METHODS AND COMPOSITIONS FOR OCULAR CELL THERAPY

• US 20200131474A1
• Filed: 10/25/2019
• Published: 04/30/2020
• Est. Priority Date: 10/26/2018
• Status: Abandoned Application

• Current Assignee: Intellia Therapeutics, Inc., Novartis Ag
• Original Assignee: Intellia Therapeutics, Inc., Novartis Ag
• Inventors: BERENSHTEYN, Frada, HAN, Bo, HAO, Xueshi, HEYDER, Jessica, HOFFMAN, Timothy Z., JIN, Qihui, LACOSTE, Arnaud, LIU, Jun, LIU, Yahu, MO, Tingting, MURRAY, Bradley Andrew, O'CONNELL, Daniel Joseph, PAN, Jianfeng, XIE, Yun Feng, YAN, Shanshan, ZOU, Yefen
• Application Number:
  US16/663,722
• Publication Number:
  US 20200131474A1
• Time in Patent Office: Days
• CPC Class Codes: A61K 31/4375 the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
• CPC Class Codes: A61K 31/4725 containing further heterocyclic rings
• CPC Class Codes: A61K 31/519 ortho- or peri-condensed with heterocyclic rings
• CPC Class Codes: A61K 35/30 Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• 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 15/1138 against receptors or cell surface proteins
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2502/085 eye cells
• CPC Class Codes: C12N 2800/80 Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
• CPC Class Codes: C12N 5/0621 Eye cells, e.g. cornea, iris pigmented cells photoreceptors C12N5/062
• CPC Class Codes: C12N 5/0623 Stem cells
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

The present invention provides ocular cells, genetically modified by a CRISPR system targeting the expression of B2M for ocular cell therapy. The invention further provides methods of generating an expanded population of genetically modified ocular cells, for example limbal stem cells (LSCs) or corneal endothelial cells (CECs), wherein the cells are expanded involving the use of a LATS inhibitor and the expression of B2M in the cells has been reduced or eliminated. The present invention also provides a cell populations, preparations, uses and methods of therapy comprising said cells.

Claims

1. A modified limbal stem cell, which has reduced or eliminated expression of beta-2-microglobulin (B2M) relative to an unmodified limbal stem cell, wherein the B2M expression is reduced or eliminated by a CRISPR system comprising:

a) a gRNA molecule comprising a targeting domain complementary to a target sequence in the B2M gene;

or

b) a nucleic acid molecule encoding a qRNA molecule comprising a targeting domain complementary to a target sequence in the B2M gene.

3. The modified limbal stem cell of claim 1, wherein the modified limbal stem cell was cultured in media comprising a large tumor suppressor kinase (“

LATS”

) inhibitor, optionally wherein the LATS inhibitor is a compound of Formula A1

4. The modified limbal stem cell according to claim 3, wherein the compound is selected from:

N-methyl-2-(pyridin-4-yl)-N-(1,1,1-trifluoropropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;

2-methyl-1-(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propoxy) propan-2-ol;

2,4-dimethyl-4-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}pentan-2-ol;

N-tert-butyl-2-(pyrimidin-4-yl)-1,7-naphthyridin-4-amine;

2-(pyridin-4-yl)-N-[1-(trifluoromethyl)cyclobutyl]pyrido[3,4-d]pyrimidin-4-amine;

N-propyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;

N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;

3-(pyridin-4-yl)-N-(1-(trifluoromethyl)cyclopropyl)-2,6-naphthyridin-1-amine;

2-(3-methyl-1H-pyrazol-4-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;

2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propan-1-ol;

2-(pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1-yl)pyrido[3,4-d]pyrimidine;

N-cyclopentyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;

N-propyl-2-(3-(trifluoromethyl)-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine;

N-(2-methylcyclopentyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;

2-(3-chloropyridin-4-yl)-N-(1,1,1-trifluoro-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;

2-(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propoxy)ethan-1-ol;

N-(1-methylcyclopropyl)-7-(pyridin-4-yl)isoquinolin-5-amine;

(1S,2S)-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclopentan-1-ol;

N-methyl-2-(pyridin-4-yl)-N-[(2S)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine;

N-methyl-N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;

N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;

3-(pyridin-4-yl)-N-(1-(trifluoromethyl)cyclopropyl)-2,6-naphthyridin-1-amine and N-methyl-2-(pyridin-4-yl)-N-[(2R)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine.

