Methods of treating hypertrophic cardiomyopathy with AAV gene therapeutic vectors and therapeutic formulations

Abstract

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

Description

[0001] Cross-references to related applications

[0002] This application claims priority to 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 herein by reference in their entirety. Technical Field

[0003] This article provides a recombinant adeno-associated virus (rAAV) gene therapy vector and viral particles, which can be used to treat and prevent hypertrophic cardiomyopathy by increasing the expression of cardiac myosin-binding protein C (cMyBP-C).

[0004] Incorporate into the sequence list by reference

[0005] This application includes a sequence list in a computer-readable form (filename: SequenceListing.xml, size: 489,630 bytes; creation date: October 17, 2023) as another part of the disclosure. The contents of the sequence list xlm file are hereby incorporated in their entirety by reference. Background Technology

[0006] Despite considerable progress in preventing heart disease caused by environmental factors (such as nicotine, high cholesterol, or diabetes) and in the symptomatic treatment of heart disorders, improvements are still needed in the treatment of hereditary cardiomyopathy. Hereditary cardiomyopathy includes hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and arrhythmogenic right ventricular cardiomyopathy (ARVC).

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

[0008] Hereditary hypertrophic cardiomyopathy is a genetic disorder known to be caused by more than 1,000 different mutations in at least 10 genes encoding components of the cardiac sarcomere, such as cardiac myosin-binding protein C (cMyBP-C), β-myosin heavy chain (MYH7), cardiac troponin T (TNNT2), cardiac troponin I (TNNI3), myosin ventricular essential light chain 1 (MYL3), myosin ventricular regulatory light chain 2 (MYL2), cardiac α-actin (ACTC), α-tropomyosin (TPM1), titin (TTN), and four-and-a-half LIM protein 1 (FHL1) (Richard et al., Circulation, 107: 2227-2232 (2003); Schlossarek et al., J. Mol. Cell Cardiol. 50: 613-20 (2011); Friedrich et al., Hum. Mol. Genet. 21: 3237-54). (2012)). Many mutations are missense mutations encoding full-length mutant peptides, while other frameshift or splice site mutations can lead to truncation (Marian et al., Circ. Res. 121: 749-70 (2017); Walsh et al., Genet. Med. 19: 192-203 (2017)). The most common truncation mutant peptides are MYBPC3 and FHL1, which mainly exhibit frameshift mutations, resulting in C-terminal truncation.

[0009] The most frequently mutated gene in HCM is MYBPC3, which encodes cardiac myosin-binding protein C (cMyBP-C) (Bonne et al., Nat. Genet. 11:438-40 (1995); Watkins et al., N. Engl. J. Med. 364:1643-56 (2011)). cMyBP-C is a major component of the sarcomere A band, in which it interacts with myosin, actin, and titin (Schlossarek et al., J. Mol. Cell. Cardiol. 50: 613-20 (2011)). In humans and mice, cMyBP-C is detected only in the heart (Fougerousse et al., Circ. Res. 82: 130-3 (1998)) and is involved in the regulation of 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)). Mutations in approximately 70% of the MYBPC3 gene result in frameshifts and the production of C-terminal truncated proteins (Carrier et al., Circ. Res. 80: 427-34 (1997)). The truncated protein is unstable and has never been detected in the 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)).

[0010] Current drug-based treatments for HCM alleviate symptoms but do not address the underlying genetic causes of the disease. Gene-based or RNA-based therapies will be the only curative treatment for HCM. Gene therapy has been successfully tested in non-hereditary heart diseases (Jessup et al., Circulation, 124: 304-13 (2011)). Summary of the Invention

[0011] The embodiments described herein relate to pharmaceutical compositions comprising recombinant AAV particles and methods of treating heart disease using recombinant AAV particles.

[0012] The pharmaceutical composition is suitable for treating or preventing hemorrhage (HCM) in mammalian subjects, preferably human subjects, who require treatment. In some embodiments, the subject requiring treatment is a subject carrying a mutation in at least one or both genes encoding cMyBP-C. After administration to the subject to treatment, the pharmaceutical composition provides expression of the encoded cardiac myosin-binding protein in the subject, preferably in the myocardium of the subject.

[0013] In one aspect, this disclosure provides pharmaceutical compositions of recombinant adeno-associated virus (rAAV) particles for use in any of the methods disclosed herein. This disclosure also provides use of the recombinant AAV particles as described herein for preparing a medicament for treatment according to any of the methods described herein.

[0014] In one related aspect, this disclosure provides a pharmaceutical composition comprising rAAV particles, a buffer, an isotonic agent, an additional magnesium chloride (MgCl2) stabilizer, a cryopreservative, and a surfactant at a concentration of at least about 6E13 vg (vector genome) / ml to about 4E14 vg / ml or any concentration within this range as disclosed herein, which is stable for at least about 1 year, 1.5 years, or 2 years during storage at about -60°C or lower. In some embodiments, the pharmaceutical composition has a formulation pH in the range of about 6 to about 9, about 7 to about 7.8. In some embodiments, the pharmaceutical composition has a formulation pH of 7.4.

[0015] In another aspect, this disclosure provides a pharmaceutical composition as disclosed herein that is stable for at least about 1 year, at least about 1.5 years, or at least about 2 years, or about 1 year to about 2 years, during storage at about -60°C (minus 60°C) or lower. In other embodiments, the pharmaceutical formulation comprises one or more of a buffer, an isotonic agent, a stabilizer (e.g., a magnesium chloride (MgCl2) stabilizer), a cryopreservative, and a surfactant.

[0016] 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 (millimolar) at a concentration of about 1 to about 4 mM, sodium chloride at a concentration of about 100 mM to about 150 mM, MgCl2 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 poloxamer or polysorbate at a concentration of about 0.05% to about 0.15% w (weight) / v (volume). In some embodiments, 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, MgCl2 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 poloxamer or polysorbate at a concentration of about 0.05% to about 0.15% w / v. In some embodiments, the concentration of rAAV particles is in the range of about 1E13 vg / ml to about 1E14 vg / ml, about 1E13 vg / ml to about 5E13 vg / ml, or about 5E13 vg / ml to about 1E14 vg / ml, or any concentration within these ranges as disclosed herein.

[0017] In some embodiments, the buffer is optionally Tris hydrochloride and / or Tris base. In some embodiments, the concentration of Tris hydrochloride is 17.6 mM and the concentration of Tris base is 2.4 mM. In some embodiments, the isotonic agent is NaCl at a concentration of 120 mM. In some embodiments, the stabilizer is MgCl2 at a concentration of about 1 mM to about 3.0 mM. In some embodiments, the concentration of the MgCl2 stabilizer is 1.0 mM or greater, more than 1.0 mM, 1.5 mM or greater, or up to 3.0 mM. In some embodiments, the cryopreservative is sugar. In some embodiments, the cryopreservative 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.

[0018] In one aspect, this 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 MgCl2 hexahydrate, 74 mM trehalose dihydrate, and 0.1% w / v poloxamer 188.

[0019] Preferably, in any of the foregoing pharmaceutical compositions, the rAAV particles comprise an AAV9-type capsid and a recombinant AAV carrier construct as described herein. Preferably, the pharmaceutical compositions described herein are aqueous solutions. Preferably, such solutions are sterile.

[0020] This disclosure provides a method of treating a subject with heart disease, comprising administering the pharmaceutical composition described herein to the subject via intravenous (IV) administration. For example, the heart disease may be hypertrophic cardiomyopathy or heart disease associated with a gene mutation in the MYBPC3 gene.

[0021] In a second aspect, this disclosure provides a method for treating a human subject with heart disease, comprising administering to the subject a single dose of recombinant AAV particles in the range of about 1E13 vector genomes / kg subject weight to about 4E14 vector genomes / kg subject weight (vg / kg), said recombinant AAV particles comprising (a) a cardiotropic AAV capsid, (b) a cardio-specific transcriptional regulatory region, and (c) a recombinant vector construct comprising nucleic acid encoding a functional cMyBP-C protein or a codon-optimized form thereof.

[0022] In any of the methods disclosed herein, the dose of rAAV particles may be from about 1E13 vg / kg to about 6E13 vg / kg, or from about 5E13 vg / kg to about 1E14 vg / kg, or from about 8E13 vg / kg to about 4E14 vg / kg, or any dose within these ranges as disclosed herein. In some embodiments, the dose of rAAV particles 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.

[0023] In a recombinant vector construct containing a nucleic acid encoding a functional cMyBP-C protein, the functional cMyBP-C protein may contain at least 95%, 98%, or 99% of the same amino acid sequence as amino acids 1 to 1274 of SEQ ID NO: 2. In some embodiments, the nucleic acid sequence encoding functional cMyBP-C contains at least 90%, 95%, 98%, or 99% of the same nucleotide sequence as SEQ ID NO: 1.

[0024] In the methods disclosed herein, the heart-specific transcriptional regulatory region may contain a fragment of the human TNNT2 promoter. The heart-specific transcriptional regulatory region may contain at least 90%, 95%, 98%, or 99% identical nucleotide sequences to SEQ ID NO: 51. The recombinant vector construct may contain introns or fragments thereof. The recombinant vector construct may further contain a polyadenylation signal, such as bovine growth hormone (bG) (SEQ ID NOs: 59-61) or human growth hormone (hGH) (SEQ ID NO: 62) polyadenylation signal.

[0025] In the methods disclosed herein, a population of rAAV particles generated by a method comprising: (a) providing one or more nucleic acid constructs suitable for host cells, preferably mammalian cells, the one or more nucleic acid constructs comprising: (i) a recombinant vector construct comprising (1) 5' AAV ITR and 3' AAV ITR, (2) a heart-specific transcriptional regulatory region, and (3) a nucleic acid encoding a functional cMyBP-C comprising an amino acid sequence that is at least 95% identical to amino acids 1 to 1274 of SEQ ID NO: 2; (ii) a nucleotide sequence encoding one or more AAV Rep proteins operatively linked to a promoter capable of driving the expression of the one or more Rep proteins in cells; and (iii) a nucleotide sequence encoding one or more AAV9 type capsid proteins operatively linked to a promoter capable of driving the expression of the one or more capsid proteins in cells; (b) culturing cells under conditions that allow expression of the Rep proteins and capsid proteins and the generation of AAV particles; and (c) recovering the AAV particles. Optionally, the population can be enriched with AAV particles containing the full-length or near-full-length vector genome by steps such as reducing the number of empty capsids, for example, regional ultracentrifugation or density gradient ultracentrifugation.

[0026] In some embodiments, the AAV capsid comprises at least 85%, 90%, or 95% of the same amino acid sequence as any of SEQ ID NO: 207-223. Preferably, the cardiotropic AAV capsid is an AAV9 type capsid, optionally at least 85%, 90%, or 95% identical to SEQ ID NO: 207. Preferably, the AAV capsid comprises at least 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO: 207.

[0027] In any of the methods described herein, rAAV particles are administered via intravenous infusion.

[0028] In any of the methods described herein, the subjects had heart disease associated with a gene mutation in the MYBPC3 gene. In some embodiments, the subjects had hypertrophic cardiomyopathy.

[0029] In any of the methods described herein, the dose preferably effectively improves cardiac function or structure, optionally as measured by a decrease in E / e' and / or a decrease in left atrial volume index and / or an improvement or normalization of ejection fraction and / or a reduction in longitudinal strain injury, and / or a decrease in left ventricular (LV) wall thickness and / or a reduction in LV mass. For example, improving or normalizing ejection fraction may include increasing or decreasing ejection fraction relative to a baseline identified prior to the administration of the effective dose. In some embodiments, the improvement may be at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (up to a 2-fold improvement) relative to baseline. For example, for a patient with an ejection fraction of 50%, the ejection fraction may be improved by about 20% or more to about 60% or more. In some embodiments, the dose effectively improves cardiac function and / or health-related quality of life for at least a period of about six months, optionally measured by one or more of the following: New York Heart Association (NYHA) Functional Classification, Kansas City Cardiomyopathy Questionnaire-23 (KCCQ-23) Total Symptom Score (TSS), Body Limitation Score and Clinical Summary Score (CSS), EuroQol-5 Dimensions Level 5 Version (EQ-5D-5L), EuroQol-Visual Analog Scale (EQ-VAS), Clinical Global Impression Severity and Variation Scale (CGI / C), and Patient Global Impression Severity and Variation Scale (PGIS / C). 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) (a cardiac stress indicator) is reduced by, for example, at least about 10%, 20%, 30%, 40%, or 50%.

[0030] The methods disclosed herein may further include administering to a subject a prophylacticly effective amount of corticosteroids or other systemic immunosuppressants. In some embodiments, the administered amount is effective in preventing or reducing biochemical or clinical signs of hepatotoxicity, liver inflammation, and / or cardiac inflammation.

[0031] In some embodiments, a preventative effective amount of corticosteroids or immunosuppressants is administered at a prednisone equivalent dose of about 0.5 mg / kg / day, optionally for a period of at least about 4 weeks, followed by administration of a gradually decreasing dose of corticosteroids for a period of about 4 weeks. In other embodiments, a preventative effective amount of corticosteroids or immunosuppressants is administered at a prednisone equivalent dose of at least about 40 mg / day.

[0032] In some embodiments, a preventatively effective amount of corticosteroids or immunosuppressants is administered concurrently with the rAAV particles of the present invention. In other embodiments, a preventatively effective amount of corticosteroids or immunosuppressants is administered concurrently with a preventatively effective amount of sirolimus. In some embodiments, a preventatively effective amount of sirolimus is administered at a dose of about 2 mg / day for a period of at least 8 weeks. In some embodiments, the amount of sirolimus effectively maintains a sirolimus blood level between about 5 and about 15 ng / mL. In some embodiments, a preventative TMP-SMX dose is administered orally three times weekly at a dose of 160 ± 800 mg.

[0033] The method may further include the following steps: (a) determining baseline levels of hepatotoxicity markers in the subject's blood prior to rAAV particle administration, and (b) subsequently, optionally, determining post-administration levels of the hepatotoxicity markers in the subject's blood weekly for at least 8 weeks. Alternatively, the method may further include the following steps: (a) determining baseline levels of hepatotoxicity, liver inflammation, and / or cardiac inflammation markers in the subject's blood prior to rAAV particle administration, and (b) subsequently, optionally, determining post-administration levels of the hepatotoxicity, liver inflammation, and / or cardiac inflammation markers in the subject's blood weekly for at least 8 weeks.

[0034] The methods disclosed herein may also include administering to a subject a preventatively effective amount of a complement pathway inhibitor or antagonist. In some embodiments, the administered amount effectively prevents or reduces biochemical or clinical signs of complement activation and / or thrombotic microangiopathy.

[0035] In some embodiments, a preventative effective amount of a complement pathway inhibitor or antagonist is administered concurrently with the administration of the rAAV particles of the present invention. The method may further include the steps of: (a) determining baseline levels of markers of complement activation and / or thrombotic microangiopathy in the blood of the subject prior to administration of the rAAV particles, and (b) subsequently, optionally, determining post-administration levels of the markers of complement activation and / or thrombotic microangiopathy in the blood of the subject weekly for at least 8 weeks. For example, markers of TMA may be D-dimer or erythrocyte fragmentation and thrombocytopenia observed in peripheral blood smears.

[0036] The methods disclosed herein may include administering a therapeutically effective amount of a glucocorticoid or other systemic immunosuppressant to a subject in response to hepatotoxicity, liver inflammation, and / or cardiac inflammation. Biochemical markers of hepatotoxicity may include ALT and / or AST elevations exceeding twice the upper limit of normal (ULN) or greater than or equal to twice the baseline level. Markers of myocarditis or cardiac inflammation may include abnormally increased levels of troponin I. The methods may include: (c) administering a therapeutically effective amount of a systemic immunosuppressant to a subject to reduce hepatotoxicity, liver inflammation, and / or cardiac inflammation after hepatotoxicity, liver inflammation, and / or cardiac inflammation has been detected by biochemical or clinical signs.

[0037] The methods disclosed herein may include administering a therapeutically effective amount of a complement pathway inhibitor or antagonist to a subject in response to complement activation, such as refractory complement activation and / or thrombotic microangiopathy. Markers of complement activation may include abnormally decreased levels of C3 or C4 in plasma (indicating consumption by complement activation), or abnormally increased levels of one or more complement cleavage products, such as C3a, C4a, Bb, and / or sC5b-9. Markers of thrombotic microangiopathy may include decreased platelet count, decreased platelet volume, decreased red blood cell (RBC) count, abnormal RBC morphology, hemolysis, and / or proteinuria. Clinical signs of thrombotic microangiopathy include complement activation, severe thrombocytopenia, microangiopathic hemolysis with cleavage cells, elevated transaminases, and / or acute kidney injury (AKI) progressing to renal failure. Methods may include: (c) administering a therapeutically effective amount of a complement pathway inhibitor or antagonist to a subject to reduce complement activation and / or thrombotic microangiopathy after complement activation, such as refractory complement activation, and / or thrombotic microangiopathy, is detected by biochemical or clinical signs.

[0038] In some embodiments, the prophylactic or therapeutic immunosuppressant is a glucocorticoid, optionally tacrolimus, mycophenolate mofetil, or eculizumab.

[0039] In some embodiments, the prophylactic or therapeutic immunosuppressant is a complement pathway inhibitor or antagonist, including any complement protein inhibitor or antagonist (e.g., inhibitors or antagonists of C3, C4, C5, etc.) or a MASP2 inhibitor (e.g., OMS721 antibody) and includes any of the following: pegcetacoplan (APL-2, EMPAVELI), eculizumab (SOLIRIS), avacopan (CCX168), vilobelimab (IFX-1), AMY-101, CINRYZE / BERINERT / HAEGARDA (human C1 esterase inhibitor, C1INH), ravulizumab (ALXN1210), coversin, cemdisiran (ALN-CC5), tesidolumab (LFG-316), iptacopan (LNP023), danicopan (ACH-4471), RUCONEST (recombinant C1 esterase inhibitor) or ENJAYMO (sutimlimab-jome, a C1s inhibitor).

[0040] The method disclosed herein may further include administering a prophylactic antimicrobial agent to the subject. In some embodiments, the prophylactic antimicrobial agent is a combination of sulfamethoxazole and trimethoprim. Attached Figure Description

[0041] Figure 1A Thermocapsid Integrity (TBCI) analysis is presented for various formulations containing AAV9-MYBPC3 heavy and superheavy capsids.

[0042] Figure 1B The TBCI analysis of various formulations is presented based on the percentage increase relative to the initial amount.

[0043] Figure 2A Shown on 8-week-old Rag2 - / - Eight weeks after administration of AAV9-hMYBPC3 to Rag2- / - mice, vector copies of each diploid genome of AAV9-MYBPC3 were measured.

[0044] Figure 2B Shown on 8-week-old Rag2 - / -Eight weeks after administration of AAV9-hMYBPC3 to the 2E14 vector genome / kg body weight (vg / kg), the mRNA / RPLP0 of AAV9-MYBPC3 in Rag2- / - mice was measured.

[0045] Figure 2C Shown on 8-week-old Rag2 - / - The percentage of human cMyBP-C protein in total AAV9-MYBPC3 in Rag2- / - mice 8 weeks after administration of AAV9-hMYBPC3 to the 2E14 vector genome / kg body weight (vg / kg).

[0046] Figure 2D Shown on 8-week-old Rag2 - / - Eight weeks after administration of AAV9-hMYBPC3 to the 2E14 vector genome / kg body weight (vg / kg), the total cMyBP-C protein of AAV9-MYBPC3 in Rag2- / - mice was measured.

[0047] Figure 3A The demonstration was based on wild-type (WT) mice and heterozygous MYBPC. + / - Mice and homozygous MYBPC3 - / - Data from natural history studies of mouse phenotypic development, specifically the ratio of left ventricular (LV) mass to body weight in mice.

[0048] Figure 3B Showing the posterior wall size of the LV in a mouse heart.

[0049] Figure 3C Demonstrates diastolic function of the mouse heart, mitral valve E wave (MV E).

[0050] Figure 4A The vector genome levels in the hearts of MYBPC3- / - mice and WT mice were depicted 8 weeks after administration of 2E14 vg / kg AAV9-MYBPC3 or the carrier agent in 2-week-old MYBPC3- / - mice and WT mice.

[0051] Figure 4B Transcript levels in the hearts of MYBPC3- / - mice and WT mice were depicted 8 weeks after administration of 2E14 vg / kg AAV9-MYBPC3 or mediator in 2-week-old MYBPC3- / - mice and WT mice.

[0052] Figure 4C Protein levels in the hearts of MYBPC3- / - mice and WT mice were depicted 8 weeks after administration of 2E14 vg / kg AAV9-MYBPC3 or a mediator in 2-week-old MYBPC3- / - mice and WT mice.

[0053] Figure 4D Total protein in the hearts of MYBPC3- / - mice and WT mice was depicted 8 weeks after administration of 2E14 vg / kg AAV9-MYBPC3 or a mediator in 2-week-old MYBPC3- / - mice and WT mice.

[0054] Figure 4E Determine the heart weight to body weight ratio of MYBPC3- / - mice and WT mice 8 weeks after administration.

[0055] Figure 5A This study demonstrates the MYBPC3 vector genome copy of each diploid genome in the heart of MYBPC3- / - mice 8 weeks after administration.

[0056] Figure 5B The levels of MYBPC3 transcripts in the hearts of MYBPC3- / - mice were shown 8 weeks after administration.

[0057] Figure 5C The cMyBP-C protein level in the heart of MYBPC3- / - mice was shown 8 weeks after administration of AAV9-MYBPC3.

[0058] Figure 5D The percentage of cardiomyocytes expressing cMyBP-C protein in the hearts of MYBPC3- / - mice 8 weeks after administration of AAV9-MYBPC3 is shown.

[0059] Figure 5E The heart weight to body weight ratio of MYBPC3- / - mice was depicted 8 weeks after administration of AAV9-MYBPC3 or the mediator.

[0060] Figure 6A The LV mass to body weight ratio of MYBPC3- / - mice and WT mice was displayed 26 weeks after administration of 6E13 or more AAV9-mMYBPC3, AAV9-hMYBPC3 or a mediator.

[0061] Figure 6B The posterior wall size of the heart in MYBPC3- / - mice and WT mice 26 weeks after administration is shown.

[0062] Figure 6C The cardiac output of the hearts of MYBPC3- / - mice and WT mice after administration is shown.

[0063] Figure 6D The cardiac output of the hearts of MYBPC3- / - mice and WT mice after administration is shown.

[0064] Figure 6EDemonstrates diastolic function, MV E, of the heart in MYBPC3- / - mice and WT mice after administration.

[0065] Figure 7A Data were collected to assess the in vivo transduction efficiency of cardiomyocytes in the hearts of MYBPC3- / - mice and wild-type mice at 0, 4, 8, 12, 20, 28, and 36 weeks after administration of 6E13 vg / kg or 2E14 vg / kg AAV9-MYBPC3 or a mediator. Figure 7A Show the LV weight to body weight ratio of MYBPC3- / - mice and wild-type mice at 0, 4, 8, 12, 20, 28 and 36 weeks after administration of 6E13 vg / kg or 2E14 vg / kg AAV9-MYBPC3 or mediator.

