Methods and compositions for increasing nuclease activity

• US 8,772,009 B2
• Filed: 04/20/2011
• Issued: 07/08/2014
• Est. Priority Date: 05/18/2009
• Status: Active Grant

• Current Assignee: Sangamo Biosciences, Inc.
• Original Assignee: Sangamo Biosciences, Inc.
• Inventors: Doyon, Yannick
• Primary Examiner(s): KIM, ALEXANDER D
• Application Number:
  US13/066,623
• Publication Number:
  US 20110269234A1
• Time in Patent Office: 1,175 Days
• Field of Search: None
• US Class Current: 435/196
• CPC Class Codes: C12N 2523/00 Culture process characterised by temperature
• CPC Class Codes: C12N 5/00 Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor; plant reproduction by tissue culture techniques A01H4/00
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

Methods and compositions for increasing nuclease activity by subjecting cells expressing the nuclease to hypothermic conditions to increase activity of the nucleases for genomic modifications.

Claims

1. A method for increasing the nuclease activity of at least one exogenous nuclease in an isolated mammalian cell, the method comprising:

(a) culturing the isolated mammalian cell at an optimal growth temperature above 33°

C.;

(b) transfecting the isolated mammalian cell with a polynucleotide encoding the at least one exogenous nuclease comprising a fusion protein comprising transcription activator-like effector (TAL-effector) domain and a cleavage domain into the isolated mammalian cell from step (a), wherein the polynucleotide expresses the exogenous nuclease and the exogenous nuclease cleaves a DNA target sequence in the isolated mammalian cell;

(c) culturing the isolated mammalian cell from step (b) at a temperature between 27°

C. and 33°

C. for between 1 and 4 days; and

(d) culturing the isolated mammalian cell following step (c) at the optimal growth temperature, such that the nuclease activity of the at least one exogenous nuclease is increased as compared to an isolated mammalian cell transfected and cultured only at the optimal growth temperature without the culturing step (c).

2. The method of claim 1, wherein the polynucleotide encoding the exogenous nuclease is transfected into the cell using a viral vector, a plasmid or an RNA.

3. The method of claim 2, wherein the viral vector is an Integration Defective Lentiviral vector (IDLV) construct.

4. The method of claim 1, wherein the cell is a eukaryotic cell.

5. The method of claim 1, further comprising introducing a donor nucleic acid sequence into the cell, wherein the cell comprises a genome and the donor nucleic acid sequence is integrated into the genome of the cell following cleavage of the endogenous target by the exogenous nuclease.