A New Specific Promoter Allow Hematopoietic Stem Cell Immunotherapy Approach Against Acute Lymphoblastic Leukemia Using Chimeric Antigen Receptor

Although efficient, Chimeric Antigen Receptor (CAR)-T cells therapy still presents several drawbacks including exhaustion and loss of engineered cells, and the cytokine release syndrome. To circumvent these issues, we envisioned to engineer hematopoietic stem cells (HSCs) to provide a continuous replenishment of CAR-T and CAR-NK cells. However, this would result in a potentially dangerous pan-hematopoietic expression of the CAR transgene. To avoid this, we designed a T-cell specific and a NK-cell specific synthetic promoter in order to restrict the CAR expression only to the cytotoxic cells progeny issued from CAR-modified HSCs. We assessed the efficacy of our promoters in vitro and in vivo in humanized mice. Methods Potential sequences for T-cell or NK-cell specific expression were designed in silico and then cloned in a GFP-reporter vector to test their specificity. We assessed in vitro the expression of the reporter gene using cell lines from various lineages and primary cells isolated from peripheral blood. We then transduced CD34+ cell and humanized NSG mice engrafted with a human thymus. Results Upon transfection with GFP under the control of our synthetic promoter, only Jurkat (T cells) for our T-specific promoter and NK-92 (NK cells) for the NK-specific promoter, expressed GFP. Consistently, nucleofection of PBMC with the T-cell specific promoter resulted in a GFP expression in T cells but not in B cells or monocytes. The promoter specificity was validated in vivo as progeny from engineered human HSCs injected in NSG showed the same T-cell specific expression pattern (Fig. 1). Using an in vitro artificial organoid thymic differentiation system, we observed that our synthetic promoter was active as early as CD7+C+D1 progenitor cells. We also showed that T cells transduced with our specific promoter retained CAR T-cell toxicity when facing their targets, despite a lower CAR expression than with classic strong promoter. Conclusion Our results show that we were able to generate new synthetic promoters with a lineage specificity. We are currently testing our strategy in vivo against human B-ALL cells lines and primary patients' blasts. We are also validating our new NK-specific promoter in vivo. This new strategy could help overcome side effects while improving CAR-T cells persistence, and could be used for both hematological malignancies and solid tumors after autologous transplantation.