CRISPR-Cas9 RAG2 Correction via Coding Sequence Replacement to Preserve Endogenous Gene Regulation and Locus Structure

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 a first-of-its-kind RAG2 correction strategy that replaces the entire endogenous coding sequence (CDS) to preserve the critical endogenous spatiotemporal gene regulation and locus architecture. Expression of the corrective transgene led to successful development into CD3+TCRαβ+ and CD3+TCRγδ+ T cells and promoted the establishment of highly diverse TRB and TRG repertoires in an in-vitro T-cell differentiation platform. We believe that a CDS replacement technique to correct tightly regulated genes, like RAG2, while maintaining critical regulatory elements and conserving the locus structure could bring safer gene therapy techniques closer to the clinic.