Methods and compositions for treating heart failure with reduced ejection fraction

Abstract

Embodiments provided herein relate to methods, compositions and uses for treating heart failure with reduced ejection fraction (HFrEF). Some embodiments relate to methods, compositions and uses of a recombinant viral vector encoding a 2a isoform of a sarco(endo)plasmic reticulum calcium ion (Ca2+) ATPase (SERCA2a) protein.

Description

SARDO.015W0 PATENT METHODS AND COMPOSITIONS FOR TREATING HEART FAILURE WITH REDUCED EJECTION FRACTIONRELATED APPLICATIONSThis application claims priority to U. S. Prov. App. No. 63 / 755525 filed February’ 7, 2025 which is incorporated by reference herein in its entirety.FIELD

[0001] Embodiments provided herein relate to methods, compositions and uses for treating heart failure with reduced ejection fraction (HFrEF). Some embodiments relate to methods, compositions and uses of a recombinant viral vector encoding a 2a isoform of a sarco(endo)plasmic reticulum calcium ion (Ca2+) ATPase (SERCA2a) protein.BACKGROUND

[0002] Heart failure is a complex pathophysiological syndrome encompassing a range of abnormalities that impair normal cardiac function and result in inability of the heart to pump a sufficient supply of blood to meet the metabolic requirements of the body, or to do so only at elevated ventricular filling pressures. Heart failure can be acute or chronic, but once established is generally progressive, irreversible, associated with debilitating symptoms, frequent re hospitalizations and high mortality rates.

[0003] Heart failure with reduced ejection fraction (HFrEF) treatment is tailored to the individual patient, based on the underlying causes, comorbidities and the patient’s stage at diagnosis and New York Heart Association (NYHA) functional classification. Treatment goals are to reduce symptoms, prolong survival, improve the quality of life and slow or prevent disease progression (stabilize disease). Despite optimal medical therapy employing a wide range of pharmacologic, device and surgical therapeutic options, over the long term most patients experience a progressive decline, ultimately succumbing to the syndrome or one of the underlying contributing conditions.SUMMARY

[0004] Some embodiments of the methods and compositions provided herein include a method of treating (including, inhibiting, stabilizing and / or ameliorating) heart failure with a reduced ejection fraction (HFrEF) in a subject, comprising: administering a polynucleotide comprising a nucleic acid encoding a sarcoplasmic / endoplasmic reticulum calcium ATPase (SERCA) polypeptide to the subject. The following numbered embodiments are non-limiting embodiments of the present disclosure.1. A method of treating heart failure with a reduced ejection fraction (HFrEF) in a subject, comprising:identifying a subject as having HFrEF, and administering a polynucleotide comprising a nucleic acid encoding a sarcoplasmic / endoplasmic reticulum calcium ATPase (SERCA) polypeptide to the subject identified as having HFrEF,2. The method of embodiment 1, wherein the subject comprises a left ventricular ejection fraction (LVEF) of less than 50%, or less than or equal to 45%, 40%, or 35%, or is in a range defined by any two of the preceding values.3. The method of embodiment 1 or 2, wherein the subject comprises an LVEF of less than 35%.4. The method of any one of embodiments 1-3, further comprising identifying and / or selecting the subject based on one or more characteristics of the subject.5. The method of embodiment 4, wherein the identifying and / or selecting comprising measuring in the subject one or more of:(a) an anti- AAV neutralizing antibody inhibition level against an AAV viral vector in a serum sample from the subject, optionally wherein the anti- AAV neutralizing antibody is an anti-AAVl neutralizing antibody, and the AAV viral vector is an AAV 1 viral vector;(b) a New York Heart Association (NYHA) class of heart failure;(c) an N-terminal prohormone brain natriuretic peptide (NT-proBNP) concentration;(d) an ability to walk a distance in a 6 minute walk test (6MWT);(e) a level of alanine aminotransferase, aspartate aminotransferase, or alkaline phosphatase in a liver function test;(f) a level of bilirubin; and / or(g) a hemoglobin level.6. The method of any one of embodiments 1-5, wherein the subject comprises:(a) an anti-AAVl neutralizing antibody inhibition level against an AAV1 viral vector less than or equal to 50% at a serum dilution of 1:4, optionally, wherein the anti- AAVl neutralizing antibody inhibition level is measured within 125 days prior to the treatment;(b) a cardiomyopathy selected from:(i) a non-ischemic cardiomyopathy, optionally wherein the nonischemic cardiomyopathy lacks a hypertrophic cardiomyopathy, an infiltrative cardiomyopathy or a restrictive cardiomyopathy;(ii) an ischemic cardiomyopathy, optionally wherein the ischemic cardiomyopathy is chronic ischemic cardiomyopathy, optionally wherein the chronic ischemic cardiomyopathy lacks a hypertrophic cardiomyopathy, an infiltrative cardiomyopathy or a restrictive cardiomyopathy, optionally wherein the subject has at least one major coronary vessel with thrombolysis in myocardial infarction (TIMI) grade 3 flow, wherein the at least one major coronary vessel is selected from left anterior descending (LAD), left coronary circumflex (LCx) and right coronary artery (RCA);(iii) a toxic cardiomyopathy or an alcoholic cardiomyopathy, optionally wherein a toxin or alcohol exposure has been eliminated from the subject for at least 6 months prior to the treatment;(iv) a cardiomyopathy due to acute myocarditis, optionally wherein a most recent clinical episode due to the cardiomyopathy due to acute myocarditis is at least 6 months prior to the treatment;(v) a viral cardiomyopathy or a peripartum cardiomyopathy, optionally wherein the subject is diagnosed with the viral cardiomyopathy or peripartum cardiomyopathy at least 6 months prior to the treatment;(c) a diagnosis of New York Heart Association (NYHA) class III or class IV heart failure, optionally wherein the diagnosis is at least 60 days prior to the treatment;(d) an N-terminal prohormone brain natriuretic peptide (NT-proBNP) concentration greater than 1200 pg / mL, optionally, greater than 1600 pg / mL;(e) an ability to walk a minimum of 50 meters in a 6 minute walk test (6MWT); (f) received medical therapy for the treatment of chronic heart failure, optionally wherein the medical therapy is selected from an oral diuretic, a beta blocker, ivabradine, hydralazine, aldosterone receptor blocker, and an inhibitor of the renin¬ angiotensin system selected from an angiotensin-converting enzyme inhibitor, and angiotensin-receptor blocker (ARB) and an angiotensin receptor-neprilysin inhibitor, optionally wherein the the beta blocker is selected from bisoprolol, carvedilol and metoprolol succinate, and / or the ARB is selected from candesartan and valsartan; and / or(g) a cardioverter defibrillator (ICD), optionally wherein the ICD has been implanted in the subject at least 30 days prior to the treatment,7. The method of any one of embodiments 1-6, wherein the subject is not characterized by one or more of:(a) a diagnosis of untreated heart failure;(b) an intravenous venous (IV) therapy with positive inotropes, vasodilators or diuretics within 30 days prior to treatment;(c) a restrictive cardiomyopathy, an infiltrative cardiomyopathy, a hypertrophic cardiomyopathy, a pericardial disease, amyloidosis, an uncorrected thyroid disease, or a discrete left ventricular (LV ) aneurysm;(d) an untreated left-sided valvular disease, optionally wherein the left-sided valvular disease is selected from moderate or severe mitral stenosis wherein a valve area is less than 1.5 cm2, a severe aortic stenosis with a valve area less than 1.0 cm2, and a moderate-severe (3-4+ / 4-r) aortic or mitral regurgitation;(e) a limited ability to perform the 6MWT due to claudication, arthritic or other conditions unrelated to heart failure;(f) cardiac surgery, percutaneous coronary intervention (PCI), valvuloplasty or valve replacement within 30 days prior to treatment;(g) myocardial infarction within 6 months prior to treatment;(h) active angina within 3 months prior to treatment;(i) a prior heart transplantation, left ventricular reduction surgery, or cardiomyoplasty;(j) a passive restraint device such as a CorCap™ Cardiac Support Device, a mechanical circulatory support device (MCSD) or cardiac shunt;(k) a likelihood of receiving cardiac resynchronization therapy, cardiomyoplasty, left ventricular reduction surgery, conventional revascularization procedure, MCSD, cardiac transplant or valvular repair within 12 months following treatment;(l) hemodynamic instability requiring hospitalization within 3 months prior to the treatment;(m) hypersensitivity to radiopaque agents used for angiography, and optionally a history of high dose corticosteroid pretreatment prior to contrast angiography;(n) a level of alanine aminotransferase, aspartate aminotransferase, or alkaline phosphatase in a liver function test which is at least 3 times greater than an upper limit of normal (ULN);(o) a total bilirubin greater than two times greater than an upper limit of normal; (p) an intrinsic liver disease, optionally wherein the liver disease is selected from cirrhosis, chronic hepatitis B, and hepatitis C virus infection;(q) a need for hemodialysis within 12 months or a current glomerular filtration rate (GFR) less than or equal to 20 mL / minute / 1.73 m2;(r) bleeding diathesis or thrombocytopenia with a platelet count less than 75,000 platelets / pL; and / or(s) anemia with a hemoglobin level less than 9 g / dL.8. The method of any one of embodiments 1-7, wherein the treatment stabilizes and / or provides an improvement in a symptom or measure of the HFrEF in the subject compared to the symptom or measure in an untreated subject for a period following administration of the polynucleotide of at least 1, 2, 3, 6, 9, or 12 months.9. The method of embodiment 8, wherein the period is at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 years.10. The method of any one of embodiment 8 or 9, wherein the symptom or measure is selected from the group consisting of a change in class of a New York Heart Association(NYHA) class, a score in a Kansas City Cardiomyopathy Questionnaire (KCCQ), a distance in a 6-minute walk test (6MWT), a concentration of N-terminal pro b-type natriuretic peptide (NT-proBNP), a concentration cardiac troponin I, a lack in a recurrent or terminal clinical outcome, and a ventricular function.11. The method of embodiment 10, wherein a recurrent or terminal clinical outcome is selected from a hospitalization, intravenous administration of a diuretic, vasodilator, or inotrope, mechanical fluid removal.12. The method of embodiment 10 or 11, wherein the stabilization and / or improvement in ventricular function comprises stabilization of or an improvement in a parameter selected from the group consisting of a change from baseline in left ventricular structure and function including left ventricular ejection fraction (LVEF), left ventricular end-diastolic volume (I. VEDV), end-systolic volume, stroke volume and / or circumferential strain; regional wall thickness; left ventricular LGE expressed as a percent of left ventricular mass; left ventricular viable mass; number of left ventricular segments with LGE; and composite outcome in change from baseline in LV function (LVESV), optionally wherein the parameter is assessed by late gadolinium enhancement (LGE) cardiac MRI.13. The method of any one of embodiments 8-12, further comprising measuring the stabilization and / or improvement in the subject after the period.14. The method of any one of embodiments 8-13, wherein the stabilization and / or improvement comprises:(a) stabilization of or a decrease in a NYHA class, optionally wherein the decrease is greater than or equal to 1 class or to 2 classes;(b) stabilization of or an increase in LVEF, optionally wherein the increase is greater than or equal to 5%, 10%, 15%, or 20%;(c) stabilization of or an increase in an KCCQ score, optionally wherein the increase is greater than or equal to 5 points, 10 points, 20 points, 30 points, 40 points, 50 points, 60 points, or 70 points;(d) stabilization of or an increase in a 6MWT distance, optionally wherein the increase is greater than or equal to 30 meters, 40 meters, 50 meters, 60 meters, 70 meters, or 80 meters;(e) stabilization of or a decrease in an NT-proBNP concentration, optionally wherein the decrease is greater than or equal to 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, or is in a range defined by any two of the preceding values; and / or(f) stabilization of or a decrease in a cardiac troponin I concentration, optionally wherein the decrease is greater than or equal to 30%, 35%, 40%, 45%, or 50%, or is in a range defined by any two of the preceding values.15. The method of embodiment 14, wherein the measured stabilization and / or improvement comprises at least 1, 2, 3, 4, 5, or 6 parameters selected from the stabilization of or decrease in a NYHA class, the stabilization of or increase in LVEF, the stabilization of or increase in an KCCQ score, the stabilization of or increase in a 6MWT distance, the stabilization of or decrease in an NT-proBNP concentration, and the stabilization of or decrease in a cardiac troponin I concentration.16. The method of any one of embodiments 1-15, wherein the SERCA polypeptide comprises a SERCA2a polypeptide,17. The method of any one of embodiments 1-16, wherein the polynucleotide comprises a vector.18. The method of embodiment 17, wherein the vector is selected from an adeno-associated virus (AAV) vector, a lentivirus vector, and a retrovirus vector.19. The method of embodiment 18, wherein the vector comprises an AAV vector.20. The method of embodiment 19, wherein the AAV vector encodes an AAV or fragment thereof having a serotype selected from any one of AAV serotypes 1-12.21. The method of embodiment 20, wherein the AAV vector encodes a capsid from AAV serotype- 1.22. The method of any one of embodiments 1-21, wherein the polynucleotide comprises a promoter operably linked to the nucleic acid encoding a SERCA polypeptide.23. The method of embodiment 22, wherein the promoter comprises a constitutive promoter.24. The method of embodiment 22 or 23, wherein the promoter comprises a cytomegalovirus (CMV) promoter.25. The method of any one of embodiments 1-25, wherein a viral capsid comprises the polynucleotide.26. The method of embodiment 25, w’herein the polynucleotide comprises a nucleic acid encoding the viral capsid.27. The method of any one of embodiments 1-26, wherein the administering a polynucleotide comprises systemic administration.28. The method of any one of embodiments 1-26, wherein the administering a polynucleotide comprises intravenous administration.29. The method of any one of embodiments 1-26, wherein the administering a polynucleotide comprises intracoronary infusion.30. The method of any one of embodiments 1-29, wherein the administering a polynucleotide consists of a single dose of the polynucleotide.31. The method of any one of embodiments 1-30, wherein the polynucleotide comprises a viral vector, and wherein the administration comprises a dose of the polynucleotide within a range from about 1 X 108viral genome particles (vg) to about 1 X 101532. The method of embodiment 31, wherein the dose is within a range from about 1 X 1013viral genome particles to about 9 X 1013vg.33. The method of embodiment 32, wherein the dose is within a range from about 2 X 1013viral genome particles to about 5 X 1013vg.34. The method of embodiment 33, wherein the dose is about 3 X 1013vg.35. The method of embodiment 33, wherein the dose is about 4.5 X 1013vg.36. The method of any one of embodiments 1-35, further comprising administering a vasodilator.37. The method of embodiment 36, wherein the vasodilator is administered prior to administering the polynucleotide.38. The method of embodiment 36 or 37, wherein the vasodilator is administered concurrently with administering the polynucleotide.39. The method of any one of embodiments 36-38, wherein the vasodilator comprises nitroglycerin.40. The method of any one of embodiments 1-39, wherein the subject is mammalian.41. The method of any one of embodiments 1-40, wherein the subject is human.42. The method of any one of the preceding embodiments, wherein the treatment comprises inhibiting, stabilizing, and / or ameliorating HFrEF in the subject.43. Use of a polynucleotide comprising a nucleic acid encoding a sarcoplasmic / endoplasmic reticulum calcium ATPase (SERCA) polypeptide to treat heart failure with a reduced ejection fraction (HFrEF) in a subject according to any one of embodiments 1-42.44. Use of a polynucleotide comprising a nucleic acid encoding a sarcoplasmic / endoplasmic reticulum calcium ATPase (SERCA) polypeptide in the manufacture of a medicament to treat heart failure with a reduced ejection fraction (HFrEF) in a subject according to any one of embodiments 1-42.BRIEF DESCRIPTION OF THE DRAWINGS

