Artificial nucleases comprising engineered cleavage half-domains

• US 10,066,242 B2
• Filed: 07/26/2017
• Issued: 09/04/2018
• 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:
  US15/660,072
• Publication Number:
  US 20170327848A1
• Time in Patent Office: 405 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, wherein the engineered cleavage half-domain comprising the amino acid sequence of SEQ ID NO;

57, which amino acid sequence includes a substitution mutation selected from the group consisting of;

substitution mutations at amino acid residues 486, 499 and 496;

substitution mutations at amino acid residues 487 and 496;

substitution mutations at amino acid residues 487, 499, and 496;

substitution mutations at amino acid residues 483 and 537;

substitution mutations at amino acid residues 490 and 537;

substitution mutations at amino acid residues 490, 537 and 538;

substitution mutations at amino acid residues 483, 496 and 537;

substitution mutations at amino acid positions 487, 496 and 537;

substitution mutations at amino acid residues 487, 499 and 496; and

substitution mutations at amino acid residues 483, 537 and 538.

2. The artificial nuclease of claim 1, wherein the engineered cleavage half-domain comprises substitution mutations at amino acid residues 486, 499 and 496 and further wherein the wild-type Gln (Q) residue at position 486 is replaced with a Glu (E) residue, the wild-type Iso (I) residue at position 499 is replaced with a Leu (L) residue and the wild-type Asn (N) residue at position 496 is replaced with an Asp (D) or a Glu (E) residue.

3. The artificial nuclease of claim 1, wherein the engineered cleavage half-domain comprises substitution mutations at amino acid residues 490, 538 and 537 and further wherein the wild-type Glu (E) residue at position 490 is replaced with a Lys (K) residue, the wild-type Iso (I) residue at position 538 is replaced with a Lys (K) residue, and the wild-type His (H) residue at position 537 is replaced with a Lys (K) residue or a Arg (R) residue.

4. The artificial nuclease of claim 1, wherein the engineered cleavage half-domain comprises substitution mutations at amino acid residues 490 and 537 and further wherein the wild-type Glu (E) residue at position 490 is replaced with a Lys (K) residue and the wild-type His (H) residue at position 537 is replaced with a Lys (K) residue or a Arg (R) residue.

5. The artificial nuclease of claim 1, wherein the engineered cleavage half-domain comprises substitution mutations at amino acid residues 487 and 496 and further wherein the wild-type Arg (R) residue at position 487 is replaced with an Asp (D) residue- and the wild-type Asn (N) residue at position 496 is replaced with an Asp (D) residue.

6. The nuclease of claim 5, further wherein the wild-type Ile (I) residue at position 499 is replaced with an Ala (A).

7. The nuclease of claim 1, wherein the engineered cleavage half-domain comprises substitution mutations at amino acid residues 483 and 537 and further wherein the wild-type Asp (D) residue at position 483 is replaced with an Arg (R) residue and the wild-type His (H) residue at position 537 is replaced with an Arg (R) residue.

8. The nuclease of claim 1, wherein the engineered cleavage half-domain comprises substitution mutations at amino acid residues 487, 496 and 537 and further wherein the wild-type Arg (R) residue at position 487 is replaced with an Asp (D) residue, the wild-type Asn (N) residue at position 496 is replaced with an Asp (D) residue and the wild-type His (H) residue at position 537 is replaced with an Arg (R) residue.

9. The nuclease of claim 1, wherein the engineered cleavage half-domain comprises substitution mutations at amino acid residues 483, 496 and 537 and further wherein the wild-type Asp (D) residue at position 483 is replaced with an Arg (R) residue, the wild-type Asn (N) residue at position 496 is replaced with an Asp (D) residue and the wild-type His (H) residue at position 537 is replaced with an Arg (R) residue.

10. The nuclease of claim 1, further comprising an additional amino acid substitution at one or more of positions 418, 432, 441, 481, 483, 486, 487, 490, 496, 499, 523, 527, 537, 538 and 559.

11. 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.

12. The method of claim 11, wherein the gene locus is inactivated.

13. The method of claim 11, wherein the genomic modification wherein the genomic modification comprises an insertion.

14. The method of claim 13, wherein a transgene is inserted into the gene locus.

15. The method of claim 11, wherein the genomic modification comprises a deletion.

16. The method of claim 15, wherein the genomic modification further comprises an insertion.

17. The method of claim 16, wherein a transgene is inserted into the gene locus.

18. The method of claim 11, wherein the cell is a T-cell or a hematopoietic stem cell.