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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.

Tumor-infiltrating lymphocytes (TILs) may express different inhibitory receptors (IRs) on the cell surface that are exploited by cancer cells to evade the immune attack. Thus, exhausted T cells display an unresponsive functional profile and fail to kill tumor cells. TIM-3 and LAG-3 represent highly expressed inhibitory receptors in exhausted T cells infiltrating solid tumors. We recently showed that a large fraction of bone marrow infiltrating CD8+ cells co-express multiple inhibitory receptors, including TIM-3, in patients with acute myeloid leukemia relapsing after allogeneic hematopoietic stem cell transplantation. These observations advocate for strategies to generate innovative tumor-specific T cell products resistant to inhibitory signals and able to persist long-term in treated patients. To reach these objectives we developed a strategy to simultaneously redirect T cell specificity by TCR gene transfer and permanently disrupt IRs by CRISPR/Cas9 system in long-living memory stem T cells (TSCM) for adoptive cell therapy. Primary T cells were activated with CD3/CD28-conjugated beads and cultured with low doses of IL7 and IL15, to preserve their TSCM phenotype, and electroporated with Cas9/gRNA ribonucleoproteins (RNPs) targeting a coding sequence of TIM-3,LAG3, 2B4 and of the TCR α and β chain constant region (TRAC and TRBC1/2) genes. Each gene was edited alone or in combination in a multiplex approach. The efficiencies of NHEJ-mediated inactivation of each gene were assessed by cytofluorimetric analysis and confirmed at molecular level by surveyor assay or ddPCR. In order to avoid mispairing between the endogenous TCR and the tumor-specific TCR, we simultaneously inactivated the constant regions of α and β chain of the endogenous TCR genes. Efficiency of TRAC and/or TRBC1/2 gene disruption was 98%, when measured as % of CD3neg cells. TCR knocked-out cells could be efficiently (70-85%) transduced with a lentiviral vector encoding for TCR specific for an HLA-A2 restricted peptide from NY-ESO1. We then focused on IR gene inactivation. Single gene editing produced up to 98%, 93% and 63% genetic knock-out of the TIM-3 , the LAG-3 and the 2B4 genes, respectively. We finally combined one IR inactivation with TCR gene editing in a single protocol. We obtained an average of 68% and 69% of TIM3neg and LAG3neg NY-ESO1-TCR redirected T cells, respectively, and more than 90% of edited cells showed a TSCM phenotype. When tested in functional assays, TCR-IR-edited TSCM cells proved highly effective and specific in killing HLA-A2+ NY-ESO1+ multiple myeloma cells. Of notice, TIM-3neg and LAG-3neg cells proved more effective in producing IFNg, TNFa and CD107a, once challenged with tumor cells. In conclusion, by combining the versatility of multiplex gene editing by CRISPR/Cas9 with culture conditions designed to engineer TSCM cells, we can generate innovative tumor-specific cellular products redirected against tumor antigens and resistant to inhibitory signals. Ongoing in vivo evaluation of tumor-specific IR disrupted memory stem T cells will be reported and discussed. Disclosures Bonini: Intellia Therapeutics: Research Funding.