[0005] FIG, 1 is an embodiment of a schematic for a timeline of a phase lb trial excluding aspects of a long-term follow-up. Events include Screening Period 1 (SP1), Screening Period 2 (SP2), Randomization of subjects (Rand), Days 1-2 (D1-D2), Weeks 1-52 (W1-W52).

[0006] FIG. 2 is an embodiment of a schematic for a timeline of a phase 2 trial excluding aspects of a long-term follow-up.DETAILED DESCRIPTION

[0007] Embodiments provided herein relate to methods, compositions and uses for treating heart failure with reduced ejection fraction (HFrEF). Some embodiments relate to methods, compositions and uses of a recombinant viral vector encoding a 2a isoform of a sarco(endo)plasmic reticulum calcium ion (Ca2+) ATPase (SERCA2a) protein.

[0008] Elucidation of a molecular basis of myocardial dysfunction in heart failure has identified a deficiency of the 2a isoform of the sarco(endo)plasmic reticulum calcium ion (Ca2+) ATPase (SERCA2a) protein in the cardiomyocytes, a key characteristic in the disease. SERCA2a is an enzyme Ca2+pump that transfers Ca2+from the cytosol of the cardiomyocyte to the lumen of the sarcoplasmic reticulum. SERCA2a not only determines the amount of Ca2+in the SR available to activate the next contraction, but also controls to a large extent the rate of cardiac relaxation (Vangheluwe, 2011). The expression level of SERCA2a significantlyaffects the overall cardiac contractile properties and ability of the heart to supply adequate amounts of oxygenated blood to the body. If the cardiac muscle becomes stressed from overwork or damaged from conditions such as chronic hypertension, myocardial infarction (MI) or idiopathic causes, a fetal gene survival program is initiated in cardiomyocytes, causing decreased expression of SERCA2a (Kawase, 2008). This leads to a poorly contracting myocardium and subsequent poor blood flow and reduced oxygen supply to the body. In addition, in coronary' arteries, SERCA2a and endothelial isoform of nitric oxide synthase (eNOS) protein expression are restored to normal levels by modulating calcium homeostasis in endothelial cells to improve coronary blood flow by SERCA2a gene transfer (Hadri, 2010),

[0009] Some embodiments provided herein relate to SRD-001 (SERCA2a / AA Vl). SRD-001 is a recombinant adeno associated viral (rAAV) vector, consisting of an AAV serotype 1 (AAVl) capsid and SERCA2a complementary deoxyribonucleic acid (cDNA) flanked by Inverted Terminal Repeats (ITRs) derived from AAV serotype 2 (AAV2). The SERCA2a protein is the only protein expressed from the vector in transduced cells and is a fully human, intracellular, endoplasmic protein that is naturally expressed in cardiomyocytes. In some embodiments provided herein, a route of administration is by antegrade epicardial coronary artery infusion with a syringe pump using commercially available guide or diagnostic catheters. In some embodiments, treatment restores Ca2’ cycling in cardiomyocytes by normalizing SERCA2a protein levels in HFrEF patients. In some embodiments, treatment results in targeted SERCA2a enzyme replacement in HFrEF patients and corrects imbalances in Ca2+cardiac metabolism. In some embodiments, treatment results in enhanced cardiac function and energetics that translate to stabilized or improved clinical outcomes. In some embodiments, treatment improves coronary blood flow, optionally by correcting the impaired endothelium dependent nitric oxide mediated vasodilatation observed in HFrEF. Some embodiments provided herein relate to use of SRD-001 at high doses to treat HFrEF. In some embodiments, the high doses are sufficient to induce a therapeutic effect.Failure of prior human clinical trials of SRD-001

[0010] Prior human clinical trials of a recombinant AAV vector encoding SERCA2a protein (also referred to herein as SRD-001, and previously known under the brand name of MYDICAR®) were inconclusive and / or unsuccessful. These trials MYDICAR®included CUPID-1, CUPID-2, SERCA-LVAD and AGENT-HF Trials as summarized in TABLE 1. The route of administration in all cases was antegrade epicardial coronary artery infusion.TABLE 1Duration of Genderobservation M / F / Study / Dosage N by Primary / median Diagnosisdesign (vg) cohort endpoint(s) long-term age (yrs)follow-up / rangeCUPID- 1 1.4 x 10113 12 months 9 / 3 HFrEF Incidence and phase 1 6 x 10113 24 months 54 NYHA severity of (Jaski, 3 x 10123 (40-78) Class AEs 2009) 1 x 10133 III / IV / openlabel, doseescalation,uncontrolledCUPID- 1 6 x 10118 12 months 34 / 5 HFrEF Incidence and phase 2a 3 x 10128 24 months 62.5 NYHA severity of (Jessup, 1 x 10139 (52-69) Class AEs and 2011) / Placebo 14 III / IV change from RCT dose baseline to 6 response and 12 months in composite of 5 domains (symptomatic, functional, biomarker, LV function and clinical outcome) CUPID-2 1 X 1013125 12 months 204 / 46 HFrEF Time to (Greenberg, Placebo 125 >12 months 60 NYHA recurrent 2016) / (23-80) Class II- cardiovascular RCT IV eventsDuration of Genderobservation M / F / Study / Dosage N by Primary / median Diagnosisdesign (vg) cohort endpoint(s) long-term age (yrs)follow-up / rangeSERCA- 1 X 10134 6 months 4 / 1 HFrEF Incidence and LVAD Placebo 1 10 years 36 supported severity of (Lyon, (29-69) with a AEs, 2020) / LVAD biodistribution phase 2a, and change RCT from baseline to 6 months in LV function, contractility and biomarker AGENT-HF 1 X 10135 12 months 8 / 1 HFrEF Change from (Hulot, Placebo 4 60 NYHA baseline to 6 2017) / (Not Class months in phase 2, disclosed) III / IV LVESV as RCT measured by cardiac CT Abbreviations: AEs, adverse events; CT, computed tomography; LV, left ventricular;LVAD, left ventricular assist device; LVESV, left ventricular end systolic volume; RCT, randomized controlled trial; UNK, unknownTotal dosage expressed in DNase Resistant Particles (equivalent to viral genome particles [vg]). Route of administration in all trials was antegrade epicardial coronary artery infusion as a one-time infusion only.Therapeutic methods and compositions

[0011] Some embodiments of the methods and compositions provided herein include therapeutic methods and agents to treat (including one or more of inhibiting, stabilizing, and / or ameliorating) HFrEF in a subject. As used herein, “treat” or “treating” has its ordinary and customary meaning read m light of the disclosure, and includes slowing (inhibiting) or preventing the worsening of (stabilizing), as well as improving (ameliorating), one or more aspects of a disease. In some embodiments, the subject is identified as having HFrEF, prior to treatment for HFrEF. In some embodiments, the subject has HFrEF m which a left ventricular ejection fraction (LVEF) is less than 50%, or less than or equal to 45%, 40%, 35%, or 30%, or any percentage between any one of the foregoing percentages, or is in a range defined by any two of the preceding values. In some embodiments, the LVEF is less than 35%.Some embodiments include administering to a subject a polynucleotide comprising a nucleic acid encoding a sarcoplasmic / endoplasmic reticulum calcium ATPase (SERCA) polypeptide to the subject. In some embodiments, the subject is identified as having HFrEF, prior to administration. Some embodiments include administering to the subject a polynucleotide comprising a nucleic acid encoding a sarcoplasmic / endoplasmic reticulum calcium ATPase (SERCA) polypeptide. In some embodiments, the SERCA polypeptide comprises a SERCA2 polypeptide. In some embodiments, the SERCA2 polypeptide comprises a SERCA2 isoform selected from a SERCA2a polypeptide, or a SERCA2c polypeptide. In some embodiments, the SERCA2 polypeptide comprises a SERCA2a polypeptide. In some embodiments, the polynucleotide comprises a promoter operably linked to the nucleic acid encoding the SERCA polypeptide. In some embodiments, the promoter comprises a constitutive promoter. In some embodiments, the promoter comprises a cytomegalovirus (CMV) promoter. In some embodiments, the polynucleotide comprises a vector. In some embodiments, the vector is selected from an adeno-associated virus (AAV) vector, a lentivirus vector, and a retrovirus vector. In some embodiments, the vector comprises an AAV vector. In some embodiments, a viral capsid comprises the polynucleotide. In some embodiments, the polynucleotide comprises a nucleic acid encoding the viral capsid. In some embodiments, the AAV vector encodes an AAV or fragment thereof having a serotype. In some embodiments, the AAV or fragment thereof comprise a viral capsid protein or fragment thereof. In some embodiments, the AAV or fragment thereof has a serotype selected from AAV serotype- 1 (AAV1), AAV serotype-2 (AAV2), AAV serotype-3 (AAV3), AAV serotype-4 (AAV4), AAV serotype-5 (AAV5), AAV serotype-6 (AAV6), AAV serotype-7 (AAV7), AAV serotype-8 (AAV8), AAV serotype-9 (AAV9), AAV serotype-10 (AAVrhlO), AAV serotype-11 (AAV11), and AAV serotype- 12 (AAV12). In some embodiments, the AAV or fragment thereof has a serotype selected from AAV1, and AAV9. In some embodiments, the AAV or fragment thereof has an AAV1 serotype. In some embodiments, the AAV or fragment thereof has an AAV9 serotype. In some embodiments, the AAV or fragment thereof can comprise a hybrid serotype of one or more of the foregoing serotypes.

