Methods of treatment of hypertrophic cardiomyopathy with AAV gene therapy vectors and therapeutic formulations

Abstract

Described herein are pharmaceutical compositions and methods for treating hypertrophic cardiomyopathy in a human subject using the same.

Description

METHODS OF TREATMENT OF HYPERTROPHIC CARDIOMYOPATHY WITHAAV GENE THERAPY VECTORS AND THERAPEUTIC FORMULATIONSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 580,741, filed September 6, 2023, and U.S. Provisional Patent Application No.63 / 591,172, filed October 18, 2023, the disclosures of which are hereby incorporated by reference herein in their entireties.FIELD OF THE INVENTION

[0002] Provided herein are recombinant adeno-associated virus (rAAV) gene therapy vectors and virus particles useful in the treatment and prevention of hypertrophic cardiomyopathy by increasing expression of cardiac myosin binding protein C (cMyBP-C).INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0003] This application includes, as a separate part of disclosure, a Sequence Listing in computer-readable form (Filename: SequenceListing.xml, Size: 489,630 bytes; Created: October 17, 2023). The contents of the Sequence Listing xlm file are hereby incorporated by reference in its entirety.BACKGROUND

[0004] While considerable progress has been made in the prevention of heart diseases that are caused by environmental factors, such as nicotine, hypercholesterolemia or diabetes, and in the symptomatic treatment of heart conditions, there is still a need for methods that improve the treatment of inherited cardiomyopathies. Among the cardiomyopathies that are caused by genetic factors are hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and arrhythmogenic right ventricular cardiomyopathy (ARVC).

[0005] Hypertrophic cardiomyopathy is the most prevalent genetic heart disease and is characterized by unexplained left ventricular hypertrophy. Hypertrophic cardiomyopathy is associated with initially normal systolic, but impaired diastolic function (Elliott et al., Eur. Heart J. 29: 270-6 (2008); Gersch et al., J. Thorac. Cardiovasc. Surg. 142: el53-203 (2011).Hypertrophic cardiomyopathy has a particularly high prevalence of about 1:500 in the general population (Maron et al., Circulation, 92: 785-9 (1995), and is the leading cause of sudden cardiac death in younger people, particularly in athletes. Although HCM is a life-threateningdisease, no curative treatment exists to date (Carrier et al., Cardiovasc. Res. 85:330-338 (2010); Schlossarek et al., J. Mol. Cell. Cardiol. 50: 613-20 (2011).

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

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

[0008] Current drug-based treatments of HCM alleviate the symptoms but do not treat the genetic cause underlying the disease. A gene-based or RNA-based therapy would be the only curative treatment for HCM. Gene therapeutic approaches have successfully been tested in connection with non-genetic cardiac diseases (Jessup et al., Circulation, 124: 304-13 (2011).SUMMARY

[0009] The embodiments described herein relate to pharmaceutical compositions comprising recombinant AAV particles and methods of using recombinant AAV particles to treat heart disease.

[0010] The pharmaceutical compositions are suitable for use in treating or preventing HCM in a mammalian subject in need of treatment, preferably a human subject. In some embodiments, the subject in need of treatment is one that carries a mutation in at least one or both genes encoding cMyBP-C. After administration into the subject to be treated, the pharmaceutical composition provides for the expression of the encoded cardiac myosin binding protein in the subject, preferably in the myocardium of said subject.

[0011] In one aspect, the disclosure provides a pharmaceutical composition of recombinant adeno-associated virus (rAAV) particles for use in any of the methods disclosed herein. The disclosure also provides uses of a recombinant AAV particle as described herein for the preparation of a medicament for treatment according to any of the methods described herein.

[0012] In a related aspect, the disclosure provides a pharmaceutical composition comprising rAAV particles at a concentration of at least about 6E13 vg (vector genomes) / ml (milliliters) to about 4E14 vg / ml, or any concentration within this range as disclosed herein, a buffering agent, an isotonicity agent, an additional magnesium chloride (MgCh) stabilizer, a cryopreservative agent and a surfactant which is stable during storage at about < -60°C (minus sixty degrees centigrade) or less for at least about 1 year, 1.5 years, or 2 years. In some embodiments, the pharmaceutical composition has a formulation pH ranging from about 6 to about 9, from about 7 to about 7.8. In some embodiments, the pharmaceutical composition has a formulation pH of 7.4.

[0013] In another aspect, the disclosure provides a pharmaceutical composition as disclosed herein that is stable during storage at about < -60°C (minus sixty degrees centigrade) or less for at least about 1 year, at least about 1.5 years, or at least about 2 years or from about 1 year to about 2 years. In further embodiments, the pharmaceutical formulation comprises one or more of a buffering agent, an isotonicity agent, a stabilizer (e.g., a magnesium chloride (MgCh) stabilizer), a cryopreservative agent and a surfactant.

[0014] In some embodiments, the pharmaceutical composition comprises rAAV particles at a concentration of at least about 6E13 vg / ml to about 4E14 vg / ml, or any concentration within this range as disclosed herein, Tris hydrochloride buffer at a concentration of about 10 to about 30 mM, Tris base at a concentration of about 1 to about 4 mM (millimolar), sodium chloride at a concentration of about 100 mM to about 150 mM, MgCh at a concentration of about 0.5 mM to about 3.0 mM, trehalose at a concentration of about 50 mM to about 90 mM, and a poloxamer or polysorbate at a concentration of about 0.05% to about 0.15% w (weight) / v (volume). In someembodiments, the pharmaceutical composition comprises rAAV particles at a concentration of about 1E14 vg / ml or less, Tris hydrochloride buffer at a concentration of about 10 to about 30 mM, Tris base at a concentration of about 1 to about 4 mM, sodium chloride at a concentration of about 100 mM to about 150 mM, MgCh at a concentration of about 0.5 mM to about 3.0 mM, trehalose at a concentration of about 50 mM to about 90 mM, and a poloxamer or polysorbate at a concentration of about 0.05% to about 0.15% w / v. In some embodiments, the concentration of rAAV particles ranges from about 1E13 vg / ml to about 1E14 vg / ml, from about 1E13 vg / ml to about 5E13 vg / ml, or from about 5E13 vg / ml to about 1E14 vg / ml, or any concentration within these ranges as disclosed herein.

[0015] In some embodiments, the buffering agent is optionally Tris hydrochloride and / or Tris base. In some embodiments, the Tris hydrochloride is at a concentration of 17.6 mM and the Tris base is at a concentration of 2.4 mM. In some embodiments, the isotonicity agent is NaCl at a concentration of 120 mM. In some embodiments, the stabilizer is MgCh at a concentration of about 1 mM to about 3.0mM. In some embodiments, the MgCh stabilizer is at a concentration of l.OmM or greater, greater than 1.0 mM, 1.5 mM or greater, or up to 3.0 mM. In some embodiments, the cryopreservative agent is a sugar. In some embodiments, the cryopreservative agent is trehalose at a concentration of 74 mM. In some embodiments, the surfactant is Poloxamer 188 at a concentration of about 0.1% w / v.

[0016] In one aspect, the disclosure provides a pharmaceutical composition comprising rAAV particles at a concentration of about 6E13 vg / ml, 17.6 mM Tris hydrochloride, 2.4 mM Tris base, 120 mM sodium chloride, 1 mM MgCh hexahydrate, 74 mM trehalose dihydrate, and 0.1% w / v poloxamer 188.

[0017] Preferably, in any of the foregoing pharmaceutical compositions, the rAAV particles comprise an AAV9 type capsid, and a recombinant AAV vector construct as described herein. Preferably, the pharmaceutical compositions described herein are a liquid aqueous solution. Preferably, such solutions are sterile.

[0018] The disclosure provides a method of treating a subject with heart disease, comprising administering by intravenous (IV) administration to the subject the pharmaceutical composition described herein. For example, the heart disease may be hypertrophic cardiomyopathy or a heart disease associated with a genetic mutation of the MYBPC3 gene.

[0019] In a second aspect, the disclosure provides a method of treating a human subject with a heart disease, comprising administering to the subject a single dose ranging from about 1E13 vector genomes / kilogram body weight of the subject to about 4E14 vector genomes / kilogram body weight of the subject (vg / kg) of recombinant AAV particles comprising (a) an AAV capsid with heart tropism, (b) a cardiac-specific transcription regulatory region, and (c) a recombinantvector construct comprising a nucleic acid encoding a functional cMyBP-C protein or codon optimized version thereof.

[0020] In any of the methods disclosed herein, the dose of rAAV particle may be about IE 13 vg / kg to about 6E13 vg / kg, or about 5E13 vg / kg to about 1E14 vg / kg, or about 8E13 vg / kg to about 4E14 vg / kg, or any doses within these ranges as disclosed herein. In some embodiments, the dose of rAAV particle is about 6E13, 7E13, 8E13, 9E13, 1E14, or 1.2E14 vg / kg. In some embodiments, the dose of rAAV particles is about 2E14 vg / kg. In some embodiments, the dose of rAAV particles is about 9E13 vg / kg. In some embodiments, the dose of rAAV particles is about 1.2E14 vg / kg. In some embodiments, the dose of rAAV particles is about 2.4E14 vg / kg.

[0021] In the recombinant vector construct comprising a nucleic acid encoding a functional cMyBP-C protein, the functional cMyBP-C protein may comprise an amino acid sequence at least 95%, 98% or 99% identical to amino acids 1 through 1274 of SEQ ID NO: 2. In some embodiments, the nucleic acid sequence encoding the functional cMyBP-C comprises a nucleotide sequence at least 90%, 95%, 98% or 99% identical to SEQ ID NO: 1.

[0022] In the methods disclosed herein, the cardiac-specific transcription regulatory region may comprise a fragment of a human TNNT2 promoter. The cardiac-specific transcription regulatory region may comprise a nucleotide sequence at least 90%, 95%, 98% or 99% identical to SEQ ID NO: 51. The recombinant vector construct may comprise an intron or fragment thereof. The recombinant vector construct may further comprise a polyadenylation signal, for example, a bovine growth hormone (bG) (SEQ ID NOs: 59-61) or human growth hormone (hGH) (SEQ ID NO: 62) polyadenylation signal.

[0023] In the methods disclosed herein, the subject may be administered a population of rAAV particles produced by a method comprising (a) providing a suitable host cell, preferably a mammalian cell, with one or more nucleic acid constructs comprising: (i) a recombinant vector construct comprising (1) a 5’ AAV ITR and a 3’ AAV ITR, (2) a cardiac-specific transcription regulatory region, and (3) a nucleic acid encoding a functional cMyBP-C comprising an amino acid sequence at least 95% identical to amino acids 1 through 1274 of SEQ ID NO: 2, (ii) a nucleotide sequence encoding one or more AAV Rep proteins which is operably linked to a promoter that is capable of driving expression of the Rep protein(s) in the cell; and (iii) a nucleotide sequence encoding one or more AAV9 type capsid protein which is operably linked to a promoter that is capable of driving expression of the capsid protein(s) in the cell; (b) culturing the cell under conditions permitting expression of the Rep and the capsid proteins and production of an AAV particle; and (c) recovering the AAV particle. Optionally, the population is enriched for AAV particles comprising full length or nearly full-length vector genomes bysteps that reduce the number of empty capsids, such as zonal ultracentrifugation or densitygradient ultracentrifugation.

[0024] In some embodiments, the AAV capsid comprises an amino acid sequence at least 85%, 90% or 95% identical to any one of SEQ ID NOs: 207-223. Preferably, the AAV capsid with cardiac tropism is an AAV9 type capsid, optionally at least 85%, 90% or 95% identical to SEQ ID NO: 207. Preferably, the AAV capsid comprises an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 207.

[0025] In any of the methods described herein the rAAV particles are administered by intravenous infusion.

[0026] In any of the methods herein, the subject has a heart disease associated with a genetic mutation of the MYBPC3 gene. In some embodiments, the subject has hypertrophic cardiomyopathy.

[0027] In any of the methods described herein, preferably the dose is effective to improve cardiac function or structure, optionally as measured by reduced E / e’ and / or reduced left atrial volume index and / or improving or normalizing ejection fraction and / or reduced impairment of longitudinal strain, and / or reduced left ventricular (LV) wall thickness and / or reduced LV mass. For example, the improving or normalizing of the ejection fraction may include increasing or decreasing the ejection fraction relative to a baseline identified prior to the administration of the effective dose. In some embodiments, the improvement may be an improvement of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% (2-fold) relative to baseline. For example, for a patient with an ejection fraction of 50%, the ejection fraction may improve by about 20% or more to an ejection fraction of about 60% or more. In some embodiments, the dose is effective to improve cardiac function and / or health-related quality of life, optionally as measured by any one or more of New York Heart Association (NYHA) functional classification, Kansas City Cardiomyopathy Questionnaire-23 (KCCQ-23) Total Symptom Score (TSS), physical limitations score and clinical summary score (CSS), EuroQol-5 dimension 5-level version (EQ-5D-5L), EuroQol-visual analog scale (EQ-VAS), Clinical Global Impression of Severity and Change scales (CGI / C), and Patient Global Impression of Severity and Change scales (PGIS / C), over a time period of at least about six months. In some embodiments, the improved cardiac function and / or health-related quality of life is maintained for at least about one year. In some embodiments, the level of N-terminal pro B-type natriuretic peptide (NT- proBNP), an indicator of cardiac stress, is reduced, e.g. by at least about 10%, 20%, 30%, 40% or 50%.

[0028] The methods of the disclosure may further comprise administering to the subject a prophylactically effective amount of a corticosteroid or other systemic immunosuppressant. Insome embodiments, the amount administered is effective to prevent or reduce biochemical or clinical signs of hepatotoxicity, hepatic inflammation, and / or cardiac inflammation.

[0029] In some embodiments, the prophylactically effective amount of corticosteroid or immunosuppressant is administered at a prednisone-equivalent dose of about 0.5 mg (milligram) / kg / day, optionally for a time period of at least about 4 weeks, followed by tapering amounts of the corticosteroid for a time period of about 4 weeks. In other embodiments, the prophylactically effective amount of corticosteroid or immunosuppressant is administered at a prednisone-equivalent dose of at least about 40 mg / day.

[0030] In some embodiments, the prophylactically effective amount of corticosteroid or immunosuppressant is administered concurrent with administration of the rAAV particles of the invention. In other embodiments, the prophylactically effective amount of corticosteroid or immunosuppressant is administered concurrently with a prophylactically effective amount of sirolimus. In some embodiments, the prophylactically effective amount of sirolimus is administered at a dose of about 2 mg / day for a time period of at least 8 weeks. In some embodiments, the amount of sirolimus is effective to maintain a blood level of sirolimus between about 5 to about 15 ng / mL. In some embodiments, the prophylactic amount of TMP-SMX is administered orally at a dose of 160+800 mg three-times per week.

[0031] The methods may further comprise the steps of (a) determining a baseline level of a marker of hepatotoxicity in the blood of the subject prior to rAAV particle administration, and (b) subsequently determining a post-administration level of said marker for hepatotoxicity in the blood of the subject, optionally every week for at least 8 weeks. Alternatively, the methods may further comprise the steps of (a) determining a baseline level of a marker of hepatotoxicity, hepatic inflammation and / or cardiac inflammation in the blood of the subject prior to rAAV particle administration, and (b) subsequently determining a post-administration level of said marker for hepatotoxicity, hepatic inflammation and / or cardiac inflammation in the blood of the subject, optionally every week for at least 8 weeks.

[0032] The methods of the disclosure may also comprise administering to the subject a prophylactically effective amount of a complement pathway inhibitor or antagonist. In some embodiments, the amount administered is effective to prevent or reduce biochemical or clinical signs of complement activation and / or thrombotic microangiopathy.

[0033] In some embodiments, the prophylactically effective amount of a complement pathway inhibitor or antagonist is administered concurrent with administration of the rAAV particles of the invention. The methods may further comprise the steps of (a) determining a baseline level of a marker of complement activation and / or thrombotic microangiopathy in the blood of the subject prior to rAAV particle administration, and (b) subsequently determining apost-administration level of said marker for complement activation and / or thrombotic microangiopathy in the blood of the subject, optionally every week for at least 8 weeks. For example, the marker of TMA can be D-dimer or observation of red blood cell fragmentation and thrombocytopenia in a peripheral blood smear.

[0034] The methods of the disclosure may comprise administering to the subject a therapeutically effective amount of glucocorticosteroid or other systemic immunosuppressant in response to hepatotoxicity, hepatic inflammation and / or cardiac inflammation. The biochemical marker for hepatotoxicity may comprise elevation of ALT and / or AST greater than 2x the upper limit of normal (ULN) or greater than or equal to twice the baseline level. The marker(s) for myocarditis or cardiac inflammation may include abnormally increased levels of Troponin I. The methods may comprise: (c) upon detection of hepatotoxicity, hepatic inflammation, and / or cardiac inflammation by biochemical or clinical signs, administering to the subject a therapeutically effective amount of a systemic immunosuppressant to reduce hepatotoxicity, hepatic inflammation, and / or cardiac inflammation.

[0035] The methods of the disclosure may comprise administering to the subject a therapeutically effective amount of complement pathway inhibitor or antagonist in response to complement activation, e.g. refractory complement activation, and / or thrombotic microangiopathy. The marker(s) for complement activation may include abnormally decreased levels of C3 or C4 in plasma (indicating its consumption by complement activation), or abnormally increased levels of one or more complement split products, e.g., C3a, C4a, Bb and / or sC5b-9. The marker(s) for thrombotic microangiopathy may comprise decreased platelet counts, decreased platelet volume, decreased red blood cell (RBC) counts, abnormal RBC morphology, hemolysis, and / or proteinuria. Clinical signs of thrombotic microangiopathy include complement activation, severe thrombocytopenia, microangiopathic hemolysis with schistocytes, transaminase elevations, and / or acute kidney injury (AKI) progressing to kidney failure. The methods may comprise: (c) upon detection of complement activation, e.g. refractory complement activation, and / or thrombotic microangiopathy by biochemical or clinical signs, administering to the subject a therapeutically effective amount of a complement pathway inhibitor or antagonist to reduce complement activation and / or thrombotic microangiopathy.

[0036] In some embodiments, the prophylactic or therapeutic immunosuppressant is a glucocorticosteroid, optionally, tacrolimus, Mycophenolate mofetil, or eculizumab.

[0037] In some embodiments, the prophylactic or therapeutic immunosuppressant is a complement pathway inhibitor or antagonist, including any complement protein inhibitor or antagonist (e g., inhibitor or antagonist of C3, C4, C5, etc.), or MASP2 inhibitor (e g., OMS721 antibody) and including any of pegcetacoplan (APL-2, EMPAVELI), eculizumab (SOLIRIS),avacopan (CCX168), vilobelimab (IFX-1), AMY-101, CINRYZE / BERINERT / HAEGARDA (human Cl esterase inhibitor, C1INH), ravulizumab (ALXN1210), coversin, cemdisiran (ALN- CC5), tesidolumab (LFG-316), iptacopan (LNP023), danicopan (ACH-4471), RUCONEST (recombinant Cl esterase inhibitor), or ENJAYMO (sutimlimab-jome, a Cis inhibitor).

[0038] The methods of the disclosure may further comprise administering to the subject a prophylactic antimicrobial agent. In some embodiments, the prophylactic antimicrobial agent is a combination of sulfamethoxazole and trimethoprim.BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1A shows thermal-based capsid integrity (TBCI) analysis of various formulations comprising AAV9-MYBPC3 heavy capsids and ultra-heavy capsids.

[0040] Figure IB shows TBCI analysis of the various formulations in terms of percent increase from initial amount.

[0041] Figure 2A shows vector copy per diploid genome of AAV9-MYBPC3 in Rag2- / - mice at 8-weeks post 2E14 vector genomes per kg body weight (vg / kg) AAV9-hMYBPC3 administration in 8-week-old Rag2~ / ~ mice.

[0042] Figure 2B shows mRNA / RPLPO of AAV9-MYBPC3 in Rag2- / - mice at 8-weeks post 2E14 vector genomes per kg body weight (vg / kg) AAV9-11MYBPC3 administration in 8-week- old Rag2 / ' mice.

[0043] Figure 2C shows percent of human cMyBP-C protein of total cMyBP-C protein of AAV9-MYBPC3 in Rag2- / - mice at 8-weeks post 2E14 vector genomes per kg body weight (vg / kg) AAV9-hMYBPC3 administration in 8-week-old Rag2~ / ~ mice.

[0044] Figure 2D shows total cMyBP-C protein of AAV9-MYBPC3 in Rag2- / - mice at 8- weeks post 2E14 vector genomes per kg body weight (vg / kg) AAV9-hMYBPC3 administration in 8-week-old Rag2 / _mice.

[0045] Figure 3A shows left ventricular (LV) mass to body weight ratios of the mice based on data from a natural history study conducted to evaluate phenotype development of wildtype (WT) mice, heterozygous MYBPC+ / ~ mice, and homozygous MYBPCB^' mice.

[0046] Figure 3B shows LV posterior wall dimensions of the hearts of the mice.

[0047] Figure 3C shows diastolic functions, mitral valve E wave (MV E) of the hearts of the mice.

[0048] Figure 4A depicts vector genome levels in the hearts of MYBPC3-I- mice and WT mice 8-weeks post 2E14 vg / kg AAV9-MYBPC3 or vehicle administration in 2-week-old MYBPC3-I- mice and WT mice.

[0049] Figure 4B depicts transcript levels in the hearts of the MYBPC3-I- mice and WT mice 8-weeks post 2E14 vg / kg AAV9-MYBPC3 or vehicle administration in 2 -week-old MYBPC3-I- mice and WT mice.

[0050] Figure 4C depicts protein levels in the hearts of the MYBPC3-I- mice and WT mice 8- weeks post 2E14 vg / kg AAV9-MYBPC3 or vehicle administration in 2 -week-old MYBPC3-I- mice and WT mice.

[0051] Figure 4D depicts total protein in the hearts of the MYBPC3- / - mice and WT mice 8- weeks post 2E14 vg / kg AAV9-MYBPC3 or vehicle administration in 2 -week-old MYBPC3-I- mice and WT mice.

[0052] Figure 4E depicts the heart weight to body weight ratios of the MYBPC3-I- mice and WT mice 8-weeks post administration.

[0053] Figure 5A shows MYBPC3 vector genome copies per diploid genome in the hearts of the MYBPC3-!- mice 8-weeks post administration.

[0054] Figure 5B shows MYBPC3 transcript levels in the hearts of the MYBPC3-I- mice 8- weeks post administration.

[0055] Figure 5C shows cMyBP-C protein levels in the hearts of the MYBPC3- / - mice 8- weeks post administration of AAV9-MYBPC3.

[0056] Figure 5D shows percentage of cardiomyocytes expressing cMyBP-C protein in the hearts of the MYBPC3- / - mice 8-weeks post administration of AAV9-MYBPC3.

[0057] Figure 5E depicts the heart weight to body weight ratios of the MYBPC3- / - mice 8- weeks post AAV9-MYBPC3 or vehicle administration.

[0058] Figure 6A shows LV mass to body weight ratios of MYBPC3- / - mice and WT mice at 26-weeks post 6E13 or more AAV9-mMYBPC3, AAV9-hMYBPC3, or vehicle administration in 2 -week-old MYBPC3-I- mice and WT mice.

[0059] Figure 6B shows LV posterior wall dimensions of the hearts o MYBPC3- / - mice and WT mice at 26-weeks post administration.

[0060] Figure 6C shows stroke volumes of the hearts o£MYBPC3-l- mice and WT mice post administration.

[0061] Figure 6D shows cardiac outputs of the hearts o£MYBPC3-l- mice and WT mice post administration.

[0062] Figure 6E shows diastolic functions, MV E of the hearts of MYBPC3-I- mice and WT mice post administration.

[0063] Figure 7A depicts data assessing the in vivo transduction efficiency of cardiomyocytes from the hearts o MYBPC3-l- mice and WT mice 0-, 4-, 8-, 12-, 20-, 28-, and 36- weeks post 6E13 vg / kg or 2E14 vg / kg AAV9-MYBPC3 or vehicle administration in 2-week-old MYBPC3-I- mice and wild type mice. Figure 7A shows LV mass to body weight ratios of MYBPC3-I- mice and WT mice 0-, 4-, 8-, 12-, 20-, 28-, and 36- weeks post 6E13 vg / kg or 2E14 vg / kg AAV9-MYBPC3 or vehicle administration in 2 -week-old MYBPC3-I - mice and wild type mice.

[0064] Figure 7B shows LV posterior wall dimensions of the hearts of the MYBPC3-I- mice and WT mice post administration.

[0065] Figure 7C shows diastolic functions, MV E of the hearts of the MYBPC3-I- mice and WT mice post administration.

