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 cell comprising a sequence encoding a Factor IX (F.IX) protein integrated into intron 1 of an endogenous albumin gene such that the F.IX protein is expressed from the albumin gene.
2. The cell of claim 1 , wherein the cell is a liver cell.
3. A method of treating a patient with hemophilia B, the method comprising generating a cell according to claim 2 in the patient, thereby treating the patient.
4. The method of claim 3 , wherein the cell is generated in the patient by administering at least one polynucleotide comprising sequences encoding a nuclease and the F.IX protein.
5. The method of claim 4 , wherein the nuclease comprises a zinc finger nuclease (ZFN), a TAL-effector domain nuclease (TALEN) or a CRISPR/Cas nuclease system.
6. The method of claim 5 , wherein the nuclease comprises a ZFN comprising 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 SEQ ID NOs: 4, 5, 6, 7, and 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 SEQ ID NOs: 9, 10, 11, 12, 13, and 14.
7. The method of claim 4 , wherein the at least one polynucleotide encoding the nuclease and the F.IX protein is administered intravenously to the patient.
8. The method of claim 4 , wherein first, second and third polynucleotides are administered to the patient, the first polynucleotide encoding a first zinc finger nuclease, the second polynucleotide encoding a second zinc finger protein and the third polynucleotide comprising the sequence encoding the F.IX protein and further wherein the first and second zinc finger nuclease dimerize to form a zinc finger nuclease.
9. The method of claim 8 , wherein the first, second and third polynucleotides are carried on AAV vectors.
10. The method of claim 8 , wherein the first, second and third polynucleotides are administered 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 9 , wherein the AAV vectors are administered 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.