[0012] Some embodiments also include determining the presence or absence in the subject of an antibody against an AAV serotype. In some embodiments, an AAV or fragmenttliereof encoded by the polynucleotide can be selected to have a serotype different from the AAV serotype recognized by an antibody identified in the subject.

[0013] In some embodiments, the polynucleotide administering the polynucleotide comprises systemic administration. In some embodiments, the administering the polynucleotide comprises an injection, an infusion, or an implantation. In some embodiments, the administering the polynucleotide comprises intravenous administration. In some embodiments, the administering the polynucleotide comprises intracoronary administration. In some embodiments, the administering a polynucleotide comprises one or more doses of the polynucleotide administered to the subject. In some embodiments, the administering a polynucleotide consists of a single dose of the polynucleotide administered to the subject. In some embodiments, the administering a polynucleotide consists of two doses of the polynucleotide administered to the subject. In some embodiments, the administering a polynucleotide consists of three doses of the polynucleotide administered to the subject. In some embodiments, the administering a polynucleotide consists of four doses of the polynucleotide administered to the subject. In some embodiments, the administering a polynucleotide consists of five doses of the polynucleotide administered to the subject.

[0014] In some embodiments, the polynucleotide comprises a viral vector. In some embodiments, a viral capsid or functional fragment thereof comprises the polynucleotide. In some such embodiments, a dose of the polynucleotide can be measured as a number of viral genome particles (vg), or as a number of DNase resistant particles (DRP). In some embodiments, a dose of the polynucleotide is at least 1 X 108vg, 1 X 109vg, 1 X IO10vg, 1 X 1011vg, 1 X 1012vg, 1 X 1013vg, 1 X 1014vg, 1 X 1015vg, 1 X 1016vg, 1 X 1017vg, 1 X 1018vg, 1 X 1019vg, 1 X 1020vg, or an amount between any two of the foregoing amounts. In some embodiments, a dose of the polynucleotide is at least 1 X 108DRP, 1 X 109DRP, 1 X 1010DRP, 1 X 10” DRP, 1 X 1012DRP, 1 X 1013DRP, 1 X 1014DRP, 1 X 1015DRP, 1 X 1016DRP, 1 X 10’7DRP, 1 X 10’8DRP, 1 X 1019DRP, 1 X 102° DRP, or an amount between any two of the foregoing amounts. In some embodiments, a dose of the polynucleotide is within a range from about 1 X 108vg to about 1 X 1020vg, from about 1 X 109vg to about 1 X 1017vg, from about 1 X 1010vg to about 1 X 1016vg, from about 1 X 10” vg to about 1 X 1 O'5vg, or from about 1 X 1013vg to about 1 X 1014vg. In some embodiments, a dose of the polynucleotide is within a range from about 1 X 1013vg to about 9 X 1013vg. In someembodiments, a dose of the polynucleotide is within a range from about 1 X 108DRP to about 1 X 1020DRP, from about 1 X 109DRP to about 1 X 1017DRP, from about 1 X IO10DRP to about 1 X 1016DRP, from about 1 X 1011DRP to about 1 X 1015DRP, or from about 1 X 1012DRP to about 1 X 1014DRP. In some embodiments, a dose of the polynucleotide is within a range from about 1 X 1013DRP to about 9 X 1013DRP.

[0015] Some embodiments also include administering a vasodilator. In some embodiments, the vasodilator is administered prior to administering the polynucleotide. In some embodiments, the vasodilator is administered concurrently with administering the polynucleotide. In some embodiments, the vasodilator comprises nitroglycerin.

[0016] In some embodiments, the subject is mammalian, such as human.

[0017] Some embodiments include selecting and / or identifying the subject based on one or more characteristics of the subject. Some such embodiments include measuring in a subject: (a) an anti-AAV neutralizing antibody inhibition level against an AAV viral vector in a serum sample from the subject, optionally wherein the anti-AAV neutralizing antibody is an anti-AAVl neutralizing antibody, and the AAV viral vector is an AAAI viral vector; (b) a New York Heart Association (NYHA) class of heart failure; (c) an N-terminal prohormone brain natriuretic peptide (NT-proBNP) concentration; (d) an ability to walk a distance in a 6 minute walk test (6MWT); (e) a level of alanine aminotransferase, aspartate aminotransferase, or alkaline phosphatase in a liver function test; (f) a level of bilirubin; and / or (g) a hemoglobin level.

[0018] In some embodiments the subject comprises (has the characteristic of) one or more of: (a) an anti-AAVl neutralizing antibody inhibition level against an AAV1 viral vector less than or equal to 50% at a serum dilution of 1:4, optionally, wherein the anti-AAVl neutralizing antibody inhibition level is measured within 125 days prior to the treatment; (b) a cardiomyopathy selected from: (i) a non-ischemic cardiomyopathy, optionally wherein the non-ischemic cardiomyopathy lacks a hypertrophic cardiomyopathy, an infiltrative cardiomyopathy or a restrictive cardiomyopathy; (ii) an ischemic cardiomyopathy, optionally wherein the ischemic cardiomyopathy is chronic ischemic cardiomyopathy, optionally wherein the chronic ischemic cardiomyopathy lacks a hypertrophic cardiomyopathy, an infiltrative cardiomyopathy or a restrictive cardiomyopathy, optionally wherein the subject has at least one major coronary vessel with thrombolysis in myocardial infarction (TIMI) grade 3 flow,wherein the at least one major coronary vessel is selected from left anterior descending (LAD), left coronary circumflex (LCx) and right coronary artery (RCA); (iii) a toxic cardiomyopathy or an alcoholic cardiomyopathy, optionally wherein a toxin or alcohol exposure has been eliminated from the subject for at least 6 months prior to the treatment; (iv) a cardiomyopathy due to acute myocarditis, optionally wherein a most recent clinical episode due to the cardiomyopathy due to acute myocarditis is at least 6 months prior to the treatment; (v) a viral cardiomyopathy or a peripartum cardiomyopathy, optionally wherein the subject is diagnosed with the viral cardiomyopathy or peripartum cardiomyopathy at least 6 months prior to the treatment, (c) a diagnosis of New York Heart Association (NYHA) class III or class IV heart failure, optionally wherein the diagnosis is at least 60 days prior to the treatment; (d) an N-terminal prohormone brain natriuretic peptide (NT-proBNP) concentration greater than 1200 pg / mL, optionally, greater than 1600 pg / mL; (e) an ability to walk a minimum of 50 meters in a 6 minute walk test (6MWT); (I) received medical therapy for the treatment of chronic heart failure, optionally wherein the medical therapy is selected from an oral diuretic, a beta blocker, ivabradine, hydralazine, aldosterone receptor blocker, and an inhibitor of the renin-angiotensin system selected from an angiotensin-converting enzyme inhibitor, and angiotensin-receptor blocker (ARB) and an angiotensin receptor-neprilysin inhibitor, optionally wherein the the beta blocker is selected from bisoprolol, carvedilol and metoprolol succinate, and / or the ARB is selected from candesartan and valsartan; and / or (g) a cardioverter defibrillator (ICD), optionally wherein the IC’D has been implanted in the subject at least 30 days prior to the treatment.

[0019] In some embodiments, the subject lacks one or more of: (a) a diagnosis of untreated heart failure; (b) an intravenous venous (IV) therapy with positive inotropes, vasodilators or diuretics within 30 days prior to treatment; (c) a restrictive cardiomyopathy, an infiltrative cardiomyopathy, a hypertrophic cardiomyopathy, a pericardial disease, amyloidosis, an uncorrected thyroid disease, or a discrete left ventricular (LV) aneurysm; (d) an untreated left-sided valvular disease, optionally wherein the left-sided valvular disease is selected from moderate or severe mitral stenosis wherein a valve area is less than 1.5 cm2, a severe aortic stenosis with a valve area less than 1.0 cm2, and a moderate-severe (3-4+ / 4+) aortic or mitral regurgitation; (e) a limited ability to perform the 6MWT due to claudication, arthritic or other conditions unrelated to heart failure; (f) cardiac surgery, percutaneouscoronary intervention (PCI), valvuloplasty or valve replacement within 30 days prior to treatment; (g) myocardial infarction within 6 months prior to treatment; (h) active angina within 3 months prior to treatment; (i) a prior heart transplantation, left ventricular reduction surgery, or cardiomyoplasty; (j) a passive restraint device such as a CorCap™ Cardiac Support Device, a mechanical circulatory’ support device (MCSD) or cardiac shunt; (k) a likelihood of receiving cardiac resynchronization therapy, cardiomyoplasty, left ventricular reduction surgery’, conventional revascularization procedure, MCSD, cardiac transplant or valvular repair within 12 months following treatment; (1) hemodynamic instability requiring hospitalization within 3 months prior to the treatment; (m) hypersensitivity to radiopaque agents used for angiography, and optionally a history / of high dose corticosteroid pretreatment prior to contrast angiography; (n) a level of alanine aminotransferase, aspartate aminotransferase, or alkaline phosphatase in a liver function test which is at least 3 times greater than an upper limit of normal (ULN); (o) a total bilirubin greater than two times greater than an upper limit of normal; (p) an intrinsic liver disease, optionally wherein the liver disease is selected from cirrhosis, chronic hepatitis B, and hepatitis C virus infection; (q) a need for hemodialysis within 12 months or a current glomerular filtration rate (GFR) less than or equal to 20 mL / minute / 1.73 m2; (r) bleeding diathesis or thrombocytopenia with a platelet count less than 75,000 platelets / pL; and / or (s) anemia with a hemoglobin level less than 9 g / dL.

[0020] In some embodiments, the treatment provides long-term stabilization and / or improvement in a treated subject in one or more symptoms or measures of HFrEF, compared to the one or more symptoms in an untreated subject having HFrEF. In some embodiments the stabilization and / or improvement is observable for at least a period of at least 3, 6, 9, or 12 months following administration of the treatment. In some embodiments the stabilization and / or improvement is observable for at least a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 years following administration of the treatment. In some embodiments, the symptom or measure is selected from a change in class of a New York Heart Association (NYHA) class, a score in a Kansas City Cardiomyopathy Questionnaire (KCCQ), a distance in a 6-minute walk test (6MWT), a concentration of N-terminal pro b-type natriuretic peptide (NT-proBNP), a concentration cardiac troponin I, a lack m a recurrent or terminal clinical outcome, and a ventricular function. In some embodiments, a recurrent or terminal clinical outcome is selected from a hospitalization, intravenous administration of a diuretic, vasodilator, or inotrope,mechanical fluid removal. In some embodiments, the stabilization and / or improvement in ventricular function comprises stabilization and / or an improvement in a parameter selected from the group consisting of a change from baseline in left ventricular structure and function including left ventricular ejection fraction (LVEF), left ventricular end-diastolic volume (LVTDV), end-systolic volume, stroke volume and / or circumferential strain; regional wall thickness; left ventricular LGE expressed as a percent of left ventricular mass; left ventricular viable mass; number of left ventricular segments with LGE; and composite outcome in change from baseline in LV function (LVESV), optionally wherein the parameter is assessed by late gadolinium enhancement (LGE) cardiac MRL Some embodiments also include measuring the stabilization and / or improvement m the subject after the period. In some embodiments, the stabilization and / or improvement comprises a stabilization of or decrease in a NYHA class, optionally wherein the decrease is greater than or equal to 1 class or to 2 classes. In some embodiments, the stabilization and / or improvement comprises a stabilization of or an increase in LVEF, optionally wherein the increase is greater than or equal to 5%, 10%, 15%, 20%, or 30%, or any percentage between any two of the foregoing percentages. In some embodiments, the stabilization and / or improvement comprises a stabilization of or an increase in an KCCQ score, optionally wherein the increase is greater than or equal to 5 points, 10 points, 20 points, 30 points, 40 points, 50 points, 60 points, or 70 points, or any number between any two of the foregoing numbers. In some embodiments, the stabilization and / or improvement comprises stabilization of or an increase in a 6MWT distance, optionally wherein the increase is greater than or equal to 30 meters, 40 meters, 50 meters, 60 meters, 70 meters, or 80 meters, or any distance between any two of the foregoing distances. In some embodiments, the stabilization and / or improvement comprises a stabilization of or decrease in an NT-proBNP concentration, optionally wherein the decrease is greater than or equal to 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, or any percentage between any two of the foregoing percentages, or is in a range defined by any two of the preceding values. In some embodiments, the stabilization and / or improvement comprises a stabilization of or decrease in a cardiac troponin I concentration, optionally wherein the decrease is greater than or equal to 30%, 35%, 40%, 45%, or 50%, or any percentage between any two of the foregoing percentages, or is in a range defined by any two of the preceding values. In some embodiments, the measured stabilization and / or improvement comprises at least 1, 2, 3, 4, 5, or 6 parameters selected from the stabilization ofor decrease in a NYHA class, the stabilization of or increase in LVEF, the stabilization of or increase in an KCCQ score, the stabilization of or increase in a 6MWT distance, the stabilization of or decrease in an NT-proBNP concentration, and the stabilization of or decrease in a cardiac troponin I concentration.Systems and kits