[0066] Figure 8A shows reduction of LV mass to body weight ratios of MYBPC3-I- mice at 36-weeks post 6E13 vg / kg or 2E14 vg / kg AAV9-MYBPC3 administration in 2-week-old MYBPC3-I- mice as compared .0MYBPC3-I- mice administered a vehicle at 2-week-old.

[0067] Figure 8B shows reduction in LV anterior wall thickness in the hearts of the MYBPC3- / - mice at 36-weeks post AAV9-MYBPC3 administration as compared to MYBPC3- / - mice administered a vehicle at 2-week-old.

[0068] Figure 8C shows reduction in LV posterior wall thickness in the hearts of the MYBPC3- / - mice at 36-weeks post AAV9-MYBPC3 administration as compared to MYBPC3- / - mice administered a vehicle at 2-week-old.

[0069] Figure 8D shows improvement in MV E (passive filling) of the hearts of the MYBPC3-I- mice 36-weeks post AAV9-MYBPC3 administration as compared to MYBPC3-I- mice administered a vehicle at 2-week-old.

[0070] Figure 9 shows cMyBP-C protein levels in the hearts of wildtype mice 4-, 8-, 12-, and 16- weeks post 2E14 vg / kg AAV9-MYBPC3 administration to 8-week-old male WT mice.

[0071] Figure 10A shows the MYBPC3 copies / nanograms (ng) DNA levels in the hearts of MYBPC3-I- mice and WT mice 13-weeks post administration.

[0072] Figure 10B shows the MYBPC3 transcript / ng RNA levels in the hearts of the MYBPC3-I- mice and WT mice 13-weeks post administration.

[0073] Figure 10C shows ng cMyBP-C / ng tissue of total cMyBP-C protein levels in the hearts of the MYBPC3- / - mice and WT mice 13-weeks post administration.

[0074] Figure 10D shows ng cMyBP-C / ng tissue of human cMyBP-C protein levels in the hearts of the MYBPC3-I- mice and WT mice 13-weeks post administration.

[0075] Figure 10E shows the percentage of cardiomyocytes from the hearts of MYBPC3-I- mice and WT mice expressing human cMyBP-C protein 13-weeks post administration.

[0076] Figure 11 A shows reductions in heart weight to tibia length ratio of MYBPC3-I- mice 13-weeks post AAV9-MYBPC3 administration as compared to MYBPC3- / - mice administered a vehicle at 2-week-old.

[0077] Figure 1 IB shows reductions in the LV mass to body weight ratio of the MYBPC3- / - mice 12-weeks post AAV9-MYBPC3administration as compared .0MYBPC3-I- mice administered a vehicle at 2-week-old.

[0078] Figure 11C shows improvement in diastolic functions, MV E in the hearts of the MYBPC3-I- mice 12-weeks post AAV9-MYBPC3administration as compared to MYBPC3-I- mice administered a vehicle at 2-week-old.

[0079] Figure 12 shows vector genome distributions in cardiac tissues from the hearts of cynomolgus monkeys 31 and 62 days post 1.2E14 vg / kg AAV9-MYBPC3 administration.

[0080] Figure 13 shows vector-derived hMYBPC3 transcript levels in cardiac tissues from the hearts of cynomolgus monkeys 31 and 62 days post 1.2E14 vg / kg AAV9-MYBPC3 administration.

[0081] Figure 14 depicts cMyBP-C protein expression in patient-derived induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) transduced with AAV9-MYBPC3 produced in either the Spodoptera frugiperda (Sf) insect cell system or the mammalian (i.e., human) embryonic kidney 293 (HEK293) cell system.DETAILED DESCRIPTION

[0082] Provided herein are formulations and methods for treating a human subject with a heart disease by administration of rAAV particles comprising a gene encoding a therapeutic protein for treating the heart disease. Preferably, the heart disease results from a deficiency in functional wild-type cMyBP-C, e.g. HCM, and the gene encoding a functional cMyBP-C. The human subject may be an adult human, e g. human of ages 18 to 65, or age at least 18, infant, child, or juvenile human, e.g. human of age up to 2, 2-4, 2-6 or 2-12, or at least age 12.

[0083] In some embodiments, the rAAV particles comprise (a) an AAV capsid with cardiac tropism, and (b) a recombinant vector construct (an AAV vector genome) comprising a gene encoding a functional cMyBP-C. In certain embodiments, this vector construct comprises (a) a nucleic acid comprising an AAV2 5’ inverted terminal repeat (ITR) (which may or may not be modified as known in the art), (b) a cardiomyocyte-specific transcription regulatory region, (c) a functional cMyBP-C protein coding region, (d) optionally, one or more introns, (e) a polyadenylation sequence, and (f) an AAV23’ ITR (which may or may not be modified as known in the art).

[0084] The functional cMyBP-C encoding sequence may be wild-type, codon optimized, or a variant. As used herein, wild-type cardiac myosin binding protein C (MYBPC3) gene has the nucleic acid sequence of SEQ ID NO: 1 (GenBank Accession No. NM_000256.2), and wild-typecardiac myosin binding protein C has the amino acid sequence of SEQ ID NO: 2 (GenBank Accession No. NP_000247.1) .Definitions

[0085] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. See, e.g. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nded., J. Wiley & Sons (New York, N.Y. 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, N.Y. 1989). For purposes of the present disclosure, the following terms are defined below.

[0086] As used herein, in the context of gene delivery, the term “vector” or “gene delivery vector” may refer to a particle that functions as a gene delivery vehicle, and which comprises nucleic acid (i.e., the vector genome comprising any of the vector constructs described herein) packaged within, for example, an envelope or capsid. A gene delivery vector may be a viral gene delivery vector or a non-viral gene delivery vector. Alternatively, in some contexts, the term “vector” may be used to refer only to the vector genome or vector construct. Viral vectors suitable for use herein may be a parvovirus, an adenovirus, a retrovirus, a lentivirus or a herpes simplex virus. The parvovirus may be an adenovirus-associated virus (AAV).

[0087] As used herein, the term “AAV” is a standard abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells in which certain functions are provided by a co-infecting helper virus. There are numerous serotypes of AAV that have been characterized. General information and reviews of AAV can be found in, for example, Carter, Handbook of Parvoviruses, Vol. 1, pp. 169-228 (1989); and Berns, Virology, pp. 1743-64, Raven Press, (New York) (1990); Gao et al., Meth. Mol. Biol. 807: 93- 118 (2011); Ojala et al., Mol. Ther. 26(1): 304-19 (2018). However, it is fully expected that these same principles will be applicable to additional AAV serotypes since it is well known that the various serotypes are quite closely related, both structurally and functionally, even at the genetic level. (See, e g., Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, J. R.Pattison, ed.; and Rose, Comprehensive Virology 3:1-61 (1974)). For example, all AAV serotypes apparently exhibit very similar replication properties mediated by homologous rep genes; and all bear three related capsid proteins. The degree of relatedness is further suggested by heteroduplex analysis which reveals extensive cross-hybridization between serotypes along the length of the genome; and the presence of analogous self-annealing segments at the termini that correspond to “inverted terminal repeat sequences” (ITRs).

[0088] As used herein, an “AAV vector construct” refers to nucleic acids, either singlestranded or double-stranded, having at least one of (i) an AAV 5’ inverted terminal repeat (ITR) sequence and (ii) an AAV 3’ ITR, flanking a protein-coding sequence (in one embodiment, a functional therapeutic protein-encoding sequence, e.g. cMyBP-C-encoding sequence) operably linked to transcription regulatory elements (also called “expression control elements”) that are heterologous to protein-encoding sequence and / or heterologous to the AAV viral genome, i.e., one or more promoters and / or enhancers and, optionally, a polyadenylation sequence and / or optionally one or more introns. A single-stranded AAV vector refers to nucleic acids that are present in the genome of an AAV virus particle, and can be either the sense strand or the antisense strand of the nucleic acid sequences disclosed herein. The size of such single-stranded nucleic acids is provided in bases. A double-stranded AAV vector refers to nucleic acids that are present in the DNA of plasmids, e.g., pUC19, or genome of a double- stranded virus, e.g., baculovirus, used to express or transfer the AAV vector nucleic acids. The size of such doublestranded nucleic acids in provided in base pairs (bp).

[0089] The AAV vector constructs provided herein in single strand form are less than about 7.0 kb in length, or are less than 6.5 kb in length, or are less than 6.4 kb in length, or are less than6.3 kb in length, or are less than 6.2 kb in length, or are less than 6.0 kb in length, or are less than5.8 kb in length, or are less than 5.6 kb in length, or are less than 5.5 kb in length, or are less than5.4 kb in length, or are less than 5.3 kb in length, or are less than 5.2 kb in length. The AAV vector constructs in single strand form are also at least about 4.0 kb in length. Preferably, the AAV vector constructs are also at least about 4.5 kb in length. In some embodiments, the AAV vector constructs provided herein in single strand form range from about 4.0 kb to about 5.8 kb in length.

[0090] While AAV particles have been reported in the literature having AAV genomes of > 5.0 kb, in many of these cases the 5’ or 3’ ends of the encoded genes appear to be truncated (see Hirsch et al., Molec. Ther. 18: 6-8 (2010), and Ghosh et al., Biotech. Genet. Engin. Rev. 24: 165- 78 (2007). It has been shown, however, that overlapping homologous recombination occurs in AAV infected cells between nucleic acids having 5’ end truncations and 3’ end truncations so that a complete nucleic acid encoding the large protein is generated, thereby reconstructing a functional, full-length gene.

[0091] Oversized AAV vectors are randomly truncated at the 5’ ends and lack a 5’ AAV ITR. Because AAV is a single-stranded DNA virus, and packages either the sense or antisense strand, the sense strand in oversized AAV vectors lacks the 5’ AAV ITR and possibly portions of the 5’ end of the target protein-coding gene, and the antisense strand in oversized AAV vectors lacks the 3’ ITR and possibly portions of the 3’ end of the target protein-coding gene. Afunctional transgene is produced in oversized AAV vector infected cells by annealing of the sense and antisense truncated genomes within the target cell. Thus, in certain embodiments, the AAV cMyBP-C vectors and / or viral particles comprise at least one ITR.

[0092] The term “inverted terminal repeat (ITR)” as used herein refers to the art-recognized regions found at the 5’ and 3’ termini of the AAV genome which function in cis as origins of DNA replication and as packaging signals for the viral genome. AAV ITRs, together with the AAV rep coding region, provide for efficient excision and rescue from, and integration of a nucleotide sequence interposed between two flanking ITRs into a host cell genome. Sequences of certain AAV-associated ITRs are disclosed by Yan et al., J. Virol. 79: 364-79 (2005) which is herein incorporated by reference in its entirety. ITR sequences that find use herein may be full length, wild-type AAV ITRs or fragments thereof that retain functional capability, or may be sequence variants of full-length, wild-type AAV ITRs that are capable of functioning in cis as origins of replication. AAV ITRs useful in the recombinant AAV cMyBP-C vectors of the embodiments provided herein may be derived from any known AAV serotype and, in certain embodiments, derived from the AAV2 or AAV5 serotype.

[0093] The term “control sequences” refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0094] A “transcription regulatory element” refers to nucleotide sequences of a gene involved in regulation of genetic transcription including a promoter, plus response elements, activator and enhancer sequences for binding of transcription factors to aid RNA polymerase binding and promote expression, and operator or silencer sequences to which repressor proteins bind to block RNA polymerase attachment and prevent expression. The term “cardiac-specific transcription regulatory element” or “cardiac-specific expression control element” refers to a regulatory element or region that produces preferred gene expression specifically in cardiac cells, e g., a promoter whose activity in cardiac cells is at least 2-fold or at least 5-fold higher than in any other non-cardiac cell type. Preferably, the cardiac-specific expression control element is cardiomyocyte-specific, and provides expression in cardiomyocytes at least 5-fold higher than in skeletal muscle cells. In some embodiments, the cardiomyocyte-specific promoter has an activity in cardiomyocytes at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25- fold, or at least 50-fold higher compared to its activity in a non-cardiomyocytes.

[0095] The cardiac-specific or cardiomyocyte-specific promoter is operably linked to the nucleic acid sequence encoding the cMyBP-C protein which means that the promoter iscombined with the coding nucleic acid so as to enable the expression of said coding nucleic acid under the control of the promoter in cardiomyocytes when integrated into the genome of the cell or present as an extragenomic nucleic acid construct in the cell.

[0096] Transcription regulatory elements optionally include an enhancer element, intron, polyadenylation sequence, or post-transcriptional regulatory elements for increasing the expression level of the myosin binding protein. Examples include the SV40 early gene enhancer and the enhancer of the long terminal repeat (LTR) of Rous Sarcoma Virus (Gorman et al. (1982) Proc. Natl. Acad. Sci. 79:6777). The vector also optionally comprises transcription termination sequences and polyadenylation sequences for improved expression of the human and / or non-human antigen(s). Suitable transcription terminator and polyadenylation signals can, for example, be derived from SV40 (Sambrook et al (1989), Molecular Cloning: A Laboratory Manual). Preferably, a bGH polyadenylation signal is used in the vector of the invention. Any other element which is known in the art to support efficiency or specificity of expression may be added to the expression vector, such as the Woodchuck hepatitis post- transcriptional regulatory element (wPRE). To increase the cardiac or cardiomyocyte specificity, other elements can be introduced to inactivate the expression of genes in other tissues, such as sequences encoding miRNAs such as miR122 (Geisler et al., Gene Ther. 18: 199-209 (2011).

[0097] As used herein, an “intron” is broadly defined as a sequence of nucleotides that is removable by RNA splicing. “RNA splicing” means the excision of introns from a pre-mRNA to form a mature mRNA. Introns may be upstream, downstream, or within the coding region of a gene. Insertion of an intron into a nucleotide sequence can be accomplished by any method known in the art. The only limitation of where the intron is inserted is in consideration of the packaging limitations of the AAV virus particles (e.g., about 5 kb).

[0098] As used herein, the term “operably linked” is used to describe the connection between regulatory elements and a gene or its coding region. Typically, gene expression is placed under the control of one or more regulatory elements, for example, without limitation, constitutive or inducible promoters, tissue-specific regulatory elements, and enhancers. A gene or coding region is said to be “operably linked to” or “operatively linked to” or “operably associated with” the regulatory elements, meaning that the gene or coding region is controlled or influenced by the regulatory element. For instance, a promoter is operably linked to a coding sequence if the promoter effects transcription or expression of the coding sequence.

[0099] The term “isolated” when used in relation to a nucleic acid molecule of the present disclosure typically refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. Isolated nucleic acid may be present in a form or setting that is different from that in which it is found innature. Isolated nucleic acid molecules therefore are distinguished from the nucleic acid molecule as it exists in natural cells.

[0100] As used herein, the term “variant” refers to a polynucleotide (or polypeptide) having a sequence substantially similar to a reference polynucleotide (or polypeptide). Variants encoding functional therapeutic protein, e.g. a functional cMyBP-C protein that maintains cMyBP-C activity, are contemplated herein. Procedures for the introduction of nucleotide and amino acid changes in a polynucleotide, protein or polypeptide are known to the skilled artisan (see, e.g., Sambrook et al. (1989)). In the case of a polynucleotide, a variant can have deletions, substitutions, additions of one or more nucleotides at the 5’ end, 3’ end, and / or one or more internal sites in comparison to the reference polynucleotide. Similarities and / or differences in sequences between a variant and the reference polynucleotide can be detected using conventional techniques known in the art, for example polymerase chain reaction (PCR) and hybridization techniques. Variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis. Generally, a variant of a polynucleotide, including, but not limited to, a DNA, can have at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to the reference polynucleotide as determined by sequence alignment programs known by skilled artisans. In the case of a polypeptide, a variant can have deletions, substitutions, additions of one or more amino acids in comparison to the reference polypeptide. Similarities and / or differences in sequences between a variant and the reference polypeptide can be detected using conventional techniques known in the art, for example Western blot.Generally, a variant of a polypeptide, can have at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to the reference polypeptide as determined by sequence alignment programs known by skilled artisans.

[0101] The amino acid substitutions can be conservative or non-conservative. It is preferred that the substitutions are conservative substitutions, i.e. a substitution of an amino acid residue by an amino acid of similar polarity, which acts as a functional equivalent. Preferably, the amino acid residue used as a substitute is selected from the same group of amino acids as the amino acid residue to be substituted. For example, a hydrophobic residue can be substituted with another hydrophobic residue, or a polar residue can be substituted with another polar residue having the same charge. Functionally homologous amino acids which may be used for a conservative substitution comprise, for example, non-polar amino acids such as glycine, valine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, and tryptophan. Examples ofuncharged polar amino acids comprise serine, threonine, glutamine, asparagine, tyrosine and cysteine. Examples of charged polar (basic) amino acids comprise histidine, arginine and lysine. Examples of charged polar (acidic) amino acids comprise aspartic acid and glutamic acid.

[0102] Also considered as variants are proteins which differ from their naturally occurring counterparts by addition, substitution or deletion of one or more (e.g. 2, 3, 4, 5, 10, or 15) additional amino acids. For example, additional amino acids may be present within the amino acid sequence of the original cMyBP-C protein (i.e. as an insertion), or they may be added to one or both termini of the protein. Such insertions, substitutions or deletions can take place at any position provided they do not impair the capability of the polypeptide to fulfill the function of the naturally occurring cMyBP-C protein and / or rescue the haploinsufficiency in the treated subject. Moreover, variants of cMyBP-C proteins also comprise proteins in which, compared to the original polypeptide, one or more amino acids are lacking. Such deletions may affect any amino acid position provided that it does not impair the ability to fulfill the normal function of the cMyBP-C protein and / or rescue the haploinsufficiency. To visualize the exogenous gene expression in the heart, other optional elements can be introduced as part of the cMyBP-C encoding sequence, such as tag sequences (myc, FLAG, HA, His, and the like), or fluorochromes such as GFP, YFP, RFP.

[0103] Finally, variants also refer to proteins which differ from the naturally occurring protein by structural modifications, such as modified amino acids. Modified amino acids are amino acids which have been modified either by natural processes, such as processing or post- translational modifications, or by chemical modification processes known in the art. Typical amino acid modifications comprise phosphorylation, glycosylation, acetylation, O-Linked N- acetylglucosamination, glutathionylation, acylation, branching, ADP ribosylation, crosslinking, disulfide bridge formation, formylation, hydroxylation, carboxylation, methylation, demethylation, amidation, cyclization and / or covalent or non-covalent bonding to hosphatidylinositol, flavine derivatives, lipoteichonic acids, fatty acids or lipids. Such modifications have been extensively described in the literature, e g., in Proteins: Structure and Molecular Properties, T. Creighton, 2ndedition, W. H. Freeman and Company, New York (1993). In a preferred embodiment of the invention, the nucleic acid sequence encodes a constitutively phosphorylated isoform of human cMyBP-C. It has been shown that these isoforms are particularly cardioprotective (Sadayappan et al. (2005), Circ Res 97: 1156-1163; Sadayappan et al., 2006; Proc Natl Acad Sci U S A 103:16918-16923).

[0104] The term “identity,” “homology” and grammatical variations thereof, mean that two or more referenced entities are the same, when they are “aligned” sequences. Thus, by way of example, when two polypeptide sequences are identical, they have the same amino acidsequence, at least within the referenced region or portion. Where two polynucleotide sequences are identical, they have the same polynucleotide sequence, at least within the referenced region or portion. The identity can be over a defined area (region or domain) of the sequence. An “area” or “region” of identity refers to a portion of two or more referenced entities that are the same. Thus, where two polypeptide or nucleic acid sequences are identical over one or more sequence areas or regions they share identity within that region. An “aligned” sequence refers to multiple polynucleotide or polypeptide (amino acid) sequences, often containing corrections for missing or additional bases or amino acids (gaps) as compared to a reference sequence. “Substantial homology” means that a molecule is structurally or functionally conserved such that it has or is predicted to have at least partial structure or function of one or more of the structures or functions (e g., a biological function or activity) of the reference molecule, or relevant / corresponding region or portion of the reference molecule to which it shares homology.

[0105] “Percent (%) nucleic acid sequence identity or homology” is defined as the percentage of nucleotides in a candidate sequence that are identical with a reference sequence after aligning the respective sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0106] “Percent (%) amino acid sequence identity or homology” with respect to the cMyBP- C amino acid sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a cMyBP-C polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0107] ‘Codon optimization” or “codon optimized” refers to changes made in the nucleotide sequence so that it is more likely to be expressed at a relatively high level compared to the noncodon optimized sequence. It does not change the amino acid for which each codon encodes.

[0108] An “AAV virion” or “AAV viral particle” or “AAV vector particle” or “AAV virus” refers to a viral particle composed of at least one AAV capsid protein and an encapsulated AAV vector construct as described herein. If the particle comprises a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell), it is typically referred to as a “recombinant AAV vector particle” or simply an “AAV vector”. Production of AAV vector particles necessarily includes production of AAV vector genome, as such a vector genome is contained within an AAV vector particle. It is understood that reference to the polynucleotide AAV vector construct encapsulated within the vector particle, and replication thereof, refers to the AAV vector genome.

[0109] As used herein “therapeutic AAV virus” refers to an AAV virion, AAV viral particle, AAV vector particle, or AAV virus that comprises a heterologous polynucleotide that encodes a therapeutic protein such as the cMyBP-C described herein. An “AAV vector construct” or “AAV vector genome” as used herein refers to a vector construct comprising one or more polynucleotide encoding a protein of interest (also called transgenes) that are flanked by at least one AAV terminal repeat sequences (ITRs) and operably linked to one or more expression control elements. Such AAV vector constructs can be replicated and packaged into infectious viral particles when present in a host cell that has been transfected with a vector encoding and expressing rep and cap gene products. The term generally refers to recombinant AAV that are capable of infecting cells such that the infected cells express (e g., by transcription and / or by translation) an element (e.g., nucleotide sequence, protein, etc.) of interest. To this extent, the therapeutically effective rAAV particles can include AAV particles having capsids or vector genomes (vgs) with different properties. For example, the therapeutically effective rAAV particles can have capsids with different post translation modifications. In other examples, the therapeutically effective AAV particles can contain vector genomes of differing sizes / lengths, plus or minus strand sequences, different flip / flop ITR configurations flip / flop, flop / flip, flip / flip, flop / flop, etc ), different number of ITRs (1, 2, 3, etc.), or truncations. For example, overlapping homologous recombination occurs in rAAV infected cells between nucleic acids having 5’ end truncations and 3’ end truncations so that a “complete” nucleic acid encoding the large protein is generated, thereby reconstructing a functional, full-length gene. In other examples, complementary nucleic acid sequences having 5’ end truncations and 3’ end truncations interact with each such that a “complete” nucleic acid is formed during second strand synthesis The “complete” nucleic acid encodes the large protein, thereby reconstructing a functional, full- length gene. Therapeutically effective rAAV particles are also referred to as heavy capsids, full capsids, or partially full capsids. Conversely, “therapeutically ineffective” AAV virus refer toempty capsids, or capsids that have unquantifiable or undetectable vector genomes, or vector genomes that are not capable of recombining into a complete functional nucleic acid.

[0110] As used herein “therapeutic protein” refers to a polypeptide that has a biological activity that replaces or compensates for the loss or reduction of activity of an endogenous protein. For example, a functional cMyBP-C protein is a therapeutic protein for HCM.

[0111] “Hypertrophic cardiomyopathy” as used herein refers to an inherited disease caused by mutations in genes encoding components of the cardiac sarcomere, such as cardiac myosin binding protein C, that is characterized, for example, by symptoms of heart failure, arrhythmias, chest pain, shortness of breath, fatigue and dizziness, increased heart size, increased cardiothoracic ratio, increased ventricular (anterior or posterior or both) wall thickness, decreased ejection time, decreased aortic peak flow velocity, and / or decreased aortic flow time. The end diastolic and end systolic dimensions can also be smaller than normal dimensions due to a smaller than normal cavity as a result from increased wall thickness.

[0112] “Cardiac myosin binding protein C deficiency” or a “deficiency in functional wildtype cardiac myosin binding protein C” as used herein refers to an inherited condition caused by reduced levels of functional cMyBP-C protein, due to absence of protein, reduced production of protein or production of protein that is nonfunctional. This includes HCM.

[0113] “Therapeutically effective for hypertrophic cardiomyopathy” or “Hypertrophic cardiomyopathy therapy” as used herein refers to any therapeutic intervention of a subject having HCM that ameliorates the characteristic deficiency in functional wild-type cMyBP-C, increases cMyBP-C protein levels, e.g. in myocardium, ameliorates HCM symptoms, or reduces the frequency, duration or severity of HCM symptoms.

[0114] “Hypertrophic cardiomyopathy gene therapy” as used herein refers to any therapeutic intervention of a subject having HCM that involves the replacement or restoration or increase of cMyBP-C through the delivery of one or more nucleic acid molecules to the cells of the subject that express functional cMyBP-C. In certain embodiments, MYBPC3 gene therapy refers to gene therapy involving an adeno associated viral (AAV) particle comprising a vector construct that expresses human cMyBP-C. In other embodiments, the gene therapy involves transfecting a plasmid that expresses human cMyBP-C.

