CRISPR/CAS-related methods and compositions for treating sickle cell disease
CRISPR/CAS-related compositions and methods for treatment of Sickle Cell Disease (SCD) are disclosed.
1. A method of altering a cell comprising contacting the cell with:
(a) a first gRNA molecule comprising a first targeting domain which is complementary with a first target domain located within a BCL11A gene, wherein the first targeting domain is configured to provide a first cleavage event in a region of the BCL11A gene which is complementary to a sequence that is the same as, or differs by no more than 3 nucleotides from, a sequence selected from the group consisting of SEQ ID NOs:16261 to 16279; and
(b) a Cas9 molecule.
2. The method of claim 1 , wherein the cell is from a subject having a mutation at an SCD target position in the HBB gene or from a subject which would benefit from having an alteration at an SCD target position in the BCL11A gene.
3. The method of claim 1 , wherein the cell is selected from the group consisting of an erythroid cell, a bone marrow cell, and a stem cell.
4. The method of claim 2 , wherein the contacting step is performed ex vivo.
5. The method of claim 4 , wherein the contacted cell is returned to the subject's body.
6. The method of claim 1 , wherein the contacting is performed in vivo.
7. The method of claim 1 , wherein the cell is from a subject that has SCD.
8. The method of claim 1 , further comprising contacting the cell with (c) a second gRNA molecule.
9. The method of claim 8 , wherein the second gRNA molecule comprises a second targeting domain that is complementary with a second target domain located within the BCL11A gene, wherein the second targeting domain is configured to provide a second cleavage event in a region of the BCL11A gene which is complementary to a sequence that is the same as, or differs by no more than 3 nucleotides from, a sequence selected from the group consisting of SEQ ID NOs:16261 to 16279.
10. The method of claim 9 , wherein the contacting step comprises contacting the cell with a nucleic acid that encodes at least one of (a) the first gRNA molecule, (b) the Cas9 molecule, and (c) the second gRNA molecule.
11. The method of claim 10 , wherein the nucleic acid is an AAV vector.
12. A method of altering a cell comprising contacting the cell with:
(a) a first gRNA molecule comprising a first targeting domain which is complementary with a first target domain located in a BCL11A gene, wherein the first targeting domain is configured to provide a first cleavage event in a region of the BCL11A gene, and wherein the first targeting domain comprises a sequence that is the same as, or differs by no more than 3 nucleotides from, a sequence selected from the group consisting of SEQ ID NOs:16261 to 16279; and
(b) a Cas9 molecule.
13. The method of claim 12 , wherein the cell is selected from the group consisting of an erythroid cell, a bone marrow cell, and a stem cell.
14. The method of claim 12 , wherein the cell is from a subject having a mutation at an SCD target position in the HBB gene or from a subject which would benefit from having an alteration at an SCD target position in the BCL11A gene.
15. The method of claim 14 , wherein the contacting step is performed ex vivo.
16. The method of claim 15 , wherein the contacted cell is returned to the subject's body.
17. The method of claim 12 , wherein the contacting is performed in vivo.
18. The method of claim 12 , wherein the cell is from a subject that has SCD.
19. The method of claim 12 , further comprising contacting the cell with (c) a second gRNA molecule.
20. The method of claim 19 , wherein the second gRNA molecule comprises a second targeting domain which is complementary with a second target domain located in a BCL11A gene, wherein the second targeting domain is configured to provide a second cleavage event in a region of the BCL11A gene, and wherein the second targeting domain comprises a sequence that is the same as, or differs by no more than 3 nucleotides from, a sequence selected from the group consisting of SEQ ID NOs:16261 to 16279.
21. The method of claim 20 , wherein the contacting step comprises contacting the cell with a nucleic acid that encodes at least one of (a) the first gRNA molecule, (b) the Cas9 molecule, and (c) the second gRNA molecule.
22. The method of claim 20 , wherein the contacting step comprises contacting the cell with a nucleic acid that encodes (a) the first gRNA molecule, (b) the Cas9 molecule, and (c) the second gRNA molecule.
23. The method of claim 20 , wherein contacting comprises delivering to the cell the Cas9 molecule of (b) and a nucleic acid which encodes (a) and (c).
24. The method of claim 20 , wherein contacting comprises delivering to the cell the Cas9 molecule of (b), the first gRNA of (a) and the second gRNA of (c).
25. The method of claim 20 , wherein contacting comprises delivering to the cell the first gRNA of (a), the second gRNA of (c) and a nucleic acid that encodes the Cas9 molecule of (b).
26. The method of claim 20 , further comprising contacting the cell with a third gRNA molecule.
27. The method of claim 26 , further comprising contacting the cell with a fourth gRNA molecule.
28. The method of claim 21 , wherein the nucleic acid is an AAV vector.