Artificial nucleases including engineered FokI cleavage half-domains

• US 10,640,790 B2
• Filed: 08/06/2018
• Issued: 05/05/2020
• Est. Priority Date: 02/08/2010
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Therapeutics, Inc.
• Inventors: Doyon, Yannick, Miller, Jeffrey C.
• Primary Examiner(s): Fronda, Christian L
• Application Number:
  US16/055,525
• Publication Number:
  US 20180334689A1
• Time in Patent Office: 638 Days
• Field of Search: None
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 2319/50 containing protease site
• CPC Class Codes: C07K 2319/80 containing a DNA binding domain, e.g. Lacl or Tet-repressor
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 9/10 Transferases (2.) ribonucleases C12N9/22
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Y 301/21004 Type II site-specific deoxyribonuclease (3.1.21.4)

Abstract

Disclosed herein are engineered cleavage half-domains; fusion polypeptides comprising these engineered cleavage half-domains; polynucleotides encoding the engineered cleavage half-domains and fusion proteins; and cells comprising said polynucleotides and/or fusion proteins. Also described are methods of using these polypeptides and polynucleotides, for example for targeted cleavage of a genomic sequence.

Claims

1. An artificial nuclease comprising:

(i) a DNA-binding domain that binds to a target site in the genome of the cell; and

(ii) a polypeptide comprising an engineered FokI cleavage half-domain consisting of substitution mutations at least two of the following positions;

483, 486, 487, 490, 496, 499, 537 or 538, numbered relative to a wild-type FokI sequence as shown in SEQ ID NO;

57, wherein if the FokI cleavage half-domain includes a mutation at position 486, position 490 is not mutated.

3. A method of making a genomic modification in a cell, the method comprising administering at least one nuclease according to claim 1 to a cell, wherein the nuclease genetically modifies a gene locus in the cell.

4. The method of claim 3, wherein the gene locus is inactivated.

5. The method of claim 3, wherein the genomic modification wherein the genomic modification comprises an insertion.

6. The method of claim 5, wherein a transgene is inserted into the gene locus.

7. The method of claim 3, wherein the genomic modification comprises a deletion.

8. The method of claim 7, wherein the genomic modification further comprises an insertion.

9. The method of claim 8, wherein a transgene is inserted into the gene locus.

10. The method of claim 3, wherein the cell is a T-cell, a hematopoietic stem cell or a liver cell.

2. An artificial nuclease comprising:

(i) a DNA-binding domain that binds to a target site in the genome of the cell; and

(ii) a polypeptide comprising an engineered FokI cleavage half-domain consisting of substitution mutations at least two of the following positions;

483, 486, 487, 490, 496, 499, 537 or 538, wherein if the FokI cleavage half-domain includes a mutation at position 486, position 490 is not mutated and an additional amino acid substitution at one or more of positions 418, 432, 441, 481, 486, 523, 527 and 559, numbered relative to a wild-type FokI sequence as shown in SEQ ID NO;

57.