice, rats, hamsters), rabbits, primates, or pigs such as inbred pigs. The terms "subject" and "patient" are used interchangeably herein.

[0105] The term "administration" to a subject refers to the procedure of introducing or applying one or more delivery agents together or separately to a subject, ultimately bringing the target cells present in the subject into contact with the drug.

[0106] As used herein, “to treat” means to deliver a drug or composition to a target in order to affect a physiological outcome.

[0107] As used herein, the term “gene product” refers to a protein or nucleic acid produced by the transcription of a polynucleotide, and in the case of a protein gene product, refers to the subsequent translation of the transcript into a protein. “Therapeutic gene product” refers to a gene product that, when expressed in a therapeutic dose in a subject, provides a therapeutic physiological effect or benefit to a subject in need.

[0108] As used herein, the term “therapeutic protein” refers to a protein or polypeptide that, when expressed or administered in therapeutic doses in a subject, provides a therapeutic physiological effect or benefit to a subject in need. In some embodiments, therapy using a therapeutic protein or a vector expressing a therapeutic protein provides a therapeutic physiological effect or benefit to a subject with heart disease (e.g., a subject with cardiomyopathy). Exemplary therapeutic proteins for the treatment of heart disease are provided in Table 2.

[0109] As used herein, the term “cardiomyopathy” refers to a deterioration in the function of the myocardium (i.e., the actual myocardium) for any reason. Subjects with cardiomyopathy are often at risk of arrhythmias, sudden cardiac death, or both.

[0110] As used herein, the term "hypertrophic cardiomyopathy" refers to a disease of the heart and myocardium in which a portion of the heart muscle becomes enlarged.

[0111] As used herein, the term “familial hypertrophic cardiomyopathy” refers to a genetic disorder characterized by increased growth (i.e., hypertrophy) of the left ventricular wall thickness.

[0112] As used herein, the term “effective dose” refers to the minimum amount of a drug or composition required to produce a particular physiological effect. The effective dose of a particular drug may be expressed in various ways based on the properties of the drug, such as mass / volume, cell number / volume, particle / volume, (mass of drug) / (mass of target), cell number / (mass of target), or particle / (mass of target). The effective dose of a particular drug may also refer to the semi-maximal effective concentration (EC2), which is the concentration of the drug that produces a particular magnitude of physiological response that is midway between the reference level and the maximum response level. 50 ) may be expressed as:

[0113] II. Polynucleotides In some embodiments, the disclosure provides polynucleotide sequences for the treatment and / or prevention of heart disease (e.g., cardiomyopathy). In some embodiments, the polynucleotide sequence includes a heart-specific promoter operably ligated to a polynucleotide encoding one or more therapeutic gene products for the treatment and / or prevention of cardiomyopathy.

[0114] Polynucleotides refer to polymeric forms of any modified form of any nucleotide, ribonucleotide, or deoxyribonucleotide, or any type of nucleotide, with lengths of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 5000, at least 10000, or at least 15000 or more nucleotides, ribonucleotides, or deoxyribonucleotides, as well as intermediate lengths. In this context, “intermediate length” means any length between the cited values, such as 6, 7, 8, 9, etc., 101, 102, 103, etc., 151, 152, 153, etc., 201, 202, 203, etc.

[0115] As a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode fragments of polypeptides or variants thereof as described herein. Some of these polynucleotides have minimal homology to the nucleotide sequences of any native gene. Nevertheless, polynucleotides that are altered by differences in codon use are particularly intended in certain embodiments, e.g., polynucleotides optimized for human and / or primate codon selection. Furthermore, alleles of genes containing the polynucleotide sequences provided herein may also be used. Alleles are endogenous genes that are modified as a result of one or more mutations, such as nucleotide deletions, additions, and / or substitutions.

[0116] The polynucleotides intended herein may be coupled with other DNA sequences, such as promoters and / or enhancers, untranslated regions (UTRs), signal sequences, Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosome entry sites (IRESs), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), stop codons, transcription termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags, regardless of the length of the coding sequence itself, as disclosed elsewhere herein or known in the art.

[0117] Polynucleotides can be prepared, manipulated, and / or expressed using any of the various established techniques known and available in the art.

[0118] In some embodiments, the polynucleotide sequence is a promoter. In some embodiments, the polynucleotide sequence is a promoter operably ligated to a polynucleotide encoding a therapeutic gene product for the treatment or prevention of heart disease (e.g., cardiomyopathy).

[0119] In some embodiments, the vector includes a cardiac-specific promoter operably ligated to a polynucleotide encoding a therapeutic gene product (e.g., a therapeutic protein, e.g., the MYBPC3 protein). As used herein, “cardiac-specific promoter” refers to a promoter whose activity in cardiac cells is at least twice as high as that of any other non-cardiac cell type. Preferably, a cardiac-specific promoter suitable for use in the vector of the present invention has cardiac cell activity at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, or at least 50 times higher than its activity in non-cardiac cell types.

[0120] In some embodiments, the vector comprises a cardiomyocyte-specific promoter operably ligated to a polynucleotide encoding a therapeutic gene product (e.g., the MYBPC3 protein). As used herein, “cardiomyocyte-specific promoter” designates a promoter whose activity in cardiomyocytes is at least twice as high as that in any other non-cardiac cell type or cardiac cell that is not a cardiomyocyte. Preferably, a cardiomyocyte-specific promoter suitable for use in the vectors of this disclosure has at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, or at least 50 times higher activity in cardiomyocytes compared to its activity in non-cardiac cell types or non-cardiac cell types.

[0121] In some embodiments, the cardiac-specific or cardiomyocyte-specific promoter is a human promoter. Examples of cardiac-specific or cardiomyocyte-specific promoters include, but are not limited to, the alpha-myosin heavy chain promoter, myosin light chain 2v promoter, alpha-myosin heavy chain promoter, alpha-cardiac actin promoter, alpha-tropomyosin promoter, cardiac troponin C promoter, cardiac troponin I promoter, cardiac myosin-binding protein C promoter, and sarco / endoplasmic reticulum Ca 2+ An example is an ATPase (SERCA) promoter (for instance, isoform 2 of SERCA2).

[0122] In some embodiments, the cardiac-specific promoter is the cardiac TNNT2 promoter. In some embodiments, the cardiac TNNT2 promoter is modified, for example, by polynucleotide deletion, insertion, or substitution. Exemplary polynucleotide sequences of the cardiac TNNT2 promoter are shown in Table 1 below. The transcription start site (TSS) of the TNNT2 promoter is shown in bold and underlined. [Table 1]

[0123] In some embodiments, the cardiac TNNT2 promoter is modified to include polynucleotide sequences of approximately 200–500 base pairs, approximately 250–500 base pairs, approximately 300–500 base pairs, approximately 350–500 base pairs, approximately 400–500 base pairs, approximately 450–500 base pairs, approximately 200–450 base pairs, approximately 200–400 base pairs, approximately 200–350 base pairs, approximately 200–300 base pairs, and approximately 200–250 base pairs in length. In some embodiments, the modified cardiac TNNT2 promoter includes polynucleotide sequences of approximately 350–450 base pairs, approximately 375–425 base pairs, approximately 375–400 base pairs, approximately 375–425 base pairs, approximately 400–425 base pairs, or approximately 400–450 base pairs. In some embodiments, the cardiac TNNT2 promoter includes a polynucleotide sequence of approximately 400 base pairs.

[0124] In certain embodiments, the modified cardiac troponin T promoter includes SEQ ID NO: 1, which is 300 bp to 500 bp long. For example, the modified cardiac troponin T promoter may also include SEQ ID NO: 3. In some embodiments, the 300 bp to 500 bp sequence may be ligated to further polynucleotide sequences, but not to additional sequences derived from SEQ ID NO: 1. For example, in one embodiment, the modified cardiac troponin T promoter may include SEQ ID NO: 1 of 500 bp or less, but may also include additional unrelated polynucleotide sequences. In another example, the modified cardiac troponin T promoter may include SEQ ID NO: 3, but not additional sequences derived from SEQ ID NO: 1, but may include additional unrelated polynucleotide sequences.

[0125] In some embodiments, the cardiac TNNT2 promoter is modified by polynucleotide deletions. The modification may include one, two, three or more internal deletions. Each deletion may be of 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, or 300 base pairs with respect to a reference cardiac TNNT2 promoter (SEQ ID NO: 1) having approximately 600 base pairs.

[0126] In some embodiments, the TNNT2 promoter is modified with respect to a reference cardiac TNNT2 promoter (SEQ ID NO: 1) having approximately 600 base pairs by a deletion of polynucleotides from the upstream end of the promoter. The modification may include deletions of 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, or 300 base pairs from the upstream end of the promoter with respect to a reference cardiac TNNT2 promoter (SEQ ID NO: 1) having approximately 600 base pairs. In some embodiments, the modification is a deletion of 200 base pairs from the upstream end of the promoter with respect to a reference cardiac TNNT2 promoter (SEQ ID NO: 1) having approximately 600 base pairs.

[0127] In some embodiments, the cardiac TNNT2 promoter is modified with respect to a reference cardiac TNNT2 promoter (SEQ ID NO: 1) having approximately 600 base pairs by deletion of polynucleotides from the downstream end of the promoter. The modification may include deletions of 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, or 300 base pairs from the downstream end of the promoter with respect to a reference cardiac TNNT2 promoter (SEQ ID NO: 1) having approximately 600 base pairs.

[0128] In some embodiments, the cardiac TNNT2 promoter is modified by polynucleotide internal deletions. The modifications may include 1-base pair, 2-base pair, 3-base pair, 4-base pair, 5-base pair, 10-base pair, 15-base pair, 20-base pair, 30-base pair, 40-base pair, 50-base pair, 60-base pair, 70-base pair, 80-base pair, 90-base pair, 100-base pair, 125-base pair, 150-base pair, 175-base pair, 200-base pair, 225-base pair, 250-base pair, 275-base pair, or 300-base pair internal deletions with respect to the reference cardiac TNNT2 promoter (SEQ ID NO: 1).

[0129] In some embodiments, the cardiac TNNT2 promoter is modified by the insertion of polynucleotides. The modification may include insertions of 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 125, 150, 175, 200, 225, 250, 275, or 300 base pairs with respect to the reference cardiac TNNT2 promoter (SEQ ID NO: 1).

[0130] In some embodiments, the cardiac TNNT2 promoter is modified by polynucleotide substitutions. The substitutions may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 base pair substitutions with respect to the reference cardiac TNNT2 promoter (SEQ ID NO: 1).

[0131] In some embodiments, the polynucleotide sequence of the TNNT2 promoter shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the polynucleotide sequence -450 base pairs to +1 base pair to the transcription start site of the human TNNT2 gene. In some embodiments, the polynucleotide sequence of the TNNT2 promoter shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the polynucleotide sequence -350 base pairs to +1 base pair to the transcription start site of the human TNNT2 gene. In some embodiments, the polynucleotide sequence of the TNNT2 promoter shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the polynucleotide sequence -250 base pairs to +1 base pair to the transcription start site of the human TNNT2 gene.

[0132] In some embodiments, the polynucleotide sequence of the cardiac TNNT2 promoter shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the polynucleotide sequence -450 to +50 base pairs to the transcription start site of the TNNT2 gene. In some embodiments, the polynucleotide sequence of the cardiac TNNT2 promoter shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the polynucleotide sequence -350 to +50 base pairs to the transcription start site of the TNNT2 gene. In some embodiments, the polynucleotide sequence of the cardiac TNNT2 promoter shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the polynucleotide sequence -250 to +5 base pairs to the transcription start site of the TNNT2 gene.

[0133] In some embodiments, the cardiac TNNT2 promoter includes a polynucleotide containing a sequence that shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NOs: 1 to 85. In some embodiments, the polynucleotide contains a sequence that shares at least 80% identity with any one of SEQ ID NOs: 1 to 85. In some embodiments, the polynucleotide contains a sequence that shares at least 90% identity with any one of SEQ ID NOs: 1 to 85. In some embodiments, the polynucleotide contains a sequence that shares at least 100% identity with any one of SEQ ID NOs: 1 to 85. In some embodiments, the polynucleotide contains a sequence that shares at least 80% identity with SEQ ID NOs: 1. In some embodiments, the polynucleotide contains a sequence that shares at least 90% identity with SEQ ID NOs: 1. In some embodiments, the polynucleotide contains a sequence that shares at least 100% identity with SEQ ID NOs: 1. In some embodiments, the polynucleotide contains a sequence that shares at least 80% identity with SEQ ID NOs: 3. In some embodiments, the polynucleotide includes a sequence that shares at least 90% identity with SEQ ID NO: 3. In some embodiments, the polynucleotide includes a sequence that shares at least 100% identity with SEQ ID NO: 3.

[0134] B. Exemplary gene products (proteins) The promoters of this disclosure may be operably ligated to a polynucleotide containing a sequence encoding a gene product (e.g., a protein or nucleic acid). In some embodiments, the gene product is a therapeutic protein. The therapeutic protein may be any of the native human proteins listed in Table 2, or their functional homologs or variants. The promoters of this disclosure are suitable for use with large genes that may be expressed at low levels or not expressed at all when delivered by a viral vector. The advantage of some embodiments disclosed herein lies in their ability to express therapeutic proteins (especially large therapeutic proteins) in viral vectors with limited packaging capabilities, such as AAV vectors. A “large” protein is any protein whose size affects its expression in a chosen vector. Generally, a “large” therapeutic protein contains at least about 1000 amino acids, i.e., the protein is encoded by a polynucleotide sequence of about 3 kbps or longer. Exemplary proteins containing large proteins are provided in Table 2 below. [Table 2]

[0135] Various therapeutic polynucleotides having a length of 3 kilobases or more, or therapeutic proteins encoded by polynucleotides, are expressed more effectively when operably ligated to the modified TNNT2 promoter of this disclosure compared to the approximately 600-base pair TNNT2 promoter. The promoters of this disclosure are useful for the expression of at least: a) large genes in which loss-of-function mutations result in cardiomyopathy (gene replacement therapy), b) large genes whose expression in cardiomyocytes is cardioprotective, c) combinations of genes whose co-expression in cardiomyocytes is beneficial, and d) tools for cardiomyocyte-specific genome editing. A “large” gene is any gene whose size affects its expression in a selected vector. Generally, a “large” therapeutic gene contains at least approximately 1000 amino acids, i.e., the gene contains a polynucleotide sequence of approximately 3 kbps or more. In further embodiments, the vectors and promoters of this disclosure are used to treat the diseases or disorders listed in Table 3, where polynucleotides encode therapeutic proteins shown in the table. [Table 3]

[0136] MYBPC3 is a gene expressed in cardiac cells. Various mutations in MYBPC3 are known to cause hypertrophic cardiomyopathy. Nearly half of the mutations causing hypertrophic cardiomyopathy result in truncation via nonsense mutations, frameshift mutations, or splice site mutations (Marian and Braunwald, Circ. Res. 121:749-770 (2017); Walsh et al., Genet. Med. 19:192-203 (2017)). mRNA containing immature stop codons is subjected to monitoring and degradation by nonsense mutation-dependent degradation mechanisms. This is consistent with the reduced levels of mutant RNA in analysis of cardiac tissue from hypertrophic cardiomyopathy patients who have undergone myectomy (Marston et al., Circ. Res. 105:219-222 (2009); van Dijk et al., Circulation 119:1473-1483 (2009); Helms et al. al., Circ.Cardiovasc.Genet.7:434-443(2014). Furthermore, any resulting cleaved polypeptides appear to be sensitive to the ubiquitin-proteasome degradation system. In patient muscle resection samples, no cleaved proteins were observed for nine different mutations (Rottbauer et al., J.Clin.Invest.100:475-482(1997); Moolman et al., Circulation 101:1396-1402(2000); Marston et al., Circ.Res.105:219-222(2009); van Dijk et al., Circ.Heart Fail 5:36-46(2012)). While the wild-type MYBPC3 allele appears to be slightly upregulated in heterozygous patients, the total amount of MYBPC3 protein incorporated into sarcomeres is significantly below normal, at approximately 65% ​​(Marston et al. al.,Circ.Res.105:219-222(2009);van Dijk et al.,Circ.Heart Fail 5:36-46(2012);McNamara et al.,PLoS One 12:e0180064(2017)).Therefore, the sarcomere pathophysiology in hypertrophic cardiomyopathy patients with MYBPC3 truncation mutations is thought to be due to haploinsufficiency.