[0066] Figure 7B The posterior wall size of the heart LV in MYBPC3- / - mice and WT mice after administration is shown.

[0067] Figure 7C Demonstrates diastolic function, MV E, of the heart in MYBPC3- / - mice and WT mice after administration.

[0068] Figure 8A The study demonstrated that, compared with MYBPC3- / - mice administered the causative agent at 2 weeks of age, the LV mass to body weight ratio was reduced in MYBPC3- / - mice 36 weeks after administration of 6E13 vg / kg or 2E14 vg / kg AAV9-MYBPC3.

[0069] Figure 8B The study showed that, compared with MYBPC3- / - mice administered the causative agent at 2 weeks of age, 36 weeks after AAV9-MYBPC3 administration, the anterior wall thickness of the left ventricle (LV) in the heart of MYBPC3- / - mice was reduced.

[0070] Figure 8C The study showed that, compared with MYBPC3- / - mice administered the causative agent at 2 weeks of age, 36 weeks after AAV9-MYBPC3 administration, the posterior wall thickness of the left ventricle (LV) in the heart of MYBPC3- / - mice was reduced.

[0071] Figure 8D The study demonstrated that, compared with MYBPC3- / - mice administered the mediator at 2 weeks of age, MYBPC3- / - mice showed improved cardiac MVE (passive filling) 36 weeks after AAV9-MYBPC3 administration.

[0072] Figure 9The cMyBP-C protein levels in the hearts of wild-type mice at 4, 8, 12 and 16 weeks after administration of 2E14 vg / kg AAV9-MYBPC3 to 8-week-old male WT mice are shown.

[0073] Figure 10A The levels of MYBPC3 copies / ng DNA in the hearts of MYBPC3- / - mice and WT mice 13 weeks after administration were shown.

[0074] Figure 10B The levels of MYBPC3 transcripts / ng RNA in the hearts of MYBPC3- / - mice and WT mice were shown 13 weeks after administration.

[0075] Figure 10C The total cMyBP-C protein level in the heart tissue of MYBPC3- / - mice and WT mice 13 weeks after administration is shown.

[0076] Figure 10D The human cMyBP-C protein level in the heart tissue of MYBPC3- / - mice and WT mice 13 weeks after administration is shown.

[0077] Figure 10E The percentage of cardiomyocytes from the hearts of MYBPC3- / - mice and WT mice expressing human cMyBP-C protein is shown 13 weeks after administration.

[0078] Figure 11A The study showed that, compared with MYBPC3- / - mice that were administered the causative agent at 2 weeks of age, the ratio of heart weight to tibia length was reduced in MYBPC3- / - mice 13 weeks after administration of AAV9-MYBPC3.

[0079] Figure 11B The study showed that, compared with MYBPC3- / - mice that were administered the causative agent at 2 weeks of age, MYBPC3- / - mice had a reduced LV mass to body weight ratio 12 weeks after AAV9-MYBPC3 administration.

[0080] Figure 11C The study demonstrated that diastolic function (MVE) in the heart of MYBPC3- / - mice was improved 12 weeks after administration of AAV9-MYBPC3 compared with MYBPC3- / - mice administered the mordant at 2 weeks of age.

[0081] Figure 12 The vector genome distribution in cardiac tissue from the heart of a cynomolgus monkey is shown at 31 and 62 days after administration of 1.2E14 vg / kg AAV9-MYBPC3.

[0082] Figure 13 The levels of vector-derived hMYBPC3 transcripts in cardiac tissue from cynomolgus monkey hearts were shown at 31 and 62 days after administration of 1.2E14 vg / kg AAV9-MYBPC3.

[0083] Figure 14 The expression of cMyBP-C protein in patient-derived induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) transduced with AAV9-MYBPC3, generated in either the fall armyworm (Spodoptera frugiperda, Sf) insect cell system or the mammalian (i.e., human) embryonic kidney 293 (HEK293) cell system. Detailed Implementation

[0084] This article provides formulations and methods for treating human subjects with heart disease by administering rAAV particles containing a gene encoding a therapeutic protein for treating heart disease. Preferably, the heart disease is caused by a deficiency of functional wild-type cMyBP-C (e.g., HCM) and a gene encoding functional cMyBP-C. Human subjects may be adults (e.g., individuals aged 18 to 65 years or at least 18 years), infants, children, or adolescents, such as individuals up to 2, 2–4, 2–6, or 2–12 years of age, or at least 12 years of age.

[0085] In some embodiments, the rAAV particle comprises (a) a cardiotropic AAV capsid and (b) a recombinant vector construct (AAV vector genome) containing a gene encoding a functional cMyBP-C. In some embodiments, this vector construct comprises (a) a nucleic acid containing an AAV2 5' inverted terminal repeat (ITR) sequence (which may be modified or unmodified, as known in the art), (b) a cardiomyocyte-specific transcriptional regulatory region, (c) a functional cMyBP-C protein-coding region, (d) optional one or more introns, (e) a polyadenylated sequence, and (f) an AAV2 3' ITR (which may be modified or unmodified, as known in the art).

[0086] The functional cMyBP-C coding sequence can be wild-type, codon-optimized, or a variant. As used herein, the wild-type cardiac myosin-binding protein C (MYBPC3) gene has the nucleic acid sequence of SEQ ID NO: 1 (GenBank accession number NM_000256.2), and the wild-type cardiac myosin-binding protein C has the amino acid sequence of SEQ ID NO: 2 (GenBank accession number NP_000247.1).

[0087] definition

[0088] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd Edition, J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). For the purposes of this disclosure, the following terms are defined as follows.

[0089] As used herein, in the context of gene delivery, the term "vector" or "gene delivery vector" can refer to a particle that acts as a gene delivery medium and contains nucleic acids (i.e., a vector genome containing any of the vector constructs described herein) packaged within, for example, an envelope or capsid. Gene delivery vectors can be viral gene delivery vectors or non-viral gene delivery vectors. Alternatively, in some contexts, the term "vector" can be used to refer only to the vector genome or vector construct. Viral vectors suitable for use herein can be parvoviruses, adenoviruses, retroviruses, lentiviruses, or herpes simplex viruses. Parvoviruses can be adenovirus-associated viruses (AAVs).

[0090] As used herein, the term “AAV” is the standard abbreviation for adeno-associated virus. Adeno-associated viruses are single-stranded DNA parvoviruses that grow only in cells where some of their functions are provided by co-infected helper viruses. Various AAV serotypes have been characterized. General information and commentary on AAV can be found, 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 entirely expected that these same principles will apply to other AAV serotypes, as it is well known that the various serotypes are very closely related structurally and functionally, even at the genetic level. (See, for example, Blacklowe, 1988, Parvoviruses and Human Disease, pp. 165-174, ed. JR Pattison; and Rose, Comprehensive Virology 3:1-61 (1974)). For instance, all AAV serotypes exhibit remarkably similar replication properties mediated by homologous rep genes; and all carry three associated capsid proteins. Heteroduplex analysis further demonstrates the degree of correlation, revealing extensive cross-hybridization along the genome length between serotypes; and similar self-annealing regions at the ends corresponding to inverted terminal repeats (ITRs).

[0091] As used herein, an “AAV vector construct” refers to a single-stranded or double-stranded nucleic acid having one of the following: (i) an AAV 5' inverted terminal repeat (ITR) sequence and (ii) an AAV 3' ITR, side-attached to a protein-coding sequence (in one embodiment, a functional therapeutic protein-coding sequence, such as the cMyBP-C coding sequence), operatively linked to a transcriptional regulatory element (also referred to as an “expression control element”) heterologous to the protein-coding sequence and / or heterologous to the AAV viral genome, i.e., one or more promoters and / or enhancers and optional polyadenylation sequences and / or optional one or more introns. A single-stranded AAV vector refers to a nucleic acid present in the genome of an AAV viral particle and may be the sense or antisense strand of the nucleic acid sequence disclosed herein. The size of such single-stranded nucleic acids is provided in base pairs. A double-stranded AAV vector refers to a nucleic acid present in the DNA of a plasmid (e.g., pUC19) used to express or transfer AAV vector nucleic acids or in the genome of a double-stranded virus (e.g., baculovirus). The size of such double-stranded nucleic acids is provided in base pairs (bp).

[0092] The AAV vector constructs provided herein in single-chain form have a length of less than about 7.0 kb, or less than 6.5 kb, or less than 6.4 kb, or less than 6.3 kb, or less than 6.2 kb, or less than 6.0 kb, or less than 5.8 kb, or less than 5.6 kb, or less than 5.5 kb, or less than 5.4 kb, or less than 5.3 kb, or less than 5.2 kb. The length of the single-chain AAV vector construct is also at least about 4.0 kb. Preferably, the length of the AAV vector construct is also at least about 4.5 kb. In some embodiments, the length of the AAV vector constructs provided herein in single-chain form is in the range of about 4.0 kb to about 5.8 kb.

[0093] Although AAV particles with AAV genomes >5.0 kb have been reported in the literature, in many such 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)). However, it has been shown that overlapping homologous recombination occurs between 5'-truncated and 3'-truncated nucleic acids in AAV-infected cells, resulting in the production of complete nucleic acids encoding large proteins, thereby reconstructing functional full-length genes.

[0094] Oversized AAV vectors are randomly truncated at the 5' end and lack a 5' AAV ITR. Since AAVs are single-stranded DNA viruses and package either a sense or antisense strand, the sense strand in an oversized AAV vector lacks a 5' AAV ITR and may lack the 5' end portion of the target protein encoding gene, while the antisense strand in an oversized AAV vector lacks a 3' ITR and may lack the 3' end portion of the target protein encoding gene. Functional transgenes are generated in cells infected with oversized AAV vectors by annealing the truncated sense and antisense genomes within the target cells. Therefore, in some embodiments, the AAV cMyBP-C vector and / or viral particles contain at least one ITR.

[0095] As used herein, the term "terminal inverted repeat (ITR)" refers to the industry-recognized regions located at the 5' and 3' ends of the AAV genome that function as a cis-initiated DNA replication origin and as a packaging signal for the viral genome. The AAV ITR, together with the AAV rep coding region, efficiently excises and rescues nucleotide sequences inserted between two flanking ITRs from the host cell genome and integrates them into the host cell genome. Sequences of certain AAV-associated ITRs are disclosed by Yan et al., J. Virol. 79: 364-79 (2005), which are incorporated herein by reference in their entirety. ITR sequences used herein may be full-length wild-type AAV ITRs or fragments thereof that retain their functional capabilities, or sequence variants of full-length wild-type AAV ITRs capable of functioning as a cis-initiated replication origin. AAV ITRs in the recombinant AAV cMyBP-C vectors used in the embodiments provided herein may be derived from any known AAV serotype, and in some embodiments, from AAV2 or AAV5 serotypes.

[0096] The term "control sequence" refers to the DNA sequence necessary for the expression of an operable coding sequence in a specific host organism. Control sequences applicable to prokaryotes include, for example, promoters, optional operon sequences, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0097] "Transcriptional regulatory element" refers to the nucleotide sequence of a gene involved in the regulation of genetic transcription, including a promoter, plus a response element, an activator and enhancer sequence for binding transcription factors to assist RNA polymerase binding and promote expression, and an operon or silencer sequence for binding repressor proteins to block RNA polymerase attachment and prevent expression. The terms "cardiac-specific transcriptional regulatory element" or "cardiac-specific expression control element" refer to a regulatory element or region that specifically produces preferred gene expression in cardiac cells, such as a promoter whose activity in cardiac cells is at least 2 to 5 times that in any other non-cardiac cell type. Preferably, the cardiac-specific expression control element is cardiomyocyte-specific and provides at least 5 times the expression in cardiomyocytes compared to skeletal muscle cells. In some embodiments, the activity of the cardiomyocyte-specific promoter in cardiomyocytes is at least 5 to 10 to 15 to 20 to 25 to 50 times that in non-cardiomyocytes.

[0098] A heart-specific or cardiomyocyte-specific promoter is operatively linked to a nucleic acid sequence encoding the cMyBP-C protein. This means that the promoter is combined with the encoding nucleic acid to enable the expression of the encoded nucleic acid under promoter control in cardiomyocytes, whether integrated into the cell's genome or present in the cell as an extragenomic nucleic acid construct.

[0099] Transcriptional regulatory elements optionally include enhancer elements, introns, polyadenylation sequences, or posttranscriptional regulatory elements for increasing the expression level of myosin-binding proteins. Examples include enhancers of the early SV40 gene and enhancers of the long terminal repeat (LTR) sequence of Rous sarcoma virus (Gorman et al. (1982) Proc. Natl. Acad. Sci. 79:6777). The vector also optionally contains transcription termination sequences and polyadenylation sequences to improve the expression of human and / or non-human antigens. Suitable transcription termination sequences and polyadenylation signals may be derived, for example, from SV40 (Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual). Preferably, the bGH polyadenylation signal is used in the vector of the present invention. Any other elements known in the art that support expression efficiency or specificity may be added to the expression vector, such as the marmot hepatitis posttranscriptional regulatory element (wPRE). To increase 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)).

[0100] As used herein, an "intron" is broadly defined as a nucleotide sequence that can be removed by RNA splicing. "RNA splicing" refers to the removal of introns from pre-mRNA to form mature mRNA. Introns can be upstream, downstream, or within the coding region of a gene. Insertion of an intron into a nucleotide sequence can be achieved by any method known in the field. The only limitation on the location of intron insertion is considering the packaging limitations of AAV viral particles (e.g., approximately 5 kb).

[0101] As used herein, the term "operably linked" describes the connection between a regulatory element and a gene or its coding region. Typically, gene expression is under the control of one or more regulatory elements, such as, but not limited to, constitutive or inducible promoters, tissue-specific regulatory elements, and enhancers. To say that a gene or coding region is "operably linked" or "operably associated" with a regulatory element means that the gene or coding region is controlled or influenced by the regulatory element. For example, if a promoter affects the transcription or expression of a coding sequence, then the promoter is operably linked to the coding sequence.

[0102] When used with respect to nucleic acid molecules of this disclosure, the term "isolated" generally refers to a nucleic acid sequence identified and isolated from at least one contaminating nucleic acid, which typically associates with contaminating nucleic acids in its natural source. Isolated nucleic acids may exist in forms or environments different from those found in nature. Therefore, isolated nucleic acid molecules differ from nucleic acid molecules present in natural cells.

[0103] As used herein, the term "variant" refers to a polynucleotide (or polypeptide) having a sequence substantially similar to that of a reference polynucleotide (or polypeptide). This document covers variants encoding functional therapeutic proteins, such as functional cMyBP-C proteins that maintain cMyBP-C activity. Procedures for introducing nucleotide and amino acid changes into polynucleotides, proteins, or polypeptides are known to those skilled in the art (see, for example, Sambrook et al. (1989)). In the case of polynucleotides, variants may have one or more nucleotide deletions, substitutions, or additions at the 5' end, 3' end, and / or one or more internal sites compared to a reference polynucleotide. Sequence similarity and / or differences between variants and reference polynucleotides can be detected using conventional techniques known in the art, such as polymerase chain reaction (PCR) and hybridization techniques. Variant polynucleotides also include synthetically derived polynucleotides, such as those produced by site-directed mutagenesis. Typically, variants of polynucleotides (including, but not limited to, DNA) may have at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater sequence identity with a reference polynucleotide, as determined by sequence alignment procedures known to those skilled in the art. In the case of peptides, variants may have one or more amino acid deletions, substitutions, or additions compared to a reference peptide. Sequence similarity and / or differences between variants and reference peptides can be detected using conventional techniques known in the art, such as Western blot. Typically, a variant of a polypeptide may have at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater sequence identity with a reference polypeptide, as determined by sequence alignment procedures known to those skilled in the art.

[0104] Amino acid substitutions can be conserved or non-conserved. Conservative substitutions are preferred, where an amino acid residue is replaced by an amino acid of similar polarity to serve as a functional equivalent. Preferably, the amino acid residue used as a substituent is selected from the same group of amino acids as the amino acid residue to be substituted. For example, a hydrophobic residue can be replaced by another hydrophobic residue, or a polar residue can be replaced by another polar residue with the same charge. Functional homologous amino acids that can be used for conserved substitution include, for example, nonpolar amino acids such as glycine, valine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, and tryptophan. Examples of uncharged polar amino acids include serine, threonine, glutamine, asparagine, tyrosine, and cysteine. Examples of charged polar (basic) amino acids include histidine, arginine, and lysine. Examples of electrically charged (acidic) amino acids include aspartic acid and glutamic acid.

[0105] It is also believed that proteins differing from their natural counterparts in that those with the addition, substitution, or deletion of one or more (e.g., 2, 3, 4, 5, 10, or 15) extra amino acids are variants. For example, the extra amino acid may be present within the amino acid sequence of the original cMyBP-C protein (i.e., as an insertion), or it may be added to one or both ends of the protein. Such insertions, substitutions, or deletions can occur at any position, provided they do not impair the peptide's ability to perform the function of the naturally occurring cMyBP-C protein and / or rescue haploid deficiency in the treated subject. Furthermore, variants of the cMyBP-C protein also include proteins lacking one or more amino acids compared to the original peptide. Such deletions can affect any amino acid position, provided they do not impair the normal function of the cMyBP-C protein and / or rescue haploid deficiency. To visualize exogenous gene expression in the heart, other optional elements can be introduced as part of the cMyBP-C coding sequence, such as tag sequences (myc, FLAG, HA, His, etc.) or fluorescent dyes, such as GFP, YFP, and RFP.

[0106] Finally, variants also refer to proteins that differ from naturally occurring proteins in terms of structural modifications, such as modified amino acids. Modified amino acids are those that have been modified by natural processes (e.g., processing or post-translational modification) or by chemical modification processes known in the art. Typical amino acid modifications include phosphorylation, glycosylation, acetylation, O-linked N-acetylglucosamineization, glutathioneization, acylation, branching, ADP ribosylation, cross-linking, disulfide bridge formation, formylation, hydroxylation, carboxylation, methylation, demethylation, amidation, cyclization, and / or covalent or non-covalent bonding to phosphatidylinositol, flavin derivatives, lipoteichoic acid, fatty acids, or lipids. Such modifications have been extensively described in the literature, for example, in *Proteins: Structure and Molecular Properties*, T. Creighton, 2nd edition, WH Freeman and Company, New York (1993). In a preferred embodiment of the invention, the nucleic acid sequence encodes a constitutive phosphorylation 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 USA 103:16918-16923).

[0107] The terms “identity,” “homology,” and their grammatical variations refer to two or more mentioned entities being identical when they are “aligned” sequences. Thus, for example, when two polypeptide sequences are identical, they have the same amino acid sequence at least within the mentioned region or portion. In the case of two identical polynucleotide sequences, they have the same polynucleotide sequence at least within the mentioned region or portion. Identity can be defined on a region (region or domain) of a sequence. A “region” or “domain” with identity refers to an identical portion of two or more reference entities. Thus, when two polypeptide or nucleic acid sequences are identical on one or more sequence regions or domains, they share identity within said regions. An “aligned” sequence refers to multiple polynucleotide or polypeptide (amino acid) sequences that, compared to a reference sequence, typically contain corrections (vacancies) for deletions or extra bases or amino acids. “Bulk homology” means that a molecule is structurally or functionally conserved such that it has, or is predicted to have, at least part of, a structure or function (e.g., biological function or activity) of one or more structures or functions of a reference molecule, or shares a related / corresponding region or portion of a reference molecule with which it shares homology.

[0108] "Nucleic acid sequence identity or homology percentage (%)" is defined as the percentage of nucleotides in a candidate sequence that are identical to a reference sequence after alignment of the corresponding sequences and, where necessary, the introduction of vacancies to achieve the maximum sequence identity percentage. Alignment for the purpose of determining the nucleic acid sequence identity percentage can be performed in various ways within the scope of the art, such as using publicly available computer software, such as ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring the alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0109] The "percentage of amino acid sequence identity or homology (%)" for the cMyBP-C amino acid sequence identified herein is defined as the percentage of amino acid residues in the candidate sequence that are identical to those in cMyBP-C, after sequence alignment and, where necessary, the introduction of vacancies to achieve the maximum percentage of sequence identity, and without considering any conserved substitutions as part of sequence identity. Alignments for determining the percentage of amino acid sequence identity can be performed in various ways within the scope of the art, such as using publicly available computer software, such as ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignments, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0110] "Codon optimization" refers to changes made to the nucleotide sequence that make it more likely to be expressed at a relatively high level compared to an unoptimized sequence. It does not change the amino acid encoded by each codon.

[0111] "AAV virion," "AAV virus particle," "AAV vector particle," or "AAV virus" refers to a viral particle composed of at least one AAV capsid protein and an AAV vector construct encapsulated as described herein. If the particle contains heterologous polynucleotides (i.e., polynucleotides other than the wild-type AAV genome, such as transgenes intended for delivery to mammalian cells), it is generally referred to as a "recombinant AAV vector particle" or simply "AAV vector." The generation of an AAV vector particle necessarily includes the generation of an AAV vector genome, as the vector genome is contained within the AAV vector particle. It should be understood that references to the polynucleotide AAV vector construct encapsulated within the vector particle and its replication refer to the AAV vector genome.

[0112] As used herein, “therapeutic AAV virus” refers to an AAV virion, AAV virus particle, AAV vector particle, or AAV virus containing a heterologous polynucleotide encoding a therapeutic protein (e.g., cMyBP-C as described herein). As used herein, “AAV vector construct” or “AAV vector genome” refers to a vector construct containing one or more polynucleotides encoding a protein of interest (also known as a transgene), said polynucleotide being side-joined with at least one AAV terminal repeat sequence (ITR) and operatively linked to one or more expression control elements. Such AAV vector constructs can replicate and be packaged into infectious viral particles when present in host cells transfected with a vector encoding and expressing the rep and cap gene products. The term generally refers to recombinant AAV capable of infecting cells, causing the infected cells to express (e.g., through transcription and / or through translation) the element of interest (e.g., nucleotide sequence, protein, etc.). In this regard, therapeutically effective rAAV particles may include AAV particles having a capsid or vector genome (vg) with different properties. For example, therapeutically effective rAAV particles may have capsids with different post-translational modifications. In other instances, therapeutically effective AAV particles may contain vector genomes of varying sizes / lengths, positive and negative strand sequences, different positive / negative ITR configurations (positive / negative, negative / positive, positive / positive, negative / negative, etc.), different numbers of ITRs (1, 2, 3, etc.), or truncated vector genomes. For instance, overlapping homologous recombination occurs between 5'-truncated and 3'-truncated nucleic acids in AAV-infected cells, resulting in a "complete" nucleic acid encoding a large protein, thereby reconstructing a functional full-length gene. In other instances, complementary nucleic acid sequences with 5' and 3' truncated ends interact to form a "complete" nucleic acid during second-strand synthesis. The "complete" nucleic acid encodes a 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, "treatment-ineffective" AAV viruses refer to those with empty capsids, or capsids containing vector genomes that cannot be quantified or detected, or vector genomes that cannot be reassembled into complete functional nucleic acids.

