High-efficiency transgene integration by homology-directed repair in human primary cells using DNA-PKcs inhibition

Sridhar Selvaraj; William N Feist; Sebastien Viel; Sriram Vaidyanathan; Amanda M Dudek; Marc Gastou; Sarah J Rockwood; Freja K Ekman; Aluya R Oseghale; Liwen Xu; Mara Pavel-Dinu; Sofia E Luna; M Kyle Cromer; Ruhi Sayana; Natalia Gomez-Ospina; Matthew H Porteus

doi:10.1038/s41587-023-01888-4

High-efficiency transgene integration by homology-directed repair in human primary cells using DNA-PKcs inhibition

Nat Biotechnol. 2023 Aug 3. doi: 10.1038/s41587-023-01888-4. Online ahead of print.

Authors

Sridhar Selvaraj^{1

2}, William N Feist^{1

2}, Sebastien Viel^{1

2

3

4}, Sriram Vaidyanathan^{1

2

5

6}, Amanda M Dudek^{1

2}, Marc Gastou^{1

2}, Sarah J Rockwood^{1

2}, Freja K Ekman^{1

2}, Aluya R Oseghale^{1

2}, Liwen Xu^{1

2}, Mara Pavel-Dinu^{1

2}, Sofia E Luna^{1

2}, M Kyle Cromer^{1

2

7}, Ruhi Sayana^{1

2}, Natalia Gomez-Ospina^{1

2}, Matthew H Porteus^{8

9}

Affiliations

¹ Department of Pediatrics, Stanford University, Stanford, CA, USA.
² Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA, USA.
³ Immunology Department, Lyon Sud University Hospital, Pierre-Bénite, France.
⁴ International Center of Research in Infectiology, Lyon University, INSERM U1111, CNRS UMR 5308, ENS, UCBL, Lyon, France.
⁵ Center for Gene Therapy, Abigail Wexner Research Institute, Nationwide Children's Hospital, Columbus, OH, USA.
⁶ Department of Pediatrics, The Ohio State University, Columbus, OH, USA.
⁷ Department of Surgery, University of California, San Francisco, San Francisco, CA, USA.
⁸ Department of Pediatrics, Stanford University, Stanford, CA, USA. mporteus@stanford.edu.
⁹ Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA, USA. mporteus@stanford.edu.

PMID: 37537500
DOI: 10.1038/s41587-023-01888-4

Abstract

Therapeutic applications of nuclease-based genome editing would benefit from improved methods for transgene integration via homology-directed repair (HDR). To improve HDR efficiency, we screened six small-molecule inhibitors of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a key protein in the alternative repair pathway of non-homologous end joining (NHEJ), which generates genomic insertions/deletions (INDELs). From this screen, we identified AZD7648 as the most potent compound. The use of AZD7648 significantly increased HDR (up to 50-fold) and concomitantly decreased INDELs across different genomic loci in various therapeutically relevant primary human cell types. In all cases, the ratio of HDR to INDELs markedly increased, and, in certain situations, INDEL-free high-frequency (>50%) targeted integration was achieved. This approach has the potential to improve the therapeutic efficacy of cell-based therapies and broaden the use of targeted integration as a research tool.