[0021] Some embodiments of the methods and compositions provided herein include systems and kits to treat (including one or more of inhibiting, stabilizing, and / or ameliorating) HFrEF in a subject. Some such embodiments include a polynucleotide encoding a SERCA2 polypeptide. Some embodiments include a pharmaceutical composition comprising the polynucleotide and a pharmaceutically acceptable excipient. In some embodiments, the SERCA polypeptide comprises a SERCA2 polypeptide. In some embodiments, the SERCA2 polypeptide comprises a SERCA2 isoform selected from a SERCA2a polypeptide, or a SERCA2c polypeptide. In some embodiments, the SERCA2 polypeptide comprises a SERCA2a polypeptide. In some embodiments, the polynucleotide comprises a promoter operably linked to the nucleic acid encoding the SERCA polypeptide. In some embodiments, the promoter comprises a constitutive promoter. In some embodiments, the promoter comprises a CMV promoter. In some embodiments, the polynucleotide comprises a vector. In some embodiments, the vector is selected from an AAV vector, a lentivirus vector, and a retrovirus vector. In some embodiments, the vector comprises an AAV vector. In some embodiments, a viral capsid comprises the polynucleotide. In some embodiments, the polynucleotide comprises a nucleic acid encoding the viral capsid. In some embodiments, the AAV vector encodes an AAV or fragment thereof having a serotype. In some embodiments, the AAV or fragment thereof comprise a viral capsid protein or fragment thereof. In some embodiments, the AAV or fragment thereof has a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. In some embodiments, the AAV or fragment thereof has a serotype selected from AAV1, and AAV9. In some embodiments, the AAV or fragment thereof has an AAV1 serotype.

[0022] Some embodiments include a container, such as a sterile vial, comprising a single dose of the polynucleotide to treat (including one or more of inhibiting, stabilizing, and / or ameliorating) HFrEF in a subject. In some embodiments, the polynucleotide comprisesa viral vector. In some embodiments, a viral capsid or functional fragment thereof comprises the polynucleotide. In some such embodiments, a dose of the polynucleotide can be measured as a number of viral genome particles (vg), or as a number of DRP. In some embodiments, a dose of the polynucleotide is at least 1 X 108vg, 1 X 109vg, 1 X IO10vg, 1 X 10” vg, 1 X 1012vg, 1 X 1013vg, 1 X 1014vg, 1 X 1015vg, 1 X 1016vg, 1 X 1017vg, 1 X 1018vg, 1 X 1019vg, 1 X 1020vg, or an amount between any two of the foregoing amounts. In some embodiments, a dose of the polynucleotide is at least 1 X 108DRP, 1 X 109DRP, 1 X IO10DRP, 1 X 10” DRP, 1 X 1012DRP, 1 X 1013DRP, 1 X 1014DRP, 1 X 1015DRP, 1 X 1016DRP, 1 X IO17DRP, 1 X 1018DRP, 1 X 1019DRP, 1 X IO20DRP, or an amount between any two of the foregoing amounts. In some embodiments, a dose of the polynucleotide is within a range from about 1 X 108vg to about 1 X 1020vg, from about 1 X 109vg to about 1 X 1017vg, from about 1 X IO10vg to about 1 X 1016vg, from about 1 X 10” vg to about 1 X 10i5vg, or from about 1 X 1013vg to about 1 X 1014vg. In some embodiments, a dose of the polynucleotide is within a range from about 1 X 1013vg to about 9 X 1013vg. In some embodiments, a dose of the polynucleotide is within a range from about 1 X 108DRP to about 1 X 1020DRP, from about 1 X 109DRP to about I X 1017DRP, from about I X IO10DRP to about 1 X 1016DRP, from about 1 X 10” DRP to about 1 X 1015DRP, or from about 1 X 1012DRP to about 1 X 1014DRP. In some embodiments, a dose of the polynucleotide is within a range from about 1 X 10” DRP to about 9 X 1013DRP.

[0023] Some embodiments also include a vasodilator, such as nitroglycerin.

[0024] Some embodiments of the methods and compositions provided herein include the use of a vasodilator. Aspects useful with some embodiments provided herein are disclosed in U. S. Pat. No. 8221738 which is incorporated by reference in its entirety, and for compositions, kits, and routes / methods of delivery. In some embodiments, the polynucleotide is administered in combination with a vasodilator. In some embodiments, a vasodilator can be administered to a subject prior to administration of the polynucleotide. In some embodiments, a vasodilator can be administered to a subject prior to and concurrently with administration of the polynucleotide. Examples of vasodilators include adenosine, histamine (or histamine-mducing agents), alpha blockers, theobromine, papaverine, ethanol, tetrahydrocannabinol (THC), minoxidil, nitric oxide (including nitric oxide increasing substances), and nitroglycerin. In some embodiments, a vasodilator is administered systemically, for exampleoraliy, transdermally, or by intravenous injection or infusion. In some embodiments, the infusion comprises an intracoronary infusion. Some embodiments of the methods and compositions provided herein include aspects disclosed in U.S. 2008 / 0076730; U.S. Pat. No.8221738; U.S. Pat. No. 11583662; U.S. 2024 / 0058475 which are each incorporated by reference in its entirety.Embodiments to assess efficacy

[0025] Some embodiments of the methods and compositions provided herein include one or more of the following assessment methods for clinical and functional outcomes, such as a treatment and / or a clinical trial.Embodiments of clinical events

[0026] Some embodiments include the observation and measurement of clinical events during a period of a treatment and / or clinical trial. For example, clinical events can be collected during the trial starting on Day 1 (phase 1 b) or day of randomization (phase 2) and continue through the long-term follow-up. Clinical events occurring in the 52-week follow-up of each subject in the mam study can be adjudicated and collected but not adjudicated in thelong-term follow-up. Clinical events can include the following: All-cause death and cause¬ specific mortality including cause-specific heart failure-related death; All-cause hospitalization including hospitalization for heart failure; Urgent outpatient heart failure- related visit (ambulatory worsening heart failure); Heart transplant; MCSD implantation; Non-fatal MI; and Non-fatal stroke. Non-adjudicated special events of interest in gene therapy trials include new oncologic, hematologic, rheumatologic, auto-immune, and neurologic conditions or worsening of pre-existing medical condition for the latter 4 conditions.Embodiments including a transthoracic echocardiography

[0027] Some embodiments include sequential transthoracic echocardiography (TIE) to assess chamber reverse remodeling and function m response to therapy. Parameters measured can include LV sizes and volumes and other relevant parameters. The primary parameter for LV remodeling can be LVESV indexed to body surface area (LVESVI), LVEF and global LV longitudinal strain. Other secondary measures can include LV diastolic functionand left atrial volume, area and strain. Additionally, subjective and objective measures of the degree of mitral regurgitation can be made.

[0028] The screening LVEF can be determined by echocardiography during a second screening period (SP2) and this examination can also serve as the baseline imaging assessment of subjects entering the study. An echocardiography core laboratory can be utilized to provide standardized data analyses. A written report issued by the echocardiography core laboratory within 72 hours of receipt can be available in the medical records documenting the quantitative measurement of LVEF that is less than or equal to 35% in order for the subject to qualify for the study. The echocardiography performed during screening serves the dual purpose of providing the LVEF measurement to determine eligibility and the baseline echocardiography for comparing serial imaging studies on study,

[0029] For a given subject, TTE can be performed at approximately the same time of the day throughout the study, if possible. If left ventricular walls cannot be well-defined, standard contrast agents may be used to improve visualization of wall motion and volumes. TTE can be performed prior to cardiopulmonary exercise testing, 6MWT, or any physical exertion.Embodiments including a six-minute walk test

[0030] Some embodiments include a 6MWT performed consistent with the protocol recommended by the American Thoracic Society (ATS, 2002). Subjects unable to walk for 6 minutes due to neuromuscular or peripheral vascular disease, other conditions unrelated to heart failure which preclude normal walking, or other medical contraindications to exercise testing may not perform the 6MWT but are otherwise eligible for the trial. The 6MWT can be performed on two separate dates during SP2. The initial 6MWT can include two studies separated by at least 2 hours. The first can serve as the teaching and training test where the subject becomes acclimated to the procedure and can stop to ask questions. Following a minimum of a 2-hour rest period, the test can be repeated following the standard 6MWT procedure.Embodiments comprising a NYHA classification

[0031] Some embodiments include measuring a subject’s HF symptoms, measured by NYHA classification. To standardize the classification across all study centers, each class is defined as follows without regard to results from any other tests or procedures: Class I: No limitation of physical activities. Ordinary physical activity does not cause undue fatigue, palpitation, dyspnea or anginal pain. Class II: Slight or mild limitation of physical activity. Comfortable at rest, but ordinary physical activity results in fatigue, palpitation, dyspnea or anginal pain. Class III: Marked limitation of physical activity. Comfortable at rest, but less than ordinary activity causes fatigue, palpitations, dyspnea or anginal pain. Class IV: Unable to carry out any physical activity without discomfort. Symptoms of cardiac insufficiency at rest. If any physical activity is undertaken, discomfort is increased.Embodiments measuring quality of life

[0032] Some embodiments include a subject completing a Kansas City Cardiomyopathy Questionnaire (KCCQ) to assess quality of life. The KCCQ is a 23-item self-administered questionnaire developed to independently measure the subject’s perception of their health status, which includes heart failure symptoms, impact on physical and social function, and how their heart failure impacts their quality of life (QOL) within a 2-week recall period.

[0033] The KCCQ tool quantifies the following six (6) distinct domains and two (2) summary scores:

[0034] KCCQ Symptom Domain quantifies the frequency and burden of clinical symptoms in heart failure, including fatigue, shortness of breath, paroxysmal nocturnal dyspnea and subjects’ edema / swelling. An overall symptom score is generally used in analyses; subscale scores for both frequency and severity are also available.

[0035] KCCQ Physical Function Domain measures the limitations subjects experience due to their heart failure symptoms, in performing routine activities. Activities are common, gender-neutral, and generalizable across cultures, while also capturing a range of exertional requirements.

[0036] KCCQ Quality of Life Domain is designed to reflect subjects’ assessments of their quality of life, given the current status of their heart failure.

[0037] KCCQ Social Limitation Domain quantifies the extent to which heart failure symptoms impair subjects’ abilities to interact in a number of gender-neutral social activities.

[0038] KCCQ Self-efficacy Domain quantifies subjects’ perceptions of how to prevent heart failure exacerbations and manage complications when they arise. This scale is not included in the summary scores.

[0039] KCCQ Symptom Stability Domain measures recent changes in subjects’ symptoms; their shortness of breath, fatigue or swelling. It compares frequency of heart failure symptoms at the time of completing the KCCQ with frequency 2 weeks ago. As a measure of change, it is most interpretable as a baseline assessment of the stability of subjects’ symptoms at the start of a study and shortly thereafter, as a measure of the acute response to treatment. This domain is not included in the summary scores,

[0040] Clinical Summary Score includes total symptom and physical function scores to correspond with NYHA Classification. Overall Summary Score includes the total symptom, physical function, social limitations and quality of life scores.

