RAG1 lentiviral gene therapy restores T-cell development of RAG1-SCID patient cells in artificial thymic organoids

Recombination activating gene 1 (RAG1) is essential for variable diversity joining recombination during early T- and B-cell development. Null mutations cause a complete block in receptor rearrangement, resulting in T-B- severe combined immunodeficiency (SCID). Patients with RAG1-SCID require hematopoietic stem cell transplantation for survival. Our phase I/II clinical trial (NCT04797260) is currently evaluating lentiviral RAG1 gene addition in autologous hematopoietic stem and progenitor cells (HSPCs). However, studying early human T-cell development is challenging due to limited access to thymic tissue. The artificial thymic organoid (ATO) system offers a promising in vitro model to study human T-cell differentiation. Here, we show that ATO cultures efficiently support T-cell development from healthy donor HSPCs derived from umbilical cord blood or mobilized peripheral blood, yielding not only αβ but also γδ T cells with a polyclonal T-cell receptor (TCR) repertoire. In contrast, noncorrected RAG1-deficient HSPCs from 3 RAG1-SCID patients show a developmental arrest before or at the aberrant CD4+CD8dim double-positive stage, characterized by minimal or absent CD1a upregulation and CD7 downregulation, absence of TCRβ rearrangement, and only partial TCRγ and TCRδ rearrangement. Lentiviral RAG1 gene addition using the clinical vector rescues T-cell development in these patient-derived HSPCs and restores TCR repertoire diversity. These findings highlight the ATO system as a valuable model for dissecting human T-cell development and for the preclinical development and evaluation of gene therapy.