[0113] As used herein, "therapeutic protein" refers to a polypeptide that has biological activity that replaces or compensates for the loss or reduction of activity of endogenous proteins. For example, functional cMyBP-C protein is a therapeutic protein for HCM.

[0114] As used herein, "hypertrophic cardiomyopathy" refers to a hereditary disease caused by gene mutations encoding components of the cardiac sarcomere (e.g., cardiac myosin-binding protein C), characterized by, for example, heart failure, arrhythmias, chest pain, shortness of breath, fatigue and dizziness, increased heart size, increased cardiothoracic ratio, increased ventricular wall thickness (anterior or posterior, or both), shortened ejection time, decreased peak aortic velocity, and / or shortened aortic blood flow time. End-diastolic and end-systolic dimensions may also be smaller than normal due to increased wall thickness leading to a smaller cavity.

[0115] As used in this article, "cardiac myosin-binding protein C deficiency" or "functional wild-type cardiac myosin-binding protein C deficiency" refers to a genetic disorder caused by a decrease in the level of functional cMyBP-C protein, the absence of the protein, reduced protein production, or the production of non-functional protein. This includes HCM.

[0116] As used in this article, “effective treatment for hypertrophic cardiomyopathy” or “hypertrophic cardiomyopathy therapy” refers to any therapeutic intervention in subjects with HCM who improve characteristic functional wild-type cMyBP-C deficiency, increase cMyBP-C protein levels, such as in the myocardium, improve HCM symptoms, or reduce the frequency, duration, or severity of HCM symptoms.

[0117] As used herein, “gene therapy for hypertrophic cardiomyopathy” refers to any therapeutic intervention for a subject with HCM involving replacement, restoration, or enhancement by delivering one or more nucleic acid molecules to the subject’s cells expressing functional cMyBP-C. In some embodiments, MYBPC3 gene therapy refers to gene therapy involving adeno-associated virus (AAV) particles comprising a vector construct expressing human cMyBP-C. In other embodiments, gene therapy involves transfecting plasmids expressing human cMyBP-C.

[0118] As used in this article, “treat / treatment” refers to preventative or therapeutic treatment administered to a subject exhibiting pathological signs or symptoms (i.e., HCM) with the aim of reducing or eliminating those signs or symptoms or improving their progression, severity, or duration. Signs or symptoms can be biochemical, cellular, histological, functional, subjective, or objective.

[0119] As used in this article, “improvement” refers to the reduction of the severity, progression, or duration of disease symptoms.

[0120] As used herein, “stably treating” or “stable treatment” refers to the use of a therapeutic vector construct, AAV particle, or cell administered to a subject, wherein the subject stably expresses a therapeutic protein expressed by the vector construct, AAV particle, or cell. Stably expressed therapeutic protein refers to a clinically significant duration of protein expression. As used herein, “clinically significant duration” means a duration of expression at a therapeutically effective level that has a meaningful impact on the subject’s quality of life, for example, demonstrated by a reduction in signs or symptoms of disease. In some embodiments, a clinically significant duration is expression for at least six months, at least eight months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, at least ten years, or the subject’s life.

[0121] As used herein, the term “effective amount” refers to an amount sufficient to achieve beneficial or desired biological and / or clinical outcomes.

[0122] Generally, a "pharmaceuticalally acceptable carrier" is a carrier that is non-toxic or excessively harmful to cells and preferably sterile. Exemplary pharmaceutically acceptable carriers include sterile pyrogenic water and sterile pyrogenic saline, tris(hydroxymethyl)aminomethane, or phosphate-buffered saline. Pharmaceutically acceptable carriers include physiologically acceptable carriers. The term "pharmaceuticalally acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents, etc.

[0123] AAV carrier

[0124] The recombinant vector construct disclosed herein can be used on its own as a gene therapy, or can be used to generate rAAV particles by means of methods described herein, the methods comprising providing the recombinant vector construct and the Rep and Cap genes to suitable host cells. The vector construct described herein contains a nucleic acid sequence encoding a functional cMyBP-C. The recombinant vector construct may contain nucleic acid encoding a functional human cMyBP-C, said nucleic acid being operatively linked to a heterologous expression control element, such as a promoter and / or enhancer; optional introns; and optional polyadenylation (poly-A) signaling. The heterologous expression control element may be, for example, a heterologous cardiomyocyte-specific transcriptional regulatory region as described herein.

[0125] The rAAV particle may comprise (a) a cardiotropic AAV capsid, (b) a recombinant vector construct containing a cardio-specific transcriptional regulatory region, and (c) nucleic acid encoding a functional cMyBP-C protein. The recombinant vector construct may further comprise (i) one or both of an AAV 5' inverted terminal repeat (ITR) sequence and (ii) an AAV 3' ITR, optionally wherein the AAV ITR is an AAV2 ITR. The vector construct may include additional expression control elements, such as: promoters and / or enhancers; introns; optional exons or fragments thereof; and polyadenylation (poly-A) signals. Such elements are further described herein.

[0126] Preferably, the nucleic acid encoding functional cMyBP-C is operatively linked to a cardiomyocyte-specific expression control element. In some embodiments, the recombinant AAV vector construct comprises a nucleic acid including (a) an AAV2 5' inverted terminal repeat (ITR) sequence (which may be modified or unmodified, as known in the art), (b) a cardiomyocyte-specific transcriptional regulatory region, a functional MYBPC3 protein-coding region, (c) one or more introns, including fragments of longer introns, (d) a heterologous exon or fragment thereof optionally adjacent to the intron, (e) a polyadenylated sequence, and (f) an AAV2 3' ITR (which may be modified or unmodified, as known in the art).

[0127] Preferably, the rAAV particles further comprise a cardiotropic AAV capsid, optionally an AAV9 type capsid. Exemplary cardiotropic capsids include AAV1, AAV6, AAV7, and AAV9.

[0128] Other embodiments provided herein are vector constructs encoding functional cMyBP-C peptides, wherein the constructs comprise one or more individual elements of the above-described constructs and combinations thereof in one or more different orientations. Another embodiment provided herein is for the above-described constructs in opposite orientations.

[0129] The single-chain AAV vector constructs provided herein are in the following length ranges: approximately 4.5 kb to approximately 6.5 kb, or approximately 4.5 kb to approximately 5.5 kb, or approximately 4 kb to approximately 5.5 kb, or approximately 4.8 kb to approximately 5.2 kb, or approximately 4.8 kb to approximately 5.1 kb, or approximately 4.9 kb to approximately 5.5 kb, or approximately 4.8 kb to approximately 6.0 kb, or approximately 5.0 kb to approximately 6.2 kb, or approximately 5.1 kb to approximately 6.3 kb, or approximately 5.2 kb to approximately 6.4 kb, or approximately 5.5 kb to approximately 6.5 kb, or approximately 4.0 kb to approximately 5.0 kb, or approximately 4 to approximately 4.5 kb, or approximately 4.5 kb to approximately 5 kb.

[0130] When AAV vectors are generated from oversized recombinant vector constructs, they may lack a portion of the 5' or 3' end of the recombinant vector construct. Since AAVs are single-stranded DNA viruses and package with sense or antisense strands, the sense strand in an oversized AAV vector lacks a 5' AAV ITR and may lack the 5' end portion of the target protein encoding gene, and the antisense strand in an oversized AAV vector lacks a 3' ITR and may lack the 3' end portion of the target protein encoding gene. Functional transgenes are generated in cells infected with oversized AAV vectors by annealing the sense and antisense truncated genomes within the target cells. Therefore, in some embodiments, the rAAV particles of the present invention may comprise a recombinant vector construct containing at least one ITR and a substantial portion of the nucleotide sequence encoding functional cMyBP-C, for example, more than 50%, 60%, 70%, 80%, or 90% of the nucleotide sequence length of a fragment of SEQ ID NO: 1 or 42-46. For example, the recombinant vector construct may contain at least one ITR, a cardiomyocyte-specific transcriptional regulatory region, and a significant portion of the nucleotide sequence encoding functional cMyBP-C.

[0131] The generation of vector constructs can be achieved using any suitable genetic engineering techniques known in the field, including but not limited to standard techniques for restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing, as described by Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY (1989)).

[0132] Vector constructs can incorporate sequences from the genome of any known organism. The sequences can be incorporated in their natural form or modified in any way to achieve the desired activity. For example, the sequences can contain insertions, deletions, or substitutions.

[0133] When the AAV vector construct is present in a host cell transfected with a polynucleotide encoding and expressing the rep and cap gene products, the AAV vector construct can replicate and be packaged into infectious AAV particles, preferably replication-defective AAV particles.

[0134] Expression control elements: promoters and enhancers:

[0135] In one or more embodiments, the nucleic acid sequence encoding cMyBP-C is operatively linked to one or more heterologous expression control elements. Preferably, the expression control element is a cardiomyocyte-specific expression control element. Examples of cardiomyocyte-specific control elements include, but are not limited to, the human cardiac troponin T (hTNNT2) promoter or fragments or variants thereof. Other promoters active in cardiomyocytes include fragments or variants of any of the following: muscle creatine kinase (MCK) promoter, cytomegalovirus enhancer + myosin light chain 2 promoter (CMV-MLC2 or CMV-MLC1.5, CMV-MLC260), phosphoglycerate kinase (PGK) promoter, sarcomere-specific promoter, α-myosin heavy chain promoter, myosin light chain 2v promoter, α-myosin heavy chain promoter, α-cardiac actin promoter, α-tropomyosin promoter, cardiac troponin C promoter, cardiac troponin I promoter, cardiac myosin-binding protein C promoter and / or sarcoplasmic / endoplasmic reticulum Ca2+ ATPase (SERCA) promoter (e.g., isoform 2 (SERCA2) of this promoter) and / or striated muscle promoters, such as desmin promoters. Enhancers derived from cardiomyocyte-specific transcription factor binding sites are also included.

[0136] Examples of fragments or variants of the hTNNT2 promoter include at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical (in length of SEQ ID NO) to any of SEq ID NOs: 49-52 and 75-205. Other examples include any of SEQ ID NOs: 1 to 85 of U.S. Patent Publication No. 2021 / 0252165, which is incorporated herein by reference in its entirety.

[0137] In some embodiments, the fragment or variant of the hTNNT2 promoter is longer than 420 and less than 544 nucleotides and contains at least 90% identical nucleic acid sequence to SEQ ID NO: 51. In any of the embodiments described herein, cardiomyocyte-specific promoters optionally exclude any of SEQ ID NO: 1 to 85 of U.S. Patent Publication No. 2021 / 0252165.

[0138] In the vector constructs disclosed herein, various promoters are operatively linked to nucleic acids containing the coding region of a protein of interest (human cardiac myosin-binding protein C). In some embodiments, the promoter drives the expression of the protein of interest in cells (e.g., target cells) infected with a virus derived from a viral vector. The promoter may be naturally occurring or non-natural. In some embodiments, the promoter is a synthetic promoter. In one embodiment, a synthetic promoter contains a sequence not found in nature and designed to regulate the activity of an operatively linked gene. In another embodiment, a synthetic promoter contains a fragment of a natural promoter to form a new segment of a DNA sequence not found in nature. Synthetic promoters typically contain regulatory elements, promoters, enhancers, introns, splice donors, and receptors 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 transcriptional regulatory region contains one or more additional individual enhancer elements in one or more different orientations.

[0139] In some embodiments, the transcriptional regulatory region includes a promoter and introns that enhance the expression of the protein of interest, located at the 5' end relative to the therapeutic protein-coding sequence. For example, a cardiomyocyte-specific transcriptional regulatory region may further include (in addition to a fragment of a variant of the hTNNT2 protein) a globulin intron adjacent to the 3' end of a fragment of β-globulin exon 3 (SEQ ID NO: 53). A combination of introns and exon fragments is, for example, SEQ ID NO: 55. A combination of promoters, exon fragments, and introns is, for example, SEQ ID NO: 56.

[0140] The promoter is operatively linked to a polynucleotide encoding one or more proteins of interest. In some embodiments, the promoter is operatively linked to a polynucleotide encoding the cMyBP-C protein.

[0141] The size of the promoter can vary. Due to the limited packaging capacity of AAV, it is preferable to use a promoter that is small in size but still allows for high-level production of the protein of interest in the host cell. For example, in some embodiments, the promoter is up to about 1.5 kb, up to about 1.4 kb, up to about 1.35 kb, up to about 1.3 kb, up to about 1.25 kb, up to about 1.2 kb, up to about 1.15 kb, up to about 1.1 kb, up to about 1.05 kb, up to about 1 kb, up to about 800 base pairs, up to about 600 base pairs, up to about 400 base pairs, up to about 200 base pairs, or up to about 100 base pairs.

[0142] Various additional regulatory elements can be used in vector constructs, such as enhancers or introns that further increase the expression level of the protein of interest in the host cell, polyadenylation signals, ribosome binding sequences, and / or shared splice acceptors or splice donor sites. In some embodiments, regulatory elements can facilitate the extrachromosomal maintenance of recombinant DNA molecules in the host cell and / or improve vector efficacy (e.g., scaffold / matrix attachment region (S / MAR)). Such regulatory elements are well known in the art and are further described below.

[0143] The protein of interest and the nucleic acid encoding the protein of interest

[0144] Polynucleotides encoding one or more proteins of interest may be inserted into the viral vectors disclosed herein, wherein the polynucleotides are operatively linked to a promoter. "Protein of interest" includes any functional cMyBP-C protein, including naturally occurring and non-naturally occurring variants. In some cases, the promoter may drive the expression of the protein of interest in a specific type of host cell (e.g., human cardiomyocytes). In one or more embodiments, the functional cMyBP-C comprises at least 90%, 95%, or 98% of the same amino acid sequence as SEQ ID NO: 2 (human cardiac myosin-binding protein C). This disclosure also provides isolated nucleic acid molecules encoding such functional wild-type cMY-BP-C proteins. In exemplary embodiments, the nucleic acid sequence encoding functional cMyBP-C is a wild-type MYBPC3 sequence, SEQ ID NO: 1 being one example, or codon-optimized, or a variant. The vector constructs described herein may comprise nucleotide sequences different from the wild-type nucleotide sequence but still encoding a functional cMyBP-C amino acid sequence that is at least 90%, 95%, or 98% the same as SEQ ID NO: 2. Accordingly, the nucleotide sequence may contain a portion having at least 80%, 85%, 90% or 95% homology to at least 100 consecutive bases of SEQ ID NO: 1 or 42-46, provided that the nucleotide sequence encodes a functional human cMyBP-C protein that is at least 90%, 95% or 98% identical to SEQ ID NO: 2.

[0145] As described herein, the nucleotide sequence encoding the cMyBP-C protein can be modified to improve protein expression efficiency. Methods for improving the transcription and / or translation of the gene described herein are not particularly limited. For example, the nucleotide sequence can be codon-optimized or undergo CpG reduction. As another non-limiting example of modification, one or more splice donors and / or splice acceptors in the nucleotide sequence of the protein of interest can be modified to reduce the likelihood of foreign splicing. As another non-limiting example of modification, one or more introns can be inserted into or near the nucleotide sequence of the protein of interest to optimize AAV vector packaging and enhance expression.

[0146] Since the generated cMyBP-C is functional, it will have at least a portion of the same conformation as wild-type cMyBP-C. In some embodiments, the functional cMyBP-C protein generated as described herein effectively treats subjects suffering from wild-type cMyBP-C protein deficiency and / or HCM.

[0147] Introns

[0148] In some embodiments, the vector comprises one or more introns. Introns may facilitate the processing of RNA transcripts in mammalian host cells, increase the expression of the protein of interest, and / or optimize vector packaging into AAV particles. Non-limiting examples of such introns include human β-globulin introns, human immunoglobulin G (IgG) introns, or native cMyBP-C introns. In some embodiments, the intron is a chimeric intron and comprises a fragment of a human IgG intron.

[0149] Examples of introns that enhance cMyBP-C protein expression include nucleotide sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to any of SEq ID NO: 53 or 58. In some embodiments, the intron and optionally an exon or a fragment thereof are located at 5' relative to the cMyBP-C coding sequence. In other embodiments, the intron is located within the nucleotide sequence encoding cMyBP-C, for example, between any exons. In some embodiments, the intron sequence is located between exons 2 and 3. In some embodiments, the intron sequence is located within the nucleic acid encoding cMyBP-C at position 293 of SEQ ID NO: 1.

[0150] 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 exemplary embodiments, the intron comprises SEQ ID NO: 53 [globulin intron] or a fragment of SEQ ID NO: 53 of about 50-150 nucleotides, 75-145 nucleotides, 100-135 nucleotides, or 120-135 nucleotides, or a variant of the fragment being at least 80%, 85%, 90%, or 95% identical to said fragment.

[0151] In some embodiments, the vector construct may further comprise an exon sequence or a fragment thereof; preferably adjacent to the 5' or 3' end of an intron sequence. In an exemplary embodiment, the vector construct comprises a globulin intron adjacent to the exon, which contains at least 80% or 85% or 90% or 95% of the same nucleotide sequence as any of SEQ ID NO: 55-57 [intron + exon]

[0152] Including intronic elements enhances expression compared to expression without intronic elements (see, for example, Kurachi et al., J. Biol. Chem. 270(10): 5276-81 (1995)). AAV vectors typically accept DNA inserts in a defined size range, usually about 4 kb to about 5.4 kb or slightly larger. However, there is no minimum packaging size, and small vector genomic packaging is very efficient. Introns and intronic fragments satisfy this requirement while also enhancing expression. Therefore, this disclosure is not limited to including the cMyBP-C intron sequence in AAV vectors, but also includes other introns or other DNA sequences that replace portions of the cMyBP-C intron. Additionally, other 5' and 3' untranslated regions of nucleic acids can be used to replace those untranslated regions listed for human cMyBP-C.

[0153] Transcription termination region and polyadenylation signal

[0154] The vector constructs disclosed herein may include regulatory elements, such as transcription initiation and / or transcription termination regions. Examples of transcription termination regions include (but are not limited to) polyadenylation signal sequences. Examples of polyadenylation signal sequences include, but are not limited to, human growth hormone (hGH) poly(A), bovine growth hormone (bGH) poly(A), SV40 late poly(A), rabbit β-globulin (rBG) poly(A), thymidine kinase (TK) poly(A) sequences, Proudfoot polyA, and any variant thereof. In some embodiments, the transcription termination region is located downstream of the posttranscriptional regulatory element. In some embodiments, the transcription termination region is a bGH polyA sequence (e.g., any one of SEq ID NO: 59-61), an hGH polyA sequence (e.g., any one of SEq ID NO: 62), an SV40 polyA sequence (e.g., SEQ ID NO: 63), a Proudfoot synthetic polyA sequence (e.g., SEQ ID NO: 65), or a rabbit β-globulin polyA sequence (e.g., SEQ ID NO: 66), or a fragment of about 40 to 200 nucleotides in length thereof.

[0155] The length of the poly-A signal can be approximately 150 to approximately 250 nucleotides, approximately 160 to approximately 240 nucleotides, approximately 170 to approximately 230 nucleotides, approximately 180 to approximately 220 nucleotides, or approximately 200 to approximately 210 nucleotides.

[0156] In some embodiments, the vector construct may include additional transcription and translation initiation sequences and / or additional transcription and translation termination sequences known in the art.

[0157] Virus particles

[0158] This disclosure provides adenovirus-associated virus (AAV) particles and formulations for use in therapeutic applications. Vector constructs suitable for generating AAV particles include polynucleotide inserts with SEq ID NOs of any one of 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, and 39, with 5' and 3' ITRs attached to them.

[0159] Therefore, this disclosure provides viral particles used as gene delivery vectors (containing the vector constructs provided herein), based on animal parvoviruses, particularly dependent viruses such as infectious human or simian AAVs and their components (e.g., animal parvovirus genomes), for the introduction and / or expression of the cMyBP-C protein in mammalian cells. Thus, the term "parvovirus" as used herein encompasses dependent viruses, such as any type of AAV. The production of AAV particles requires AAV "rep" and "cap" genes, which encode 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 cited references. In wild-type AAV, the rep and cap genes are typically adjacent to each other in the viral genome (i.e., they are "coupled" together as adjacent or overlapping transcription units) and are generally conserved across AAV serotypes. The AAV rep and cap genes are also individually and collectively referred to as "AAV packaging genes." The AAV cap gene used in this article encodes a cap protein capable of packaging AAV vectors and binding to target cell receptors in the presence of rep and glandular accessory function. In some embodiments, the AAV cap gene encodes a capsid protein having an amino acid sequence derived from a specific AAV serotype.

[0160] The AAV sequence used to generate AAV can be derived from the genome of any AAV serotype. Generally, AAV serotypes have genome sequences that are significantly homologous at the amino acid and nucleic acid levels, provide a similar set of genetic functions, produce essentially physically and functionally equivalent virions, and replicate and assemble through virtually the same mechanisms. Discussions on the genomic sequences and genomic similarities of AAV serotypes (see, for example, GenBank accession number U89790; GenBank accession number J01901; GenBank accession number AF043303; GenBank accession number AF085716; Chiorini et al., J. Virol. 71: 6823-33 (1997); Srivastava et al., J. Virol. 45: 555-64 (1983); Chiorini et al., J. Virol. 73: 1309-19 (1999); Rutledge et al., J. Virol. 72: 309-19 (1998); and Wu et al., J. Virol. 74: 8635-47 (2000)).

[0161] The genomes of all known AAV serotypes are very similar. The AAV genome is a linear single-stranded DNA molecule less than about 5,000 nucleotides (nt) in length. Inverted terminal repeats (ITRs) flank unique nucleotide sequences encoding non-structural replication (Rep) and structural (VP) proteins. VP proteins form the capsid. Assembly activator protein (AAP) rapidly accompanies capsid assembly and prevents degradation of free capsid proteins (Grosse et al., J. Virol. 91(20): e01198-17 (2017)). The terminal 145 nt is self-complementary and organized to form an energy-stable intramolecular double helix, thus forming T-shaped hairpins. These hairpin structures act as the starting point for viral DNA replication and as primers for the cellular DNA polymerase complex. The Rep gene encodes the Rep proteins Rep78, Rep68, Rep52, and Rep40. Rep78 and Rep68 are transcribed by the p5 promoter, and Rep52 and Rep40 are transcribed by the p19 promoter. The cap gene encodes VP proteins VP1, VP2, and VP3. The cap gene is transcribed by the p40 promoter. The ITR used in the vectors of this invention may correspond to the same serotype as the associated cap gene or may be different. In one embodiment, the ITR used herein corresponds to the AAV2 serotype and the cap gene corresponds to the AAV5 serotype.