5. The modified limbal stem cell according to claim 3, wherein the compound is selected from:

3-(pyridin-4-yl)-N-(1-(trifluoromethyl)cyclopropyl)-2,6-naphthyridin-1-amine N-(1-methylcyclopropyl)-7-(pyridin-4-yl)isoquinolin-5-amine;

2-(pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1-yl)pyrido[3,4-d]pyrimidine;

N-(tert-butyl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine; and

N-methyl-2-(pyridin-4-yl)-N-[(2S)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine.

9. The modified limbal stem cell according to claim 3, wherein the compound is present in a concentration of 3 to 10 micromolar.

10. The modified limbal stem cell of claim 1, wherein the targeting domain of the gRNA molecule is complementary to a sequence within a genomic region selected from:

chr15;

44711469-44711494, chr15;

44711472-44711497, chr15;

44711483-44711508, chr15;

44711486-44711511, chr15;

44711487-44711512, chr15;

44711512-44711537, chr15;

44711513-44711538, chr15;

44711534-44711559, chr15;

44711568-44711593, chr15;

44711573-44711598, chr15;

44711576-44711601, chr15;

44711466-44711491, chr15;

44711522-44711547, chr15;

44711544-44711569, chr15;

44711559-44711584, chr15;

44711565-44711590, chr15;

44711599-44711624, chr15;

44711611-44711636, chr15;

44715412-44715437, chr15;

44715440-44715465, chr15;

44715473-44715498, chr15;

44715474-44715499, chr15;

44715515-44715540, chr15;

44715535-44715560, chr15;

44715562-44715587, chr15;

44715567-44715592, chr15;

44715672-44715697, chr15;

44715673-44715698, chr15;

44715674-44715699, chr15;

44715410-44715435, chr15;

44715411-44715436, chr15;

44715419-44715444, chr15;

44715430-44715455, chr15;

44715457-44715482, chr15;

44715483-44715508, chr15;

44715511-44715536, chr15;

44715515-44715540, chr15;

44715629-44715654, chr15;

44715630-44715655, chr15;

44715631-44715656, chr15;

44715632-44715657, chr15;

44715653-44715678, chr15;

44715657-44715682, chr15;

44715666-44715691, chr15;

44715685-44715710, chr15;

44715686-44715711, chr15;

44716326-44716351, chr15;

44716329-44716354, chr15;

44716313-44716338, chr15;

44717599-44717624, chr15;

44717604-44717629, chr15;

44717681-44717706, chr15;

44717682-44717707, chr15;

44717702-44717727, chr15;

44717764-44717789, chr15;

44717776-44717801, chr15;

44717786-44717811, chr15;

44717789-44717814, chr15;

44717790-44717815, chr15;

44717794-44717819, chr15;

44717805-44717830, chr15;

44717808-44717833, chr15;

44717809-44717834, chr15;

44717810-44717835, chr15;

44717846-44717871, chr15;

44717945-44717970, chr15;

44717946-44717971, chr15;

44717947-44717972, chr15;

44717948-44717973, chr15;

44717973-44717998, chr15;

44717981-44718006, chr15;

44718056-44718081, chr15;

44718061-44718086, chr15;

44718067-44718092, chr15;

44718076-44718101, chr15;

44717589-44717614, chr15;

44717620-44717645, chr15;

44717642-44717667, chr15;

44717771-44717796, chr15;

44717800-44717825, chr15;

44717859-44717884, chr15;

44717947-44717972, chr15;

44718119-44718144, chr15;

44711563-44711585, chr15;

44715428-44715450, chr15;

44715509-44715531, chr15;

44715513-44715535, chr15;

44715417-44715439, chr15;

44711540-44711562, chr15;

44711574-44711596, chr15;

44711597-44711619, chr15;

44715446-44715468, chr15;

44715651-44715673, chr15;

44713812-44713834, chr15;

44711579-44711601, chr15;

44711542-44711564, chr15;

44711557-44711579, chr15;

44711609-44711631, chr15;

44715678-44715700, chr15;

44715683-44715705, chr15;

44715684-44715706, chr15;

44715480-44715502.

