Compositions for linking zinc finger modules

• US 9,163,245 B2
• Filed: 06/04/2014
• Issued: 10/20/2015
• Est. Priority Date: 05/03/2010
• Status: Active Grant

• Current Assignee: Sangamo Biosciences, Inc.
• Original Assignee: Sangamo Biosciences, Inc.
• Inventors: Paschon, David, Rebar, Edward J.
• Primary Examiner(s): Saidha, Tekchand
• Application Number:
  US14/296,159
• Publication Number:
  US 20140287500A1
• Time in Patent Office: 503 Days
• Field of Search: 435/375, 530/350, 536/23.4
• US Class Current: 1/1
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins

Abstract

Disclosed herein are compositions for linking DNA binding modules to allow for specific and selective binding to module subsites separated by 1 or more base pairs. Also described are methods of making and using compositions comprising these linkers.

Claims

1. An isolated cell or cell line in which expression of a gene is modified by a zinc finger protein comprising a plurality of non-naturally occurring zinc finger modules, wherein at least two of the non-naturally occurring zinc finger DNA-binding modules are joined by an amino acid linker sequence of 5 to 20 amino acid residues between the last residue of the N-terminal zinc finger module and the first residue of C-terminal zinc finger module, the amino acid linker comprising an N-terminal amino acid residue adjacent to the N-terminal zinc finger module, a C-terminal amino acid residue adjacent to the C-terminal zinc finger module, and amino acid residues internal to the N- and C-terminal amino acid linker residues, wherein

(i) the N-terminal amino acid linker residue or the internal amino acid linker residues comprises at least one proline residue;

(ii) the zinc finger protein binds to a target site comprising target subsites bound by each zinc finger module; and

(iii) there is a gap of 0, 1, 2 or 3 base pairs between the target subsites bound by the zinc finger DNA-binding modules joined by the amino acid linker.

2. The cell or cell line of claim 1, wherein the amino acid linker further comprises at least two basic amino acid residues.

3. The cell or cell line of claim 2, wherein the basic amino acid residues are selected from the group consisting of an arginine residue, a histidine residue, a lysine residue or combinations thereof.

4. The cell or cell line of claim 1, wherein one or more of the zinc finger proteins further comprises a regulatory domain.

5. The cell or cell line of claim 4, wherein the regulatory domain is a transcriptional modulating domain.

6. The cell or cell line of claim 5, wherein the regulatory domain is an activation domain or a repression domain.

7. The cell or cell line of claim 4, wherein the regulatory domain is a cleavage domain or cleavage half-domain.

8. The cell or cell line of claim 1, wherein the zinc finger protein is administered as a polynucleotide.

9. The cell or cell line of claim 1, wherein the modification comprises up or down regulation of the gene.

10. The cell or cell line of claim 1, wherein the modification comprises cleavage of the gene.

11. The cell or cell line of claim 1, wherein the gene is an endogenous cellular gene.