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1. WO2016154579 - CRISPR/CAS-MEDIATED GENE CONVERSION

Note: Text based on automatic Optical Character Recognition processes. Please use the PDF version for legal matters

[ EN ]

CLAIMS

What is claimed is:

1. A method of modifying an endogenous target gene in a cell, the method comprising:

contacting the cell with a first gRNA molecule, a first enzymatically active Cas9

(eaCas9) molecule, a second gRNA molecule, and a second eaCas9 molecule;

wherein the first gRNA molecule and the first eaCas9 molecule associate with the target gene and generate a first single strand cleavage event on a first strand of the target gene;

wherein the second gRNA molecule and the second eaCas9 molecule associate with the target gene and generate a second single strand cleavage event on a second strand of the target gene, thereby forming a double strand break having a first overhang and a second overhang; and

wherein the first overhang and the second overhang in the target gene are repaired using an endogenous homologous region, thereby modifying the endogenous target gene in the cell.

2. The method of claim 1, wherein, after repair of the first overhang and the second overhang, the target gene comprises the sequence of the endogenous homologous region.

3. The method of any one of claims 1-2, wherein the cell is not contacted with an exogenous nucleic acid homologous to the target gene.

4. The method of any one of claims 1-3, wherein the first overhang and the second overhang are repaired by homology directed repair (HDR).

5. The method of claim 4, wherein the HDR is gene conversion.

6. The method of any one of claims 1-5, wherein the first overhang is a 5' overhang, and the second overhang is a 5' overhang.

7. The method of claim 6, wherein the first overhang and the second overhang undergo processing, exposing a first 3' overhang and a second 3' overhang.

8. The method of any one of claims 1-7, wherein the method is used to correct a mutation in the endogenous target gene, and wherein the mutation is located between the first single strand break and the second single strand break.

9. The method of any one of claims 1-8, wherein the method is used to correct a mutation in the endogenous target gene, and wherein wherein the mutation is located within fewer than 50 nucleotides of the first single strand break, or within fewer than 50 nucleotides of the second single strand break.

10. The method of any one of claims 1-9, wherein the endogenous target gene has at least 80% sequence homology with the endogenous homologous region.

11. The method any of claims 1-10, wherein the method is used to correct a mutation in the endogenous target gene, and wherein the endogenous homologous region comprises a domain which is homologous to a domain in the endogenous target gene but which does not comprise the mutation.

12. The method of any one of claims 1-11, wherein the endogenous homologous region comprises at least one of: a control region which is homologous to a control region of the target gene; a coding region which is homologous to a coding region of the target gene; a non-coding region which is homologous to a non-coding region of the target gene; an intron which is homologous to an intron of the target gene; or an exon which is homologous to an exon of the target gene.

13. The method of any one of claims 1-12, wherein the first eaCas9 molecule is a first nickase molecule and the second eaCas9 molecule is a second nickase molecule.

14. The method of claim 13, wherein each nickase molecule forms a single strand break in the target gene.

15. The method of any one of claims 1-14, wherein the first eaCas9 molecule and the second eaCas9 molecule are the same species of eaCas9 molecule.

16. The method of claim 15, wherein the eaCas9 molecule comprises HNH-like domain cleavage activity but has no N-terminal RuvC-like domain cleavage activity.

17. The method of claim 15, wherein the eaCas9 molecule is an HNH-like domain nickase.

18. The method of any one of claims 15-17, wherein the eaCas9 molecule comprises a mutation at an amino acid position corresponding to amino acid position D10 of Streptococcus pyogenes Cas9.

19. The method of any one of claims 1-18, wherein the target gene is an HBB gene.

20. The method of claim 19, wherein the first gRNA molecule is a gRNA molecule comprising SEQ ID NO:387, and wherein the second gRNA molecule is a gRNA molecule comprising SEQ ID NO: 16318.

