US 8,956,828 B2 · Targeted disruption of T cell receptor genes using engineered zinc finger protein nucleases

Abstract

Disclosed herein are methods and compositions for inactivating TCR genes, using zinc finger nucleases (ZFNs) comprising a zinc finger protein and a cleavage domain or cleavage half-domain in conditions able to preserve cell viability. Polynucleotides encoding ZFNs, vectors comprising polynucleotides encoding ZFNs and cells comprising polynucleotides encoding ZFNs and/or cells comprising ZFNs are also provided. Disclosed herein are also methods and compositions for expressing a functional exogenous TCR in the absence of endogenous TCR expression in T lymphocytes, including lymphocytes with a central memory phenotype. Polynucleotides encoding exogenous TCR, vectors comprising polynucleotides encoding exogenous TCR and cells comprising polynucleotides encoding exogenous TCR and/or cells comprising exogenous TCR are also provided.

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.

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

Life of this patent

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

Life of this patent

Abstract

Claims15