METHODS AND COMPOSITIONS FOR OCULAR CELL THERAPY
First Claim
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;
orb) a nucleic acid molecule encoding a qRNA molecule comprising a targeting domain complementary to a target sequence in the B2M gene.
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0 Petitions
Accused Products
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.
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Citations
81 Claims
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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:
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a) a gRNA molecule comprising a targeting domain complementary to a target sequence in the B2M gene;
orb) a nucleic acid molecule encoding a qRNA molecule comprising a targeting domain complementary to a target sequence in the B2M gene. - View Dependent Claims (3, 4, 5, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 32, 33, 41, 42, 54, 55, 56, 57, 58, 59, 61, 70, 71, 78, 79, 80, 81)
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2. (canceled)
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6. (canceled)
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7. (canceled)
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8. (canceled)
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25. (canceled)
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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. - View Dependent Claims (27, 28, 31, 34, 35, 36, 40)
- 141 to 159, thereby eliminating surface expression of MHC Class I molecules in the cell, or
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29. A modified limbal stem cell comprising a genome in which the b2 microglobulin (B2M) gene on chromosome 15 has been edited:
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(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. - View Dependent Claims (30)
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37. (canceled)
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38. (canceled)
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39. (canceled)
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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; andb) 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). - View Dependent Claims (44, 45, 46, 50, 51, 52, 60, 62)
- 23-105 or 108-119, or 134 to 140, or
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47. (canceled)
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48. (canceled)
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49. (canceled)
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53. (canceled)
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63. (canceled)
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64. (canceled)
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72. (canceled)
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73. (canceled)
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74. (canceled)
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77. (canceled)
Specification