CRISPR-Cas9 engineering of the RAG2 locus via complete coding sequence replacement for therapeutic applications

Daniel Allen, Orli Knop, Bryan Itkowitz, Nechama Kalter, Michael Rosenberg, Ortal Iancu, Katia Beider, Yu Nee Lee, Arnon Nagler, Raz Somech and Ayal Hendel ()
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Daniel Allen: Bar-Ilan University
Orli Knop: Bar-Ilan University
Bryan Itkowitz: Bar-Ilan University
Nechama Kalter: Bar-Ilan University
Michael Rosenberg: Bar-Ilan University
Ortal Iancu: Bar-Ilan University
Katia Beider: The Division of Hematology and Bone Marrow Transplantation, Chaim Sheba Medical Center, Tel-Hashomer
Yu Nee Lee: Tel Aviv University
Arnon Nagler: The Division of Hematology and Bone Marrow Transplantation, Chaim Sheba Medical Center, Tel-Hashomer
Raz Somech: Tel Aviv University
Ayal Hendel: Bar-Ilan University

Nature Communications, 2023, vol. 14, issue 1, 1-16

Abstract: Abstract RAG2-SCID is a primary immunodeficiency caused by mutations in Recombination-activating gene 2 (RAG2), a gene intimately involved in the process of lymphocyte maturation and function. ex-vivo manipulation of a patient’s own hematopoietic stem and progenitor cells (HSPCs) using CRISPR-Cas9/rAAV6 gene editing could provide a therapeutic alternative to the only current treatment, allogeneic hematopoietic stem cell transplantation (HSCT). Here we show an innovative RAG2 correction strategy that replaces the entire endogenous coding sequence (CDS) for the purpose of preserving the critical endogenous spatiotemporal gene regulation and locus architecture. Expression of the corrective transgene leads to successful development into CD3+TCRαβ+ and CD3+TCRγδ+ T cells and promotes the establishment of highly diverse TRB and TRG repertoires in an in-vitro T-cell differentiation platform. Thus, our proof-of-concept study holds promise for safer gene therapy techniques of tightly regulated genes.

Date: 2023
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DOI: 10.1038/s41467-023-42036-5

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