METHODS AND COMPOSITIONS FOR MODIFYING GENOMIC DNA

• US 20180112235A1
• Filed: 04/13/2016
• Published: 04/26/2018
• Est. Priority Date: 04/13/2015
• Status: Active Grant

• Current Assignee: Maxcyte, Inc. (Entremed, Incorporated)
• Original Assignee: Maxcyte, Inc. (Entremed, Incorporated)
• Inventors: Li, Linhong, PESHWA, Madhusudan
• Granted Patent: US 11,608,511 B2
• Time in Patent Office: Days
• CPC Class Codes: A61K 35/17 Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes when activated by a specific antigen A61K39/00
• CPC Class Codes: A61K 35/33 Fibroblasts
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• 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 15/902 using homologous recombination
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0636 T lymphocytes
• CPC Class Codes: C12N 5/0646 Natural killers cells [NK], NKT cells
• CPC Class Codes: C12N 5/0652 Cells of skeletal and connective tissues; Mesenchyme
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates

Abstract

Compositions and methods concern the sequence modification of an endogenous genomic DNA region. Certain aspects relate to a method for site-specific sequence modification of a target genomic DNA region in cells comprising: contacting the cells with an activating composition; transfecting the cells with a transfection composition comprising (a) donor DNA and (b) a DNA digesting agent; wherein the donor DNA comprises: (i) a homologous region comprising nucleic acid sequence homologous to the target genomic DNA region; and (ii) a sequence modification region; and wherein the genomic DNA sequence is modified specifically at the target genomic DNA region.

Claims

1. A method for site-specific sequence modification of a target genomic DNA region in cells comprising:

contacting the cells with an activating composition;

transfecting the cells with a non-viral transfection composition comprising (a) donor DNA and (b) a DNA digesting agent;

wherein the donor DNA comprises;

(i) a homologous region comprising nucleic acid sequence homologous to the target genomic DNA region; and

(ii) a sequence modification region; and

wherein the genomic DNA sequence is modified specifically at the target genomic DNA region.

2. The method of claim 1, wherein the cells are immune cells.

3. The method of claim 2, wherein the immune cells are T cells.

4. The method of claim 3, wherein the T cells are primary T cells.

5. The method of claim 4, wherein the activating composition comprises anti-CD3 and anti-CD28 antibodies.

6. The method of claim 5, wherein the transfection of the cells comprises flow electroporation of the cells.

7. A method for site-specific sequence modification of a target genomic DNA region in cells comprising:

contacting the cells with an activating composition;

transfecting the cells with a transfection composition comprising (a) donor DNA and (b) a DNA digesting agent;

wherein the donor DNA comprises;

(i) a homologous region comprising nucleic acid sequence homologous to the target genomic DNA region; and

(ii) a sequence modification region; and

wherein the genomic DNA sequence is modified specifically at the target genomic DNA region.

8. The method of claim 7, wherein the transfection of the cells comprises electroporation of the cells.

9. The method of claim 8, wherein the electroporation is flow electroporation using a flow electroporation device.

10. The method of any one of claims 7-9, wherein the cells are transfected at a time period of less than seven days after contacting the cells with the activating composition.

11. The method of claim 10, wherein the cells are transfected at a time period of less than 3 days after contacting the cells with the activating composition.

12. The method of claim 11, wherein the cells are transfected at a time period of 2 days or less after contacting the cells with the activating composition.

13. The method of claim 12, wherein the cells are transfected two days after contacting the cells with the activating composition.

14. The method of any one of claims 7-13, wherein the DNA digesting agent is selected from a TALEN, transposase, integrase and nuclease.

15. The method of any one of claims 7-14, wherein the DNA digesting agent is encoded on one or more RNAs.

16. The method of any one of claims 7-15, wherein the DNA digesting agent is a nuclease.

17. The method of claim 16, wherein the DNA digesting agent is Cas9.

18. The method of any one of claims 7-17, wherein the transfection composition further comprises a guide RNA.

19. The method of claim 16, wherein the nuclease is a site-specific nuclease.

20. The method of any one of claims 7-19, wherein the donor DNA is a plasmid.

21. The method of any one of claims 7-19, wherein the donor DNA comprises a transgene.

22. The method of any one of claims 7-19, wherein the donor DNA is an oligo.

23. The method of any one of claims 7-19, wherein the donor DNA is a single-stranded oligo.

24. The method of any one of claims 7-23, wherein the concentration of the donor DNA in the transfection composition is from about 10 to about 500 μ

g/mL.