[0041] KCCQ responses are provided along a rating scale continuum with equal spacing from worst to best. On average, the 23-item version takes 4-6 minutes to complete. The concepts quantified in the KCCQ are designed to be relevant and appreciable by all heart failure subjects specified in the qualified context of use. The Flesch Reading Ease is 76 and the Flesch-Kincaid Grade level is 6.7. The tool can be used to evaluate the effectiveness of a heart failure medical device studied in a clinical trial.Embodiments of efficacy evaluation

[0042] Some embodiments include an efficacy evaluation. In some embodiments, an efficacy evaluation is a secondary objective of the phase 2 trial and exploratory for the phase lb trial. Efficacy parameters include changes from baseline in LVESVI, 6MWT distance, NT-proBNP, quality of life assessed by KCCQ and NYHA classification, hs-cTnl, LVEF, and LV global longitudinal strain; and rate of recurrent clinical events. Additional imaging, hematologic and clinical metrics will be analyzed in an exploratory fashion.

[0043] Summaries for the first 4 parameters as well as hs-cTnl, LVEF, and LV global longitudinal strain can include means of observed values and changes from baseline.For NYHA classification, change from baseline can be summarized as a categorical variable (eg, Class III to Class IV) as well as binary responder variable with response defined as at least al -class improvement. KCCQ listings can include individual, domain and summary scores. KCCQ can be summarized by domain - Physical Limitation, Symptom Stability, Symptom Frequency, Symptom Burden, Symptom Overall, Social Limitation, Self-Efficacy and Quality of Life – as well as by the KCCQ summary categories of Clinical and Overall Recurrent events are defined as hospitalizations for failure of the native heart that has not been implanted with an MCSD and / or ambulatory worsening failure of the native heart that has not been implanted with an MCSD. Terminal events are defined as all-cause death, MCSD or transplant. Event rates can be summarized as number of events per patient-year. Graphical depictions can include mean cumulative function plots (recurrent events) and Kaplan-Meier plots (terminal events).EXAMPLESExample 1—A phase 1b / 2 trial of the safety and efficacy of SRD-001 (AAV1 / SERCA2a) in subjects with heart failure with reduced ejection fractionObjectives

[0044] Phase lb: Primary objective: To evaluate the safety and tolerability of a one¬ time intracoronary administration of SRD-001 (AAV1 / SERCA2a) in subjects with heart failure with reduced ejection fraction (HFrEF). Secondary objective: To evaluate viral kinetics and shedding of AAV1 / SERCA2a. Exploratory objective: To evaluate the efficacy of SRD-001 in the phase lb trial.

[0045] Phase 2: Primary objective: To evaluate the effect of SRD-001 on left ventricular function and remodeling, exercise tolerance, quality of life and associated biomarkers. Secondary objective: To further evaluate safety as well as the efficacy of SRD-001 as assessed by symptomatic parameters, physical activity, biomarkers of disease, cardiac function and remodeling, and rate of clinical events.

[0046] Study Design: This is a phase lb sequential, uncontrolled, dose escalation trial followed by a seamless phase 2 randomized, controlled trial.Methodology

[0047] FIG. 1 and FIG. 2 summarize events of the phase lb trial and the phase 2 trial, respectively, each exclude aspects of long term follow up.

[0048] Screening Periods: In the phase lb and subsequent phase 2 trials, there will be two sequential screening periods In the initial screening period (SP1), which can last up to 90 days prior to the second Screening Period 2 (SP2), subjects who have been identified with New York Heart Association (NYHA) Class III / TV HFrEF and a left ventricular ejection fraction (LVEF) < 35% will undergo chart review followed by a blood draw to assess their anti-AAVl neutralizing antibody (NAb) levels and, in the case of subjects considered for the phase 2 cohort, a blood draw to assess qualifying N-terminal prohormone brain natriuretic peptide (NT-proBNP) levels if they do not meet other recent heart failure criteria (Inclusion criteria 6a and b). Subjects with < 50% inhibition of the viral vector at a serum dilution of 1:4 (negative) and, in the case of potential phase 2 subjects, an appropriate NT-proBNP level, both drawn within 90 days of SP2, will enter SP2, which can last up to 35 days prior to treatment. In SP2, subjects will undergo a battery of tests and procedures to further determine eligibility and to establish baseline measurements of certain parameters.

[0049] Phase lb: In the phase lb trial, 3-4 subjects each in 2 ascending dose cohorts -----Cohort A at a fixed SRD-001 dose of 3 × 1013viral genome particles (vg) and Cohort B at a fixed dose of 4.5 × 1013vg - -will be sequentially treated in a staggered fashion. At least 4 weeks will separate treatment between each subject (as well as stopping / pausing rules) in order to provide for individual evaluation of safety. The contract research organization will organize subject recruitment and sequencing of treatment among investigative sites so that there is no duplication of treatment. Following each 4- week evaluation period post-infusion, the sponsor’s medical monitor will determine if it is safe, based on cumulative safety data on all treated subjects to date, to proceed with treatment of the next subject; provided, however, that no prespecified stopping / pausing rule has been triggered in which case the stopping / pausing rule will be followed. Other subjects may enter SP1 while this determination is being made. After the fourth subject in Cohort A is followed for at least 4 weeks following infusion and at least 1 subject has been followed for at least 12 weeks following infusion, the independent data monitoring committee (iDMC) will review all available safety data to make a recommendation as to whether to (a) add up to 3 subjects in a concurrent or staggered mannerin order to better characterize the safety profile of SRD-001 or for any other reason, or (b) proceed with dose escalation according to the dose escalation rules and open Cohort B to enrollment. The process will be repeated for Cohort B with 3-4 subjects after which the iDMC will make a recommendation as to whether to (a) add up to 3 additional subjects m a concurrent or staggered manner in order to better characterize the safety profile of SRD-001 or for any other reason, or (b) proceed to the phase 2 trial.

[0050] Phase 2: Subjects meeting study inclusion / exclusion criteria will be competitively enrolled at up to 20 participating sites. Fifty subjects meeting eligibility criteria will be randomized 1:1. Final baseline testing not required to establish eligibility, such as the 6 minute walk test (6MWT), can be completed up to Day 1 prior to treatment. Subject assignment will be blinded to the subject, all site personnel, the contract research organization and the sponsor. The iDMC members will be unblinded and all attempts will be made to sequester all information regarding treatment assignment to committee members only.

[0051] Treatment: All subjects in both phases of the trial will first undergo diagnostic angiography without required concomitant left or right heart catheterization to determine the presence and severity of coronary artery disease. If adequate coronary patency is established, intracoronary infusion of the study product will proceed. In phase lb, all subjects will receive intra-coronary injection of SRD-001 at a fixed concentration of 3 * 1013vg (Cohort A) or 4.5 x io1’ vg (Cohort B). In phase 2, subjects will be randomly allocated to receive either SRD-001 or placebo where the fixed dose of SRD-001 will be either the maximum tolerated dose (MTD) established in phase 1 b or the highest tested dose in phase 1 b. After the procedure, subjects will be monitored continuously for a minimum of 4 hours and then discharged to home that same day if medically appropriate. Subjects who have fluid overload or experience periprocedural complications may be hospitalized at the discretion of the investigator. During the trial, the decision to hospitalize a subject for any reason will be totally and independently left to the discretion of the investigator or treating physician without any consultation with the sponsor or any of its consultants or designees.

[0052] Trial Procedures: In both phases of the trial, subjects will be followed during the conduct of the trial at prespecified study visits. At Day 2 and Week 1, a telephone evaluation will be performed, and if clinically indicated, an in-person evaluation and assessment will be performed at the soonest available time. At Weeks 2, 4, 12, 24, 36 and 52,subjects will undergo a battery of safety and efficacy assessments including physical examination, NYHA classification, laboratory’ tests, interrogation of implantable cardioverter defibrillator (ICD), 12-lead electrocardiogram (ECG), biomarkers of disease and collection of adverse events (AEs) including clinical events that will be adjudicated by the Clinical Events Committee. At Weeks 24 and 52, subjects will also undergo echocardiography, 6MWT and complete the Kansas City Cardiomyopathy Questionnaire (KCCQ).

[0053] In phase lb Cohort A, bodily fluids for viral vector kinetics and shedding will be collected on or before Day 1 and on Days 2 and 3, Week 1 (Day 7) and Day 10 in addition to the in-person visits; the sponsor will arrange specimen collection and pick-up by a home health nurse or technician,

[0054] Investigators and research personnel will also be trained to identify potential cellular immune responses which could occur in the Week 2 to Week 16 timeframe.

[0055] Four-Year Follow-Up: The end of the main study for each subject will occur at the Week 52 visit after which each subject will enter into the 4-year long-term follow-up for biannual semi-structured telephone questionnaire that will collect information on overall status, status of heart failure, other comorbid conditions of interest in gene therapy trials, and relevant clinical events with supporting source documentation. Formal adjudication of clinical events will not occur in the four-year follow-up period.Sample Size Justification

[0056] The phase lb sample size of 6-14 subjects and the phase 2 sample size of 50 are considered adequate sample sizes for the primary and secondary objectives of the trial. With 25 subjects infused with SRD-001, the trial will have > 0.8 probability of observing an AE with an incidence rate of at least 6.4% and 50-65% power, depending on the correlation among endpoints, to detect efficacy based on a similar analytical model to that specified in this protocol. Number of Subjects (planned): N=56-64.Diagnosis and Main Criteria for Inclusion

[0057] Unless otherwise specified, screening must be performed within 35 days prior to treatment except as noted herein. Subjects must meet the following criteria to be eligible for the study:

[0058] 1. Anti- AAV 1 NAb inhibition of the viral vector by <50% at a serum dilution of 1:4 within 125 days prior to infusion.

[0059] 2. Age 18 to 80 years, inclusive, at the time of signing the first informed consent.

[0060] 3. Chronic ischemic or non-ischemic cardiomyopathy, except for hypertrophic cardiomyopathy or infiltrative or restrictive cardiomyopathies. Toxic or alcoholic cardiomyopathies and cardiomyopathies due to acute myocarditis are allowed as long as toxin / alcohol exposure or myocarditis has been eliminated and there has been > 6 months since the end of the last clinical episode in order to rule-out spontaneous recovery. Similarly, subjects with viral or peripartum cardiomyopathy will not be enrolled until > 6 months from initial diagnosis. Subjects with ischemic cardiomyopathy must have at least 1 major coronary vessel (Left anterior descending (LAD), Left coronary circumflex (LCx) or Right coronary artery (RCA)) with Thrombolysis in Myocardial Infarction (TIMI) grade 3 flow. If a subject has not undergone recent coronary angiography, TIMI flow may be assessed during the study angiography just prior to the administration of investigational medicine product.

[0061] 4. LVEF <35% as determined by the echocardiography core laboratory.

[0062] 5. Diagnosis of NYHA class III / IV heart failure for a minimum of 60 days prior to screening.

[0063] 6. For phase 2 only, the presence of at least one of the following: a. Hospitalization for heart failure within 6 months of screening, b. At least 2 outpatient interventions for the intended treatment of signs and symptoms of worsening heart failure requiring intravenous (IV) diuretics in the past 12 months prior to screening, c. NT-proBNP >1200 pg / mL during screening; if subject is in atrial fibrillation, NT-proBNP >1600 pg / mL during screening.

[0064] 7. Ability to walk a minimum of 50 meters in the 6MWT.

[0065] 8. Individualized, maximal, optimized HF therapy consistent with American College of Cardiology / American Heart Association / Heart Failure Society of America practice guidelines for the treatment of chronic heart failure (ACC / AHA'HFSA guidelines) and as updated from time to time: a. Maximal guideline-recommended medical therapy as tolerated, which can include oral diuretic, inhibitor of the renin-angiotensin system (angiotensin-converting enzyme [ACE] inhibitor; angiotensin-receptor blocker [ARB]; orangiotensin receptor-neprilysin inhibitor [ARNI]), beta blocker, ivabradine where appropriate, hydralazine, aldosterone receptor blocker and SGLT2i. i. The choice of ARB is limited to those approved for heart failure (candesartan or valsartan). ii. The choice of beta blocker is limited to those approved for heart failure (bisoprolol, carvedilol or metoprolol succinate). Metoprolol tartrate is not approved for HF and is not allowed, iii. Dosing of the above medications must be stable for a minimum of 30 days prior to screening, although up- or down- titration of diuretics, as medically indicated, is permitted, iv. Subjects requiring IV diuretics during this period will be required to undergo an additional 30-day period of stabilization on oral diuretics following the last day of IV diuresis, v. Enrollment of any subject with any deviation from these criteria must be preapproved by the sponsor, b. Resynchronization therapy, if clinically indicated according to ACC / AHA HF guidelines, must have been initiated at least 6 months prior to screening, c. If the subject is already participating in a cardiac rehabilitation program, it should have been routinely adhered to for at least 3 months prior to study entry and be continued at least through the 52-week main study,

[0066] 9. Presence of a cardioverter defibrillator (ICD) is required and must have been implanted a minimum of 30 days prior to screening.

