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

• US 10,689,717 B2
• Filed: 09/28/2018
• Issued: 06/23/2020
• Est. Priority Date: 08/22/2008
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Therapeutics, Inc.
• Inventors: Wang, Jianbin
• Primary Examiner(s): Raghu, Ganapathirama
• Application Number:
  US16/146,637
• Publication Number:
  US 20190032158A1
• Time in Patent Office: 634 Days
• 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 method of making a point mutation in a genomic sequence in an isolated cell, the method comprising introducing a polynucleotide encoding an artificial nuclease into the cell, the 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 wherein the genomic sequence is modified.

2. The method of claim 1, wherein the point mutation results in inactivation of an endogenous gene.

3. The method of claim 2, wherein the point mutation is made in a mutant genomic sequence.

4. The method of claim 1 wherein the artificial nuclease further comprises one or more additional enzymes.

5. The method of claim 1, further comprising providing a second nuclease that makes a second single-stranded break in the genomic sequence.

6. The method of claim 1, wherein the artificial nuclease comprises a first fusion protein comprising a first zinc finger protein DNA-binding domain and the first cleavage domain and a second fusion protein comprising a second fusion protein comprising a second zinc finger protein DNA-binding domain and the second cleavage domain.

7. The method of claim 6, wherein the first and second cleavage domains are FokI cleavage domains.

8. An isolated cell made by the method of claim 1.