Methods and compositions for targeted single-stranded cleavage and targeted integration

• US 9,200,266 B2
• Filed: 03/06/2014
• Issued: 12/01/2015
• Est. Priority Date: 08/22/2008
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Biosciences, Inc.
• Inventors: Wang, Jianbin
• Primary Examiner(s): RAGHU, GANAPATHIRAM
• Application Number:
  US14/199,753
• Publication Number:
  US 20140212928A1
• Time in Patent Office: 635 Days
• Field of Search: 435/440, 435/455, 435/463, 435/91.1, 424/94.1, 536/23.4, 530/350
• US Class Current: 1/1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• 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: C07K 2319/70 containing domain for protein-protein interaction
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 1/706 for hepatitis
• CPC Class Codes: C12Q 1/708 for papilloma
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C12Y 301/00 Hydrolases acting on ester bonds (3.1)

Abstract

Disclosed herein are methods and compositions for generating a single-stranded break in a target sequence, which facilitates targeted integration of one or more exogenous sequences.

Claims

1. An artificial nuclease comprising

(i) first and second cleavage domains from an endonuclease, wherein the first cleavage domain is catalytically inactive and the second cleavage domain is catalytically active;

and

(ii) a DNA-binding molecule that is heterologous to the first and second cleavage domains, and further wherein the DNA-binding molecule of the nuclease binds to a target sequence in a double-stranded genome and the nuclease induces a site-specific single-stranded break at or near the target sequence in the double-stranded genome such that the double-stranded genome is modified.

2. The nuclease of claim 1, wherein the modification is selected from the group consisting of insertions, deletions and combinations thereof.

3. The nuclease of claim 2, wherein modification results in inactivation of an endogenous gene.

4. The nuclease of claim 2, wherein the modification comprises targeted integration of an exogenous sequence.

5. A composition comprising the nuclease of claim 1.

6. An isolated cell or cell line comprising a nuclease of claim 1.

7. A method of generating a single-stranded break in a target double-stranded sequence in cellular chromatin of a cell, the method comprising:

providing a nuclease according to claim 1 to the cell such that the nuclease generates a single-stranded break in the target double-stranded sequence in cellular chromatin.

8. The method of claim 7, further comprising providing an exogenous sequence to the cell such that the exogenous sequence is inserted into the cellular chromatin at or near the single-stranded break.

9. The method of claim 8, wherein the exogenous sequence replaces a wild-type genomic sequence.

10. The method of claim 7, wherein a target sequence in the cellular chromatin is inactivated.

11. The method of claim 7, further comprising providing a second nuclease that makes a second single-stranded break in the double-stranded cellular chromatin.

12. A cell made by the method of claim 7.