[0067] 10. All male subjects regardless of fertility status or the fertility status of their partner must agree to use a condom and spermicide during any sexual relations for 6 months following administration of investigational medicine product to protect their partner from potential viral shedding.

[0068] 11. All subjects regardless of fertility status or the fertility status of their partner must agree to have any male partner use a condom and spermicide during any sexual relations for 6 months following administration of investigational medicine product to protect their partner from potential viral shedding.

[0069] 12. All subjects capable of procreation with their partners must agree to use adequate contraception for 6 months following administration of investigational medicinal product to avoid pregnancy (defined as oral or injectable contraceptives, intrauterine devices, surgical sterilization in addition to / or a combination of a condom and spermicide).

[0070] 13. Agree to not donate sperm or oocytes for 6 months following administration of investigational medicinal product.

[0071] 14. Ability to sign Informed Consent Form and Release of Medical Information.Main Criteria for Subject Exclusion

[0072] Unless otherwise specified, screening must be performed within 35 days prior to treatment except as noted below. Subjects meeting any of the following criteria will be excluded from the study:

[0073] 1, New diagnosis of currently untreated heart failure.

[0074] 2. Any IV therapy with positive inotropes, vasodilators or diuretics within 30 days prior to screening.

[0075] 3. Restrictive or infiltrative cardiomyopathy, hypertrophic cardiomyopathy, pericardial disease, amyloidosis, uncorrected thyroid disease or discrete left ventricular (LV) aneurysm.

[0076] 4, Untreated left-sided valvular disease including moderate or severe mitral stenosis (valve area <1.5 cm2), severe aortic stenosis with valve area <1.0 cm2, or moderate- severe (3 -4+ / 4+) aortic or mitral regurgitation.

[0077] 5. Limited ability to perform the 6MWT due to claudication, arthritic or other conditions unrelated to heart failure.

[0078] 6. Cardiac surgery, percutaneous coronary intervention (PCI), valvuloplasty or valve replacement within 30 days prior to screening.

[0079] 7. Myocardial infarction within 6 months prior to screening or active angina in the 3 months prior to screening.

[0080] 8. Prior heart transplantation, left ventricular reduction surgery (LVRS), cardiomyoplasty, passive restraint device (e.g. CorCap™ Cardiac Support Device), mechanical circulatory support device (MCSD) or cardiac shunt.

[0081] 9. Likely to receive cardiac resynchronization therapy, cardiomyoplasty, LVRS, conventional revascularization procedure, MCSD, cardiac transplant or valvular repair in the 12 months following treatment.

[0082] 10. Hemodynamic instability requiring hospitalization in the past 3 months.

[0083] 11. Prior coronary artery bypass grafting (CABG) is not necessarily exclusionary. Review of the most recent coronary angiography should be performed, takinginto account the dominance of the system, the accessibility of the graft(s) orifice and the contribution of the graft vessel(s) and native coronary arteries to viable myocardial perfusion. The case and tentative infusion strategy must be discussed with the medical monitor prior to enrollment of the subject into the study.

[0084] 12. Known hypersensitivity to radiopaque agents used for angiography; history of or likely need for, high dose corticosteroid pretreatment prior to contrast angiography.

[0085] 13, Significant, in the opinion of the investigator, left main or ostial right coronary luminal stenosis

[0086] 14, Liver function tests (alanine aminotransferase [ALT], aspartate aminotransferase [AST], alkaline phosphatase) >3 x upper limit of normal (ULN), total bilirubin >2 x ULN or known intrinsic liver disease (e.g., cirrhosis, chronic hepatitis B or hepatitis C virus infection).

[0087] 15, Current or likely need for hemodialysis within 12 months or current glomerular filtration rate (GFR) <20 mL / minute / 1.73 m2 estimated by Modification of Diet in Renal Disease (MDRD) formula for calculating the GFR MDRD calculation.

[0088] 16. Bleeding diathesis or thrombocytopenia defined as platelet count <75,000 platelets / p.

[0089] 17. Anemia defined as hemoglobin <9 g / dL.

[0090] 18. Diagnosis of, or treatment for, any cancer within the last 5 years except for Grade 1 prostate cancer, basal cell carcinoma or carcinomas in situ where surgical excision was considered curative. (Past medical history of cancer is not exclusionary as long as the subject has been disease free for at least 5 years since the time of diagnosis and treatment).

[0091] 19. Previous participation in a study of gene transfer; however, if the study was unblinded or documentation otherwise exists that the subject was randomized to the placebo control group and did not receive active gene transfer agent, the subject may be considered for this study.

[0092] 20. Receiving investigational intervention or participating in another clinical study within 30 days or within 5 half-lives of the drug prior to screening. Exception may be made if the individual is enrolled in a non-therapeutic observational study (registry), the observational portion of a therapeutic study where the sponsoring authority authorizesenrollment, or received or will receive a COVID-19 vaccine under Emergency Use Authorization or similar regulatory authorization.

[0093] 21. Pregnancy or lactation / breastfeeding.

[0094] 22. Recent history of psychiatric disease (including drug or alcohol abuse) that is likely to impair subject’s ability to comply with protocol-mandated procedures, in the opinion of the investigator.

[0095] 23. Other unstable / uncontrolled comorbidity (e.g., diabetes mellitus with hemoglobin Ale >8%) or concurrent medical condition(s) that, while not explicitly excluded by the protocol, could jeopardize the safety of the patient or objectives of the study.Investi gational Product. Dosage and Mode of Administration

[0096] SRD-001 at a fixed dose of either 3 x 1013vg or 4.5 × 1013vg administered into the left and / or right coronary artery via antegrade epicardial coronary artery infusion with commercially available guide or diagnostic cardiac catheters and the B. Braun Perfusor® Space Syringe Pump set at a flow rate of 300 mL per hour over 10 minutes. Nitroglycerin IV infusion should be administered for a minimum of 10 minutes at the highest tolerated dose prior to infusion and concomitantly with the infusion of SRD-001.Duration of Treatment

[0097] One-time intracoronary infusion.Reference Therapy, Dosage and Mode of Administration

[0098] In the phase 2 trial, SRD-001 matching placebo will be the reference therapy. It is composed of the same excipients as SRD-001 without the AAVl / SERCA2a active ingredient and is administered in the same manner as SRD-001.Criteria for Evaluation

[0099] Safety: Adverse events; electrocardiography (ECG); ICD interrogation; and laboratory tests including complete blood count (CBC) with white blood cell (WBC) differential and platelets, basic metabolic and comprehensive hepatic serum chemistry panels, lactate dehydrogenase (LDH) and uric acid, high sensitivity cardiac troponin I (hs-Tnl),creatine phosphokinase (CPK), Enzyme-linked ImmunoSpot (ELISpot), urinalysis, and urine pregnancy test. Efficacy: NYHA classification, KCCQ, 6MWT, NT-pro-BNP, transthoracic echocardiography, and clinical events.Statistical methods

[0100] Analysis Populations: The phase lb population will be all subjects who received SRD-001 in the phase lb trial. The intent-to-treat (ITT) population will be all subjects randomized in the phase 2 trial, summarized and analyzed according to the randomized treatment assignment. The modified ITT (mITT) population will be all subjects treated in the phase 2 trial, summarized and analyzed according to the randomized treatment assignment. The per- protocol (PP) population will be subjects treated in the phase 2 trial with no protocol deviations that meaningfully impact the completeness, accuracy and / or reliability of the study data. The safety population will be subjects treated in the phase 2 trial, summarized and analyzed according to the treatment received,

[0101] Safety and efficacy data will be listed for the phase lb population and for the phase 2 ITT population. Efficacy analyses will be done using the mITT population at the Week 24 and Week 52 analysis data cutoffs. Efficacy analyses will also be done using the ITT and PP populations.

[0102] Methods: Summaries will be by treatment group and study visit. Categorical variables will be summarized as frequencies and percentages in each category. Continuous variables will be summarized as numbers of subjects, means, standard deviations, medians and ranges. Clinical event rates will be summarized as events per patient year of follow-up. Treatment effect on clinical event rates will be summarized as hazard ratios as estimated by a semi-parametric joint frailty model to account for competing risk from terminal events. Hospitalization duration will be summarized per patient year of follow-up.

[0103] Both group- and subject-level overall treatment effect in the phase 2 trial will be explored using a composite outcome approach. For the group-level analysis, outcomes will be classified into one of five domains: (1) Symptomatic: NYHA class and KCCQ score; (2) Functional: 6MWT; (3) Biomarkers: NT-proBNP (log-normal); (4) LV Function / Remodeling: LVEF, LVESV (percentage and absolute change), global longitudinal strain; and (5) Clinical Outcome: recurrent and terminal events.

[0104] Treatment effect on changes from baseline will be analyzed using analysis of covariance or, for change in NYHA class, by comparing distributions of change scores using a chi square test of association (or Fisher’s exact test if any change category’ within treatment group contains less than 5 subjects). Treatment effect on clinical outcomes will be analyzed using the joint frailty model. Hypothesis tests will be one-sided.

[0105] Within treatment groups, a “success” domain will meet the following criteria: (1) For at least one outcome within the domain, SRD-001 superiority is demonstrated at the 0.10 significance level; and (2) For other outcomes within the domain, SRD-001 superiority is demonstrated descriptively.

[0106] Group level investigational medicine improvement will be concluded if: (1) There are at least 2 “success” domains; and (2) There is not clinically significant worsening (pre-defined) in the SRD-001 group in any domain,

[0107] For the subject-level analysis, each subject will be scored on each outcome as clinically significantly improved (+1), clinically significantly worsened (-1), or unchanged (0) and a total subject level activity score will be calculated as the sum across all outcomes; clinically significant change will be pre-defined. A one-sided t test will be used to test for a treatment effect based on mean scores. Subject-level investigational medicine improvement will be concluded if SRD-001 superiority is demonstrated at the 0.10 significance level.

[0108] SRD-001 activity will be considered “significant” if: (1) Improvement in the SRD-001 group is detected at either the group or subject level; and (2) Descriptive improvement in the SRD-001 group is evident at both the group and subject level.

[0109] Because evaluation of efficacy is a secondary objective in a phase 2 trial, there will be no adjustment for multiplicity.Example 2 — Preliminary results of trial

[0110] The clinical trial disclosed in Example 1was performed. Preliminary results from the clinical trial are listed in TABLE 2, TABLE 3 and TABLE 4 for tested parameters at day 1 (baseline), 6 and 12 months after treatment. ND: not determined; NR: normal range.TABLE 2Subject NYHA classification LVEF (%)(dose [vg]) Baseline 6 months 12 months Baseline 6 months 12 months 1001III II II 31 35 36 (3 el 3)1015III II II 26 30 24 (3 el 3)1020III II I 34 39 25.3 (3 el3)1021III II ND 25 20.4 ND (3 e!3)1006in I II 30 36 34 (3 el 3)1002III ND ND 23 ND ND (3 el 3)1023III II 34.3 39(4.5 el 3)NR → I ≥ 55TABLE 3Subject KCCQ (total score) 6MWT (m)(dose [vg]) Baseline 6 months 12 months Baseline 6 months 12 months 100128 54 87 375.2 353.6 431.7 (3 el 3)1015100 100 99 600 563.3 579.1 (3 el 3)102068 89 85 382.2 411.5 450 (3 el3)102169 78 ND 53.6 9.0 ND* (3 el 3)100680 87 67 378.1 415.9 420 (3 el 3)100243 ND ND 328 368 ND* (3 el 3)Subject KCCQ (total score) 6MWT (m)(dose [vg]) Baseline 6 months 12 months Baseline 6 months 12 months 102398 398.6 408.4(4.5 el 3)NR → 100 400-700TABLE 4Subject NT-ProBNP (pg / mL) Cardiac troponin I (pg / mL) (dose [vg]) Baseline 6 months 12 months Baseline 6 months 12 months 1001194 170.5 157.8 25.8 17.2 13.9(3 el 3)10151325 1693 1411 10.7 9.0 7.4(3 el 3)1020570.9 98.5 228.7 7.1 6.5 10.6(3 el 3)10215764 6871 ND 82.2 97.0 ND(3 el 3)10061397 1105 1513 8.2 8.6 7.5(3 el3)1002548 422.6 ND 18.4 30.7 ND(3 e!3)1023551.1 219.1 8.6 6.4(4.5 el 3)NR → ≤ 198 < 19.8

[0111] Subject 1021 died 31 weeks post-treatment. Subject 1002 died 45 weeks post- treatment. Deaths not related to dug product as adjudicated by medical monitors.

