Targeted disruption of T cell receptor genes using engineered zinc finger protein nucleases

1. An isolated zinc finger nuclease (ZFN) comprising

a zinc finger protein that binds to a target site in a T-cell receptor (TCR) gene, wherein the zinc finger protein comprises four, five or six zinc finger domains ordered F1 to F4 for four-finger proteins, F1 to F5 for five-finger proteins and F1 to F6 for six-finger proteins, wherein the zinc finger protein is selected from the group consisting of a protein comprising the following amino acid sequences in the recognition regions:

(i) F1: QSGDLTR (SEQ ID NO:2);

F2: QRTHLKA (SEQ ID NO:3);

F3: QSGDRNK (SEQ ID NO: 4);

F4: DRSNLSR (SEQ ID NO:5); and

F5: RSDALTQ (SEQ ID NO:6);

(ii) F1: TSGSLSR (SEQ ID NO:8);

F2: QSSVRNS (SEQ ID NO:9);

F3: RSDNLST (SEQ ID NO:10);

F4: DRSALAR (SEQ ID NO:11); and

F5: LKQNLDA (SEQ ID NO:12);

(iii) F1: DRSALSR (SEQ ID NO:14);

F2: QSGHLSR (SEQ ID NO:15);

F3: DRSDLSR (SEQ ID NO:16);

F4: RSDALSR (SEQ ID NO:17); and

F5: DRSDLSR (SEQ ID NO:16);

(iv) F1: DRSNLSR (SEQ ID NO:5);

F2: QKTSLQA (SEQ ID NO:19);

F3: DRSALSR (SEQ ID NO:14);

F4: QSGNLAR (SEQ ID NO:20);

F5: GKEELNE (SEQ ID NO:21); and

F6: RSSDLSR (SEQ ID NO:22);

(v) F1: GNVDLIE (SEQ ID NO:24);

F2: RSSNLSR (SEQ ID NO:25);

F3: RSDALSV (SEQ ID NO:26);

F4: DSSHRTR (SEQ ID NO:27); and

F5: WRSCRSA (SEQ ID NO:28);

(vi) F1: DSSDRKK (SEQ ID NO:30);

F2: RSDNLSV (SEQ ID NO:31);

F3: RRFILRG (SEQ ID NO:32);

F4: QSGDLTR (SEQ ID NO:2); and

F5: TSGSLTR (SEQ ID NO:33);

(vii) F1: QSGDLTR (SEQ ID NO:2);

F2: QTSTLSK (SEQ ID NO:35);

F3: QSGHLSR (SEQ ID NO:15);

F4: DRSDLSR (SEQ ID NO:16); and

F5: RSDALAR (SEQ ID NO:36);

(viii) F1: QSGDLTR (SEQ ID NO:2);

F2: WRSSLAS (SEQ ID NO:38);

F3: QSGDLTR (SEQ ID NO:2);

F4: HKWVLRQ (SEQ ID NO:39); and

F5: DRSNLTR (SEQ ID NO:40);

(ix) F1: QSGDLTR (SEQ ID NO:2);

F2: QWGTRYR (SEQ ID NO:42);

F3: ERGTLAR (SEQ ID NO:43);

F4: RSDNLRE (SEQ ID NO:44);

F5: QSGDLTR (SEQ ID NO:2); and

F6: TSGSLTR (SEQ ID NO:33);

(x) F1: RSAHLSR (SEQ ID NO:46);

F2: DRSDLSR (SEQ ID NO:16);

F3: RSDHLSV (SEQ ID NO:47); and

F4: QNNHRIT (SEQ ID NO:48);

(xi) F1: QRSNLVR (SEQ ID NO:50);

F2: RNDDRKK (SEQ ID NO:51);

F3: TSGNLTR (SEQ ID NO:52); and

F4: TSANLSR (SEQ ID NO:53);

(xii) F1: DRSTLRQ (SEQ ID NO:55);

F2: QRSNLVR (SEQ ID NO:50);

F3: RNDDRKK (SEQ ID NO:51);

F4: RSAHLSR (SEQ ID NO:46); and

F5: QSGHLSR (SEQ ID NO:15);

(xiii) F1: QRSNLVR (SEQ ID NO:50);

F2: RNDDRKK (SEQ ID NO:51);

F3: QSGHLAR (SEQ ID NO:56); and

F4: QSGHLSR (SEQ ID NO:15);

(xiv) F1: RSAHLSR (SEQ ID NO:46);

F2: DRSDLSR (SEQ ID NO:16);

F3: RSDHLSV (SEQ ID NO:47); and

F4: QNNHRIT (SEQ ID NO:48);