11. The modified limbal stem cell of claim 10, wherein the targeting domain of the gRNA molecule is complementary to a sequence within a genomic region selected from:

chr15;

44715513-44715535, chr15;

44711542-44711564, chr15;

44711563-44711585, chr15;

44715683-44715705, chr15;

44711597-44711619, or chr15;

44715446-44715468.

12. The modified limbal stem cell of claim 10, wherein the targeting domain of the gRNA molecule is complementary to a sequence within a genomic region chr15:

44711563-44711585.

13. The modified limbal stem cell of claim 1, wherein the targeting domain of the gRNA molecule to B2M comprises a targeting domain comprising the sequence of any one of SEQ ID NOs:

23-105 or 108-119 or 134-140.

14. The modified limbal stem cell of claim 13, wherein the targeting domain of the gRNA molecule to B2M comprises a targeting domain comprising the sequence of any one of SEQ ID NOs:

108, 111, 115, 116, 134 or 138.

15. The modified limbal stem cell of claim 13, wherein the targeting domain of the gRNA molecule to B2M comprises a targeting domain comprising the sequence of SEQ ID NO:

108.

16. The modified limbal stem cell of claim 13, wherein the targeting domain of the gRNA molecule to B2M comprises a targeting domain comprising the sequence of SEQ ID NO:

115.

17. The modified limbal stem cell of claim 13, wherein the targeting domain of the gRNA molecule to B2M comprises a targeting domain comprising the sequence of SEQ ID NO:

116.

18. The modified limbal stem cell of claim 1, wherein the gRNA comprises the sequence of any one of SEQ ID NO:

120, 160-177.

19. The modified limbal stem cell of claim 18, wherein the gRNA comprises the sequence of any one of SEQ ID NO:

120, 162, 166, 167, 171, and 175.

20. The modified limbal stem cell of claim 18, wherein the gRNA comprises the sequence of SEQ ID NO:

120.

21. The modified limbal stem cell of claim 18, wherein the gRNA comprises the sequence of SEQ ID NO:

166.

22. The modified limbal stem cell of claim 18, wherein the gRNA comprises the sequence of SEQ ID NO:

167.

23. The modified limbal stem cell of claim 1, wherein the CRISPR system is an S. pyogenes Cas9 CRISPR system.

24. The modified limbal stem cell of claim 23, wherein the CRISPR system comprises a Cas9 molecule comprising SEQ ID NO:

106 or 107 or any of SEQ ID NO;

124 to 134.

32. The modified limbal stem cell of claim 1, 2, 26, or 29, wherein the modified limbal stem cell comprises an indel formed at or near the target sequence complementary to the targeting domain of the gRNA molecule.

33. The modified limbal stem cell of claim 32, wherein the indel comprises a deletion of 10 or greater than 10 nucleotides, optionally 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides.

41. The modified limbal stem cell of claim 1, 2, 26, or 29, wherein the cell is autologous with respect to a patient to be administered said cell.

42. The modified limbal stem cell of claim 1, 2, 26, or 29, wherein the cell is allogeneic with respect to a patient to be administered said cell.

54. A cell population comprising the modified limbal stem cell of claim 1.

55. The cell population of claim 54, wherein the modified limbal stem cell comprises an indel formed at or near the target sequence complementary to the targeting domain of the gRNA molecule domain.

56. The cell population of claim 55, wherein the indel comprises a deletion of 10 or greater than 10 nucleotides, optionally 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides.

57. The cell population of claim 55, wherein the indel is formed in at least about 40%, of the cells of the cell population.

58. The cell population of claim 55, wherein an off-target indel is detected in no more than about 5% of the cells of the cell population.

59. A composition comprising the modified limbal stem cell of claim 1.

61. A composition comprising the cell population of claim 54.

70. A method of treating a patient suffering from an ocular disease comprising the step of administering to the patient in need thereof the modified limbal stem cell of claim 1.

