Institute of Nanotechnology and Advanced Materials, The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, 5290002, Israel.
The Division of Hematology and Bone Marrow Transplantation, Chaim Sheba Medical Center, Tel-Hashomer, Ramat Gan, 5266202, Israel.
Nat Commun. 2023 Oct 27;14(1):6771. doi: 10.1038/s41467-023-42036-5.
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 CD3TCRαβ and CD3TCRγδ 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.
RAG2-SCID 是一种由重组激活基因 2（RAG2）突变引起的原发性免疫缺陷病，该基因与淋巴细胞成熟和功能过程密切相关。使用 CRISPR-Cas9/rAAV6 基因编辑对患者自身造血干细胞和祖细胞（HSPC）进行体外操作，可以为唯一的现有治疗方法，异基因造血干细胞移植（HSCT）提供治疗选择。在这里，我们展示了一种创新的 RAG2 纠正策略，该策略替代了整个内源性编码序列（CDS），目的是保留关键的内源性时空基因调控和基因座结构。纠正转基因的表达导致 CD3TCRαβ 和 CD3TCRγδ T 细胞的成功发育，并在体外 T 细胞分化平台中促进高度多样化的 TRB 和 TRG 库的建立。因此，我们的概念验证研究为受严格调控基因的更安全的基因治疗技术提供了希望。