[0112] In TABLE 2, TABLE 3 and TABLE 4, clinically meaningful improvements from baseline (BL) include: NYHA decrease, > 1 point (class change); LVEF increase, > 5%; KCCQ increase > 5 points; 6MWT increase > 30 meters; NT-proBNP decrease > 35%; and Cardiac troponin I decrease > 30%. Improvements below clinically meaningful levels indicated stabilization of IIFrEF.

[0113] In subjects where a NYHA class had been measured: at 6 months, clinically meaningful improvements were observed for 5 / 6 subjects, and stabilizing improvements were observed for 1 / 6 subjects; at 12 months, clinically meaningful improvements were observed for 4 / 4 subjects. From baseline to 12 months, subject 1020 had a significant improvement of a decrease in a NYHA class from class III to class I.

[0114] In subjects where an LVEF percentage had been measured: at 6 months, clinically meaningful improvements were observed for 5 / 6 subjects, and stabilizing improvements were observed for 1 / 6 subjects; at 12 months, clinically meaningful improvements were observed for 2 / 4 subjects, and stabilizing improvements were observed for 1 / 4 subjects. From baseline to 12 months, subjects 1001 and 1006 had significant improvements in an increase in LVEF of about 16% and about 13%, respectively.

[0115] In subjects where a KCCQ score had been measured: at 6 months, clinically meaningful improvements were observed for 4 / 5 subjects, and stabilizing improvements were observed for 1 / 5 subjects; at 12 months, clinically meaningful improvements were observed for 2 / 4 subjects, and stabilizing improvements were observed for 1 / 4 subjects. From baseline to 12 months, subjects 1001 and 1020 had significant improvements in an increase in a KCCQ score of 59 points and 17 points, respectively.

[0116] In subjects where a 6MWT distance had been measured: at 6 months, clinically meaningful improvements were observed for 3 / 7 subjects, and stabilizing improvements were observed for 2 / 7 subjects; at 12 months, clinically meaningful improvements were observed for 3 / 4 subjects, and stabilizing improvements were observed for 1 / 4 subjects. From baseline to 12 months, subjects 1001, 1020, 1006 had significant improvements in an increase in a 6MWT distance of 56 meters, 68 meters, and 42 meters, respectively.

[0117] In subjects where an NT-ProBMP concentration had been measured: at 6 months, clinically meaningful improvements were observed for 4 / 7 subjects, and stabilizing improvements were observed for 3 / 7 subjects; at 12 months, clinically meaningful improvements were observed for 1 / 4 subjects, and stabilizing improvements were observed for 3 / 4 subjects. From baseline to 12 months, subject 1020 had a significant improvement in an decrease in a NT-ProBMP concentration of 60%.

[0118] In subjects where a cardiac troponin I concentration had been measured: at 6 months, clinically meaningful improvements were observed for 1 / 7 subjects, and stabilizing improvements were observed for 5 / 7 subjects; at 12 months, clinically meaningful improvements were observed for 2 / 4 subjects, and stabilizing improvements were observed for 1 / 4 subjects. From baseline to 12 months, subjects 1001 and 1015 had significant improvements in a decrease in cardiac troponin I concentration of about 46% and 31%, respectively.Example 3 — Methods and materialsAssessment of Efficacy

[0119] The following section describes assessment methods for clinical and functional outcomes included in the trial.Clinical Events

[0120] Clinical Events will be collected during the trial starting on Day 1 (phase lb) or day of randomization (phase 2) and continue through the long-term follow-up. Clinical events occurring in the 52-week follow-up of each subject in the mam study will be adjudicated by the CEC and collected but not adjudicated in the long-term follow-up. Clinical events will include the following: All-cause death and cause-specific mortality including cause-specific heart failure-related death; All-cause hospitalization including hospitalization for heart failure; Urgent outpatient heart failure-related visit (ambulatory worsening heart failure); Heart transplant; MCSD implantation; Non-fatal MI; and Non-fatal stroke.

[0121] Non-adjudicated special events of interest in gene therapy trials include new oncologic, hematologic, rheumatologic, auto-immune, and neurologic conditions or worsening of pre-existing medical condition for the latter 4 conditions.Transthoracic Echocardiography

[0122] Sequential transthoracic echocardiography (TTE) will be used to assess chamber reverse remodeling and function in response to therapy. Parameters measured will include LV sizes and volumes and other relevant parameters. The primary parameter for LV remodeling will be LVESV indexed to body surface area (LVESVI), LVEF and global LVlongitudinal strain. Other secondary measures will include LV diastolic function and left atrial volume, area and strain. Additionally, subjective and objective measures of the degree of mitral regurgitation will be made.

[0123] The screening LVEF will be determined by echocardiography during SP2 and this examination will also serve as the baseline imaging assessment of subjects entering the study. An echocardiography core laboratory will be utilized to provide standardized data analyses. A written report issued by the echocardiography core laboratory within 72 hours of receipt must be available in the medical records documenting the quantitative measurement of LVEF that is less than or equal to 35% in order for the subject to qualify for the study. The echocardiography performed during screening serves the dual purpose of providing the LVEF measurement to determine eligibility and the baseline echocardiography for comparing serial imaging studies on study.

[0124] For a given subject, TTE should be performed at approximately the same time of the day throughout the study, if possible. If left ventricular walls cannot be well-defined, standard contrast agents may be used to improve visualization of wall motion and volumes. TTE should always be performed prior to cardiopulmonary exercise testing, 6MWT, or any physical exertion.Six Minute Walk Test

[0125] A 6MWT will be performed consistent with the protocol recommended by the American Thoracic Society (ATS, 2002). Subjects unable to walk for 6 minutes due to neuromuscular or peripheral vascular disease, other conditions unrelated to heart failure which preclude normal walking, or other medical contraindications to exercise testing should not perform the 6MWT but are otherwise eligible for the trial. The 6MWT will be performed on two separate dates during SP2. The initial 6MWT will include two studies separated by at least 2 hours. The first will serve as the teaching and training test where the subject becomes acclimated to the procedure and can stop to ask questions. Following a minimum of a 2-hour rest period, the test will be repeated following the standard 6MWT procedure.NYHA Classification

[0126] The subject’s HF symptoms, measured by NYHA classification, will be determined by the investigator. For the purpose of this study and to standardize the classification across all study centers, each class is defined as follows without regard to results from any other tests or procedures: Class I: No limitation of physical activities. Ordinary physical activity does not cause undue fatigue, palpitation, dyspnea or anginal pain. Class II: Slight or mild limitation of physical activity. Comfortable at rest, but ordinary physical activity results in fatigue, palpitation, dyspnea or anginal pain. Class III: Marked limitation of physical activity. Comfortable at rest, but less than ordinary activity causes fatigue, palpitations, dyspnea or anginal pain. Class IV: Unable to carry out any physical activity without discomfort. Symptoms of cardiac insufficiency at rest. If any physical activity is undertaken, discomfort is increased.Quality of Life

[0127] The Kansas City Cardiomyopathy Questionnaire (KCCQ) will be completed by the subject in order to assess quality of life. The KCCQ is a 23 -item self-administered questionnaire developed to independently measure the subject’s perception of their health status, which includes heart failure symptoms, impact on physical and social function, and how their heart failure impacts their quality of life (QOL) within a 2-week recall period.

[0128] The KCCQ tool quantifies the following six (6) distinct domains and two (2) summary scores:

[0129] KCCQ Symptom Domain quantifies the frequency and burden of clinical symptoms in heart failure, including fatigue, shortness of breath, paroxysmal nocturnal dyspnea and subjects’ edema / swelling. An overall symptom score is generally used in analyses; subscale scores for both frequency and severity are also available.

[0130] KCCQ Physical Function Domain measures the limitations subjects experience due to their heart failure symptoms, in performing routine activities. Activities are common, gender- neutral, and generalizable across cultures, while also capturing a range of exertional requirements.

[0131] KCCQ Quality of Life Domain is designed to reflect subjects’ assessments of their quality of life, given the current status of their heart failure.

[0132] KCCQ Social Limitation Domain quantifies the extent to which heart failure symptoms impair subjects’ abilities to interact in a number of gender-neutral social activities.

[0133] KCCQ Self-efficacy Domain quantifies subjects’ perceptions of how to prevent heart failure exacerbations and manage complications when they arise. This scale is not included in the summary scores.

[0134] KCCQ Symptom Stability Domain measures recent changes in subjects’ symptoms; their shortness of breath, fatigue or swelling. It compares frequency of heart failure symptoms at the time of completing the KCCQ with frequency 2 weeks ago. As a measure of change, it is most interpretable as a baseline assessment of the stability of subjects’ symptoms at the start of a study and shortly thereafter, as a measure of the acute response to treatment. This domain is not included in the summary scores,

[0135] Clinical Summary Score includes total symptom and physical function scores to correspond with NYHA Classification. Overall Summary Score includes the total symptom, physical function, social limitations and quality of life scores.

[0136] KCCQ responses are provided along a rating scale continuum with equal spacing from worst to best. On average, the 23-item version takes 4-6 minutes to complete. The concepts quantified in the KCCQ are designed to be relevant and appreciable by all heart failure subjects specified in the qualified context of use. The Flesch Reading Ease is 76 and the Flesch-Kincaid Grade level is 6.7. The tool can be used to evaluate the effectiveness of a heart failure medical device studied in a clinical trial.Statistics — Efficacy Parameters

[0137] Efficacy evaluation is a secondary objective of the phase 2 trial and exploratory for the phase lb trial. Efficacy parameters include changes from baseline in LVESVI, 6MWT distance, NT-proBNP, quality of life assessed by KCCQ and NYHA classification, hs-cTnl, LVEF, and LV global longitudinal strain; and rate of recurrent clinical events. Additional imaging, hematologic and clinical metrics will be analyzed in an exploratory fashion.

[0138] Summaries for the first 4 parameters as well as hs-cTnl, LVEF, and LV global longitudinal strain will include means of observed values and changes from baseline. For NYHA classification, change from baseline will be summarized as a categorical variable (eg, Class III to Class IV) as well as binary responder variable with response defined as at least al -class improvement. KCCQ listings will include individual, domain and summary’ scores. KCCQ will be summarized by domain - Physical Limitation, Symptom Stability, Symptom Frequency, Symptom Burden, Symptom Overall, Social Limitation, Self-Efficacy and Quality of Life – as well as by the KCCQ summary categories of Clinical and Overall Recurrent events are defined as hospitalizations for failure of the native heart that has not been implanted with an MCSD and / or ambulatory worsening failure of the native heart that has not been implanted with an MCSD. Terminal events are defined as all-cause death, MCSD or transplant. 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[0139] The term “comprising” as used herein is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0140] The above description discloses several methods and materials of the present disclosure. The disclosed embodiments are susceptible to modifications in the methodsand materials, as well as alterations in the fabrication methods and equipment. Such modifications will become apparent to those skilled in the art from a consideration of this disclosure or practice of the embodiments disclosed herein. Consequently, it is not intended that this disclosure be limited to the specific embodiments disclosed herein, but that it cover all modifications and alternatives coming within the true scope and spirit of the disclosure.

[0141] All references cited herein, including but not limited to published and unpublished applications, patents, and literature references, are incorporated herein by reference in their entirety and are hereby made a part of this specification. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.

Claims

WHAT IS CLAIMED IS:

1. A method of treating heart failure with a reduced ejection fraction (HFrEF) in a subject, comprising:identifying a subject as having HFrEF, and administering a polynucleotide comprising a nucleic acid encoding a sarcoplasmic / endoplasmic reticulum calcium ATPase (SERCA) polypeptide to the subject identified as having HFrEF.

2. The method of claim 1, wherein the subject comprises a left ventricular ejection fraction (LVEF) of less than 50%, or less than or equal to 45%, 40%, or 35%, or is in a range defined by any two of the preceding values.

3. The method of claim 1 or 2, wherein the subject comprises an LVEF of less than 5%.

4. The method of any one of claims 1-3, further comprising identifying and / or selecting the subject based on one or more characteristics of the subject.