21. The method of claim 19, wherein the first gRNA molecule is a gRNA molecule comprising any one of SEQ ID NOs: 387-485, 6803-6871, or 16010-16256, and wherein the second gRNA molecule is a gRNA molecule comprising any one of SEQ ID NOs: 387-485, 6803-6871, or 16010-16256.

22. The method of claim 19, wherein the cell is in, or is taken from, a patient having sickle cell disease or beta thalassemia.

23. The method of claim 19, wherein the endogenous homologous region comprises a region of an endogenous HDB gene.

24. The method of any one of claims 1-18, wherein the target gene is an SMN1 gene.

25. The method of claim 24, wherein the cell is in, or is taken from, a patient having spinal muscular atrophy.

26. The method of claim 24, wherein the endogenous homologous region includes a region of an endogenous SMN2 gene.

27. The method of any one of claims 1-18, wherein the target gene is an NCF1 (p47-PHOX) gene.

28. The method of claim 27, wherein the cell is in, or is taken from, a patient having chronic granulomatous disease.

29. The method of claim 27, wherein the endogenous homologous region includes a region of a p47-PHOX pseudogene.

30. The method of any one of claims 1-29, wherein the cell is a population of cells, and wherein the first overhang and the second overhang in the target gene are repaired by gene conversion in about 12% to about 45% of the cells in the population of cells.

31. The method of any one of claims 1-29, wherein the cell is a population of cells, and wherein the first overhang and the second overhang in the target gene are repaired by non-homologous end joining (NHEJ) in less than 40% of the cells in the population of cells.

32. The method of claim 31, wherein the NHEJ results in a deletion in the target gene.

33. The method of claim 31, wherein the NHEJ results in an insertion of the target gene.

34. The method of any one of claims 1-33, wherein the cell is selected from the group consisting of a blood cell, a neuronal cell, an immune cell, a muscle cell, a stem cell, a progenitor cell, and a diseased cell.

35. The method of any one of claims 1-34, wherein the cell is a mammalian cell.

36. The method of any one of claims 1-35, wherein the cell is a human cell.

37. A cell altered by the method of any one of claims 1-36.

38. A pharmaceutical composition comprising the cell of claim 37.

39. A composition, comprising.

a first non-naturally occurring gRNA molecule;

a first non-naturally occurring enzymatically active Cas9 (eaCas9) molecule a second non-naturally occurring gRNA molecule; and

a second non-naturally occurring eaCas9 molecule;

wherein the first gRNA molecule and the first eaCas9 molecule are designed to associate with a target gene and generate a first single strand cleavage event on a first strand of the target gene;

wherein the second gRNA molecule and the second eaCas9 molecule are designed to associate with the target gene and generate a second single strand cleavage event on a second strand of the target gene, thereby forming a double strand break in the target gene having a first overhang and a second overhang; and

wherein the first gRNA molecule, the first eaCas9 molecule, the second gRNA molecule and the second eaCas9 molecule are designed such that the first overhang and the second overhang in the target gene are repaired by gene conversion using an endogenous homologous region.

40. The composition of claim 39, wherein the first non-naturally occurring eaCas9 molecule is an HNH-like domain nickase, and wherein the second non-naturally occurring eaCas9 molecule is an HNH-like domain nickase.

41. The composition of claim 39 or claim 40, for use as a medicament.

42. The composition of claim 39 or claim 40, for use in the treatment of sickle cell disease or beta thalassemia.

43. The composition of claim 39 or claim 40, for use in the treatment of spinal muscular atrophy.

44. The composition of claim 39 or claim 40, for use in the treatment of chronic granulomatous disease.

45. A method of modifying a target region of a target gene in a mammalian cell, the method comprising:

generating, within the cell, a first single strand break on a first strand of the target gene and second single strand break on a second strand of the target gene, thereby forming a double strand break in the target gene having a first 5' overhang and a second 5' overhang;

wherein the target region of the target gene is located

(a) between the first single strand break and the second single strand break,

(b) within fewer than 50 nucleotides of the first single strand break, or

(c) within fewer than 50 nucleotides of the second single strand break;

wherein the double strand break is repaired by gene conversion,

thereby modifying the target region of the target gene in the mammalian cell.

