Biological pacemakers including mutated hyperpolarization-activated cyclic nucleotide-gated (HCN) channels
A composition for implantation into cardiac tissue includes a biological pacemaker that, when implanted, expresses an effective amount of a mutated hyperpolarization-activated and cyclic nucleotide-gated (HCN) isoform to modify I h when compared with wild-type HCN. Methods for implementing each of the biologocal pacemakers include implanting each of biologocal pacemakers into cardiac tissue.
1. A composition for implantation into cardiac tissue, comprising a biological pacemaker comprising a polynucleotide sequence encoding a mutated hyperpolarization-activated and cyclic nucleotide-gated (HCN) isoform that, when implanted, expresses an effective amount of the mutated HCN isoform to modify I h activation kinetics, wherein the HCN isoform is HCN-4, the mutation comprises a S3-S4 linker mutation, and the mutated HCN-4 demonstrates increased I h activation kinetics compared to wild type HCN-4.
2. The composition according to claim 1 , comprising genetically engineered cells comprising the polynucleotide sequence encoding the mutated HCN-4 isoform.
3. The composition according to claim 1 , wherein the S3-S4 linker mutation is selected from the group consisting of T360A, Δ363-367, T360A+Δ363-367, TRI360-362AGM, TRI360-362KGM, T360A+I362M, T360A+Δ365-367, E365G, E365A, R361G, TR360-361AA, 1362C, I362S, I362T, and TRI360-362AGM+Δ363-367.
4. The composition according to claim 1 , wherein the polynucleotide sequence encoding the mutated HCN-4 isoform further comprises a delivery vector.
5. The composition according to claim 4 , wherein the delivery vector is an adeno-associated virus (AAV) vector.
6. The composition according to claim 1 , wherein the mutated HCN-4 isoform further comprises a C-terminal region truncation.
7. The composition according to claim 6 , wherein the truncation begins 16 amino acid residues past the cyclic nucleotide-binding domain (CNBD).
8. The composition according to claim 6 , wherein the truncated, mutated HCN-4 isoform comprises a cyclic nucleotide-binding domain (CNBD) and is truncated past the CNBD.
9. A method for modifying pacemaker current in cardiac tissue, comprising implanting into heart tissue of a subject in need a composition of claim 1 .
10. A method for modifying pacemaker current in cardiac tissue, the method comprising implanting into heart tissue of a subject in need a composition according to claim 2 .
11. The method according to claim 9 , wherein the S3-S4 linker mutation is selected from the group consisting of T360A, Δ363-367, T360A+Δ363-367, TRI360-362AGM, TRI360-362KGM, T360A+I362M, T360A+Δ365-367, E365G, E365A, R361G, TR360-361AA, I362C, I362S, I362T, and TRI360-362AGM +Δ363-367.
12. The method according to claim 9 , wherein the polynucleotide sequence encoding the mutated HCN-4 isoform further comprises a delivery vector.
13. The method according to claim 12 , wherein the delivery vector comprises an adeno-associated virus (AAV) vector.
14. The method according to claim 9 , wherein the mutated HCN-4 isoform further comprises a C-terminal region truncation.
15. The method according to claim 14 , wherein the truncated HCN-4 isoform includes a cyclic nucleotide-binding domain (CNBD) and is truncated past the CNBD.
16. The method according to claim 15 , wherein the HCN-4 is truncated 16 amino acid residues past the CNBD.
17. A composition for implantation into cardiac tissue, comprising a biologocal pacemaker comprising a polynucleotide sequence encoding a mutated hyperpolarization-activated and cyclic nucleotide-gated (HCN) isoform that, when implanted, expresses an effective amount of the mutated HCN isoform to modify I h activation kinetics, wherein the HCN isoform is HCN-4 and the mutation comprises a S3-S4 linker mutation and wherein the mutated HCN-4 demonstrates increased I h activation kinetics compared to wild type HCN-4 and demonstrates V 1/2 kinetics that are unchanged compared to wild type HCN-4.
18. The composition according to claim 17 , wherein the mutated HCN-4 comprises a S3-S4 linker mutation selected from the group consisting of T360A, T360A+Δ363-367, TRI360-362KGM, and TRI360-362AGM+Δ363-367.
19. A method for modifying pacemaker current in cardiac tissue, comprising implanting into heart tissue of a subject in need a composition according to claim 17 .
20. The composition of claim 17 wherein the mutated HCN-4 further demonstrates deactivation kinetics that are unchanged compared to wild type HCN-4.
21. A composition for implantation into cardiac tissue comprising a polynucleotide sequence encoding a mutated hyperpolarization-activated and cyclic nucleotide-gated-4 (HCN-4) isoform, wherein the mutated HCN-4 comprises a S3-S4 linker mutation selected from the group consisting of T360A, T360A+Δ363-367, TRI360-362KGM, and TRI360-362AGM+Δ363-367.
22. A method for modifying pacemaker current in cardiac tissue, comprising implanting into heart tissue of a subject in need a composition according to claim 21 .
23. The composition according to claim 21 , wherein the mutated HCN-4 comprises the S3-S4 linker mutation T360A+Δ363-367.
24. The composition according to claim 21 , wherein the mutated HCN-4 isoform further comprises a C-terminal region truncation.
25. The composition according to claim 23 , wherein the mutated HCN-4 isoform further comprises a C-terminal region truncation.
26. The composition according to claim 23 , wherein the polynucleotide sequence encoding the mutated HCN-4 isoform further comprises a delivery vector.
27. An isolated polynucleotide sequence that encodes a mutated HCN-4 comprising a S3-S4 linker mutation selected from the group consisting of T360A, T360A+Δ363-367, TRI360-362KGM, and TRI360-362AGM + 66 363-367.
28. The isolated polynucleotide sequence of claim 27 , wherein the mutated HCN-4 further comprises a C-terminal region truncation.
29. A method for modifying pacemaker current in cardiac tissue, the method comprising implanting into heart tissue of a subject in need an isolated polynucleotide sequence of claim 27 .
30. The isolated polynucleotide sequence of claim 27 , wherein the isolated polynucleotide sequence further comprises a delivery vector.
31. The isolated polynucleotide sequence of claim 30 , wherein the delivery vector comprises an adeno-associated virus (AAV) vector.
32. An isolated genetically engineered cell comprising the isolated polynucleotide sequence of claim 27 .
33. The isolated polynucleotide sequence of claim 28 , wherein the truncation begins 16 amino acid residues past the cyclic nucleotide-binding domain (CNBD).
34. The isolated polynucleotide sequence of claim 28 , wherein the truncated, mutated HCN-4 comprises a cyclic nucleotide-binding domain (CNBD) and is truncated past the CNBD.
35. The isolated polynucleotide sequence of claim 28 , wherein the isolated polynucleotide sequence further comprises a delivery vector.
36. The isolated polynucleotide sequence of claim 35 , wherein the delivery vector comprises an adeno-associated virus (AAV) vector.
37. The isolated polynucleotide sequence of claim 33 , wherein the isolated polynucleotide sequence further comprises a delivery vector.
38. The isolated polynucleotide sequence of claim 37 , wherein the delivery vector comprises an adeno-associated virus (AAV) vector.
39. The isolated polynucleotide sequence of claim 34 , wherein the isolated polynucleotide sequence further comprises a delivery vector.
40. The isolated polynucleotide sequence of claim 39 , wherein the delivery vector comprises an adeno-associated virus (AAV) vector.