METHODS FOR INTRACELLULAR DELIVERY AND ENHANCED GENE TARGETING

• US 20190330628A1
• Filed: 11/15/2017
• Published: 10/31/2019
• Est. Priority Date: 11/15/2016
• Status: Active Grant

• Current Assignee: City of Hope
• Original Assignee: City of Hope
• Inventors: CASTANOTTO, Daniela, STEIN, Cy, ZHANG, Xiaowei
• Granted Patent: US 11,643,655 B2
• Time in Patent Office: Days
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 31/13 Amines A61K31/04 takes precedence
• CPC Class Codes: A61K 31/137 Arylalkylamines, e.g. amphetamine, epinephrine, salbutamol, ephedrine or methadone
• CPC Class Codes: A61K 31/194 having two or more carboxyl groups, e.g. succinic, maleic or phthalic acid
• CPC Class Codes: A61K 31/20 having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
• CPC Class Codes: A61K 31/201 having one or two double bonds, e.g. oleic, linoleic acids
• CPC Class Codes: A61K 31/7105 Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
• CPC Class Codes: A61K 31/713 Double-stranded nucleic acids or oligonucleotides
• CPC Class Codes: A61K 33/02 Ammonia; Compounds thereof
• CPC Class Codes: A61K 33/36 Arsenic; Compounds thereof
• CPC Class Codes: A61K 33/40 Peroxides
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/3231 having an additional ring, e.g. LNA, ENA
• CPC Class Codes: C12N 2320/31 Combination therapy

Abstract

Disclosed herein are methods and compositions for enhancing gene targeting. The method entails co-administrating to a cell a targeting molecule and a means of enhancing the function of the targeting molecule upon delivery to the cell. The means of enhancing the function of the targeting molecule including one or more of a stressor that induces cellular stress, a proton sponge molecule, and an endosome or lysosome inhibitor. Compositions disclosed include a targeting molecule and one or more of a stressor that induces cellular stress, a proton sponge molecule, and an endosome or lysosome inhibitor.

Claims

1. A method of enhancing gene targeting comprising:

administering a targeting molecule to a cell, wherein the targeting molecule binds a target molecule in the cell; and

administering a stressor to the cell, wherein the stressor induces a cellular stress response;

wherein co-administration of the stressor with the targeting molecule enhances the function of the targeting molecule.

2. The method of claim 1, further comprising administering a proton sponge molecule or an endosome or lysosome inhibitor to the target cell, wherein co-administration of the proton sponge molecule or the endosome or lysosome inhibitor with the targeting molecule and the stressor further enhances the function of the targeting molecule.

3. The method of claim 1 or 2, wherein the targeting molecule includes an oligonucleotide.

4. The method of claim 3, wherein the targeting molecule includes an antisense oligonucleotide (ASO), a splicing switch oligonucleotide (SSO), siRNA, miRNA, or shRNA.

5. The method of claim 1 or 2, wherein the targeting molecule includes a small molecule.

6. The method of any of claims 1-5, wherein the stressor includes arsenic, H₂O₂, glutathione, lipofectamine, or heat shock.

7. The method of any of claims 2-6, wherein the proton sponge molecule includes ammonium, and the endosome or lysosome inhibitor includes Ambroxol (Amb), Cyclohexylamine (CHA) or oleic acid (OA).

8. The method of any of claims 1-7, wherein the cell is a cultured cell.

9. The method of claim 8, wherein the cell is part of a tissue or organ.

10. The method of any of claims 1-9, wherein the target molecule includes a nucleotide sequence.

11. The method of claim 10, wherein the nucleotide sequence is found in the cell nucleus or the cell nucleolus.

12. The method of claim 10, herein the nucleotide sequence is found in the cell cytoplasm.

13. The method of claim 11 or 12, wherein the nucleotide sequence includes an mRNA, ncRNA, piRNA, miRNA, viralRNA, or a promoter sequence.

14. The method of any of claims 1-13, wherein the targeting molecule, the stressor, the proton sponge molecule, or the endosome or lysosome inhibitor is administered as a liquid, solid, vapor or any other formulation, or is administered orally, via injection, absorption, or inhalation.

15. The method of any of claims 1-14, wherein the targeting molecule and the stressor are part of a composition.

16. The method of claim 15, wherein the composition further comprises the proton sponge molecule or the endosome or lysosome inhibitor.

17. A method of enhancing gene targeting comprising:

administering a targeting molecule to a cell, wherein the targeting molecule binds a target molecule in the cell; and

administering a proton sponge molecule or an endosome or lysosome inhibitor to the cell;

wherein co-administration of the proton sponge molecule or the endosome or lysosome inhibitor with the targeting molecule enhances the function of the targeting molecule.

18. The method of claim 17, further comprising administering a stressor to the target cell, wherein co-administration of the stressor with the targeting molecule and the proton sponge molecule or the endosome or lysosome inhibitor further enhances the function of the targeting molecule.

19. The method of claim 17 or 18, wherein the targeting molecule includes an oligonucleotide.

20. The method of claim 19, wherein the targeting molecule includes an antisense oligonucleotide (ASO), a splicing switch oligonucleotide (SSO), siRNA, miRNA, or shRNA.