71. The method of claim 70, wherein the ocular disease is limbal stem cell deficiency.

78. A method of treating a patient suffering from an ocular disease comprising the step of administering to the patient in need thereof the cell population of claim 54.

79. The method of claim 78, wherein the ocular disease is limbal stem cell deficiency.

80. A method of treating a patient suffering from an ocular disease comprising the step of administering to the patient in need thereof the composition of claim 59, 60, 61, or 62.

81. The method of claim 80, wherein the ocular disease is limbal stem cell deficiency.

2. (canceled)

6. (canceled)

7. (canceled)

8. (canceled)

25. (canceled)

26. A modified limbal stem cell comprising a genome in which the b2 microglobulin (B2M) gene on chromosome 15 has been edited

(a) to delete a contiguous stretch of genomic DNA comprising the sequence of any one of SEQ ID NOs:

141 to 159, thereby eliminating surface expression of MHC Class I molecules in the cell, or

(b) to form an indel at or near the target sequence complementary to the targeting domain of the gRNA molecule comprising the sequence of any one of SEQ ID NOs;

23-105 or 108-119 or 134-140, thereby eliminating surface expression of MHC Class I molecules in the cell.

27. The modified limbal stem cell of claim 26 comprising a genome in which the b2 microglobulin (B2M) gene on chromosome 15 has been edited:

(a) to delete a contiguous stretch of genomic DNA comprising the sequence of any one of SEQ ID NOs;

141, 148 or 149, thereby eliminating surface expression, of MHC Class I molecules in the cell, or

(b) to form an indel at or near the target sequence complementary to the targeting domain of the gRNA molecule domain comprising the sequence of any one of SEQ ID NOs;

108, 111, 115, 116, 134 or 138, thereby eliminating surface expression of MHC Class I molecules in the cell.

28. The modified limbal stem cell of claim 26 comprising a genome in which the b2 microglobulin (B2M) gene on chromosome 15 has been:

(a) edited to delete a contiguous stretch of genomic DNA comprising the sequence of SEQ ID NOs;

141, thereby eliminating surface expression, of MHC Class I molecules in the cell, or

(b) to form an indel at or near the target sequence complementary to the targeting domain of the gRNA molecule domain comprising the sequence of any one of SEQ ID NOs;

108, thereby eliminating surface expression of MHC Class I molecules in the cell.

31. The modified limbal stem cell of claim 28 comprising a genome in which the b2 microglobulin (B2M) gene on chromosome 15 has been edited

(a) to delete a contiguous stretch of genomic DNA region chr15:

44711563-44711585, thereby eliminating surface expression of MHC Class I molecules in the cell, or;

(b) to form an indel at or near the genomic DNA region, thereby eliminating surface expression of MHC Class I molecules in the cell.

34. The modified limbal stem cell of claim 26 or 29, wherein the modified limbal stem cell was cultured in media comprising a large tumor suppressor kinase (“

LATS”

) inhibitor, optionally wherein the LATS inhibitor is a compound of Formula A1

35. The modified limbal stem cell according to claim 34, wherein the compound is selected from:

N-methyl-2-(pyridin-4-yl)-N-(1,1,1-trifluoropropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;

2-methyl-1-(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propoxy) propan-2-ol;

2,4-dimethyl-4-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}pentan-2-ol;

N-tert-butyl-2-(pyrimidin-4-yl)-1,7-naphthyridin-4-amine;

2-(pyridin-4-yl)-N-[1-(trifluoromethyl)cyclobutyl]pyrido[3,4-d]pyrimidin-4-amine;

N-propyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;

N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;

3-(pyridin-4-yl)-N-(1-(trifluoromethyl)cyclopropyl)-2,6-naphthyridin-1-amine;

2-(3-methyl-1H-pyrazol-4-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;

2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propan-1-ol;

2-(pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1-yl)pyrido[3,4-d]pyrimidine;

N-cyclopentyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;

N-propyl-2-(3-(trifluoromethyl)-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine;

N-(2-methylcyclopentyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;

2-(3-chloropyridin-4-yl)-N-(1,1,1-trifluoro-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;

2-(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propoxy)ethan-1-ol;

N-(1-methylcyclopropyl)-7-(pyridin-4-yl)isoquinolin-5-amine;

(1S,2S)-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclopentan-1-ol;

N-methyl-2-(pyridin-4-yl)-N-[(2S)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine;

N-methyl-N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;

N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;

3-(pyridin-4-yl)-N-(1-(trifluoromethyl)cyclopropyl)-2,6-naphthyridin-1-amine and N-methyl-2-(pyridin-4-yl)-N-[(2R)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine.

