Methods and compositions for treating hemophilia B
Disclosed herein are methods and compositions for insertion of Factor IX (FIX) sequences into the genome of a cell for treating hemophilia B.
1. A protein comprising an engineered zinc finger protein DNA-binding domain that binds to a target site in an endogenous Factor IX (FIX) gene, wherein the DNA-binding domain comprises four or five zinc finger recognition regions ordered F1 to F4 or F1 to F5 from N-terminus to C-terminus, and wherein
(i) when the DNA-binding domain comprises five zinc finger recognition regions, F1 to F5 comprise the following amino acid sequences:
(SEQ ID NO: 4)
F1: QSGDLTR
(SEQ ID NO: 5)
F2: RSDVLSE
(SEQ ID NO: 6)
F3: DRSNRIK
(SEQ ID NO: 7)
F4: RSDNLSE
(SEQ ID NO: 8)
F5: QNATRIN,
wherein the DNA-binding domain binds to the target site shown in SEQ ID NO:3; and
(ii) when the DNA-binding domain comprises four zinc finger recognition regions, F1 to F4 comprise the following amino acid sequences:
(SEQ ID NO: 10)
F1: RSDSLSV
(SEQ ID NO: 11)
F2: TSGHLSR
(SEQ ID NO: 12)
F3: RSDHLSQ
(SEQ ID NO: 13)
F4: HASTRHC,
wherein the DNA-binding domain binds to the target site shown in SEQ ID NO:9.
2. The protein according to claim 1 , further comprising a cleavage domain or cleavage half-domain.
3. The protein of claim 2 , wherein the cleavage half-domain is a wild-type or engineered FokI cleavage half-domain.
4. A polynucleotide encoding the protein of claim 1 .
5. A gene delivery vector comprising a polynucleotide according to claim 4 .
6. An isolated cell comprising the protein of claim 1 .
7. An isolated cell comprising the polynucleotide of claim 4 .
8. A method of integrating an exogenous sequence into an endogenous FIX gene in a cell, the method comprising:
expressing at least one polynucleotide according to claim 4 in the cell such that the exogenous sequence is integrated into the endogenous FIX gene.
9. A cell made by the method of claim 8 .
10. The cell of claim 9 , wherein the cell is a hepatic cell or a stem cell.
11. The cell of claim 10 , wherein the stem cell is selected from the group consisting of an embryonic stem cell (ESC), an induced pluripotent stem cell (iPSC), a CD34+ hematopoietic stem cell and a hepatic stem cell.