21. The method of claim 17 or 18, wherein the targeting molecule includes a small molecule.

22. The method of any of claims 17-21, wherein the proton sponge molecule includes ammonium, and the endosome or lysosome inhibitor includes Ambroxol (Amb), Cyclohexylamine (CHA) or oleic acid (OA).

23. The method of any of claims 18-22, wherein the stressor includes arsenic, H₂O₂, glutathione, lipofectamine, or heat shock.

24. The method of any of claims 17-23, wherein the cell is a cultured cell.

25. The method of claim 24, wherein the cell is part of a tissue or organ.

26. The method of any of claims 17-25, wherein the target molecule includes a nucleotide sequence.

27. The method of claim 26, wherein the nucleotide sequence is found in the cell nucleus or the cell nucleolus.

28. The method of claim 26, herein the nucleotide sequence is found in the cell cytoplasm.

29. The method of claim 27 or 28, wherein the nucleotide sequence includes an mRNA, ncRNA, piRNA, miRNA, viralRNA, or a promoter sequence.

30. The method of any of claims 17-29, wherein the targeting molecule, the stressor, the proton sponge molecule, or the endosome or lysosome inhibitor is administered as a liquid, solid, vapor or any other formulation, or is administered orally, via injection, absorption, or inhalation.

31. The method of any of claims 17-30, wherein the targeting molecule and the proton sponge molecule or the endosome or lysosome inhibitor are part of a composition.

32. The method of claim 31, wherein the composition further comprises the stressor.

33. A composition comprising:

a targeting molecule, wherein the targeting molecule binds a target molecule in the cell; and

a stressor, wherein the stressor induces a cellular stress response;

wherein the composition enhances the function of the targeting molecule when administered to a cell.

34. The composition of claim 33, further comprising a proton sponge molecule or an endosome or lysosome inhibitor.

35. The composition of claim 33 or 34, wherein the stressor includes arsenic, H₂O₂, glutathione, lipofectamine, or heat shock.

36. The composition of claim 34 or 35, wherein the proton sponge molecule includes ammonium, and the endosome or lysosome inhibitor includes Ambroxol (Amb), Cyclohexylamine (CHA) or oleic acid (OA).

37. The composition of any of claims 33-36, wherein the targeting molecule includes an antisense oligonucleotide (ASO), a splicing switch oligonucleotide (SSO), siRNA, miRNA, or shRNA.

38. The composition of any of claims 33-37, wherein the composition is administered to a cell in vitro.

39. The composition of claim 38, wherein the cell is a cultured cell.

40. The composition of any of claims 33-37, wherein the composition is administered to a cell in vivo.

41. The composition of claim 40, wherein the cell is part of an organ or tissue.

42. The composition of claim 40 or 41, wherein the targeting molecule, the stressor, the proton sponge molecule, or the endosome or lysosome inhibitor is administered as a liquid, solid, vapor or any other formulation, or is administered orally, via injection, absorption, or inhalation.

43. The composition of any of claims 33-37, the composition is administered to a cell ex vivo.

44. The composition of claim 43, wherein the cell is part of an organ or tissue.

45. The composition of claim 43 or 44, wherein the cell is found in a subjects circulation.

46. A composition comprising:

a targeting molecule, wherein the targeting molecule binds a target molecule in the cell; and

a proton sponge molecule or an endosome or lysosome inhibitor;

wherein the composition enhances the function of the targeting molecule when administered to a cell.

47. The composition of claim 46, further comprising a stressor, wherein the stressor induces a cellular stress response.

48. The composition of claim 46 or 47, wherein the proton sponge molecule includes ammonium, and the endosome or lysosome inhibitor includes Ambroxol (Amb), Cyclohexylamine (CHA) or oleic acid (OA).

49. The composition of claim 47 or 48, wherein the stressor includes arsenic, H₂O₂, glutathione, lipofectamine, or heat shock.

50. The composition of any of claims 46-49, wherein the targeting molecule includes an antisense oligonucleotide (ASO), a splicing switch oligonucleotide (SSO), siRNA, miRNA, or shRNA.

51. The composition of any of claims 46-50, wherein the composition is administered to a cell in vitro.

52. The composition of claim 51, wherein the cell is a cultured cell.

53. The composition of any of claims 46-50, wherein the composition is administered to a cell in vivo.

54. The composition of claim 53, wherein the cell is part of an organ or tissue.

55. The composition of claim 53 or 54, wherein the targeting molecule, the stressor, the proton sponge molecule, or the endosome or lysosome inhibitor is administered as a liquid, solid, vapor or any other formulation, or is administered orally, via injection, absorption, or inhalation.

56. The composition of any of claims 46-50, the composition is administered to a cell ex vivo.

57. The composition of claim 56, wherein the cell is part of an organ or tissue.

58. The composition of claim 56 or 57, wherein the cell is found in a subjects circulation.

59. A method of controlling the cellular localization of an siRNA or an endogenous miRNA, comprising administering a stressor, a proton sponge molecule, or both to a cell, such that the siRNA or the endogenous miRNA translocates from the cytoplasm to the nucleus, thereby increasing the function of the siRNA or the miRNA against its nuclear RNA target, wherein the stressor induces a cellular stress response.

60. The method of claim 59, wherein the stressor includes arsenic, H₂O₂, glutathione, lipofectamine, or heat shock.

61. The method of claim 59, wherein the proton sponge molecule includes ammonium.