[0115] ‘Treat” or “treatment” as used herein refers to preventive or therapeutic treatment which refers to a treatment administered to a subject who exhibits signs or symptoms of pathology, i.e., HCM, for the purpose of diminishing or eliminating those signs or symptoms or ameliorating their progression, severity or duration. The signs or symptoms can be biochemical, cellular, histological, functional, subjective or objective.

[0116] “Ameliorate” as used herein refers to the action of lessening the severity of symptoms, progression, or duration of a disease.

[0117] As used herein “stably treating” or “stable treatment” refers to using a therapeutic vector construct, AAV particle or cell administered to a subject where the subject stably expresses a therapeutic protein expressed by the vector construct, AAV particle or cell. Stably expressed therapeutic protein means that the protein is expressed for a clinically significant length of time. “Clinically significant length of time” as used herein means expression at therapeutically effective levels for a length of time that has a meaningful impact on the quality of life of the subject, e.g., demonstrated by reduced signs or symptoms of disease. In certain embodiments clinically, significant length of time is expression for at least six months, for at least eight months, for at least one year, for at least two years, for at least three years, for at least four years, for at least five years, for at least six years, for at least seven years, for at least eight years, for at least nine years, for at least ten years, or for the life of the subject.

[0118] As used herein, the term “effective amount” refers to an amount sufficient to effect beneficial or desirable biological and / or clinical results.

[0119] In general, a “pharmaceutically acceptable carrier” is one that is not toxic or unduly detrimental to cells and is preferably sterile. Exemplary pharmaceutically acceptable carriers include sterile, pyrogen-free water and sterile, pyrogen-free, saline, tris(hydroxymethyl)aminomethane, or phosphate buffered saline. Pharmaceutically acceptable carriers include physiologically acceptable carriers. The term “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatibleAAV Vectors

[0120] The recombinant vector construct of the disclosure may be used itself as gene therapy or may be used to produce rAAV particles by methods described herein, comprising providing to a suitable host cell the recombinant vector construct, together with Rep and Cap genes. The vector constructs described herein comprise a nucleic acid sequence that encodes a functional cMyBP-C. The recombinant vector construct may comprise a nucleic acid encoding functional human cMyBP-C operably linked to a heterologous expression control element, e.g. a promoter and / or enhancer; optionally an intron; and optionally a polyadenylation (poly A) signal. The heterologous expression control element may be a heterologous cardiomyocyte-specific transcription regulatory region, e g., as described herein.

[0121] The rAAV particles may comprise (a) an AAV capsid with heart tropism, and (b) a recombinant vector contract comprising a cardiac-specific transcription regulatory region, and(c) a nucleic acid encoding a functional cMyBP-C protein. The recombinant vector construct may further comprise one or both of (i) an AAV 5’ inverted terminal repeat (ITR) sequence and (ii) an AAV 3’ ITR, optionally wherein the AAV ITRs are AAV2 ITRs. The vector construct may include additional expression control elements, for example: a promoter and / or enhancer; an intron; optionally an exon or fragment thereof; and a polyadenylation (polyA) signal. Such elements are further described herein.

[0122] Preferably, the nucleic acid encoding the functional cMyBP-C is operably linked to cardiomyocyte-specific expression control elements. In certain embodiments, the recombinant AAV vector construct comprises a nucleic acid comprising (a) an AAV2 5’ inverted terminal repeat (ITR) (which may or may not be modified as known in the art), (b) a cardiomyocyte - specific transcription regulatory region, a functional MYBPC3 protein coding region, (c) one or more introns including fragments of longer introns, (d) optionally a heterologous exon or fragment thereof adjacent to the intron, (e) a polyadenylation sequence, and (f) an AAV23’ ITR (which may or may not be modified as known in the art).

[0123] Preferably, the rAAV particles also comprise an AAV capsid with cardiac tropism, optionally an AAV9 type capsid. Example capsids with cardiac tropism include AAV1, 6, 7 and 9.

[0124] Other embodiments provided herein are directed to vector constructs encoding a functional cMyBP-C polypeptide, wherein the constructs comprise one or more of the individual elements of the above-described constructs and combinations thereof, in one or more different orientation(s). Another embodiment provided herein is directed to the above-described constructs in an opposite orientation.

[0125] The AAV vector constructs provided herein in single strand form range from about 4.5 kb to about 6.5 kb in length, or from about 4.5 kb to about 5.5 kb in length, or from about 4 kb to about 5.5 kb in length, or range from about 4.8 kb to about 5.2 k in length, or 4.8 kb to 5.1 kb in length, or range from about 4.9 kb to about 5.5 kb in length, or about 4.8 kb to about 6.0 kb in length, or about 5.0 kb to 6.2 kb in length or about 5.1 kb to about 6.3 kb in length, or about 5.2 kb to about 6.4 kb in length, or about 5.5 kb to about 6.5 kb in length, or range from about 4.0 kb to about 5.0 kb in length, or range from about 4 to about 4.5 kb in length, or range from about 4.5 kb to about 5 kb in length.

[0126] When AAV vectors are produced from oversized recombinant vector constructs, they may lack a portion of the 5’ or 3’ ends of the recombinant vector construct. Because AAV is a single-stranded DNA virus, and packages either the sense or antisense strand, the sense strand in oversized AAV vectors lacks the 5’ AAV ITR and possibly portions of the 5’ end of the target protein-coding gene, and the antisense strand in oversized AAV vectors lacks the 3’ ITR andpossibly portions of the 3’ end of the target protein-coding gene. A functional transgene is produced in oversized AAV vector infected cells by annealing of the sense and antisense truncated genomes within the target cell. Thus, in certain embodiments, the rAAV particles of the invention may comprise recombinant vector constructs that comprise at least one ITR, and a substantial portion of a nucleotide sequence encoding a functional cMyBP-C, such as a fragment of SEQ ID NO: 1 or 42-46 that is greater than 50%, 60%, 70%, 80%, or 90% of the length of the nucleotide sequence. For example, the recombinant vector construct may comprise at least one ITR, a cardiomyocyte-specific transcription regulatory region, and a substantial portion of a nucleotide sequence encoding a functional cMyBP-C.

[0127] Generation of the vector constructs can be accomplished using any suitable genetic engineering techniques well known in the art, including, without limitation, the standard techniques of restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing, for example as described in Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, N.Y. (1989)).

[0128] The vector constructs can incorporate sequences from the genome of any known organism. The sequences can be incorporated in their native form or can be modified in any way to obtain a desired activity. For example, the sequences can comprise insertions, deletions or substitutions.

[0129] AAV vector constructs can be replicated and packaged into infectious AAV particles, preferably replication deficient AAV particles, when present in a host cell that has been transfected with a polynucleotide encoding and expressing rep and cap gene product.Expression Control Elements: Promoters and Enhancers:

[0130] In one or more embodiments, the nucleic acid sequence encoding cMyBP-C is operably linked to one or more heterologous expression control elements. Preferably, the expression control element is a cardiomyocyte-specific expression control element. Examples of cardiomyocyte-specific control elements include, but are not limited, to human cardiac troponin T (hTNNT2) promoter or fragments or variants thereof. Other promoters with activity in cardiomyocytes include fragments or variants of any of: muscle creatine kinase (MCK) promoter, cytomegalovirus enhancer + myosin light chain 2 promoter (CMV-MLC2, or CMV- MLC1.5, CMV-MLC260), a phosphoglycerate kinase (PGK) promoter, a sarcomere-specific promoters, 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 / or sarco / endoplasmic reticulum Ca2+”ATPase (SERCA) promoter (e.g. iso-form 2 of thispromoter (SERCA2)), and / or a striated muscle promoter, such as the desmin promoter. Enhancers derived from cardiomyocyte -specific transcriptional factor binding sites are also contemplated.

[0131] Examples of fragments or variants of hTNNT2 promoter include a cardiomyocyte- specific promoter sequence at least, or more than, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of SEq ID NOs: 49-52 and 75-205 (over the length of the SEQ ID NO). Other examples include any one of SEQ ID NO: 1 to 85 of U.S. Pat. Pub. No. 2021 / 0252165, incorporated by reference in its entirety.

[0132] In some embodiments, the fragment or variant of the hTNNT2 promoter is more than 420 and less than 544 nucleotides in length and comprises a nucleic acid sequence at least 90% identical to SEQ ID NO: 51. In any of the embodiments described herein, the cardiomyocyte- specific promoter optionally excludes any one of SEQ ID NO: 1 to 85 of U.S. Pat. Pub. No. 2021 / 0252165.

[0133] Various promoters can be operably linked with a nucleic acid comprising the coding region of the protein of interest, human cardiac myosin binding protein C, in the vector constructs disclosed herein. In some embodiments, the promoter can drive the expression of the protein of interest in a cell infected with a virus derived from the viral vector, such as a target cell. The promoter can be naturally occurring or non-naturally occurring. In some embodiments the promoter is a synthetic promoter. In one embodiment the synthetic promoter comprises sequences that do not exist in nature and which are designed to regulate the activity of an operably linked gene. In another embodiment the synthetic promoter comprises fragments of natural promoters to form new stretches of DNA sequence that do not exist in nature. Synthetic promoters are typically comprised of regulatory elements, promoters, enhancers, introns, splice donors and acceptors that are designed to produce enhanced tissue specific expression. Examples of promoters include, but are not limited to, viral promoters, plant promoters and mammalian promoters. In another embodiment the promoter is a cardiomyocyte specific promoter. In some embodiments, the transcription regulatory region comprises one or more additional individual enhancer elements, in one or more different orientation(s).

[0134] In some embodiments, the transcription regulatory region comprises a promoter and an intron that enhances expression of the protein of interest, located 5’ to the therapeutic proteincoding sequence. For example, the cardiomyocyte-specific transcription regulatory region may further comprise (in addition to the fragment of variant of hTNNT2 protein) a globin intron adjacent to the 3’ end of a fragment of beta globin exon 3 (SEQ ID NO: 53). The combination of the intron and exon fragment is, for example, SEQ ID NO: 55. The combination of the promoter, exon fragment, and intron is, for example, SEQ ID NO: 56.

[0135] The promoter is operably linked with a polynucleotide encoding one or more proteins of interest. In some embodiments, the promoter is operably linked with a polynucleotide encoding the cMyBP-C protein.

[0136] The size of the promoter can vary. Because of the limited packaging capacity of AAV, it is preferred to use a promoter that is small in size, but at the same time allows high level production of the protein(s) of interest in host cells. For example, in some embodiments the promoter is at most about 1.5 kb, at most about 1.4 kb, at most about 1.35 kb, at most about 1.3 kb, at most about 1.25 kb, at most about 1.2 kb, at most about 1.15 kb, at most about 1.1 kb, at most about 1.05 kb, at most about 1 kb, at most about 800 base pairs, at most about 600 base pairs, at most about 400 base pairs, at most about 200 base pairs, or at most about 100 base pairs.

[0137] Various additional regulatory elements can be used in the vector constructs, for example enhancers or introns to further increase expression level of the protein of interest in a host cell, a polyadenylation signal, a ribosome binding sequence, and / or a consensus splice acceptor or splice donor site. In some embodiments, the regulatory element can facilitate maintenance of the recombinant DNA molecule extrachromosomally in a host cell and / or improve vector potency (e.g. scaffold / matrix attachment regions (S / MARs)). Such regulatory elements are well known in the art and are described further below.Protein of Interest and Nucleic Acids Encoding the Protein of Interest.

[0138] A polynucleotide encoding one or more proteins of interest can be inserted into the viral vectors disclosed herein, wherein the polynucleotide is operably linked with the promoter. A “protein of interest” includes any functional cMyBP-C protein, including naturally-occurring and non-naturally occurring variants thereof. In some instances, the promoter can drive the expression of the protein(s) of interest in a specific type of host cell (e.g., human myocardiocyte). In one or more embodiments, the functional cMyBP-C comprises an amino acid sequence at least 90%, 95% or 98% identical to SEQ ID NO: 2 (a human cardiac myosin binding protein C). The present disclosure also provides an isolated nucleic acid molecule which encodes such functional wild-type cMY-BP-C protein. In example embodiments, the nucleic acid sequence encoding the functional cMyBP-C is a wild-type MYBPC3 sequence, of which SEQ ID NO: 1 is one example, or is codon optimized, or is a variant. The vector constructs described herein may comprise a nucleotide sequence that differs from wild type nucleotide sequence but still encodes a functional cMyBP-C amino acid sequence at least 90%, 95% or 98% identical to SEQ ID NO: 2. According to this aspect, the nucleotide sequence may comprise a portion having at least 80%, 85%, 90% or 95% homology to at least 100 consecutive bases of SEQ ID NO: 1 or42-46, as long as the nucleotide sequence encodes functional human cMyBP-C protein at least 90%, 95% or 98% identical to SEQ ID NO: 2.

[0139] As described herein, the nucleotide sequence encoding the cMyBP-C protein can be modified to improve expression efficiency of the protein. The methods that can be used to improve the transcription and / or translation of a gene herein are not particularly limited. For example, the nucleotide sequence can be codon optimized, or can undergo CpG reduction. As another non-limiting example for the modification, one or more of the splice donors and / or splice acceptors in the nucleotide sequence of the protein of interest is modified to reduce the potential for extraneous splicing. As another non-limiting example for the modification, one or more introns can be inserted within or adjacent to the nucleotide sequence of the protein of interest to optimize AAV vector packaging and enhance expression.

[0140] Since the cMyBP-C that is produced is functional, it will have a conformation which is the same as at least a portion of the wild type cMyBP-C. In certain embodiments, a functional cMyBP-C protein produced as described herein effectively treats a subject suffering from deficiency in wild-type cMyBP-C protein and / or HCM.Introns

[0141] In some embodiments, the vector comprises one or more introns. The introns may facilitate processing of the RNA transcript in mammalian host cells, increase expression of the protein of interest and / or optimize packaging of the vector into AAV particles. Non-limiting examples of such an intron are a human beta globin intron, a human immunoglobulin G (IgG) intron or a native cMyBP-C intron. In some embodiments, the intron is a chimeric intron and comprises a fragment of a human IgG intron.

[0142] Examples of introns that enhances expression of the cMyBP-C protein include a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any of SEq ID NOs: 53 or 58 . In some embodiments, the intron and optionally an exon or fragment thereof is located 5’ to the cMyBP-C coding sequence. In further examples, the intron located within the nucleotide sequence encoding cMyBP-C, for example, between any of the exons. In some embodiments, the intron sequence is located between exon 2 and 3. In some embodiments, the intron sequence is located at a position within the nucleic acid encoding cMyBP-C that corresponds to position 293 of SEQ ID NO: 1.

[0143] In one or more embodiments, the intron may be about 50 to about 150 nucleotides in length, or about 100 to about 135 nucleotides in length. In example embodiments, the intron comprises SEQ ID NO: 53 [globin intron] or a fragment thereof that is that is about 50-150nucleotides, 75-145 nucleotides, 100-135 nucleotides, or 120-135 nucleotides of SEQ ID NO: 53 or a variant of said fragment that is at least 80%, 85%, 90%, or 95% identical to said fragment.

[0144] In some embodiments, the vector constructs may further comprise an exon sequence or fragment thereof; preferably adjacent to the 5’ or 3’ end of an intron sequence. In an example embodiment, the vector construct comprises a globin intron adjacent to an exon comprising a nucleotide sequence at least 80% or 85% or 90% or 95% identical to any of SEQ ID NO: 55-57 [intron + exon],

[0145] Inclusion of an intron element may enhance expression compared with expression in the absence of the intron element (see e.g. Kurachi et al., J. Biol. Chem. 270(10): 5276-81 (1995). AAV vectors typically accept inserts of DNA having a defined size range which is generally about 4 kb to about 5.4 kb, or slightly more. However, there is no minimum size for packaging and small vector genomes package very efficiently. Introns and intron fragments fulfill this requirement while also enhancing expression. Thus, the present disclosure is not limited to the inclusion of cMyBP-C intron sequences in the AAV vector and include other introns or other DNA sequences in place of portions of a cMyBP-C intron. Additionally, other 5’ and 3’ untranslated regions of nucleic acid may be used in place of those recited for human cMyBP-C.Transcription termination regions and polyadenylation signals

[0146] The vectors constructs disclosed herein may include regulatory elements such as a transcription initiation region and / or a transcriptional termination region. Examples of a transcription termination region include, but are not limited to, polyadenylation signal sequences. Examples of polyadenylation signal sequences include, but are not limited to, human growth hormone (hGH) poly(A), bovine growth hormone (bGH) poly(A), SV40 late poly(A), rabbit beta-globin (rBG) poly(A), thymidine kinase (TK) poly(A) sequences, Proudfoot poly A, and any variants thereof. In some embodiments, the transcriptional termination region is located downstream of the posttranscriptional regulatory element. In some embodiments, the transcriptional termination region is a bGH polyA (e g., any of SEq ID NOs: 59-61), a hGH polyA (e.g. any of SEq ID NOs: 62), a SV40 polyA (e.g. SEQ ID NO: 63), a Proudfoot synthetic polyA (e.g. SEQ ID NO: 65) or a rabbit beta-globin polyA (e.g. SEQ ID NO: 66) sequence or a fragment thereof about 40 to 200 nucleotides in length.

[0147] The polyA signal may be about 150 to about 250 nucleotides in length, about 160 to about 240 nucleotides in length, about 170 to about 230 nucleotides in length, about 180 to about 220 nucleotides in length, or about 200 to about 210 nucleotides in length.

[0148] In some embodiments, the vector constructs can include additional transcription and translation initiation sequences, and / or additional transcription and translation terminator sequences, which are known in the art.Viral Particles.

[0149] The present disclosure provides adenovirus-associated virus (AAV) particles and formulations for use in methods of therapy. Vector constructs suitable for production of AAV particles include the polynucleotide inserts of any of SEq ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36 and 39, flanked by 5’ and 3’ ITRs.

[0150] Accordingly, the present disclosure provides viral particles for use as gene delivery vectors (comprising a vector construct provided herein) based on animal parvoviruses, in particular dependoviruses such as infectious human or simian AAV, and the components thereof (e g., an animal parvovirus genome) for introduction and / or expression of a cMyBP-C protein in a mammalian cell. The term “parvoviral” as used herein thus encompasses dependoviruses such as any type of AAV. Production of AAV particles requires AAV “rep” and “cap” genes, which are genes encoding replication and encapsulation proteins, respectively. AAV rep and cap genes have been found in all AAV serotypes examined to date and are described herein and in the references cited. In wild-type AAV, the rep and cap genes are generally found adjacent to each other in the viral genome (i.e., they are “coupled” together as adjoining or overlapping transcriptional units), and they are generally conserved among AAV serotypes. AAV rep and cap genes are also individually and collectively referred to as “AAV packaging genes.” The AAV cap genes for use herein encode Cap proteins which are capable of packaging AAV vectors in the presence of rep and adeno helper function and are capable of binding target cellular receptors. In some embodiments, the AAV cap gene encodes a capsid protein having an amino acid sequence derived from a particular AAV serotype.

[0151] The AAV sequences employed for the production of AAV can be derived from the genome of any AAV serotype. Generally, the AAV serotypes have genomic sequences of significant homology at the amino acid and the nucleic acid levels, provide a similar set of genetic functions, produce virions which are essentially physically and functionally equivalent, and replicate and assemble by practically identical mechanisms. For the genomic sequence of AAV serotypes and a discussion of the genomic similarities. (See, e g., GenBank Accession number U89790; GenBank Accession number J0190I; GenBank Accession number AF043303; GenBank Accession number AF085716; Chiorini et al., J. Virol. 71 : 6823-33 (1997); Srivastava et al., J. Virol. 45: 555-64 (1983); Chiorini et al., J. Virol. 73: 1309-19 (1999); Rutledge et al., J. Virol. 72: 309-19 (1998); and Wu et al., J. Virol. 74: 8635-47(2000)).

[0152] The genomic organization of all known AAV serotypes is very similar. The genome of AAV is a linear, single-stranded DNA molecule that is less than about 5,000 nucleotides (nt) in length. Inverted terminal repeats (ITRs) flank the unique coding nucleotide sequences for the non- structural replication (Rep) proteins and the structural (VP) proteins. The VP proteins form the capsid. The assembly-activating protein (AAP) rapidly chaperones capsid assembly and prevents degradation of free capsid proteins (Grosse et al., J. Virol. 91(20): eOl 198-17 (2017). The terminal 145 nt are self-complementary and are organized so that an energetically stable intramolecular duplex forming a T-shaped hairpin may be formed. These hairpin structures function as an origin for viral DNA replication, serving as primers for the cellular DNA polymerase complex. The Rep genes encode the Rep proteins, Rep78, Rep68, Rep52, and Rep40. Rep78 and Rep68 are transcribed from the p5 promoter, and Rep 52 and Rep40 are transcribed from the pl9 promoter. The cap genes encode the VP proteins, VP1, VP2, and VP3. The cap genes are transcribed from the p40 promoter. The ITRs employed in the vectors of the present embodiment may correspond to the same serotype as the associated cap genes or may differ. In one embodiment, the ITRs employed herein correspond to an AAV2 serotype and the cap genes correspond to an AAV5 serotype.

[0153] The AAV VP proteins are known to determine the cellular tropicity of the AAV virion. The VP protein-encoding sequences are significantly less conserved than Rep proteins and genes among different AAV serotypes. The ability of Rep and ITR sequences to crosscomplement corresponding sequences of other serotypes allows for the production of pseudotyped AAV particles comprising the capsid proteins of a serotype (e.g., AAV1, 5 or 8) and the Rep and / or ITR sequences of another AAV serotype (e g., AAV2). Such pseudotyped rAAV particles are a part of the present disclosure.

[0154] The AAV particles described herein (and the encoding AAV vector genomes) may comprise any of the capsid proteins described in WO-2018 / 022608, WO-2019 / 222136, WO 2023 / 034989, WO 2023 / 034990, WO 2023 / 034980, WO 2023 / 034997, WO 2023 / 034994, or WO 2023 / 034996, incorporated by reference herein in its entirety for its disclosure of human and simian AAV capsids and their properties such as transduction efficiency, tissue tropism, glycan- binding, and resistance to neutralization by IVIG, including but not limited to any of the capsids in the sequence listing and variants thereof, e.g. with chimeric swapped variable regions and / or glycan binding sequences and / or GH loop.

[0155] In one embodiment, the AAV ITR sequences for use in the context of the present disclosure are derived from AAV1, AAV2, AAV4 and / or AAV6. Likewise, the Rep (e.g., Rep78 and Rep52) coding sequences are in one embodiment derived from AAV1, AAV2, AAV4 and / or AAV6. The sequences coding for the VP1, VP2, and VP3 capsid proteins for use in the contextof the present disclosure may however be taken from any serotype, such as from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or AAV12, or from simian AAVs, including any of the capsid proteins described in WO 2018 / 022608 or PCT / US 19 / 32097, or newly developed AAV-like particles obtained by e.g. capsid shuffling techniques and AAV capsid libraries, or any capsid at least 90% identical to any of SeQ ID NOs: 207-223.

[0156] For example, the amino acid sequences of various capsids are published. See, e.g., AAVRh.l / hu.14 / AAV9 AAS99264.1 (SEQ ID NO: 207)AAVRh.8 SEQ97 of U.S. Pat. Pub. 2013 / 0045186 (SEQ ID NO: 208) AAVRh.10 SEQ81 of U.S. Pat. Pub. 2013 / 0045186 (SEQ ID NO: 209) AAVRh.74 SEQ 1 of Int’l. Pat. Pub. WO 2013 / 123503(SEQ ID NO: 210) AAV1 AAB_95452.1 (SEQ ID NO: 211) AAV2 YP_680426.1 (SEQ ID NO: 212) AAV3 NP_043941.1 (SEQ ID NO: 213) AAV3B AAB95452.1 (SEQ ID NO: 214) AAV4 NP_044927.1 (SEQ ID NO: 215) AAV5 YP_068409.1 (SEQ ID NO: 216) AAV6 AAB95450.1 (SEQ ID NO: 217) AAV7 YP_077178.1 (SEQ ID NO: 218) AAV8 YP_077180.1 (SEQ ID NO: 219) AAV10 AAT46337.1 (SEQ ID NO: 220) AAV11 AAT46339 1 (SEQ ID NO: 221) AAV12 ABI16639. 1 (SEQ ID NO: 222)AAV13 ABZ10812.1 (SEQ ID NO: 223)

[0157] Modified “AAV” sequences also can be used in the context of the present disclosure, e.g. for the production of AAV gene therapy vectors. Such modified sequences e.g. sequences having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more nucleotide and / or amino acid sequence identity (e g., a sequence having about 75-99% nucleotide sequence identity) to an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9 ITR, Rep, or VP, can be used in place of wildtype AAV ITR, Rep, or VP sequences.

