Targeted Gene Therapies for Pain and Other Neuro-Related Disorders

• US 20200131537A1
• Filed: 03/20/2018
• Published: 04/30/2020
• Est. Priority Date: 03/20/2017
• Status: Active Grant

• Current Assignee: University of Pittsburgh of The Commonwealth System of Higher Education
• Original Assignee: University of Pittsburgh of The Commonwealth System of Higher Education
• Inventors: Seal, Rebecca, Arokiaraj, Cynthia Mary
• Granted Patent: US 11,713,470 B2
• Time in Patent Office: Days
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61K 47/6425 the peptide or protein in the drug conjugate being a receptor, e.g. CD4, a cell surface antigen, i.e. not a peptide ligand targeting the antigen, or a cell surface determinant, i.e. a part of the surface of a cell
• CPC Class Codes: A61K 48/0075 characterised by an aspect of the delivery route, e.g. oral, subcutaneous
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12N 2750/14143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
• CPC Class Codes: C12N 2830/008 cell type or tissue specific enhancer/promoter combination

Abstract

Provided herein are nucleic acids for expressing modified ligand-gated ion channel proteins in excitable cells or secretory cells, such as nerves and neurons and optionally including viral sequences, such as Adeno-associated virus sequences, for delivery to excitable cells or secretory cells of a patient. Also provided herein are methods of modulating cell membrane potentials in an excitable cell or secretory cell, and for treatment of a disease or disorder associated with the nervous system in a patient, such as chronic pain or itch.

Claims

1. A nucleic acid comprising a gene for expressing a modified ligand-gated ion channel, comprising an open reading frame encoding a modified ligand-gated ion channel under transcriptional control of transcriptional control elements governing cell-specific expression in CNS neurons, such as dorsal horn neurons, spinal cord cells, or brain cells, or in inhibitory neurons or nerve cells, such as a CCK promoter, a Tac1 promoter, an NTS promoter, an NMU promoter, a Calb1 promoter, an SST promoter, a GRPR promoter, a parvalbumin promoter, a Gal promoter, an NPY promoter, a PKCγ

promoter, or a Calb2 promoter, wherein the modified ligand-gated ion channel comprises a modified ligand binding domain activatable by an exogenous ligand, and optionally selective to the exogenous ligand, and an ion pore domain.

5. The nucleic acid of claim 1, wherein the transcription control element comprises a human promoter sequence.

6. The nucleic acid of claim 5, wherein the transcription control element is a promoter having at least 75 percent sequence identity to a sequence set forth in SEQ ID NOs:

1-17.

7. The nucleic acid of claim 1, wherein the modified ligand binding domain is a modified α

7 nicotinic acetylcholine ligand binding domain.

8. The nucleic acid of claim 7, wherein the modified α

7 nicotinic acetylcholine ligand binding domain comprises a sequence having at least 75 percent sequence identity to a sequence set forth in SEQ ID NO;

18, SEQ ID NO;

19, or SEQ ID NO;

20.

9. The nucleic acid of claim 7, wherein the modified α

7 nicotinic acetylcholine ligand binding domain comprises an amino acid substitution at one or more of amino acid residues 77, 79, 115, 131, 139, 141, 175, 210, 216, 217, or 219 of SEQ ID NO;

18, SEQ ID NO;

19, or SEQ ID NO;

20, such as Q79A, Q79G, L141A, L141F, L141P, W77F, W77Y, or W77M.

11. The nucleic acid of claim 7, wherein the modified α

7 nicotinic acetylcholine ligand binding domain comprises;

a. a L131G amino acid substitution, a Q139L amino acid substitution, and a Y217F amino acid substitution;

b. a W77F amino acid substitution, a Q79G amino acid substitution, and a G175K amino acid substitution;

c. a Q79G amino acid substitution, a Y115F amino acid substitution, and a G175K amino acid substitution;

d. a Y115F amino acid substitution and a G175K amino acid substitution;

e. a Q79G amino acid substitution and a 216I amino acid substitution;

or

f. a R27D amino acid substitution and/or a E41R amino acid substitution.

13. The nucleic acid of claim 7, wherein the modified α

7 nicotinic acetylcholine ligand binding domain has reduced binding with endogenous acetylcholine (ACh) as compared to unmodified α

7-nAChR LBD.

14. The nucleic acid of claim 1, wherein the ion pore domain is anion-selective, or cation-selective, and optionally is an ion pore domain of an ionotropic nicotinic acetylcholine receptor, an ionotropic serotonin receptor, an ionotropic glycine receptor, an ionotropic GABA receptor, a serotonin 3 receptor (5HT3) ion pore domain, a glycine receptor (GlyR) ion pore domain, a gramma-aminobutyric acid (GABA) receptor ion pore domain, or an α

7 nicotinic acetylcholine receptor.

16. The nucleic acid of claim 14, wherein the ion pore domain is a GlyR ion pore domain comprising an amino acid substitution at residue 298, such as an A298G amino acid substitution.

18. The nucleic acid of claim 1, wherein the exogenous ligand is selected from the group consisting of a quinuclidine, a tropane, a 9-azabicyclo[3.3.1]nonane, a 6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine, and a 1,4-diazabicyclo[3.2.2]nonane.

