Transient inhibition of 53BP1 increases the frequency of targeted integration in human hematopoietic stem and progenitor cells

Ron Baik, M. Kyle Cromer, Steve E. Glenn, Christopher A. Vakulskas, Kay O. Chmielewski, Amanda M. Dudek, William N. Feist, Julia Klermund, Suzette Shipp, Toni Cathomen, Daniel P. Dever and Matthew H. Porteus ()
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Ron Baik: Stanford University School of Medicine
M. Kyle Cromer: Stanford University School of Medicine
Steve E. Glenn: Integrated DNA Technologies, Inc.
Christopher A. Vakulskas: Integrated DNA Technologies, Inc.
Kay O. Chmielewski: Medical Center – University of Freiburg
Amanda M. Dudek: Stanford University School of Medicine
William N. Feist: Stanford University School of Medicine
Julia Klermund: Medical Center – University of Freiburg
Suzette Shipp: Stanford University School of Medicine
Toni Cathomen: Medical Center – University of Freiburg
Daniel P. Dever: Stanford University School of Medicine
Matthew H. Porteus: Stanford University School of Medicine

Nature Communications, 2024, vol. 15, issue 1, 1-14

Abstract: Abstract Genome editing by homology directed repair (HDR) is leveraged to precisely modify the genome of therapeutically relevant hematopoietic stem and progenitor cells (HSPCs). Here, we present a new approach to increasing the frequency of HDR in human HSPCs by the delivery of an inhibitor of 53BP1 (named “i53”) as a recombinant peptide. We show that the use of i53 peptide effectively increases the frequency of HDR-mediated genome editing at a variety of therapeutically relevant loci in HSPCs as well as other primary human cell types. We show that incorporating the use of i53 recombinant protein allows high frequencies of HDR while lowering the amounts of AAV6 needed by 8-fold. HDR edited HSPCs were capable of long-term and bi-lineage hematopoietic reconstitution in NSG mice, suggesting that i53 recombinant protein might be safely integrated into the standard CRISPR/AAV6-mediated genome editing protocol to gain greater numbers of edited cells for transplantation of clinically meaningful cell populations.

Date: 2024
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DOI: 10.1038/s41467-023-43413-w

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