EP-4739352-A1 - HUMAN THERAPY UTILIZING NOVEL PROKARYOTIC VOLTAGE GATED SODIUM CHANNELS

Abstract

A gene therapy approach is described whereby augmentation of both peak sodium current and calcium transient amplitude in cardiomyocytes effectively alleviates pathologies of heart failure. Prokaryotic sodium channel gene delivery is described as a new therapy for reduced ejection fraction-associated heart failure.

Inventors

• BURSAC, NENAD
• WU, Tianyau

Assignees

• Duke University

Dates

Publication Date

20260513

Application Date

20240705

Claims (1)

1. Atty Docket No.: 1449507-DU8236PCT WHAT IS CLAIMED IS: 1. A method for treating reduced ejection fraction in a subject with heart failure, the method comprising: administering to the subject a recombinant vector comprising a polynucleotide sequence encoding a prokaryotic sodium ion channel (BacNav) polypeptide operably linked to a promoter, wherein the promoter directs expression of the BacNa v polypeptide in cardiomyocytes of the subject. 2. The method of claim 1, wherein the BacNav polypeptide is h2SheP. 3. The method of claim 1 or 2, wherein the vector is a recombinant adeno-associated virus (AAV) vector. 4. The method of claim 3, wherein the recombinant AAV vector serotype is rAAV6, rAAV9, rAAVrh74, cc47, myoAAV2A, myoAAV4A, myoAAV4E, AAV2i8, rAAV.KK04, Anc80L65, rAAVM41, AAV2-THGTPAD, or AAV2-NLPGSGD. 5. The method of any one of claims 1-4, wherein the promoter is a cardiomyocyte-specific promoter. 6. The method of claim 5, wherein the cardiomyocyte-specific promoter is α-myosin heavy chain (α-MHC) promoter, myosin light chain (MLC2v) promoter, cardiac troponin T (cTnT) promoter, or atrial natriuretic factor (ANF, atrial-specific) promoter. 7. The method of any one of claims 1-4, wherein the promoter is a chimeric muscle-specific promoter. 8. The method of claim 7, wherein the chimeric muscle-specific promoter is MHCK7, CK8, or SPc5-12. 55 30155659V.1 Atty Docket No.: 1449507-DU8236PCT 9. The method of any one of claims 1-8, wherein the administering to the subject is by intracoronary injection or direct injection into cardiac muscle walls. 10. The method of any one of claims 1-8, wherein the administering to the subject is by intravenous injection. 11. The method of any one of claims 3-10, wherein anti-AAV antibodies have been reduced in the subject’s blood prior to administering the vector. 12. The method of any one of claims 1-11, wherein the subject does not have a voltage gated ion channel-related condition. 13. The method of any one of claims 1-12, wherein the subject is a mammal. 14. The method of claim 13, wherein the mammal is a human. 15. The method of any one of claims 1-14, wherein the subject is in stage I heart failure, stage II heart failure, stage III heart failure, or stage IV heart failure. 16. A method for improving both contractile and electrical dysfunction in a subject with heart failure, the method comprising: administering to the subject a recombinant vector comprising a polynucleotide sequence encoding a prokaryotic sodium ion channel (BacNa v ) polypeptide operably linked to a promoter, wherein the promoter directs expression of the BacNav polypeptide in cardiomyocytes of the subject. 17. A method for increasing subsarcolemmal Ca 2+ levels in cardiomyocytes of a subject with heart failure, the method comprising: administering to the subject a recombinant vector comprising a polynucleotide sequence encoding a prokaryotic sodium ion channel (BacNa v ) polypeptide operably 56 30155659V.1 Atty Docket No.: 1449507-DU8236PCT linked to a promoter, wherein the promoter directs expression of the BacNav polypeptide in cardiomyocytes of the subject. 18. A method for inhibiting the Na + /Ca 2+ exchanger (NCX) in cardiomyocytes of a subject with heart failure, the method comprising: administering to the subject a recombinant vector comprising a polynucleotide sequence encoding a prokaryotic sodium ion channel (BacNa v ) polypeptide operably linked to a promoter, wherein the promoter directs expression of the BacNav polypeptide in cardiomyocytes of the subject. 19. A method for reducing the incidence of arrythmia in a subject with heart failure, the method comprising: administering to the subject a recombinant vector comprising a polynucleotide sequence encoding a prokaryotic sodium ion channel (BacNav) polypeptide operably linked to a promoter, wherein the promoter directs expression of the BacNav polypeptide in cardiomyocytes of the subject. 20. A method for reversing reduced ejection fraction in a subject with heart failure, the method comprising: administering to the subject a recombinant vector comprising a polynucleotide sequence encoding a prokaryotic sodium ion channel (BacNav) polypeptide operably linked to a promoter, wherein the promoter directs expression of the BacNa v polypeptide in cardiomyocytes of the subject. 21. A method for augmenting peak Na+ current and Ca2+ transient amplitude in cardiomyocytes of a subject with heart failure, the method comprising: administering to the subject a recombinant vector comprising a polynucleotide sequence encoding a prokaryotic sodium ion channel (BacNav) polypeptide operably linked to a promoter, wherein the promoter directs expression of the BacNa v polypeptide in cardiomyocytes of the subject. 