METHOD OF INACTIVATING A GLUCOCORTICOID RECEPTOR GENE IN AN ISOLATED CELL

• US 20160076056A1
• Filed: 11/20/2015
• Published: 03/17/2016
• Est. Priority Date: 11/13/2006
• Status: Active Grant

• Current Assignee: City of Hope, Sangamo Therapeutics, Inc.
• Original Assignee: City of Hope, Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.)
• Inventors: Reik, Andreas, Jensen, Michael, Holmes, Michael C., Gregory, Philip D., Ando, Dale
• Granted Patent: US 10,907,175 B2
• Time in Patent Office: Days
• 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 zinc finger nuclease (ZFN) fusion protein comprising a cleavage domain and a zinc-finger protein (ZFP) that binds to a target site in a glucocorticoid receptor (GR) gene, wherein the ZFP comprises 4 or 5 engineered zinc-finger binding domains, each zinc finger domain comprising a recognition helix region designated F1 to F4 or F1 to F5, wherein the zinc finger protein comprises the recognition helix regions as shown in a single row of Table 2 and further wherein the target site is selected from the group consisting of SEQ ID NOs:

1 to 7.

2. A polynucleotide encoding the fusion protein of claim 1.

3. An expression vector comprising the isolated polynucleotide of claim 2.

4. The expression vector of claim 3, wherein the expression vector is a viral delivery vector.

5. The expression vector of claim 4, wherein the viral delivery vector is an adenovirus, a hybrid adenovirus or a non-integrating lentivirus.

6. An isolated cell comprising one or more fusion proteins according to claim 1.

7. An isolated cell comprising one or more polynucleotides according to claim 2.

8. The cell of claim 7, wherein the cell is a T-cell.

9. A cell or cell line in which an endogenous GR gene is partially or fully inactivated using a fusion protein according to claim 1.

10. A method of inactivating a GR gene, the method comprising the step of contacting a cell with the polynucleotide of claim 2.

11. A method of selecting cells comprising an exogenous sequence that has been integrated into a GR gene, the method comprising

expressing first and second fusion proteins according to claim 1 in the cell such that the first and second fusion proteins dimerize and catalyze a double-strand break in the GR gene;

introducing a polynucleotide into the cell, wherein the polynucleotide comprises a first region of homology to sequences upstream of the double-strand break, a second region of homology to sequences downstream of the double-strand break and the exogenous sequence; and

treating the cells with a natural or synthetic corticosteroid under conditions such that cells not comprising the integrated exogenous sequence are killed, thereby selecting cells into which an exogenous sequence has been introduced into a GR gene.

12. The method of claim 11, wherein the corticosteroid is synthetic.

14. The method of claim 11, wherein the exogenous sequence comprises a transgene.

15. The method of claim 11, wherein the expressing step comprises contacting the cell with a viral delivery vector.

16. The method of claim 15, wherein the vector is replication-defective.

17. The method of claim 15, wherein the viral delivery vector is an adenovirus, a hybrid adenovirus or a non-integrating lentivirus.

13. The method of claim 13, wherein the corticosteriod is dexamethasone.