36. The modified limbal stem cell according to claim 34, wherein the compound is selected from:

3-(pyridin-4-yl)-N-(1-(trifluoromethyl)cyclopropyl)-2,6-naphthyridin-1-amine;

N-(1-methylcyclopropyl)-7-(pyridin-4-yl)isoquinolin-5-amine;

2-(pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1-yl)pyrido[3,4-d]pyrimidine;

N-(tert-butyl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine; and

N-methyl-2-(pyridin-4-yl)-N-[(2S)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine.

40. The modified limbal stem cell according to claim 34, wherein the compound is present in a concentration of 3 to 10 micromolar.

29. A modified limbal stem cell comprising a genome in which the b2 microglobulin (B2M) gene on chromosome 15 has been edited:

(a) to delete a contiguous stretch of genomic DNA region selected from any one of;

chr15;

44711469-44711494, chr15;

44711472-44711497, chr15;

44711483-44711508, chr15;

44711486-44711511, chr15;

44711487-44711512, chr15;

44711512-44711537, chr15;

44711513-44711538, chr15;

44711534-44711559, chr15;

44711568-44711593, chr15;

44711573-44711598, chr15;

44711576-44711601, chr15;

44711466-44711491, chr15;

44711522-44711547, chr15;

44711544-44711569, chr15;

44711559-44711584, chr15;

44711565-44711590, chr15;

44711599-44711624, chr15;

44711611-44711636, chr15;

44715412-44715437, chr15;

44715440-44715465, chr15;

44715473-44715498, chr15;

44715474-44715499, chr15;

44715515-44715540, chr15;

44715535-44715560, chr15;

44715562-44715587, chr15;

44715567-44715592, chr15;

44715672-44715697, chr15;

44715673-44715698, chr15;

44715674-44715699, chr15;

44715410-44715435, chr15;

44715411-44715436, chr15;

44715419-44715444, chr15;

44715430-44715455, chr15;

44715457-44715482, chr15;

44715483-44715508, chr15;

44715511-44715536, chr15;

44715515-44715540, chr15;

44715629-44715654, chr15;

44715630-44715655, chr15;

44715631-44715656, chr15;

44715632-44715657, chr15;

44715653-44715678, chr15;

44715657-44715682, chr15;

44715666-44715691, chr15;

44715685-44715710, chr15;

44715686-44715711, chr15;

44716326-44716351, chr15;

44716329-44716354, chr15;

44716313-44716338, chr15;

44717599-44717624, chr15;

44717604-44717629, chr15;

44717681-44717706, chr15;

44717682-44717707, chr15;

44717702-44717727, chr15;

44717764-44717789, chr15;

44717776-44717801, chr15;

44717786-44717811, chr15;

44717789-44717814, chr15;

44717790-44717815, chr15;

44717794-44717819, chr15;

44717805-44717830, chr15;

44717808-44717833, chr15;

44717809-44717834, chr15;

44717810-44717835, chr15;

44717846-44717871, chr15;

44717945-44717970, chr15;

44717946-44717971, chr15;

44717947-44717972, chr15;

44717948-44717973, chr15;

44717973-44717998, chr15;

44717981-44718006, chr15;

44718056-44718081, chr15;

44718061-44718086, chr15;

44718067-44718092, chr15;

44718076-44718101, chr15;

44717589-44717614, chr15;

44717620-44717645, chr15;

44717642-44717667, chr15;

44717771-44717796, chr15;

44717800-44717825, chr15;

44717859-44717884, chr15;