19. The nucleic acid of claim 1, comprising a sequence of a packageable viral genome comprising the gene for expressing a modified ligand-gated ion channel.

20. The nucleic acid of claim 19, comprising Adeno-associated virus ITR sequences flanking the gene for expressing a modified ligand-gated ion channel, producing a sequence of a packageable recombinant AAV genome or a self-complementary AAV genome comprising the gene for expressing a modified ligand-gated ion channel.

40. A method of treating a disease or disorder associated with the nervous system in a patient comprising:

delivering a nucleic acid according to claim 1 to the patient, and administering the exogenous ligand to the patient in an amount effective to activate the modified ligand gated ion channel in a patient thereby treating the disease or disorder associated with the nervous system in the patient.

2. (canceled)

3. (canceled)

4. (canceled)

10. (canceled)

12. (canceled)

15. (canceled)

17. (canceled)

21. A method of modulating the membrane potential of an excitable cell or a secretory cell, comprising expressing in the cell a genetic construct comprising a gene for expressing a modified ligand-gated ion channel, comprising an open reading frame encoding a modified ligand-gated ion channel under transcriptional control of transcriptional control elements governing cell-specific expression in CNS neurons, such as dorsal horn neurons, spinal cord cells, or brain cells, or in inhibitory neurons or nerve cells, such as a CCK promoter, a Tac1 promoter, an NTS promoter, an NMU promoter, a Calb1 promoter, an SST promoter, a GRPR promoter, a parvalbumin promoter, a Gal promoter, an NPY promoter, a PKCγ

promoter, or Calb2 promoter and a modified ligand-gated ion channel comprising a modified ligand binding domain activatable by an exogenous ligand, and optionally selective to the exogenous ligand, and an ion pore domain, and contacting the cell with an amount of the exogenous ligand effective to activate the modified ligand gated ion channel thereby modulating the membrane potential of the cell.

25. The method of claim 21, wherein the transcription control element comprises a human promoter sequence.

26. The method of claim 21, wherein the transcription control element is a promoter having at least 75 percent sequence identity to a sequence set forth in SEQ ID NOs:

1-17.

27. The method of any one of claim 21, wherein the modified ligand binding domain is a modified α

7 nicotinic acetylcholine ligand binding domain.

28. The method of claim 27, wherein the modified α

7 nicotinic acetylcholine ligand binding domain comprises a sequence having at least 75 percent sequence identity to a sequence of SEQ ID NO;

18, SEQ ID NO;

19, or SEQ ID NO;

20, wherein the modified α

7 nicotinic acetylcholine ligand binding domain optionally comprises an amino acid substitution at one ore more of amino acid residues 77, 79, 115, 131, 139, 141, 175, 210, 216, 217, or 219 of SEQ ID NO;

18, SEQ ID NO;

19, or SEQ ID NO;

20, such as a Q79A, Q79G, L141A, L141F, L141P, W77F, W77Y, or W77M substitution.

31. The method of claim 27, wherein the modified α

7 nicotinic acetylcholine ligand binding domain comprises, with reference to a sequence of SEQ ID NO;

18, SEQ ID NO;

19, or SEQ ID NO;

20;

a. a L131G amino acid substitution, a Q139L amino acid substitution, and a Y217F amino acid substitution;

b. a W77F amino acid substitution, a Q79G amino acid substitution, and a G175K amino acid substitution;

c. a Q79G amino acid substitution, a Y115F amino acid substitution, and a G175K amino acid substitution;

d. a Y115F amino acid substitution and a G175K amino acid substitution;

e. a Q79G amino acid substitution and a 216I amino acid substitution;

or

f. a R27D amino acid substitution and/or a E41R amino acid substitution.

33. The method of claim 27, wherein the modified α

7 nicotinic acetylcholine ligand binding domain has reduced binding with endogenous acetylcholine (ACh) as compared to unmodified α

7-nAChR LBD.

34. The method of claim 21, wherein the ion pore domain is an ion pore domain of an ionotropic nicotinic acetylcholine receptor, an ionotropic serotonin receptor, an ionotropic glycine receptor, an ionotropic GABA receptor, a serotonin 3 receptor (5HT3) ion pore domain, a glycine receptor (GlyR) ion pore domain, a gamma-aminobutyric acid (GABA) receptor ion pore domain, or an α

7 nicotinic acetylcholine receptor.

38. The method of claim 21, wherein the exogenous ligand-gated ion channel ligand is selected from the group consisting of a quinuclidine, a tropane, a 9-azabicyclo[3.3.1]nonane, a 6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine, and a 1,4-diazabicyclo[3.2.2]nonane.

39. The method of claim 21, wherein the genetic construct comprises a sequence of a packageable viral genome comprising the gene for expressing a modified ligand-gated ion channel.

22. (canceled)

23. (canceled)

24. (canceled)

29. (canceled)

30. (canceled)

32. (canceled)

35. (canceled)

36. The method of claim 35, wherein the ion pore domain is a GlyR ion pore domain comprising an amino acid substitution at residue 298, such as an A298G amino acid substitution.

37. (canceled)

41. (canceled)

42. (canceled)

43. (canceled)

44. (canceled)

45. (canceled)