X-linked lymphoproliferative disease (XLP1) results from SH2D1A gene mutations affecting the SLAM-associated protein (SAP). A regulated lentiviral vector (LV), XLP-SMART LV, designed to express SAP at therapeutic levels in T, NK, and NKT cells, is crucial for effective gene therapy. We experimentally identified 34 genomic regulatory elements of the SH2D1A gene and designed XLP-SMART LVs to emulate the lineage and stage-specific control of SAP. We screened them for their on-target enhancer activity in T, NK, and NKT cells and their off-target enhancer activity in B cell and myeloid populations. In combination, three enhancer elements increased SAP promoter expression up to 4-fold in on-target populations in vitro. NSG-Tg(Hu-IL15) xenograft studies with XLP-SMART LVs demonstrated up to 7-fold greater expression in on-target cells over a control EFS-LV, with no off-target expression. The XLP-SMART LVs exhibited stage-specific T and NK cell expression in peripheral blood, bone marrow, spleen, and thymic tissues (mimicking expression patterns of SAP). Transduction of XLP1 patient CD8+ T cells or BM CD34+ cells with XLP-SMART LVs restored restimulation-induced cell death and NK cytotoxicity to wild-type levels, respectively. These data demonstrate that it is feasible to create a lineage and stage-specific LV to restore the XLP1 phenotype by gene therapy.

CD3δ SCID is a devastating inborn error of immunity caused by mutations in CD3D, encoding the invariant CD3δ chain of the CD3/TCR complex necessary for normal thymopoiesis. We demonstrate an adenine base editing (ABE) strategy to restore CD3δ in autologous hematopoietic stem and progenitor cells (HSPCs). Delivery of mRNA encoding a laboratory-evolved ABE and guide RNA into a CD3δ SCID patient's HSPCs resulted in a 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 the preclinical efficacy of ABE in HSPCs for the treatment of CD3δ SCID, providing a foundation for the development of a one-time treatment for CD3δ SCID patients.