44717947-44717972, chr15;

44718119-44718144, chr15;

44711563-44711585, chr15;

44715428-44715450, chr15;

44715509-44715531, chr15;

44715513-44715535, chr15;

44715417-44715439, chr15;

44711540-44711562, chr15;

44711574-44711596, chr15;

44711597-44711619, chr15;

44715446-44715468, chr15;

44715651-44715673, chr15;

44713812-44713834, chr15;

44711579-44711601, chr15;

44711542-44711564, chr15;

44711557-44711579, chr15;

44711609-44711631, chr15;

44715678-44715700, chr15;

44715683-44715705, chr15;

44715684-44715706, chr15;

44715480-44715502, thereby eliminating surface expression of MHC Class I molecules in the cell, or

(b) to form an indel at or near the genomic DNA region selected from any one of;

chr15;

44711469-44711494, chr15;

44711472-44711497, chr15;

44711483-44711508, chr15;

44711486-44711511, chr15;

44711487-44711512, chr15;

44711512-44711537, chr15;

44711513-44711538, chr15;

44711534-44711559, chr15;

44711568-44711593, chr15;

44711573-44711598, chr15;

44711576-44711601, chr15;

44711466-44711491, chr15;

44711522-44711547, chr15;

44711544-44711569, chr15;

44711559-44711584, chr15;

44711565-44711590, chr15;

44711599-44711624, chr15;

44711611-44711636, chr15;

44715412-44715437, chr15;

44715440-44715465, chr15;

44715473-44715498, chr15;

44715474-44715499, chr15;

44715515-44715540, chr15;

44715535-44715560, chr15;

44715562-44715587, chr15;

44715567-44715592, chr15;

44715672-44715697, chr15;

44715673-44715698, chr15;

44715674-44715699, chr15;

44715410-44715435, chr15;

44715411-44715436, chr15;

44715419-44715444, chr15;

44715430-44715455, chr15;

44715457-44715482, chr15;

44715483-44715508, chr15;

44715511-44715536, chr15;

44715515-44715540, chr15;

44715629-44715654, chr15;

44715630-44715655, chr15;

44715631-44715656, chr15;

44715632-44715657, chr15;

44715653-44715678, chr15;

44715657-44715682, chr15;

44715666-44715691, chr15;

44715685-44715710, chr15;

44715686-44715711, chr15;

44716326-44716351, chr15;

44716329-44716354, chr15;

44716313-44716338, chr15;

44717599-44717624, chr15;

44717604-44717629, chr15;

44717681-44717706, chr15;

44717682-44717707, chr15;

44717702-44717727, chr15;

44717764-44717789, chr15;

44717776-44717801, chr15;

44717786-44717811, chr15;

44717789-44717814, chr15;

44717790-44717815, chr15;

44717794-44717819, chr15;

44717805-44717830, chr15;

44717808-44717833, chr15;

44717809-44717834, chr15;

44717810-44717835, chr15;

44717846-44717871, chr15;

44717945-44717970, chr15;

44717946-44717971, chr15;

44717947-44717972, chr15;

44717948-44717973, chr15;

44717973-44717998, chr15;

44717981-44718006, chr15;

44718056-44718081, chr15;

44718061-44718086, chr15;

44718067-44718092, chr15;

44718076-44718101, chr15;

44717589-44717614, chr15;

44717620-44717645, chr15;

44717642-44717667, chr15;

44717771-44717796, chr15;

44717800-44717825, chr15;

44717859-44717884, chr15;

44717947-44717972, chr15;

44718119-44718144, chr15;

44711563-44711585, chr15;

44715428-44715450, chr15;

44715509-44715531, chr15;

44715513-44715535, chr15;

44715417-44715439, chr15;

44711540-44711562, chr15;

44711574-44711596, chr15;

44711597-44711619, chr15;

44715446-44715468, chr15;

44715651-44715673, chr15;

44713812-44713834, chr15;

44711579-44711601, chr15;

44711542-44711564, chr15;

44711557-44711579, chr15;

44711609-44711631, chr15;

44715678-44715700, chr15;

44715683-44715705, chr15;

44715684-44715706, chr15;

44715480-44715502, thereby eliminating surface expression, of MHC Class I molecules in the cell.

