Method of inactivating a glucocorticoid receptor gene in an isolated cell

• US 9,217,026 B2
• Filed: 11/13/2007
• Issued: 12/22/2015
• Est. Priority Date: 11/13/2006
• Status: Active Grant

• Current Assignee: City of Hope, Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Biosciences, Inc.
• Inventors: Reik, Andreas, Jensen, Michael, Holmes, Michael C., Gregory, Philip D., Ando, Dale
• Primary Examiner(s): WILSON, MICHAEL C
• Application Number:
  US11/983,888
• Publication Number:
  US 20080188000A1
• Time in Patent Office: 2,961 Days
• Field of Search: 435/325, 424/93.21, 514/44
• US Class Current: 1/1
• CPC Class Codes: A61P 11/06 Antiasthmatics
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 9/12 Antihypertensives
• CPC Class Codes: C07K 14/721 Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2710/10043 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2740/15043 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: G01N 2333/723 Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
• CPC Class Codes: G01N 33/743 Steroid hormones

Abstract

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.

Claims

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.