Abstract Hyper-IgM Type 1 (HIGM1) is caused by mutations of CD40L, whose absence in CD4 T cells impairs signaling for B cell activation and Ig class-switching. Since unregulated CD40L expression leads to lymphoproliferations/lymphomas in the mouse model of the disease, gene correction must preserve the physiological regulation of the gene. Gene editing of either autologous T cells or hematopoietic stem cells (HSC) held promise for treating HIGM1. We developed a "one size fits all" editing strategy to insert a 5'-truncated corrective CD40L cDNA in the first intron of the native human gene, effectively making expression conditional to targeted insertion in the intended locus. By exploiting a protocol that preserves T stem memory cells (TSCM), we reproducibly obtained ~40% of editing efficiency in healthy donor and patients derived T cells, restoring regulated, although partial, CD40L surface expression. The reconstituted level of expression, however, was sufficient to fully restore helper function to B cells. In order to select, track and potentially deplete edited T cells, we coupled the corrective cDNA with a clinically compatible selector gene and confirmed that enriched T cells preserved their engraftment capacity in NSG mice. Unexpectedly, the presence of an IRES-linked downstream coding frame counteracted the shorter half-life of transcript from the edited locus, allowing replenishment of intracellular stores and surface translocation of physiological amounts of CD40L upon activation. We also tailored the CD40L editing strategy to human HSC, reaching up to 15-30% editing in HSC long term engrafting NSG mice, depending on the HSC source. We then modelled the therapeutic potential of both T cell and HSC gene therapy by infusing increasing proportions of WT murine cells, as surrogates of edited cells, in HIGM1 mice. Administration of functional T cells at clinically relevant doses in HIGM1 mice, preconditioned or not with different lymphodepleting regimens, achieved long term stable T cell engraftment and partial rescue of antigen specific IgG response and germinal center formation in splenic follicles after vaccination with a thymus dependent antigen. Remarkably, infusion of T cells from mice pre-exposed to the antigen, mimicking treatment of chronically infected patients, was effective even in absence of conditioning and protected the mice from a disease relevant infection induced by the opportunistic pathogen Pneumocystis murina. Transplantation of functional T cells admixed with an equal number of HIGM1 T cells resulted in lower vaccination response, indicating competition between WT and HIGM1 cells and implying that increasing the fraction of corrected cells in the graft by selection would improve immune reconstitution. Concerning HSC gene therapy, transplanting 25% WT cells along with HIGM1 ones in HIGM1 mice - mirroring the editing efficiencies achieved in human HSC - rescued antigen specific IgG response and established protection from pathogen comparably to T cell therapy. These findings suggest that autologous edited T cells can provide immediate and substantial benefits to HIGM1 patients and position T cell as competitive strategy to HSC gene therapy, because of more straightforward translation, lower safety challenges and potentially comparable clinical benefits. We thus embarked in assessing GMP compliant reagents and protocols for T cell activation, culture and editing and developed a scalable manufacturing process. Optimization of clinical grade culture conditions allowed further increasing editing efficiency, total cellular yield and maintenance of TSCM thus paving the way to the design of a clinical trial. Disclosures Naldini: Genenta Science: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Other: Founder.