30. The modified limbal stem cell of claim 29 comprising a genome in which the b2 microglobulin (B2M) gene on chromosome 15 has been edited:

(a) to delete a contiguous stretch of genomic DNA region selected from;

chr15;

44715513-44715535, chr15;

44711542-44711564, chr15;

44711563-44711585, chr15;

44715683-44715705, chr15;

44711597-44711619, or chr15;

44715446-44715468, or

(b) to form an indel at or near the genomic DNA region selected from any one of;

chr15;

44715513-44715535, chr15;

44711542-44711564, chr15;

44711563-44711585, chr15;

44715683-44715705, chr15;

44711597-44711619, or chr15;

44715446-44715468.

37. (canceled)

38. (canceled)

39. (canceled)

43. A method of preparing a modified limbal stem cell or a population of modified limbal stem cells for ocular cell therapy comprising,

a) modifying a limbal stem cell or a population of limbal stem cells by reducing or eliminating expression of B2M comprising introducing into the limbal stem cell or the population of limbal stem cells a CRISPR system comprising a gRNA molecule with a targeting domain

(i) comprising the sequence of any one of SEQ ID NOs:

23-105 or 108-119, or 134 to 140, or

(ii) complementary to a sequence within a genomic region selected from;

chr15;

44711469-44711494, chr15;

44711472-44711497, chr15;

44711483-44711508, chr15;

44711486-44711511, chr15;

44711487-44711512, chr15;

44711512-44711537, chr15;

44711513-44711538, chr15;

44711534-44711559, chr15;

44711568-44711593, chr15;

44711573-44711598, chr15;

44711576-44711601, chr15;

44711466-44711491, chr15;

44711522-44711547, chr15;

44711544-44711569, chr15;

44711559-44711584, chr15;

44711565-44711590, chr15;

44711599-44711624, chr15;

44711611-44711636, chr15;

44715412-44715437, chr15;

44715440-44715465, chr15;

44715473-44715498, chr15;

44715474-44715499, chr15;

44715515-44715540, chr15;

44715535-44715560, chr15;

44715562-44715587, chr15;

44715567-44715592, chr15;

44715672-44715697, chr15;

44715673-44715698, chr15;

44715674-44715699, chr15;

44715410-44715435, chr15;

44715411-44715436, chr15;

44715419-44715444, chr15;

44715430-44715455, chr15;

44715457-44715482, chr15;

44715483-44715508, chr15;

44715511-44715536, chr15;

44715515-44715540, chr15;

44715629-44715654, chr15;

44715630-44715655, chr15;

44715631-44715656, chr15;

44715632-44715657, chr15;

44715653-44715678, chr15;

44715657-44715682, chr15;

44715666-44715691, chr15;

44715685-44715710, chr15;

44715686-44715711, chr15;

44716326-44716351, chr15;

44716329-44716354, chr15;

44716313-44716338, chr15;

44717599-44717624, chr15;

44717604-44717629, chr15;

44717681-44717706, chr15;

44717682-44717707, chr15;

44717702-44717727, chr15;

44717764-44717789, chr15;

44717776-44717801, chr15;

44717786-44717811, chr15;

44717789-44717814, chr15;

44717790-44717815, chr15;

44717794-44717819, chr15;

44717805-44717830, chr15;

44717808-44717833, chr15;

44717809-44717834, chr15;

44717810-44717835, chr15;

44717846-44717871, chr15;

44717945-44717970, chr15;

44717946-44717971, chr15;

44717947-44717972, chr15;

44717948-44717973, chr15;

44717973-44717998, chr15;

44717981-44718006, chr15;

44718056-44718081, chr15;

44718061-44718086, chr15;

44718067-44718092, chr15;

44718076-44718101, chr15;

44717589-44717614, chr15;

44717620-44717645, chr15;

44717642-44717667, chr15;

44717771-44717796, chr15;

44717800-44717825, chr15;

44717859-44717884, chr15;

44717947-44717972, chr15;

44718119-44718144, chr15;

44711563-44711585, chr15;

