Method of inactivating a glucocorticoid receptor gene in an isolated cell
Disclosed herein are methods and compositions for inactivation of the human glucocorticoid receptor (GR) gene by targeted cleavage of genomic DNA encoding the GR. Such methods and compositions are useful, for example, in therapeutic applications which require retention of immune function during glucocorticoid treatment.
1. A method of inactivating a glucocorticoid receptor (GR) gene in an isolated cell, the method comprising:
A)(i) expressing a first fusion protein in an isolated cell, wherein the first fusion protein comprises:
a) a cleavage half-domain; and
b) a zinc finger DNA-binding domain that recognizes the nucleic acid sequence of SEQ ID NO: 4, wherein the zinc finger DNA-binding domain comprises the following recognition helices in the following order:
TSRALTA (SEQ ID NO: 23);
DRANLSR (SEQ ID NO: 17);
RSDNLSE (SEQ ID NO: 24); and
QNANRKT (SEQ ID NO: 25); and
(ii) expressing a second fusion protein in the isolated cell, wherein the second fusion protein comprises:
a) a cleavage half-domain; and
b) a zinc finger DNA-binding domain that recognizes the nucleic acid sequence of SEQ ID NO: 5, wherein the zinc finger DNA-binding domain comprises the following recognition helices in the following order:
RSDVLSE (SEQ ID NO: 14);
RSANLTR (SEQ ID NO: 27);
RSDNLST (SEQ ID NO: 28);
HSHARIK (SEQ ID NO: 29),
such that the first and second fusion proteins catalyze a double stranded break and inactivate the GR gene; or
B) i) expressing a first fusion protein in an isolated cell, wherein the first fusion protein comprises:
a) a cleavage half-domain; and
b) a first zinc finger DNA-binding domain that recognizes the nucleic acid sequence of SEQ ID NO: 4, wherein the zinc finger DNA-binding domain comprises the following recognition helices in the following order:
TSRALTA (SEQ ID NO: 23);
DRANLSR (SEQ ID NO: 17);
RSDNLSE (SEQ ID NO: 24); and
ERANRNS (SEQ ID NO: 26); and
(ii) expressing a second fusion protein in the isolated cell, wherein the second fusion protein comprises:
a) a cleavage half-domain; and
b) a zinc finger DNA-binding domain that recognizes the nucleic acid sequence of SEQ ID NO: 5, wherein the zinc finger DNA-binding domain comprises the following recognition helices in the following order:
DGWNRDC (SEQ ID NO: 32) or DSWNLQV (SEQ ID NO: 33);
RSANLTR (SEQ ID NO: 27);
TSGNLTR (SEQ ID NO: 30);
TSGSLTR (SEQ ID NO: 31),
such that the first and second fusion proteins catalyze a double stranded break and inactivate the GR gene.