[0137] In some embodiments, the disclosure provides a vector comprising a polynucleotide sequence encoding the MYBPC3 protein operably ligated to a modified cardiac TNNT2 promoter. Similarly, in some embodiments, the polynucleotide sequence operably ligated to a cardiac-specific promoter (e.g., a modified cardiac TNNT2 promoter) is MYBPC3, or a variant, variant, or fragment thereof. In humans, the MYBPC3 gene encodes the MYBPC3 protein (also known as MyBP-C), which regulates the cardiac sarcomere, the basic unit of muscle contraction. The cardiac sarcomere consists of thick and thin filaments, and MYBPC3 adheres to the thick filament to prevent premature degradation. Exemplary MYBPC3 polynucleotide sequences are shown in Table 4A below. In some embodiments, the polynucleotide encoding MYBPC3 shares a sequence that shares at least 85%, 90%, 95%, 99%, or 100% identity with one of sequence numbers 86-89. Exemplary MYBPC3 protein sequences are shown in Table 4B. In some embodiments, the vector genome encoding MYBPC3 encodes a MYBPC3 protein that shares at least 85%, 90%, 95%, 99%, or 100% identity with one of sequence numbers 103–106. [Table 4A-1] [Table 4A-2] [Table 4A-3] [Table 4A-4] [Table 4A-5] [Table 4B-1] [Table 4B-2]

[0138] In some embodiments, the disclosure provides a vector comprising a polynucleotide sequence encoding a potassium voltage-gated channel subfamily H member 2 (KCNH2) protein, operably ligated to a modified cardiac TNNT2 promoter. Similarly, in some embodiments, the polynucleotide sequence operably ligated to a cardiac-specific promoter (e.g., a modified cardiac TNNT2 promoter) is KCNH2, or a variant, variant, or fragment thereof (e.g., SEQ ID NO: 107). In humans, the KCNH2 gene encodes the KCNH2 protein (hERG1, also known as SEQ ID NO: 108, e.g.,) which, together with other KCNH2 proteins, forms a potassium channel to transport potassium from cells. The KCNH2 protein is abundantly expressed in the cardiomyocyte, which recharges cardiac tissue after each heartbeat to maintain a regular rhythm. In some embodiments, the polynucleotide encoding KCNH2 shares at least 90%, 95%, 99%, or 100% identity with SEQ ID NO: 107. In some embodiments, the KCNH2 protein shares at least 90%, 95%, 99%, or 100% identity with SEQ ID NO: 108.

[0139] In some embodiments, the disclosure provides a vector comprising a polynucleotide sequence encoding a transient receptor potential channel subfamily M membrane 4 (TRPM4) protein operably ligated to a modified cardiac TNNT2 promoter. Similarly, in some embodiments, the polynucleotide sequence operably ligated to a cardiac-specific promoter (e.g., a modified cardiac TNNT2 promoter) is TRPM4, or a variant, variant, or fragment thereof (e.g., SEQ ID NO: 109). In humans, the TRPM4 gene encodes the TRPM4 protein (e.g., SEQ ID NO: 110), which functions as a channel that controls the flow of cations into and from cells. TRPM4 channels are abundantly expressed in cardiac cells and play a crucial role in the generation and transmission of electrical signals. In some embodiments, the polynucleotide encoding TRPM4 shares at least 90%, 95%, 99%, or 100% identity with SEQ ID NO: 109. In some embodiments, the TRPM4 protein shares at least 90%, 95%, 99%, or 100% identity with SEQ ID NO: 110.

[0140] In some embodiments, the disclosure provides a vector comprising a polynucleotide sequence encoding the desmoglein 2 (DSG2) protein operably ligated to a modified cardiac TNNT2 promoter. Similarly, in some embodiments, the polynucleotide sequence operably ligated to a cardiac-specific promoter (e.g., a modified cardiac TNNT2 promoter) is DSG2, or a variant, variant, or fragment thereof (e.g., SEQ ID NO: 111). In humans, the DSG2 gene encodes the DSG2 protein (e.g., SEQ ID NO: 112), which is a transmembrane glycoprotein and a component of desmosomes. Desmosomes are intercellular junctions that provide strong cell-cell adhesion, giving tissue mechanical strength. In some embodiments, the polynucleotide encoding DSG2 shares at least 90%, 95%, 99%, or 100% identity with SEQ ID NO: 111. In some embodiments, the DSG2 protein shares at least 90%, 95%, 99%, or 100% identity with SEQ ID NO: 112.

[0141] In some embodiments, the disclosure provides a vector comprising a polynucleotide sequence encoding the ATPase sarcoplasmic reticulum / endoplasmic reticulum calcium transport 2 (ATP2A2) protein, operably ligated to a modified cardiac TNNT2 promoter. Similarly, in some embodiments, the polynucleotide sequence operably ligated to a cardiac-specific promoter (e.g., a modified cardiac TNNT2 promoter) is ATP2A2, or a variant, variant, or fragment thereof (e.g., SEQ ID NO: 113). In humans, the ATP2A2 gene encodes the sarco(endo)endoplasmic reticulum calcium-ATPase 2 (SERCA2) protein (e.g., SEQ ID NO: 114), which catalyzes the hydrolysis of ATP that transposes and ligates calcium from the cytosol to the lumen of the endoplasmic reticulum. The regulation of calcium ions into and out of the sarcoplasmic reticulum assists in muscle contraction and relaxation. In some embodiments, the polynucleotide encoding ATP2A2 shares at least 90%, 95%, 99%, or 100% identity with SEQ ID NO: 113. In some embodiments, the ATP2A2 protein shares at least 90%, 95%, 99%, or 100% identity with SEQ ID NO: 114.

[0142] In some embodiments, the disclosure provides a vector comprising a polynucleotide sequence encoding the calcium voltage-gated channel subunit alpha-1C (CACNA1C) protein, operably ligated to a modified cardiac TNNT2 promoter. Similarly, in some embodiments, the polynucleotide sequence operably ligated to a cardiac-specific promoter (e.g., a modified cardiac TNNT2 promoter) is CACNA1C, or a variant, variant, or fragment thereof (e.g., SEQ ID NO: 115). In humans, the CACNA1C gene encodes the alpha-1 subunit of a voltage-gated calcium channel protein (e.g., SEQ ID NO: 116), which functions to mediate the influx of calcium ions into cells via membrane polarization. In some embodiments, the polynucleotide encoding CACNA1C shares at least 90%, 95%, 99%, or 100% identity with SEQ ID NO: 115. In some embodiments, the CACNA1C protein shares at least 90%, 95%, 99%, or 100% identity with SEQ ID NO: 116.

[0143] In some embodiments, the disclosure provides a vector comprising a polynucleotide sequence encoding a dystrophin (DMD) protein operably ligated to a modified cardiac TNNT2 promoter. Similarly, in some embodiments, the polynucleotide sequence operably ligated to a cardiac-specific promoter (e.g., a modified cardiac TNNT2 promoter) is DMD, or a variant, variant, or fragment thereof (e.g., SEQ ID NO: 117). In humans, the DMD gene encodes the DMD protein (e.g., SEQ ID NO: 118), which forms a component of the dystrophin-glycoprotein complex (DGC). The DGC acts as an anchor, connecting the cytoskeleton to the extracellular matrix, thereby strengthening muscle fibers and protecting them from damage as muscles contract and relax. In some embodiments, the polynucleotide encoding DMD shares at least 90%, 95%, 99%, or 100% identity with SEQ ID NO: 117. In some embodiments, the DMD protein shares at least 90%, 95%, 99%, or 100% identity with SEQ ID NO: 118.

[0144] In some embodiments, the disclosure provides a vector comprising a polynucleotide sequence encoding a DM1 protein kinase (DMPK) protein operably ligated to a modified cardiac TNNT2 promoter. Similarly, in some embodiments, the polynucleotide sequence operably ligated to a cardiac-specific promoter (e.g., a modified cardiac TNNT2 promoter) is DMPK, or a variant, variant, or fragment thereof (e.g., SEQ ID NO: 119). In humans, the DMPK gene encodes a myotonic dystrophy protein kinase protein (e.g., SEQ ID NO: 120), which plays a crucial role in brain, muscle, and cardiac development and homeostasis. Myotonic dystrophy protein kinase inhibits myosin phosphatases, which play a role in muscle tone and relaxation. In some embodiments, the polynucleotide encoding DMPK shares at least 90%, 95%, 99%, or 100% identity with SEQ ID NO: 119. In some embodiments, the DMPK protein shares at least 90%, 95%, 99%, or 100% identity with SEQ ID NO: 120.

[0145] In some embodiments, the disclosure provides a vector comprising a polynucleotide sequence encoding an ectopic P-granule protein 5 homolog (EPG5) protein operably ligated to a modified cardiac TNNT2 promoter. Similarly, in some embodiments, the polynucleotide sequence operably ligated to a cardiac-specific promoter (e.g., a modified cardiac TNNT2 promoter) is EPG5, or a variant, variant, or fragment thereof (e.g., SEQ ID NO: 121). In humans, the EPG5 gene encodes the EPG5 protein (e.g., SEQ ID NO: 122), which functions in autophagy to facilitate the interaction between autophagosomes and lysosomes. In some embodiments, the polynucleotide encoding EPG5 shares at least 90%, 95%, 99%, or 100% identity with SEQ ID NO: 121. In some embodiments, the EPG5 protein shares at least 90%, 95%, 99%, or 100% identity with SEQ ID NO: 122.

[0146] In some embodiments, the disclosure provides a vector comprising a polynucleotide sequence encoding the EvC ciliary complex subunit 1 (EVC) protein, operably ligated to a modified cardiac TNNT2 promoter. Similarly, in some embodiments, the polynucleotide sequence operably ligated to a cardiac-specific promoter (e.g., a modified cardiac TNNT2 promoter) is EVC, or a variant, variant, or fragment thereof (e.g., SEQ ID NO: 123). In humans, the EVC gene encodes the EVC protein (e.g., SEQ ID NO: 124), primarily found in pili, which plays a role in intercellular communication. The EVC protein also regulates sonic hedgehog, which is involved in cell growth and differentiation. In some embodiments, the polynucleotide encoding EVC shares at least 90%, 95%, 99%, or 100% identity with SEQ ID NO: 123. In some embodiments, the EVC protein shares at least 90%, 95%, 99%, or 100% identity with SEQ ID NO: 124.

[0147] In some embodiments, the disclosure provides a vector comprising a polynucleotide sequence encoding a lymphin protein operably ligated to a modified cardiac TNNT2 promoter. Similarly, in some embodiments, the polynucleotide sequence operably ligated to a cardiac-specific promoter (e.g., a modified cardiac TNNT2 promoter) is EVC2, or a variant, variant, or fragment thereof (e.g., SEQ ID NO: 125). In humans, the EVC2 gene encodes a lymphin protein (e.g., SEQ ID NO: 126). The function of lymphin is unknown, but it is important for normal growth and development, particularly bone and tooth development. In some embodiments, the polynucleotide encoding lymphin shares at least 90%, 95%, 99%, or 100% identity with SEQ ID NO: 125. In some embodiments, the lymphin protein shares at least 90%, 95%, 99%, or 100% identity with SEQ ID NO: 126.

[0148] In some embodiments, the disclosure provides a vector comprising a polynucleotide sequence encoding the fibrillin-1 protein, operably ligated to a modified cardiac TNNT2 promoter. Similarly, in some embodiments, the polynucleotide sequence operably ligated to a cardiac-specific promoter (e.g., a modified cardiac TNNT2 promoter) is FBN1, or a variant, variant, or fragment thereof (e.g., SEQ ID NO: 127). In humans, the FBN1 gene encodes fibrillin-1 and asprosin proteins (e.g., SEQ ID NO: 128). Fibrillin-1 is a glycoprotein that acts as a structural component of calcium-bound microfibrils, providing support that gives force to elastic and inelastic connective tissue throughout the body. Asprosin is a hormone normally secreted by white adipose tissue to regulate glucose homeostasis. In some embodiments, the polynucleotide encoding fibrillin-1 shares at least 90%, 95%, 99%, or 100% identity with SEQ ID NO: 127. In some embodiments, the fibrillin-1 protein shares at least 90%, 95%, 99%, or 100% identity with SEQ ID NO: 128.

[0149] In some embodiments, the disclosure provides a vector comprising a polynucleotide sequence encoding a neurofibromin (NF1) protein operably ligated to a modified cardiac TNNT2 promoter. Similarly, in some embodiments, the polynucleotide sequence operably ligated to a cardiac-specific promoter (e.g., a modified cardiac TNNT2 promoter) is NF1, or a variant, variant, or fragment thereof (e.g., SEQ ID NO: 129). In humans, the NF1 gene encodes the NF1 protein (e.g., SEQ ID NO: 130), which functions as a tumor suppressor and negative regulator of the Ras signaling pathway that stimulates cell growth and division. In some embodiments, the polynucleotide encoding NF1 shares at least 90%, 95%, 99%, or 100% identity with SEQ ID NO: 129. In some embodiments, the NF1 protein shares at least 90%, 95%, 99%, or 100% identity with SEQ ID NO: 130.

