Retroviral UNC13D Gene Transfer Restores Cytotoxic Activity of T Cells Derived from Familial Hemophagocytic Lymphohistiocytosis Type 3 Patients In Vitro.

Familial hemophagocytic lymphohistiocytosis (FHL) is a group of life-threatening, autosomal recessive disorders of severe hyperinflammation. FHL type 3 (FHL-3) accounts for about 30% of FHL cases. It is characterized by mutations in the UNC13D gene that give rise to functionally impaired or absent Munc13-4 protein, resulting in impaired secretion of lytic granules by cytotoxic lymphocytes. Etoposide-based therapy is currently used as the standard of care that results in around 60% 5-year survival, illustrating the need for novel treatment approaches. Key problems include treatment toxicity and failure to induce or maintain remission of the hyperinflammation. Instead of immunosuppression, transplantation of autologous gene-corrected T cells can be envisaged as an approach to restore the impaired immune reaction. This study established a protocol that enabled hyperactivated, FHL-3 patient-derived T cells to be cultured and a codon-optimized UNC13D expression cassette to be delivered by either alpha- or gamma-retroviral gene transfer. The data demonstrate that the established protocol can be applied to FHL-3 patient cells with various genetic backgrounds and that gamma-retroviral UNC13D transfer restored expression of functional Munc13-4, as well as degranulation capacity and cell-mediated cytotoxicity of those patient-derived CD8+ T cells. Furthermore, the study shows that the co-introduction of a truncated low-affinity nerve growth factor receptor coding sequence enabled the therapeutic effect to be optimized by enriching transduced cells in a Good Manufacturing Practice-compliant manner. In conclusion, this study lays the foundation for an adaptive immune cell therapy approach aiming at immunological stabilization of FHL-3 patients with autologous, immune-competent T cells prior to hematopoietic stem-cell transplantation.