Compositions for linking DNA-binding domains and cleavage domains

• US 10,538,787 B2
• Filed: 02/09/2017
• Issued: 01/21/2020
• Est. Priority Date: 08/28/2013
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Therapeutics, Inc.
• Inventors: Paschon, David, Zhang, Lei
• Primary Examiner(s): Gebreyesus, Kagnew H
• Application Number:
  US15/428,762
• Publication Number:
  US 20170152527A1
• Time in Patent Office: 1,076 Days
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• CPC Class Codes: C12N 15/01 Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• 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

a DNA-binding domain having an N-terminus and a C-terminus, wherein the DNA-binding domain binds to a nucleotide target site;

a truncated FokI cleavage domain in which the N-terminal residues QLVKS (residues 158 to 162 of SEQ ID NO;

1) are deleted; and

a linker between the C-terminus of the DNA-binding domain and the N-terminus of the cleavage domain, wherein the linker comprises a sequence selected from the group consisting of (N)GICPPPRPRTSPP (L8a, SEQ ID NO;

2);

(T)GTAPIEIPPEVYP (L8b, SEQ ID NO;

3);

(N)GSYAPMPPLALASP (L8c, SEQ ID NO;

4);

(P)GIYTAPTSRPTVPP (L8d, SEQ ID NO;

5);

(N)GSQTPKRFQPTHPSA (L8e, SEQ ID NO;

6);

TGLMPPSHPRQPIHINF (L8g, SEQ ID NO;

234);

TGTVHTSPICPQTYP (L8i, SEQ ID NO;

161);

TGSGTPTRPHPPLPP (L8j, SEQ ID NO;

235);

(H)LPKPANPFPLD (L7-1, SEQ ID NO;

7);

(H)RDGPRNLPPTSPP (L7-2, SEQ ID NO;

8);

(H)RLPDSPTALAPDTL (L7-6, SEQ ID NO;

9);

(D)PNSPISRARPLNPHP (L7-3, SEQ ID NO;

10);

(Y)GPRPTPRLRCPIDSLIFR (L7-5, SEQ ID NO;

11);

(H)CPASRPIHP (L6-2, SEQ ID NO;

12);

(G)LQSLIPQQLL (L6-6, SEQ ID NO;

13);

(G)LQPTVNHEYNN (L6-7, SEQ ID NO;

14); and

(P)ANIHSLSSPPPL (L6-1, SEQ ID NO;

15) and further wherein the amino acid residue shown in round parentheses is optionally present.

2. The fusion protein of claim 1, wherein the linker further comprises a ZC sequence (SEQ ID NO:

35).

3. The fusion protein of claim 2, wherein the ZC sequence is at the N-terminus of the linker.

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

5. A dimer comprising two fusion proteins according to claim 1.

6. The dimer of claim 5, wherein the dimer is a homodimer or heterodimer.

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

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

9. A cell comprising a polynucleotide according to claim 7.

10. A method for targeted cleavage of cellular chromatin in a region of interest in a 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.

11. The method of claim 10, wherein both nucleases comprise fusions proteins according to claim 1.

12. The method of claim 10, wherein the target sites for the zinc finger nucleases are 3 to 20 base pairs apart.

13. The method of claim 10, 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.

14. 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.

15. A kit for producing a nuclease, the kit comprising a polynucleotide according to claim 7 contained in one or more containers, optional hardware, and instructions for use of the kit.

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