Methods for increasing efficiency of gene editing in cells

Patent metadata

Field	Value
Publication number	US-12325865-B2
Application number	US-201816482294-A
Country	US
Kind code	B2
Filing date	Jan 31, 2018
Priority date	Feb 1, 2017
Publication date	Jun 10, 2025
Grant date	Jun 10, 2025

Abstract

The present disclosure provides methods of improving nuclease-mediated gene targeting frequencies in cells using PFTα or bFGF.

First claim

The invention claimed is:

1. A method of making a population of PERV-free porcine cells, the method comprising:
(a) genetically modifying a primary porcine cell which comprises porcine endogenous retrovirus (PERV) genes by providing a vector comprising a nucleic acid sequence which encodes a nuclease and three sgRNAs to the primary porcine cell, wherein the nuclease is expressed and cuts the PERV genes, thereby producing a genetically modified, PERV-free, primary porcine cell,
(b) providing an exogenous pifithrin α (PFTα) and a basic fibroblast growth factor (bFGF) to the primary porcine cell concomitant with step (a),
(c) contacting the primary porcine cell with a large SV40 T antigen to increase targeting efficiency,
(d) isolating the genetically modified, PERV-free primary porcine cell, and expanding the genetically modified, PERV-free porcine cell to a population of PERV-free porcine cells, wherein the population of PERV-free porcine cells exhibits a lack of PERV transmission as evidenced by co-culturing a cell with the population of PERV-free porcine cells.

2. The method of claim 1 , wherein the nuclease is a Cas nuclease, a Cas nickase, or a nuclease null Cas bound to a nuclease.

3. The method of claim 2 , wherein the nuclease is the Cas nuclease.

4. The method of claim 3 , wherein the Cas nuclease is Cas9.

5. The method of claim 1 , wherein the PERV genes comprise PERV pol genes.

6. The method of claim 1 , wherein of the PFTα and the bFGF inhibit apoptosis in the primary porcine cell.

7. The method of claim 1 , wherein the PFTα and the bFGF increase gene editing rates in the primary porcine cell.

8. The method of claim 1 , wherein the PFTα and the bFGF increase a cell survival rate in the primary porcine cell.

Assignees

• Harvard College

Inventors

• Church George M
• Yang Luhan

Classifications

• C12N2800/80

  Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites · CPC title

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title

• C07K14/503

  basic FGF [bFGF] · CPC title

• C12N15/111

  General methods applicable to biologically active non-coding nucleic acids · CPC title