A Rapamycin-Activated Caspase 9-Based Suicide Gene

Abstract

Engineered T cell therapies show considerable promise in the treatment of refractory malignancies. Given the ability of engineered T cells to engraft and persist for prolonged periods along with unpredicted toxicities, incorporation of a suicide gene to allow selective depletion after administration is desirable. Rapamycin is a safe and widely available immunosuppressive pharmaceutical that acts by heterodimerization of FKBP12 with the FRB fragment of mTOR. The apical caspase caspase 9 is activated by homodimerization through its CARD domain. We developed a rapamycin-induced caspase 9 suicide gene. First, we showed that caspase 9 could be activated by a two-protein format with replacement of the CARD domain with both FRB and FKBP12. We next identified an optimal compact single-protein rapamycin caspase 9 (rapaCasp9) by fusing both FRB and FKBP12 with the catalytic domain of caspase 9. Functionality of rapaCasp9 when co-expressed with a CD19 CAR was demonstrated in vitro and in vivo.

Keywords: cancer; immunotherapy; suicide genes.