57 30155659V.1 Atty Docket No.: 1449507-DU8236PCT 22. The method of any one of claims 16-21, wherein the BacNav polypeptide is h2SheP. 23. The method of claim 22, wherein the vector is a recombinant adeno-associated virus (AAV) vector. 24. The method of claim 23, wherein the recombinant AAV vector serotype is rAAV6, rAAV9, rAAVrh74, cc47, myoAAV2A, myoAAV4A, myoAAV4E, AAV2i8, rAAV.KK04, Anc80L65, rAAVM41, AAV2-THGTPAD, or AAV2-NLPGSGD. 25. The method of any one of claims 16-24, wherein the promoter is a cardiomyocyte- specific promoter. 26. The method of claim 25, wherein the cardiomyocyte-specific promoter is α-myosin heavy chain (α-MHC) promoter, myosin light chain (MLC2v) promoter, cardiac troponin T (cTnT) promoter, or atrial natriuretic factor (ANF, atrial-specific) promoter. 27. The method of any one of claims 16-24, wherein the promoter is a chimeric muscle- specific promoter. 28. The method of claim 27, wherein the chimeric muscle-specific promoter is MHCK7, CK8, or SPc5-12. 29. The method of any one of claims 16-28, wherein the administering to the subject is by intracoronary injection or direct injection into cardiac muscle walls. 30. The method of any one of claims 16-28, wherein the administering to the subject is by intravenous injection. 31. The method of any one of claims 23-30, wherein anti-AAV antibodies have been reduced in the subject’s blood prior to administering the vector. 58 30155659V.1 Atty Docket No.: 1449507-DU8236PCT 32. The method of any one of claims 16-31, wherein the subject does not have a voltage gated ion channel-related condition. 33. The method of any one of claims 16-32, wherein the subject is a mammal. 34. The method of claim 33, wherein the mammal is a human. 35. The method of any one of claims 16-34, wherein the subject is in stage I heart failure, stage II heart failure, stage III heart failure, or stage IV heart failure. 36. A recombinant vector comprising a first polynucleotide sequence encoding a prokaryotic sodium ion channel (BacNaV) polypeptide and a second polynucleotide sequence encoding SERCA2a, I-1C, SUMO-1, or BAG3, wherein the first and the second polynucleotides are operably linked to a promoter. 37. The recombinant vector of claim 36, wherein the vector is a recombinant adeno- associated virus (AAV) vector. 38. The recombinant AAV vector of claim 37, wherein the vector serotype is rAAV6, rAAV9, rAAVrh74, cc47, myoAAV2A, myoAAV4A, myoAAV4E, AAV2i8, rAAV.KK04, Anc80L65, rAAVM41, AAV2-THGTPAD, or AAV2-NLPGSGD. 39. The vector of any one of claims 36-38, wherein the first polynucleotide sequence is upstream of the second polynucleotide sequence. 40. The vector of any one of claims 36-38, wherein the second polynucleotide sequence is upstream of the first polynucleotide sequence. 41. The vector of any one of claims 36-40, wherein the first polynucleotide sequence and the second polynucleotide sequence are operably linked to the same promoter. 59 30155659V.1 Atty Docket No.: 1449507-DU8236PCT 42. The vector of any one of claims 36-40, wherein the first polynucleotide sequence and the second polynucleotide sequence are each operably linked to a different promoter. 43. A method for treating reduced ejection fraction in a subject with heart failure, the method comprising: administering to the subject a recombinant vector of any one of claims 36-42, wherein the promoter directs expression of the BacNa v polypeptide in cardiomyocytes of the subject. 44. A method for improving both contractile and electrical dysfunction in a subject with heart failure, increasing subsarcolemmal Ca 2+ levels in cardiomyocytes of a subject with heart failure, inhibiting the Na + /Ca 2+ exchanger (NCX) in cardiomyocytes of a subject with heart failure, reducing the incidence of arrythmia in a subject with heart failure, reversing reduced ejection fraction in a subject with heart failure, or augmenting peak Na+ current and Ca2+ transient amplitude in cardiomyocytes of a subject with heart failure, the method comprising: administering to the subject a recombinant vector of any one of claims 36-42, wherein the promoter directs expression of the BacNav polypeptide in cardiomyocytes of the subject. 45. The method of claim 43 or 44, wherein the administering to the subject is by intracoronary injection or direct injection into cardiac muscle walls. 46. The method of any one of claims 43-45, wherein the administering to the subject is by intravenous injection. 47. The method of any one of claims 43-46, wherein anti-AAV antibodies have been reduced in the subject’s blood prior to administering the vector. 48. The method of any one of claims 43-47, wherein the subject does not have a voltage gated ion channel-related condition. 60 30155659V.1 Atty Docket No.: 1449507-DU8236PCT 49. The method of any one of claims 43-48, wherein the subject is a mammal. 50. The method of claim 49, wherein the mammal is a human. 51. The method of any one of claims 43-50, wherein the subject is in stage I heart failure, stage II heart failure, stage III heart failure, or stage IV heart failure. 61 30155659V.1

