325. Targeted Gene Addition Strategies for the Treatment of X-Linked Lymphoproliferative Disease

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X-linked lymphoproliferative disease is an immunodeficiency arising from mutations in the SH2D1A gene encoding SAP, a key regulator of immune function expressed in T cells, natural killer, and natural killer T cells. Haemopoietic stem-cell transplantation is curative, and we have shown proof of concept of haemopoietic stem-cell gene therapy in a mouse model. Preliminary data suggests that adoptive transfer of T cells corrected using lentiviral gene therapy can correct immune defects. However, targeted gene editing of T cells may allow for physiologically regulated SAP expression. We constructed SAP-specific TALE-effector nucleases (TALENs) capable of generating a site-specific double-stranded break close to the translation start codon, allowing correction of most described SH2D1A mutations. TALEN activity was validated with a surveyor assay in HEK293T cells. To facilitate optimisation of delivery methods, we designed a fluorescent reporter assay that, when stably integrated into a cellular DNA, provides a fluorescence signal when repair of DNA double-stranded breaks (DSB) created by TALENs occurs. Using the reporter assay in Jurkat cells we observed reduced toxicity and highly efficient delivery using an mRNA nucleofection platform, with 23% of treated cells showing repair mediated fluorescence. We therefore proceeded to deliver TALEN mRNA to human primary T cells, and demonstrated the generation of double-stranded breaks in 25% of the bulk population. To harness the repair of DSB, we constructed a homologous recombination donor in a non-integrating lentiviral vector format to mediate delivery of cDNA with low toxicity. Combined with TALEN mRNA nucleofection, homologous recombination-driven incorporation of donor in T cells was possible. This methodology could be applied to an autologous T-cell gene therapy strategy, although the low level of homologous recombination remains a limiting factor. We plan to test our strategy in patient cell lines to restore SAP expression and cytotoxic function of cytotoxic T lymphocytes.