232. High Levels of iCasp9 Expression Are Needed for Suicide Gene Transfer

Abstract

Primary central nervous system lymphomas are aggressive tumors with no efficient treatment. In this study, we wanted to take advantage of a lentiviral vector (LV) specifically designed to target murine MHC II expressing cells (eg. lymphoma B-cells) to evaluate the feasibility of an in vivo targeted suicide gene therapy in a murine model of primary intraocular lymphoma. The inducible caspase 9 (iCasp9) (*) strategy used efficiently in T cells was tested as a suicide gene for this approach. As a first step, we showed that 15-20% of A20. IIA lymphoma tumor cells were transduced in vitro with the MHC II targeted LV expressing iCasp9, however, apoptosis could not be induced in these cells even after enrichment and cloning. None of the stably-expressing clones could be induced to apoptose by the inductor of dimerization. These clones had integrated only 1 copy of the transgene suggesting that levels of iCasp9 were insufficient or that cells were resistant to treatment. The same results were obtained with LV expressing iCasp9 from the cellular promoters (PGK or EF1α). In contrast, clones transduced with only 1 copy of a gammaretroviral LTR-driven-iCasp9 vector were efficiently induced to apoptose. The MLV clones expressed higher levels of transgene than LV clones and 90% of the cells died 24 hours after iCasp9 activation showing that A20.IIA tumor cells were not resistant to the mechanism. Clearly, a threshold of iCasp9 expression must be achieved to induce apoptosis by gene transfer. In other cells such as 3T3 cells, Jurkat T cells, HCT116 cells and Raji cells, more than 2 copies of the LV PGK-iCasp9 must be integrated to induce apoptosis since cells expressing only 1 copy survive. Whereas this threshold of LV transduction may be easily attained in T cells, it is very difficult to integrate more than 2 copies of LV per B cell in spite of repeated cycles of infection coupled with various preactivation stimuli. We are currently exploring solutions to augment B cell transduction and to increase iCasp9 expression from LV. Altogether, our results provide benchmark values for iCasp9 efficacy and show that highly expressing vectors must be developed to apply iCasp9 suicide gene therapy strategies in B-lymphoma but also with other target cells, to avoid escape to treatment.