Description

Atty Docket No.: 1449507-DU8236PCT HUMAN THERAPY UTILIZING NOVEL PROKARYOTIC VOLTAGE GATED SODIUM CHANNELS CROSS-REFERENCES TO RELATED APPLICATIONS [0001] The present application claims priority to U.S. Provisional Application No.63/512,038 filed July 5, 2023, the full disclosure of which is incorporated by reference in its entirety for all purposes. STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT [0002] This invention was made with government support under Grant No. RO1EB032726 awarded by the National Institutes of Health. The government has certain rights in the invention. REFERENCE TO SEQUENCE LISTING [0003] The contents of the electronic sequence listing (1449507-DU8236PCT-SL; Size: 56.0 kilobytes; and Date of Creation: July 5, 2024) is herein incorporated by reference in its entirety. BACKGROUND [0004] In cardiac muscle cells sodium channels are primarily responsible for generating the rapid upstroke of the action potential (AP). In this manner sodium channels are essential to the initiation and conduction of electrical signals, and the proper function of sodium channels is therefore necessary for normal function of the heart. Reduced sodium current density and slow action potential conduction can arise from altered extracellular environment, cell morphology, or channel regulation that occur in conditions such as myocardial infarction, heart failure, and cardiac ischemia. In particular, these conditions lead to damage of cardiac tissue and the development of fibrosis, characterized by excessive fibroblast proliferation. Increased amounts of fibroblasts can separate cardiac muscle cells causing slow or discontinuous conduction. Further, genetic mutations that result in loss of function in voltage-gated sodium channels (VGSCs) can cause reduced tissue excitability, leading to various cardiac disorders. 1 30155659V.1 Atty Docket No.: 1449507-DU8236PCT [0005] Heart failure (HF) is a condition or a collection of symptoms that weaken or stiffen the heart. HF constitutes a tremendous health and socioeconomic burden (Tsao, 2023), affecting ~2% of the adult population worldwide and over 6 million people in the United States (Tsao, 2023; Metra, 2017). Both acute and chronic ischemic insults to the heart cause irreversible cardiomyocyte (CM) loss and fibrotic remodeling that impair not only cardiac contraction but also action potential (AP) conduction, often leading to sudden cardiac death (SCD) (Tsao, 2023). Despite considerable progress, current therapies for HF and SCD are limited, with a 5-year patient mortality rate surpassing 50% (Tsao, 2023; Marijon, 2022; Cleland, 1998). Congestive heart failure (CHF is a chronic progressive condition that affects the pumping power of the heart muscle. CHF specifically refers to the stage in which fluid builds up in the heart and causes it to pump inefficiently. Without sufficient blood flow, all major body functions are disrupted. [0006] The growing understanding of the complex molecular mechanisms underlying cardiac contractile and electrical dysfunction makes gene therapy a promising strategy to mitigate the high mortality of HF patients (Kieserman, 2019; Argiro, 2024; Mundisugih, 2024). Several gene therapy approaches have been proposed to rescue electrical abnormalities (Sasano, 2006; Greener, 2012) or restore Ca2+-handling deficits (Greenberg, 2016; Fish, 2013; Tilemann, 2013) associated with HF. Recently, these efforts have been expanded by using precise genome editing tools to prevent the development of acquired or genetic cardiomyopathies (Lebek, 2023; Chai, 2023; Reichart, 2023). However, there are currently no approaches that directly and stably increase both peak Na+ current and Ca2+ transient amplitude in CMs, which could simultaneously provide antiarrhythmic (via faster AP conduction) and inotropic (via stronger contraction) benefits to the failing heart. therapies for cardiac conditions could greatly benefit from approaches that enhance electrical excitability and AP conduction in the heart via delivery of functional VGSCs. However, gene-based therapies involving VGSCs are largely hampered by the inability to stably express mammalian channels using viral delivery methods as the genes encoding the VGSCs are too large (>6 kb) to be efficiently incorporated into viral vectors. SUMMARY [0007] The Summary is provided to introduce a selection of concepts that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used as an aid in limiting the scope of the claimed subject matter. 2 30155659V.1 Atty Docket No.: 1449507-DU8236PCT [0008] The present disclosure is based, in part, on the discovery by the inventors that expression of prokaryotic voltage-gated sodium channels (BacNav) in mammalian CMs can simultaneously target sodium and calcium dysregulation in failing CMs and provide robust inot