[0162] The AAV VP protein is known to determine the cellularity of AAV virions. The VP protein coding sequence is significantly less conserved than the Rep protein and gene across different AAV serotypes. The ability of the Rep and ITR sequences to cross-complement the corresponding sequences of other serotypes allows for the generation of pseudotyped AAV particles containing the capsid protein of one serotype (e.g., AAV1, 5, or 8) and the Rep and / or ITR sequences of another AAV serotype (e.g., AAV2). Such pseudotyped rAAV particles are part of this disclosure.

[0163] The AAV particles (and genomes encoding AAV vectors) described herein may comprise any capsid protein 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. All disclosures of human and primate AAV capsids and their properties (e.g., transduction efficiency, tissue orientation, glycan binding, and IVIG neutralization resistance) are incorporated herein by reference, including (but not limited to) any capsids and variants thereof in the sequence listings, such as variable regions with chimeric exchanges and / or glycan binding sequences and / or GH loops.

[0164] In one embodiment, the AAV ITR sequence used in the context of this disclosure is derived from AAV1, AAV2, AAV4, and / or AAV6. Similarly, in one embodiment, the Rep (e.g., Rep78 and Rep52) coding sequence is derived from AAV1, AAV2, AAV4, and / or AAV6. However, the sequences encoding the VP1, VP2, and VP3 capsid proteins used in the context of this disclosure may be derived from any serotype, such as from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12, or from simian AAV, including any capsid protein described in WO 2018 / 022608 or PCT / US19 / 32097, or newly developed AAV-like particles obtained by, for example, capsid truncation techniques and AAV capsid libraries, or any capsid that is at least 90% identical to any of SeQ ID NO: 207-223.

[0165] For example, the amino acid sequences of various capsids are disclosed. See, for example...

[0166] AAVRh.1 / hu.14 / AAV9 AAS99264.1 (SEQ ID NO: 207)

[0167] AAVRh.8 SEQ97 (SEQ ID NO: 208) of U.S. Patent Publication No. 2013 / 0045186

[0168] AAVRh.10 SEQ81 (SEQ ID NO: 209) of U.S. Patent Publication No. 2013 / 0045186

[0169] AAVRh.74 SEQ 1 (SEQ ID NO: 210) of International Patent Publication WO 2013 / 123503

[0170] AAV1 AAB_95452.1 (SEQ ID NO: 211)

[0171] AAV2 YP_680426.1 (SEQ ID NO: 212)

[0172] AAV3 NP_043941.1 (SEQ ID NO: 213)

[0173] AAV3B AAB95452.1 (SEQ ID NO: 214)

[0174] AAV4 NP_044927.1 (SEQ ID NO: 215)

[0175] AAV5 YP_068409.1 (SEQ ID NO: 216)

[0176] AAV6 AAB95450.1 (SEQ ID NO: 217)

[0177] AAV7 YP_077178.1 (SEQ ID NO: 218)

[0178] AAV8 YP_077180.1 (SEQ ID NO: 219)

[0179] AAV10 AAT46337.1 (SEQ ID NO: 220)

[0180] AAV11 AAT46339.1 (SEQ ID NO: 221)

[0181] AAV12 ABI16639.1 (SEQ ID NO: 222)

[0182] AAV13 ABZ10812.1 (SEQ ID NO: 223)

[0183] The modified “AAV” sequence can also be used in the context of this disclosure, for example, for generating AAV gene therapy vectors. Such modified sequences, such as 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 greater nucleotide and / or amino acid sequence identity with the ITR, Rep or VP of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9 (e.g., sequences having about 75-99% nucleotide sequence identity), can be used to replace wild-type AAV ITR, Rep or VP sequences.

[0184] In some embodiments, the nucleic acid sequence encoding the AAV capsid protein is operatively linked to an expression control sequence for expression in a specific cell type (e.g., Sf9 or HEK cells). Techniques known to those skilled in the art for expressing foreign genes in insect or mammalian host cells can be used to practice the embodiments described. Methods for molecular engineering and expression of peptides in insect cells are described in, for example, Summers and Smith (1986), A Manual of Methods for Baculovirus Vectors and Insect Culture Procedures, Texas Agricultural Experimental Station Bull. 7555, College Station, Tex.; Luckow (1991), Prokop et al., Cloning and Expression of Heterologous Genes in Insect Cells with Baculovirus Vectors' Recombinant DNA Technology and Applications, 97-152; King, LA and RD Possee (1992), The baculovirus expression system, Chapman and Hall, United Kingdom; O'Reilly, DR, LK Miller, VA Luckow (1992), Baculovirus Expression Vectors: A Laboratory Manual, New York; WH Freeman and Richardson, CD (1995), Baculovirus Expression Protocols, Methods in Molecular Biology. Volume 39; U.S. Patent No. 4,745,051; US-2003148506; and WO-03 / 074714, all incorporated herein by reference in their entirety. Promoters particularly suitable for transcription of nucleotide sequences encoding AAV capsid proteins are, for example, polyhedral promoters. However, other promoters active in insect cells are known in the art, such as p10, p35, or IE-1 promoters, and other promoters described in the foregoing references are also considered.

[0185] The use of insect cells for expressing heterologous proteins has been well demonstrated, as have methods for introducing nucleic acids (e.g., vectors, such as insect cell-compatible vectors) into such cells and methods for maintaining said cells in cultures. (See, for example, *METHODS IN MOLECULAR BIOLOGY*, edited by Richard, Humana Press, NJ (1995); O'Reilly et al., *BACULOVIRUS EXPRESSION VECTORS*, *A LABORATORY MANUAL*, Oxford Univ. Press (1994); Samulski et al., *J. Virol.* 63: 3822-8 (1989); Kajigaya et al., *Proc. Nat'l. Acad. Sci. USA*, 88: 4646-50 (1991); Ruffing et al., *J. Virol.* 66: 6922-30 (1992); Kirnbauer et al., *Virol.* 219: 37-44 (1996); Zhao et al., *Virol.* 272: 382-93) (2000); and U.S. Patent No. 6,204,059). In some embodiments, the nucleic acid construct encoding an AAV protein (e.g., AAV rep or cap protein) in insect cells is an insect cell-compatible vector. As used herein, “insect cell-compatible vector” or “vector” means a nucleic acid molecule capable of productively transforming or transfecting an insect or insect cell. Exemplary biological vectors include plasmids, linear nucleic acid molecules, and recombinant viruses. Any vector may be used, provided it is compatible with insect cells. The vector may be integrated into the insect cell genome, but the presence of the vector in the insect cell is not necessarily permanent, and transiently attached vectors are also included. The vector may be introduced by any known means, such as through chemical treatment of the cell, electroporation, or infection. In some embodiments, the vector is a baculovirus, a viral vector, or a plasmid. In one embodiment, the vector is a baculovirus, i.e., the construct is a baculovirus vector. Baculovirus vectors and methods of using them are described in the foregoing references on molecular engineering of insect cells.

[0186] A method for generating recombinant adeno-associated virus (AAV) particles in insect or mammalian cells containing any AAV vector construct provided herein comprises the following steps: culturing cells transfected with any AAV vector construct provided herein (in combination with various AAV cap and rep genes), and recovering recombinant therapeutic AAV particles from said transfected cells or the supernatant of said transfected cells. Generation of AAV particles in mammalian cells (e.g., HEK293 cells) is preferred because it produces a population of AAV particles with better efficacy and more consistent effects on multiple parameters of contractile function.

[0187] The cells used for recombinant AAV generation provided herein include any cell type susceptible to baculovirus infection, including vertebrate or insect cells. For example, the insect cell lines used may be derived from fall armyworm, such as Sf9, Sf21, SF900+, Drosophila cell lines, mosquito cell lines (e.g., Aedes albopictus), silkworm cell lines (e.g., Bombyx mori), Trichoplusia ni cell lines (e.g., High Five cells), or Lepidoptera cell lines (e.g., Ascalaphaodorata cell lines); for example, High Five, Sf9, Se301, SeIZD2109, SeUCR1, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAm1, BM-N, Ha2302, Hz2E5, and Ao38. In another embodiment, mammalian cells such as HEK293, HeLa, CHO, NSO, SP2 / 0, PER.C6, Vero, RD, BHK, HT 1080, A549, Cos-7, ARPE-19, and MRC-5 can be used.

[0188] In some embodiments, helper plasmids or helper viruses, vector constructs, and plasmids encoding the AAV cap gene can be used to transfect insect or mammalian cells; and recombinant AAV viruses can be collected at different time points after co-transfection. For example, recombinant AAV viruses can be collected at approximately 12 hours, approximately 24 hours, approximately 36 hours, approximately 48 hours, approximately 72 hours, approximately 96 hours, approximately 120 hours, or any of these time points, after co-transfection.

[0189] Recombinant AAV particles can also be produced using any conventional method known in the art suitable for producing infectious recombinant AAV. In some cases, recombinant AAV can be produced by using insect or mammalian cells that stably express some of the essential components for AAV particle production. For example, plasmids (or plasmids) containing the AAV rep and cap genes, along with a selection marker (e.g., a neomycin resistance gene), can be integrated into the cell's genome. The insect or mammalian cells can then be co-transfected with a helper virus (e.g., an adenovirus or baculovirus providing the helper function) and a viral vector construct containing 5' and 3' AAV ITRs (and, if necessary, a nucleotide sequence encoding a heterologous protein). The advantage of this method is that the cells are selectable and suitable for large-scale production of recombinant AAV particles. As another non-limiting example, the rep and cap genes can be introduced into packaging cells using adenovirus or baculovirus instead of plasmids. As yet another non-limiting example, a viral vector construct containing 5' and 3' AAV ITRs and the rep-cap gene can be stably integrated into the DNA of the production cell, and the helper function can be provided by wild-type adenovirus to produce recombinant AAV.

[0190] The manufacturing process of AAV particles may produce empty capsids, heavy capsids (e.g., intermediate capsids), and ultraheavy capsids (e.g., full capsids). Ultraheavy capsids contain the desired full-length or near-full-length AAV vector genome. Heavy capsids may contain a truncated AAV vector genome of intermediate length. Manufacturing steps may include reducing the number of empty capsids to enrich the population with rAAV particles containing ultraheavy capsids, or enriching the population with rAAV particles containing both heavy and ultraheavy capsids, for example, by pooling them together. Such steps are known in the art and include regional ultracentrifugation or density gradient ultracentrifugation.

[0191] Mixtures

[0192] In some embodiments, the pharmaceutical formulations provided herein are liquid formulations comprising rAAV particles, preferably rAAV9 particles, said rAAV particles comprising any of the vector constructs disclosed herein, capable of delivering therapeutic proteins for the treatment of heart disease. The concentration of rAAV virions in the formulation may vary. In some embodiments, in any formulation herein, the concentration of rAAV particles in the formulation may be in the range of 1E13 to about 4E14 vg / ml, for example, at least about 6E13 vg / ml to about 4E14 vg / ml, or about 1E13 vg / ml to about 1E14 vg / ml, or about 1E13 vg / ml to about 5E13 vg / ml, or about 5E13 vg / ml to about 1E14 vg / ml.

[0193] In any specific embodiment of any formulation described herein, the concentration of rAAV particles in the formulation may be approximately any of the following vg / ml: approximately 1E13, 1.1E13, 1.2E13, 1.3E13, 1.4E13, 1.5E13, 1.6E13, 1.7E13, 1.8E13, 1.9E13, 2E13, 2.1E13, 2.2E13, 2.3E13, 2.4E13, 2.5E13, 2.6E13, 2.7E13, 2.8E13, 2.9E13, 3E13, 3.1E13, 3.2E13, 3.3E13, 3.4E13, 3.5E13, 3.6E13, 3.7E13, 3.8E13, 3.9E13, 4E13, 4.1E13, 4.2E13, 4.3E13, 4.4E13, 4.5E13, 4.6E13, 4.7E13, 4.8E13, 4.9E13, 5E13, 5.1E13, 5.2E13, 5.3E13, 5.4E13, 5.5E13, 5.6E13, 5.7E13, 5.8E13, 5.9E13, 6E13, 6.1E13, 6.2E13, 6.3E13, 6.4E13, 6.5E13, 6.6E13 , 6.7E13, 6.8E13, 6.9E13, 7E13, 7.1E13, 7.2E13, 7.3E13, 7.4E13, 7.5E13, 7.6E13, 7.7E13, 7.8E13, 7.9E13, 8E13, 8.1E13, 8.2E13 , 8.3E13, 8.4E13, 8.5E13, 8.6E13, 8.7E13, 8.8E13, 8.9E13, 9E13, 9.1E13, 9.2E13, 9.3E13, 9.4E13, 9.5E13, 9.6E13, 9.7E13, 9.8E1 3. 9.9E13, 1E14, 1.1E14, 1.2E14, 1.3E14, 1.4E14, 1.5E14, 1.6E14, 1.7E14, 1.8E14, 1.9E14, 2E14, 2.1E14, 2.2E14, 2.3E14, 2.4E1 4. 2.5E14, 2.6E14, 2.7E14, 2.8E14, 2.9E14, 3E14, 3.1E14, 3.2E14, 3.3E14, 3.4E14, 3.5E14, 3.6E14, 3.7E14, 3.8E14, 3.9E14 or 4E14 vg / ml.

[0194] In other embodiments, the AAV particle pharmaceutical formulations provided herein comprise one or more sterile, pharmaceutically acceptable excipients to provide the formulation with advantageous properties for storage and / or administration to subjects to treat genetic conditions. In some embodiments, the pharmaceutical formulations provided herein are capable of being stored at temperatures below about -60°C (minus 60°C), -40°C, or -20°C for at least 6 months, 1.5 years, or 2 years without significant change in stability. Preferably, the pharmaceutical formulation is stable at freezer temperatures for at least one year.

[0195] Furthermore, the drug formulations described herein are stable under suitable accelerated storage conditions. Examples of stress conditions include durations of approximately 6, 9, 12, 18, and / or 24 months at approximately 25°C and approximately 60% humidity, or (for active pharmaceutical ingredients intended for storage in a freezer) durations of approximately -20°C for approximately 12 months. Examples of accelerated conditions include durations of approximately 6, 9, 12, 18, and / or 24 months at approximately 2°C to approximately 8°C, or (for active pharmaceutical ingredients intended for storage in a freezer) durations of approximately -20°C for approximately 12 months. See, for example, FDA Guidance for Industry: Stability Testing of New Drug Substances and Products, November 2003.

[0196] In this regard, the term "stable" means that the rAAV particles present in the formulation substantially maintain their physical stability, chemical stability, and / or biological activity during storage. In some embodiments, the recombinant AAV particles present in the pharmaceutical formulation retain at least about 80% of their vg / ml in human subjects (or at least about 80% of their infectious rAAV particles) during a defined period of storage at -65°C; in other embodiments, they retain at least about 85%, 90%, 95%, 98%, or 99% of their vg / ml in human subjects or their infectious rAAV particles. Preferably, the rAAV particles retain at least about 98% of their vg / ml.

[0197] The AAV9 capsid is less stable than the AAV5 capsid and requires additional excipient formulation to achieve a desirable shelf life. Specifically, the overweight AAV9 capsid exhibits significant instability at 25°C or room temperature.

[0198] In some respects, the formulation of rAAV particles includes one or more buffers. For example, citrate, phosphate, tris(hydroxymethyl)aminomethane (Tris), Tris base, or other buffers are well known in the art. In some embodiments, a 10 to 30 mM Tris buffer with a pH of about 7-8 can improve capsid stability and / or potency while reducing deamidation. Tris buffers are superior to other buffers, such as phosphate or citrate buffers, in improving or maintaining capsid stability and / or potency.

[0199] In some embodiments, the rAAV particle formulations provided herein may contain one or more isotonic agents, such as sodium chloride. Sodium chloride, within certain concentration ranges, can maintain the colloidal stability of the coating and the clarity of the solution.

[0200] In some embodiments, the rAAV particle formulations provided herein may contain stabilizers, such as MgCl2. Such stabilizers help maintain capsid integrity and / or capsid potency and / or the effectiveness of heavy and / or super-heavy capsids after storage at frozen or room temperature, preferably for at least one year. Such stabilizers preferably help maintain capsid integrity and / or capsid potency and / or effectiveness under stress conditions such as high temperature or agitation. In some embodiments, such stabilizers also reduce the formation of aggregates (e.g., visible or subvisible particles (SVP)). Studies have shown that the addition of MgCl2 is the most important formulation factor for increasing capsid stability, especially for unstable super-heavy capsids.

[0201] In some embodiments, the concentration of MgCl2 is from about 0.5 mM to about 1.5 mM, for example, about 0.5 mM, about 1.0 mM, or about 1.5 mM. In some embodiments, the concentration of MgCl2 is from about 0.5 mM to about 3 mM, or from 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 3.0 mM. In some embodiments, the concentration of MgCl2 is >1.5 mM to about 3 mM. In some embodiments, the concentration of MgCl2 (e.g., 1 mM MgCl2) provides capsid stability and prevents or reduces the occurrence of SVPs that may appear in some 3 mM MgCl2 formulations under various types of stress.

[0202] Capsid integrity can be measured, for example, by size exclusion chromatography combined with multi-angle light scattering (SEC-MALS) and thermally based capsid integrity (TBCI). SEC-MALS also analyzes capsid potency and aggregation. TBCI measures capsid thermal stability. Potency can be measured in cardiomyocytes in 2D or 3D format according to Example 2 below.

[0203] Other buffers and isotonic agents / stabilizers known in the art are suitable and can be routinely used in the formulations presented herein.

[0204] In another embodiment, the rAAV particulate formulations provided herein may include one or more build-up agents, including cryoprotectants. Exemplary build-up agents include, but are not limited to, mannitol, sucrose, dextran, lactose, trehalose, and povidone (PVPK24). Cryoprotectants can maintain the stability of the liquid formulation at freezing temperature conditions.

[0205] In another embodiment, the rAAV particle formulations provided herein may comprise one or more surfactants, which may be nonionic surfactants. Exemplary surfactants include ionic surfactants, nonionic surfactants, and combinations thereof. For example, surfactants may be, but are not limited to, TWEEN 80 (also known as polysorbate 80, or its chemical name polyoxyethylene dehydrated sorbitan monooleate), sodium dodecyl sulfate, sodium stearate, ammonium lauryl sulfate, TRITON AG 98 (Rhone-Poulenc), poloxamer 407, poloxamer 188, and combinations thereof. Surfactants reduce the adsorption of rAAV C1-INH carrier particles to contact surfaces and / or reduce precipitation, and thus reduce the loss of viral particles.

[0206] In another aspect, this disclosure provides a pharmaceutical composition comprising rAAV particles at a concentration of at least 1E13 vg / ml, or at least 6E13 vg / ml to about 4E14 vg / ml, a buffer, an isotonic agent, a stabilizer, a cryoprotectant, and a surfactant, which is stable for at least about 1 year, 1.5 years, or 2 years when stored at about -60°C (minus 60°C) or lower. In some embodiments, the buffer is a Tris buffer. In some embodiments, the cryoprotectant is a sugar, such as trehalose or a suitable hydrate thereof. In some embodiments, the stabilizer is preferably, for example, MgCl2 in hexahydrate form. In some embodiments, the surfactant is poloxamer, such as poloxamer 188, or alternatively, 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.

[0207] In some embodiments, the pharmaceutical composition is aqueous and comprises rAAV particles 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 1 mM to about 4 mM, sodium chloride at a concentration of about 100 mM to about 150 mM, magnesium chloride 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 polysorbate at a concentration of about 0.05% to about 0.15% w / v.

[0208] In some embodiments, the concentration of Tris is about 10 to about 50 mM, about 10 to about 30 mM, or about 15 to about 25 mM. In some embodiments, the concentration of the Tris base is about 1.5 mM to about 3.5 mM or about 2 mM to about 3 mM. In some embodiments, the concentration of sodium chloride is about 100 to about 140 mM or about 110 to about 130 mM. In some embodiments, the concentration of magnesium chloride is about 0.75 mM to about 1.25 mM, or about 1 mM to about 1.5 mM, or about 0.5 mM to about 3 mM, or about 1 mM to about 3 mM, or about 1.5 to about 3 mM, or more than 1.5 mM to about 3 mM. In some embodiments, the concentration of magnesium chloride is >1.5 mM. In some embodiments, the concentration of trehalose is about 60 mM to about 80 mM or about 65 mM to about 75 mM. In some embodiments, poloxamer is poloxamer 188 at a concentration of about 0.05% to 0.15% w / v, optionally about 0.1% w / v.

[0209] In some embodiments, the pharmaceutical composition comprises rAAV particles at a concentration of about 6E13 vg / ml, 17.6 mM Tris, 2.4 mM Tris base, 120 mM sodium chloride, 1 mM magnesium chloride (hexahydrate), 74 mM trehalose (dihydrate), and 0.1% w / v poloxamer 188.

[0210] The rAAV particle formulations provided herein are stable and can be stored for extended periods without unacceptable variations 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 one month, such as at least one month, at least three months, at least six months, at least 12 months, at least 18 months, at least 24 months, or longer. In another embodiment, the formulation is stable at a temperature below or equal to about -20°C for at least six months, such as at least six months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or longer. In another embodiment, the formulation is stable at a temperature below or equal to about -40°C for at least six months, such as at least six months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or longer. In yet another embodiment, the formulation is stable at a temperature below or equal to about -60°C for at least six months, such as at least six months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or longer. For example, the formulation is stable at temperatures below or equal to approximately -20°C, -40°C, or -60°C for 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 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.

[0211] Preferably, the pharmaceutical composition is an aqueous solution and is intended for storage at freezing temperatures. In various embodiments, the pharmaceutical composition is stored at temperatures ≤-60°C or lower, wherein the stability or potency of the pharmaceutical composition is maintained at least substantially at the storage temperature. In other embodiments, the pharmaceutical composition is stored at temperatures ≤-20°C or about -20°C or lower without unacceptable changes in quality, potency, or purity. In various instances, the pharmaceutical compositions were stored at the following temperatures: -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, - Temperatures can be set at 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 -100°C without unacceptable variations in quality, potency, or purity. In any of these embodiments, the composition is used for intravenous administration of rAAV particles to a patient with heart disease (e.g., HCM).

[0212] Treatment

[0213] In any embodiment, the subject has heart disease, optionally hypertrophic cardiomyopathy (HCM). HCM is the most commonly inherited heart disease as an autosomal dominant trait, caused by mutations in genes encoding cardiac sarcomere (or sarcomere-associated) proteins. HCM is characterized by thickening of the ventricular septum and left ventricular wall that cannot be explained by another cardiac, systemic, or metabolic disease. In some embodiments, prior to administration of the rAAV particles as described herein, the subject has a clinical diagnosis of non-obstructive hypertrophic cardiomyopathy (nHCM) or obstructive hypertrophic cardiomyopathy (oHCM) conforming to the current American College of Cardiology (ACC) / American Heart Association (AHA) heart failure classification system 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 III heart failure.

[0214] In any embodiment, the subject may have a mutation in the MyBPC-3 gene encoding MYBPC3, optionally detected by PCR and / or genome sequencing and / or restriction fragment length polymorphism (RFLP) analysis.