[0158] In some embodiments, a nucleic acid sequence encoding an AAV capsid protein is operably linked to expression control sequences for expression in a specific cell type, such as Sf9 or HEK cells. Techniques known to one skilled in the art for expressing foreign genes in insect host cells or mammalian host cells can be used to practice the embodiment. Methodology formolecular engineering and expression of polypeptides in insect cells is described, for example, in Summers and Smith (1986) A Manual of Methods for Baculovirus Vectors and Insect Culture Procedures, Texas Agricultural Experimental Station Bull. No. 7555, College Station, Tex.; Luckow (1991) In Prokop et al., Cloning and Expression of Heterologous Genes in Insect Cells with Baculovirus Vectors' Recombinant DNA Technology and Applications, 97-152; King, L. A. and R. D. Possee (1992) The baculovirus expression system, Chapman and Hall, United Kingdom; O'Reilly, D. R., L. K. Miller, V. A. Luckow (1992) Baculovirus Expression Vectors: A Laboratory Manual, New York; W.H. Freeman and Richardson, C. D. (1995) Baculovirus Expression Protocols, Methods in Molecular Biology, volume 39; U.S. Pat. No. 4,745,051; US- 2003148506; and WO-03 / 074714, all of which are incorporated by reference in their entireties. A particularly suitable promoter for transcription of a nucleotide sequence encoding an AAV capsid protein is e.g. the polyhedron promoter. However, other promoters that are active in insect cells are known in the art, e.g. the pl 0, p35 or IE-1 promoters and further promoters described in the above references are also contemplated.

[0159] Use of insect cells for expression of heterologous proteins is well documented, as are methods of introducing nucleic acids, such as vectors, e.g., insect-cell compatible vectors, into such cells and methods of maintaining such cells in culture. (See, e.g., METHODS IN MOLECULAR BIOLOGY, ed. Richard, Humana Press, N J (1995); O'Reilly et al., BACULOVIRUS EXPRESSION VECTORS, A LABORATORY MANUAL, Oxford Univ Press (1994); Samulski et al., J. Virol. 63: 3822-8 (1989); Kajigaya et al., Proc. Nat'l. Acad. Sci. USA, 88: 4646-50 (1991); Ruffing et al., J. Virol. 66: 6922-30 (1992); Kimbauer et al., Virol. 219: 37-44 (1996); Zhao et al., Virol. 272: 382-93 (2000); and U.S. Pat. No. 6,204,059). In some embodiments, the nucleic acid construct encoding AAV proteins (e.g. AAV rep or cap proteins) in insect cells is an insect cell-compatible vector. An “insect cell-compatible vector” or “vector” as used herein refers to a nucleic acid molecule capable of productive transformation or transfection of an insect or insect cell. Exemplary biological vectors include plasmids, linear nucleic acid molecules, and recombinant viruses. Any vector can be employed as long as it is insect cell-compatible The vector may integrate into the insect cells genome but the presence of the vector in the insect cell need not be permanent and transient episomal vectors are also included. The vectors can be introduced by any means known, for example by chemical treatment of the cells, electroporation, or infection. In some embodiments, the vector is a baculovirus, a viral vector, or a plasmid. In one embodiment, the vector is a baculovirus, i.e. the construct is a baculoviral vector. Baculoviral vectors and methods for their use are described in the above cited references on molecular engineering of insect cells.

[0160] Methods of producing recombinant adeno-associated virus (AAV) particles in insect or mammalian cells that comprises any of the AAV vector constructs provided herein comprise the steps of culturing a cell that has been transfected with any of the AAV vector constructs provided herein (in association with various AAV cap and rep genes) and recovering recombinant therapeutic AAV particles from the transfected cell or supernatant of the transfected cell. Production of AAV particles in mammalian cells, e.g. HEK293 cells, is preferred because it results in a population of AAV particles with better potency and more consistent effects on multiple parameters of contractile function.

[0161] The cells useful for recombinant AAV production provided herein include any cell type susceptible to baculovirus infection, including vertebrate or insect cells. For example, the insect cell line used can be from Spodoptera frugiperda such as Sf9, Sf21 , SF900+, drosophila cell lines, mosquito cell lines, e.g., Aedes albopictus derived cell lines, domestic silkworm cell lines, e.g. Bombyx mori cell lines, Trichoplusia ni cell lines such as High Five cells or Lepidoptera cell lines such as Ascalapha odorata cell lines; for example, High Five, Sf9, Se301, SeIZD2109, SeUCRl, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAml, BM-N, Ha2302, Hz2E5, and Ao38. In another embodiment, mammalian cells such as HEK293, HeLa, CHO, NSO, SP2 / 0, PER.C6, Vero, RD, BHK, HT 1080, A549, Cos-7, ARPE-19, and MRC-5 can be used.

[0162] In some embodiments, the insect or mammalian cell can be transfected with the helper plasmid or helper virus, the vector construct and the plasmid encoding the AAV cap genes; and the recombinant AAV virus can be collected at various time points after cotransfection. For example, the recombinant AAV virus can be collected at about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 96 hours, about 120 hours, or a time between any of these two time points after the co-transfection.

[0163] Recombinant AAV particles can also be produced using any conventional methods known in the art suitable for producing infectious recombinant AAV. In some instances, a recombinant AAV can be produced by using an insect or mammalian cell that stably expresses some of the necessary components for AAV particle production For example, a plasmid (or multiple plasmids) comprising AAV rep and cap genes, and a selectable marker, such as a neomycin resistance gene, can be integrated into the genome of the cell. The insect or mammalian cell can then be co-infected with a helper virus (e g., adenovirus or baculovirus providing the helper functions) and the viral vector construct comprising the 5' and 3' AAV ITR (and the nucleotide sequence encoding the heterologous protein, if desired). The advantages of this method are that the cells are selectable and are suitable for large-scale production of the recombinant AAV particle. As another non-limiting example, adenovirus or baculovirus ratherthan plasmids can be used to introduce rep and cap genes into packaging cells. As yet another non-limiting example, both the viral vector construct containing the 5' and 3' AAV ITRs and the rep-cap genes can be stably integrated into the DNA of producer cells, and the helper functions can be provided by a wild-type adenovirus to produce the recombinant AAV.

[0164] The manufacturing process for AAV particles may result in empty capsids, heavy capsids (e.g., intermediate capsids) and ultra-heavy capsids (e.g., full capsids). Ultra-heavy capsids contain the desired full-length or nearly full-length AAV vector genomes. Heavy capsids may contain truncated AAV vector genomes of intermediate length. Manufacturing steps may include steps that reduce the number of empty capsids, in order to enrich the population for rAAV particles comprising ultra-heavy capsids, or to enrich the population for rAAV particles comprising heavy capsids and ultra-heavy capsids, e.g. pooled together. Such steps are known in the art and include zonal ultracentrifugation or density -gradient ultracentrifugation.Formulations

[0165] In certain embodiments, the pharmaceutical formulations provided herein are liquid formulations that comprise rAAV particles, preferably rAAV9 particles, comprising any of the vector constructs disclosed herein, capable of delivering a therapeutic protein for treatment of cardiac disease. The concentration of rAAV virions in the formulation may vary. In certain embodiments, in any of the formulations herein, the concentration of rAAV particle in the formulation may range from 1E13 to about 4E14 vg / ml, for example, from at least about 6E13 vg / ml to about 4E14 vg / ml, or from about 1E13 vg / ml to about 1E14 vg / ml, or from about 1E13 vg / ml to about 5E13 vg / ml, or from about 5E13 vg / ml to about 1E14 vg / ml.

[0166] In specific embodiments of any of the formulations herein, the concentration of rAAV particle in the formulation may be approximately any one of the following vg / ml: about 1 E13, 1.1 E13, 1.2 E13, 1.3 E13, 1.4 E13, 1.5 E13, 1.6 E13, 1.7 E13, 1.8 E13, 1.9 E13, 2 E13, 2.1 E13, 2.2 E13, 2.3 E13, 2.4 E13, 2.5 E13, 2.6 E13, 2.7 E13, 2.8 E13, 2.9 E13, 3 E13, 3.1 E13, 3.2E13, 3.3 E13, 3.4 E13, 3.5 E13, 3.6 E13, 3.7 E13, 3.8 E13, 3.9 E13, 4 E13, 4 1 E13, 4.2 E13, 4.3E13, 4.4 E13, 4.5 E13, 4.6 E13, 4.7 E13, 4.8 E13, 4.9 E13, 5 E13, 5.1 E13, 5.2 E13, 5.3 E13, 5.4E13, 5.5 E13, 5.6 E13, 5.7 E13, 5.8 E13, 5.9 E13, 6 E13, 6.1 E13, 6.2 E13, 6.3 E13, 6.4 E13, 6.5E13, 6.6 E13, 6.7 E13, 6.8 E13, 6.9 E13, 7 E13, 7.1 E13, 7.2 E13, 7.3 E13, 7.4 E13, 7.5 E13, 7.6 E13, 7.7 E13, 7.8 E13, 7.9 E13, 8 E13, 8.1 E13, 8.2 E13, 8.3 E13, 8.4 E13, 8.5 E13, 8.6 E13, 8.7E13, 8.8 E13, 8.9 E13, 9 E13, 9.1 E13, 9.2 E13, 9.3 E13, 9.4 E13, 9.5 E13, 9.6 E13, 9.7 E13, 9.8E13, 9.9 E13, 1 E14, 1.1 E14, 1.2 E14, 1.3 E14, 1.4 E14, 1.5 E14, 1.6 E14, 1.7 E14, 1.8 E14, 1.9E14, 2 E14, 2.1 E14, 2.2 E14, 2.3 E14, 2.4 E14, 2.5 E14, 2.6 E14, 2.7 E14, 2.8 E14, 2.9 E14, 3E14, 3.1 E14, 3.2 E14, 3.3 E14, 3.4 E14, 3.5 E14, 3.6 E14, 3.7 E14, 3.8 E14, 3.9 E14, or 4 E14 vg / ml.

[0167] In other embodiments, the AAV particle pharmaceutical formulation provided herein comprises one or more sterile pharmaceutically acceptable excipients to provide the formulation with advantageous properties for storage and / or administration to subjects for the treatment of the genetic disorder. In certain embodiments, the pharmaceutical formulations provided herein are capable of being stored at less than about -60°C (minus 60 degrees centigrade), -40°C, or - 20°C for a period of at least 6 months, 1.5 years, or 2 years, with no appreciable change in stability. Preferably, the pharmaceutical formulations are stable for at least one year at freezer temperatures.

[0168] In addition, the pharmaceutical formulations provided herein are stable under suitable accelerated storage conditions. Examples of stressed conditions include at about 25°C and about 60% humidity for a time period of, e.g., 6, 9, 12, 18 and / or 24 months, or (for drug substances intended for storage in a freezer) at about -20°C for a time period of, e.g. 12 months. Examples of accelerated conditions include at about 2°C to about 8°C for a time period of, e.g., 6, 9, 12, 18 and / or 24 months, or (for drug substances intended for storage in a freezer) at about -20°C for a time period of, e.g. 12 months. See, e.g., FDA Guidance for Industry: Stability Testing of New Drug Substances and Products, Nov 2003.

[0169] In this regard, the term “stable” means that the rAAV particle present in the formulation essentially retains its physical stability, chemical stability and / or biological activity during storage. In certain embodiments, the recombinant AAV particle present in the pharmaceutical formulation retains at least about 80% of its vg / ml (or at least about 80% of its infectious rAAV particles) in a human patient during storage for a determined period of time at - 65°C, in other embodiments at least about 85%, 90%, 95%, 98% or 99% of its vg / ml, or alternatively infectious rAAV particles, in a human subject. Preferably, the rAAV particle retains at least about 98% of its vg / ml.

[0170] AAV9 capsids are less stable than AAV5 capsids, requiring additional formulation excipients to attain desirable shelf life. In particular, ultra-heavy AAV9 capsids show significant instability at 25°C or room temperature.

[0171] In certain aspects, the formulation of rAAV particles comprises one or more buffering agents. For example, citrate, phosphate, tri s(hydroxymethyl)aminom ethane (Tris), Tris base or other buffers are well known in the art. In some embodiments, 10 to 30 mM Tris buffer at pH about 7-8 may improve capsid stability and / or potency with reduced deamidation. Tris buffer is superior to other buffers, such as phosphate or citrate buffer, in improving or maintaining capsid stability and / or potency.

[0172] In some embodiments, the rAAV particle formulation provided herein may comprise one or more isotonicity agents, such as sodium chloride. Sodium chloride within certain concentration ranges may maintain capsid colloidal stability and solution clarity.

[0173] In some embodiments, the rAAV particle formulation provided herein may comprise a stabilizer, such as MgCh. Such stabilizers help maintain capsid integrity, and / or capsid titer and / or potency of heavy and / or ultra heavy capsid(s) after storage at freezing temperature or room temperature, preferably for at least a year. Such stabilizers preferably help maintain capsid integrity, and / or capsid titer and / or potency under stressed conditions such as high temperature or agitation. In some embodiments, such stabilizers also reduce the formation of aggregates, e.g. visible particulates or subvisible particulates (SVP). Studies showed the addition of MgCh is the most significant formulation factor to increase capsid stability, especially for unstable ultraheavy capsids.

[0174] In some embodiments, the concentration of MgCh is about 0.5 mM to about 1.5 mM, for example, about 0.5 mM, about 1.0 mM, or about 1.5mM. In some embodiments, the concentration of MgCh is about 0.5 mM to about 3 mM, or about 1 mM to about 3 mM, for example, about 0.5 mM, about 1.0 mM, about 1.5 mM, about 2.0 mM, about 2.5 mM or about 3.0mM. In some embodiments the concentration of MgCh is >1.5mM to about 3 mM. In some embodiments, the concentration of MgCh (for example, 1 mM MgCh) provides capsid stability and prevents or reduces the appearance of SVP that may appear in some 3 mM MgCh formulations under various types of stress.

[0175] Capsid integrity can be measured, for example, by size exclusion chromatography with multi-angle light scattering (SEC-MALS) and thermal-based capsid integrity (TBCI). SECMALS also analyzes capsid titer and aggregation. TBCI measures capsid thermal stability. Potency can be measured by testing in cardiomyocytes in 2D or 3D format according to Example 2 below.

[0176] Other buffering agents and isotonicity / stabilizer agents known in the art are suitable and may be routinely employed for use in the formulations provided herein.

[0177] In another embodiment, the rAAV particle formulations provided herein may comprise one or more bulking agents, including cryoprotective agents Exemplary bulking agents include without limitation mannitol, sucrose, dextran, lactose, trehalose, and povidone (PVP K24). Cryoprotective agents may maintain stability of the liquid formulation under freezing temperature conditions.

[0178] In yet another embodiment, the rAAV particle formulations provided herein may comprise one or more surfactants, which may be non-ionic surfactants. Exemplary surfactants include ionic surfactants, non-ionic surfactants, and combinations thereof. For example, thesurfactant can be, without limitation, TWEEN 80 (also known as polysorbate 80, or its chemical name polyoxyethylene sorbitan monooleate), sodium dodecyl sulfate, sodium stearate, ammonium lauryl sulfate, TRITON AG 98 (Rhone-Poulenc), poloxamer 407, poloxamer 188 and the like, and combinations thereof. Surfactants reduce adsorption of rAAV Cl -INH vector particles to contact surfaces and / or reduce precipitation, and thus reduce loss of viral particles.

[0179] In yet another aspect, the disclosure provides a pharmaceutical composition comprising rAAV particle at a concentration of at least IE 13 vg / ml, or at least 6E13 vg / ml to about 4E14 vg / ml, a buffering agent, an isotonicity agent, stabilizer agent, a cryopreservative agent and a surfactant which is stable during storage at about -60°C (minus sixty degrees centigrade) or less for at least about 1 year, 1.5 years, or 2 years. In some embodiments, the buffer is a Tris buffer. In some embodiments, the cryopreservative agent is a sugar, for example, trehalose or a suitable hydrate thereof. In some embodiments, the stabilizer is preferably MgCE, for example in the hexahydrate form. In some embodiments, the surfactant is a poloxamer, e.g., poloxamer 188, or alternatively a polysorbate at a concentration of less than 0.2% w / v, or less than 0.15% w / v, for example, about 0.1% w / v.

[0180] In some embodiments, the pharmaceutical composition is aqueous and comprises rAAV particle at a concentration of at least 6E13 vg / ml, Tris buffer at a concentration of about 10 to about 30 mM, Tris base at a concentration of about ImM to about 4mM, sodium chloride at a concentration of about 100 mM to about 150 mM, magnesium chloride at a concentration of about 0.5mM to about 3.0mM, trehalose at a concentration of about 50mM to about 90mM, and a poloxamer or polysorbate at a concentration of about 0.05% to about 0.15% w / v.

[0181] In some embodiments, the Tris is at a concentration of about 10 to about 50 mM, about 10 to about 30 mM, or about 15 to about 25 mM. In some embodiments, the Tris base is at a concentration of about 1.5mM to about 3.5mM, or about 2mM to about 3mM. In some embodiments, the sodium chloride is at a concentration of about 100 to about 140 mM, or about 110 to about 130 mM. In some embodiments, the magnesium chloride is at a concentration of about 0.75mM to about 1.25mM, or about 1 mM to about 1.5 mM, or about 0.5 mM to about 3mM, or about 1 mM to about 3 mM, or about 1.5 to about 3 mM, or greater than 1.5 mM to about 3 mM. In some embodiments, the magnesium chloride is at a concentration of > I.5mM. In some embodiments, the trehalose is at a concentration of about 60mM to about 80mM, or about 65mM to about 75mM In some embodiments, the poloxamer is poloxamer 188 at a concentration of about 0.05% to 0. 15% w / v, optionally about 0.1% w / v.

[0182] In some embodiments, the pharmaceutical composition comprises rAAV particle at a concentration of about 6E13 vg / ml, 17.6 mM Tris, 2.4mM Tris base, 120 mM sodium chloride,ImM magnesium chloride (hexahydrate), 74mM trehalose (dihydrate), and 0. 1% w / v poloxamer 188.

[0183] The rAAV particle formulations provided herein are stable and can be stored for extended periods of time without an unacceptable change in quality, potency, or purity. In one aspect, the formulation is stable at a temperature of about 5°C (e.g., 2°C to 8°C) for at least 1 month, for example, at least 1 month, at least 3 months, at least 6 months, at least 12 months, at least 18 months, at least 24 months, or more. In another embodiment, the formulation is stable at a temperature of less than or equal to about -20°C for at least 6 months, for example, at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or more. In another embodiment, the formulation is stable at a temperature of less than or equal to about - 40°C for at least 6 months, for example, at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or more. In another embodiment, the formulation is stable at a temperature of less than or equal to about -60°C for at least 6 months, for example, at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or more. For example, the formulation is stable at a temperature of less than or equal to about -20°C, - 40°C, or -60°C for 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months, 34 months, 35 months, 36 months, 37 months, 38 months, 39 months, 40 months, 41 months, 42 months, 43 months, 44 months, 45 months, 46 months, 47 months, 48 months, 49 months, 50 months, 51 months, 52 months, 53 months, 54 months, 55 months, 56 months, 57 months, 58 months, 59 months, 60 months, 61 months, 62 months, 63 months, 64 months, 65 months, 66 months, 67 months, 68 months, 69 months, 70 months, 71 months, 72 months, 73 months, 74 months, 75 months, 76 months, 77 months, 78 months, 79 months, 80 months, 81 months, 82 months, 83 months, 84 months, 85 months, 86 months, 87 months, 88 months, 89 months, 90 months, 91 months, 92 months, 93 months, 94 months, 95 months, 96 months, 97 months, 98 months, 99 months, 100 months, 101 months, 102 months, 103 months, 104 months, 105 months, 106 months, 107 months, 108 months, 109 months, 110 months, 111 months, 112 months, 113 months, 114 months, 115 months, 116 months, 117 months, 118 months, 119 months, or 120 months.

[0184] Preferably the pharmaceutical composition is a liquid aqueous solution and is for storage at freezing temperature. In different embodiments, the pharmaceutical composition is stored at a temperature of < -60 °C / -60 degrees Celsius (°C) or less, where the stability or potency of the pharmaceutical composition is at least substantially maintained at the storage temperature. In other embodiments, the pharmaceutical composition is stored at a temperature of < -20 °C orabout -20 °C or less without an unacceptable change in quality, potency, or purity. In different examples, the pharmaceutical composition is stored at a temperature of -20 °C, -21 °C, -22 °C, -23 °C, -24 °C, -25 °C, -26 °C, -27 C, -28 °C, -29 °C, -30 °C, -31 C, -32 °C, -33 °C, -34 °C, -35 °C, - 36 °C, -37 °C, -38 °C, -39 °C, -40 C, -41 °C, -42 °C, -43 °C, -44 C, -45 °C, -46 °C, -47 °C, -48 °C, - 49 °C, -50 °C, -51 °C, -52 °C, -53 C, -54 °C, -55 °C, -56 °C, -57 C, -58 °C, -59 °C, -60 °C, -61 °C, - 62 °C, -63 °C, -64 °C, -65 °C, -66 C, -67 °C, -68 °C, -69 °C, -70 C, -71 °C, -72 °C, -73 °C, -74 °C, - 75 °C, -76 °C, -77 °C, -78 °C, -79 C, -80 °C, -81 °C, -82 °C, -83 C, -84 °C, -85 °C, -86 °C, -87 °C, - 88 °C, -89 °C, -90 °C, -91 °C, -92 °C, -93 °C, -94 °C, -95 °C, -96 °C, -97 °C, -98 °C, -99 °C, or -100C without an unacceptable change in quality, potency, or purity. In any of these embodiments, the composition is for use in intravenous administration of rAAV particle to a patient with cardiac disease, e.g. HCM.Treatment methods

[0185] In any of the embodiments, the subject has heart disease, optionally hypertrophic cardiomyopathy (HCM). (HCM) is a genetic cardiac disease most commonly transmitted as an autosomal dominant trait, caused by mutations in genes encoding cardiac sarcomere (or sarcomere-related) proteins. HCM is characterized by intraventricular septum and left ventricular wall thickening, not explained by another cardiac, systemic, or metabolic disease. In some embodiments, prior to administration of rAAV particles as described herein, the subject has a clinical diagnosis of non-obstructive hypertrophic cardiomyopathy (nHCM) or obstructive hypertrophic cardiomyopathy (oHCM) consistent with current American College of Cardiology (ACC) / American Hearth Associate (AHA) classification systems for heart failure and / or European Society of Cardiology (ESC) guidelines. For example, the subject has a New York Heart Association (NYHA) functional classification of class II or class III heart failure.

[0186] In any of the embodiments, the subject may have a mutation in a MyBPC-3 gene encoding MYBPC3, optionally detected by PCR and / or genome sequencing and / or restriction fragment length polymorphism (RFLP) analysis.

[0187] Echocardiography can determine the presence and magnitude of obstruction to blood flow through the left ventricular outflow tract (LVOT). A pressure gradient of > 30 mmHg, either at rest or with provocation, is indicative of obstruction and patients are considered to have oHCM Those without obstruction are considered to have nHCM.

[0188] In some embodiments, the subject to be treated has a non-obstructive phenotype, for example, as determined by peak LVOT gradient <30 mmHg at rest and / or peak LVOT gradient < 50 mmHg with Valsalva maneuver.

[0189] In some embodiments, the subject has an ejection fraction of >50%. In some embodiments, the subject has elevated levels of NT -proBNP (>300 picograms (pg) / mL). In some embodiments, the subject has peak oxygen consumption (PVO2) of <80% or 90% predicted on cardiopulmonary exercise test (CPET) with respiratory exchange ratio (RER) >1.0 on screening CPET. In some embodiments, the subject has more than one of the foregoing characteristics.

[0190] In some embodiments, the subject is an adult, for example, age at least 18, or age 18 to 65. The subject may be a male or a female, e.g. a nonpregnant female. In some embodiments, the subject is ajuvenile, for example, age 12 to 18, or age 6 to 12, or age 6 to 18, or age 2 tol2, or age 0 to 6, or age 0 to 2, or age 2 to 6, or age at least 12.