25. The method of any one of claims 7-24, wherein the cells are mammalian cells.

26. The method of claim 25, wherein the cells are human cells.

27. The method of claim 25, wherein the cells are fibroblasts.

28. The method of claim 25, wherein the mammalian cells are peripheral blood lymphocytes.

29. The method of claim 25, wherein the mammalian cells are primary T cells.

30. The method of claim 25, wherein the cells are natural killer (NK) cells.

31. The method of claim 29 or 30, wherein the activation composition comprises anti-CD3 and anti-CD28 antibodies.

32. The method of claim 25, wherein the mammalian cells are stem cells.

33. The method of claim 32, wherein the stem cells are hematopoietic stem cells.

34. The method of claim 32, wherein the cells are mesenchymal stem cells.

35. The method of claim 25, wherein the mammalian cells are primary cells.

36. The method of any one of claims 7-35, wherein the target genomic DNA sequence comprises a disease-associated gene.

37. The method of any one of claim 7-36, wherein the donor DNA comprises a disease-associated gene.

38. The method of any one of claims 7-37, wherein the donor DNA comprises a chimeric antigen receptor (CAR).

39. The method of claim 38, wherein the sequence modification region of the donor DNA comprises a CAR.

40. The method of any one of claims 7-36, wherein the donor DNA comprises a homologous region comprising at least 10 nucleic acids of homologous sequence.

41. The method of claim 40, wherein the donor DNA comprises a homologous region comprising at least 20 nucleic acids of homologous sequence.

42. The method of claim 41, wherein the donor DNA comprises a homologous region comprising at least 30 nucleic acids of homologous sequence.

43. The method of any one of claims 7-42, wherein the efficiency of the sequence modification is greater than 0.2%.

44. The method of claim 43, wherein the efficiency of the sequence modification is greater than 1%.

45. The method of claim 44, wherein the efficiency of the sequence modification is greater than 5%.

46. The method of any one of claims 7-45, wherein the cell viability after transfection is at least 30%.

47. The method of claim 46, wherein the cell viability after transfection is at least 40%.

48. The method of claim 47, wherein the cell viability after transfection is at least 50%.

49. The method of any one of claims 7-48, wherein the DNA sequence modification is one or more stop codons.

50. The method of any one of claims 7-48, wherein the sequence modification changes one or more base pairs of the genomic sequence.

51. The method of any one of claims 7-48, wherein the sequence modification adds one or more base pairs of the genomic sequence.

52. The method of any one of claims 7-48, wherein the sequence modification deletes one or more base pairs of the genomic sequence.

53. The method of any one of claims 7-48, wherein the DNA sequence modification is site-specific targeted integration of a transgene.

54. The method of any one of claims 7-53, wherein the transfection composition comprises two or more donor DNAs with different complimentary sequence.

55. The method of claim 54, wherein the transfection composition comprises two or more DNA digesting agents.

56. The method of claim 55, wherein the transfection composition comprises two or more site-specific DNA digesting agents;

wherein the DNA digesting agents are targeted to different genomic sites.

57. The method of any of claims 7-56, further comprising expanding a clonal isolated and selected cell to produce clonal cells having the DNA sequence modification.

58. The method of claim 57, wherein cells are expanded for large scale manufacturing.

59. The method of any of claim 57 or 58, wherein cells are expanded in a volume greater than 1 L.

60. The method of claim 59, wherein cells are expanded in a volume of 3 L or more.

61. The method of any of claims 7-60, wherein the cells are cultured in serum-free media.

62. The method of any of claims 7-61, further comprising screening the cells for the sequence modification.

63. The method of any of claims 7-62, further comprising freezing transfected cells.

64. The method of any of claims 7-63, further comprising expanding transfected cells that were previously frozen.

67. A transfected cell produced using the methods of any one of claims 7-65.

65. A method for producing a stable cell line comprising a genomic DNA sequence modification of a target genomic DNA sequence, the method comprising:

contacting the cells with an activating composition;

transfecting the cells with a transfection composition comprising (a) donor DNA and (b) a DNA digesting agent;

wherein the donor DNA comprises;

(i) a homologous region comprising nucleic acid sequence homologous to the target genomic DNA region; and

(ii) a sequence modification region; and

screening transfected cells for the genomic DNA sequence modification at the target genomic DNA region;

isolating screened transfected cells by limiting dilution to obtain clonal cells;

expanding isolated transfected cells to produce a stable cell line comprising the genomic DNA sequence modification.

66. A cell line produced by the method of claim 65.

68. A method of treating a subject having or suspected of having a disease or condition by administering an effective amount of the transfected cell of claim 67 or the cell line of claim 66.

69. A clinical research method comprising administering an effective amount of the transfected cell of claim 67 or the cell line of claim 66.

70. A method of treating a cancer in a subject in need thereof comprising

contacting cells with an activating composition;

transfecting the cells with a transfection composition comprising (a) donor DNA and (b) a DNA digesting agent;

wherein the donor DNA comprises;

(i) a homologous region comprising nucleic acid sequence homologous to the target genomic DNA region; and

(ii) a chimeric antigen receptor (CAR); and

wherein the genomic DNA sequence is modified specifically at the target genomic DNA region to integrate the CAR; and

administering the cells to the patient.

71. The method of claim 70, wherein the transfection composition is non-viral.

72. The method of claim 70 or 71, wherein the cells are autologous.

73. The method of claim 70 or 72, wherein the cells are T cells or NK cells.

74. The method of any one of claims 70-73, wherein the cancer is leukemia, a B-cell malignancy or acute lymphoblastic leukemia.

75. The method of any one of claims 70-74, wherein the cells are isolated from the blood of the patient.

76. The method of claim 75, wherein the cells are isolated by apheresis.

77. The method of any one of claims 70-76 further comprising isolating cells from the patient.