5. The method of claim 4, wherein the identifying and / or selecting comprising measuring in the subject one or more of:(a) an anti-AAV neutralizing antibody inhibition level against an AAV viral vector in a serum sample from the subject, optionally wherein the anti-AAV neutralizing antibody is an anti-AAV1 neutralizing antibody, and the AAV viral vector is an AAV1 viral vector;(b) a New York Heart Association (NYHA) class of heart failure;(c) an N- terminal prohormone brain natriuretic peptide (NT-proBNP) concentration;(d) an ability to walk a distance in a 6 minute walk test (6MWT);(e) a level of alanine aminotransferase, aspartate aminotransferase, or alkaline phosphatase in a liver function test;(f) a level of bilirubin; and / or(g) a hemoglobin level.

6. The method of any one of claims 1-5, wherein the subject comprises:(a) an anti-AAVl neutralizing antibody inhibition level against an AAV1 viral vector less than or equal to 50% at a serum dilution of 1:4, optionally, wherein the anti-AAV1 neutralizing antibody inhibition level is measured within 125 days prior to the treatment;(b) a cardiomyopathy selected from:(i) a non-ischemic cardiomyopathy, optionally wherein the non¬ ischemic cardiomyopathy lacks a hypertrophic cardiomyopathy, an infiltrative cardiomyopathy or a restrictive cardiomyopathy;(ii) an ischemic cardiomyopathy, optionally wherein the ischemic cardiomyopathy is chronic ischemic cardiomyopathy, optionally wherein the chronic ischemic cardiomyopathy lacks a hypertrophic cardiomyopathy, an infiltrative cardiomyopathy or a restrictive cardiomyopathy, optionally wherein the subject has at least one major coronary vessel with thrombolysis in myocardial infarction (TIMI) grade 3 flow, wherein the at least one major coronary vessel is selected from left anterior descending (LAD), left coronary circumflex (LCx) and right coronary artery (RCA);(iii) a toxic cardiomyopathy or an alcoholic cardiomyopathy, optionally wherein a toxin or alcohol exposure has been eliminated from the subject for at least 6 months prior to the treatment;(iv) a cardiomyopathy due to acute myocarditis, optionally wherein a most recent clinical episode due to the cardiomyopathy due to acute myocarditis is at least 6 months prior to the treatment;(v) a viral cardiomyopathy or a peripartum cardiomyopathy, optionally wherein the subject is diagnosed with the viral cardiomyopathy or peripartum cardiomyopathy at least 6 months prior to the treatment;(c) a diagnosis of New York Heart Association (NYHA) class 111 or class IV heart failure, optionally wherein the diagnosis is at least 60 days prior to the treatment;(d) an N-terminal prohormone brain natriuretic peptide (NT-proBNP) concentration greater than 1200 pg / mL, optionally, greater than 1600 pg / mL;(e) an ability to walk a minimum of 50 meters in a 6 minute walk test (6MWT); (f) received medical therapy for the treatment of chronic heart failure, optionally wherein the medical therapy is selected from an oral diuretic, a beta blocker, ivabradine, hydralazine, aldosterone receptor blocker, and an inhibitor of the renin-angiotensin system selected from an angiotensin-converting enzyme inhibitor, and angiotensin-receptor blocker (ARB) and an angiotensin receptor-neprilysin inhibitor, optionally wherein the the beta blocker is selected from bisoprolol, carvedilol and metoprolol succinate, and / or the ARB is selected from candesartan and valsartan; and / or(g) a cardioverter defibrillator (ICD), optionally wherein the ICD has been implanted in the subject at least 30 days prior to the treatment.

7. The method of any one of claims 1-6, wherein the subject is not characterized by one or more of:(a) a diagnosis of untreated heart failure;(b) an intravenous venous (IV) therapy with positive inotropes, vasodilators or diuretics within 30 days prior to treatment;(c) a restrictive cardiomyopathy, an infiltrative cardiomyopathy, a hypertrophic cardiomyopathy, a pericardial disease, amyloidosis, an uncorrected thyroid disease, or a discrete left ventricular (LV) aneurysm;(d) an untreated left-sided valvular disease, optionally wherein the left-sided valvular disease is selected from moderate or severe mitral stenosis wherein a valve area is less than 1.5 cm2, a severe aortic stenosis with a valve area less than 1.0 cm2, and a moderate-severe (3-4+ / 4+) aortic or mitral regurgitation;(e) a limited ability to perform the 6MWT due to claudication, arthritic or other conditions unrelated to heart failure;(f) cardiac surgery, percutaneous coronary intervention (PCI), valvuloplasty or valve replacement within 30 days prior to treatment;(g) myocardial infarction within 6 months prior to treatment;(h) active angina within 3 months prior to treatment;(i) a prior heart transplantation, left ventricular reduction surgery, or cardiomyoplasty;(j) a passive restraint device such as a CorCap™ Cardiac Support Device, a mechanical circulatory support device (MCSD) or cardiac shunt;(k) a likelihood of receiving cardiac resynchronization therapy, cardiomyoplasty, left ventricular reduction surgery, conventional revascularizationprocedure, MCSD, cardiac transplant or valvular repair within 12 months following treatment;(l) hemodynamic instability requiring hospitalization within 3 months prior to the treatment;(m) hypersensitivity to radiopaque agents used for angiography, and optionally a history of high dose corticosteroid pretreatment prior to contrast angiography;(n) a level of alanine aminotransferase, aspartate aminotransferase, or alkaline phosphatase in a liver function test which is at least 3 times greater than an upper limit of normal (ULN);(o) a total bilirubin greater than two times greater than an upper limit of normal; (p) an intrinsic liver disease, optionally wherein the liver disease is selected from cirrhosis, chronic hepatitis B, and hepatitis C virus infection;(q) a need for hemodialysis within 12 months or a current glomerular filtration rate (GFR) less than or equal to 20 mL / minute / 1.73 m2;(r) bleeding diathesis or thrombocytopenia with a platelet count less than 75,000 platelets / μL; and / or(s) anemia with a hemoglobin level less than 9 g / dL.

8. The method of any one of claims 1-7, wherein the treatment stabilizes and / or provides an improvement in a symptom or measure of the HFrEF in the subject compared to the symptom or measure in an untreated subject for a period following administration of the polynucleotide of at least 1, 2, 3, 6, 9, or 12 months.

9. The method of claim 8, wherein the period is at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 years.

10. The method of any one of claim 8 or 9, wherein the symptom or measure is selected from the group consisting of a change in class of a New York Heart Association (NYHA) class, a score in a Kansas City Cardiomyopathy Questionnaire (KCCQ), a distance in a 6-minute walk test (6MWT), a concentration of N-terminal pro b-type natriuretic peptide (NT-proBNP), a concentration cardiac troponin I, a lack in a recurrent or terminal clinical outcome, and a ventricular function.

11. The method of claim 10, wherein a recurrent or terminal clinical outcome is selected from a hospitalization, intravenous administration of a diuretic, vasodilator, or inotrope, mechanical fluid removal.

12. The method of claim 10 or 11, wherein the stabilization and / or improvement in ventricular function comprises stabilization of or an improvement in a parameter selected from the group consisting of a change from baseline in left ventricular structure and function including left ventricular ejection fraction (LVEF), left ventricular end-diastolic volume (LVEDV), end-systolic volume, stroke volume and / or circumferential strain; regional wall thickness; left ventricular LGE expressed as a percent of left ventricular mass; left ventricular viable mass; number of left ventricular segments with LGE; and composite outcome in change from baseline in LV function (LVESV), optionally wherein the parameter is assessed by late gadolinium enhancement (LGE) cardiac MRI.

13. The method of any one of claims 8-12, further comprising measuring the stabilization and / or improvement in the subject after the period.

14. The method of any one of claims 8-13, wherein the stabilization and / or improvement comprises:(a) stabilization of or a decrease in a NYHA class, optionally wherein the decrease is greater than or equal to 1 class or to 2 classes;(b) stabilization of or an increase in LVEF, optionally wherein the increase is greater than or equal to 5%, 10%, 15%, or 20%;(c) stabilization of or an increase in an KCCQ score, optionally wherein the increase is greater than or equal to 5 points, 10 points, 20 points, 30 points, 40 points, 50 points, 60 points, or 70 points;(d) stabilization of or an increase in a 6MWT distance, optionally wherein the increase is greater than or equal to 30 meters, 40 meters, 50 meters, 60 meters, 70 meters, or 80 meters;(e) stabilization of or a decrease in an NT-proBNP concentration, optionally wherein the decrease is greater than or equal to 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, or is in a range defined by any two of the preceding values; and / or(f) stabilization of or a decrease in a cardiac troponin I concentration, optionally wherein the decrease is greater than or equal to 30%, 35%, 40%, 45%, or 50%, or is in a range defined by any two of the preceding values.

15. The method of claim 14, wherein the measured stabilization and / or improvement comprises at least 1, 2, 3, 4, 5, or 6 parameters selected from the stabilization of or decrease in a NYHA class, the stabilization of or increase in LVEF, the stabilization of or increase in an KCCQ score, the stabilization of or increase in a 6MWT distance, the stabilization of or decrease in an NT-proBNP concentration, and the stabilization of or decrease in a cardiac troponin I concentration.

16. The method of any one of claims 1-15, wherein the SERCA polypeptide comprises a SERCA2a polypeptide.

17. The method of any one of claims 1-16, wherein the polynucleotide comprises a vector,18. The method of claim 17, wherein the vector is selected from an adeno-associated virus (AAV) vector, a lenti virus vector, and a retrovirus vector.

19. The method of claim 18, wherein the vector comprises an AAV vector.

20. The method of claim 19, wherein the AAV vector encodes an AAV or fragment thereof having a serotype selected from any one of AAV serotypes 1-12.

21. The method of claim 20, wherein the AAV vector encodes a capsid from AAV serotype-1.

22. The method of any one of claims 1-21, wherein the polynucleotide comprises a promoter operably linked to the nucleic acid encoding a SERCA polypeptide.

23. The method of claim 22, wherein the promoter comprises a constitutive promoter.

24. The method of claim 22 or 23, wherein the promoter comprises a cytomegalovirus (CMV) promoter.

25. The method of any one of claims 1-25, wherein a viral capsid comprises the polynucleotide.

26. The method of claim 25, wherein the polynucleotide comprises a nucleic acid encoding the viral capsid.

27. The method of any one of claims 1-26, wherein the administering a polynucleotide comprises systemic administration.

28. The method of any one of claims 1-26, wherein the administering a polynucleotide comprises intravenous administration.

29. The method of any one of claims 1-26, wherein the administering a polynucleotide comprises intracoronary infusion.

30. The method of any one of claims 1-29, wherein the administering a polynucleotide consists of a single dose of the polynucleotide.

31. The method of any one of claims 1 -30, wherein the polynucleotide comprises a viral vector, and wherein the administration comprises a dose of the polynucleotide within a range from about 1 X 108viral genome particles (vg) to about 1 X 1015vg.

32. The method of claim 31, wherein the dose is within a range from about 1 X 1013viral genome particles to about 9 X 1013vg.

33. The method of claim 32, wherein the dose is within a range from about 2 X 1013viral genome particles to about 5 X 1013vg.

34. The method of claim 33, wherein the dose is about 3 X 1013vg.

35. The method of claim 33, wherein the dose is about 4.5 X 1013vg.

36. The method of any one of claims 1-35, further comprising administering a vasodilator.

37. The method of claim 36, wherein the vasodilator is administered prior to administering the polynucleotide.

38. The method of claim 36 or 37, wherein the vasodilator is administered concurrently with administering the polynucleotide.

39. The method of any one of claims 36-38, wherein the vasodilator comprises nitroglycerin.

40. The method of any one of claims 1-39, wherein the subject is mammalian.

41. The method of any one of claims 1-40, wherein the subject is human.

42. The method of any one of the preceding claims, wherein the treatment comprises inhibiting, stabilizing, and / or ameliorating HFrEF in the subject.

43. Use of a polynucleotide comprising a nucleic acid encoding a sarcoplasmic / endoplasmic reticulum calcium ATPase (SERCA) polypeptide to treat heartfailure with a reduced ejection fraction (HFrEF) in a subject according to any one of claims 1-42.

44. Use of a polynucleotide comprising a nucleic acid encoding a sarcoplasmic / endoplasmic reticulum calcium ATPase (SERCA) polypeptide in the manufacture of a medicament to treat heart failure with a reduced ejection fraction (HFrEF) in a subject according to any one of claims 1-42.

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