46. The method of claim 45, wherein the step of generating the first single strand break and the second single strand break comprises contacting the cell with at least one eaCas9 molecule, a first gRNA molecule, and a second gRNA molecule.

47. The method of claim 45, wherein the first gRNA molecule and the at least one eaCas9 molecule associate with the target gene and generate the first single strand break, and wherein the second gRNA molecule and the at least one eaCas9 molecule associate with the target gene and generate the second single strand break.

48. The method of any one of claims 45-47, wherein the double strand break is repaired by gene conversion using an endogenous homologous region.

49. The method of claim 48, wherein the endogenous homologous region is located within a gene cluster comprising the target gene.

50. The method of claim 48 or claim 49, wherein the target gene is an HBB gene, and wherein the endogenous homologous region is a region of an HBD gene.

51. The method of claim 50, wherein the first gRNA molecule comprises a targeting sequence comprising SEQ ID NO:387 and the second gRNA molecule comprises a targeting sequence comprising SEQ ID NO: 16318.

52. The method of claim 47, wherein the at least one eaCas9 molecule is at least one eaCas9 nickase molecule.

53. The method of claim 52, wherein the at least one eaCas9 nickase molecule is at least one HNH-type nickase molecule.

54. A method of increasing the percentage of cells in a population of cells that modify a target region of a target gene by gene conversion using an endogenous homologous region, the method comprising

contacting the population of cells with a first gRNA molecule, a first enzymatically active Cas9 (eaCas9) molecule, a second gRNA molecule, and a second eaCas9 molecule; wherein the first gRNA molecule and the first eaCas9 molecule associate with the target gene and generate a first single strand cleavage event on a first strand of the target gene;

wherein the second gRNA molecule and the second eaCas9 molecule associate with the target gene and generate a second single strand cleavage event on a second strand of the target gene, thereby forming a double strand break in the target gene having a first 5' overhang and a second 5' overhang; and

wherein the first 5' overhang and the second 5' overhang in the target gene are repaired by gene conversion using the endogenous homologous region,

thereby increasing the percentage of cells in the population of cells that modify the target region of the target gene by gene conversion using the endogenous homologous region.

55. The method of claim 54, wherein the first 5' overhang and the second 5' overhang in the target gene are repaired by gene conversion in about 12% to about 45% of the cells in the population of cells.

56. The method of claim 54, wherein the percentage of cells in the population of cells that modify the target region of the target gene by gene conversion using the endogenous homologous region is increased, as compared to a percentage of cells in a population of cells which would modify the target region of the target gene by gene conversion using the endogenous homologous region in the absence of the contacting.

57. The method of claim 54, wherein the percentage of cells in the population of cells that modify the target region of the target gene by gene conversion using the endogenous homologous region is increased, as compared to a percentage of cells in a population of cells which would modify the target region of the target gene by gene conversion using the endogenous homologous region after contacting the population of cells with a first gRNA molecule, a first enzymatically active Cas9 (eaCas9) molecule, a second gRNA molecule, and a second eaCas9 molecule which would generate a first 3' overhang and a second 3' overhang in the target gene.

58. The method of claim 54, wherein the percentage of cells in the population of cells that modify the target region of the target gene by gene conversion using the endogenous homologous region is increased, as compared to a percentage of cells in a population of cells which would modify the target region of the target gene by gene conversion using the endogenous homologous region after contacting the population of cells with a first gRNA molecule, a first enzymatically active Cas9 (eaCas9) molecule, a second gRNA molecule, and a second eaCas9 molecule which would generate a double stranded blunt end brake in the target gene.