Compositions for linking DNA-binding domains and cleavage domains

• US 9,394,531 B2
• Filed: 05/28/2009
• Issued: 07/19/2016
• Est. Priority Date: 05/28/2008
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.)
• Inventors: Miller, Jeffrey C.
• Primary Examiner(s): HUTSON, RICHARD G
• Application Number:
  US12/455,143
• Publication Number:
  US 20090305419A1
• Time in Patent Office: 2,609 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/14 for treating abnormal movements, e.g. chorea, dyskinesia
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 5/38 of the suprarenal hormones
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/04 Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/80 containing a DNA binding domain, e.g. Lacl or Tet-repressor
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

Disclosed herein are compositions for linking DNA binding domains and cleavage domains (or cleavage half-domains) to form non-naturally occurring nucleases. Also described are methods of making and using compositions comprising these linkers.

Claims

1. A fusion protein comprising from N-terminus to C-terminus:

a DNA-binding domain that binds to a nucleotide target site;

a ZC linker (SEQ ID NO;

2); and

a FokI cleavage domain, wherein the FokI cleavage domain comprises the sequence shown in residues 158 to 356 of SEQ ID NO;

3 or residues 158 to 360 of SEQ ID NO;

4.

2. The fusion protein of claim 1, wherein the DNA-binding domain is a zinc finger protein.

3. A polynucleotide encoding at least one fusion protein according to claim 1.

4. A cell comprising a fusion protein according to claim 1.

5. A cell comprising a polynucleotide according to claim 3.

6. A method for targeted cleavage of cellular chromatin in a region of interest in an isolated cell, the method comprising:

expressing a pair of nucleases in the cell under conditions such that cellular chromatin is cleaved at the region of interest, wherein the nucleases bind to target sites in the region of interest and further wherein at least one nuclease comprises a fusion protein according to claim 1.

7. The method of claim 6, wherein both nucleases comprise fusions proteins according to claim 1.

8. The method of claim 6, wherein the target sites for the zinc finger nucleases are 3 to 9 base pairs apart.

9. The method of claim 6,

wherein the nucleases binds to sequences between 2 and 50 nucleotides apart, such that cellular chromatin is cleaved in the region of interest.

10. The method of claim 9, wherein both nucleases comprise fusion proteins according to claim 1.

11. The method of claim 9, further comprising the step of introducing a donor polynucleotide into the cell, wherein all or part of the donor polynucleotide is incorporated into the region of interest following cleavage.

12. A kit for producing a nuclease, the kit comprising a fusion protein according to claim 1 contained in one or more containers, optional hardware, and instructions for use of the kit.

13. A kit for producing a nuclease, the kit comprising a polynucleotide according to claim 3 contained in one or more containers, optional hardware, and instructions for use of the kit.

14. The kit of claim 13, further comprising a donor polynucleotide.