44715428-44715450, chr15;

44715509-44715531, chr15;

44715513-44715535, chr15;

44715417-44715439, chr15;

44711540-44711562, chr15;

44711574-44711596, chr15;

44711597-44711619, chr15;

44715446-44715468, chr15;

44715651-44715673, chr15;

44713812-44713834, chr15;

44711579-44711601, chr15;

44711542-44711564, chr15;

44711557-44711579, chr15;

44711609-44711631, chr15;

44715678-44715700, chr15;

44715683-44715705, chr15;

44715684-44715706, chr15;

44715480-44715502, wherein the limbal stem cell or the population of limbal stem cells have optionally been cultured in the presence of a LATS inhibitor; and

b) further expanding the modified limbal stem cell or the population of modified limbal stem cells in cell culture media comprising a LATS inhibitor; and

c) optionally, enriching the population of limbal stem cells with the limbal stem cells having reduced or eliminated expression of B2M by fluorescene activated cell sorting (FACS) or magnetic activated cell sorting (MACS).

44. The method of claim 43, wherein the LATS inhibitor is a compound of Formula A1

45. The method according to claim 44, wherein the compound is selected from:

N-methyl-2-(pyridin-4-yl)-N-(1,1,1-trifluoropropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;

2-methyl-1-(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propoxy)propan-2-ol;

2,4-dimethyl-4-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}pentan-2-ol;

N-tert-butyl-2-(pyrimidin-4-yl)-1,7-naphthyridin-4-amine;

2-(pyridin-4-yl)-N-[1-(trifluoromethyl)cyclobutyl]pyrido[3,4-d]pyrimidin-4-amine;

N-propyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;

N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;

3-(pyridin-4-yl)-N-(1-(trifluoromethyl)cyclopropyl)-2,6-naphthyridin-1-amine;

2-(3-methyl-1H-pyrazol-4-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;

2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propan-1-ol;

2-(pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1-yl)pyrido[3,4-d]pyrimidine;

N-cyclopentyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;

N-propyl-2-(3-(trifluoromethyl)-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine;

N-(2-methylcyclopentyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;

2-(3-chloropyridin-4-yl)-N-(1,1,1-trifluoro-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;

2-(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propoxy)ethan-1-ol;

N-(1-methylcyclopropyl)-7-(pyridin-4-yl)isoquinolin-5-amine;

(1S,2S)-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclopentan-1-ol;

N-methyl-2-(pyridin-4-yl)-N-[(2S)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine;

N-methyl-N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;

N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;

3-(pyridin-4-yl)-N-(1-(trifluoromethyl)cyclopropyl)-2,6-naphthyridin-1-amine and N-methyl-2-(pyridin-4-yl)-N-[(2R)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine.

46. The method according to claim 44, wherein the compound is selected from 3-(pyridin-4-yl)-N-(1-(trifluoromethyl)cyclopropyl)-2,6-naphthyridin-1-amine;

N-(1-methylcyclopropyl)-7-(pyridin-4-yl)isoquinolin-5-amine;

2-(pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1-yl)pyrido[3,4-d]pyrimidine;

N-(tert-butyl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine; and

N-methyl-2-(pyridin-4-yl)-N-[(2S)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine.

50. The method according to claim 44, wherein the compound is present in a concentration of 3 to 10 micromolar.

51. The method of claim 43, wherein the CRISPR system is an S. pyogenes Cas9 CRISPR system.

52. The method of claim 51, wherein the CRISPR system comprises a Cas 9 molecule comprising SEQ ID NO:

106 or 107 or any of SEQ ID NO;

124 to 134.

60. A composition comprising a modified limbal stem cell obtained by the method of claim 43.

62. A composition comprising a population of modified limbal stem cells obtained by the method of claim 43.

47. (canceled)

48. (canceled)

49. (canceled)

53. (canceled)

63. (canceled)

64. (canceled)

65. (canceled)

66. (canceled)

67. (canceled)

68. (canceled)

69. (canceled)

72. (canceled)

73. (canceled)

74. (canceled)

75. (canceled)

76. (canceled)

77. (canceled)