[0191] In some embodiments, the subject has not received prior treatment with gene therapy or an investigational medicinal treatment concurrently or within 6 months prior to screening. In some embodiments, the subject does not have evidence of an active or chronic infection, or immunosuppressive disorder. In some embodiments, the subject does not have a contraindication to glucocorticosteroids. In some embodiments, the subject does not have a significant or uncontrolled chronic condition(s) defined as requiring regular medical attention and treatment including but not limited to acute or chronic liver disease, chronic obstructive pulmonary disease, uncontrolled diabetes, etc. In some embodiments, the subject does not have a history of clinically relevant arrhythmogenic syncope, sustained ventricular tachycardia, resuscitated sudden cardiac arrest, or appropriate ICD discharge within 6 months of screening. In some embodiments, the subject does not have paroxysmal atrial fibrillation at the time of screening. In some embodiments, the subject does not have persistent or permanent atrial fibrillation In some embodiments, the subject does not have significant valvular heart disease (e.g., moderate-severe valvular aortic stenosis, moderate- severe mitral regurgitation not due to systolic anterior motion of the mitral valve) or other heart disease including but not limited to structural heart disease (not related to nHCM), congenital heart disease, coronary heart disease, pericardial disease, acute myocarditis, infiltrative cardiomyopathy, obstructive hypertrophic cardiomyopathy, or history of pericarditis / myocarditis / pericardial effusion In some embodiments, the subject does not have one or more of the following: 1) detectable antibodies to AAV9 capsid (i.e. seropositivity); or (2) evidence of clinically abnormal laboratory values including: a) Systolic blood pressure > 150 mmHg (millimeters of mercury) or < 90 mmHg, b) Diastolic blood pressure > 100 mmHg or < 60 mmHg, c) Resting pulse rate < 40 bpm (beats per minute) or > 100 bpm, d) Resting respiratory rate < 9 bpm or > 20 bpm, e) Pulse oximetry < 94% on room air, f) Temperature > 100.4 degree Fahrenheit, g) Liver enzymes > 2 times the upper limit of normal, h) Abnormalbilirubin, i) eGFR < 45 ml / min (minutes) / 1.73m2(square meters) by CKD-EPI, j) Platelet count < 100,000, k) Hemoglobin < 10 g / dL (grams per deciliter), and / or 1) Hemoglobin A1C > 8.

[0192] In some embodiments, the subject does not have treatment related, Grade 3 or greater adverse events such as left ventricular systolic dysfunction, myocarditis, hepatic failure, immune system disorder, thrombotic microangiopathy, or infusion-related reaction.

[0193] Prior to infusion of the rAAV particles, the baseline status of the subject is evaluated by physical examination and clinical laboratory tests. In some embodiments, prior to administration of the rAAV particles, the subject is tested for antigenic and functional levels of MYBPC3. In some embodiments, prior to administration of the rAAV particles, the subject is tested for baseline AAV9 antibody detection. In some embodiments, prior to administration of the rAAV particles, the subject is tested for baseline echocardiogram and electrocardiogram, and optionally cardiac magnetic resonance imaging (MRI). In some embodiments, prior to administration of the rAAV particles, the subject is tested for baseline N-terminal pro hormone BNP (NT-proBNP) levels. In some embodiments, prior to administration of the rAAV particles, the subject is tested for high sensitivity Troponin I (hsTNI) levels. In some embodiments, prior to administration of the rAAV particles, the subject is tested for baseline exercise capacity assessed as measured by PVO2 via cardiopulmonary exercise testing (CPET).

[0194] Examples of assessments include the Kansas City Cardiomyopathy Questionnaire-23 (KCCQ-23), e.g. Total Symptom Score (TSS), physical limitations score and clinical summary score (CSS) used to assess symptoms and health related quality of life (HRQoL) impacts of subjects; EuroQol-5 dimension 5-level version (EQ-5D-5L) and EuroQol -visual analog scale (EQ-VAS) to assess health status; Clinical Global Impression of Severity and Change scales (CGI / C), and Patient Global Impression of Severity and Change scales (PGIS / C).

[0195] In the methods of the disclosure, the rAAV particle is administered intravenously in a single dose administration. In some embodiments, the vector construct or rAAV particle is administered by intravenous injection either as a single bolus or via infusion over a prolonged time period, which may be at least about 1, 5, 10, 15, 30, 45, 60, 75, 90, 120, 150, 180, 210 or 240 minutes, or more. In some embodiments, rAAV particle is administered by infusion at an infusion rate of about 20, 30, 40, 50, 60, 70, or 80 mL / hour (1.33 mL / min).

[0196] In some embodiments, the rAAV particle is administered at a dose ranging from about 1E13 to about 4E14 vector genomes per kilogram body weight of the subject (vg / kg), or about 1E13 to about 6E13 vg / kg, or about 5E13 to about 1E14, or about 8E13 to about 4E14, for example, a dose of about 2E13 vg / kg, or a dose of about 6E13 vg / kg, or a dose of about 1E14 vg / kg, or a dose of about 2E14 vg / kg.

[0197] In some embodiments, the doses may be approximately any one of the following: about 1 E13 vg / kg, about 1.1 E13 vg / kg, about 1.2 E13 vg / kg, about 1.3 E13 vg / kg, about 1.4 E13 vg / kg, about 1.5 E13 vg / kg, about 1.6 E13 vg / kg, about 1.7 E13 vg / kg, about 1.8 E13 vg / kg, about 1.9 E13 vg / kg, about 2 E13 vg / kg, about 2.1 E13 vg / kg, about 2.2 E13 vg / kg, about 2.3 E13 vg / kg, about 2.4 E13 vg / kg, about 2.5 E13 vg / kg, about 2.6 E13 vg / kg, about 2.7 E13 vg / kg, about 2.8 E13 vg / kg, about 2.9 E13 vg / kg, about 3 E13 vg / kg, about 3.1 E13 vg / kg, about 3.2 E13 vg / kg, about 3.3 E13 vg / kg, about 3.4 E13 vg / kg, about 3.5 E13 vg / kg, about 3.6 E13 vg / kg, about 3.7 El 3 vg / kg, about 3.8 El 3 vg / kg, about 3.9 E13 vg / kg, about 4 E13 vg / kg, about 4.1 E13 vg / kg, about 4.2 E13 vg / kg, about 4.3 E13 vg / kg, about 4.4 E13 vg / kg, about 4.5 E13 vg / kg, about 4.6 E13 vg / kg, about 4.7 E13 vg / kg, about 4.8 E13 vg / kg, about 4.9 E13 vg / kg, about 5 E13 vg / kg, about 5.1 E13 vg / kg, about 5.2 E13 vg / kg, about 5.3 E13 vg / kg, about 5.4 E13 vg / kg, about 5.5 E13 vg / kg, about 5.6 E13 vg / kg, about 5.7 E13 vg / kg, about 5.8 E13 vg / kg, about 5.9 E13 vg / kg, about 6 E13 vg / kg, about 6.1 E13 vg / kg, about 6.2 E13 vg / kg, about 6.3 E13 vg / kg, about 6.4 E13 vg / kg, about 6.5 E13 vg / kg, about 6.6 E13 vg / kg, about 6.7 E13 vg / kg, about 6.8 E13 vg / kg, about 6.9 E13 vg / kg, about 7 E13 vg / kg, about 7.1 E13 vg / kg, about 7.2 E13 vg / kg, about 7.3 E13 vg / kg, about 7.4 E13 vg / kg, about 7.5 E13 vg / kg, about 7.6 E13 vg / kg, about 7.7 E13 vg / kg, about 7.8 E13 vg / kg, about 7.9 E13 vg / kg, about 8 E13 vg / kg, about 8.1 E13 vg / kg, about 8.2 E13 vg / kg, about 8.3 E13 vg / kg, about 8.4 E13 vg / kg, about 8.5 E13 vg / kg, about 8.6 E13 vg / kg, about 8.7 E13 vg / kg, about 8.8 E13 vg / kg, about 8.9 E13 vg / kg, about 9 E13 vg / kg, about 9.1 E13 vg / kg, about 9.2 E13 vg / kg, about 9.3 E13 vg / kg, about 9.4 E13 vg / kg, about 9.5 E13 vg / kg, about 9.6 E13 vg / kg, about 9.7 E13 vg / kg, about 9.8 E13 vg / kg, about 9.9 E13 vg / kg, about 1 E14 vg / kg, about 1.1 E14 vg / kg, about 1.2 E14 vg / kg, about 1.3 E14 vg / kg, about 1.4 E14 vg / kg, about 1.5 E14 vg / kg, about 1.6 E14 vg / kg, about 1.7 E14 vg / kg, about 1.8 E14 vg / kg, about 1.9 E14 vg / kg, about 2 E14 vg / kg, about 2.1 E14 vg / kg, about 2.2 E14 vg / kg, about 2.3 E14 vg / kg, about 2.4 E14 vg / kg, about 2.5 E14 vg / kg, about 2.6 E14 vg / kg, about 2.7 E14 vg / kg, about 2.8 E14 vg / kg, about 2.9 E14 vg / kg, about 3 E14 vg / kg, about 3.1 E14 vg / kg, about 3.2 E14 vg / kg, about 3.3 E14 vg / kg, about 3.4 E14 vg / kg, about 3.5 E14 vg / kg, about 3.6 E14 vg / kg, about 3.7 E14 vg / kg, about 3.8 E14 vg / kg, about 3.9 E14 vg / kg, or about 4 E14 vg / kg.

[0198] In some embodiments, the rAAV particle is administered at a dose of about 6E13 vg / kg, 9E 13 vg / kg or 1.2E14 vg / kg.

[0199] In some embodiments, subjects are concurrently administered BACTRIM® (sulfamethoxazole and trimethoprim) as a precaution against pneumocystis jiroveci pneumonia (PJP).

[0200] After infusion of the rAAV particles, the methods may further comprise the step of monitoring various parameters of the subject’s health, e.g. measuring the parameters on a weeklybasis. Measuring can alternatively occur every 1, 2, 3, 4, 5 or 6 days or every week or every two weeks or every three weeks or every month. Parameters may be monitored through Week 16, 20, 24, 28, 32, 36, 40, 44, 48 or longer. The methods may include measuring NT-proBNP and troponin I levels of the subject as well as echocardiogram and electrocardiogram assessments.

[0201] The methods of the disclosure may result in clinically significant improvement in cardiac structure / morphology and / or function. The methods of the disclosure may result in improvement in cardiac diastolic function as measured by, for example, decreased E / e’ and / or decreased left atrial volume index. The methods of the disclosure may result in improvement in cardiac systolic function as measured by, for example, improving or normalizing ejection fraction and / or reduced impairment of longitudinal strain. For example, the improving or normalizing of the ejection fraction may include increasing or decreasing the ejection fraction relative to a baseline identified prior to the administration of rAAV vectors. The methods of the disclosure may result in improvement in cardiac structure as evidenced by, for example, reduced left ventricular (LV) wall thickness and / or reduced LV mass. The methods of the disclosure may result in improvement in diastolic and / or systolic function, and / or improved cardiac structure.

[0202] The methods of the disclosure may also result in clinically significant improvement in exercise capacity as measured by, for example, improved PVO2 during cardiopulmonary exercise testing (CPET)

[0203] The methods of the disclosure may result in improved cardiac function, e.g. moving from class III to class II heart failure, or moving from class II to class I heart failure, or moving from class III to class I heart failure according to the New York Heart Association (NYHA) functional classification system.

[0204] The methods of the disclosure may further result in improved health-related quality of life (HRQoL), e g as measured by, for example, the Kansas City Cardiomyopathy Questionnaire-23 (KCCQ-23) Total Symptom Score (TSS), Physical Limitations Score, and Clinical Summary Score (CSS) and / or the Clinician Global Impression of Severity and Change scales (CGIS / C), and Patient Global Impression of Severity and Change scales (PGIS / C), EuroQol-5 dimension 5-level version (EQ-5D-5L) and / or EuroQol -visual analog scale (EQ- VAS). In some embodiments, the dose may be effective to improve one or more of these measures of health-related quality of relief by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% over a period of at least about six months. In some embodiments, the dose may be effective to improve one or more of these measures of health-rel ted quality of relief by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or100% over a period of at least about one year. In some embodiments, the improvement is maintained for at least about one year, or 2, 3, 4 or 5 years.

[0205] In some embodiments, the dose may be effective to reduce levels of, or prevent increased levels of, the cardiac biomarker NT-proBNP. In some embodiments, the dose may be effective to prevent increased levels of cardiac biomarker troponin I, or reduce the incidence of increased levels of troponin I. For example, the dose is effective to reduce NT-proBNP levels by at least about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 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%. In these examples, the reduction in NT- proBNP levels is a percentage reduction from an initial threshold (e.g., > 300 pg / mL).Additionally in these examples, the reduction in NT-proBNP levels is seen at 16 weeks, 20 weeks, 24 weeks, 28 weeks, 32 weeks, 36 weeks, 40 weeks, 44 weeks, 48 weeks, 52 weeks, 56 weeks, or longer post administration of the dose.

[0206] In some embodiments, the dose may be effective to increase peak oxygen consumption (PVO2) by at least about 0.1 mL / kg / minutes, 0.2 mL / kg / minutes, 0.3 mL / kg / minutes, 0.4 mL / kg / minutes, 0.5 mL / kg / minutes, 0.6 mL / kg / minutes, 0.7 mL / kg / minutes, 0.8 mL / kg / minutes, 0.9 mL / kg / minutes, 1 mL / kg / minutes, 1.1 mL / kg / minutes, 1.2 mL / kg / minutes, 1.3 mL / kg / minutes, 1.4 mL / kg / minutes, 1.5 mL / kg / minutes, 1.6 mL / kg / minutes, 1.7 mL / kg / minutes, 1.8 mL / kg / minutes, 1.9 mL / kg / minutes, 2 mL / kg / minutes, 2.1 mL / kg / minutes, 2.2 mL / kg / minutes, 2.3 mL / kg / minutes, 2.4 mL / kg / minutes, 2.5 mL / kg / minutes, 2.6 mL / kg / minutes, 2.7 mL / kg / minutes, 2.8 mL / kg / minutes, 2.9 mL / kg / minutes, or 3 mL / kg / minutes. Additionally in these examples, the increase in peak oxygen consumption is seen at 16 weeks, 20 weeks, 24 weeks, 28 weeks, 32 weeks, 36 weeks, 40 weeks, 44 weeks, 48 weeks, 52 weeks, 56 weeks, or longer post administration of the dose.

[0207] The methods of the disclosure provide administration of rAAV particles in a manner that is safe, e g , no or low incidence of clinically significant treatment-emergent serious adverse events; no or low incidence of clinically significant changes in standard clinical laboratory values; no or low incidence of complement activation or hypersensitivity; no or low incidence of elevation to Grade 2 or higher or Grade 3 or higher of markers of hepatotoxicity such as AST and / or ALT (or if changes occur, most are transient or resolve after treatment with systemic immunosuppressant). Another alternative example of methods of the disclosure provide administration of rAAV particles in a manner that is safe include no or low incidence of abnormalities in coagulation markers. The methods may also provide a reduced immuneresponse against the AAV capsid. The methods may also provide improved blood biodistribution, or reduced vector shedding in urine, stool, semen, or saliva.

[0208] In an aspect of the disclosure, hepatotoxicity, e.g. as detected through transient hepatic transaminase enzyme elevations, may be reduced or avoided by prophylactic immunosuppression treatment or therapeutic immunosuppression treatment. According to these aspects, in addition to administration of a therapeutically effective amount of AAV virus, the subject may be treated either prophylactically, therapeutically, or both with a glucocorticosteroid or other immunosuppressant to prevent and / or treat any hepatotoxicity associated with administration of the AAV virus.

[0209] In a related aspect of the disclosure, hepatic inflammation and / or cardiac inflammation may be reduced or avoided by prophylactic or therapeutic immunosuppression treatment. According to this aspect, in addition to administration of a therapeutically effective amount of AAV virus, the subject may be treated either prophylactically, therapeutically, or both with a glucocorticosteroid or other immunosuppressant to prevent and / or treat any hepatic inflammation and / or cardiac inflammation associated with administration of the AAV virus.

[0210] In another aspect of the disclosure, complement activation, e g. refractory complement activation, and / or thrombotic microangiopathy may be reduced or avoided by prophylactic or therapeutic administration of complement pathway inhibitor or antagonist. According to this aspect, in addition to administration of a therapeutically effective amount of AAV virus, the subject may be treated either prophylactically, therapeutically, or both with a complement pathway inhibitor or antagonist to prevent and / or treat any complement activation, e g. refractory complement activation, and / or thrombotic microangiopathy associated with administration of the AAV virus.Prophylactic immunosuppression treatment

[0211] The methods of disclosure may further comprise administering to the subject a prophylactically effective amount of an immunosuppressant (e.g. glucocorticosteroid or complement pathway inhibitor or antagonist). In some embodiments, the subject is prophylactically administered a glucocorticosteroid and sirolimus to prevent or reduce hepatotoxicity (e g. as detected by ALT elevation above the upper limit of normal (ULN), or at least 2 times baseline ALT) and / or hepatic inflammation and / or myocarditis and / or cardiac inflammation. In some embodiments, the subject is prophylactically administered a complement pathway inhibitor or antagonist to prevent or reduce complement activation, e.g. refractory complement activation, and / or thrombotic microangiopathy. In some embodiments, the prophylactically effective amount of immunosuppressant (e.g. glucocorticosteroids, complementpathway inhibitor or antagonist) is administered concurrent with administration of the rAAV particles of the invention. “Concurrent” as used herein means the same day, for example, or within one day or one week of (prior to or after) administration of the rAAV particles. In other embodiments, the administration of the prophylactically effective amount of immunosuppressant (e g. glucocorticosteroids, complement pathway inhibitor or antagonist) begins after administration of the rAAV particles, e.g. starting at 1, 2, 3, 4, 5, 6, 7, or 8 weeks after administration of the rAAV particles, but prior to detection of hepatotoxicity. In further embodiments, prophylactically effective amount of immunosuppressant (e.g. sirolimus) is administered beginning two times a day for two-days prior to administration and continuing until about 8 weeks after administration of rAAV particles, e.g. administered two times a day for two days at -5 day, -4 day, -3 days, -2 day, -1 day, week 0, week 1, week 2, week 3, week 4, week 5, week 6, week 7 and week 8.

[0212] The glucocorticosteroids or other immunosuppressant such as a complement pathway inhibitor or antagonist may be administered for a prophylactic treatment time period, e.g., for a time period of at least about 1 to 4 weeks (1, 2, 3, 4 weeks), and is preferably followed by tapering period during which tapering amounts of the corticosteroids or other immunosuppressant are administered, e.g., for a time period of about 2 to 4 weeks, or about 2, 3, or 4 weeks. For example, the prophylactically effective amount of the glucocorticosteroids is a dose equivalent to a prednisone dose of about 40 mg / day, administered for a time period of at least about 1 to 8 weeks (1, 2, 3, 4, 5, 6, 7, 8), followed by tapering amounts of the glucocorticosteroids for a time period of about 2, 3 or 4 weeks. In some embodiments, the prophylactically effective amount of the glucocorticosteroids is administered for a time period of about 4 weeks, followed by tapering amounts of the glucocorticosteroids for a time period of about 4 weeks. For example, a glucocorticosteroid is administered at a dose equivalent to a prednisone dose of about 40 mg / day concurrent with said administration for a time period of about 4 weeks, followed by tapering amounts of the glucocorticosteroid for a time period of about 4 weeks.

[0213] The sirolimus may be administered for a prophylactic treatment time period, e.g. for a time period of at least about -3 days to 8 weeks (loading dose at -3 days to day -1, followed by dosing during weeks 1, 2, 3, 4, 5, 6, 7, 8) and is preferably administered concurrently with glucocorticosteroids beginning at week 1. In some embodiments, the prophylactically effective amount of the glucocorticosteroids and sirolimus are administered concurrently for a time period of about 4 weeks followed by a taper of the glucocorticosteroid. For example, the sirolimus is loaded at a dose of 6 mg / day for two days prior to rAAV administration, followed by a dose of sirolimus 2 mg / day every day, while the glucocorticosteroid is administered on the same day asrAAV administration, followed by a dose equivalent to a prednisone dose of about 40 mg / day every day (during weeks 1, 2, 3 and 4). Following this, sirolimus continues to be administered at a dose of 2 mg / day during tapering of the dose of glucocorticosteroid for a time period of about 4 weeks (during weeks 5, 6, 7, 8 post-rAAV administration).

[0214] ALT and AST levels are monitored weekly. If there is ALT elevation to greater than upper limit of normal (ULN) or greater than 2x baseline ALT value, during the first 4 weeks, adjustments to glucocorticosteroid dosing are based on clinical judgment, and liver enzymes may be monitored more frequently.

[0215] Prophylactic immunosuppressant therapy, for example, may include the administration of a complement pathway inhibitor or antagonist. Examples of complement pathway inhibitors or antagonists include complement protein inhibitors or antagonists (e.g. inhibitors or antagonists of C3, C4, C5, etc.), or MASP2 inhibitor (e.g., OMS721 antibody) and including any of pegcetacoplan (APL-2, EMPAVELI), eculizumab (SOLIRIS), avacopan (CCX168), vilobelimab (IFX-1), AMY-101, CINRYZE / BERINERT / HAEGARDA (Cl INH), ravulizumab (ALXN1210), coversin, cemdisiran (ALN-CC5), tesidolumab (LFG-316), iptacopan (LNP023), danicopan (ACH-4471), RUCONEST (recombinant Cl esterase inhibitor), or ENJAYMO (sutimlimab-jome, a Cis inhibitor). In preferred embodiments, the prophylactic complement pathway inhibitor or antagonist is C3 inhibitor (e g., pegcetacoplan), Cl esterase inhibitor, recombinant Cl esterase inhibitor, or Cis inhibitor (e.g. sutimlimab).

[0216] In some embodiments, subjects are administered prophylactic immunosuppressants as described below. In further embodiments, prophylactic antimicrobials are administered concurrently with glucocorticosteroid / sirolimus immunosuppressant administration.Therapeutic / Reactive immunosuppression treatment

[0217] Administration of an AAV particle of the present disclosure may, in some cases, result in an observable degree of hepatotoxicity and / or hepatic inflammation and / or cardiac inflammation and / or myocarditis and / or complement activation, e.g. refractory complement activation and / or thrombotic microangiopathy. Hepatotoxicity or hepatic inflammation may be measured by a variety of well-known and routinely used techniques for example, measuring concentrations of certain liver-associated enzyme(s) (e.g., alanine transaminase, ALT) in the bloodstream of a subject both prior to AAV administration (i.e., baseline) and after AAV administration. An observable increase in ALT concentration after AAV administration (as compared to prior to administration) is indicative of drug-induced hepatotoxicity. The methods of the disclosure may comprise administering to the subject a therapeutically effective amount ofa glucocorticosteroid or other systemic immunosuppressant to treat hepatotoxicity, upon detection of hepatotoxicity.

[0218] Myocarditis and / or cardiac inflammation may be detected by, for example, an abnormally increased level of troponin I, high sensitivity troponin I (hsTNI), and / or N-terminal pro hormone BNP (NT-proBNP). Other signs include ejection fraction (EF) less than about 50%, arrhythmias, myopericarditis, and / or regional wall motion abnormalities.

[0219] Complement activation may be detected by, for example, abnormally decreased levels of C3 or C4 in plasma (indicating its consumption by complement activation), or abnormally increased levels of one or more complement split products, e.g., C3a, C4a, Bb and / or sC5b-9. Refractory complement activation is characterized by complement activation that has not been inhibited by prophylactic immunosuppression or complement activation in the context of ongoing thrombotic angiopathy.

[0220] Thrombotic microangiopathy is characterized by anemia due to red blood cell destruction within the microvasculature, accompanied by thrombocytopenia due to platelet activation and consumption. Thrombotic microangiopathy may be detected by, for example, decreased platelet counts, decreased platelet volume, decreased red blood cell (RBC) counts, abnormal RBC morphology, hemolysis, and / or proteinuria. Clinical signs of thrombotic microangiopathy include complement activation, severe thrombocytopenia, microangiopathic hemolysis with schistocytes, transaminase elevations, and / or acute kidney injury (AKI) progressing to kidney failure.

[0221] Reactive immunosuppressant therapy, for example, administration of glucocorticosteroids at a dose equivalent to a prednisone dose of at least about 0.5 mg / kg / day or at (e.g., 60 mg / day) or at least about 40 mg / day, and optionally further administering tacrolimus or Mycophenolate mofetil, may be initiated after the prophylactic steroids / sirolimus regimen completion, or in response to mild ALT elevations that meet pre-specified criteria, or based on clinical judgment. For example, 60 mg / day of glucocorticosteroids can be administered if there is a breakthrough in liver function test despite 40 mg / day glucocorticosteroid administration. In some embodiments, it is initiated if ALT is greater than the ULN or greater than 2x baseline in two consecutive assessments within 72 hours, or 3x ULN in two consecutive assessments within 48 hours. In some embodiments, the reactive immunosuppressive regimen has a total duration of 8 weeks with 5 weeks of a dose equivalent to a prednisone dose of > 0.5 mg / kg / day glucocorticosteroid dosing, followed by a 3-week dose taper if ALT is both less than or equal to ULN and less than or equal to 2x baseline value. Liver enzymes are monitored weekly over 4 weeks in the period following discontinuation of reactive immunosuppression therapy, or morefrequently if ALT values are above the ULN. In some embodiments, tacrolimus is administered for break-through myocarditis.