[0150] In some embodiments, the disclosure provides a vector comprising a polynucleotide sequence encoding a sodium channel protein type 5 subunit alpha (SCN5A) protein operably ligated to a modified cardiac TNNT2 promoter. Similarly, in some embodiments, the polynucleotide sequence operably ligated to a cardiac-specific promoter (e.g., a modified cardiac TNNT2 promoter) is SCN5A, or a variant, variant, or fragment thereof (e.g., SEQ ID NO: 131). In humans, the SCN5A gene encodes the SCN5A protein (e.g., SEQ ID NO: 132), which is a tetrodotoxin-resistant voltage-gated sodium channel subunit. SCN5A is primarily found in cardiomyocytes and is involved in the initial upstroke of action potentials in electrocardiograms. In some embodiments, the polynucleotide encoding SCN5A shares at least 90%, 95%, 99%, or 100% identity with SEQ ID NO: 131. In some embodiments, the SCN5A protein shares at least 90%, 95%, 99%, or 100% identity with SEQ ID NO: 132.

[0151] In some embodiments, the disclosure provides a vector comprising a polynucleotide sequence encoding the SOS1 (son of sevenless homolog 1) protein, operably ligated to a modified cardiac TNNT2 promoter. Similarly, in some embodiments, the polynucleotide sequence operably ligated to a cardiac-specific promoter (e.g., a modified cardiac TNNT2 promoter) is SOS1, or a variant, variant, or fragment thereof (e.g., SEQ ID NO: 133). In humans, the SOS1 gene encodes the SOS1 protein (e.g., SEQ ID NO: 134), which functions as a component of a trimer complex involved in the transduction signal from Ras to Rac by promoting Rac-specific guanine nucleotide exchanger activity. In some embodiments, the polynucleotide encoding SOS1 shares at least 90%, 95%, 99%, or 100% identity with SEQ ID NO: 133. In some embodiments, the SOS1 protein shares at least 90%, 95%, 99%, or 100% identity with SEQ ID NO: 134.

[0152] In some embodiments, this disclosure provides a vector comprising a polynucleotide sequence encoding the natriuretic peptide receptor 1 (NPR1) protein operably ligated to a modified cardiac TNNT2 promoter. Similarly, in some embodiments, the polynucleotide sequence operably ligated to a cardiac-specific promoter (e.g., a modified cardiac TNNT2 promoter) is NPR1, or a variant, variant, or fragment thereof (e.g., SEQ ID NO: 135). In humans, the NPR1 gene encodes the NPR1 protein (also referred to as GC-A) (e.g., SEQ ID NO: 136), a transmembrane catalytic receptor with intracellular guanylyl cyclase activity. NPR1 acts as a receptor for both atrioventricular and cerebral natriuretic peptides, vasoactive hormones that play a crucial role in cardiovascular homeostasis. In some embodiments, the polynucleotide encoding NPR1 shares at least 90%, 95%, 99%, or 100% identity with SEQ ID NO: 135. In some embodiments, the NPR1 protein shares at least 90%, 95%, 99%, or 100% identity with SEQ ID NO: 136.

[0153] In some embodiments, the disclosure provides a vector comprising a polynucleotide sequence encoding the receptor tyrosine protein kinase erbB-4 (ERBB4) protein, operably ligated to a modified cardiac TNNT2 promoter. Similarly, in some embodiments, the polynucleotide sequence operably ligated to a cardiac-specific promoter (e.g., a modified cardiac TNNT2 promoter) is ERBB4, or a variant, variant, or fragment thereof (e.g., SEQ ID NO: 137). The human ERBB4 gene encodes the human ERBB4 protein (e.g., SEQ ID NO: 138), which is a transmembrane receptor for the epidermal growth factor family. Signaling via the ERBB4 receptor induces a variety of cellular responses, including mitosis and differentiation. In some embodiments, the polynucleotide encoding ERBB4 shares at least 90%, 95%, 99%, or 100% identity with SEQ ID NO: 137. In some embodiments, the ERBB4 protein shares at least 90%, 95%, 99%, or 100% identity with SEQ ID NO: 138.

[0154] In some embodiments, the disclosure provides a vector comprising a polynucleotide sequence encoding a vascularly active intestinal peptide (VIP) operably ligated to a modified cardiac TNNT2 promoter. Similarly, in some embodiments, the polynucleotide sequence operably ligated to a cardiac-specific promoter (e.g., a modified cardiac TNNT2 promoter) is VIP, or a variant, variant, or fragment thereof (e.g., SEQ ID NO: 139). In humans, the VIP gene encodes a vascularly active intestinal peptide (e.g., SEQ ID NO: 140) that functions as a neuromodulator and neurotransmitter. VIP is a potent vasodilator that modulates smooth muscle activity, epithelial cell secretion, and blood flow in the gastrointestinal tract. In some embodiments, the polynucleotide encoding VIP shares at least 90%, 95%, 99%, or 100% identity with SEQ ID NO: 139. In some embodiments, the VIP protein shares at least 90%, 95%, 99%, or 100% identity with SEQ ID NO: 140.

[0155] In some embodiments, the disclosure provides a vector comprising a polynucleotide sequence encoding a beta-myosin heavy chain (MyHC-β) operably ligated to a modified cardiac TNNT2 promoter. Similarly, in some embodiments, the polynucleotide sequence operably ligated to a cardiac-specific promoter (e.g., a modified cardiac TNNT2 promoter) is MYH7, or a variant, variant, or fragment thereof (e.g., SEQ ID NO: 141). In humans, the MYH7 gene encodes the MyHC-β protein (e.g., SEQ ID NO: 142), which is a hexamer asymmetry motility that forms the majority of the thick filaments of the cardiomyocyte. The enzymatic activity of ATPase in the myosin head fuels the process of hydrolyzing ATP and shortening the sarcomere to generate intraventricular pressure and power. In some embodiments, the polynucleotide encoding MyHC-β shares at least 90%, 95%, 99%, or 100% identity with SEQ ID NO: 141. In some embodiments, the MyHC-β protein shares at least 90%, 95%, 99%, or 100% identity with SEQ ID NO: 142.

[0156] III. Vectors In some embodiments, this disclosure provides vectors for the treatment or prevention of cardiomyopathy. In particular, the vectors described herein include a cardiac-specific promoter operably ligated to a polynucleotide encoding a therapeutic protein, and the expression of the therapeutic protein treats a subject in need of it (e.g., a subject with cardiomyopathy). For example, in some embodiments, the vector is an AAV-based vector containing a cardiac TNNT2 promoter operably ligated to a polynucleotide encoding a MYBPC3 protein for the treatment or prevention of cardiomyopathy.

[0157] In some embodiments, the vector includes, in addition to the cardiac-specific promoter (e.g., a modified cardiac troponin T promoter) and therapeutic gene product (e.g., MYBPC3 protein) described herein, a marker gene to facilitate the identification or selection of transfected, transduced, or infected cells. Examples of marker genes include, but are not limited to, fluorescent proteins such as enhanced green fluorescent protein, Ds-Red (DsRed: Discosoma sp. red fluorescent protein (RFP); Bevis et al. (2002) Nat. Biotechnol. 20(11):83-87), yellow fluorescent protein, mCherry, and cyanofluorescent proteins, as well as genes encoding proteins that confer resistance to selectors, such as neomycin resistance genes, puromycin resistance genes, and blastosidine resistance genes.

[0158] In some embodiments, the vector includes a polynucleotide sequence having a maximum size of about 4.0 kilobases, about 4.5 kilobases, about 5 kilobases, about 5.1 kilobases, about 5.2 kilobases, about 5.3 kilobases, about 5.4 kilobases, or about 5.5 kilobases. In some embodiments, the vector includes a polynucleotide sequence having a maximum size of about 4.5 kilobases. In some embodiments, the vector includes a polynucleotide sequence having a maximum size of about 5 kilobases. In some embodiments, the vector includes a polynucleotide sequence having a maximum size of about 5.5 kilobases. In some embodiments, the vector comprises a polynucleotide sequence having a maximum size of about 6 kilobases.

[0159] Methods for introducing polynucleotides into host cells are known in the art, and any known method can be used to introduce the polynucleotides described herein into cells. Suitable methods include, for example, viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and microfluidic delivery methods.

[0160] A. Nonviral vectors In some embodiments, the polynucleotides described herein are delivered to cells in non-viral vectors such as transposons, nanoparticles (e.g., lipid nanoparticles), liposomes, exosomes, attenuated bacteria, or virus-like particles. In some embodiments, the non-viral vector is a mammalian virus-like particle. For example, mammalian virus-like particles can be generated (e.g., by "empty" purification of mammalian virus-like particles, followed by ex vivo assembly of the mammalian virus-like particles with the desired cargo). The non-viral vector may be manipulated to incorporate a targeted ligand to alter the specificity of the target tissue.

[0161] B. Viral vectors In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a retroviral vector, such as a lentiviral vector. As used herein, the term “retrovirus” refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and then covalently binds its genomic DNA to the host genome. Retroviral vectors are a common tool for gene delivery (Miller, Nature .357:455-460(2000)). Once a virus is integrated into the host genome, it is called a “provirus.” The provirus functions as a template for RNA polymerase II, directing the expression of the RNA molecule encoded by the virus. In some embodiments, the retroviral vector is modified so as not to be integrated into the host cell genome.

[0162] Examples of retroviruses (Retroviridae) include (1) genera such as Molony mouse leukemia virus (M-MuLV), Molony mouse sarcoma virus (MoMSV), mouse mammary tumor virus (MuMTV), Gibbon ape leukemia virus (GaLV), and feline leukemia virus (FLV); (2) genus Supmavirus, such as monkey foam virus; and (3) genus Lentivirus, such as human immunodeficiency virus-1 and monkey immunodeficiency virus.

[0163] As used herein, the term “lentiviral” or “lentivirus” refers to a group (or genus) of complex retroviruses. Exemplary lentiviruses include, but are not limited to, HIV (human immunodeficiency virus, including HIV-1 and HIV-2), Visna-Maedivirus (VMV) virus, canine arthritis encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV).

[0164] In some embodiments, the viral vector is an adenovirus vector. The genetic makeup of an adenovirus contains a linear double-stranded DNA virus of approximately 36 kb, which allows for the substitution of large fragments of adenovirus DNA with foreign sequences up to 7 kb (Grunhaus et al., Seminar in Virology 200(2):535-546, 1992).

[0165] In some embodiments, the viral vector is an AAV vector selected from the group consisting of adeno-associated virus (AVV) vectors, such as serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, or chimeric AAVs derived therefrom.

[0166] In some embodiments, AAV expression vectors are pseudotyped to enhance targeting. Pseudotyping strategies can facilitate gene transfer and maintain expression in target cell types. For example, the AAV2 genome can be packaged into a capsid of another AAV serotype, such as AAV5, AAV7, or AAV8, producing pseudotyped vectors such as AV2 / 5, AAV2 / 7, and AAV2 / 8, respectively, as described in Balaji et al. J Surg Res. Sep;184(1):691-698 (2013). In some embodiments, AAV9 can be used to target the expression of myofibroblast-like lineages, as described in Piras et al. Gene Therapy 23:469-478 (2016). In some embodiments, AAV1, AAV6, or AAV9 are used, and in some embodiments, the AAV is engineered as described in Asokari et al. Hum Gene Ther. Nov;24(11):906-913(2013); Pozsgai et al. Mol Ther. Apr 5;25(4):855-869(2017); Kotterman, MA and DVS. Schaffer Engineering Adeno-Associated Viruses for Clinical Gene Therapy. Nature Reviews Genetics, 15:445-451(2014); and US20160340393A1. In some embodiments, the viral vector is an AAV engineered to increase target cell infectivity as described in US20180066285A1.

[0167] C. Adjustment element In some embodiments, the disclosure provides a vector comprising one or more regulatory elements operably conjugated to a polynucleotide encoding a therapeutic protein or nucleic acid. In some embodiments, the regulatory element is a heart-specific promoter (e.g., a modified TNNT2 promoter) operably conjugated to a therapeutic protein or nucleic acid for the treatment of heart disease.

[0168] As used herein, the term “regulatory element” refers to the untranslated region of a vector that interacts with host cell proteins to carry out transcription and translation (e.g., the origin of replication, selection cassette, promoter, enhancer, translation initiation signal (Shine Dalgarno sequence or Kozak sequence), intron, polyadenylated sequence, 5' and 3' untranslated regions). These elements may vary in their intensity and specificity. Transcriptional regulatory elements may function in either eukaryotic cells (e.g., mammalian cells) or prokaryotic cells (e.g., bacterial cells or primitive cells). In some embodiments, a polynucleotide sequence encoding a therapeutic gene product (e.g., a therapeutic protein or nucleic acid) described herein is operably ligated to multiple regulatory elements that enable polynucleotide expression in both prokaryotic and eukaryotic cells.

[0169] As used herein, the term “transcription start site” or “TSS” refers to the first base pair transcribed by RNA polymerase when RNA polymerase initiates transcription. The TSS is distinct from the start codon (quinonical, ATG), which must be downstream of the TSS in the transcription region of a polynucleotide. The location of the transcription start site can be determined experimentally or by prediction using one of various prediction algorithms. Annotated TSSs are available from the Eukaryotic Promoter Database and the UCSC Genome Browser. Multiple TSSs for TNNT2 are identified in the UCSC Genome Browser.

[0170] As used herein, the TSS of TNNT2 is defined as the sequence identified by C at the 5' end of the motif identified by dbTSS: C TCCATC.

[0171] As used herein, the term “modified cardiac TNNT2 promoter” means a promoter comprising a polynucleotide sequence of at least 200 base pairs, each comprising one or more continuous or discontinuous polynucleotide segments that share 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the corresponding segment of the TNNT2p-600 segment provided in Table 1 as Sequence ID No. 1. If it is a “promoter”, the modified cardiac TNNT2 promoter must be able to promote RNA polymerase-mediated transcription initiation in a host or target cell at or near the TSS within the promoter (i.e., at or near the TTS of TNNT2 as defined herein), or if the endogenous TSS of TNNT2 is not present in the modified cardiac TNNT2 promoter, then RNA polymerase-mediated transcription initiation at the downstream (3' of the sense strand) to downstream (3') end of the modified cardiac TNNT2 promoter at a heterologous TSS of up to 100 base pairs. Similarly, the modified cardiac TNNT2 promoter may contain only the sequence upstream of the TNNT2 TSS, or it may contain the TNNT2 TSS.