[0215] Echocardiography can determine the presence and extent of blood flow obstruction through the left ventricular outflow tract (LVOT). A pressure gradient of ≥30 mmHg at rest or upon provocation indicates obstruction, and the patient is considered to have oHCM. Those without obstruction are considered to have nHCM.

[0216] In some embodiments, the subject to be treated has a non-obstructive phenotype, for example, determined by a peak LVOT gradient of <30 mmHg at rest and / or a peak LVOT gradient of <50 mmHg during the Valsalva maneuver.

[0217] In some embodiments, the subject has an ejection fraction ≥50%. In some embodiments, the subject has elevated levels of NT-proBNP (≥300 picograms (pg) / mL). In some embodiments, the subject has ≤80% or 90% of peak oxygen consumption (PVO2) predicted by cardiopulmonary exercise testing (CPET), and a respiratory exchange ratio (RER) ≥1.0 according to screening CPET. In some embodiments, the subject has more than one of the foregoing characteristics.

[0218] In some embodiments, the subject is an adult, such as being at least 18 years old or between 18 and 65 years of age. The subject may be male or female, such as a non-pregnant woman. In some embodiments, the subject is a teenager, such as 12 to 18 years old, or 6 to 12 years old, or 6 to 18 years old, or 2 to 12 years old, or 0 to 6 years old, or 0 to 2 years old, or 2 to 6 years old, or at least 12 years old.

[0219] In some embodiments, the subject has not received prior treatment with gene therapy or investigational medical treatment concurrently or within 6 months prior to screening. In some embodiments, the subject has no evidence of active or chronic infection or immunosuppressive disorder. In some embodiments, the subject has no contraindications to glucocorticoids. In some embodiments, the subject does not have a significant or uncontrolled chronic disease 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 has no clinically relevant history of arrhythmogenic syncope, sustained ventricular tachycardia, cardiac arrest resuscitation, or appropriate ICD discharge within 6 months prior to 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 major valvular heart disease (e.g., moderate to severe valvular aortic stenosis, moderate to severe mitral regurgitation not caused by systolic forward motion of the mitral valve) or other heart disease, including but not limited to structural heart disease (unrelated to nHCM), congenital heart disease, coronary artery disease, pericardial disease, acute myocarditis, infiltrative cardiomyopathy, obstructive hypertrophic cardiomyopathy, or a history of pericarditis / myocarditis / pericardial effusion. In some embodiments, the subject does not have one or more of the following: 1) detectable antibodies against the 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% in room air, f) temperature >100.4 degrees Fahrenheit, g) liver enzymes >2 times the upper limit of normal, h) abnormal bilirubin, i) eGFR <45 ml / min / 1.73 m according to CKD-EPI. 2 (square meters), j) platelet count <100,000, k) hemoglobin ≤10 g / dL, and / or l) hemoglobin A1C ≥8.

[0220] In some embodiments, the subject does not have treatment-related grade 3 or greater adverse events, such as left ventricular systolic dysfunction, myocarditis, liver failure, immune system disorders, thrombotic microangiopathy, or infusion-related reactions.

[0221] Prior to rAAV particle infusion, the subject's baseline status is assessed through physical examination and clinical laboratory tests. In some embodiments, the subject's MYBPC3 antigen and functional levels are tested prior to rAAV particle administration. In some embodiments, the subject's baseline AAV9 antibody level is tested prior to rAAV particle administration. In some embodiments, the subject's baseline echocardiography and electrocardiography are performed prior to rAAV particle administration, and optionally cardiac magnetic resonance imaging (MRI) is performed. In some embodiments, the subject's baseline N-terminal pro-BNP (NT-proBNP) level is tested prior to rAAV particle administration. In some embodiments, the subject's high-sensitivity troponin I (hsTNI) level is tested prior to rAAV particle administration. In some embodiments, the subject's baseline exercise capacity is tested prior to rAAV particle administration, measured via cardiopulmonary exercise testing (CPET) using PVO2.

[0222] Examples of assessments include the Kansas City Cardiomyopathy Questionnaire-23 (KCCQ-23), with features such as the Total Symptom Score (TSS), Physical Limitations Score, and Clinical Summary Score (CSS) to assess the impact of symptoms on health-related quality of life (HRQoL); the EuroQol-5 Dimensions Level 5 Version (EQ-5D-5L) and the EuroQol-Visual Analog Scale (EQ-VAS) to assess health status; the Clinical Global Impression Severity and Variation Scale (CGI / C) and the Patient Global Impression Severity and Variation Scale (PGIS / C).

[0223] In the methods of this disclosure, rAAV particles are administered intravenously as a single dose. In some embodiments, the carrier construct or rAAV particles are administered by intravenous injection as a single bolus or via infusion over a prolonged period, which may be at least about 1, 5, 10, 15, 30, 45, 60, 75, 90, 120, 150, 180, 210, or 240 minutes or longer. In some embodiments, the rAAV particles are administered by infusion at an infusion rate of about 20, 30, 40, 50, 60, 70, or 80 mL / hour (1.33 mL / min).

[0224] In some embodiments, rAAV particles are administered at a dose ranging from about 1E13 to about 4E14 vector genomes per kilogram of subject body weight (vg / kg), or from about 1E13 to about 6E13 vg / kg, or from about 5E13 to about 1E14, or from 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.

[0225] In some embodiments, the dosage may be approximately any of the following: about 1E13 vg / kg, about 1.1E13 vg / kg, about 1.2E13 vg / kg, about 1.3E13 vg / kg, about 1.4E13 vg / kg, about 1.5E13 vg / kg, about 1.6E13 vg / kg, about 1.7E13 vg / kg, about 1.8E13 vg / kg, about 1.9E13 vg / kg, about 2E13 vg / kg, about 2.1E13 vg / kg, about 2.2E13 vg / kg, about 2.3E13 vg / kg, about 2.4E13 vg / kg, about 2.5E13 vg / kg, about 2.6E13 vg / kg, about 2.7E13 vg / kg, about 2.8E13 vg / kg, about 2.9E13 vg / kg. vg / kg, approximately 3E13 vg / kg, approximately 3.1E13 vg / kg, approximately 3.2E13 vg / kg, approximately 3.3E13 vg / kg, approximately 3.4E13 vg / kg, approximately 3.5E13 vg / kg, approximately 3.6E13 vg / kg, approximately 3.7E13 vg / kg, approximately 3.8E13 vg / kg, approximately 3.9E13 vg / kg, approximately 4E13 vg / kg, approximately 4.1E13 vg / kg, approximately 4.2E13 vg / kg, approximately 4.3E13 vg / kg, approximately 4.4E13 vg / kg, approximately 4.5E13 vg / kg, approximately 4.6E13 vg / kg, approximately 4.7E13 vg / kg, approximately 4.8E13 vg / kg, approximately 4.9E13 vg / kg, approximately 5E13 vg / kg, approximately 5.1E13 vg / kg, approximately 5.2E13 vg / kg, approximately 5.3E13 vg / kg, approximately 5.4E13 vg / kg, approximately 5.5E13 vg / kg, approximately 5.6E13 vg / kg, approximately 5.7E13 vg / kg, approximately 5.8E13 vg / kg, approximately 5.9E13 vg / kg, approximately 6E13 vg / kg, approximately 6.1E13 vg / kg, approximately 6.2E13 vg / kg, approximately 6.3E13 vg / kg, approximately 6.4E13 vg / kg, approximately 6.5E13 vg / kg, approximately 6.6E13 vg / kg, approximately 6.7E13 vg / kg, approximately 6.8E13 vg / kg, approximately 6.9E13 vg / kg, approximately 7E13 vg / kg, approximately 7.1E13 vg / kg, approximately 7.2E13 vg / kg, approximately 7.3E13 vg / kg, approximately 7.4E13 vg / kg, approximately 7.5E13 vg / kg, approximately 7.6E13 vg / kg, approximately 7.7E13 vg / kg, approximately 7.8E13 vg / kg, approximately 7.9E13 vg / kg, approximately 8E13 vg / kg, approximately 8.1E13 vg / kg, approximately 8.2E13 vg / kg, approximately 8.3E13 vg / kg, approximately 8.4E13 vg / kg, approximately 8.5E13 vg / kg, approximately 8.6E13 vg / kg, approximately 8.7E13 vg / kg, approximately 8.8E13 vg / kg, approximately 8.9E13 vg / kg, approximately 9E13 vg / kg, approximately 9.1E13 vg / kg, approximately 9.2E13 vg / kg, approximately 9.3E13 vg / kg, approximately 9.4E13 vg / kg, approximately 9.5E13 vg / kg, approximately 9.6E13 vg / kg, approximately 9.7E13 vg / kg, approximately 9.8E13 vg / kg, approximately 9.9E13 vg / kg, approximately 1E14 vg / kg, approximately 1.1E14 vg / kg, approximately 1.2E14 vg / kg, approximately 1.3E14 vg / kg, approximately 1.4E14 vg / kg, approximately 1.5E14 vg / kg, approximately 1.6E14 vg / kg, approximately 1.7E14 vg / kg, approximately 1.8E14 vg / kg, approximately 1.9E14 vg / kg, approximately 2E14 vg / kg, approximately 2.1E14 vg / kg, approximately 2.2E14 vg / kg, approximately 2.3E14 vg / kg, approximately 2.4E14 vg / kg, approximately 2.5E14 vg / kg, approximately 2.6E14 vg / kg, approximately 2.7E14 vg / kg, approximately 2.8E14 vg / kg, approximately 2.9E14 vg / kg, approximately 3E14 Vg / kg, approximately 3.1E14 Vg / kg, approximately 3.2E14 Vg / kg, approximately 3.3E14 Vg / kg, approximately 3.4E14 Vg / kg, approximately 3.5E14 Vg / kg, approximately 3.6E14 Vg / kg, approximately 3.7E14 Vg / kg, approximately 3.8E14 Vg / kg, approximately 3.9E14 Vg / kg, or approximately 4E14 Vg / kg.

[0226] In some embodiments, rAAV particles are administered at a dose of approximately 6E13 vg / kg, 9E13 vg / kg, or 1.2E14 vg / kg.

[0227] In some embodiments, BACTRIM® (sulfamethoxazole and trimethoprim) was administered to subjects simultaneously as a preventative measure against Pneumocystis jiroveci pneumonia (PJP).

[0228] Following the infusion of rAAV particles, the method may further include steps to monitor various parameters of the subject's health, such as weekly measurements. Alternatively, measurements may be performed every 1, 2, 3, 4, 5, or 6 days, weekly, bi-weekly, tri-weekly, or monthly. Parameters may be monitored at weeks 16, 20, 24, 28, 32, 36, 40, 44, 48, or longer. The method may include measuring the subject's NT-proBNP and troponin I levels, as well as echocardiographic and electrocardiographic assessments.

[0229] The methods disclosed herein can cause clinically significant improvements in cardiac structure / morphology and / or function. The methods disclosed herein can improve diastolic function, as measured by, for example, a decrease in E / e' and / or a decrease in left atrial volume index. The methods disclosed herein can improve cardiac systolic function, as measured by, for example, improvement or normalization of ejection fraction and / or reduction in longitudinal strain injury. For example, improvement or normalization of ejection fraction may include an increase or decrease in ejection fraction relative to a baseline identified prior to administration of the rAAV carrier. The methods disclosed herein can improve cardiac structure, as demonstrated by, for example, a decrease in left ventricular (LV) wall thickness and / or LV mass. The methods disclosed herein can improve diastolic and / or systolic function, and / or improve cardiac structure.

[0230] The methods disclosed herein can also result in clinically significant improvements in exercise capacity, as measured by, for example, improved PVO2 during cardiopulmonary exercise testing (CPET).

[0231] The methods disclosed herein can improve cardiac function, such as moving from Class III to Class II heart failure, or from Class II to Class I heart failure, or from Class III to Class I heart failure according to the New York Heart Association (NYHA) functional classification system.

[0232] The methods disclosed herein can further improve health-related quality of life (HRQoL), for example, 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 Severity and Variation Scale (CGIS / C) and the Patient Global Impression Severity and Variation Scale (PGIS / C), the EuroQol-5 Dimensions 5-Level Version (EQ-5D-5L), and / or the EuroQol-Visual Analogue Scale (EQ-VAS). In some embodiments, the dose can effectively improve one or more of these measures of health-related remission quality 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 dosage is effective in improving one or more of these measures of quality of health-related remission 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 one year. In some embodiments, the improvement is maintained for at least about one year, or for two, three, four, or five years.

[0233] In some embodiments, the dose can effectively reduce the level of the cardiac biomarker NT-proBNP or prevent an increase in the level of the cardiac biomarker NT-proBNP. In some embodiments, the dose can effectively prevent an increase in the level of the cardiac biomarker troponin I or reduce the incidence of an increase in the level of troponin I. For example, the dosage can effectively reduce NT-proBNP levels by at least approximately 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 instances, the reduction in NT-proBNP levels was a percentage decrease from the initial threshold (e.g., ≥300 pg / mL). Additionally, in these instances, reductions in NT-proBNP levels were observed at 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56 weeks or longer after dose administration.

[0234] In some embodiments, the dose can effectively increase peak oxygen consumption (PVO2) by at least about 0.1 mL / kg / min, 0.2 mL / kg / min, 0.3 mL / kg / min, 0.4 mL / kg / min, 0.5 mL / kg / min, 0.6 mL / kg / min, 0.7 mL / kg / min, 0.8 mL / kg / min, 0.9 mL / kg / min, 1 mL / kg / min, 1.1 mL / kg / min, 1.2 mL / kg / min, 1.3 mL / kg / min, and 1.4 mL / kg / min. The doses were 1.5 ml / kg / min, 1.6 ml / kg / min, 1.7 ml / kg / min, 1.8 ml / kg / min, 1.9 ml / kg / min, 2 ml / kg / min, 2.1 ml / kg / min, 2.2 ml / kg / min, 2.3 ml / kg / min, 2.4 ml / kg / min, 2.5 ml / kg / min, 2.6 ml / kg / min, 2.7 ml / kg / min, 2.8 ml / kg / min, 2.9 ml / kg / min, or 3 ml / kg / min. Additionally, in these cases, an increase in peak oxygen consumption was observed at 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56 weeks, or longer after dose administration.

[0235] The methods disclosed herein provide for the safe administration of rAAV particles, for example, without the occurrence of clinically significant treatment-induced serious adverse events or at a low incidence; without clinically significant changes in standard clinical laboratory values ​​or at a low incidence; without the occurrence of complement activation or hypersensitivity reactions or at a low incidence; and without the occurrence of or at a low incidence of elevations to grade 2 or higher or grade 3 or higher in hepatotoxicity markers (or, if changes occur, most are transient or resolve after treatment with systemic immunosuppressants). Another alternative example of the methods disclosed herein provides for the safe administration of rAAV particles, including without or at a low incidence of coagulation marker abnormalities. The methods may also provide an immune response against AAV capsid reduction. The methods may also provide improved blood biodistribution or reduced carrier shedding in urine, feces, semen, or saliva.

[0236] In one aspect of this disclosure, hepatotoxicity (e.g., detected by transient elevation of liver transaminases) can be reduced or avoided by prophylactic or therapeutic immunosuppressive therapy. According to these aspects, in addition to administration of a therapeutically effective dose of AAV virus, subjects may be treated prophylactically, therapeutically, or both with glucocorticoids or other immunosuppressants to prevent and / or treat any hepatotoxicity associated with AAV virus administration.

[0237] In one relevant aspect of this disclosure, hepatic and / or cardiac inflammation can be reduced or avoided through prophylactic or therapeutic immunosuppressive therapy. According to this aspect, in addition to administration of a therapeutically effective dose of AAV virus, subjects may be treated prophylactically, therapeutically, or both with glucocorticoids or other immunosuppressants to prevent and / or treat any hepatic and / or cardiac inflammation associated with AAV virus administration.

[0238] In another aspect of this disclosure, complement activation (e.g., refractory complement activation) and / or thrombotic microangiopathy can be reduced or avoided through prophylactic or therapeutic administration of complement pathway inhibitors or antagonists. According to this aspect, in addition to administration of a therapeutically effective amount of AAV virus, subjects may be treated prophylactically, therapeutically, or both with complement pathway inhibitors or antagonists to prevent and / or treat any complement activation associated with AAV virus administration, such as refractory complement activation and / or thrombotic microangiopathy.

[0239] Prophylactic immunosuppressive therapy

[0240] The methods disclosed herein may further include administering a prophylactic effective amount of an immunosuppressant (e.g., a glucocorticoid or complement pathway inhibitor or antagonist) to a subject. In some embodiments, a subject is prophylactically administered glucocorticoids and sirolimus to prevent or reduce hepatotoxicity (e.g., detected by an ALT elevation at least twice the upper limit of normal (ULN) or baseline ALT) and / or liver inflammation and / or myocarditis and / or cardiac inflammation. In some embodiments, a subject is prophylactically administered a complement pathway inhibitor or antagonist to prevent or reduce complement activation, such as refractory complement activation, and / or thrombotic microangiopathy. In some embodiments, a prophylactic effective amount of an immunosuppressant (e.g., a glucocorticoid, complement pathway inhibitor or antagonist) is administered concurrently with the administration of the rAAV particles of the present invention. As used herein, “concurrently” means, for example, on the same day as the administration of the rAAV particles, or within one day or one week (before or after) the administration of the rAAV particles. In other embodiments, administration of a preventative effective dose of an immunosuppressant (e.g., glucocorticoids, complement pathway inhibitors, or antagonists) is initiated after administration of the rAAV particles, for example, at weeks 1, 2, 3, 4, 5, 6, 7, or 8 after rAAV particle administration, but before hepatotoxicity is detected. In other embodiments, a preventative effective dose of an immunosuppressant (e.g., sirolimus) is initiated twice daily for two days prior to administration and continued for approximately 8 weeks after rAAV particle administration, for example, twice daily for two days on days -5, -4, -3, -2, -1, week 0, week 1, week 2, week 3, week 4, week 5, week 6, week 7, and week 8.

[0241] Glucocorticoids or other immunosuppressants (e.g., complement pathway inhibitors or antagonists) may be administered for a prophylactic treatment period, for example, at least about 1 to 4 weeks (1, 2, 3, 4 weeks), and preferably followed by a tapering period during which a tapering dose of corticosteroids or other immunosuppressants is administered, for example, for about 2 to 4 weeks, or for about 2, 3, or 4 weeks. For example, a prophylactic effective dose of glucocorticoids is equivalent to about 40 mg / day of prednisone, administered for at least about 1 to 8 weeks (1, 2, 3, 4, 5, 6, 7, 8), followed by a tapering dose of glucocorticoids for about 2, 3, or 4 weeks. In some embodiments, a prophylactic effective dose of glucocorticoids is administered for about 4 weeks, followed by a tapering dose of glucocorticoids for about 4 weeks. For example, concurrently with the administration, glucocorticoids are administered at a dose equivalent to approximately 40 mg / day of prednisone for approximately 4 weeks, followed by a gradual reduction in the amount of glucocorticoids for approximately 4 weeks.

[0242] Sirolimus can be administered for a prophylactic treatment period, such as for at least approximately 3 days to 8 weeks (with a loading dose from day 3 to day 1, followed by administration during weeks 1, 2, 3, 4, 5, 6, 7, and 8), and preferably co-administered with glucocorticoids starting in week 1. In some embodiments, a prophylacticly effective amount of glucocorticoids and sirolimus are co-administered for approximately 4 weeks, followed by a gradual tapering of the glucocorticoids. For example, prior to rAAV administration, sirolimus is loaded at a dose of 6 mg / day for two days, followed by a dose of 2 mg / day of sirolimus, while glucocorticoids are administered on the same day as rAAV administration, followed daily (during weeks 1, 2, 3, and 4) at a dose equivalent to approximately 40 mg / day of prednisone. Subsequently, sirolimus was continued at a dose of 2 mg / day for approximately 4 weeks during the gradual reduction of glucocorticoid dosage (during weeks 5, 6, 7, and 8 after rAAV administration).

[0243] Monitor ALT and AST levels weekly. If ALT rises to above the upper limit of normal (ULN) or more than twice the baseline ALT value, adjust glucocorticoid dosage based on clinical judgment during the first 4 weeks, and monitor liver enzymes more frequently.

[0244] Prophylactic immunosuppressive therapy may include, for example, the administration of complement pathway inhibitors or antagonists. 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 inhibitors (e.g., OMS721 antibody) and include any of the following: percicobulin (APL-2, EMPAVELI), eculizumab (SOLIRIS), avacopan (CCX168), virorumab (IFX-1), AMY-101, CINRYZE / BERINERT / HAEGARDA (C1INH), rivolizumab (ALXN1210), carvedin, sedaslan (ALN-CC5), terdurumab (LFG-316), ipcopan (LNP023), danicopan (ACH-4471), RUCONEST (recombinant C1 esterase inhibitor), or ENJAYMO (sutemozolomab-jumi, a C1s inhibitor). In a preferred embodiment, the prophylactic complement pathway inhibitor or antagonist is a C3 inhibitor (e.g., percecobulum), a C1 esterase inhibitor, a recombinant C1 esterase inhibitor, or a C1s inhibitor (e.g., sutimlimab).

[0245] In some embodiments, a prophylactic immunosuppressant as described below is administered to the subject. In other embodiments, a prophylactic antimicrobial agent is administered concurrently with a glucocorticoid / sirolimus immunosuppressant.

[0246] Therapeutic / Reactive Immunosuppressive Therapy

[0247] In some cases, administration of the AAV particles of this disclosure can cause observable hepatotoxicity and / or liver inflammation and / or cardiac inflammation and / or myocarditis and / or complement activation, such as refractory complement activation and / or thrombotic microangiopathy. Hepatotoxicity or liver inflammation can be measured using a variety of well-known and routinely used techniques, such as measuring the concentration of certain liver-related enzymes (e.g., alanine aminotransferase, ALT) in the subject's bloodstream before (i.e., baseline) and after AAV administration. An observable increase in ALT concentration after AAV administration (compared to pre-administration) indicates drug-induced hepatotoxicity. The methods of this disclosure may include administering a therapeutically effective amount of glucocorticoids or other systemic immunosuppressants to a subject to treat hepatotoxicity upon detection of hepatotoxicity.

[0248] Myocarditis and / or cardiac inflammation can be detected by, for example, abnormally elevated levels of troponin I, high-sensitivity troponin I (hsTNI), and / or N-terminal pro-BNP (NT-proBNP). Other signs include an ejection fraction (EF) of less than approximately 50%, arrhythmias, myocarditis, pericarditis, and / or regional wall motion abnormalities.

[0249] Complement activation can be detected by, for example, abnormally low levels of C3 or C4 in plasma (indicating the consumption of these by complement activation), or abnormally high levels of one or more complement cleavage products, such as C3a, C4a, Bb, and / or sC5b-9. Refractory complement activation is characterized by complement activation that has not been suppressed by prophylactic immunosuppression or occurs against the backdrop of progressive thrombotic vascular disease.