[0222] Therapeutic / reactive immunosuppressant therapy, for example, may include the administration of a complement pathway inhibitor or antagonist. Examples of complement pathway inhibitors or antagonists include complement protein inhibitors or antagonists (e.g. inhibitors or antagonists of C3, C4, C5, etc ), or MASP2 inhibitor (e.g., OMS721 antibody), and including any of pegcetacoplan (APL-2, EMPAVELI), eculizumab (SOLIRIS), avacopan (CCX168), vilobelimab (IFX-1), AMY-101, CINRYZE / BERINERT / HAEGARDA (C1INH), ravulizumab (ALXN1210), coversin, cemdisiran (ALN-CC5), tesidolumab (LFG-316), iptacopan (LNP023), danicopan (ACH-4471) RUCONEST (recombinant Cl esterase inhibitor), or ENJAYMO (sutimlimab-jome, a Cis inhibitor). The administration of the reactive immunosuppressant can continue for, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks or 1, 2 or 3 months. In some embodiments, eculizumab is administered for refractory complement activation in the context of thrombotic microangiography.

[0223] The methods of disclosure may further comprise the step of (a) determining a baseline level of a marker of hepatotoxicity, hepatic inflammation, cardiac inflammation, complement activation, and / or thrombotic microangiography in the blood of the subject prior to said administration, optionally about one month prior to said administration, and (b) determining a post-administration level of said marker for hepatotoxicity, hepatic inflammation, cardiac inflammation, complement activation, and / or thrombotic microangiography in the blood of the subject after said administration, optionally every week, or every 1, 2, 3, 4, 5, or 6 days.

[0224] Such methods may further comprise the step of: (c) upon detection of hepatotoxicity, hepatic inflammation, cardiac inflammation, complement activation, and / or thrombotic microangiography by biochemical or clinical signs, administering to the subject a therapeutically effective amount of an immunosuppressant(s) (e g. glucocorticosteroid and optionally tacrolimus or Mycophenolate mofetil and optionally or additionally complement pathway inhibitor or antagonist) for a therapeutic treatment time period, e.g., at least about 5 to about 8 weeks (e.g., 5, 6, 7 or 8 weeks), and is preferably followed by a tapering time period during which tapering amounts of the immunosuppressant(s) (e.g. glucocorticosteroid and optionally tacrolimus or Mycophenolate mofetil and optionally or additionally complement pathway inhibitor or antagonist) are administered for a time period of about 2 to 4 weeks (e.g. 3 weeks). For example, the step (c) comprises, upon detection of hepatotoxicity by (i) a post-administration level of said marker of hepatotoxicity greater than the upper limit of normal (ULN), or (ii) a postadministration level of said marker of hepatotoxicity greater than or equal to twice the baseline level of said marker of hepatotoxicity, administering to the subject a therapeutically effectiveamount of a glucocorticosteroid for a time period of at least about 5 to about 8 weeks or longer (e g., 5, 6, 7 or 8 weeks or longer), optionally together with tacrolimus or Mycophenolate mofetil, followed by tapering amounts of the immunosuppressant for a time period of about 2, 3 or 4 weeks. In any of such embodiments, the marker of hepatotoxicity is ALT and / or AST, preferably ALT. In some embodiments, upon said detection, a prednisone equivalent is administered at a prednisone-equivalent dose of 40 mg / day for a time period of about 5 weeks, followed by tapering amounts of the prednisone equivalent for a time period of about 3 weeks.

[0225] “Prophylactic” immunosuppressant treatment refers to the administration of a glucocorticosteroid, combination steroid / sirolimus and / or other immunosuppressant such as complement pathway inhibitor or antagonist to prevent or reduce the adverse event, for example, hepatotoxicity (e g. as detected by an increase in measured ALT levels in the subject), hepatic inflammation, cardiac inflammation, myocarditis, complement activation, including refractory complement activation, and / or thrombotic microangiography. “Therapeutic” glucocorticosteroid, immunosuppressant treatment refers to the administration of immunosuppressant(s) such as a glucocorticosteroid, combination steroid / sirolimus and / or other immunosuppressant such as complement pathway inhibitor or antagonist, to treat the adverse event, for example, reduce hepatotoxicity, hepatic inflammation, cardiac inflammation, myocarditis, complement activation, including refractory complement activation, and / or thrombotic microangiography caused by administration of an AAV virus, for example, to reduce an elevated ALT concentration in the bloodstream of the subject caused by administration of an AAV virus.

[0226] In certain embodiments, prophylactic or therapeutic glucocorticosteroid treatment may comprise administration of a prednisone-equivalent dose of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more mg / day, e.g. a prednisone-equivalent dose of between about 10 mg / day and about 60 mg / day of the glucocorticosteroid to the subject. In certain embodiments, prophylactic or therapeutic glucocorticosteroid treatment of a subject may occur over a continuous period of at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 weeks, or more, followed by a period of administering tapering amounts. Corticosteroid that find use in the methods described herein include any known or routinely-employed glucocorticosteroid including, for example, dexamethasone, prednisone, prednisolone, fludrocortisone, hydrocortisone, budesonide and the like, at the equivalent doses for the same time periods.

[0227] Other systemic immunosuppressants that may be administered in prophylactically effective or therapeutically effective doses to prevent or reduce hepatotoxicity include (1) calcineurin inhibitors, e.g. tacrolimus or cyclosporine, (2) antiproliferative agents or IMDH inhibitors, e.g. mycophenolate, leflunomide or azathioprine, (3) mTOR inhibitors, e.g., sirolimus or everolimus. (4) janus kinase inhibitors, e.g. tofacitinib, or (5) immunosuppressant antibodies.EXAMPLES

[0228] The following examples are set forth to assist in understanding the disclosure and should not, of course, be construed as specifically limiting the embodiments described and claimed herein. Such variations of the embodiments, including the substitution of all equivalents now known or later developed, which would be within the purview of those skilled in the art, and changes in formulation or minor changes in experimental design, are to be considered to fall within the scope of the embodiments incorporated herein.EXAMPLE 1: STABILITY EVALUATION OF AAV PARTICLES

[0229] rAAV particles having an AAV9 capsid were tested for long-term stability in various formulations.

[0230] Various formulations of heavy and ultra-heavy AAV9 capsids containing a vector construct described herein were tested for capsid integrity, aggregation and potency. The formulations were subjected to various stressed conditions, including elevated temperature, agitation, and freeze-thaw cycles. In general, AAV9 capsids are less stable than AAV5 capsids, and the ultra-heavy AAV9 capsids were less stable than the heavy AAV9 capsids. Formulations were tested comparing Tris buffer to other buffers, such as citrate buffer. A thermal-based capsid integrity (TBCI) assay as described in PCT Publication No WO2021 / 062164 was performed under accelerated conditions. Briefly, the TBCI assay includes the steps of combining AAV particles with a fluorescent dye that fluoresces when bound to nucleic acid in samples, exposing the samples to different conditions such as accelerated conditions or stress conditions, and evaluating the integrity of the capsids under the different conditions by measuring fluorescence. Fluorescence emissions increase as the dye binds to nucleic acid that escapes degraded capsids such that increased fluorescence emissions exemplify decreased capsid integrity. Results indicated the stability of AAV9 particles in Tris buffer were substantial greater than AAV9 particles in Citrate buffer when assessed using the thermal-based capsid integrity (TBCI) assay.

[0231] The TBCI assay also was used to monitor stability of formulations comprising heavy capsids or ultra-heavy capsids at room temperature 25°C, comprising 20mM Tris, pH 7.4, 74mM Trehalose, 120mM NaCl, and 0.1% Pl 88. Both the heavy and the ultra-heavy capsids exhibited reduced capsid integrity over 2 weeks. The ultra-heavy capsids were more unstable than the heavy capsids, exhibiting degradation of nearly 2 logs. Formulations were also tested with and without MgCh stabilizer. The addition of MgCh (for example, 3 mM MgCh) completelymitigated this instability, maintaining thermal stability and capsid integrity to greater than 95%, or greater than 98% (Figures 1A and IB).

[0232] Ultra-heavy AAV9 capsid formulations were tested for capsid titer and aggregation by SEC-MALS after storage at 30°C for 4 days. Results showed the ultra-heavy variant exhibited dramatic aggregation and main peak area loss when temperature stressed. However, the addition of MgCh (for example, 3 mM MgCh) prevented this aggregation for the ultra-heavy variant at elevated temperatures. The integrity of stressed ultra-heavy capsids with and without MgCh was also analyzed by negative stain TEM (nsTEM) as a visual confirmation of SEC and TBCI results. Both samples were held at 30°C for 1 day prior to grid preparation. The ultraheavy capsids formulated without MgCh had -60% internally stained capsids compared to -0.5% capsids formulated with MgCh.

[0233] Formulations comprising pooled heavy and ultra-heavy capsids in formulations comprising various concentrations of stabilizer, 0.5 mM, 1.75 mM and 3mM MgCh were subjected to various stressors including: 2 weeks at 25°C, end-over-end stress for 48 hours and freeze-thaw stress xlO. Overall, concentrations of 0.5-3.0 mM MgCh in formulation was shown to maintain capsid integrity over time across these stressors.

[0234] Maintenance of subvisible particulate (SVP) was analyzed for formulations comprising ImM and 3 mM MgCh. Particle concentration 2-10 pm (p / mL) for formulations at 5°C and 25°C was determined at T=0, T=1 week and T=2 weeks. No increase in SVP was observed over time in the presence of MgCh. However, SVP randomly appear in some 3mM MgCh formulations under various types of stress. Thus, results showed that ImM MgCh was able to provide capsid stability and also mitigate SVP appearance.

[0235] Testing of formulations with and without MgCh for protein expression and function generally as described above showed that MgCh had no significant impact on protein expression levels in vitro in cardiomyocytes or in vivo in a MYBPC3 knockout mouse model.

[0236] The results showed the addition of MgCh is the most significant formulation factor to increase capsid stability, especially for the unstable ultra-heavy variant. Concentrations of 0.5- 3mM MgCh provided similar stabilization effect

[0237] A formulation of 17.6 mM Tris hydrochloride, 2.4 mM Tris base, 120 mM NaCl, ImM MgCh hexahydrate, 74 mM Trehalose dihydrate, 0.1% w / v Poloxamer 188 at pH 7.4 provided the best long-term stability.EXAMPLE 2: ADMINISTRATION OF AAV PARTICLES TO MICE

[0238] An in vivo study was conducted to evaluate administration of AAV9-hMYPBC3 particles comprising an AAV9 capsid and an AAV vector genome comprising (a) a hTNNT2promoter, (b) a wild type or codon optimized cMyBP-C gene, (c) a polyadenylation signal, and optionally (d) a globin intron (for example, any of SEQ ID NO: 3-41). Data demonstrated that administration of 2E14 vg / kg of AAV9-MYBPC3 via a single IV dose resulted in an increased and sustained production of functional MYPBC3 expression to levels expected to be therapeutic in human subjects with HCM.

[0239] The Rag2 mice strain harbored a pan deletion of the entire protein coding region of the recombination activating gene 2 (Rag2) locus and exhibited arrested B- and T-cell differentiation. A single IV dose of 2E14 of AAV9-hMYBPC3 vector genomes per kg body weight (vg / kg) was administered to 8-week-old Rag2~ / ~ mice. Heart homogenates were assessed for total and human-specific cMyBP-C protein levels. Results indicated measurable levels of transduction in whole heart homogenates at 8 weeks post-dose, as assessed by digital droplet polymerase chain reaction (ddPCR) analysis of vector genome and transcript (Figures 2A and 2B) as well as liquid chromatography-mass spectrometry (LC / MS) evaluation of protein levels (Figures 2C and 2D). Measurement of total protein levels, as well as human specific protein levels revealed that human cMyBP-C accounts for 15% of the total protein present in the AAV9- hMYBPC3 transduced Rag2' / ' mouse heart. Measurement of total levels of protein in mouse heart indicated that transduction with AAV9-hMYBPC3 vectors does not increase the total amount of protein within the heart, due to conservation of stoichiometry between sarcomeric proteins. Incorporation of the human protein within the sarcomere is likely dependent on the rate of protein turnover within the cells. These data indicated expression and incorporation of the human protein into a mouse heart with the full complement of murine protein is achievable.

[0240] Human cMyBP-C protein expression was confirmed in cardiomyocytes by immunohistochemistry. Immunohistochemical assessment of heart tissue was conducted and showed that on average, 65% of cardiomyocytes across longitudinal heart tissue sections including left and right ventricular walls, interventricular septum and right atrial wall were positive for human cMyBP-C. The total amount of human protein produced with AAV9- MYBPC3 in a mouse that has the full complement of endogenous murine cMyBP-C is on average 15% of total levels and total levels of cMyBP-C (murine + human protein) did not change with vector administration.

[0241] This data confirms AAV9-hMYPBC3 vector can achieve widespread distribution throughout the mouse heart and is able to transduce and express protein that is incorporated into the sarcomere even in the background of a full complement of endogenous cMyBP-C in WT mice. This finding also supports the use of C57BL / 6 WT mice, which have the full complement of murine cMyBP-C (see Figures 2A-2D). Data is presented as the mean + / - standard deviation (SD).EXAMPLE 3: ADMINISTRATION IN A MOUSE MODEL OF HCM

[0242] MYBPCS^' mice were created via ere recombination during in vitro fertilization allowing for the generation of heterozygous knock-out embryos. The homozygous progeny had a deletion of the mouse MYBPC3 exons 3-5 leading to a frameshift. The splice between exons 2 and 6 was out of frame and resulted in a premature stop codon in exon 6. It is expected that the resulting mRNA was unstable and was a target for nonsense mediated decay mechanisms. Thus, the MYBPC3 / ' mice are null for functional cMyBP-C protein. A natural history study was conducted to evaluate phenotype development within a 20-week period. The mice were characterized by echocardiography using a Vevo 3100 preclinical imaging system capable of 4 imaging formats including B-mode, M-mode, doppler imaging, and three-dimensional (3D) imaging. Left ventricular (LV) mass, LV wall measurements, and parameters of systolic and diastolic function were quantified. The mice separated into two cohorts. Analysis of cohort 1 began at 4 weeks of age, but a significant phenotype was already present. Therefore, analysis of cohort 2 began at day 4 of the postnatal period to determine the age of phenotype onset. The MYBPC3~ / ~ mice develop hypertrophy as evidenced by increased LV mass (Figure 3 A). By 2 weeks of age, MYBPC3- / - mice exhibited increased left ventricular anterior wall diameter and left ventricular posterior wall diameter compared to heterozygous and WT mice (Figure 3B). Both increases in interventricular septal diameter and left ventricular posterior wall diameter are clinical manifestations of HCM in adult patients, indicating that this mouse model replicates the characteristics of HCM. MYBPC3'1' mice exhibited impaired ventricular relaxation as evidenced by decreased measures of diastolic function such as mitral inflow velocity E-wave (MV E), representing the early, passive fdling of the left ventricle, which was a clinically relevant endpoint readily assessed nMYBPC3'1' mice (Figure 3C). (Jiang J, Burgon PG, Wakimoto H, Onoue K et al. Cardiac myosin binding protein C regulates postnatal myocyte cytokinesis. Proc Natl Acad Sci USA. 2015; 112(29):9046-9051.) MYBPC+ / ~ mice heterozygous hearts are indistinguishable from WT in terms of cMyBP-C protein expression.

[0243] MYBPC3'1' mice developed early and severe clinically relevant disease manifestations of human HCM. In particular, left ventricle hypertrophy, increased left ventricle wall measurements and impaired diastolic function were phenotypes that mimic the human HCM condition which make the MYBPC3'1' mouse an appropriate model to study the disease modifying effects of the vector constructs described herein.

[0244] In Figures 3A, 3B, and 3C, **** indicates p-value <0.0001 according to one-way analysis of variance (ANOVA) with a Tukey’s multiple comparison. Data is presented as the mean ± SD from MYBPC3 ' *.EXAMPLE 4:

[0245] A short-term 8-week pharmacology study was performed to assess transduction, expression and efficacy. 2-week-old MYBPC3 ~ mice were dosed with 2E14vg / kg of AAV9- MYBPC3 For this study, the AAV9-MYBPC3 included human MYBPC3 driven by a hTNNT2 promoter encapsulated in AAV9 (AAV9-MYBPC3). Vector DNA and RNA transcripts were measured in whole heart homogenates by ddPCR, and cMyBP-C levels were also measured by LC / MS. The DNA and RNA levels 8 weeks post-dose were approximately 10- and 5-fold higher, respectively, than what was measured in the adult Rag2~ / ~ mice that received the same 2E14 vg / kg dose (Figures 4A and 4B). Both human specific protein as well as total protein (mouse and human) were measured in order to make the comparison between the WT and the MYBPC31' mice. Result show AAV9-MYBPC3 at the 2E14vg / kg dose restores cMyBP-C levels in the MYBPC3'1' mouse to WT levels (Figures 4C and 4D) Protein levels were measured by LC / MS but cannot be compared directly to protein levels in the previous Rag2~ / ~ studies because different peptide and protein standards were used for the quantitation. Cardiac hypertrophy, as evidenced by heart to body weight ratios, in the MYBPC3'1' mice was markedly reduced and nearly normalized to WT levels (Figure 4E). In Figure 4E, **** indicates value <0.0001 according to one-way ANOVA with a Tukey’s multiple comparison. Data is presented as the mean + / - SD or the mean + / - standard error of the mean (SEM). Data is presented as the mean + / - SD or the mean + / - standard error of the mean (SEM).

[0246] The improved transduction and expression levels in the MYBPC3 / ' mouse compared to the Rag2~f~ mouse (see Figures 4A-4E) is likely due to the higher vector genome transduction per cell, but also due to the lack of any endogenous cMyBP-C, which can limit the transgene expression in a wildtype setting.

[0247] An initial pilot study was performed to assess the in vivo transduction efficiency of cardiomyocytes after AAV9-MYBPC3 administration. Specifically, an 8-week pharmacology study was performed to assess transduction, expression and impact of a 1.2E14 vg / kg dose of AAV9-MYPBC3 on hypertrophy. 2-week-old MYBPCS^' mice (N = 10 per group) were administered a vehicle control or 1.2E14 vg / kg dose of AAV9-MYBPC3 and tissue was collected 8 weeks post dose to assess MYBPC3 vector genome copy number in heart tissue homogenate (n = 6-7) and cMyBP-C protein expression by immunofluorescence staining (n = 3). Vector DNA and RNA transcripts were measured in whole heart homogenate by ddPCR, and cMyBP-C levels were also measured by LC / MS. The percentage of cardiomyocytes expressing cMyBP-C protein was determined by immunohistochemistry. There was an average of 4.46 vector genome copies per diploid genome in the mouse heart (Figure 5A) and increasedexpression of MYBPC3 transcripts (Figure 5B). AAV9-MYBPC3 administration also induced widespread expression of human cMyBP-C protein in cardiomyocytes throughout the mouse myocardium (Figure 5C). To this end, cMyBP-C protein correctly incorporated into the sarcomere. Heart tissue sections were also stained for human cMyBP-C by immunohistochemistry. Whole heart tissues sections from AAV9-MYPBC3 treated mice were analyzed by immunohistochemistry and showed on average 94% of cardiomyocytes across the ventricular myocardium were positive for human cMyBP-C staining (Figure 5D). These results illustrate widespread transduction and expression of the vector. Heart weight to body weight ratio was measured and shown to be significantly reduced at 8 weeks post-dose (Figure 5E). Thus, AAV9-MYBPC3 administration demonstrated excellent distribution and expression in cardiomyocytes within the mouse myocardium and a statistically significant reduction in hypertrophy.

[0248] In Figure 5E, * indicates p-value <0.05 according to one-way ANOVA with a Tukey’s multiple comparison. Data is presented as the mean + / - SD or mean + / - SEM.EXAMPLE 5: DOSAGE

[0249] A long-term study evaluated the effects of AAV9-MYBPC3 at different dose ranges. Two vectors, human MYBPC3 driven by a hTNNT2 promoter encapsulated in AAV9 (AAV9- MYBPC3) and an analogous vector encoding murine cMyBP-C protein driven by a hTNNT2 promoter encapsulated in AAV9 (AAV9-mMYBPC3) were analyzed for the pharmacodynamic effect and pharmacologic activity in the C57BL / 6J- FC C3 / _mouse model of HCM. 2-week- old MYBPC3 ~ male mice were administered lE14vg / kg AAV9-MYBPC3 and also doses of 6E13, 1E14 and 2E14vg / kg of a AAV9-mMYBPC3 vector. Additionally, a mid-dose 1E14 vg / kg dose of AAV9-MYBPC3 was also included as a comparator since both the 6E13vg / kg and 2E14vg / kg dose of this construct showed sustained efficacy in earlier studies The rAAV particles were in a formulation of 17.6 mM Tris hydrochloride, 2.4 mM Tris base, 120 mM NaCl, ImM MgCh hexahydrate, 74 mM Trehalose dihydrate, 0.1% w / v Poloxamer 188 at pH 7.4. The results of this study indicates species specificity of the construct and assess the impact on cardiovascular function and hypertrophy over time. Preliminary echocardiography data collected up to 26 weeks post dose shows that beginning as early as 4 weeks post dose, hypertrophy was reduced in MYBPC3' ' mice treated with all doses of the AAV9-mMYBPC3 construct as well as the 1E14 vg / kg dose of AAV9-MYBPC3 resulting in an early and sustained reduction in hypertrophy (left ventricular mass and wall dimensions) in MYBPC3 '' mice that persisted throughout the study duration of 26 weeks. Effects were seen as early as 4 weeks post dose with the 2E14vg / kg dose of AAV9-mMYBPC3 and the 1E14 vg / kg dose of AAV9-MYBPC3 At 4 weeks post dose, the left ventricular mass to body weight ratio was reduced compared to vehicle treated MYBPC3- / - mice after administration of the lE14vg / kg dose of AAV9-MYBPC3 and the 2E14 vg / kg dose of the AAV9-mMYBPC3 vector. All vector administrations resulted in reduced left ventricular mass to body weight ratios compared to vehicle treated mice 26 weeks post administration (Figure 6A). Reductions in left ventricular posterior and anterior wall dimensions during diastole were also achieved with all vector administration as compared to vehical treated mice 26 weeks post administration (Figure 6B). A trend towards normalization of cardiac output and stroke volume (SV) was also observed at all doses 26 weeks post administration (Figure 6C and 6D). An improvement in ejection fraction (EF) and fractional shortening (FS) was observed at all doses 26 weeks post administration. A trend towards MV E and E / A ratio normalization was observed at all dose s26 weeks post administration, although there was a high degree of variability within the measurements obtained (see Figure 6E). This data suggests an overall improvement in ventricular relaxation in these treated MYBPC3'' mice. In Figures 6A, 6B, 6C, 6D, and 6E, **** indicates p-value <0.0001, *** indicates p-value <0.001, ** indicates p-value <0.01, and * indicates p-value <0.05 according to one-way ANOVA with a Tukey’s multiple comparison. Data is presented as the mean + / - SEM.

[0250] The results of this 26-week study illustrate both an early and sustained reduction in hypertrophy after all doses of the AAV9-mMYBPC3 construct, as well as with the lE14vg / kg dose of AAV9-MYBPC3. Additionally, improvements in systolic function were achieved with high doses of the AAV9-mMYBPC3 construct. These results demonstrate the efficacy and safety of these constructs in this mouse model and point to know structural and functional differences between human and murine protein (Shaffer 2009).

[0251] A long-term 42-week study was conducted to assess the longitudinal impact of AAV9-MYBPC3 on cardiovascular function and left ventricular hypertrophy. 2-week-old MYBPC3'' male mice were administered a 6E13 vg / kg or 2E14vg / kg dose of AAV9-MYPBC3. A single bolus injection of vehicle control, 6E13 vg / kg of AAV9-MYPBC3, or 2E14 vg / kg of AAV9-MYPBC3 was administered to 2-week-old MYBPC3'' mice (N = 10-13 per group); a cohort of 10 wildtype mice were also evaluated. The rAAV particles were in a formulation of 17.6 mM Tris hydrochloride, 2.4 mM Tris base, 120 mM NaCl, ImM MgCh hexahydrate, 74 mM Trehalose dihydrate, 0. 1% w / v Pol oxamer 188 at pH 7.4. Tissues were collected at 0-, 4-, 8- , 12-, 20-, 28-, 36-, and 42-weeks post dose. Cardiovascular function and left ventricular hypertrophy were assessed by echocardiography. Indices of left ventricular hypertrophy such as left ventricular mass / body weight, left ventricular anterior wall dimensions and left ventricular posterior wall dimensions, showed early and sustained reductions with treatment. Beginning asearly as 4 weeks post dose, left ventricular hypertrophy in MYBPC3 ' mice treated with both low and high dose AAV9-MYBPC3 was reduced as evidenced by left ventricular mass to body weight ratio. Several deaths in vehicle treated group (3 / 10) between 36-44 weeks of age contributed to the reduction the statistical significance of the measurement at the 42-week timepoint. Data reported at 0-, 4-, 8-, 12-, 20-, 28-, and 36-weeks post administration is shown in Figures 7A-7C. Data is presented as the mean + / - SD.

