Methods and compositions for regulation of transgene expression
Nucleases and methods of using these nucleases for expressing a transgene from a safe harbor locus in a secretory tissue, and clones and animals derived therefrom.
1. One or more polynucleotides encoding first and/or second zinc finger nucleases, wherein
(i) the first zinc finger nuclease comprises a cleavage domain and a first zinc finger protein DNA-binding domain that binds to a target site in SEQ ID NO:127 in an endogenous albumin gene, the first zinc finger protein DNA-binding domain comprising 6 zinc finger domains ordered F1 to F6, wherein F1 to F6 each comprise a recognition helix region as follows:
(a) F1: QSSDLSR (SEQ ID NO:46);
F2: LRHNLRA (SEQ ID NO:105);
F3: DQSNLRA (SEQ ID NO:106);
F4: RPYTLRL (SEQ ID NO:107);
F5: QSSDLSR (SEQ ID NO:46); and
F6: HRSNLNK (SEQ ID NO:108); or
(b) F1: QSSDLSR (SEQ ID NO:46);
F2: HRSNLNK (SEQ ID NO:108);
F3: DQSNLRA (SEQ ID NO:106);
F4: RPYTLRL (SEQ ID NO:107);
F5: QSSDLSR (SEQ ID NO:46); and
F6: HRSNLNK (SEQ ID NO:108); or
(c) F1: QSSDLSR (SEQ ID NO:46);
F2: LKWNLRT (SEQ ID NO:109);
F3: DQSNLRA (SEQ ID NO:106);
F4: RPYTLRL (SEQ ID NO:107);
F5: QSSDLSR (SEQ ID NO:46); and
F6: HRSNLNK (SEQ ID NO:108); or
(d) F1: QSSDLSR (SEQ ID NO:46);
F2: WKWNLRA (SEQ ID NO:110);
F3: DQSNLRA (SEQ ID NO:106);
F4: RPYTLRL (SEQ ID NO:107);
F5: QSSDLSR (SEQ ID NO:46); and
F6: HRSNLNK (SEQ ID NO:108); and
(ii) the second zinc finger nuclease comprises a second zinc finger protein DNA-binding domain that binds to a target site in SEQ ID NO:128 in the endogenous albumin gene, the second zinc finger protein DNA-binding domain comprising 5 zinc finger domains ordered F1 to F5, wherein F1 to F5 each comprise a recognition helix region as follows:
(a) F1: QSGNLAR (SEQ ID NO: 5);
F2: LMQNRNQ (SEQ ID NO: 97);
F3: LKQHLNE (SEQ ID NO: 111);
F4: TSGNLTR (SEQ ID NO: 11); and
F5: RRYYLRL (SEQ ID NO: 112); or
(b) F1: QSGNLAR (SEQ ID NO: 5);
F2: HLGNLKT (SEQ ID NO: 94);
F3: LKQHLNE (SEQ ID NO: 111);
F4: TSGNLTR (SEQ ID NO: 11); and
F5: RRDWRRD (SEQ ID NO: 113); or
(c) F1: QSGNLAR (SEQ ID NO: 5);
F2: LMQNRNQ (SEQ ID NO: 97);
F3: LKQHLNE (SEQ ID NO: 111);
F4: TSGNLTR (SEQ ID NO: 11); and
F5: RRDWRRD (SEQ ID NO: 113); or
(d) F1: QRSNLVR (SEQ ID NO: 99);
F2: TSSNRKT (SEQ ID NO: 53);
F3: LKHHLTD (SEQ ID NO: 95);
F4: TSGNLTR (SEQ ID NO: 11); and
F5: RRDWRRD (SEQ ID NO: 113).
2. One or more adeno-associated virus (AAV) vectors comprising the one or more polynucleotides of claim 1 .
3. An isolated cell comprising the one or more polynucleotides according to claim 1 .
4. The isolated cell of claim 3 , wherein the cell is a liver cell or a stem cell.
5. The isolated cell of claim 4 , wherein the stem cell is selected from the group consisting of an embryonic stem cell (ESC), an induced pluripotent stem cell (iPSC), a hepatic stem cell and a liver stem cell.
6. A kit comprising one or more expression vectors comprising the one or more polynucleotides according to claim 1 and instructions for using the expression vectors to cleave an endogenous albumin gene.
7. A pharmaceutical composition comprising the one or more polynucleotides of claim 1 and a pharmaceutically acceptable carrier and/or excipient.
8. The pharmaceutical composition of claim 7 further comprising a donor polynucleotide comprising a transgene encoding a therapeutic protein.