Precise Correction of Patient-Derived HSPCs Enables T Cell Differentiation in a Personalized SCID Gene Editing Strategy

Severe Combined Immunodeficiency (SCID) is a fatal inborn error of immunity that demands rapid diagnosis and early therapeutic intervention. With newborn screening programs, we can now identify causative mutations within days after birth and isolate hematopoietic stem and progenitor cells (HSPCs) from peripheral blood—before bone marrow transplantation is initiated. This creates a unique opportunity for fast, personalized gene correction, and in our platform, the entire process can be completed within 12 weeks from birth. We have developed a CRISPR-Cas9–based gene editing strategy that employs mutation-specific sgRNAs with short single-stranded oligodeoxynucleotide (ssODN) templates encoding the corrected sequence. These templates also include silent mutations that prevent re-cutting and enhance the precision of homology-directed repair (HDR). By preserving the endogenous regulatory elements, this approach maintains physiological gene expression levels, which are critical for achieving durable immune reconstitution—an advantage over conventional gene addition methods. To date, we have successfully applied this strategy to a patient with ARTEMIS deficiency, demonstrating efficient correction and restored T cell differentiation in vitro. Ongoing projects are focused on targeting RAG1, RAG2, IL7Rα, and RFX5. We also improve HDR efficiency using inhibitors of non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ), while minimizing toxicity. Corrected HSPCs give rise to mature CD3⁺, CD4⁺, and CD8⁺ T cells, and TCR repertoire analysis supports robust polyclonal immune regeneration. While further studies are needed to establish the safety and scalability of this approach, our findings offer a compelling proof-of-concept for rapid, precise, and personalized CRISPR gene editing for SCID, directly utilizing patient-derived cells.