[0252] Data reported at 36-weeks post administration is shown in Figures 8A-8D. In Figures 8A, 8B, 8C, and 8D, **** indicates p-value <0.0001, *** indicates p-value <0.001, ** indicates p-value <0.01, and * indicates p-value <0.05 according to one-way ANOVA with a Tukey’s multiple comparison. Data is presented as the mean + / - SD. For both doses, AAV9-MYPBC3 administration induced early and sustained reductions in LV mass and anterior wall dimensions as compared to vehicle treated MYBPC3'- mice. Specifically, early and sustained reduction in interventricular septum and posterior wall dimensions were achieved beginning 4 weeks post dose and sustained throughout the study duration (42 weeks post-dose). Diastolic function was assessed by 4-chamber doppler imaging and included measurements of passive ventricular filling (mitral velocity E-wave - MV E). A total of 7 echocardiography assessments were made throughout the study, and at 42 weeks post dose, mice were sacrificed to quantify cMyBP-C protein in the myocardium. MYBPC3'1' mice show impairment in MV E as early as 2 weeks of age. At 4 weeks post dose, both low and high dose MYBPC3'1' mice show a significant improvement in MV E, which is sustained throughout the duration of the study. Both doses improved diastolic function as quantified by changes in MV E measurements. These data also indicate an overall improvement in ventricular relaxation as early as 4 weeks post-dose. Severity of the model and the significantly impaired contractile function led to several deaths both in the vehicle (30%; 3 / 10) and treated groups (23%; 6 / 26). Histopathology revealed left atrial thrombus as the cause of morbidity in all hearts evaluated and showed no evidence of immune-mediated inflammation. No macroscopic liver lesions were observed in any mouse on study.Immunohistochemistry for cMyBP-C in high dose euthanized mouse hearts shows that mice were successfully transduced and are expressing human cMyBP-C in the majority of cells in the myocardium.

[0253] The results of this long-term study illustrated both an early and sustained reduction in hypertrophy after a 6E13 and 2E14 vg / kg dose of AAV9-MYBPC3 as well as improvement in diastolic function. Thus, a single administration of AAV9-MYBPC3 in 2 -week-old MYBPC3 / ~ mice led to sustained reductions in hypertrophy and LV wall thickness, as well as improved diastolic function.EXAMPLE 6:

[0254] A peak protein expression study was performed in WT mice to determine the GLP mouse toxicity study duration. The toxicology of AAV9-MYPBC3 vector was analyzed in adult C57BL / 6 male mice to determine the timing of peak expression of the transgene and proper study duration in which to assess toxicity. 8-week-old male C57BL / 6 mice were administered AAV9-MYPBC3 vector. Protein in heart homogenate were assessed at 4-, 8-, 12-, and 16-weeks post dose. Protein in heart homogenate will also be assessed at 1 month, 2 months, 3 months and 4 months post dose and liver samples were be archived for future analyses. As shown in Figure 9, administration of 2E14 vg / kg of AAV9-MYPBC3 via a single intravenous dose to 8-week-old male WT mice resulted in near peak expression of the human protein by 8-weeks post administration with a trend toward slight increase by 12-weeks post administration. In Figure 9, **** indicates p-value <0.0001, *** indicates p-value <0.001, and ** indicates p-value <0.01 according to one-way ANOVA analysis with a Tukey’s multiple comparison. Data is presented as the mean + / - SD.

[0255] No adverse hepatic toxicity or DRG histopathological findings were observed up to 4E14 vg / kg 3-months post injection.EXAMPLE 7: DOSE RANGE

[0256] A dose ranging study was conducted in 2-week-old MYBPC3~f~ mice to determine the minimum efficacious dose of AAV9-MYBPC3 and to test AAV9-MYBPC3 vector produced using the final vector production process. The rAAV particles were in a formulation of 17.6 mM Tris hydrochloride, 2.4 mM Tris base, 120 mM NaCl, ImM MgCh hexahydrate, 74 mM Trehalose dihydrate, 0.1% w / v Poloxamer 188 at pH 7.4. Group 1 included 10 WT mice (control) and groups 2-7 each included 10 MYBPC3 ' mice to be analyzed. Group 2 were the MYBPC^ mouse control administered vehicle. Groups 3, 4, 5, 6 and 7 were administered a dose of 6E12 vg / kg, 2E13 vg / kg, 6E13 vg / kg, 1.2E14 vg / kg and 1.2E14 vg / kg of AAV9-MYPBC3, respectively. Analysis by echocardiography was performed at baseline, 13-weeks post dose and at termination of the study. Heart weight to body weight, cardiac transduction and expression - DNA, RNA, protein levels and protein distribution was also assessed to determine the minimally efficacious dose. Mice were then euthanized at 13- or 36-weeks post-dose for tissue collection. DNA, RNA, and protein were extracted from the heart homogenates. Cardiac transduction and distribution were assessed by DNA, RNA, protein, and immunohistochemical analysis to correlate efficacy to tissue levels and distribution. Vector DNA, RNA, and expressed protein levels showed a straightforward dose-dependent increase that depended on the dose of AAV9- MYBPC3 administered (Figures 10A-10D). Human cMyBP-C protein expression was confirmedin cardiomyocytes by immunohistochemistry. A trend was also noted in that an increase in the percentage of cells expressing cMyBP-C with increasing dose (Figure 10E). The administered AAV9-MYBPC3 transduced 61-94% of the cardiomyocytes depending on the dose. Staining intensity from human cMyBP-C positive cardiomyocytes also trended higher with increasing dose. In Figures 10A, 10B, 10C, 10D, and 10E, **** indicates p-value <0.0001, *** indicates p-value <0.001, ** indicates p-value <0.01, and * indicates p-value <0.05 according to one-way ANOVA with a Tukey’s multiple comparison. Data is presented as the mean + / - SD.

[0257] It was also noted that the 6E13vg / kg dose of AAV9-MYPBC3 was the lowest dose that showed a reduction in hypertrophy and improvement in diastolic function at 13 weeks post dose (Figures 11 A-l 1C). Thus, AAV9-MYBPC3 induced dose-responsive transduction and established durable expression of MYBPC3 RNA and cMyBP-C protein throughout the MYBPC3''' mouse hearts and restored cMyBP-C protein expression and function in a dosedependent manner. In Figures 1A, 11B, and 11C, **** indicates p-value <0.0001, *** indicates p-value <0.001, ** indicates p-value <0.01, and * indicates p-value <0.05 according to one-way ANOVA with a Tukey’s multiple comparison. Data is presented as the mean + / - SD.EXAMPLE 8: NON-GLP PILOT NHP STUDY

[0258] Cynomolgus monkeys were used to demonstrate translatability of transduction and expression of AAV9-MYBPC3 as well as safety and tolerability. The transgene expression in the non-human primate (NHP) tissues, tolerability, and biodistribution was evaluated for AAV9- hMYPBC3 particles comprising an AAV9 capsid and an AAV vector genome comprising (a) a hTNNT2 promoter, (b) a wild type or codon optimized cMyBP-C gene, (c) a polyadenylation signal, and optionally (d) a globin intron (for example, any of SEQ ID NO: 3-41) at various time periods of 31, 62, 91 and 153 days after administration. The NHPs were euthanized at 31 days for tissue collection. DNA and RNA were extracted from the collected tissues.

[0259] Preliminary results show no overt differences in clinical observations, body weight, macroscopic observations, or ECG were noted among the 3 vector groups. No changes were attributed to AAV9-hMYBPC3 administration during the first 62 days. A transient, minimally to mildly increased ALT in animals on Days 8 and / or 15 (males only) was observed. No DRG histopathological findings were observed up to 1.2E14 vg / kg dose in NHP.

[0260] AAV9-MYPBC3 vector DNA transduction was detected in necropsied tissues using ddPCR and measured separately in five striated muscle tissue sections: left atrium, right atrium, left ventricle, right ventricle, and septum. Vector DNA was also measured in heart vascular tissues, aorta, and pulmonary artery (Figure 12). Data is presented as the mean + / - SEM. Vector transduction was evenly distributed across heart tissue sections, with no significant difference invector distribution measured at 31- or 62-days post-dose. There was also no significant difference in vector DNA levels in any tissue section between 31-day and 62-day cohorts. Accordingly, a single AAV9-MYPBC3 dose efficiently delivered the MYBPC3 cDNA to the heart of the NHPs and was detectable 31 days post-dose.

[0261] AAV9-MYPBC3 vector-derived hMYBPC3 mRNA was detected in necropsied tissues using RT-ddPCR and measured separately in five cardiac muscle tissue sections, left atrium, right atrium, left ventricle, right ventricle, and septum. Vector-derived transcripts were also measured in heart vascular tissues, aorta, and pulmonary artery (Figure 13). Data is presented as the mean + / - SEM. Significantly less RNA transcription was measured in aorta than left ventricle in the 31-day cohort, but no other significant difference in vector transcription was measured between any other tissue sections in the same cohort. There was no significant difference in vector transcript levels measured in any tissue section between 31-day and 62-day cohorts. Accordingly after administration of a single AAV9-MYPBC3 dose, MYBPC3 RNA transcripts were detected in multiple heart tissues at 31 days post-dose showing that AAV9- MYPBC3 demonstrated efficient transduction and expression of MYBPC3 RNA in heart tissue from the NHPs.EXAMPLE 9:

[0262] Several different vector designs were produced using two independent manufacturing methods: the Spodoptera frugiperda insect cell system (Sf) and the mammalian (i.e., human) embryonic kidney 293 (HEK293) cell system. Different AAV9-MYBPC3 vectors or a vehicle control produced by each manufacturing system were screened for their ability to produce cMyBP-C protein in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). The human iPSC-CMs carried a bi-allelic truncating MYBPC3 mutation that results in complete absence of cMyBP-C protein. The screened AAV9-MYBPC3 vectors included vector Al (SEQ ID NO: 3-5), vector A2 (SEQ ID NO: 6-8), vector A3 (SEQ ID NO: 9-11), vector A4 (SEQ ID NO: 12-14), vector A5 (SEQ ID NO: 15-17), and vector A6 (SEQ ID NO: 18-20). The different AAV9-MYBPC3 vectors produced with the HEK293 cell manufacturing system yielded the strongest protein expression across multiple vector designs in iPSC-CMs as compared to the AAV vectors produced with the Sf cell manufacturing system (Figure 14). Also, engineered heart tissue derived from the human iPSC-CMs transduced with the AAV vectors produced with the HEK293 cell manufacturing system exhibited improved contractile function. Accordingly, the HEK293 cell manufacturing system produced AAV vectors exhibiting superior in vitro and in vivo expression and functionality compared with other candidate vectors and manufacturing systems.EXAMPLE 10: ADMINISTRATION OF AAV PARTICLES TO HUMAN SUBJECTS Non-obstructive Hypertrophic Cardiomyopathy

[0263] A clinical study is performed on subjects with non-obstructive Hypertrophic Cardiomyopathy (nHCM) caused by MYBPC3 gene mutations to demonstrate safety, tolerability, and potential efficacy of a single intravenous infusion of AAV9-MYPBC3. The study will provide evidence of effectiveness in improving functional outcomes and / or morbidities in patients with HCM.

[0264] Human subjects are administered rAAV particles comprising an AAV9 type capsid and a recombinant AAV vector construct described herein (e.g., any of SEQ ID NOs: 3-41), at one of three dose levels (6E13 vg / kg, 9E13 vg / kg or 1.2E14 vg / kg) to assess the efficacy, safety and tolerability of the rAAV particles at the indicated doses. Subjects are also administered prophylactic immune modulation through administration of steroids and sirolimus as described below. Optionally, antimicrobial prophylaxis is employed, e.g. sulfamethoxazole / trimethoprim prophylaxis is administered to protect against pneumocystis jiroveci pneumonia (PJP). Subjects are assessed for TB and vaccination.

[0265] Subjects with nHCM and no neutralizing titers or predetermined titers of anti-AAV9 antibodies (Part A) are administered the rAAV particles at the desired dose, in a single intravenous infusion at an infusion rate of 20 mL / hour (0.33 mL / min) with a maximum of 80 mL / hr (1.33 mL / min), and are followed for 5 years to evaluate durability of the response. AAV9- seropositive nHCM participants, who meet all other inclusion and exclusion criteria, will be enrolled into a concurrent comparator study arm (Part B)

[0266] One objective of the study is to evaluate the safety of AAV9-MYBPC3. A number of safety endpoints have been identified, most notably number of participants with targeted adverse, cardiac events and targeted adverse, immunological events (transaminitis, thrombotic microangiopathy [TMA]), based primarily on prior safety findings associated with other gene therapies using AAV vectors.

[0267] Several efficacy endpoints are measured to fully characterize the effect of AAV9- MYPBC3 on the heart and subject. Changes in cardiac systolic and diastolic function are assessed by echocardiograms and serum biomarkers of cardiac stress. Changes in cardiac morphology are assessed by echocardiographic measures of left ventricular mass and wall thickness. Changes in exercise capacity will be assessed by peak oxygen consumption (PVO2) obtained during cardiopulmonary exercise testing (CPET) as well as other maximal and submaximal exercise parameters. Changes in patient symptoms and functional status are assessed by KCCQ-23 and NYHA, respectively. Optionally, myocardial cell transduction andRNA and protein expression are assessed from myocardial tissue obtained by endomyocardial biopsy, compared to pre-dose levels.

[0268] One efficacy endpoint is change in PVO2 as measured by a CPET. An increase in PVO2 of 1 ml / kg / min has previously been shown to represent a clinically relevant threshold of improvement in patients with HCM and has been associated with a reduced risk of all-cause mortality or cardiac transplantation (Coats et al., Circ Heart Fail. 8(6): 1022-31 (2015); Masri et al., J Am Heart Assoc. 7:e007944 (2018)). Further, in previous studies in patients with HCM, an improvement in PVO2 has been associated with improvement in functional classification (Olivotto et al., Lancet, 396(10253):759-69 (2020)).Inclusion and exclusion criteria

[0269] The clinical study inclusion criteria may include one or more of the following: (1) adult subject 18-65 years of age, (2) clinical diagnosis of non-obstructive hypertrophic cardiomyopathy (nHCM) consistent with current ACC / AHA and / or ESC guidelines; (3) pathogenic, heterozygous truncating mutations in the MYBPC3 gene with genetic testing confirmed at a CLIA-approved laboratory, (4) non-obstructive phenotype defined as peak left ventricular outflow tract (LVOT) gradient <30 mmHg at rest and / or peak LVOT gradient < 50 mmHg with Valsalva maneuver, (5) Class II or Class III heart failure according to the New York Heart Association (NYHA) functional classification system, on optimal therapy for HCM; (4) ejection fraction of > 50%, (5) elevated levels of NT -proBNP (>300 pg / mL); (6) currently using a beta-blocker and / or calcium channel blocker at optimal dose(s) with stable medication doses for at least 3 months prior to screening; optionally with body mass index (BMI) < 35 kg / m2. Other inclusion criteria may include peak oxygen consumption (PVO2) of < 80% predicted on cardiopulmonary exercise test (CPET) with respiratory exchange ratio (RER) >1.0 on screening CPET. Patients may also have an implantable cardioverter defibrillator (ICD).

[0270] The clinical study exclusion criteria may include one or more of the following: (1) evidence of an active infection or chronic infection, or immunosuppressive disorder; (2) contraindication to corticosteroids, (3) significant or uncontrolled chronic condition(s) defined as requiring regular medical attention and treatment including but not limited to acute or chronic liver disease, chronic obstructive pulmonary disease, uncontrolled diabetes, etc., (4) a history of clinically relevant arrhythmogenic syncope, sustained ventricular tachycardia, resuscitated sudden cardiac arrest, or appropriate ICD discharge within 6 months of screening, (5) Paroxysmal atrial fibrillation at the time of screening, (6) Persistent or permanent atrial fibrillation, (7) Significant valvular heart disease (moderate- severe valvular aortic stenosis, moderate-severe mitral regurgitation not due to systolic anterior motion of the mitral valve) orother heart disease including but not limited to structural heart disease (not related to nHCM), congenital heart disease, coronary heart disease, pericardial disease, acute myocarditis, infiltrative cardiomyopathy, obstructive hypertrophic cardiomyopathy, and history of pericarditis / myocarditis / pericardial effusion, and (8) prior treatment with gene therapy or an investigational medicinal treatment concurrently or within 6 months prior to screening. Other exclusion criteria include history of obstructive HCM (resolved with surgery), history of alcohol septal ablation for oHCM, currently listed for heart transplantation or plan to be listed, having one or more risk factors for sudden cardiac death, history or known genetic predisposition to thrombotic microangiopathy (TMA), hemolytic-uremic syndrome or complement deficiency, history of thromboembolic (TW) event or known thrombophilia, significant liver fibrosis (> F 3), having an active or chronic infection or any immunosuppressive disorder, any contraindication to use of corticosteroids (including diagnosis of glaucoma, untreated osteoporosis or diabetes), history of clinically significant malignant disease in last 10 years, weight or BMI exclusion, borderline AAV9 seropositivity, controlled Atrial Fibrillation, a heart rate exclusion value, a blood pressure value, a pulse oximetry value, an eGFR value, or myocardial infarction. Myectomies are not identified as an exclusion criterion and may be allowed.

[0271] One or more of the following diagnostic clinical study exclusion criteria apply: (1) detectable antibodies to AAV9 capsid (i.e. seropositivity); or (2) evidence of clinically abnormal laboratory values including: a) Systolic blood pressure > 150 mmHg or < 90 mmHg, b) Diastolic blood pressure > 100 mmHg or < 60 mmHg, c) Resting pulse rate < 40 bpm or > 100 bpm, d) Resting respiratory rate < 9 bpm or > 20 bpm, e) Pulse oximetry < 94% on room air, f) Temperature > 100.4 degree Fahrenheit, g) Liver enzymes > 2 times the upper limit of normal, h) Abnormal bilirubin, i) eGFR < 45 ml / min / 1.73m2 by CKD-EPI, j) Platelet count < 100,000, k) Hemoglobin < 10 g / dL, and / or 1) Hemoglobin A1C > 8.Monitoring of safety and efficacy

[0272] Prior to infusion of the rAAV particles, subjects are evaluated for: (1) baseline physical examination; (2) baseline clinical laboratory tests, including antigenic and functional levels of MYBPC3, and (b) baseline AAV9 antibody detection.

[0273] After infusion of the rAAV particles, safety parameters that are monitored include: (1) incidence of adverse events and serious adverse events, (2) incidence of dose limited toxicities (DLTs), (3) incidence of targeted adverse cardiac events: a) EF<50%, b) arrhythmias, c) myopericarditis, d) regional wall motion abnormalities, e) increase in troponin I, and (4) incidence of targeted adverse immunological events, defined as: a) transaminitis, b) thrombotic microangiopathy (TMA), or c) increase in markers of complement activation (i.e. sC5b-9).

[0274] The following additional indicators of efficacy are monitored: (1) myocardial stress and injury, (2) cardiac structure / morphology and function, (3) exercise capacity, (4) functional cardiac status, (5) symptoms and health-related quality of life (HRQoL) impacts, (6) health issues, and (7) global anchor measures.

[0275] Myocardial stress and injury are monitored by blood biomarker assays which assess changes in N-terminal pro hormone BNP (NT-proBNP) and high sensitivity troponin I (hsTNI). Other endpoints may include assaying changes in Pl Cl. Cardiac structure / morphology and function are monitored by echocar di ography / cMRI which assesses changes in diastolic function (E / e’ and left atrial volume index), systolic function (ejection fraction and longitudinal strain), and structure (LV wall thickness, LV mass). Exercise capacity is monitored by assessing change in PVO2 via cardiopulmonary exercise testing (CPET). The New York Heart Association (NYHA) functional classification is used to assess functional cardiac status and the Kansas City Cardiomyopathy Questionnaire-23 (KCCQ-23) Total Symptom Score (TSS), physical limitations score and clinical summary score (CSS) are used to assess symptoms and health related quality of life (HRQoL) impacts of subjects. Health status is also assessed by EuroQol-5 dimension 5- level version (EQ-5D-5L) and EuroQol-visual analog scale (EQ-VAS), and global anchor measures are assessed by Clinical Global Impression of Severity and Change scales (CGI / C), and Patient Global Impression of Severity and Change scales (PGIS / C). The incidence of adverse events and serious adverse events are monitored.Immunogenicity and Safety Assessments

[0276] Immunogenicity of subjects is assessed throughout the study. A complement panel is performed 2 days prior to infusion, daily for 7 days post-infusion, and weekly for the duration of the study (i.e. weeks 2-10). Additionally, an anti-AAV9 Tab assay is performed 2-days prior to infusion, on the day of infusion, and weekly for the duration of the study (i.e. weeks 1-10). Adverse events and LFTs are assessed 2 days prior to infusion, daily for 7 days post-infusion, and weekly for the duration of the study (i.e. weeks 2-10). Vector shedding is assessed the day of infusion and weeks 4 and 8.Cardiac Assessment

[0277] Subjects undergo baseline screening beginning 8-week prior to infusion. Cardiac function and structure are assessed 4-week prior to infusion by echocardiogram and electrocardiogram. NYHA, KCCQ-23, CPET cardiac MR and ambulatory ECG monitoring is performed. Subject levels of NT-proBNP and troponin I are assessed 8-weeks and 4 weeks prior to infusion.

[0278] Subjects undergo aggressive cardiac, immunologic and hepatic surveillance early on in the study that reduce in frequency based on clinical status. Adverse event assessment begins on day of infusion and continue for 16 weeks. Cardiac assessment of subjects by echocardiogram and electrocardiogram as well as NT-proBNP and Troponin I levels is performed on the day of infusion, weekly for weeks 2-16, and weeks 24, 32, 40 and 48. NYHA and KCCQ-23 is performed on the day of infusion and weeks 4, 8, 16, 24 and 48. Ambulatory ECG monitoring is performed on weeks 2, 4, 8, 16, 24 and 48.

[0279] Other endpoints may include acquiring and assessing biopsies, having the patient undertake a six-minute walk test, or noting other patient-reported outcome measures. A CGI short form may also be used as an overall patient assessment tool.

[0280] Other safety endpoints may include identifying transaminitis, identifying ejection fractions of 40% or 45%, identifying arrhythmias, or evaluating wall motion.Prophylactic Immune Modulation

[0281] Subjects receive prophylactic immunosuppressant therapy with glucocorticosteroid, optionally sirolimus, and optionally, a complement pathway inhibitor or antagonist. A 30-day prophylactic glucocorticosteroid course is administered to subjects with a starting dose of 40 mg / day, beginning on Day 1 of infusion, followed by a 30-day dose taper beginning at Week 5 provided tests indicate normal liver function and no evidence of myocarditis. Additionally, 3- days prior to infusion subjects will be prophylactically administered 6mg / d x2d Sirolumus followed by 2mg / d for 60-days. Sirolimus drug monitoring will be implemented to ensure target sirolimus blood levels are maintained between 5 to 15 ng / mL. Additionally, participants who are on more than one immunosuppressive medication will be administered 160+800 mg trimethoprim-sulfamethoxazole (TMP-SMX) (e g. 1 double strength tablet) orally three-times per week beginning 1-day prior to infusion for prophylaxis against Pneumocystis jirovecci.Reactive Immune Modulation

[0282] Reactive immunosuppressant therapy for example glucocorticosteroid optionally in combination with a complement pathway inhibitor or antagonist may be initiated in response to liver or cardiac inflammation observed after the prophylactic regimen is completed. In response an extended full-dose glucocorticosteroid treatment is administered beyond the initial 30-days prophylactic period. Tacrolimus or Mycophenolate mofetil will be added as an alternative immunosuppressant upon elevated liver function tests despite steroids / sirolimus administration. Tacrolimus is administered for break-through myocarditis and Eculizumab is administered for refractory complement activation in the context of thrombotic microangiography.Obstructive Hypertrophic Cardiomyopathy

[0283] A clinical study is performed on subjects with obstructive Hypertrophic Cardiomyopathy (oHCM) caused by MYBPC3 gene mutations to demonstrate safety, tolerability, and potential efficacy of a single intravenous infusion of AAV9-MYPBC3. The study will provide evidence of effectiveness in improving functional outcomes and / or morbidities in patients with HCM.