[0250] Thrombotic microangiopathy is characterized by anemia caused by the destruction of red blood cells within the microvascular system, accompanied by thrombocytopenia due to platelet activation and consumption. Thrombotic microangiopathy can be detected by, for example, decreased platelet count, decreased platelet volume, decreased red blood cell (RBC) count, abnormal RBC morphology, hemolysis, and / or proteinuria. Clinical signs of thrombotic microangiopathy include complement activation, severe thrombocytopenia, microangiopathic hemolysis with cleavage cells, elevated transaminases, and / or acute kidney injury (AKI) progressing to renal failure.

[0251] Reactive immunosuppressive therapy may be initiated upon completion of a prophylactic steroid / sirolimus regimen, in response to a mild ALT elevation meeting pre-specified criteria, or based on clinical judgment. This may involve administering a glucocorticoid at a dose equivalent to at least about 0.5 mg / kg / day of prednisone, or at least about 40 mg / day (e.g., 60 mg / day), optionally followed by tacrolimus or mycophenolate mofetil. For example, if liver function tests fail despite administration of 40 mg / day of glucocorticoids, then 60 mg / day of glucocorticoids may be administered. In some embodiments, the administration may be initiated if ALT is greater than the ULN or more than twice the baseline in two consecutive assessments within 72 hours, or greater than three times the 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 doses equivalent to a glucocorticoid dose of >0.5 mg / kg / day prednisone, followed by a 3-week dose reduction if ALT is less than or equal to the ULN and less than or equal to twice the baseline value. During the period following discontinuation of reactive immunosuppressive therapy, liver enzymes are monitored weekly for 4 weeks, or more frequently if ALT values ​​are higher than the ULN. In some embodiments, tacrolimus is administered for breakthrough myocarditis.

[0252] Therapeutic / reactive immunosuppressive therapies may include, for example, the administration of complement pathway inhibitors or antagonists. 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 inhibitors (e.g., OMS721 antibody), and include any of the following: percicobulin (APL-2, EMPAVELI), eculizumab (SOLIRIS), avacopan (CCX168), virorumab (IFX-1), AMY-101, CINRYZE / BERINERT / HAEGARDA (C1INH), rivolizumab (ALXN1210), carvedin, sedaslan (ALN-CC5), terdurumab (LFG-316), ipcopan (LNP023), danicopan (ACH-4471), RUCONEST (recombinant C1 esterase inhibitor), or ENJAYMO (sutemozolomab-jumi, a C1s inhibitor). The administration of reactive immunosuppressants can be sustained for, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 or 12 weeks, or 1 month, 2 months or 3 months. In some embodiments, eculizumab is administered for refractory complement activation in the context of thrombotic microangiography.

[0253] The method disclosed herein may further include the following steps: (a) determining baseline levels of biomarkers for hepatotoxicity, liver inflammation, cardiac inflammation, complement activation, and / or thrombotic microangiography in the subject's blood, optionally about one month prior to the administration; and (b) determining post-administration levels of biomarkers for hepatotoxicity, liver inflammation, cardiac inflammation, complement activation, and / or thrombotic microangiography in the subject's blood, optionally weekly or every 1, 2, 3, 4, 5, or 6 days after the administration.

[0254] Such methods may further include the following steps: (c) after hepatotoxicity, liver inflammation, cardiac inflammation, complement activation, and / or thrombotic microangiography is detected by biochemical or clinical signs, administering to the subject a therapeutically effective amount of immunosuppressants (e.g., glucocorticoids and optionally tacrolimus or mycophenolate mofetil, and optionally or otherwise complement pathway inhibitors or antagonists) for a duration of continuous therapeutic treatment, for example at least about 5 to about 8 weeks (e.g., 5, 6, 7, or 8 weeks), and subsequently preferably for a duration of gradually decreasing amounts of immunosuppressants (e.g., glucocorticoids and optionally tacrolimus or mycophenolate mofetil, and optionally or otherwise complement pathway inhibitors or antagonists) for a duration of about 2 to 4 weeks (e.g., 3 weeks). For example, step (c) includes administering a therapeutically effective amount of glucocorticoids to the subject for at least about 5 to about 8 weeks or longer (e.g., 5, 6, 7, or 8 weeks or longer) after hepatotoxicity is detected by (i) a level greater than the upper limit of normal (ULN) after administration of the hepatotoxicity marker, or (ii) a level greater than or equal to twice the baseline level of the hepatotoxicity marker after administration of the hepatotoxicity marker, optionally together with tacrolimus or mycophenolate mofetil, followed by administration of a gradually decreasing dose of an immunosuppressant for about 2, 3, or 4 weeks. In any of such embodiments, the hepatotoxicity marker is ALT and / or AST, preferably ALT. In some embodiments, after the detection, a prednisone equivalent is administered at a prednisone equivalent dose of 40 mg / day for about 5 weeks, followed by administration of a gradually decreasing dose of the prednisone equivalent for about 3 weeks.

[0255] "Prophylactic" immunosuppressive therapy refers to the administration of glucocorticoids, combination steroids / sirolimus, and / or other immunosuppressants (e.g., complement pathway inhibitors or antagonists) to prevent or reduce adverse events such as hepatotoxicity (e.g., detected by an increase in ALT levels measured in the subject), liver inflammation, cardiac inflammation, myocarditis, complement activation (including refractory complement activation), and / or thrombotic microangiography. "Therapeutic" glucocorticoid immunosuppressive therapy refers to the administration of immunosuppressants, such as glucocorticoids, combination steroids / sirolimus, and / or other immunosuppressants, such as complement pathway inhibitors or antagonists, to treat adverse events such as reducing hepatotoxicity, liver inflammation, cardiac inflammation, myocarditis, complement activation (including refractory complement activation), and / or thrombotic microangiography caused by AAV administration, such as reducing the increase in ALT concentration in the bloodstream of the subject caused by AAV administration.

[0256] In some embodiments, prophylactic or therapeutic glucocorticoid treatment may comprise administering to a subject a prednisone equivalent dose of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more mg / day, for example, a prednisone equivalent dose of glucocorticoid between about 10 mg / day and about 60 mg / day. In some embodiments, prophylactic or therapeutic glucocorticoid treatment to a subject may be administered over a continuous period of at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 weeks or longer, followed by periods of gradually decreasing doses. Corticosteroids that can be used in the methods described herein include any known or conventionally used glucocorticoids in equivalent doses over the same time period, including, for example, dexamethasone, prednisone, prednisolone, fludrocortisone, hydrocortisone, budesonide, etc.

[0257] Other systemic immunosuppressants that can prevent or reduce hepatotoxicity by administering effective doses or therapeutic doses include (1) calcineurin inhibitors, such as tacrolimus or cyclosporine, (2) antiproliferators or IMDH inhibitors, such as mycophenolate, leflunomide or azathioprine, (3) mTOR inhibitors, such as sirolimus or everolimus, (4) januskinase inhibitors, such as tofacitinib, or (5) immunosuppressive antibodies.

[0258] Example

[0259] The following examples are provided to aid in understanding this disclosure and should not, of course, be construed as specifically limiting the embodiments described and claimed herein. Such variations of the embodiments (including substitutions for all equivalents now known or developed hereafter that are within the capabilities of a person skilled in the art), as well as minor variations in formulation or experimental design, should be considered to fall within the scope of the embodiments incorporated herein.

[0260] Example 1: Stability assessment of AAV particles

[0261] The long-term stability of rAAV particles with AAV9 caps was tested in various formulations.

[0262] The capsid integrity, aggregation, and potency of various formulations containing heavy and superheavy AAV9 capsids of the vector constructs described herein were tested. The formulations were subjected to various stress conditions, including elevated temperatures, agitation, and freeze-thaw cycles. Generally, AAV9 capsids are less stable than AAV5 capsids, and superheavy AAV9 capsids are less stable than heavy AAV9 capsids. The formulations were tested by comparing Tris buffer with other buffers (e.g., citrate buffer). A thermal capsid integrity (TBCI) assay, as described in PCT Publication No. WO2021 / 062164, was performed under accelerated conditions. In short, the TBCI assay comprises the following steps: combining AAV particles with a fluorescent dye that fluoresces upon binding to nucleic acids in a sample; exposing the sample to different conditions, such as accelerated or stress conditions; and assessing capsid integrity under different conditions by measuring fluorescence. As the dye binds to nucleic acids escaping capsid degradation, fluorescence emission increases, indicating decreased capsid integrity. The results showed that when evaluated using a heat-based capsid integrity (TBCI) assay, the stability of AAV9 particles in Tris buffer was significantly greater than that in citrate buffer.

[0263] TBCI assays were also used to monitor the stability of formulations containing heavy or super-heavy capsids at room temperature (25°C), the formulations containing 20 mM Tris pH 7.4, 74 mM trehalose, 120 mM NaCl, and 0.1% P188. Both heavy and super-heavy capsids exhibited reduced capsid integrity within 2 weeks. The super-heavy capsid was more unstable than the heavy capsid, exhibiting near-2 logarithmic degradation. Formulations were also tested with and without MgCl2 stabilizer. The addition of MgCl2 (e.g., 3 mM MgCl2) completely mitigated this instability, maintaining thermal stability and capsid integrity at greater than 95% or greater than 98%. Figure 1A and 1B ).

[0264] After storage at 30°C for 4 days, the capsid titer and aggregation of the overweight AAV9 capsid formulations were tested by SEC-MALS. Results showed that the overweight variant exhibited significant aggregation and loss of main peak area under temperature stress. However, the addition of MgCl2 (e.g., 3 mM MgCl2) prevented this aggregation of the overweight variant at high temperatures. The integrity of the stressed overweight capsids with and without MgCl2 was also analyzed by negative staining TEM (nsTEM) as a visual confirmation of the SEC and TBCI results. Both samples were held at 30°C for 1 day prior to grid preparation. The overweight capsid without MgCl2 formulation had approximately 60% internally stained capsid compared to approximately 0.5% capsid with MgCl2 formulation.

[0265] Formulas containing aggregated heavy and superheavy capsids in formulations containing various concentrations of stabilizer (0.5 mM, 1.75 mM, and 3 mM MgCl2) were subjected to various stressors, including 2 weeks and 48 hours of reverse stress at 25°C and freeze-thaw stress ×10. Overall, MgCl2 concentrations of 0.5–3.0 mM in the formulations showed that capsid integrity was maintained over time under these stressors.

[0266] Maintenance of subvisible particles (SVP) in formulations containing 1 mM and 3 mM MgCl2 was analyzed. The concentrations (p / mL) of 2–10 µm particles in the formulations at 5 °C and 25 °C were determined at T=0, T=1 week, and T=2 weeks. No increase in SVP over time was observed in the presence of MgCl2. However, SVP randomly appeared in some 3 mM MgCl2 formulations under various types of stress. Therefore, the results indicate that 1 mM MgCl2 provides capsid stability and also reduces SVP occurrence.

[0267] As typically tested above, the protein expression and function of formulations with and without MgCl2 showed that MgCl2 had no significant effect on protein expression levels in in vitro cardiomyocytes or in vivo MYBPC3 knockout mouse models.

[0268] The results showed that the addition of MgCl2 was the most important regulating factor for increasing capsid stability, especially for unstable hyperheavy variants. Concentrations of 0.5–3 mM MgCl2 provided similar stabilizing effects.

[0269] The formulation containing 17.6 mM Tris hydrochloride, 2.4 mM Tris base, 120 mM NaCl, 1 mM MgCl2 hexahydrate, 74 mM trehalose dihydrate, and 0.1% w / v poloxamer 188 at pH 7.4 provides the best long-term stability.

[0270] Example 2: Administration of AAV granules to mice

[0271] In vivo studies were conducted to evaluate the administration of AAV9-hMYPBC3 particles comprising an AAV9 capsid and an AAV vector genome containing (a) an hTNNT2 promoter, (b) a wild-type or codon-optimized cMyBP-C gene, (c) a polyadenylation signal, and optionally (d) globulin introns (e.g., any of SEQ ID NO: 3-41). Data showed that administration of a single IV dose of 2E14 vg / kg AAV9-MYPBC3 increased and sustained functional MYPBC3 expression to levels expected to be therapeutic in human subjects with HCM.

[0272] Rag2 - / - The mouse strain exhibited complete deletion of the entire protein-coding region of the recombinant activation gene 2 (Rag2) locus and displayed arrested B cell and T cell differentiation. Rag2 mice at 8 weeks of age... - / - Mice were administered a single IV dose of the 2E14AAV9-hMYBPC3 vector genome / kg body weight (vg / kg). Total cMyBP-C protein levels and human-specific cMyBP-C protein levels in cardiac homogenates were assessed. Results indicated measurable levels of transduction in the entire cardiac homogenate at 8 weeks post-administration, as analyzed by digital droplet polymerase chain reaction (ddPCR) of the vector genome and transcripts. Figure 2A and 2B ) and assessment of protein levels by liquid chromatography-mass spectrometry (LC / MS) Figure 2C and 2D The assessment included measurements of total protein levels and human-specific protein levels, showing that human cMyBP-C accounts for a significant portion of Rag2 transduced by AAV9-hMYBPC3. - / - The total protein content in the mouse heart is 15%. Measurements of total protein levels in the mouse heart indicate that transduction with the AAV9-hMYBPC3 vector does not increase the total amount of protein in the heart due to stoichiometry between sarcomere proteins. Human protein incorporation into the sarcomere may depend on intracellular protein turnover. These data suggest that the expression and incorporation of human proteins into the mouse heart is achievable with a complete set of mouse proteins.

[0273] Human cMyBP-C protein expression was confirmed in cardiomyocytes by immunohistochemistry. Immunohistochemical evaluation of cardiac tissue showed that, on average, 65% of cardiomyocytes in longitudinal cardiac tissue sections (including left and right ventricular walls, interventricular septum, and right atrial wall) were positive for human cMyBP-C. In mice with a complete set of endogenous mouse cMyBP-C, the total amount of human protein produced by AAV9-MYBPC3 was, on average, 15% of the total level, and the total cMyBP-C (mouse + human protein) level did not change with vector administration.

[0274] This data confirms that the AAV9-hMYPBC3 vector can achieve widespread distribution throughout the mouse heart and can transduce and express proteins incorporated into the sarcomere even in the context of a complete set of endogenous cMyBP-C in WT mice. This finding also supports the use of C57BL / 6 WT mice with a complete set of mouse cMyBP-C (see [link to relevant documentation]). Figure 2A-2D The data are presented as mean + / - standard deviation (SD).

[0275] Example 3: Administration in HCM mouse model

[0276] MYBPC3 - / - Mice were generated via cre recombination during in vitro fertilization, allowing for the production of heterozygous knockout embryos. Homozygous offspring exhibited deletions of exons 3-5 in mouse MYBPC3, resulting in a frameshift. There was also a splicing loss between exons 2 and 6, and a premature stop codon was produced in exon 6. The resulting mRNA was expected to be unstable and a target of unintentionally mediated decay mechanisms. Therefore, MYBPC3... - / - Mice lacked functional cMyBP-C protein. A natural history study was conducted to assess phenotypic development over a 20-week period. Mice were characterized by echocardiography using the Vevo 3100 preclinical imaging system, which supports four 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. Mice were divided into two cohorts. Analysis of cohort 1 began at 4 weeks of age, but significant phenotypes were already present. Therefore, analysis of cohort 2 began on day 4 after birth to determine the age of phenotypic onset. MYBPC3 - / - The mice exhibited hypertrophy, evidenced by an increase in LV mass. Figure 3A By 2 weeks of age, compared with heterozygous and WT mice, MYBPC3- / - mice showed increased diameters of the anterior and posterior walls of the left ventricle. Figure 3B Increased interventricular septal diameter and left ventricular posterior wall diameter are clinically expressed in HCM in adult patients, indicating that this mouse model replicates the characteristics of HCM. As demonstrated by measures of diastolic function, such as a decrease in mitral valve inflow velocity E wave (MVE), MYBPC3... - / - Mice exhibited impaired ventricular relaxation, with MV E representing early passive filling of the left ventricle, which is characteristic of MYBPC3. - / - Clinically relevant endpoints that are easily assessed in mice ( Figure 3C ). (Jiang J, Burgon PG, Wakimoto H, Onoue K, et al. Cardiac myosinbinding protein C regulates postnatal myocyte cytokinesis. Proc Natl Acad SciUSA. 2015;112(29):9046-9051.) MYBPC + / - The mouse heterozygous heart was indistinguishable from the WT heart in terms of cMyBP-C protein expression.

[0277] MYBPC3 - / -Mice exhibited early and severe clinically relevant disease expression of human hemorrhage (HCM). Specifically, left ventricular hypertrophy, increased left ventricular wall measurements, and impaired diastolic function were phenotypes mimicking human HCM, which led to the expression of MYBPC3. - / - Mice are a suitable model for studying the disease-modifying effects of the vector constructs described in this paper.

[0278] exist Figure 3A , 3B In 3C, **** indicates that the p-value is ≤0.0001 based on one-way ANOVA and Tukey's multiple comparisons. Data is presented relative to MYBPC3. - / - *Average ±SD.

[0279] Example 4:

[0280] A short-term, 8-week pharmacological study was conducted to evaluate transduction, expression, and efficacy. The study was performed on 2-week-old MYBPC3 cells. - / - Mice were administered AAV9-MYBPC3 at a dose of 2E14 vg / kg. For this study, AAV9-MYBPC3 comprised human MYBPC3 (AAV9-MYBPC3) driven by the hTNNT2 promoter encapsulated in AAV9. Vector DNA and RNA transcripts in whole heart homogenates were measured by ddPCR, and cMyBP-C levels were also measured by LC / MS. At 8 weeks post-administration, DNA and RNA levels were comparable to those of adult Rag2 mice receiving the same 2E14 vg / kg dose. - / - Approximately 10-fold and 5-fold higher than the levels measured in mice. Figure 4A and 4B Human-specific proteins and total proteins (mouse and human) were measured in WT and MYBPC3. - / - Comparisons were made between mice. Results showed that a dose of 2E14 vg / kg of AAV9-MYBPC3 reduced MYBPC3 levels. - / - cMyBP-C levels in mice returned to WT levels. Figure 4C and 4D Protein levels were measured by LC / MS, but could not be directly compared with previous Rag2 studies. - / - Protein levels were compared in the study because quantification was performed using different peptide and protein standards. The heart-to-body weight ratio demonstrated that MYBPC3... - / - Cardiac hypertrophy was significantly reduced in mice and almost normalized relative to WT levels. Figure 4E ).exist Figure 4EIn this context, **** indicates a value ≤0.0001 based on one-way ANOVA and multiple comparisons. Data is presented as mean + / - SD or mean + / - standard error of the mean (SEM).

[0281] With Rag2 - / - Compared to mice, MYBPC3 - / - Improved transduction and expression levels in mice (see) Figures 4A-4E This may be attributed to higher vector genome transduction per cell, but also to the lack of any endogenous cMyBP-C, which could limit transgene expression in wild-type environments.

[0282] An initial pilot study was conducted to evaluate the in vivo transduction efficiency of AAV9-MYBPC3 in cardiomyocytes after administration. Specifically, an 8-week pharmacological study was performed to evaluate the transduction, expression, and effects on hypertrophy of AAV9-MYBPC3 at a dose of 1.2E14 vg / kg. MYBPC3 was introduced into 2-week-old cardiomyocytes. - / - Mice (N=10 mice / group) were administered either the vector control or a dose of 1.2E14 vg / kg of AAV9-MYBPC3, and tissues were collected 8 weeks post-administration to assess the MYBPC3 vector genome copy number in cardiac tissue homogenates (n=6-7) and to evaluate cMyBP-C protein expression by immunofluorescence staining (n=3). Vector DNA and RNA transcripts in whole-heart homogenates were measured by ddPCR, and cMyBP-C levels were also measured by LC / MS. The percentage of cardiomyocytes expressing cMyBP-C protein was determined by immunohistochemistry. An average of 4.46 vector genome copies were found per diploid genome in the mouse heart. Figure 5A And the expression of MYBPC3 transcripts is increased. Figure 5B AAV9-MYBPC3 administration also induced widespread expression of human cMyBP-C protein in cardiomyocytes throughout mouse myocardium. Figure 5C Therefore, cMyBP-C protein is correctly incorporated into the sarcomere. Immunohistochemistry was also used to stain heart tissue sections for human cMyBP-C. Immunohistochemical analysis of whole heart tissue sections from AAV9-MYPBC3-treated mice showed that an average of 94% of cardiomyocytes in the entire ventricular myocardium were positive for human cMyBP-C staining. Figure 5D These results demonstrate the extensive transduction and expression of the vector. Heart weight to body weight ratio was measured, and a significant decrease in the heart weight to body weight ratio was observed 8 weeks after administration. Figure 5E Therefore, AAV9-MYBPC3 showed excellent distribution and expression in cardiomyocytes of mouse myocardium, and hypertrophy was statistically significantly reduced.

[0283] exist Figure 5E In the figures, * indicates a p-value ≤ 0.05 for both one-way ANOVA and graphical multiple comparisons. Data are presented as mean + / - SD or mean + / - SEM.

[0284] Example 5: Dosage

[0285] A long-term study evaluated the effects of AAV9-MYBPC3 at different dose ranges. In HCM, C57BL / 6J-MYCPC3 - / - In a mouse model, the pharmacodynamic effects and pharmacological activities of two vectors were analyzed. The vectors were human MYBPC3 (AAV9-MYBPC3) driven by the hTNNT2 promoter encapsulated in AAV9 and a similar vector encoding mouse cMyBP-C protein (AAV9-mMYBPC3) driven by the hTNNT2 promoter encapsulated in AAV9. Two-week-old MYBPC3 mice were subjected to... - / - Male mice were administered 1E14 vg / kg of AAV9-MYBPC3, and also AAV9-mMYBPC3 vectors at doses of 6E13, 1E14, and 2E14 vg / kg. A moderate dose of 1E14 vg / kg of AAV9-MYBPC3 was also included as a comparison, as both 6E13 vg / kg and 2E14 vg / kg doses of this construct showed sustained efficacy in earlier studies. rAAV particles were formulated with 17.6 mM Tris hydrochloride, 2.4 mM Tris base, 120 mM NaCl, 1 mM MgCl2 hexahydrate, 74 mM trehalose dihydrate, and 0.1% w / v poloxamer 188 at pH 7.4. The results of this study indicate the species specificity of the construct and assess its effects on cardiovascular function and hypertrophy over time. Preliminary echocardiographic data collected up to 26 weeks post-dose showed that, as early as 4 weeks post-dose, MYBPC3 was effectively treated with all doses of the AAV9-mMYBPC3 construct and AAV9-MYBPC3 at a dose of 1E14 vg / kg. - / - In mice, reduced hypertrophy leads to MYBPC3 - / -Early and sustained reductions in hypertrophy (left ventricular mass and wall size) were observed in mice throughout the 26-week study duration. Effects were visible as early as 4 weeks after administration of AAV9-mMYBPC3 at doses of 2E14 vg / kg and 1E14 vg / kg. At 4 weeks post-administration, compared to mediated MYBPC3- / - mice, administration of both 1E14 vg / kg and 2E14 vg / kg AAV9-mMYBPC3 vectors resulted in a lower left ventricular mass-to-body weight ratio. At 26 weeks post-administration, all vector administrations resulted in a lower left ventricular mass-to-body weight ratio compared to mediated mice. Figure 6A At 26 weeks post-administration, compared with mice treated with the carrier, all carrier administrations also resulted in a reduction in the size of the left ventricular posterior and anterior walls during diastole. Figure 6B At 26 weeks post-administration, a trend toward normalization of cardiac output and stroke volume (SV) was also observed at all doses. Figure 6C and 6D At 26 weeks post-administration, improvements in ejection fraction (EF) and fractional shortening (FS) were observed at all doses. A trend toward normalization of MV E and E / A ratio was observed at all doses at 26 weeks post-administration, but high variability was observed within the obtained measurements (see [link to relevant documentation]). Figure 6E This data indicates that these processed MYBPC3 - / - Overall improvement in ventricular relaxation in mice. Figure 6A , 6B In categories 6C, 6D, and 6E, based on univariate ANOVA and graphical multiple comparisons, **** indicates a p-value ≤ 0.0001, *** indicates a p-value ≤ 0.001, ** indicates a p-value ≤ 0.01, and * indicates a p-value ≤ 0.05. Data are presented as mean + / - SEM.