[0172] The length of a promoter (e.g., a modified cardiac TNNT2 promoter), which "has" a very large number of base pairs, is defined, as used herein, according to the number of base pairs in the polynucleotide sequence from the 5' end to its 3' end of the promoter, including any intervening sequences that do not align with the reference promoter sequence (e.g., the endogenous cardiac TNNT2 promoter of a human or other organism). The 5' and 3' ends of the promoter are defined as the last base pairs in either direction to match the corresponding sequence of the reference promoter sequence when the sequence is aligned by the BLAST algorithm or equivalent. Thus, the length of the promoter in a vector can be determined by using the polynucleotide sequence of the vector to search a nucleotide database containing the genome of the reference organism and by identifying one or more aligned regions containing or within the range of an endogenous gene of approximately 1–5 kb, or by aligning the vector to a given reference promoter. When the promoter aligns with the reference genome or reference promoter sequence as a continuous segment, the length of the promoter is the reported length alignment (3' end position minus 5' end position, + 1 unless TSS is included). If a promoter aligns across multiple segments (e.g., 2, 3, 4, or 5 segments), the length of the promoter can be calculated by subtracting the 5' end position of the 5' segment of the reference genome or reference promoter sequence from the 3' end position of the 3' segment of the reference genome or reference promoter sequence, and adding 1 unless the TSS is included (so that the calculated length includes both endpoints). For example, the length of a promoter extending from 100 bp before TSS(-100 bp) to 5 bp before TSS(-5 bp) is -5 - (-100) + 1 = 100 - 5 + 1 = 96 bp. TSSs are numbered +1 bp. Therefore, the length of a promoter extending from 100 bp before TSS(-100 bp) to 5 bp after TSS(+5 bp) is +5 - (-100) = 100 + 5 = 105 bp.

[0173] The term “enhancer” refers to a segment of DNA containing a sequence that can result in transcriptional enhancement, and in some examples, may function independently of its orientation relative to another regulatory sequence. Enhancers can function cooperatively or additively with promoters and / or other enhancer elements. Enhancers may overlap with promoters, or may be upstream or downstream of promoters. In some embodiments, a modified cardiac TNNT2 promoter includes one or more enhancers. In some embodiments, a modified cardiac TNNT2 promoter does not include enhancers.

[0174] In addition to, or instead of, the modified cardiac TNNT2 promoter, some embodiments employ other eukaryotic promoters, including, but not limited to, the following: early cytomegalovirus (CMV), herpes simplex virus (HSV) thymidine kinase, viral simian virus 40 (SV40) (e.g., early and late SV40), splenic focal-forming virus (SFFV) promoter, long-term repeat (LTR) from retroviruses (e.g., Moloney's mouse leukemia virus (MoMLV) LTR promoter or Rous sarcoma virus (RSV) LTR, herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, It also includes the vaccinia virus-derived P11 promoter, elongation factor 1-alpha (EF1α) promoter, early growth response 1 (EGR1) promoter, ferritin H (FerH) promoter, ferritin L (FerL) promoter, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, eukaryotic translation initiation factor 4A1 (EIF4A1) promoter, heat shock 70kDa protein 5 (HSPA5) promoter, heat shock protein 90kDa beta, member 1 (HSP90B1) promoter, heat shock protein 70kDa (HSP70) promoter, β-kinesin (β-KIN) promoter, and human ROSA 26 loci (Irions et al., Nature Biotechnology 25, 1477-1482 (2007), ubiquitin C (UBC) promoter, phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken β-actin (CAG) promoter, β-actin promoter and myeloproliferative sarcoma virus enhancer, deleted negative control region, dl587rev primer-binding site substitution (MND) promoter, and mouse metallothionein-1). The vector may also contain a ribosome-binding site and transcription terminator for translation initiation. The vector may also contain a polynucleotide sequence for expression amplification.The vector may also contain polynucleotide sequences encoding protein tags (e.g., 6xHis tags, hemagglutinin tags, green fluorescent protein, etc.) fused to site-directed modified polypeptides, thus resulting in chimeric polypeptides.

[0175] In some embodiments, the promoters of this disclosure are tissue-specific. The term “tissue-specific promoter” means a polynucleotide sequence that functions as a promoter, i.e., modulates the expression of a selected polynucleotide sequence operably ligated to a promoter, thereby affecting the expression of the selected polynucleotide sequence in specific cells of a tissue, such as muscle cells or cardiomyocytes. In some embodiments, the tissue-specific promoter is a cardiac-specific promoter. In some embodiments, the cardiac-specific promoter is a TNNT2 or modified TNNT2 promoter. The tissue-specific promoter causes the expression of the operably ligated polynucleotide, or the gene product encoded by that polynucleotide, at a level of 5-fold, 10-fold, 20-fold, 25-fold, or more in the tissue of interest than in the reference tissue.

[0176] In some embodiments, the vectors described herein include a transcription termination signal. Elements that direct the efficient termination and polyadenylation of heterologous nucleic acid transcripts increase heterologous gene expression. Transcription termination signals are generally found downstream of polyadenylation signals. In some embodiments, the vector includes a polyadenylation sequence 3' of the polynucleotide encoding the polypeptide to be expressed. As used herein, the terms “polyA site” or “polyA sequence” refer to a DNA sequence that directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase II. Polyadenylation sequences can enhance mRNA stability by adding a polyA tail to the 3' end of the coding sequence, thus contributing to improved translation efficiency. Cleavage and polyadenylation are directed by poly(A) sequences in RNA. The core poly(A) sequence of mammalian premRNA has two recognition elements adjacent to the cleavage-polyadenylation site. Typically, a nearly immutable AAUAAA hexamer is located 20–50 nucleotides upstream of a more variable element rich in U or GU residues. Cleavage of the nascent transcript occurs between these two elements, linked to the addition of up to 250 adenosines to the 5' cleavage product. In certain embodiments, the core poly(A) sequence is an ideal polyA sequence (e.g., AATAAA, ATTAAA, AGTAAA). In certain embodiments, the poly(A) sequence is the SV40 polyA sequence, the bovine growth hormone polyA sequence (BGHpA), the rabbit β-globin polyA sequence (rβgpA), its variants, or another suitable heterologous or endogenous polyA sequence known in the art.

[0177] IV. Recombinant adeno-associated virus (rAAV) viral genome, expression cassette, and rAAV virion This disclosure provides an expression cassette comprising a polynucleotide encoding a sequence encoding a transgene, such as the MYBPC3 polypeptide, or a functional variant thereof. The transgene polynucleotide sequence in the expression cassette may, for example, be an open reading frame encoding a protein. The expression cassette may optionally include a promoter operably linked to the transgene, optionally an intron region, optionally a polyadenylation (Poly(A)) signal, optionally a Woodchuck hepatitis virus post-transcription factor (WPRE), and optionally a transcription termination signal. The expression cassette may be flanked by one or more inverted terminal repeats (ITRs). An expression cassette flanked by one or more ITRs is referred to herein as a “viral genome.” The ITRs in the expression cassette serve as markers used for viral packaging of the expression cassette (Clark et al. Hum Gene Ther. 6:1329-41 (1995)). Exemplary and non-limiting embodiments of the viral genomes of this disclosure are shown in Figures 1A, 1C, and 2A. The polynucleotide encoding the expression cassette provides the function of expressing the transgene within the host cell. The expression cassette can be integrated into the host cell genome, for example, by infecting the host cell with an rAAV virion containing the capsid protein and a viral genome containing the expression cassette.

[0178] The promoter sequence of the expression cassette, if present, controls the expression of the polynucleotide encoding the transgene, such as the sequence encoding MYBPC3 or a functional variant thereof. Various promoters can be used. Promoters may be cell type specific. Constitutive promoters used in expression cassettes may be, for example, the cytomegalovirus enhancer fused to the chicken β-actin promoter (CAG), the monkey virus 40 (SV40) promoter, and the herpes simplex virus thymidine kinase (HSV-TK) promoter (Damdindorj et al. PLoS One. 9:e106472 (2014)). Other cell type specific promoters may also be used. Examples of cardiac cell-specific promoters include the MLC2v promoter (Phillips et al. Hypertension. 39:651-5 (2002)) and the cardiac troponin-T (cTnT) promoter (Konkalmatt et al. Circ Cardiovasc Imaging. 6:478-486 (2013)).

[0179] In some embodiments, the present disclosure is optimized for cardiac cell-specific expression and length so as to accommodate a transgene of a specific size. In one embodiment, the promoter of the rAAV vector genome described herein is a polynucleotide having a length of 300 bp to 500 bp.

[0180] Examples of expression cassette sequences and viral genome sequences in this disclosure can be found in Table 5. In some embodiments, the expression cassette includes a polynucleotide sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 95, SEQ ID NO: 99, or SEQ ID NO: 101. In some embodiments, the viral genome includes a polynucleotide sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 98, SEQ ID NO: 100, or SEQ ID NO: 102. In other embodiments, the expression cassette may be segmented according to a polynucleotide region adjacent to the transgene. A polynucleotide sequence extending from the 5' end of the cassette to the 5' end of the transgene is referred to herein as the 5' segment of the expression cassette. A polynucleotide sequence extending from the 3' end of the transgene to the 3' end of the expression cassette is referred to herein as the 3' segment of the expression cassette. In one embodiment, the 5' segment of the expression cassette contains a polynucleotide sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 93. In one embodiment, the 3' segment of the expression cassette contains a polynucleotide sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 94.

[0181] The ability to express large transgenes delivered by rAAV vectors or rAAV virions is limited. The rAAV vector genome size has a maximum sequence length of approximately 5 kb and therefore provides a limit on the length of all elements required for an expression cassette, including regulatory elements, e.g., promoters, and transgenes, e.g., MYBPC3. If the rAAV vector genome exceeds 5 kb, it results in a vector genome break during the rAAV virion package or cleaves the expression of the transgene (Wu et al. Mol Ther. 18:80-86 (2010)). In some embodiments, this disclosure provides an rAAV vector genome optimized for carrying large transgenes. The elements of the vector genome are shortened in length to accommodate larger transgenes. In one embodiment, the 5' and 3' segments of the expression cassette both contain a maximum of 0.8 kbp or a maximum of 0.9 kbp. In another embodiment, the 5'ITR, 5' segment, 3' segment, and 3'ITR all contain 1.2 kbp or up to 1.3 kbp. In one embodiment, the 5' segment contains up to 500 bp or up to 480 bp. In one embodiment, the 3' segment contains up to 200 bp or up to 150 bp. In another embodiment, the vector genome contains up to 4.7 kbp, 4.8 kbp, 4.9 kbp, or 5.0 kbp. In some embodiments, the polynucleotide encoding the gene product contains 3 kb to 11 kb, 3 kbp to 5 kbp, 3.5 kbp to 4.5 kbp, or 3.7 kbp to 4 kbp. In some embodiments, the polynucleotide encoding the gene product contains 3.7 kbp to 3.9 kbp. In some embodiments, the polynucleotide encoding the gene product contains 3.8 kbp. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5]

[0182] In certain aspects of this disclosure, rAAV virions are used to deliver the expression cassettes described herein to target cardiac cells, for example, to treat cardiomyopathy. Accordingly, this disclosure provides rAAV virions, rAAV virions comprising an AAV capsid, and an expression cassette comprising a polynucleotide encoding a transgene operably linked to a promoter.

[0183] The rAAV virions of this disclosure include a capsid protein. The capsid protein is a structural protein that constitutes the assembled icosahedral package of the rAAV virion, which includes an expression cassette. The capsid protein is classified by serotype. The wild-type capsid serotype in the rAAV virion may be, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 (Naso et al. BioDrugs 31:317-334 (2017)). Manipulated capsid types include chimeric capsids and mosaic capsids (Choi et al. Curr Gene Ther. 5:299-310 (2005)). Capsids are selected for rAAV virions based on their ability to transduce specific tissues or cell types (Liu et al. Curr Pharm Des. 21:3248-56 (2015)).

[0184] Any capsid protein capable of promoting rAAV virion gene delivery into cardiac cells can be used for the delivery of the transgenes described herein. Capsid proteins used for rAAV virions for transgene delivery into cardiac cells resulting in high expression may include, for example, AAV4, AAV6, AAV7, AAV8, and AAV9 (Zincarelli et al. Mol. Ther. 16:P1073-1080 (2008)). Artificial capsids, such as chimeric capsids generated via a complex library, may also be used for transgene delivery into cardiac cells resulting in high expression (see U.S. Patent No. 63 / 012,703, the contents of which are incorporated herein by reference). Other capsid proteins with various characteristics may also be used in the rAAV virions of this disclosure. AAV vectors and capsids are provided in U.S. Patent Application Publication Nos. US10011640B2;US7892809B2, US8632764B2, US8889641B2, US9475845B2, US10889833B2, US10480011B2 and US10894949B2, the contents of which are incorporated herein by reference, and International Patent Publication Nos. WO2020198737A1, WO2019028306A2, WO2016054554A1, WO2018152333A1, WO2017106236A1, WO2008124724A1, WO2017212019A1, WO2020117898A1, WO2017192750A1, WO2020191300A1, and WO2017100671A1, the contents thereof are incorporated herein by reference.

[0185] In some embodiments, the rAAV virions of this disclosure include an engineered capsid protein. The engineered capsid protein may be derived from a parent, e.g., a wild-type capsid, and may include, for example, a variant polypeptide sequence with respect to the parent capsid sequence at one or more sites. For example, the variant site of the parent capsid may occur at the VR-IV, VR-V, VR-VII, and / or VR-VIII sites (see, for example, Buning and Srivastava. Mol Ther Methods Clin Dev. 12:248-265 (2019)).

[0186] In some embodiments, the capsid protein is an AAV5 / AAV9 chimeric capsid protein. In some embodiments, the chimeric capsid protein contains at least one, two, three, four, or five polypeptide segments derived from the AAV5 capsid protein (SEQ ID NO: 144). In some embodiments, the chimeric capsid protein contains at least one, two, three, four, or five polypeptide segments derived from the AAV9 capsid protein (SEQ ID NO: 143). In some embodiments, at least one polypeptide segment is derived from the AAV5 capsid protein, and at least one polypeptide segment is derived from the AAV9 capsid protein.

[0187] In some embodiments, the capsid protein is a combination capsid protein. As used herein, “combination capsid protein” refers to an AAV5 / AAV9 chimeric capsid protein, which further comprises amino acid mutations to the chimeric parent sequence at one or more sites. In some embodiments, one or more sites of the chimeric parent sequence are selected from the sites corresponding to the VR-IV, VR-V, VR-VII, and VR-VIII sites of the AAV9 capsid protein.

[0188] In some embodiments, the rAAV virion comprises an engineered capsid protein selected from Table 6.

Table 6-1

Table 6-2

[0189] In some embodiments, rAAV is replication-deficient in that the rAAV virions cannot independently further replicate and package their genomes. For example, when heart cells are targeted with rAAV virions, the transgene is expressed in the target heart cells, but rAAV cannot replicate because the target heart cells lack the AAV rep and cap genes and accessory function genes.