(xv) F1: RSDVLSA (SEQ ID NO:59);

F2: DRSNRIK (SEQ ID NO:60);

F3: RSDVLSE (SEQ ID NO:61);

F4: QSGNLAR (SEQ ID NO:20); and

F5: QSGSLTR (SEQ ID NO:62);

(xvi) F1: RSDHLST (SEQ ID NO:64);

F2: RSDNLTR (SEQ ID NO:65);

F3: DRSNLSR (SEQ ID NO:5);

F4: TSSNRKT (SEQ ID NO:66);

F5: RSANLAR (SEQ ID NO:67);

F6: RNDDRKK (SEQ ID NO:51);

(xvii) F1: RSDHLST (SEQ ID NO:64);

F2: RSDNLTR (SEQ ID NO:65);

F3: DRSNLSR (SEQ ID NO:5);

F4: LQFNRNQ (SEQ ID NO:68);

F5: RSANLAR (SEQ ID NO:67); and

F6: RNDDRKK (SEQ ID NO:51);

(xviii) F1: RSDHLST (SEQ ID NO:64);

F2: RSDNLTR (SEQ ID NO:65);

F3: DSSNLSR (SEQ ID NO:69):

F4: LRFNLSN (SEQ ID NO:70);

F5: RSANLAR (SEQ ID NO:67); and

F6: RNDDRKK (SEQ ID NO:51);

(xix) F1: RSDHLST (SEQ ID NO:64);

F2: RSDNLTR (SEQ ID NO:65);

F3: DSSNLSR (SEQ ID NO:69);

F4: LHFQLTG (SEQ ID NO:71);

F5: RSANLAR (SEQ ID NO:67); and

F6: RNDDRKK (SEQ ID NO:51);

(xx) F1: RSDVLSA (SEQ ID NO:59);

F2: DRSNRIK (SEQ ID NO:60);

F3: RSDVLSE (SEQ ID NO:61);

F4: QSGNLAR (SEQ ID NO:20); and

F5: QSGSLTR (SEQ ID NO:62);

(xxi) F1: DRSNLSR (SEQ ID NO:5);

F2: LKFALAN (SEQ ID NO:72);

F3: RSDVLSE (SEQ ID NO:61);

F4: QSGNLAR (SEQ ID NO:20); and

F5: QSGSLTR (SEQ ID NO:62);

(xxii) F1: RSDVLSA (SEQ ID NO:59);

F2: DRSNRIK (SEQ ID NO:60);

F3: RSDVLSE (SEQ ID NO:61);

F4: QSGNLAR (SEQ ID NO:20); and

F5: QSGALAR (SEQ ID NO:73); and

(xxiii) F1: RLSVLTI (SEQ ID NO:74);

F2: DRANLTR (SEQ ID NO:75);

F3: RSDVLSE (SEQ ID NO:61);

F4: QSGNLAR (SEQ ID NO:20); and

F5: QSGALAR (SEQ ID NO:73);

and a cleavage domain.

2. The zinc finger nuclease of claim 1 , wherein the TCR gene is a human TCR α gene or human TCR β gene.

3. The zinc finger nuclease of claim 1 , wherein the cleavage domain comprises a wild-type or engineered FokI cleavage half-domain.

4. A polynucleotide encoding a zinc finger nuclease according to claim 1 .

5. A gene delivery vector comprising the polynucleotide of claim 4 .

6. The gene delivery vector of claim 5 , wherein the vector is an adenoviral or lentiviral vector.

7. The gene delivery vector of claim 6 , wherein the adenoviral vector is an AdF5/F35 vector or the lentiviral vector is an integrase-defective lentiviral vector (IDLV).

8. An isolated cell or a cell line comprising a zinc finger nuclease according to claim 1 .

9. An isolated cell or a cell line comprising a polynucleotide according to claim 4 .

10. An isolated cell in which a TCR gene has been inactivated, wherein the cell is descended from the isolated cell or cell line of claim 8 .

11. A method of inactivating a TCR gene in an isolated cell, the method comprising expressing a zinc finger nuclease according to claim 1 in the isolated cell.

12. The method of claim 11 , further comprising

introducing into the isolated cell a sequence encoding one or more TCR genes specific for an MHC/antigen complex or a chimeric antigen receptor.

13. The method of claim 12 , wherein the sequence is integrated at or near the cleavage site.

14. The method of claim 12 , wherein the sequence is integrated into a safe-harbor locus.

15. The method of claim 11 , wherein the isolated cell is selected from the group consisting of a stem cell, a progenitor cell, and a T-cell.