[0284] Human subjects are administered rAAV particles comprising an AAV9 type capsid and a recombinant AAV vector construct described herein (e.g., any of SEQ ID NOs: 3-41), at one of three dose levels (6E13 vg / kg, 9E13 vg / kg and 1.2E14 vg / kg) to assess the efficacy, safety and tolerability of the rAAV particles at the indicated doses. Subjects are also administered prophylactic immune modulation through administration of steroids and sirolimus as described below. Optionally, antimicrobial prophylaxis is employed, e.g. sulfamethoxazole / trimethoprim prophylaxis is administered to protect against PJP. Subjects are assessed for TB and vaccination.

[0285] Subjects with oHCM and no neutralizing titers or predetermined titers of anti-AAV9 antibodies (Part A) are administered the rAAV particles at the desired dose, in a single intravenous infusion at an infusion rate of 20 mL / hr (0.33 mL / min) with a maximum of 80 mL / hr (1.33 mL / min), and are followed for 5 years to evaluate durability of the response. AAV9- seropositive oHCM participants, who meet all other inclusion and exclusion criteria, will be enrolled into a concurrent comparator study arm (Part B).

[0286] One objective of the study is to evaluate the safety of AAV9-MYBPC3. A number of safety endpoints have been identified, most notably number of participants with targeted adverse, cardiac events and targeted adverse, immunological events (transaminitis, TMA), based primarily on prior safety findings associated with other gene therapies using AAV vectors.

[0287] Several efficacy endpoints are measured to fully characterize the effect of AAV9- MYPBC3 on the heart and subject. Changes in cardiac systolic and diastolic function are assessed by echocardiograms and serum biomarkers of cardiac stress. Changes in cardiac morphology are assessed by echocardiographic measures of left ventricular mass and wall thickness. Changes in left ventricular outflow tract obstruction is assessed by echocardiographic meausre of left ventricular outflow pressure gradient. Changes in exercise capacity will be assessed by PVO2 obtained during CPET as well as other maximal and submaximal exercise parameters. Changes in patient symptoms and functional status are assessed by KCCQ-23 and NYHA, respectively. Optionally, myocardial cell transduction and RNA and protein expressionare assessed from myocardial tissue obtained by endomyocardial biopsy, compared to pre-dose levels.

[0288] One efficacy endpoint is change in PVO2 as measured by a CPET. An increase in PVO2 of 1 ml / kg / min has previously been shown to represent a clinically relevant threshold of improvement in patients with HCM and has been associated with a reduced risk of all-cause mortality or cardiac transplantation (Coats et al., Circ Heart Fail. 8(6): 1022-31 (2015); Masri et al., J Am Heart Assoc. 7:e007944 (2018)). Further, in previous studies in patients with HCM, an improvement in PVO2 has been associated with improvement in functional classification (Olivotto et al., Lancet, 396(10253):759-69 (2020)).Inclusion and exclusion criteria

[0289] The clinical study inclusion criteria may include one or more of the following: (1) clinical diagnosis of oHCM consistent with current ACC / AHA and / or ESC guidelines; (2) pathogenic, heterozygous truncating mutations in the MYBPC3 gene with genetic testing confirmed at a CLIA-approved laboratory; (3) Class II or Class III heart failure according to the NYHA functional classification system, on optimal therapy for HCM; (4) ejection fraction of > 50%, (5) elevated levels ofNT-proBNP (>300 pg / mL). Other inclusion criteria may include PVO2 of < 80% predicted on CPET with RER >1.0 on screening CPET. Patients also may have an ICD.

[0290] Echocardiography may be used to determine the presence and magnitude of obstruction to blood flow through the LVOT. A pressure gradient of > 30 mmHg, either at rest or with provocation, is indicative of obstruction and patients are considered to have oHCM. Thus, patient may be diagnosed with oHCM using this method and selected for the clinical study.

[0291] The clinical study exclusion criteria may include one or more of the following: (1) evidence of an active infection or chronic infection, or immunosuppressive disorder; (2) contraindication to glucocorticosteroids, (3) significant or uncontrolled chronic condition(s) defined as requiring regular medical attention and treatment including but not limited to acute or chronic liver disease, chronic obstructive pulmonary disease, uncontrolled diabetes, etc., (4) a history of clinically relevant arrhythmogenic syncope, sustained ventricular tachycardia, resuscitated sudden cardiac arrest, or appropriate ICD discharge within 6 months of screening, (5) Paroxysmal atrial fibrillation at the time of screening, (6) Persistent or permanent atrial fibrillation, (7) Significant valvular heart disease (moderate- severe valvular aortic stenosis, moderate-severe mitral regurgitation not due to systolic anterior motion of the mitral valve) or other heart disease including but not limited to structural heart disease (not related to oHCM), congenital heart disease, coronary heart disease, pericardial disease, acute myocarditis,infiltrative cardiomyopathy, and history of pericarditis / myocarditis / pericardial effusion, and (8) prior treatment with gene therapy or an investigational medicinal treatment concurrently or within 6 months prior to screening. Other exclusion criteria include weight or BMI exclusion, borderline AAV9 seropositivity, controlled Atrial Fibrillation, a heart rate exclusion value, a blood pressure value, a pulse oximetry value, an eGFR value, or myocardial infarction.Myectomies are not identified as an exclusion criterion and may be allowed.

[0292] One or more of the following diagnostic clinical study exclusion criteria apply: (1) detectable antibodies to AAV9 capsid (i.e. seropositivity); or (2) evidence of clinically abnormal laboratory values including: a) Systolic blood pressure > 150 mmHg or < 90 mmHg, b) Diastolic blood pressure > 100 mmHg or < 60 mmHg, c) Resting pulse rate < 40 bpm or > 100 bpm, d) Resting respiratory rate < 9 bpm or > 20 bpm, e) Pulse oximetry < 94% on room air, f) Temperature > 100.4 degree Fahrenheit, g) Liver enzymes > 2 times the upper limit of normal, h) Abnormal bilirubin, i) eGFR < 45 ml / min / 1.73m2 by CKD-EPI, j) Platelet count < 100,000, k) Hemoglobin < 10 g / dL, and / or 1) Hemoglobin A1C > 8.Monitoring of safety and efficacy

[0293] Prior to infusion of the rAAV particles, subjects are evaluated for: (1) baseline physical examination; (2) baseline clinical laboratory tests, including antigenic and functional levels of MYBPC3, and (b) baseline AAV9 antibody detection.

[0294] After infusion of the rAAV particles, safety parameters that are monitored include: (1) incidence of adverse events and serious adverse events, (2) incidence of DLTs, (3) incidence of targeted adverse cardiac events: a) EF<50%, b) arrhythmias, c) myopericarditis, d) regional wall motion abnormalities, e) increase in troponin I, and (4) incidence of targeted adverse immunological events, defined as: a) transaminitis, b) TMA, or c) increase in markers of complement activation (i.e. sC5b-9).

[0295] The following additional indicators of efficacy are monitored: (1) myocardial stress and injury, (2) cardiac structure / morphology, left ventricular outflow tract obstruction, and function, (3) exercise capacity, (4) functional cardiac status, (5) symptoms and HRQoL impacts, (6) health issues, and (7) global anchor measures.

[0296] Myocardial stress and injury are monitored by blood biomarker assays which assess changes in NT -proBNP and hsTNI. Other endpoints may include assaying changes in PIC 1. Cardiac structure / morphology and function are monitored by echocardiography / cMRI which assesses changes in diastolic function (E / e’ and left atrial volume index), systolic function (ejection fraction and longitudinal strain), and structure (LV wall thickness, LV mass). Exercise capacity is monitored by assessing change in PVO2 via CPET. The NYHA functionalclassification is used to assess functional cardiac status and the KCCQ-23 TSS, physical limitations score and CSS are used to assess symptoms and HRQoL impacts of subjects. Health status is also assessed by EQ-5D-5L and EQ-VAS, and global anchor measures are assessed by CGI / C, and PGIS / C. The incidence of adverse events and serious adverse events are monitored.Immunogenicity and Safety Assessments

[0297] Immunogenicity of subjects is assessed throughout the study. A complement panel is performed 2 days prior to infusion, daily for 7 days post-infusion, and weekly for the duration of the study (i.e. weeks 2-10). Additionally, an anti-AAV9 Tab assay is performed 2-days prior to infusion, on the day of infusion, and weekly for the duration of the study (i.e. weeks 1-10).Adverse events and LFTs are assessed 2 days prior to infusion, daily for 7 days post-infusion, and weekly for the duration of the study (i.e. weeks 2-10). Vector shedding is assessed the day of infusion and weeks 4 and 8.Cardiac Assessment

[0298] Subjects undergo baseline screening beginning 8-week prior to infusion. Cardiac function and structure are assessed 4-week prior to infusion by echocardiogram and electrocardiogram. NYHA, KCCQ-23, CPET cardiac MR and ambulatory ECG monitoring is performed. Subject levels of NT-proBNP and troponin I are assessed 8-weeks and 4 weeks prior to infusion.

[0299] Subjects undergo aggressive cardiac, immunologic and hepatic surveillance early on in the study that reduce in frequency based on clinical status. Adverse event assessment begins on day of infusion and continue for 16 weeks. Cardiac assessment of subjects by echocardiogram and electrocardiogram as well as NT-proBNP and Troponin I levels is performed on the day of infusion, weekly for weeks 2-16, and weeks 24, 32, 40 and 48. NYHA and KCCQ-23 is performed on the day of infusion and weeks 4, 8, 16, 24 and 48. Ambulatory ECG monitoring is performed on weeks 2, 4, 8, 16, 24 and 48.

[0300] Other endpoints may include acquiring and assessing biopsies, having the patient undertake a six-minute walk test, or noting other patient-reported outcome measures. A CGI short form may also be used as an overall patient assessment tool.

[0301] Other safety endpoints may include identifying transaminitis, identifying ejection fractions of 40% or 45%, identifying arrhythmias, or evaluating wall motion.Prophylactic Immune Modulation

[0302] Subjects receive prophylactic immunosuppressant therapy with glucocorticosteroid, optionally sirolimus, and optionally, a complement pathway inhibitor or antagonist. A 30-day prophylactic glucocorticosteroid course is administered to subjects with a starting dose of 40 mg / day, beginning on Day 1 of infusion, followed by a 30-day dose taper beginning at Week 5 provided tests indicate normal liver function and no evidence of myocarditis. Additionally, 3- days prior to infusion subjects will be prophylactically administered 6mg / d x2d Sirolumus followed by 2mg / d for 60-days. Additionally, subjects on more than one immunosuppressive medication will be prophylactically administered 160+800mg (i.e. one double-strength tablet) of TMP-SMX orally three-times per week.Reactive Immune Modulation

[0303] Reactive immunosuppressant therapy for example glucocorticosteroid optionally in combination with a complement pathway inhibitor or antagonist may be initiated in response to liver or cardiac inflammation observed after the prophylactic regimen is completed. In response an extended full-dose glucocorticosteroid treatment is administered beyond the initial 30-days prophylactic period. Tacrolimus or Mycophenolate mofetil will be added as an alternative immunosuppressant upon elevated liver function tests despite steroids / sirolimus administration. Tacrolimus is administered for break-through myocarditis and Eculizumab is administered for refractory complement activation in the context of thrombotic microangiography.

[0304] The embodiments described herein are intended to be merely exemplary, and those skilled in the art will recognize, or will be able to ascertain using no more than routine experimentation, numerous equivalents of specific compounds, materials, and procedures. All such equivalents are considered to be within the scope of the disclosure.

[0305] All of the patents, patent applications and publications referred to herein are incorporated by reference herein in their entireties. Citation or identification of any reference in this application is not an admission that such reference is available as prior art to this application. The full scope of the disclosure is better understood with reference to the appended claims.

Claims

CLAIMS1. A pharmaceutical composition comprising recombinant adeno-associated virus (rAAV) particles, optionally with AAV9 serotype capsid, at a concentration of at least about 6E13 vg / ml to about 4E14 vg / ml which comprises a buffering agent, an isotonicity agent, an additional MgCh stabilizer, a cryopreservative agent and a surfactant which is stable during storage at about < -60°C (minus sixty degrees centigrade) or less for at least about 1 year, 1.5 years, or 2 years.

2. The pharmaceutical composition of claim 1, wherein the pH of the formulation ranges from about 6 to about 9, optionally ranging from about 7 to about 7.8.

3. The pharmaceutical composition of claim 2, wherein the pH is 7.4.

4. A pharmaceutical composition comprising rAAV particles, optionally with AAV9 serotype capsid, at a concentration of at least about 6E13 vg / ml to about 4E14 vg / ml, or about IE 14 vg / ml or less, Tris hydrochloride buffer at a concentration of about 10 to about 30 mM, Tris base at a concentration of about 1 to about 4 mM, sodium chloride at a concentration of about 100 mM to about 150 mM, MgCE at a concentration of about 0.5mM to about 3.0mM, trehalose at a concentration of about 50mM to about 90mM, and a poloxamer or polysorbate at a concentration of about 0.05% to about 0.15% w / v.

5. The pharmaceutical composition of claim 4, wherein the concentration of rAAV particles ranges from about IE 13 vg / ml to about 1E14 vg / ml.

6. The pharmaceutical composition of claim 4, wherein the concentration of rAAV particles ranges from about IE 13 vg / ml to about 5E13 vg / ml, or from about 5E13 vg / ml to about 1E14 vg / ml.

7. The pharmaceutical composition of any one of claims 1-6, wherein the buffering agent comprises Tris hydrochloride and / or Tris base.

8. The pharmaceutical composition of claim 7, wherein the Tris hydrochloride is at a concentration of 17.6 mM and the Tris base is at a concentration of 2.4 mM.

9. The pharmaceutical composition of any one of claims 1-8, wherein the isotonicity agent is NaCl at a concentration of 120 mM.

10. The pharmaceutical composition of any one of claims 1-9, wherein the stabilizer is MgCI? at a concentration of 1 mM.

11. The pharmaceutical composition of any one of claims 1-10, wherein the stabilizer is MgCI? at a concentration of greater than 1.5mM.

12. The pharmaceutical composition of any one of claims 1-11, wherein the stabilizer is MgCL at a concentration of up to 3.0mM.

13. The pharmaceutical composition of any one of claims 1-12, wherein the cryopreservative agent is a sugar.

14. The pharmaceutical composition of any one of claims 1-13, wherein the cryopreservative agent is trehalose at a concentration of 74mM.

15. The pharmaceutical composition of any one of claims 1-14, wherein the surfactant is Pol oxamer 188.

16. The pharmaceutical composition of claim 15, wherein the surfactant is at a concentration of about 0.1% w / v.

17. The pharmaceutical composition of any of claims 1-16, wherein the composition is liquid.

18. A pharmaceutical composition comprising rAAV particles at a concentration of about 6E13 vg / ml, 17.6mM Tris hydrochloride, 2.4mM Tris base, 120mM sodium chloride, ImM MgCE hexahydrate, 74mM trehalose dihydrate, and 0. 1% w / v poloxamer 188.

19. The pharmaceutical composition of claim 18, wherein the rAAV particles comprise an AAV9 type capsid.

20. The pharmaceutical composition of claim 18, wherein the composition is liquid.

21. A method of using the pharmaceutical composition of any one of claims 1-20 to treat a subject with heart disease by intravenous administration of said pharmaceutical composition.

22. The method of claim 21, wherein the heart disease is associated with a genetic mutation of the MYBPC3 gene.

23. The method of claim 21, wherein the heart disease is hypertrophic cardiomyopathy.

24. A method of treating a human subject with a heart disease, comprising administering to the subject a single dose ranging from about 1E13 vg / kg to about 4E14 vg / kg of rAAV particles comprising (a) an AAV capsid with heart tropism, (b) a cardiac-specific transcription regulatory region, and (c) a recombinant vector construct comprising a nucleic acid encoding a functional c- MyBPC protein or codon optimized version thereof.

25. The method of claim 24, wherein the dose is about 1E13 to about 6E13 vg / kg, or about 5E13 to about 1E14, or about 8E13 to about 4E14.

26. The method of claim 24, wherein the dose is about 9E13 vg / kg.

27. The method of claim 24, wherein the dose is about 1E14 vg / kg.

28. The method of claim 24, wherein the dose is about 2E14 vg / kg.

29. The method of claim 24, wherein the dose is about 1.2E14 vg / kg.

30. The method of claim 24, wherein the dose is about 2.4E14 vg / kg.

31. The method of any one of claims 24-30, wherein the functional c-MyBPC protein comprises an amino acid sequence at least 95%, 98% or 99% identical to amino acids 1 through 1274 of SEQ ID NO: 2.

32. The method of any one of claims 24-31, wherein the nucleic acid encoding the functional MyBPC3 comprises a nucleotide sequence at least 90%, 95%, 98% or 99% identical to SEQ ID NO: 1.

33. The method of any one of claims 24-32, wherein the cardiac-specific transcription regulatory region comprises a fragment of a human TNNT2 promoter.

34. The method of any one of claims 24-33, wherein the cardiac-specific transcription regulatory region comprises a nucleotide sequence at least 90%, 95%, 98% or 99% identical to SEQ ID NO: 51.

35. The method of any one of claims 24-34, wherein the recombinant vector construct comprises an intron.

36. The method of any one of claims 24-35, wherein the recombinant vector construct comprises a polyadenylation signal.

37. The method of any one of claims 24-36, wherein the subject is administered a population of rAAV particles produced by a method comprising (a) providing a mammalian cell comprising one or more nucleic acid constructs comprising: (i) a recombinant vector construct comprising (1) a 5’ AAV ITR and a 3’ AAV ITR, (2) a cardiac-specific transcription regulatory region, and (3) a nucleic acid encoding a functional MyBPC3 comprising an amino acid sequence at least 95% identical to amino acids 1 through 1274 of SEQ ID NO: 2, (ii) a nucleotide sequence encoding one or more AAV Rep proteins which is operably linked to a promoter that is capable of driving expression of the Rep protein(s) in the cell; and (iii) a nucleotide sequence encoding one or more AAV9 type capsid protein which is operably linked to a promoter that is capable of driving expression of the capsid protein(s) in the cell; (b) culturing the mammalian cell under conditions permitting expression of the Rep and the capsid proteins and production of an AAV particle; and (c) recovering the AAV particle.

38. The method of claim 37, wherein the population is enriched for rAAV particles comprising full length, nearly full-length, or therapeutically effective length vector genomes by steps that reduce the number of empty capsids.

39. The method of any one of claims 24-38, wherein the AAV capsid comprises an amino acid sequence at least 85%, 90% or 95% identical to any one of SEQ ID NOs: 207-223.

40. The method of any one of claims 24-39, wherein the AAV capsid with cardiac tropism is an AAV9 type capsid, optionally at least 85%, 90% or 95% identical to SEQ ID NO: 207.

41. The method of any one of claims 24-40, wherein the rAAV particles are administered intravenously.

42. The method of any one of claims 24-41, wherein the subject has a heart disease associated with a genetic mutation of the MYBPC3 gene.

43. The method of claim 42, wherein the heart disease is hypertrophic cardiomyopathy.

44. The method of any one of claims 21-43, wherein the dose is effective to improve cardiac function or structure, optionally as measured by decreased E / e’ and / or decreased left atrial volume index and / or improving or normalizing ejection fraction and / or reduced impairment of longitudinal strain, and / or reduced left ventricular (LV) wall thickness and / or reduced LV mass.

45. The method of any one of claims 21-44, wherein the dose is effective to improve cardiac function and / or health-related quality of life, optionally as measured by any one or more of New York Heart Association (NYHA) functional classification, Kansas City Cardiomyopathy Questionnaire-23 (KCCQ-23) Total Symptom Score (TSS), physical limitations score and clinical summary score (CSS), EuroQol-5 dimension 5-level version (EQ-5D-5L), EuroQol- visual analog scale (EQ-VAS), Clinical Global Impression of Severity and Change scales (CGI / C), and Patient Global Impression of Severity and Change scales (PGIS / C), over time period of at least about six months.

46. The method of either claim 44 or 45, wherein the improved cardiac function and / or health-related quality of life is maintained for at least about one year.

47. The method of any one of claims 21-46, further comprising administering to the subject a prophylactically effective amount of immunosuppressant, in an amount effective to prevent or reduce biochemical or clinical signs of hepatotoxicity, hepatic inflammation, and / or cardiac inflammation.

48. The method of claim 47, wherein the prophylactic immunosuppressant is a corticosteroid.

49. The method of claim 48, wherein a prophylactically effective amount of corticosteroid is administered at a prednisone-equivalent dose of about 0.5 mg / kg / day, about 40 mg / day, or about 60 mg / kg, optionally for a time period of at least about 4 weeks, followed by tapering amounts of the corticosteroid for a time period of about 4 weeks.

50. The method of claim 48, wherein the prophylactic immunosuppressant is concurrently administered with a prophylactically effective amount of sirolimus.

51. The method of claim 50, wherein the sirolimus is administered at a dose of about 2 mg / day for a time period of at least 8 weeks, or in an amount effective to maintain a blood level of sirolimus between about 5 to about 15 ng / mL.

52. The method of claim 48, wherein the prophylactic immunosuppressant is concurrently administered with a prophylactically effective amount of TMP-SMX.

53. The method of claim 52, wherein the TMP-SMX is administered orally at a dose of 160+800 mg three-times per week.

54. The method of any of claims 21-53, further comprising the step of (a) determining a baseline level of a marker of hepatotoxicity, hepatic inflammation and / or cardiac inflammation in the blood of the subject prior to rAAV particle administration, and (b) subsequently determining a post-administration level of said marker for hepatotoxicity, hepatic inflammation and / or cardiac inflammation in the blood of the subject every week for at least 8 weeks.

55. The method of claim 54, further comprising administering to the subject a therapeutic amount of immunosuppressant in response to hepatotoxicity, hepatic inflammation or cardiac inflammation, optionally in response to an elevation of ALT or AST greater than 2 x the upper limit of normal (ULN) or greater than or equal to twice the baseline level.

56. The method of claim 55, wherein the therapeutic amount of immunosuppressant administration comprises the step of: (c) upon detection of hepatotoxicity, hepatic inflammation and / or cardiac inflammation by biochemical or clinical signs, administering to the subject a therapeutically effective amount of a systemic immunosuppressant to reduce hepatotoxicity, hepatic inflammation and / or cardiac inflammation.

57. The method of any one of claims 55-56, wherein the therapeutic immunosuppressant is tacrolimus, Mycophenolate mofetil, or eculizumab.

58. The method of any of claims 21-57, further comprising administering to the subject a prophylactic antimicrobial agent.

59. The method of claim 58, wherein the prophylactic antimicrobial agent is a combination of sulfamethoxazole and trimethoprim.

60. The method of any one of claims 21-59, further comprising administering to the subject a prophylactically effective amount of a complement pathway inhibitor or antagonist, in an amount effective to prevent or reduce biochemical or clinical signs of complement activation and / or thrombotic microangiopathy.

61. The method of claim 60, wherein the complement pathway inhibitor or antagonist is OMS721 antibody, pegcetacoplan (APL-2), eculizumab (Soliris), avacopan (CCX168), vilobelimab (IFX-1), AMY-101, , Cl esterase inhibitor (CINRYZE / BERINERT / HAEGARDA), recombinant Cl esterase inhibitor (RUCONEST), ravulizumab (ALXN1210), coversin, cemdisiran (ALN-CC5), tesidolumab (LFG-316), iptacopan (LNP023), danicopan (ACH-4471) or ENJAYMO (sutimlimab-jome).

62. The method of any one of claims 21-61, further comprising the step of (a) determining a baseline level of a marker of complement activation and / or thrombotic microangiopathy in the blood of the subject prior to rAAV particle administration, and (b) subsequently determining a post-administration level of said marker for complement activation and / or thrombotic microangiopathy in the blood of the subject every week for at least 8 weeks..

63. The method of claim 62, further comprising administering to the subject a therapeutic amount of a complement pathway inhibitor or antagonist in response to complement activation and / or thrombotic microangiopathy.

64. The method of claim 63, wherein the therapeutic amount of immunosuppressant administration comprises the step of: (c) upon detection of complement activation and / or thrombotic microangiopathy by biochemical or clinical signs, administering to the subject atherapeutically effective amount of a complement pathway inhibitor or antagonist to reduce complement activation and / or thrombotic microangiopathy.

65. The method of any one of claims 63-64, wherein the complement pathway inhibitor or antagonist is OMS721 antibody, pegcetacoplan (APL-2), eculizumab (Soliris), avacopan (CCX168), vilobelimab (IFX-1), AMY-101, Cl esterase inhibitor (CINRYZE / BERINERT / HAEGARDA), recombinant Cl esterase inhibitor (RUCONEST), ravulizumab (ALXN1210), coversin, cemdisiran (ALN-CC5), tesidolumab (LFG-316), iptacopan (LNP023), danicopan (ACH-4471), or ENJAYMO (sutimlimab-jome).

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