[0190] In some embodiments, the rAAV virions of the present disclosure encapsulating the expression cassettes described herein can be produced using helper-free production. rAAV is a replication-deficient virus and typically requires components from a live helper virus, such as an adenovirus, in host cells for the packaging of infective rAAV virions. The rAAV helper-free production system enables the production of infective rAAV virions without a live helper virus. In the helper-free system, a host packaging cell line is co-transfected with three plasmids. The first plasmid may contain adenoviral gene products (e.g., E2A, E4, and VA RNA genes) necessary for the packaging of rAAV virions. The second plasmid may contain the necessary AAV genes (e.g., REP gene and CAP gene). The third plasmid contains a polynucleotide sequence encoding the transgene of interest and a promoter adjacent to the ITR. The host cell packaging cell line may be, for example, an AAV-293 host cell. Suitable host cells contain additional components necessary for the packaging of infective rAAV virions not supplied by the plasmids. In some embodiments, the CAP gene may encode, for example, the AAV capsid protein described herein.

[0191] IV. Treatment method This disclosure also provides pharmaceutical compositions comprising an rAAV vector genome or rAAV virion disclosed herein, and one or more pharmaceutically acceptable carriers, diluents, or excipients. In certain embodiments, the pharmaceutical composition comprises an rAAV vector genome or rAAV virion described herein, comprising a polynucleotide sequence encoding a therapeutic protein or nucleic acid operably ligated to a cardiac-specific promoter (e.g., a modified TNNT2 promoter). For example, in some embodiments, the pharmaceutical composition is an AAV9 vector comprising a modified cardiac TNNT2 promoter (SEQ ID NO: 3) operably ligated to a MYBPC3 protein (SEQ ID NO: 86). For example, a pharmaceutical composition is provided comprising a therapeutically effective amount of a vector comprising a polynucleotide sequence encoding a therapeutic protein or nucleic acid capable of restoring cardiac contractility for use in the prevention or treatment of cardiomyopathy.

[0192] This disclosure provides a method for expressing polynucleotides in cells. The method may include, for example, transducing target cells using rAAV virions, rAAV vector genomes, or expression cassettes as described herein. Target cells may include, for example, cardiac cells, muscle cells, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), cardiomyocytes, or MYBPC3 - / - This may include, but is not limited to, iPSC-CMs. In one embodiment, a method for expressing the MYBPC3 protein in cells includes transducing a target cell or population of target cells using an rAAV virion or rAAV vector genome as described herein. In one embodiment, the cells are MYBPC3 - / - The cell is a cell. In one embodiment, the cell contains an inactivating mutation in one or both copies of the endogenous MYBPC3 gene.

[0193] The compositions described herein can be used in methods for treating subjects having a heart disease or condition. "To treat" or "to treat a condition or subject that requires it" means (1) taking measures to obtain beneficial or desirable outcomes, including clinical outcomes such as a reduction in symptoms; (2) preventing the disease, e.g., preventing the clinical symptoms of the disease from occurring in a patient susceptible to the disease, but not experiencing or exhibiting symptoms of the disease; (3) inhibiting the disease, e.g., stopping or reducing the occurrence of the disease or its clinical symptoms; (4) mitigating the disease, e.g., causing regression of the disease or its clinical symptoms; or (5) delaying the disease. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, promoting the contraction of cardiac sarcomeres.

[0194] Subjects requiring treatment with the compositions and methods of this disclosure include, but are not limited to, individuals with congenital heart disease, individuals with degenerative muscle disease, and individuals with conditions resulting in ischemic cardiac tissue (e.g., individuals with coronary artery disease). In some cases, the methods are useful for treating degenerative muscle disease or conditions (e.g., familial cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, or coronary artery disease with resulting ischemic cardiomyopathy). In some embodiments, the method in question is useful for treating individuals with heart or cardiovascular disease or disorder, such as cardiovascular disease, aneurysm, stenosis, arrhythmia, atherosclerosis, cerebrovascular disorder (stroke), cerebrovascular disease, congenital heart disease, congestive heart failure, myocarditis, coronary artery disease, dilating artery disease, diastolic disorder, endocarditis, hypertension, cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, coronary artery disease with resulting ischemic cardiomyopathy, mitral valve prolapse, myocardial infarction (heart attack), or venous thromboembolism. In some embodiments, the subjects have cardiomyopathy or are at risk of developing cardiomyopathy.

[0195] In some embodiments, the compositions and methods disclosed herein can be used to prevent and / or treat cardiomyopathy in a subject. In some embodiments, the compositions and methods described herein can be used to treat cardiomyopathy associated with mutations in cardiac myosin-binding protein C (MYBPC3), such as hypertrophic cardiomyopathy and familial hypertrophic cardiomyopathy. Cardiomyopathy treated by the compositions and methods described herein may also include cardiomyopathy associated with pulmonary embolism, venous thrombosis, myocardial infarction, transient ischemic attack, peripheral vascular disease, atherosclerosis, ischemic heart disease, and / or other myocardial injury or vascular disease. In certain embodiments, cardiomyopathy treated by the compositions and methods described herein may include heart diseases associated with hypercontraction of myocardial tissue, such as heart failure associated with left ventricular hypercontraction.

[0196] In some embodiments, the compositions and methods described herein can induce the detectable expression of a therapeutic protein or nucleic acid (e.g., MYBPC3 protein), or its variant, variant, or fragment, to modulate the contractile function of myocardial tissue in a subject requiring such modification. In some embodiments, the amount, concentration, and quantity of the composition that modulates contractile function in myocardial tissue administered to the subject can be controlled and / or optimized to substantially improve cardiac functional parameters while mitigating adverse side effects.

[0197] The amount of the composition that modulates contractility administered to myocardial tissue may be the amount necessary to produce detectable expression in the heart of a therapeutic protein or nucleic acid (e.g., MYBPC3 protein) or variant, variant, or fragment thereof; preserve and / or improve contractility; delay the onset of cardiomyopathy or reverse the pathological course of the disease; increase muscle cell viability; improve muscle fiber function; inhibit left ventricular hypertrophy; regress cardiac hypertrophy; normalize cardiac systolic and diastolic function; or restore normal cross-bridge behavior at the muscle fiber layer level.

[0198] In some embodiments, the compositions and methods disclosed herein result in the detectable expression of the MYBPC3 protein, or its variants, variants, or fragments, in cardiac cells of a subject being treated. In some embodiments, administration of the rAAV vector genome or rAAV virion described herein induces specific expression of the MYBPC3 protein in the heart of a subject. In some embodiments, administration of the rAAV vector genome or rAAV virion described herein results in low or undetectable expression of MYBPC3 in the skeletal tissue, brain, and / or liver of a subject, and optionally, the subject has or is at risk of developing cardiomyopathy.

[0199] In some embodiments, the compositions and methods disclosed herein result in the detectable expression of the KCNH2 protein or its variants, variants, or fragments in cardiac cells being treated.

[0200] In some embodiments, the compositions and methods disclosed herein result in the detectable expression of the TRPM4 protein or its variants, variants, or fragments in cardiac cells being treated.

[0201] In some embodiments, the compositions and methods disclosed herein result in the detectable expression of the DSG2 protein or its variants, variants, or fragments in cardiac cells being treated.

[0202] In some embodiments, the compositions and methods disclosed herein result in the detectable expression of the ATP2A2 protein, or its variants, variants, or fragments, in cardiac cells being treated.

[0203] In some embodiments, the compositions and methods disclosed herein result in detectable expression of CACNA1C, DMD, DMPK, EPG5, EVC, EVC2, FBN1, NF1, SCN5A, SOS1, NPR1, ERBB4, VIP, or MYH7, or variants, mutants, or fragments thereof, in the heart cells of a subject being treated.

[0204] "Detectable expression" generally refers to at least 5%, 10%, 15%, 20% or more expression compared to a control subject or tissue not treated with a vector. In some embodiments, detectable expression means an expression 1.5-fold, 2-fold, 2.5-fold, or 3-fold greater than the vector-free control. Expression can be evaluated by Western blot, as described in the following examples, or by enzyme-linked immunosorbent assay (ELISA), or other methods known in the art. In some cases, expression is measured quantitatively using a standard curve. The standard curve can be generated using purified protein, e.g., purified MYBPC3 protein, by the methods described in the examples or methods known in the art. Alternatively, expression of a therapeutic gene product can be evaluated by quantification of the corresponding mRNA.

[0205] In some embodiments, detectable expression of a therapeutic gene product in heart tissue occurs at a dose of vector genome per kilogram (kg) of subject body weight of 3×10 14 vg / kg or less, 2×10 14 vg / kg or less, 1×10 14 vg / kg or less, 9×10 13 vg / kg or less, 8×10 13 vg / kg or less, 7×10 13 vg / kg or less, 6×10 13 vg / kg or less, 5×10 13 vg / kg or less, 4×10 13 vg / kg or less, 3×10 13 vg / kg or less, 2×10 13 vg / kg or less or 1×... 13 vg / kg or less.

[0206] In various embodiments, the compositions described herein contain the rAAV virion or vector genome described herein, as well as one or more pharmaceutically acceptable excipients. The pharmaceutically acceptable excipients may include pharmaceutically acceptable vehicles (e.g., carriers, diluents, and excipients) in an injectable formulation. These may be, in particular, isotonic sterile saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium, or magnesium chloride, etc., or mixtures of such salts), or, depending on the case of sterile water or saline, dry, particularly lyophilized, compositions that enable the formation of an injectable solution upon addition. Exemplary pharmaceutical forms suitable for injectable use include, for example, sterile aqueous solutions or dispersions, formulations containing sesame oil, peanut oil, or aqueous propylene glycol, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions.

[0207] In various embodiments, the pharmaceutical compositions of this disclosure are approximately 1 × 10 8 Genome copies / milliliter (GC / mL), approximately 5 × 10⁻⁶ 8 GC / mL, approximately 1×10 9 GC / mL, approximately 5×10 9 GC / mL, approximately 1×10 10 GC / mL, approximately 5×10 10 GC / mL, approximately 1×10 11 GC / mL, approximately 5×10 11 GC / mL, approximately 1×10 12 GC / mL, approximately 5×10 12 GC / mL, approximately 5×10 13 GC / mL, approximately 1×10 14 GC / mL, or approximately 5 × 10 14 Contains a GC / mL viral vector (e.g., rAAV virion).

[0208] In various embodiments, the pharmaceutical compositions of this disclosure are approximately 1 × 10 8 Viral genome copies / milliliter (GC / mL), 5 × 10 8 vg / mL, approximately 1×10 9vg / mL, approximately 5×10 9 vg / mL, approximately 1×10 10 vg / mL, approximately 5×10 10 vg / mL, approximately 1×10 11 vg / mL, approximately 5×10 11 vg / mL, approximately 1×10 12 vg / mL, approximately 5×10 12 vg / mL, approximately 5×10 13 vg / mL, approximately 1×10 14 vg / mL, or approximately 5 × 10 14 Contains a vg / mL viral vector (e.g., rAAV virion).

[0209] In some embodiments, the pharmaceutical compositions of the present disclosure are available in quantities of approximately 1 mL, 5 mL, 10 mL, approximately 20 mL, approximately 25 mL, approximately 30 mL, approximately 35 mL, approximately 40 mL, approximately 45 mL, approximately 50 mL, approximately 55 mL, approximately 60 mL, 65 mL, approximately 70 mL, approximately 75 mL, approximately 80 mL, approximately 85 mL, approximately 90 mL, approximately 95 mL, approximately 100 mL, approximately 105 mL, approximately 110 mL, and approximately 115 mL. It will be paid in a total amount of mL, approximately 120 mL, approximately 125 mL, approximately 130 mL, approximately 135 mL, approximately 140 mL, approximately 145 mL, approximately 150 mL, approximately 155 mL, approximately 160 mL, approximately 165 mL, approximately 170 mL, approximately 175 mL, approximately 180 mL, approximately 185 mL, approximately 190 mL, approximately 200 mL, approximately 205 mL, approximately 210 mL, approximately 215 mL, or approximately 220 mL.

[0210] In some embodiments, the method disclosed herein is approximately 1 × 10 8 Genome copies / milliliter (GC / mL), approximately 5 × 10⁻⁶ 8 GC / mL, approximately 1×10 9 GC / mL, approximately 5×10 9 GC / mL, approximately 1×10 10 GC / mL, approximately 5×10 10 GC / mL, approximately 1×10 11 GC / mL, approximately 5×10 11 GC / mL, approximately 1×10 12 GC / mL, approximately 5×10 12 GC / mL, approximately 5×10 13 GC / mL, approximately 1×10 14 GC / mL, or approximately 5 × 1014 Administering rAAV virions encoding MYBPC3 at a dose of rAAV virions in GC / mL.

[0211] In a preferred embodiment, the method of the present disclosure is about 3×10 12 GC / mL, about 3×10 13 GC / mL, about 1×10 14 GC / mL, or about 3×10 14 GC / mL, and administering rAAV virions encoding MYBPC3 intravenously.

[0212] In a preferred embodiment, the method of the present disclosure is about 3×10 11 GC / mL, about 3×10 12 [[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​13 vg / mL, about 1×10 14 vg / mL, or about 3×10 14 vg / mL of rAAV virions, and includes intravenously administering rAAV virions encoding MYBPC3.

[0215] In a preferred embodiment, the method of the present disclosure is about 3×10 11 vg / mL, about 3×10 12 vg / mL, about 1×10 13 vg / mL, or about 3×10 13 vg / mL of rAAV virions, and includes administering rAAV virions encoding MYBPC3 by local delivery to the heart.

[0216] Genome copies per milliliter can be determined by quantitative polymerase change reaction (qPCR) using a standard curve generated using a reference sample having a known concentration of the viral polynucleotide genome. For AAV, the reference sample is often the transfer plasmid used for the production of rAAV virions, but other reference samples may be used.

[0217] Alternatively, or additionally, the concentration of the viral vector can be determined by measuring the titer of the vector in a cell line. Viral titer is generally expressed as viral particles per unit volume (e.g., vp / mL). In various embodiments, the pharmaceutical composition of the present disclosure is about 1×10 8 viral particles / milliliter (vp / mL), about 5×10 8 vp / mL, about 1×10 9 vp / mL, about 5×10 9 vp / mL, about 1×10 10 vp / mL, about 5×10 10 vp / mL, about 1×10 11 vp / mL, about 5×10 11 vp / mL, about 1×10 12 vp / mL, about 5×10 12 vp / mL, about 5×10 13vp / mL, or approximately 1 × 10⁻⁶ 14 vp / mL, or approximately 5 × 10⁻⁶ 14 It includes a viral vector (e.g., rAAV virion).

[0218] In one embodiment, the present disclosure provides a kit comprising a container for housing a pharmaceutical composition described herein.

[0219] The rAAV virions or vector genomes of this disclosure can be administered to subjects in need by systemic application, for example, by intravenous, intra-arterial, or intraperitoneal delivery of the vector, similar to those shown in animal models (Katz et al., 2012, Gene Ther. 19:659-669). In some embodiments, the rAAV virions or vector genomes of this disclosure treat or prevent hypertrophic cardiomyopathy, in which case the vector is administered systemically.

