Adenine Base Editing of Hematopoietic Stem Cells Rescues T-Cell Development for CD3d Severe Combined Immune Deficiency

CD3d SCID is a devastating inborn error of immunity caused by mutations in CD3D, encoding the invariant CD3d chain of the CD3/TCR complex necessary for normal thymopoiesis. We demonstrate an adenine base editing (ABE) strategy to restore CD3d in autologous hematopoietic stem and progenitor cells (HSPC). Delivery of mRNA encoding a laboratory-evolved ABE and guide RNA into CD3d SCID patient's HSPCs resulted in 71.2±7.85% (n=3) correction of the pathogenic mutation. Edited HSPCs differentiated in artificial thymic organoids produced mature T cells exhibiting diverse TCR repertoires and TCR-dependent functions. Edited human HSPCs transplanted into immunodeficient mice showed 88% reversion of the CD3D defect in human CD34+ cells isolated from mouse bone marrow after 16 weeks, indicating correction of long-term repopulating HSCs. These findings demonstrate preclinical efficacy of ABE in HSPC for the treatment of CD3d SCID, providing a foundation for the development of a one-time treatment for CD3d SCID patients. Overall design: scRNA-sequencing of HSPC-derived T-lineage