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, Ayal Hendel

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

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.

Original languageEnglish
Article number6771
JournalNature Communications
Volume14
Issue number1
DOIs
StatePublished - 27 Oct 2023

Bibliographical note

Publisher Copyright:
© 2023, Springer Nature Limited.

Funding

We would like to thank the members of the Somech and the Hendel Labs for reading the manuscript and providing practical advice. Additionally, we would like to thank D. Russell for providing the pDGM6 plasmid. We give special thanks to the Technion Genomics Center team, and especially Dr. Nitsan Fourier, for excellent technical assistance and support with the long-read sequencing. This study was supported in part by research funding from the European Research Council (ERC) under the European Union Horizon 2020 research and innovation program (Grant No. 755758 A.H.) as well as the Israel Science Foundation (ISF)—Israel Precision Medicine Partnership (IPMP) (Grant No. 3115/19 A.H.) and Israel Science Foundation (ISF)—Individual Research Grants (Grant No. 2031/19 A.H.). We would like to thank the members of the Somech and the Hendel Labs for reading the manuscript and providing practical advice. Additionally, we would like to thank D. Russell for providing the pDGM6 plasmid. We give special thanks to the Technion Genomics Center team, and especially Dr. Nitsan Fourier, for excellent technical assistance and support with the long-read sequencing. This study was supported in part by research funding from the European Research Council (ERC) under the European Union Horizon 2020 research and innovation program (Grant No. 755758 A.H.) as well as the Israel Science Foundation (ISF)—Israel Precision Medicine Partnership (IPMP) (Grant No. 3115/19 A.H.) and Israel Science Foundation (ISF)—Individual Research Grants (Grant No. 2031/19 A.H.).

FundersFunder number
Hendel Labs
IPMP3115/19 A.H.
Israel Science Foundation (ISF)—Israel Precision Medicine Partnership
Technion Genomics Center
European Research Council
Israel Science Foundation2031/19 A.H.
Horizon 2020755758

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