[0220] In some embodiments, the rAAV virions or vector genomes of this disclosure may be delivered, for example, by direct administration to cardiac tissue via intra-coronary artery delivery. In some embodiments, the vector is administered as a single dose by antegrade epicardial infusion over 10 minutes in a cardiac catheterization laboratory after angiography (percutaneous coronary delivery without vascular balloon occlusion) using a standard 5F or 6F guide or diagnostic catheter (Jaski et al., 2009, J Card Fail. 15:171-181).

[0221] Subjects suitable for treatment using the compositions, compositions, and methods of this disclosure include individuals with cardiac conditions (e.g., mammalian subjects such as humans, non-human primates, domestic mammals, and experimental non-human mammalian subjects such as mice and rats).

[0222] In some embodiments, the rAAV virion or vector genome of this disclosure can be used to treat subjects in need. In some embodiments, the viral vector can be administered to subjects in need of treatment for cardiovascular disease. In some embodiments, the rAAV virion or vector genome is administered to subjects to treat cardiomyopathy. In some embodiments, the viral vector is administered systemically. In other embodiments, the viral vector is delivered by direct administration to cardiac tissue.

[0223] rAAV virions or vector genomes can be administered by a variety of routes, including but not limited to direct injection into the heart or cardiac catheterization. In preferred embodiments, a pharmaceutical composition containing rAAV virions encoding MYBPC3 is administered by intracardiac catheter delivery via retrograde coronary sinus infusion (RCSI). Alternatively, viral vectors can be administered systemically, such as by intravenous infusion. When direct injection is used, it can be performed during either open-heart surgery or minimally invasive surgery. In some cases, viral vectors are delivered into the pericardial cavity by injection or infusion.

[0224] Viral vectors administered to subjects can be tracked by various methods. For example, recombinant viruses labeled or expressed with markers (such as green fluorescent protein or beta-galactosidase) can be easily detected. Recombinant viruses may be engineered to cause target cells to express marker proteins, such as surface expression proteins or fluorescent proteins. Alternatively, infection of target cells with recombinant viruses can be detected by the expression of cell markers not expressed by the animals used in the study (e.g., human-specific antigens when cells are injected into experimental animals). The presence and phenotype of target cells can be assessed by fluorescence microscopy (e.g., green fluorescent protein or beta-galactosidase), immunohistochemistry (e.g., using antibodies against human antigens), ELISA (e.g., using antibodies against human antigens), or RT-PCR analysis using hybridization conditions that induce amplification specific to primers and RNA exhibiting a cardiac phenotype.

[0225] All patents, patent publications, and other publications referenced and identified herein are incorporated herein individually and expressly by reference in their entirety for all purposes. [Examples]

[0226] Example 1: Design of a vector genome for large cargo The objective of this study is to evaluate vectors with deletions in their non-coding regions against their parent vectors. Surprisingly, this shows that deletions in the non-coding region increase the capabilities of the vector.

[0227] With two intact contiguous ITR sequences (approximately 130 bp each) for optimal transgene expression, a promoter, introns, WPRE and polyadenylation signals, and standard cis-regulatory sequences, a typical AAV vector genome requires approximately 1.8–2.0 kbp of non-coding DNA sequence. Transgenes larger than approximately 3.0 kbp, such as the 3.8 kbp transgene MYBPC3, result in a vector genome exceeding 5.0 kbp. For example, Mearini et al., Nat Commun 5:5515 (2014) reported an AAV vector encoding MYBPC3 with a vector genome size of 5.4 kbp. Without the ITR sequence, this would be approximately 5.2 kbp.

[0228] A reporter system was created to test whether AAV vectors tolerate shortened non-coding regions. A conventional AAV vector with a CAG promoter, intron, WPRE, and standard polyA sequence was modified by removing the WPRE (589 bp) and shortening the polyA sequence (removing 170 bp) (Figure 1A). Expression of the GFP reporter cloned at multiple cloning sites (MCS) was maintained, but slightly decreased when these vector elements were deleted or shortened (Figure 1B). The vector was further cleaved by deleting an intron and from part of the 3' sequence to the 5' ITR (Figure 1C). The final vector genome was approximately 1.1 kpb (0.8 kpb without the ITR).

[0229] Example 2: Expression of the MYBPC3 transgene in induced cardiomyocytes in vitro The objective of this study was to provide improved tissue-specific promoters for the expression of therapeutic gene products in cardiomyocytes induced in vitro using an AAV-based vector system. Human induced pluripotent stem cells (iPSCs) were differentiated into cardiomyocytes using mesoderm induction, cardiac specification, and metabolic selection, as previously described (Tohyama et al. Cell Stem Cell. 2013;12(1):127-37; Lian et al. Proc Natl Acad Sci U A. 2012;109(27):E1848-57; Burridge PW, Holmstrom A, Wu JC. Curr Protoc Hum Genet. 2015;87:21 3 1-15.). iPSC-CM viral transduction was performed using AAV6 with specified multiple infections.

[0230] The gene expression cassette shown in Figure 2A was constructed for an AAV-based vector system for the treatment of cardiomyopathy. The vector-based AAV vector shown in Figure 1C contained several cis-regulatory elements, including two inverted terminal repeats (ITRs, 260 bp each), a polyadenylation signal (A, 49 bp), and either the full-length (SEQ ID NO: 1) or modified cardiac troponin T (TNNT2) promoter (SEQ ID NOs: 2-4). It did not contain a WPRE, and both the poly-A signal and the 3'-5' ITR were shortened. The modified TNNT2 promoter contained a 100-200 bp deletion at the 5' (upstream) end of the wild-type TNNT2 promoter. Human myosin-binding protein C (MYBPC3, SEQ ID NO: 86), with a polynucleotide sequence length of 3.825 kb, was tested as a therapeutic gene product in iPSC-derived cardiomyocytes.

[0231] To determine whether wild-type or modified TNNT2 promoters can induce detectable expression of the MYBPC3 protein, - / - iPSC-derived cardiomyocytes, 6 × 10 4Cells were transduced with AAV6 particles at MOI. Cell expression for MYBPC3 protein was analyzed 5–15 days after infection by immunofluorescence or Western blotting.

[0232] Figure 2B shows that the AAV vector containing the wild-type TNNT2 promoter (SEQ ID NO: 1) is MYBPC3 - / - This study demonstrates that it drives the expression of the MYBPC3 protein in sarcomeres of iPSC-derived cardiomyocytes.

[0233] Figures 3A-3C show MYBPC3 genetically modified using an AAV vector containing a 400 bp modified TNNT2 promoter (SEQ ID NO: 3). - This shows that in / -iPSC-derived cardiomyocytes, it drives higher MYBPC3 protein expression than either the 600 bp wild-type TNNT2 (SEQ ID NO: 1) or the 500 bp modified TNNT2 (SEQ ID NO: 2) promoter. In contrast, MYBPC3 transfected with the plasmid encoding MYBPC3 (rather than transfected by a virus) under the control of either the 600 bp wild-type TNNT2 (SEQ ID NO: 1) or the 400 bp modified TNNT2 promoter (SEQ ID NO: 3). - / - iPSC-derived cardiomyocytes showed similar MYBPC3 protein expression.

[0234] Example 3: Mybpc3 - / - Mouse model of hypertrophic severe cardiomyopathy Homozygous Mybpc3 knockout mice (KOs) were generated in a C57Bl / 6 background by deleting CRISPR-Cas9 vs. gRNA in exons 1 and 2 (Figure 4A). Despite normal Mendelian ratios and body weight comparable to wild-type littermates, the KO mice showed severe deficits in cardiac function (Figures 4C and 4D) and marked cardiac hypertrophy (Figures 4E-4G) at 2 weeks of age (Figure 4B). This model exhibits more severe cardiac hypertrophy than other models (Schlossarek et al. Basic Res. Cardiol. 107:1-13 (2012)). Our KO mice exhibit severe, early-onset HCM in young adults (2-week-old mice) modeling the development of HCM in human children, as well as in late-stage HCM in adults. Lekanne Deprez et al., J Med Genet 43:829-832(2006); 102:254-258 (2012); Wessels et al., Eur J Hum Genet 23:922-928 (2015).

[0235] Example 4: Expression of MYBPC3 transgene in cardiac tissue in vivo The objective of this study was to investigate the in vivo expression of therapeutic proteins using an AAV-based vector system containing a modified heart-specific promoter.

[0236] AAV9 recombinant virus containing either a 600 bp wild-type TNNT2 (SEQ ID NO: 1) or a 400 bp modified TNNT2 (SEQ ID NO: 3) promoter operably ligated to a polynucleotide encoding MYBPC3 was injected into adult mice postorbitally. Tissue samples were collected from the heart, skeletal muscle (tibialis anterior), liver, and whole brain two weeks after infection. RNA was extracted from all tissues, synthesized into cDNA, and analyzed by qRT-PCR using primers specific to human MYBPC3.

[0237] As shown in Figure 5, mice injected with AAV9-based vectors containing either the wild-type or modified TNNT2 promoter showed higher levels of MYBPC3 mRNA in cardiac tissue compared to skeletal, brain, or liver tissue. The 400 bp modified TNNT2 promoter showed increased MYBPC3 mRNA expression in cardiac tissue compared to the 600 bp wild-type TNNT2 promoter.

[0238] As shown in Figures 6A-6B, adult mice were intravenously administered an AAV9 vector containing a modified 400 bp TNNT2 promoter cassette via tail vein injection. Tissue samples were collected from the heart and liver four weeks after injection. Absolute quantification of viral genome per microgram of genomic DNA was evaluated by qPCR using linearized standards. RNA was extracted from all tissues, synthesized into cDNA, and analyzed by qRT-PCR using primers specific to human MYBPC3. Surprisingly, the 400 bp TNNT2 promoter maintained high selectivity for the heart, and in adult mice, liver expression of the transgene was less than 1 / 10,000 of cardiac expression, even though the vector genome detected in the liver was 100 times larger than that in the heart four weeks after administration (Figure 6A, logarithmic scale) (Figure 6B, logarithmic scale).

[0239] In summary, these results suggest that a 200bp deletion from the wild-type TNNT2 promoter, i.e., the 400bp modified TNNT2 promoter (SEQ ID NO: 3), effectively drives highly selective expression of the MYBPC3 protein in cardiomyocytes, despite the substantial deletion of the promoter sequence.

[0240] Example 5: Recovery of cardiac function in Mybpc3 null mice This embodiment demonstrates functional relief of loss of function in mouse Mybpc3 using the vector designed for large cargo described in Example 1, as well as the 400 bp modified hTNNT2 promoter described in Examples 2 and 3.

[0241] A 400 bp hTNNT2 promoter and the mouse Mybpc3 gene were cloned into the vector shown in Figure 1C. This vector was packaged using an AAV9 capsid to generate a test vector.

[0242] In the first experiment, homozygous Mybpc3 - / - Mice are given Mybpc3 or vehicle, encoding HBSS, 1E14 vg·kg -1 The test vector was injected postorbitally at 2 weeks of age. Cardiac tissue was collected 2 weeks later (4 weeks later) along with tissue from wild-type littermates. The experimental vector was Mybpc3 2 weeks after injection. - / - Wild-type levels of MYBPC3 protein expression were achieved in mice (Figure 7). The test vector was 1E14 vg·kg. -1 We concluded that MYBPC3 could be expressed at physiological levels in young animals at such low doses.

[0243] In the second experiment, the first experiment was repeated with a lower dose of 3E13 vg·kg. -1 And it was repeated. Homozygous Mybpc3 - / - Mice are given Mybpc3 or Vehicle, code 3E13 vg·kg, which are associated with HBSS. -1 and 1E14 vg·kg -1The test vector was injected postorbitally at 2 weeks of age. Wild-type levels of cardiac MYBPC3 protein expression were observed 2 weeks and 6 weeks after injection. - / - It was detected in mice by ELISA (Figure 8). The test vector was 3E13 vg·kg. -1 We concluded that MYBPC3 could be expressed at physiological levels in young animals at such low doses.

[0244] In the third experiment, cardiac function was evaluated using an assay related to hypertrophic cardiomyopathy. Hypertrophic cardiomyopathy is physiologically characterized by (1) mg g -1 (1) This is presented as an increase in cardiac size, reported as the ratio of left ventricular weight to total weight (LVM / BW), and (2) as fractional shortening (FAS) measured by echocardiography.

[0245] Homozygous Mybpc3 - / - Mice were given 1E13 vg·kg of test vector encoding HBSS, which is either Mybpc3 or the vehicle. -1 , 3E13 vg·kg -1 and 1E14 vg·kg -1 It was injected posteriorly into the orbit at 2 weeks of age. All doses tested (1E13 vg·kg) -1 , 3E13 vg·kg -1 and 1E14 vg·kg -1 Dose-dependent recovery of cardiac function was observed. LVM / BM decreased from vehicle control at 6 weeks after injection at all tested doses (Figure 9A). FAS (expressed as the percentage change in the internal dimension of the LV between systolic and diastolic pressure, Figure 9B) and ejection fraction (Figure 9C) increased from vehicle control at all tested doses at 6 weeks after injection. Further improvements in LVM / BM (Figure 9D), FAS (Figure 9E), and EF (Figure 9F) were observed at 31 weeks after injection. 3E13 vg·kg -1 and 1E14 vg·kg -1 Consistent with the equal levels of MYBPC3 protein expression observed at the dose (see Figure 8), 3E13 vg·kg -1 or 1E14 vg·kg -1Animals treated with this method show similar improvements in hypertrophy, FAS, or EF. 1E13 vg·kg -1 Even with dose alone, hypertrophy, FAS, and EF all improve compared to the vehicle control.

[0246] The test vector is 1E13 vg·kg -1 They concluded that even such a low dose had the ability to restore cardiac function in young animals.

[0247] Restoring function in symptomatic juvenile mice is more difficult in cases of hypertrophic cardiomyopathy than in infants, because hypertrophic cardiomyopathy is a progressive disorder. Older animals exhibit more severe disease than younger animals. To our knowledge, restoration of MYBPC3 function loss in symptomatic juvenile animals has not been demonstrated in AAV. Our model also includes complete loss of function caused by deletion of the Mybpc3 gene, not just partial loss of function due to mutation.

[0248] We compared our results with those reported by Mearini et al., Nat.Commun. 5:5515 (2014), using a 5.4 kb expression cassette encoding Mybpc3 in mice with a single nucleotide polymorphism in the endogenous Mybpc3 gene. Mearni et al. based their very high dose analysis (1E12 vg, 3E12 vg, 7E14 vg·kg) on ​​an average neonatal mass of 1.5 g for an AAV9 vector encoding the same Mybpc3 gene. -1 and 2E15 vg·kg -1 In mice injected as neonates (not symptomatic juveniles) of the same age (corresponding to [specific condition]), the prevention of high LVM / BW at 2 weeks of age was reported. Mearni et al. used the 550 bp hTNNT2 promoter instead of the 400 bp modified hTNNT2 promoter of the present invention. The vector by Mearni et al. was 2E15 vg·kg -1 However, this does not significantly prevent the reduction of FAS. In contrast, the vector of the present invention has at least 1E13 vg·kg -1 ~1E14 vg·kg -1Even at such low doses, it shows improvement in physiological parameters in young animals (not just neonates).

