Methods and compositions for treating hemophilia
Disclosed herein are methods and compositions for insertion of transgene sequences encoding proteins involved in clotting into the genome of a cell for treating conditions including hemophilias.
1. A method of integrating a donor sequence encoding a Factor IX (F.IX) protein into an endogenous albumin gene in a cell, the method comprising:
cleaving the endogenous albumin gene in the cell using at least one zinc finger nuclease (ZFN), at least one TAL-effector domain nuclease (TALEN) or at least one CRISPR/Cas nuclease system that binds to a target site in the endogenous albumin gene comprising at least 15 base pairs of SEQ ID NO:2 or SEQ ID NO:3 in the presence of the donor sequence such that the donor sequence is integrated into the endogenous albumin gene, the F.IX protein is expressed and secreted from the cell.
2. The method of claim 1 , wherein the F.IX protein is a human F.IX protein.
3. The method of claim 2 , wherein the donor sequence is comprised within an AAV vector.
4. The method of claim 3 , wherein the AAV vector is an AAV2/6 vector.
5. The method of claim 1 , wherein the cell is a liver cell.
6. A method of treating a patient with hemophilia B, the method comprising modifying an endogenous albumin gene according to the method of claim 1 , wherein the Factor IX protein is expressed in the patient, thereby treating the patient.
7. The method of claim 6 , wherein the at least one nuclease and the donor sequences are comprised within AAV vectors that are administered.
8. The method of claim 7 , wherein the nuclease comprises first and second zinc finger nucleases, the first zinc finger nuclease comprising a FokI cleavage domain and a zinc finger protein comprising 5 zinc finger domains ordered F1 to F5, wherein each zinc finger domain comprises a recognition helix region and wherein the recognition helix regions of the zinc finger protein are shown in the first row of Table 1 (SEQ ID NOs:4-8) and the second zinc finger nuclease comprising a FokI cleavage domain and a zinc finger protein comprising 6 zinc finger domains ordered F1 to F6, wherein each zinc finger domain comprises a recognition helix region and wherein the recognition helix regions of the zinc finger protein are shown in the second row of Table 1 (SEQ ID NOs:9-14).
9. The method of claim 8 , wherein the AAV vectors comprising the first nuclease, the second nuclease and the donor are provided in a ratio about 1:1:1, about 1:1:2, about 1:1:3, about 1:1:4, about 1:1:5, about 1:1:6, about 1:1:7, about 1:1:8, about 1:1:9, about 1:1:10, about 1:1:11, about 1:1:12, about 1:1:13, about 1:1:14, about 1:1:15, about 1:1:16, about 1:1:17, about 1:1:18, about 1:1:19, or about 1:1:20.
10. The method of claim 7 , wherein the nuclease comprises first and second zinc finger nucleases, wherein the first and second zinc finger nucleases and the donor sequence are provided in the composition in a ratio of about 1:1:1, about 1:1:2, about 1:1:3, about 1:1:4, about 1:1:5, about 1:1:6, about 1:1:7, about 1:1:8, about 1:1:9, about 1:1:10, about 1:1:11, about 1:1:12, about 1:1:13, about 1:1:14, about 1:1:15, about 1:1:16, about 1:1:17, about 1:1:18, about 1:1:19, or about 1:1:20.
11. The method of claim 6 , wherein the at least one nuclease and the donor sequence are administered intravenously.