425. Production of Gene Corrected T Cell Precursors for Therapy of SCID X1

Gene therapy for SCID and CID usually leads to a more efficient T-cell reconstitution as compared to HSCT from a mismatched related donor. However, as observed in ADA, SCID X1 and WAS gene therapy trials, T cell recovery is sometimes slow and associated with morbidity and mortality due to viral infections. In order to shorten post-transplant immunodeficiency and to reduce the frequency of these complications, one possible strategy is to transplant gene-corrected T-cell committed precursors. We have recently set up a protocol for transplanting in vitro-committed T-cell precursors able to seed the thymus and generate a wave of mature and polyclonal T-cells significantly faster than is usually observed. This protocol is based on a 3 to 7 day-culture of CD34+ cells in the presence of immobilized Notch ligand Delta-like-4 (DL-4), an adhesion molecule and a combination of cytokines; this culture system enables the in vitro generation of large amounts of T-cell precursor cells that (i) display the phenotypic and molecular signatures (i.e. gene expression pattern and TCR rearrangement pattern) of early thymic progenitor cells and (ii) have a high T-lymphopoietic potential in vitro and in vivo when transferred into NOD/SCPD/γc knock-out (NSG) mice (Reimann C, Stem Cells, 2012). The goal of the present project was to combine exposure to DL-4 and gene therapy approaches (DL-4/GT). For this purpose, gene correction of CD34+ cells from SCID-X1 patients was performed on DL-4 coated plates and the ex vivo transduced cells were either seeded on OP9/DL-1 culture or injected into NSG mice to determine their in vitro and in vivo capacity to generate mature and diverse T cells. In the DL-4/GT condition, the frequency and number of CD7+CD5+commited T-cell precursors was increased as compared to GT condition alone. Experiments to assess if the DL-4/GT condition accelerate T cell differentiation of gene corrected cells are currently ongoing. Altogether, our results suggest that a short exposure to the Notch ligand DL-4 might be an useful tool to generate large amounts of corrected T-cell precursors.