Crispr/cas-related methods and compositions for treating sickle cell disease

Abstract

CRISPR/CAS-related compositions and methods for treatment of Sickle Cell Disease (SCD) are disclosed.

1 - 189 . (canceled) 
     
     
         190 . A gRNA molecule comprising a targeting domain which is complementary with a target domain from the HBB or BCL11A gene. 
     
     
         191 . The gRNA molecule of  claim 190 , wherein said targeting domain is configured to provide a cleavage event selected from a double strand break and a single strand break, within 500, 400, 300, 200, 100, 50, 25, or 10 nucleotides of an SCD target point position or an SCD target knockout position. 
     
     
         192 . The gRNA molecule of  claim 190 , wherein said targeting domain is configured to target an early coding region or an enhancer region of the BCL11A gene. 
     
     
         193 . The gRNA molecule of  claim 190 , wherein said targeting domain is configured to target a mutation in the HBB gene. 
     
     
         194 . The gRNA molecule of  claim 190 , wherein said targeting domain is configured to target the promoter region of the BCL11A gene. 
     
     
         195 . The gRNA molecule of  claim 190 , wherein said targeting domain comprises or consists of a sequence that is the same as, or differs by no more than 3 nucleotides from, a targeting domain sequence from any of Tables 1A-1D, 2A-2F, 3A-3C, 4A-4E, 5A-5E, 6A-6B, 7A-7D, 8A-8D, 9, 10A-10D, 11A-11D, 12, 13A-13D, 14A-14C, 15A-15D, 16A-16E, 17A-17B, 18A-18C, 19A-19E, 20A-20C, 21A-21E, 22A-22E, 23A-23C, 24A-24D, 25A-25B, 26, or 31. 
     
     
         196 . A nucleic acid that comprises a nucleotide sequence that encodes a gRNA molecule comprising a targeting domain that is complementary with an SCD target domain in the HBB gene or BCL11A gene. 
     
     
         197 . The nucleic acid of  claim 196  wherein said targeting domain is configured to provide a cleavage event selected from a double strand break and a single strand break, within 500, 400, 300, 200, 100, 50, 25, or 10 nucleotides of the SCD target point position or the SCD target knockout position. 
     
     
         198 . The nucleic acid of  claim 196 , wherein said targeting domain is configured to target an early coding region or an enhancer region of the BCL11A gene. 
     
     
         199 . The nucleic acid of  claim 196 , wherein said targeting domain is configured to target a mutation in the HBB gene. 
     
     
         200 . The nucleic acid of  claim 196 , wherein said targeting domain is configured to target the promoter region of the BCL11A gene. 
     
     
         201 . The nucleic acid of  claim 196 , wherein said targeting domain comprises or consists of a sequence that is the same as, or differs by no more than 3 nucleotides from, a targeting domain sequence from any of Tables 1A-1D, 2A-2F, 3A-3C, 4A-4E, 5A-5E, 6A-6B, 7A-7D, 8A-8D, 9, 10A-10D, 11A-11D, 12, 13A-13D, 14A-14C, 15A-15D, 16A-16E, 17A-17B, 18A-18C, 19A-19E, 20A-20C, 21A-21E, 22A-22E, 23A-23C, 24A-24D, 25A-25B, 26, or 31. 
     
     
         202 . The nucleic acid of  claim 196 , further comprising a sequence that encodes a Cas9 molecule. 
     
     
         203 . A method of altering a cell comprising contacting said cell with:
 (a) a gRNA molecule comprising a targeting domain which is complementary with a target domain from the HBB or BCL11A gene; and   (b) a Cas9 molecule.   
     
     
         204 . The method of  claim 203 , wherein said targeting domain is configured to target an early coding region or an enhancer region of the BCL11A gene. 
     
     
         205 . The method of  claim 203 , wherein said targeting domain is configured to target a mutation in the HBB gene. 
     
     
         206 . The method of  claim 203 , wherein said targeting domain is configured to target the promoter region of the BCL11A gene. 
     
     
         207 . The method of  claim 203 , wherein said targeting domain comprises or consists of a sequence that is the same as, or differs by no more than 3 nucleotides from, a targeting domain sequence from any of Tables 1A-1D, 2A-2F, 3A-3C, 4A-4E, 5A-5E, 6A-6B, 7A-7D, 8A-8D, 9, 10A-10D, 11A-11D, 12, 13A-13D, 14A-14C, 15A-15D, 16A-16E, 17A-17B, 18A-18C, 19A-19E, 20A-20C, 21A-21E, 22A-22E, 23A-23C, 24A-24D, 25A-25B, 26, or 31. 
     
     
         208 . The method of  claim 203 , wherein said cell is selected from the group consisting of an erythroid cell, a bone marrow cell, and a stem cell. 
     
     
         209 . The method of  claim 203 , wherein said contacting step is performed ex vivo.