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

• US 8,703,489 B2
• Filed: 08/18/2009
• Issued: 04/22/2014
• Est. Priority Date: 08/22/2008
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.)
• Inventors: Wang, Jianbin
• Primary Examiner(s): RAGHU, GANAPATHIRAM
• Application Number:
  US12/583,329
• Publication Number:
  US 20100047805A1
• Time in Patent Office: 1,708 Days
• Field of Search: 435/463, 435/440, 435/6.1, 536/23.1, 536/23.2, 530/350
• US Class Current: 435/463
• 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. A protein complex comprising:

(a) a first fusion protein comprising a DNA-binding domain and at least one catalytically inactive FokI cleavage domain or cleavage half-domain, wherein the cleavage domain or cleavage half-domain comprises a mutation at one or more of amino acid residues 450, 467 and 469; and

(b) a second fusion protein comprising a zinc finger domain and a FokI catalytically active cleavage half domain,

wherein the catalytically inactive FokI cleavage half domain forms a heterodimer with the catalytically active FokI cleavage half-domain of the second fusion protein and further wherein the protein complex induces a single-stranded break in a target double-stranded sequence.

2. A composition comprising one or more polynucleotides encoding the fusion proteins of the protein complex of claim 1.

3. An isolated cell or cell line comprising a protein complex according to claim 1.

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

providing a composition according to claim 2, wherein the heterodimer generates a single-stranded break in the target double-stranded sequence.

5. The method of claim 4, wherein the target sequence is in cellular chromatin.

6. A method of inserting an exogenous sequence into a region of interest in a cell, the method comprising

generating a single-stranded break in the region of interest according to the method of claim 4;

and

providing an exogenous sequence that is inserted into the region of interest following generation of a single-stranded break.

7. The method of claim 6, wherein the exogenous sequence replaces a wild-type genomic sequence.

8. A method of inactivating a target sequence in a region of interest of a cell, the method comprising

generating a single-stranded break in the region of interest according to the method of claim 4, and

providing an exogenous sequence that replaces the wild-type sequence in the region of interest and wherein the exogenous sequence partially or fully inactivates the target sequence.