[0249] Modifications to vectors and promoters dramatically and remarkably increase the potency of the vectors.

[0250] Example 6: Direct comparison of 5.4kbp cassette and 4.7kbp cassette In this example, a 5.4 kbp cassette encoding the Mybpc3 gene is directly compared with a 4.7 kbp cassette encoding the Mybpc3 gene in mature (2.5 months old) homozygous mice with advanced disease.

[0251] Homozygous Mybpc3 - / - Mice were given 3E13 vg·kg of AAV9 vector encoding Mybpc3 in association with a 5.4kbp or 4.7kbp cassette (Figure 10A). -1 or 1E14 vg·kg -1 The 4.7kbp cassette was injected posteriorly into the orbit, or the vehicle control, HBSS, was injected. Even when administered in this advanced stage of cardiac dysfunction, the 4.7kbp cassette significantly improved cardiac function based on ejection fraction (EF) (Figure 10B) and showed a clear functional recovery above pre-administration baseline (Figure 10C), unlike animals treated with the vehicle (Veh) or the 5.4kbp cassette. Furthermore, compared to the 5.4kbp cassette, the 4.7kbp cassette was also able to significantly reduce hypertrophy, as indicated by LVM / BM 18 weeks after injection (Figure 10D).

[0252] This example demonstrates the functional recovery of loss of function in mouse Mybpc3 using the vector skeleton and promoter modifications described in Examples 1-3.

[0253] This example also deals with the challenging disease model of adult homozygous Mybpc3 - / - In mice, a low dose of the 5.4 kbp vector (SEQ ID NO: 201) did not produce physiological improvement, while the 4.7 kbp vector according to this disclosure produced 3E13 vg·kg-1 This demonstrates that even at such low doses, it induces statistically significant improvements in physiological parameters associated with cardiomyopathy.

[0254] Example 7: Greater efficacy with an improved AAV capsid encoding MYBPC3 This example shows a large cargo vector (Example 1) and Mybpc3 - / - This demonstrates how the improved capabilities of the modified promoters (Examples 2 and 3), based on the recovery of mice (Example 5), can be further enhanced by the use of the manipulated AAV capsid.

[0255] The AAV9 capsid variant CR9-10 showed significantly higher cardiac phenotype upon systemic delivery in adult mice compared to AAV9 with a GFP-coded cassette determined by ELISA (p<0.05, one-way ANOVA; Dunnett's multiple comparison test) (Figure 11A).

[0256] In the second experiment, the expression cassette encoding the mouse Mybpc3 gene was packaged in either AAV9 or CR9-10, and Mybpc3 - / - We compared the ability of homozygous Mybpc3 to save the heart. - / - Mice were fed 1E13 vg·kg of AAV9 vector, CR9-10 vector, or vehicle control HBSS. -1 and 3E13 vg·kg -1 The test substances were injected posteriorly into the orbit at 2 weeks of age. All test substances significantly improved cardiac function based on ejection fraction (EF) (Figure 11B), with a clear recovery of function (ΔEF) above pre-administration baseline (Figure 11C). Consistent with improved cardiac transduction, CR9-10 resulted in a greater improvement in EF than AAV9.

[0257] Example 8. Non-human primate studies of manipulated AAV capsid variants. The biodistribution of AAV vectors with manipulated capsids (U.S. Provisional Patent Application No. 63 / 012,703, the entirety of which is incorporated herein by reference) was evaluated in male cynomolgus monkeys (Macaca fascicularis) after intravenous delivery.

[0258] AAV vectors generated with 14 different capsids, including AAV9, were pooled and injected into NHP at a dose of 1E13 vg·kg-1 (n=3). Viral DNA was extracted from the left ventricle and liver one month after systemic delivery. Consistent with mouse results, CR9-10 showed increased cardiac transduction compared to AAV9 (Figure 12A). Furthermore, many variants reduced hepatic viral load compared to AAV9 (Figure 12B) and improved the ratio of left ventricular transduction to liver infection (Figure 12C). Example 10: Restoration of cardiac function in Mybpc3c null mice using the human MYBPC3 gene.

[0259] This example uses Mybpc3 - / - This demonstrates the ability of the human MYBPC3 gene to restore mice.

[0260] AAV9 encoding the mouse Mybpc3 gene (mMybpc3) (1E14 vg·kg-1), AAV9 encoding the human MYBPC3 gene (hMYBPC3) (1E14 vg·kg-1), or HBSS as a vehicle, homozygous Mybpc3 - / - Mice were injected into the posterior orbit at 2 weeks of age. Cardiac size and function were monitored by echocardiography until 8 months after injection. These results are from Mybpc3. - / -This study demonstrates that AAV9-mediated cardiac MYBPC3 replacement with either mMybpc3 or hMYBPC3 in mice resulted in recovery of cardiac size and function. Ejection fraction (EF) was significantly improved by both human and mouse orthologues of MYBPC3, with mMybpc3 yielding a greater improvement in EF compared to hMYBPC3 (Figure 13A). Importantly, hMYBPC3 was just as potent as mMybpc3 in reducing cardiac hypertrophy over time, as evidenced by left ventricular mass normalized to body weight (LVM / BW) (Figure 13B). This was further validated by a comparable reduction in left ventricular posterior wall thickness during diastole (LVPW;d) (Figure 13C). Definitively, all improvements showed robust stability up to 8 months post-injection.

[0261] In the second study, the effectiveness of hMYBPC3 was evaluated across a range of viral doses. Homozygous Mybpc3 - / - Mice were injected postorbitally at 2 weeks of age with test vectors encoding the human MYBPC3 gene or the vehicle HBSS: 1E13 vg·kg-1, 1E14 vg·kg-1, and 3E14 vg·kg-1. At 14 weeks post-injection, dose-dependent improvements in cardiac function were observed for all test doses (1E13 vg·kg-1, 1E14 vg·kg-1, and 3E14 vg·kg-1), with significant improvements above pre-administration baseline for treatment with 1E14 vg·kg-1 and 3E14 vg·kg-1, as shown in EF (Figure 14A) (Figure 14B). Based on LVM / BW (Figure 14C), a significant reduction in cardiac hypertrophy was also observed for treatment with 1E14 vg·kg-1 and 3E14 vg·kg-1. The hMYBPC3 test vector was concluded to have the ability to preserve cardiac function in adult animals at low doses of approximately 1E13 vg·kg-1.

[0262] Example 11: Treatment of hypertrophic cardiomyopathy (HCM) Cardiomyopathy is the leading cause of sudden cardiac arrest in children under 18 years of age. Hypertrophic cardiomyopathy (HCM) affects 500,000 Americans and can lead to heart failure or sudden death. Loss-of-function mutations in myosin-binding protein C3, MYBPC3, are the most common genetic cause of HCM. The majority of MYBPC3 mutations causing HCM are cleaved via nonsense mutations, frameshift mutations, or splice site mutations. The sarcomere pathophysiology in the majority of HCM patients with MYBPC3 mutations is thought to be due to haploinsufficiency, as the total amount of MYBPC3 protein incorporated into the sarcomere is significantly below normal. Reduced sarcomere levels of MYBPC3 result in reduced myosin inhibition, which allows more myosin heads to engage on actin filaments, leading to hypercontraction.

[0263] The clearest pathway to treating haploinsufficiency is the restoration of the deficient gene product, in this case, wild-type MYBPC3. Therefore, we successfully designed an AAV vector (TN-201) with superior properties for selectively restoring MYBPC3 to cardiomyocytes upon systemic delivery. Crucially, we demonstrated for the first time, the ability of both the mouse surrogate and TN-201 to restore cardiac dysfunction and hypertrophy in a disease symptom mouse model using AAV.

[0264] Dose-range efficacy studies demonstrated the restoration of wild-type MYBPC3 protein and saturation of cardiac improvement at a clinically relevant dose of 3E13 vg / kg. Furthermore, pilot safety studies in adult and infant mice injected with more than 10-fold effective doses showed no clinical findings, changes in cardiac function, or histopathological findings. Importantly, TN-201, produced using the highly scalable Sf9 platform, has proven effective against Mybpc3 disease. - / - It was found to yield similarly potent efficacy in the model. Finally, we established that the efficacy we observed was sufficiently significant for stable benefits up to 8 months post-injection, as well as for the recovery of cardiac dysfunction even in late homozygous disease.

[0265] Example 12: Clinical Trial Pharmaceutical compositions comprising rAAV virion encoding MYBPC3 as described herein are administered intravenously or via retrograde coronary sinus (RCSI). Functional efficacy is determined by assessment of cardiac function (e.g., New York Heart Association Functional Classification, NYHA; cardiopulmonary exercise testing, CPET), quality of life questionnaires (e.g., Kansas City Cardiomyopathy Questionnaire Clinical Quality Score, KCCQ-CSS), cardiac imaging (e.g., echocardiography), cardiac biomarkers (e.g., troponin and NT-proBNP), cardiac rhythm and immunological assessment, assessment of cardiac function (e.g., Pediatric Interagency Registry for Mechanically Assisted Circulatory Support, PEDIMACS; Ross classification), and / or Major Adverse Cardiac Events (MACE) (all-cause death, heart transplantation, induction, initiation of ventilation, or mechanical circulatory support). Clinical trials may include annual monitoring of safety and continued efficacy (e.g., adverse events, severe adverse events, electrocardiogram, cardiac enzymes, biomarkers, functional status, left ventricular (LV) function / mass, quality of life, serological chemistry tests, and liver function tests) for up to 10 years. In certain embodiments, for example, the following are provided: (Item 1) A modified cardiac troponin T promoter containing polynucleotides with lengths of 300 bp to 500 bp. (Item 2) The promoter according to item 1, wherein the polynucleotide comprises a sequence that shares at least 80%, at least 90%, or 100% identity with any one of sequence numbers 1 to 85. (Item 3) The promoter according to item 1, wherein the polynucleotide comprises a sequence that shares at least 80%, at least 90%, or 100% identity with SEQ ID NO: 3. (Item 4) The aforementioned polynucleotide is (i) Located upstream of the transcription start site of the troponin T gene, and containing it, or (ii) The transcription start site of the troponin T gene is -450 bp to +1 bp, -350 bp to +1 bp, -250 bp to +1 bp, -450 bp to +50 bp, -350 bp to +50 bp, or -250 bp to +50 bp. The promoter described in item 1, which shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a genomic polynucleotide sequence, and optionally the troponin T gene is a human troponin T gene. (Item 5) The aforementioned promoter, (i) muscle-specific promoters, and / or (ii) Cardiac cell-specific promoters, and / or (iii) A promoter that is a cardiomyocyte-specific promoter, as described in any one of items 1 to 4. (Item 6) The aforementioned promoter, (i) a natural troponin T promoter of approximately 600 bp, and / or (ii) A promoter according to any one of items 1 to 5, having the same cell type specificity as the reference promoter containing Sequence ID No. 1. (Item 7) The promoter according to any one of items 1 to 6, wherein the promoter expresses a gene product operably linked to it at least about 10%, at least about 20%, and at least about 30% more than the natural troponin T promoter, and optionally the natural troponin T promoter is a reference promoter containing SEQ ID NO: 1. (Item 8) A vector comprising a promoter described in any one of items 1 to 7, operably bound to a polynucleotide encoding a gene product. (Item 9) The aforementioned vector is a viral vector, and optionally, The viral vector is an adeno-associated virus vector (AAV), and / or the viral vector has a packaging limit of approximately 5.5 kB at most. The vector according to item 8, wherein the AAV vector is optionally AAV9 or a variant thereof. (Item 10) The aforementioned gene product (i) MYBPC3, KCNH2, TRPM4, DSG2 and ATP2A2 proteins, or (ii) A vector as described in item 8 or 9, selected from the proteins CACNA1C, DMD, DMPK, EPG5, EVC, EVC2, FBN1, NF1, SCN5A, SOS1, NPR1, ERBB4, VIP, and MYH7. (Item 11) The vector according to any one of items 8 to 10, wherein the gene product is Cas9, which is arbitrarily selected from SpCas9, St1Cas9, and SaCas9. (Item 12) The vector according to any one of items 8 to 11, wherein the vector comprises a polynucleotide encoding a second gene product, and optionally the second gene product is a functional RNA, optionally a microRNA, or a guide RNA. (Item 13) Isolated cells containing a promoter described in any one of items 1-7. (Item 14) The isolated cells described in item 13, wherein the isolated cells are induced pluripotent stem cells or isolated cardiomyocytes. (Item 15) A pharmaceutical composition comprising a vector as described in any one of items 8 to 12. (Item 16) A cell therapy composition comprising the cells described in item 13 or item 14. (Item 17) A recombinant adeno-associated virus (AAV) vector genome comprising a MYBPC3 protein or a MYBPC3 polynucleotide encoding a functional variant thereof, and a promoter, wherein the promoter is a modified cardiac troponin T promoter as described in any one of items 1 to 7. (Item 18) The rAAV vector genome described in item 17, wherein the MYBPC3 polynucleotide is full-length MYBPC3 containing at least about 3.5 kB, about 3.8 kB, or truncated MYBPC3. (Item 19) The rAAV vector genome described in item 17 or item 18, wherein the aforementioned MYBPC3 is human MYBPC3. (Item 20) The rAAV vector genome described in any one of items 17-19, wherein the vector expresses MYBPC3 at approximately the same level as a reference AAV vector containing a native troponin T promoter of approximately 600 bp, at least about 10% higher, or at least about 20% higher. (Item 21) A recombinant adeno-associated virus (AAV) vector genome comprising an expression cassette comprising, in the order of 5' to 3', a 5' segment containing a promoter, a polynucleotide encoding a gene product, and a 3' segment containing a polyA signal, wherein the expression cassette is optionally adjacent to one or both of the 5' inverted terminal repeat (ITR) and the 3' ITR. The polynucleotide encoding the gene product includes 3kb-11kb, 3kb-5kb, 3.5kb-4.5kb, or 3.7kb-4kb. a) Both the 5' segment and the 3' segment contain a maximum of 0.8kbp or a maximum of 0.9kbp, a) The 5'ITR, 5' segment, 3' segment, and 3'ITR all contain a maximum of 1.2kbp or a maximum of 1.3kbp, and / or c) The vector genome is a recombinant adeno-associated virus (AAV) vector genome containing up to 4.7 kbp, up to 4.8 kbp, up to 4.9 kbp, up to 5.0 kbp, or up to 5.2 kbp. (Item 22) The rAAV vector genome described in item 21, wherein the 5' segment comprises up to 500 bp or up to 480 bp, and / or the 3' segment comprises up to 200 bp or up to 150 bp. (Item 23) The polynucleotide encoding the gene product is an rAAV vector genome as described in item 21 or item 22, comprising 3.7 kbp to 3.9 kbp, optionally 3.8 kbp. (Item 24) The gene product is MYBPC3 or a functional variant thereof, and optionally, the polynucleotide encoding MYBPC3 shares at least 90% identity with SEQ ID NO: 86, at least 95% identity with SEQ ID NO: 86, or is SEQ ID NO: 86, and / or An rAAV vector genome as described in any one of items 21-23, wherein the MYBPC shares at least 90% identity with the polypeptide sequence of SEQ ID NO: 103, at least 95% identity with the polypeptide sequence of SEQ ID NO: 103, or is the polypeptide sequence of SEQ ID NO: 103. (Item 25) The rAAV vector genome according to any one of items 21 to 24, wherein the promoter is a modified cardiac troponin T promoter according to any one of items 1 to 8. (Item 26) (i) The promoter includes a sequence that shares at least 80% identity, at least 90% identity, or 100% identity with sequence number 91, and / or (ii) The polyA signal essentially consists of or comprises a sequence that shares at least 90% identity with sequence number 92, shares at least 95% identity with sequence number 92, or is sequence number 92. (iii) The 5' segment shares at least 80% identity, at least 90% identity, at least 95% identity with sequence number 93, or is sequence number 93, and / or (iii) The rAAV vector genome according to item 25, wherein the 3' segment shares at least 80% identity with, at least 90% identity with, or at least 95% identity with, sequence number 94. (Item 27) The rAAV vector genome according to any one of items 21 to 26, wherein the expression cassette shares at least 80% identity, at least 90% identity, or at least 95% identity with SEQ ID NO: 95, and / or the rAAV vector genome shares at least 80% identity, at least 90% identity, or at least 95% identity with SEQ ID NO: 102, or at least 95% identity with SEQ ID NO: 102. (Item 28) The AAV vector genome according to any one of items 21 to 27, wherein the 5'ITR contains a sequence that shares 95% identity with SEQ ID NO: 96, and / or the 3'ITR contains a sequence that shares at least 95% identity with SEQ ID NO: 97. (Item 29) A recombinant AAV (rAAV) virion comprising the rAAV vector genome and AAV capsid protein described in any one of items 17-28, Recombinant AAV(rAAV) virions wherein, optionally, the rAAV virion is serotype AAV9 virion or a variant thereof, and / or the AAV capsid protein is AAV9 capsid protein or a variant thereof. (Item 30) An in vitro or ex vivo method for expressing the MYBPC3 protein in cells, comprising transducing the cells with an rAAV virion as described in item 29 or an rAAV vector genome as described in any one of items 17-28. (Item 31) The aforementioned cells are MYBPC3 - / - The method described in item 30, which is a cell. (Item 32) The method according to item 30 or item 31, wherein the cells contain an inactivating mutation in one or both copies of the endogenous MYBPC3 gene. (Item 33) An rAAV virion as described in item 29 or an rAAV vector genome as described in any one of items 17-28, for use as a drug. (Item 34) An rAAV virion as described in item 29, or an rAAV vector genome as described in any one of items 17-28, used in a manner to treat and / or prevent cardiomyopathy in a subject, wherein the cardiomyopathy is, optionally, hypertrophic cardiomyopathy. (Item 35) An rAAV virion as described in item 29 or an rAAV vector genome as described in any one of items 17-28, for use in methods of treating and / or preventing a disease or disorder caused by the target MYBPC3 mutation. (Item 36) The method comprises expressing the MYBPC3 protein and / or increasing MYBPC3 activity and / or increasing the cardiac function of the heart of the subject, for use of an rAAV virion or rAAV vector genome as described in item 34 or item 35. (Item 37) The rAAV virion or rAAV vector genome for use according to any one of items 33 to 36, wherein administration of the rAAV virion or rAAV vector genome causes specific expression of MYBPC3 in the heart of the subject. (Item 38) The rAAV virion or rAAV vector genome for use as described in any one of items 33 to 37, wherein administration of the rAAV virion or rAAV vector genome causes low or undetectable expression of MYBPC3 in the skeletal tissue, brain, and / or liver of the subject. (Item 39) An rAAV virion or rAAV vector genome for use as described in any one of items 34-38, wherein the subject contains the MYBPC3 mutation. (Item 40) The method described above is (i) Intravenously administer the rAAV virion or rAAV vector genome to the subject. (i) administer the rAAV virion or rAAV vector genome to the subject intracardiac, (iii) rAAV virion or rAAV vector genome for use according to any one of items 34 to 39, comprising directly injecting the subject with the rAAV virion or rAAV vector genome. (Item 41) rAAV virions or rAAV vector genomes for use as described in any one of items 34-40, wherein the subject is a mammal, and optionally the subject is human. (Item 42) The subject is young, and the rAAV virion or rAAV vector genome for use as described in any one of items 34-41. (Item 43) The subject is an adult, and the rAAV virion or rAAV vector genome for use as described in any one of items 34-41. (Item 44) The aforementioned rAAV virion or rAAV vector genome is approximately 10 11 vg / kg~about 10 14 rAAV virions or rAAV vector genomes for use as described in any one of items 33-43, administered at a dose of vg / kg.