[0286] The results of this 26-week study demonstrate an early and sustained reduction in hypertrophy following all doses of the AAV9-mMYBPC3 construct and after administration of 1E14 vg / kg of AAV9-MYBPC3. Additionally, improved contractile function was 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 understanding the structural and functional differences between human and mouse proteins (Shaffer 2009).

[0287] A long-term 42-week study was conducted to evaluate the longitudinal effects of AAV9-MYBPC3 on cardiovascular function and left ventricular hypertrophy. The study included MYBPC3 in 2-week-old infants. - / -Male mice were administered AAV9-MYPBC3 at doses of 6E13 vg / kg or 2E14 vg / kg. MYBPC3 was introduced into 2-week-old mice. - / - Mice (N=10-13 mice / group) were administered a single bolus of either the mediator control, 6E13 vg / kg AAV9-MYPBC3, or 2E14 vg / kg AAV9-MYPBC3; a cohort of 10 wild-type mice was also evaluated. rAAV particles were formulated with 17.6 mM Tris hydrochloride, 2.4 mM Tris base, 120 mM NaCl, 1 mM MgCl2 hexahydrate, 74 mM trehalose dihydrate, and 0.1% w / v poloxamer 188 at pH 7.4. Tissues were collected at 0, 4, 8, 12, 20, 28, 36, and 42 weeks post-administration. Cardiovascular function and left ventricular hypertrophy were assessed by echocardiography. Indices of left ventricular hypertrophy, such as left ventricular mass / body weight, left anterior wall size, and left posterior wall size, showed early and sustained reductions under treatment. As early as 4 weeks after administration, MYBPC3 treated with low and high doses of AAV9-MYBPC3 was used. - / - Left ventricular hypertrophy was reduced in all mice, as evidenced by the left ventricular mass-to-body weight ratio. Several deaths in the mediator-treated group (3 / 10) between 36 and 44 weeks of age resulted in a statistically significant decrease in measurements at the 42-week time point. Data reported at 0, 4, 8, 12, 20, 28, and 36 weeks post-administration are shown in… Figures 7A-7C The data is presented as the average plus or minus SD.

[0288] Data reported 36 weeks after application are shown in Figures 8A-8D In. Figure 8A , 8B In 8C and 8D, based on one-way ANOVA and multiple comparisons, **** indicates p-value ≤ 0.0001, *** indicates p-value ≤ 0.001, ** indicates p-value ≤ 0.01, and * indicates p-value ≤ 0.05. Data are presented as mean + / - SD. For both doses, compared with MYBPC3 treated with the mordant... - / - Compared to mice, AAV9-MYPBC3 administration induced an early and sustained reduction in ventricular mass and anterior wall size. Specifically, an early and sustained reduction in interventricular septum and posterior wall size began at 4 weeks post-administration and persisted throughout the study duration (42 weeks post-administration). Diastolic function was assessed by four-chamber Doppler imaging and included measurements of passive ventricular filling (mitral valve velocity E wave - MVE). A total of seven echocardiographic assessments were performed throughout the study, and mice were sacrificed at 42 weeks post-administration to quantify cMyBP-C protein in the myocardium. MYBPC3 - / -Mice showed MV E impairment as early as 2 weeks of age. At 4 weeks post-administration, low and high doses of MYBPC3... - / - Mice showed significant improvement in ventricular ventricular activity (MVE), which persisted throughout the study duration. Both doses improved diastolic function, as quantified by changes in MVE measurements. These data also indicated an overall improvement in ventricular relaxation as early as 4 weeks post-administration. In the mediator (30%; 3 / 10) and treatment groups (23%; 6 / 26), the severity of the model and significantly impaired systolic function led to several deaths. Histopathology revealed left atrial thrombosis as the cause of all assessed cardiac pathogenesis, and no evidence of immune-mediated inflammation was shown. No gross hepatic lesions were observed in any of the mice studied. Immunohistochemistry of cMyBP-C in the hearts of high-dose euthanized mice showed successful transduction and expression of human cMyBP-C in most cells of the myocardium.

[0289] The results of this long-term study demonstrate an early and sustained reduction in hypertrophy and improvement in diastolic function following AAV9-MYBPC3 at doses of 6E13 and 2E14 vg / kg. Therefore, MYBPC3 in 2-week-old infants... - / - In mice, a single administration of AAV9-MYBPC3 resulted in a sustained reduction in hypertrophy and LV wall thickness, as well as improved diastolic function.

[0290] Example 6:

[0291] Peak protein expression studies were conducted in WT mice to determine the duration of studies investigating GLP-mediated toxicity. The toxicology of the AAV9-MYPBC3 vector was analyzed in adult male C57BL / 6 mice to determine the timing of peak transgene expression and the appropriate duration of studies to assess toxicity. The AAV9-MYPBC3 vector was administered to 8-week-old male C57BL / 6 mice. Proteins in heart homogenates were assessed at 4, 8, 12, and 16 weeks post-administration. Proteins in heart homogenates will also be assessed at 1, 2, 3, and 4 months post-administration, and liver samples will be archived for future analysis. Figure 9 As shown, administration of 2E14 vg / kg of AAV9-MYPBC3 to 8-week-old male WT mice via a single intravenous dose resulted in near-peak expression of human protein at 8 weeks post-administration, with a tendency to slightly increase up to 12 weeks post-administration. Figure 9 In the data, based on one-way ANOVA and graphical multiple comparisons, **** indicates a p-value ≤ 0.0001, *** indicates a p-value ≤ 0.001, and ** indicates a p-value ≤ 0.01. Data are presented as mean + / - SD.

[0292] No adverse hepatotoxicity or DRG histopathological findings were observed 3 months after injection, up to 4E14 vg / kg.

[0293] Example 7: Dosage Range

[0294] MYBPC3 at 2 weeks old - / - A dose-range study was conducted in mice to determine the minimum effective dose of AAV9-MYBPC3 and to test the AAV9-MYBPC3 vector produced using the final vector production process. The rAAV particles were formulated with 17.6 mM Tris hydrochloride, 2.4 mM Tris base, 120 mM NaCl, 1 mM MgCl2 hexahydrate, 74 mM trehalose dihydrate, and 0.1% w / v poloxamer 188 at pH 7.4. Group 1 consisted of 10 WT mice (control), and groups 2–7 each consisted of 10 mice to be analyzed for MYBPC3. - / - Mice. Group 2 consisted of MYBPC mice treated with the causative agent. - / - Mouse control. Groups 3, 4, 5, 6, and 7 were administered AAV9-MYPBC3 at doses of 6E12 vg / kg, 2E13 vg / kg, 6E13 vg / kg, 1.2E14 vg / kg, and 1.2E14 vg / kg, respectively. Echocardiographic analysis was performed at baseline, 13 weeks post-administration, and at the end of the study. Heart weight, body weight, cardiac transduction and expression—DNA, RNA, protein levels, and protein distribution were also assessed to determine the minimum effective dose. Mice were then euthanized at 13 or 36 weeks post-administration for tissue collection. DNA, RNA, and protein were extracted from the heart homogenate. Cardiac transduction and distribution were assessed by DNA, RNA, protein, and immunohistochemical analysis to correlate efficacy with tissue levels and distribution. Vector DNA, RNA, and expressed protein levels showed a direct dose-dependent increase, depending on the dose of AAV9-MYPBC3 administered. Figure 10A-10D Human cMyBP-C protein expression was confirmed in cardiomyocytes by immunohistochemistry. A trend was also observed where the percentage of cells expressing cMyBP-C increased with increasing dose. Figure 10E The administered AAV9-MYBPC3 transduced 61%-94% of cardiomyocytes, depending on the dose. The staining intensity of human cMyBP-C positive cardiomyocytes also tended to increase with increasing dose. Figure 10A , 10B In 10C, 10D, and 10E, based on one-way ANOVA and graphical multiple comparisons, **** indicates a p-value ≤ 0.0001, *** indicates a p-value ≤ 0.001, ** indicates a p-value ≤ 0.01, and * indicates a p-value ≤ 0.05. Data are presented as mean + / - SD.

[0295] It was also noted that the dose of AAV9-MYPBC3 at 6E13 vg / kg was the lowest dose that showed a reduction in hypertrophy and improvement in diastolic function after 13 weeks of administration. Figure 11A-11C Therefore, AAV9-MYBPC3 induces dose-responsive transduction and throughout MYBPC3. - / - Durable expression of MYBPC3 RNA and cMyBP-C protein was established in mouse hearts, and cMyBP-C protein expression and function were restored in a dose-dependent manner. Figure 1A , 11B In 11C, based on one-way ANOVA and Tuki multiple comparisons, **** indicates p-value ≤ 0.0001, *** indicates p-value ≤ 0.001, ** indicates p-value ≤ 0.01, and * indicates p-value ≤ 0.05. Data are presented as mean + / - SD.

[0296] Example 8: Non-GLP Pilot NHP Study

[0297] Cynomolgus monkeys were used to demonstrate the translatability, safety, and tolerability of AAV9-MYBPC3 transduction and expression. Transgenic expression, tolerability, and biodistribution of AAV9-hMYPBC3 particles containing the AAV9 capsid and the following AAV vector genomes were evaluated in non-human primate (NHP) tissues at multiple time points at days 31, 62, 91, and 153 post-administration. These included: (a) the hTNNT2 promoter, (b) the wild-type or codon-optimized cMyBP-C gene, (c) the polyadenylation signal, and optionally (d) globulin introns (e.g., any one of SEQ ID NO: 3-41). NHPs were euthanized at day 31 for tissue collection. DNA and RNA were extracted from the collected tissues.

[0298] Preliminary results showed no significant differences in clinical observation, body weight, macroscopic observation, or ECG among the three vector groups. No changes induced by AAV9-hMYBPC3 administration occurred during the first 62 days. Transient, minimal to mild increases in ALT were observed in males only on days 8 and / or 15. No DRG histopathological findings were observed in NHP up to a dose of 1.2E14 vg / kg.

[0299] AAV9-MYPBC3 vector DNA transduction was detected in autopsy tissues using ddPCR, and was measured individually in five skeletal muscle tissue sections: left atrium, right atrium, left ventricle, right ventricle, and diaphragm. Vector DNA was also measured in cardiac vascular tissue, aorta, and pulmonary artery. Figure 12Data are presented as mean ± SEM. Vector transduction was uniformly distributed in cardiac tissue sections, with no significant difference in vector distribution measured at 31 or 62 days post-dose. There was also no significant difference in vector DNA levels in any tissue section between the 31-day and 62-day cohorts. Therefore, a single dose of AAV9-MYPBC3 effectively delivers MYBPC3 cDNA to the heart of NHP and is detectable at 31 days post-dose.

[0300] hMYBPC3 mRNA derived from the AAV9-MYPBC3 vector was detected in autopsy tissues using RT-ddPCR, and was measured individually in five myocardial tissue sections (left atrium, right atrium, left ventricle, right ventricle, and septum). Vector-derived transcripts were also measured in cardiac vascular tissue, the aorta, and the pulmonary artery. Figure 13 Data are presented as mean + / - SEM. In the 31-day cohort, RNA transcription measured in the aorta was significantly lower than in the left ventricle, but no other significant differences in vector transcription were measured between any other tissue sections within the same cohort. There were no significant differences in vector transcript levels measured in any tissue sections between the 31-day and 62-day cohorts. Therefore, the detection of MYBPC3 RNA transcripts in multiple cardiac tissues at 31 days post-administration following a single dose of AAV9-MYPBC3 indicates that AAV9-MYPBC3 demonstrates efficient transduction and expression of MYBPC3 RNA from NHP-derived cardiac tissue.

[0301] Example 9:

[0302] Several different vector designs were produced using two independent manufacturing methods: the fall armyworm insect cell system (Sf) and the mammalian (i.e., human) embryonic kidney 293 (HEK293) cell system. Different AAV9-MYBPC3 vectors or mediator controls produced by each manufacturing system were screened to assess their ability to produce cMyBP-C protein in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM). Human iPSC-CM carried a biallelic truncated MYBPC3 mutation that resulted in the complete absence of cMyBP-C protein. The screened AAV9-MYBPC3 vectors included vector A1 (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). Compared to AAV vectors produced using the Sf cell manufacturing system, different AAV9-MYBPC3 vectors produced using the HEK293 cell manufacturing system yielded the strongest protein expression across various vector designs in iPSC-CM. Figure 14 Furthermore, engineered heart tissue derived from human iPSC-CM transduced with AAV vectors produced using the HEK293 cell manufacturing system exhibited improved contractile function. Therefore, the AAV vectors produced using the HEK293 cell manufacturing system demonstrate superior in vitro and in vivo expression and function compared to other candidate vectors and manufacturing systems.

[0303] Example 10: Administration of AAV particles to human subjects

[0304] Non-obstructive hypertrophic cardiomyopathy

[0305] A clinical study will be conducted in subjects with non-obstructive hypertrophic cardiomyopathy (nHCM) caused by a mutation in the MYBPC3 gene to demonstrate the safety, tolerability, and potential efficacy of a single intravenous infusion of AAV9-MYPBC3. This study will provide evidence of effective improvement in functional outcomes and / or morbidity in patients with HCM.

[0306] Human subjects were administered rAAV particles containing an AAV9 capsid and a carrier construct described herein (e.g., any one of SEQ ID NO: 3-41) at one of three dose levels (6E13 vg / kg, 9E13 vg / kg, or 1.2E14 vg / kg) to evaluate the efficacy, safety, and tolerability of the rAAV particles at the indicated dose. Prophylactic immunomodulation was also administered to subjects via administration of steroids and sirolimus, as described below. Optionally, antimicrobial prophylaxis, such as administration of sulfamethoxazole / methoxypyridazine, was employed to prevent Pneumocystis pneumonia (PJP). Subjects were assessed for TB and vaccination.

[0307] Subjects with nHCM and no anti-AAV9 antibody at a neutralizing titer or predetermined titer (Part A) will receive the required dose of rAAV particles in a single intravenous infusion at a rate of 20 mL / hr (0.33 mL / min), up to a maximum of 80 mL / hr (1.33 mL / min), and will be followed for 5 years to assess the durability of response. AAV9 seropositive nHCM participants who meet all other inclusion and exclusion criteria will be recruited into the parallel comparative study group (Part B).

[0308] One objective of the study was to assess the safety of AAV9-MYBPC3. Based primarily on previous safety findings associated with other gene therapies using AAV vectors, several safety endpoints were identified, most notably the number of participants with targeted adverse cardiac events and targeted adverse immune events (elevated transaminases, thrombotic microangiopathy [TMA]).

[0309] Several efficacy endpoints were measured to fully characterize the effects of AAV9-MYPBC3 on the heart and subjects. Changes in cardiac systolic and diastolic function were assessed by echocardiography and serum biomarkers of cardiac stress. Changes in cardiac morphology were assessed by echocardiographic measurements of left ventricular mass and wall thickness. Changes in exercise capacity were assessed by peak oxygen consumption (PVO2) and other maximal and submaximal exercise parameters obtained during cardiopulmonary exercise testing (CPET). Changes in patient symptoms and functional status were assessed using KCCQ-23 and NYHA, respectively. Optionally, cardiomyocyte transduction and RNA and protein expression were assessed from myocardial tissue obtained via endocardial myocardial biopsy compared to pre-drug levels.

[0310] One efficacy endpoint was the change in PVO2 as measured by CPET. Previous studies have shown that an increase in PVO2 of 1 ml / kg / min represents a threshold of clinically relevant improvement in HCM patients and reduces the risk of all-cause mortality or heart transplantation (Coats et al., Circ Heart Fail. 8(6):1022-31 (2015); Masri et al., J Am Heart Ass. 7:e007944 (2018)). Furthermore, previous studies in HCM patients have shown that improvements in PVO2 lead to improvements in functional classification (Olivotto et al., Lancet, 396(10253):759-69 (2020)).

[0311] Inclusion and Exclusion Criteria

[0312] Clinical study inclusion criteria may include one or more of the following: (1) adult subjects aged 18–65 years; (2) a clinical diagnosis of non-obstructive hypertrophic cardiomyopathy (nHCM) in accordance with current ACC / AHA and / or ESC guidelines; (3) a pathogenic heterozygous truncated mutation in the MYBPC3 gene, confirmed by genetic testing at a CLIA-approved laboratory; (4) a non-obstructive phenotype defined as a peak left ventricular outflow tract (LVOT) gradient at rest <30 mmHg and / or a peak LVOT gradient during Valsalva maneuver <50 mmHg; (5) optimal therapy for HCM according to the New York Heart Association (NYHA) functional classification system (Class II or III); (6) an ejection fraction ≥50%; (7) elevated NT-proBNP levels (>300 pg / mL); (8) currently using a β-blocker and / or calcium channel blocker at optimal doses and a stable drug dose for at least 3 months prior to screening; and optionally, a body mass index (BMI) ≤35. kg / m 2Other inclusion criteria may include a peak oxygen consumption (PVO2) ≤80% predicted on cardiopulmonary exercise testing (CPET) and a respiratory exchange rate (RER) ≥1.0 at the time of CPET screening. Patients may also have an implantable cardioverter defibrillator (ICD).

[0313] Exclusion criteria for clinical studies may include one or more of the following: (1) evidence of active or chronic infection or immunosuppressive illness; (2) contraindications to corticosteroids; (3) a major or uncontrolled chronic condition defined as requiring regular medical care 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 arrhythmic syncope, sustained ventricular tachycardia, cardiac arrest resuscitation or appropriate ICD discharge within 6 months of screening; and (5) paroxysmal atrial fibrillation at the time of screening. (6) Persistent or permanent atrial fibrillation, (7) Major valvular heart disease (moderate to severe valvular aortic stenosis, moderate to severe mitral regurgitation not caused by systolic forward motion of the mitral valve) or other heart disease, including but not limited to structural heart disease (unrelated to nHCM), congenital heart disease, coronary heart disease, pericardial disease, acute myocarditis, infiltrative cardiomyopathy, obstructive hypertrophic cardiomyopathy and a history of pericarditis / myocarditis / pericardial effusion, and (8) Prior treatment with gene therapy or investigational drug therapy concurrently or within 6 months prior to screening. Other exclusion criteria include a history of obstructive HCM (resolved surgically), a history of alcohol diaphragmatic ablation for oHCM, currently listed for or planned for heart transplantation, having one or more risk factors for sudden cardiac death, a history of thrombotic microangiopathy (TMA) or a known genetic predisposition to thrombotic microangiopathy, hemolytic uremic syndrome or complement deficiency, a history of thromboembolic (TW) events or a known thrombophilia, significant liver fibrosis (≥F 3), active or chronic infection or any immunosuppressive disorder, any contraindications to corticosteroid use (including glaucoma, untreated osteoporosis, or a diagnosis of diabetes), a history of clinically significant malignancy within the past 10 years, exclusion based on weight or BMI, borderline AAV9 seropositivity, controlled atrial fibrillation, heart rate exclusion value, blood pressure value, pulse oximetry value, eGFR value, or myocardial infarction. Myotomy was not identified as an exclusion criterion and is permissible.

[0314] The following diagnostic clinical exclusion criteria apply: 1) detectable antibody against the 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% in room air, f) temperature >100.4 degrees Fahrenheit, g) liver enzymes >2 times the upper limit of normal, h) abnormal bilirubin, i) eGFR <45 ml / min / 1.73 m2 according to CKD-EPI, j) platelet count <100,000, k) hemoglobin ≤10 g / dL, and / or l) hemoglobin A1C ≥8.

[0315] Monitoring safety and efficacy

[0316] Prior to infusion of rAAV particles, the following assessments were performed on the subject: (1) baseline physical examination; (2) baseline clinical laboratory tests, including antigen and functional levels of MYBPC3; and (b) baseline AAV9 antibody testing.

[0317] Safety parameters monitored after infusion of rAAV particles included: (1) the incidence of adverse events and serious adverse events, (2) the incidence of dose-limiting toxicity (DLT), (3) the incidence of targeted adverse cardiac events: a) EF < 50%, b) arrhythmia, c) myocarditis / pericarditis, d) regional wall motion abnormalities, e) increased troponin I, and (4) the incidence of targeted adverse immune events, defined as: a) elevated transaminases, b) thrombotic microangiopathy (TMA), or c) increased complement activation markers (i.e., sC5b-9).

[0318] Monitor the following additional efficacy indicators: (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) impact, (6) health problems, and (7) global anchoring metric.

[0319] Myocardial stress and injury were monitored using blood biomarker assays, which assessed changes in N-terminal pro-BNP (NT-proBNP) and high-sensitivity troponin I (hsTNI). Other endpoints may include measurements of P1C1. Cardiac structure / morphology and function were monitored by echocardiography / cMRI, assessing 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 was monitored by assessing changes in PVO2 via cardiopulmonary exercise testing (CPET). Functional cardiac status was assessed using the New York Heart Association (NYHA) functional classification, and the Kansas City Cardiomyopathy Questionnaire-23 (KCCQ-23) Total Symptom Score (TSS), Physical Limitation Score, and Clinical Summary Score (CSS) were used to assess the subjects' symptoms and health-related quality of life (HRQoL). Health status was also assessed using the EuroQol-5 Dimensions Level 5 (EQ-5D-5L) and the EuroQol-Visual Analogue Scale (EQ-VAS), and global anchoring metric was assessed using the Clinical Global Impression Severity and Variation Scale (CGI / C) and the Patient Global Impression Severity and Variation Scale (PGIS / C). The incidence of adverse events and serious adverse events was monitored.

