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 language | English |
|---|---|
| Article number | 6771 |
| Journal | Nature Communications |
| Volume | 14 |
| Issue number | 1 |
| DOIs | |
| State | Published - 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.).
| Funders | Funder number |
|---|---|
| Hendel Labs | |
| IPMP | 3115/19 A.H. |
| Israel Science Foundation (ISF)—Israel Precision Medicine Partnership | |
| Technion Genomics Center | |
| European Commission | |
| Israel Science Foundation | 2031/19 A.H. |
| Horizon 2020 | 755758 |