Claims

1. A modified cardiac troponin T promoter comprising a polynucleotide having 375 bp to 425 bp of the nucleic acid sequence of Sequence ID No. 1, wherein the polynucleotide has a sequence that shares at least 90% or 100% identity with the full-length nucleic acid sequence of Sequence ID No.

3.

2. The promoter according to claim 1, wherein the polynucleotide has the sequence described in Sequence ID No.

3.

3. The promoter according to claim 1 or claim 2, wherein the polynucleotide has 400 bp.

4. The promoter according to any one of claims 1 to 3, wherein the promoter expresses the gene product operably linked to it at least 10%, at least 20%, and at least 30% more than the natural troponin T promoter.

5. The promoter according to claim 4, wherein the natural troponin T promoter is a reference promoter containing the nucleic acid sequence of SEQ ID NO:

1.

6. A vector comprising a promoter according to any one of claims 1 to 5, operably bound to a polynucleotide encoding a gene product.

7. The vector according to claim 6, wherein the vector is a viral vector.

8. The vector according to claim 7, wherein the viral vector is an adeno-associated virus (AAV) vector and / or the viral vector has a packaging limit of up to 5.5 kB.

9. The vector according to claim 8, wherein the AAV vector is AAV9.

10. The aforementioned gene product (i) MYBPC3, KCNH2, TRPM4, DSG2 and ATP2A2 proteins, or (ii) A vector according to any one of claims 6 to 9, selected from the proteins CACNA1C, DMD, DMPK, EPG5, EVC, EVC2, FBN1, NF1, SCN5A, SOS1, NPR1, ERBB4, VIP, and MYH7.

11. The vector according to any one of claims 6 to 10, wherein the gene product is Cas9.

12. The vector according to claim 11, wherein Cas9 is selected from SpCas9, St1Cas9, and SaCas9.

13. The vector according to any one of claims 6 to 12, wherein the vector comprises a polynucleotide encoding a second gene product.

14. The vector according to claim 13, wherein the second gene product is a functional RNA selected from microRNA or guide RNA.

15. Isolated cells comprising the promoter according to any one of claims 1 to 5.

16. The isolated cell according to claim 15, wherein the isolated cell is an induced pluripotent stem cell or an isolated cardiomyocyte.

17. A pharmaceutical composition comprising the vector described in any one of claims 6 to 14.

18. A cell therapy composition comprising the cells described in claim 15 or claim 16.

19. A recombinant adeno-associated virus (AAV) vector genome comprising a MYBPC3 polynucleotide encoding the MYBPC3 protein, and a promoter, wherein the promoter is the modified cardiac troponin T promoter described in any one of claims 1 to 5.

20. The rAAV vector genome according to claim 19, wherein the MYBPC3 polynucleotide is full-length MYBPC3 containing at least 3.5 kB, containing 3.8 kB, or is a truncated MYBPC3.

21. The rAAV vector genome according to claim 19 or claim 20, wherein the MYBPC3 is human MYBPC3.

22. The rAAV vector genome according to any one of claims 19 to 21, wherein the vector expresses MYBPC3 at the same level as a reference AAV vector containing a 600 bp native troponin T promoter, at least 10% higher or at least 20% higher.

23. A recombinant adeno-associated virus (AAV) vector genome comprising an expression cassette comprising, in the order of 5' to 3', a 5' segment containing a promoter according to any one of claims 1 to 3, a polynucleotide encoding a gene product, and a 3' segment containing a polyA signal, The polynucleotide encoding the gene product includes 3kb to 11kb, 3kb to 5kb, 3.5kb to 4.5kb, or 3.7kb to 4kb. a) Both the 5' segment and the 3' segment have a maximum of 0.8 kbp or Includes up to 0.9 kbp, b) The 5' ITR, the 5' segment, the 3' segment, and the 3' ITR all contain a maximum of 1.2 kbp or a maximum of 1.3 kbp, and / or c) The vector genome is a recombinant adeno-associated virus (AAV) vector genome containing up to 4.7 kbp, up to 4.8 kbp, up to 4.9 kbp, up to 5.0 kbp, or up to 5.2 kbp.

24. The rAAV vector genome according to claim 23, wherein the expression cassette is adjacent to one or both of the 5' inverted terminal repeat (ITR) and the 3' ITR.

25. The rAAV vector genome according to claim 23 or 24, wherein the 5' segment comprises up to 500 bp or up to 480 bp, and / or the 3' segment comprises up to 200 bp or up to 150 bp.

26. The rAAV vector genome according to claim 23, claim 24, or claim 25, wherein the polynucleotide encoding the gene product comprises 3.7 kbp to 3.9 kbp.

27. The rAAV vector genome according to claim 26, wherein the polynucleotide encoding the gene product comprises 3.8 kbp.

28. The rAAV vector genome according to any one of claims 23 to 27, wherein the gene product is MYBPC3.

29. The polynucleotide encoding MYBPC3 shares at least 90% identity with the nucleic acid sequence of SEQ ID NO: 86, at least 95% identity with the nucleic acid sequence of SEQ ID NO: 86, or the nucleic acid sequence of the polynucleotide encoding MYBPC3 is the nucleic acid sequence of SEQ ID NO: 86, and / or The rAAV vector genome according to claim 28, wherein MYBPC3 shares at least 90% identity with the polypeptide sequence of SEQ ID NO: 103, at least 95% identity with the polypeptide sequence of SEQ ID NO: 103, or the polypeptide sequence of MYBPC3 is the polypeptide sequence of SEQ ID NO:

103.

30. The rAAV vector genome according to any one of claims 23 to 28, wherein the promoter is the modified cardiac troponin T promoter according to any one of claims 1 to 5.

31. (i) The polyA signal includes or consists of a sequence that shares at least 90% identity or at least 95% identity with the nucleic acid sequence of SEQ ID NO: 92, or the nucleic acid sequence of the polyA signal is the nucleic acid sequence of SEQ ID NO:

92. (ii) The 5' segment shares at least 90% identity, at least 95% identity, with the nucleic acid sequence of SEQ ID NO: 93, or the nucleic acid sequence of the 5' segment is the nucleic acid sequence of SEQ ID NO: 93, and / or (iii) The rAAV vector genome according to claim 30, wherein the 3' segment shares at least 90% identity with the nucleic acid sequence of SEQ ID NO: 94, shares at least 95% identity with SEQ ID NO: 94, or the nucleic acid sequence of the 3' segment is the nucleic acid sequence of SEQ ID NO:

94.

32. The rAAV vector genome according to any one of claims 23 to 31, wherein the expression cassette shares at least 90% identity and at least 95% identity with the nucleic acid sequence of SEQ ID NO: 95, or the nucleic acid sequence of the expression cassette is the nucleic acid sequence of SEQ ID NO: 95, and / or the rAAV vector genome shares at least 90% identity and at least 95% identity with the nucleic acid sequence of SEQ ID NO: 102, or the nucleic acid sequence of the rAAV vector genome is the nucleic acid sequence of SEQ ID NO:

102.

33. The rAAV vector genome according to any one of claims 23 to 32, wherein the 5'ITR includes a sequence that shares 95% identity with the nucleic acid sequence of SEQ ID NO: 96, and / or the 3'ITR includes a sequence that shares at least 95% identity with the nucleic acid sequence of SEQ ID NO:

97.

34. A recombinant AAV (rAAV) virion comprising the rAAV vector genome and AAV capsid protein according to any one of claims 19 to 33.

35. The rAAV virion according to claim 34, wherein the rAAV virion is a serotype AAV9 virion and / or the AAV capsid protein is an AAV9 capsid protein.

36. An in vitro or ex vivo method for expressing the MYBPC3 protein in cells, comprising transducing the cells with the rAAV virion described in claim 34 or the rAAV vector genome described in any one of claims 19 to 33.

37. The aforementioned cells are MYBPC3 －／－ The method according to claim 36, wherein the cell is a cell.

38. The method according to claim 36 or claim 37, wherein the cells contain an inactivating mutation in one or both copies of the endogenous MYBPC3 gene.

39. A composition for use as a pharmaceutical agent, comprising the rAAV virion described in claim 34 or the rAAV vector genome described in any one of claims 19 to 33.

40. A composition for use in a method for treating and / or preventing cardiomyopathy in a subject, wherein the composition comprises an rAAV virion according to claim 34, or an rAAV vector genome according to any one of claims 19 to 33.

41. The composition according to claim 40, wherein the cardiomyopathy is hypertrophic cardiomyopathy.

42. A composition for use in a method of treating and / or preventing a disease or disorder caused by a target MYBPC3 mutation, comprising the rAAV virion described in claim 34 or the rAAV vector genome described in any one of claims 19 to 33.

43. The composition according to claim 40, claim 41, or claim 42, wherein the method comprises expressing the MYBPC3 protein and / or increasing MYBPC3 activity and / or increasing the cardiac function of the target heart.

44. The composition according to any one of claims 40 to 43, characterized in that administration of the composition causes specific expression of MYBPC3 in the heart of the subject.

45. The composition according to any one of claims 40 to 44, characterized in that administration of the composition causes low or undetectable expression of MYBPC3 from the composition in the skeletal tissue, brain, and / or liver of the subject.

46. The composition according to any one of claims 40 to 45, wherein the subject includes the MYBPC3 mutation.

47. The method described above is (i) administering the composition intravenously to the subject, (ii) administering the composition intracardiac to the subject, or (iii) The composition according to any one of claims 40 to 46, comprising directly injecting the composition into the subject.

48. The composition according to any one of claims 40 to 47, wherein the subject is a mammal.

49. The composition according to claim 48, wherein the subject is a human.

50. The composition according to any one of claims 40 to 49, wherein the subject is young.

51. The composition according to any one of claims 40 to 49, wherein the subject is an adult.

52. The rAAV virion or rAAV vector genome is 10 １１ vg / kg ~ 10 １４ The composition according to any one of claims 39 to 51, characterized in that it is administered at a dose of vg / kg.

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