[0320] Immunogenicity and safety assessment

[0321] Immunogenicity in subjects was assessed throughout the study. Complement combination assays were performed daily for 2 days prior to infusion, daily for 7 days post-infusion, and weekly throughout the study duration (weeks 2–10). Additionally, anti-AAV9 Tab assays were performed weekly for 2 days prior to infusion, on the day of infusion, and weekly throughout the study duration (weeks 1–10). Adverse events and LFTs were assessed daily for 2 days prior to infusion, daily for 7 days post-infusion, and weekly throughout the study duration (weeks 2–10). Vector detachment was assessed on the day of infusion and at weeks 4 and 8.

[0322] Cardiac assessment

[0323] Subjects underwent baseline screening starting 8 weeks prior to infusion. Cardiac function and structure were assessed by echocardiography and electrocardiography 4 weeks prior to infusion. NYHA, KCCQ-23, CPET cardiac MR, and ambulatory ECG monitoring were performed. NT-proBNP and troponin I levels were assessed at 8 weeks and 4 weeks prior to infusion.

[0324] Subjects underwent active cardiac, immune, and liver monitoring early in the study, with monitoring frequency decreasing based on clinical status. Adverse event assessment began on the day of infusion and continued for 16 weeks. Cardiac assessments were performed on the day of infusion, weekly from week 2 to week 16, and at weeks 24, 32, 40, and 48 by echocardiography and electrocardiography, as well as NT-proBNP and troponin I levels. NYHA and KCCQ-23 were administered on the day of infusion and at weeks 4, 8, 16, 24, and 48. Ambulatory ECG monitoring was performed at weeks 2, 4, 8, 16, 24, and 48.

[0325] Other endpoints may include obtaining and evaluating biopsies, having patients perform a six-minute walk test, or recording other patient-reported outcome measures. The abbreviated form of CGI can also be used as a holistic patient assessment tool.

[0326] Other safety endpoints may include identifying elevated transaminase levels, identifying 40% or 45% ejection fraction, identifying arrhythmias, or assessing ventricular wall motion.

[0327] Preventive immune regulation

[0328] Subjects received prophylactic immunosuppressive therapy with glucocorticoids, optionally sirolimus, and optionally a complement pathway inhibitor or antagonist. Subjects received a 30-day prophylactic glucocorticoid regimen, starting at 40 mg / day from day 1 of infusion, followed by a 30-day dose reduction starting at week 5, provided tests indicated normal liver function and no evidence of myocarditis. Additionally, sirolimus was administered prophylactically at 6 mg / day for 2 days prior to infusion, followed by 2 mg / day for 60 days. Sirolimus monitoring was performed to ensure target sirolimus blood levels were maintained between 5 and 15 ng / mL. Furthermore, participants taking more than one immunosuppressive drug received 160 + 800 mg trimethoprim-sulfamethoxazole (TMP-SMX) (e.g., one double-strength tablet) orally three times weekly starting one day prior to infusion to prevent Jepserella pneumoniae pneumonia.

[0329] Reactive immune regulation

[0330] Reactive immunosuppressive therapy (e.g., glucocorticoids optionally in combination with complement pathway inhibitors or antagonists) may be initiated in response to liver or cardiac inflammation observed after completion of the prophylaxis program. In response, extended full-dose glucocorticoid therapy is administered after the initial 30-day prophylaxis period. Following elevated liver function tests, tacrolimus or mycophenolate mofetil will be added as an alternative immunosuppressant, despite the administration of steroid / sirolimus. For breakthrough myocarditis, tacrolimus is administered, and for refractory complement activation in the context of thrombotic microangiography, eculizumab is administered.

[0331] Obstructive hypertrophic cardiomyopathy

[0332] A clinical study will be conducted in subjects with obstructive hypertrophic cardiomyopathy (oHCM) caused by a mutation in the MYBPC3 gene to demonstrate the safety, tolerability, and potential efficacy of a single intravenous infusion of AAV9-MYPBC3. This study will provide evidence of effective improvement in functional outcomes and / or morbidity in patients with HCM.

[0333] Human subjects were administered rAAV particles comprising an AAV9 capsid and a carrier construct described herein (e.g., any one of SEQ ID NO: 3-41) at one of three dose levels (6E13 vg / kg, 9E13 vg / kg, and 1.2E14 vg / kg) to evaluate the efficacy, safety, and tolerability of the rAAV particles at the indicated dose. Prophylactic immunomodulation was also administered to subjects via administration of steroids and sirolimus, as described below. Optionally, antimicrobial prophylaxis, such as administration of sulfamethoxazole / methoxypyridazine, was employed to prevent PJP. Subjects were assessed for TB and vaccination.

[0334] Part A subjects with oHCM and no anti-AAV9 antibody at a neutralizing or predetermined titer will be administered the required dose of rAAV particles in a single intravenous infusion at a rate of 20 mL / hr (0.33 mL / min), up to a maximum of 80 mL / hr (1.33 mL / min), and will be followed for 5 years to assess the durability of response. AAV9 seropositive oHCM participants who meet all other inclusion and exclusion criteria will be recruited into the parallel comparison study group (Part B).

[0335] One objective of the study was to assess the safety of AAV9-MYBPC3. Based primarily on previous safety findings associated with other gene therapies using AAV vectors, several safety endpoints were identified, most notably the number of participants experiencing adverse cardiac events and adverse immune events (elevated transaminases, TMA).

[0336] Several efficacy endpoints were measured to fully characterize the effects of AAV9-MYPBC3 on the heart and subjects. Changes in cardiac systolic and diastolic function were assessed by echocardiography and serum biomarkers of cardiac stress. Changes in cardiac morphology were assessed by echocardiographic measurements of left ventricular mass and wall thickness. Changes in left ventricular outflow tract obstruction were assessed by echocardiographic measurements of left ventricular outflow tract pressure gradient. Changes in exercise capacity were assessed by PVO2 and other maximal and submaximal exercise parameters obtained during CPET. Changes in patient symptoms and functional status were assessed using KCCQ-23 and NYHA, respectively. Optionally, cardiomyocyte transduction and RNA and protein expression were assessed from myocardial tissue obtained via endocardial myocardial biopsy compared to pre-drug levels.

[0337] One efficacy endpoint was the change in PVO2 as measured by CPET. Previous studies have shown that an increase in PVO2 of 1 ml / kg / min represents a threshold of clinically relevant improvement in HCM patients and reduces the risk of all-cause mortality or heart transplantation (Coats et al., Circ Heart Fail. 8(6):1022-31 (2015); Masri et al., J Am Heart Ass. 7:e007944 (2018)). Furthermore, previous studies in HCM patients have shown that improvements in PVO2 lead to improvements in functional classification (Olivotto et al., Lancet, 396(10253):759-69 (2020)).

[0338] Inclusion and Exclusion Criteria

[0339] Clinical trial inclusion criteria may include one or more of the following: (1) a clinical diagnosis of oHCM that meets current ACC / AHA and / or ESC guidelines; (2) a pathogenic heterozygous truncated mutation in the MYBPC3 gene, confirmed by genetic testing at a CLIA-approved laboratory; (3) optimal therapy for HCM according to NYHA functional classification of class II or III heart failure; (4) ejection fraction ≥50%; and (5) elevated NT-proBNP levels (>300 pg / mL). Other inclusion criteria may include PVO2 ≤80% predicted on CPET and RER ≥1.0 at the time of CPET screening. Patients may also have ICD.

[0340] Echocardiography can be used to determine the presence and size of obstruction of blood flow through the LVOT. A pressure gradient of ≥30 mmHg at rest or upon provocation indicates obstruction, and the patient is considered to have oHCM. Therefore, this method can be used to diagnose patients with oHCM and select them for clinical research.

[0341] Exclusion criteria for clinical studies may include one or more of the following: (1) evidence of active or chronic infection or immunosuppressive disorder; (2) contraindications to corticosteroids; (3) a major or uncontrolled chronic condition defined as requiring regular medical care 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 arrhythmic syncope, sustained ventricular tachycardia, cardiac arrest resuscitation, or appropriate ICD discharge within 6 months of screening; and (5) paroxysmal position at the time of screening. (6) Persistent or permanent atrial fibrillation, (7) major valvular heart disease (moderate to severe valvular aortic stenosis, moderate to severe mitral regurgitation not due to systolic forward motion of the mitral valve) or other heart disease, including but not limited to structural heart disease (unrelated to oHCM), congenital heart disease, coronary artery disease, pericardial disease, acute myocarditis, infiltrative cardiomyopathy and a history of pericarditis / myocarditis / pericardial effusion, and (8) prior treatment with gene therapy or investigational drug therapy concurrently or within 6 months prior to screening. Other exclusion criteria include weight or BMI exclusion, borderline AAV9 seropositivity, controlled atrial fibrillation, heart rate exclusion value, blood pressure value, pulse oximetry value, eGFR value, or myocardial infarction. Myotomy was not identified as an exclusion criterion and is permissible.

[0342] The following diagnostic clinical exclusion criteria apply: 1) detectable antibody against the 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% in room air, f) temperature >100.4 degrees Fahrenheit, g) liver enzymes >2 times the upper limit of normal, h) abnormal bilirubin, i) eGFR <45 ml / min / 1.73 m2 according to CKD-EPI, j) platelet count <100,000, k) hemoglobin ≤10 g / dL, and / or l) hemoglobin A1C ≥8.

[0343] Monitoring safety and efficacy

[0344] Prior to infusion of rAAV particles, the following assessments were performed on the subject: (1) baseline physical examination; (2) baseline clinical laboratory tests, including antigen and functional levels of MYBPC3; and (b) baseline AAV9 antibody testing.

[0345] Safety parameters monitored after infusion of rAAV particles included: (1) the incidence of adverse events and serious adverse events, (2) the incidence of DLT, (3) the incidence of targeted adverse cardiac events: a) EF < 50%, b) arrhythmia, c) myocarditis and pericarditis, d) regional wall motion abnormalities, e) increased troponin I, and (4) the incidence of targeted adverse immune events, defined as: a) elevated transaminase, b) TMA, or c) increased complement activation marker (i.e., sC5b-9).

[0346] Monitor the following additional efficacy indicators: (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 impact, (6) health problems, and (7) global anchoring metric.

[0347] Myocardial stress and injury were monitored using blood biomarkers assessing changes in NT-proBNP and hsTNI. Other endpoints may include measuring changes in P1C1. Cardiac structure / morphology and function were monitored by echocardiography / cMRI, assessing 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 was monitored by assessing changes in PVO2 via CPET. Functional cardiac status was assessed using the NYHA functional classification, and subject symptoms and HRQoL impact were assessed using the KCCQ-23 TSS, physical limitation score, and CSS. Health status was also assessed using EQ-5D-5L and EQ-VAS, and global anchoring metric was assessed using CGI / C and PGIS / C. The incidence of adverse events and serious adverse events was monitored.

[0348] Immunogenicity and safety assessment

[0349] Immunogenicity in subjects was assessed throughout the study. Complement combination assays were performed daily for 2 days prior to infusion, daily for 7 days post-infusion, and weekly throughout the study duration (weeks 2–10). Additionally, anti-AAV9 Tab assays were performed weekly for 2 days prior to infusion, on the day of infusion, and weekly throughout the study duration (weeks 1–10). Adverse events and LFTs were assessed daily for 2 days prior to infusion, daily for 7 days post-infusion, and weekly throughout the study duration (weeks 2–10). Vector detachment was assessed on the day of infusion and at weeks 4 and 8.

[0350] Cardiac assessment

[0351] Subjects underwent baseline screening starting 8 weeks prior to infusion. Cardiac function and structure were assessed by echocardiography and electrocardiography 4 weeks prior to infusion. NYHA, KCCQ-23, CPET cardiac MR, and ambulatory ECG monitoring were performed. NT-proBNP and troponin I levels were assessed at 8 and 4 weeks prior to infusion.

[0352] Subjects underwent active cardiac, immune, and liver monitoring early in the study, with monitoring frequency decreasing based on clinical status. Adverse event assessment began on the day of infusion and continued for 16 weeks. Cardiac assessments were performed on the day of infusion, weekly from week 2 to week 16, and at weeks 24, 32, 40, and 48 by echocardiography and electrocardiography, as well as NT-proBNP and troponin I levels. NYHA and KCCQ-23 were administered on the day of infusion and at weeks 4, 8, 16, 24, and 48. Ambulatory ECG monitoring was performed at weeks 2, 4, 8, 16, 24, and 48.

[0353] Other endpoints may include obtaining and evaluating biopsies, having patients perform a six-minute walk test, or recording other patient-reported outcome measures. The abbreviated form of CGI can also be used as a holistic patient assessment tool.

[0354] Other safety endpoints may include identifying elevated transaminase levels, identifying 40% or 45% ejection fraction, identifying arrhythmias, or assessing ventricular wall motion.

[0355] Preventive immune regulation

[0356] Subjects received prophylactic immunosuppressive therapy with glucocorticoids, optionally sirolimus, and optionally a complement pathway inhibitor or antagonist. Subjects received a 30-day prophylactic course of glucocorticoids, starting at a dose of 40 mg / day from day 1 of infusion, followed by a 30-day dose reduction starting in week 5, provided that liver function was normal and there was no evidence of myocarditis. Additionally, sirolimus was prophylactically administered 6 mg / day for 2 days prior to infusion, followed by 2 mg / day for 60 days. Furthermore, subjects taking more than one immunosuppressive drug received a prophylactic dose of 160 + 800 mg (i.e., one double-strength tablet) of TMP-SMX orally three times weekly.

[0357] Reactive immune regulation

[0358] Reactive immunosuppressive therapy (e.g., glucocorticoids optionally in combination with complement pathway inhibitors or antagonists) may be initiated in response to liver or cardiac inflammation observed after completion of the prophylaxis program. In response, extended full-dose glucocorticoid therapy is administered after the initial 30-day prophylaxis period. Following elevated liver function tests, tacrolimus or mycophenolate mofetil will be added as an alternative immunosuppressant, despite the administration of steroid / sirolimus. For breakthrough myocarditis, tacrolimus is administered, and for refractory complement activation in the context of thrombotic microangiography, eculizumab is administered.

[0359] The embodiments described herein are intended to be illustrative only, and those skilled in the art will recognize or be able to determine many equivalents of particular compounds, materials, and procedures using only conventional experiments. All such equivalents are considered to be within the scope of this disclosure.

[0360] All patents, patent applications, and publications mentioned herein are incorporated herein by reference in their entirety. Any reference or designation of any reference in this application does not constitute an admission that such reference is prior art. The full scope of this disclosure can be better understood by referring to the appended claims.

Claims

1. A pharmaceutical composition comprising recombinant adeno-associated virus (rAAV) particles optionally having an AAV9 serotype capsid at a concentration of at least about 6E13 vg / ml to about 4E14 vg / ml, said pharmaceutical composition comprising a buffer, an isotonic agent, an additional MgCl2 stabilizer, a cryopreservative, and a surfactant, which is stable for at least about 1 year, 1.5 years, or 2 years when stored at about -60°C or lower.

2. The pharmaceutical composition according to claim 1, wherein the pH value of the formulation is in the range of about 6 to about 9, optionally in the range of about 7 to about 7.

8.

3. The pharmaceutical composition according to claim 2, wherein the pH value is 7.

4.

4. A pharmaceutical composition comprising rAAV particles optionally having an AAV9 serotype capsid at a concentration of at least about 6E13 vg / ml to about 4E14 vg / ml or 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, MgCl2 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 poloxamer or polysorbate at a concentration of about 0.05% to about 0.15% w / v.

5. The pharmaceutical composition according to claim 4, wherein the concentration of rAAV particles is in the range of about 1E13 vg / ml to about 1E14 vg / ml.

6. The pharmaceutical composition of claim 4, wherein the concentration of rAAV particles is in the range of about 1E13 vg / ml to about 5E13 vg / ml, or about 5E13 vg / ml to about 1E14 vg / ml.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the buffer comprises Tris hydrochloride and / or Tris base.

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

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

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the stabilizer is MgCl2 at a concentration of 1 mM.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the stabilizer is MgCl2 at a concentration of more than 1.5 mM.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the stabilizer is MgCl2 at a concentration of up to 3.0 mM.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the cryopreservative is sugar.

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

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the surfactant is poloxamer 188.

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

17. The pharmaceutical composition according to any one of claims 1 to 16, wherein the composition is a liquid.

18. 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 MgCl2 hexahydrate, 74 mM 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 according to claim 18, wherein the composition is a liquid.

21. A method of treating a subject with heart disease using a pharmaceutical composition according to any one of claims 1 to 20, wherein the pharmaceutical composition is administered intravenously.

22. The method of claim 21, wherein the heart disease is associated with a gene mutation in 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 heart disease, comprising administering to the subject a single dose of rAAV particles in the range of about 1E13 vg / kg to about 4E14 vg / kg, said rAAV particles comprising (a) a cardiotropic AAV capsid, (b) a cardio-specific transcriptional regulatory region and (c) a recombinant vector construct comprising a nucleic acid encoding a functional c-MyBPC protein or a codon-optimized form 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 according to any one of claims 24 to 30, wherein the functional c-MyBPC protein comprises at least 95%, 98%, or 99% of the same amino acid sequence as amino acids 1 to 1274 of SEQ ID NO:

2.

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

1.

33. The method according to any one of claims 24 to 32, wherein the heart-specific transcriptional regulatory region comprises a fragment of the human TNNT2 promoter.

34. The method according to any one of claims 24 to 33, wherein the heart-specific transcriptional regulatory region comprises at least 90%, 95%, 98% or 99% of the same nucleotide sequence as SEQ ID NO:

51.

35. The method according to any one of claims 24 to 34, wherein the recombinant vector construct comprises introns.

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

37. The method of any one of claims 24 to 36, wherein the subject is administered a population of rAAV particles generated by a method comprising: (a) providing mammalian cells comprising one or more nucleic acid constructs, the 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 heart-specific transcriptional regulatory region, and (3) a nucleic acid encoding a functional MyBPC3 comprising an amino acid sequence that is at least 95% identical to amino acids 1 to 1274 of SEQ ID NO: 2; (ii) a nucleotide sequence encoding one or more AAV Rep proteins operatively linked to a promoter capable of driving the expression of the one or more Rep proteins in the cells; and (iii) a nucleotide sequence encoding one or more AAV9 type capsid proteins operatively linked to a promoter capable of driving the expression of the one or more capsid proteins in the cells; (b) culturing the mammalian cells under conditions that allow expression of the Rep proteins and the capsid proteins and the generation of AAV particles; and (c) recovering the AAV particles.

38. The method of claim 37, wherein the population is enriched with rAAV particles containing a vector genome of full length, near full length, or therapeutically effective length by the step of reducing the number of empty capsids.

39. The method according to any one of claims 24 to 38, wherein the AAV capsid comprises at least 85%, 90%, or 95% of the same amino acid sequence as any one of SEQ ID NO:207-223.

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

207.

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

42. The method according to any one of claims 24 to 41, wherein the subject suffers from heart disease associated with a gene mutation in the MYBPC3 gene.

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

44. The method according to any one of claims 21 to 43, wherein the dose effectively improves cardiac function or structure, optionally as measured by a decrease in E / e' and / or a decrease in the left atrial volume index and / or an improvement or normalization of the ejection fraction and / or a reduction in longitudinal strain damage, and / or a decrease in left ventricular (LV) wall thickness and / or a decrease in LV mass.

45. The method of any one of claims 21 to 44, wherein said dose effectively improves cardiac function and / or health-related quality of life for a period of at least about six months, optionally measured by one or more of the following: New York Heart Association (NYHA) functional classification, Kansas City Cardiomyopathy Questionnaire-23 (KCCQ-23) Total Symptom Score (TSS), Body Limitation Score and Clinical Summary Score (CSS), EuroQol-5 Dimensions 5-Level Version (EQ-5D-5L), EuroQol-Visual Analog Scale (EQ-VAS), Clinical Global Impression Severity and Variation Scale (CGI / C), and Patient Global Impression Severity and Variation Scale (PGIS / C).

46. ​​The method of 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 according to any one of claims 21 to 46, further comprising administering to the subject a preventatively effective amount of an immunosuppressant in an amount that effectively prevents or reduces biochemical or clinical signs of hepatotoxicity, liver 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 preventative effective amount of corticosteroid is administered at a dose equivalent to prednisone of about 0.5 mg / kg / day, about 40 mg / day, or about 60 mg / kg, optionally for a period of at least about 4 weeks, followed by administration of a gradually decreasing amount of the corticosteroid for a period of about 4 weeks.

50. The method of claim 48, wherein the prophylactic immunosuppressant is administered simultaneously with a prophylacticly 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 period of at least 8 weeks, or at an amount that effectively maintains the blood level of sirolimus between about 5 and about 15 ng / mL.

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

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

54. The method according to any one of claims 21 to 53, further comprising the following steps: (a) Baseline levels of markers of hepatotoxicity, liver inflammation, and / or cardiac inflammation in the blood of the subject prior to administration of rAAV particles, and (b) post-administration levels of the markers of hepatotoxicity, liver inflammation, and / or cardiac inflammation in the blood of the subject subsequently measured weekly for at least 8 weeks.

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

56. The method of claim 55, wherein the administration of the therapeutic dose of the immunosuppressant comprises the following steps: (c) After hepatotoxicity, liver inflammation and / or cardiac inflammation are detected by biochemical or clinical signs, the subject is given a therapeutically effective amount of systemic immunosuppressant to reduce hepatotoxicity, liver inflammation and / or cardiac inflammation.

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

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

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

60. The method according to any one of claims 21 to 59, further comprising administering to the subject a preventatively effective amount of a complement pathway inhibitor or antagonist in an amount that effectively prevents or reduces 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, a C1 esterase inhibitor (CINRYZE / BERINERT / HAEGARDA), a recombinant C1 esterase inhibitor (RUCONEST), ravulizumab (ALXN1210), coversin, cemdisiran (ALN-CC5), tesidolumab (LFG-316), iptacopan (LNP023), danicopan (ACH-4471), or ENJAYMO. (sutimlimab-jome).

62. The method according to any one of claims 21 to 61, further comprising the following steps: (a) Baseline levels of markers of complement activation and / or thrombotic microangiopathy in the blood of the subject prior to administration of rAAV particles, and (b) post-administration levels of the markers of complement activation and / or thrombotic microangiopathy in the blood of the subject subsequently measured weekly for at least 8 weeks.

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

64. The method of claim 63, wherein the administration of the therapeutic dose of the immunosuppressant comprises the following steps: (c) After complement activation and / or thrombotic microangiopathy is detected by biochemical or clinical signs, the subject is given a therapeutically effective amount of a complement pathway inhibitor or antagonist to reduce complement activation and / or thrombotic microangiopathy.

65. The method according to any one of claims 63 to 64, wherein the complement pathway inhibitor or antagonist is OMS721 antibody, percicobulin (APL-2), eculizumab (Soliris), avacopan (CCX168), virolimab (IFX-1), AMY-101, C1 esterase inhibitor (CINRYZE / BERINERT / HAEGARDA), recombinant C1 esterase inhibitor (RUCONEST), rivolizumab (ALXN1210), carvedin, sedeslam (ALN-CC5), terdurumab (LFG-316), ipcopan (LNP023), danicopan (ACH-4471), or ENJAYMO (sutemozolomab-Jumi).

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