PARENT SESSION
1:30 PM to 3:30 PM
Tuesday, April 23, 2002
Poster Session 16 Gene Therapy
Room: Nevada Exhibition Center
(P21-222) A Novel EBV-Targeted Adenoviral Vector Utilizing a Non-Cleavable FasL for Nasopharyngeal Cancer Therapy.
LI, Jian-Hua1, SHI, Willa1, CHIA, Marie1, MEDIN, Jeff1, HUANG, Dolly2, KLAMUT, Henry1, YEH, Wen-Chen3, LIU, Fei-Fei*,1, 1 Ontario Cancer Institute, Toronto, Ontario2 Chinese University of Hong Kong, Hong Kong3 Amgen Research Institute, Toronto
ABSTRACT-
Purpose: We had achieved nasopharyngeal carcinoma (NPC)-specific gene expression by modifying the promoter with the addition of family of repeat (FR) sequences, which upon binding by EBNA1 will activate transcription (denoted as oriP). This targeting strategy now allows us to utilize the death gene FasL, which was improved by deleting its putative cleavage sites (AA 103-136), thereby reducing the risk of hepatotoxicity. Materials & Methods Two novel replication deficient adenoviruses were thus made: adv.oriP.luc and adv.oriPncFasL (non-cleavable FasL). EBV-negative CNE-2Z, and EBV-positive C666-1 NPC cells were utilized. FasL protein expression was examined using Western blot (Santa Cruz); cell viability was assessed using the MTT assay. Apoptosis was quantitated with acridine-orange/ethidium bromide. Caspase 3 and 8 activities were measured using a fluorometric technique (Clontech); caspase 8 activity was inhibited using Z-LETD (Enzymesys). Results Infection with adv.oriPncFasL resulted in a dose-dependent increase in FasL expression only in C666-1 cells. A dose-dependent reduction in viability was observed for the C666-1 cells, with 9% survival 6 days after exposure to adv.oriPncFasL (25 pfu/cell), which was further reduced to 0.8% with the addition of 6 Gy. The proportion of apoptotic cells increased from 3% (control) to 41% with adv.oriPncFasL (25 pfu/cell), which increased slightly to 47% with the addition of 6 Gy. Minimal cytotoxicity (<10%) was observed when C666-1 cells were treated with adv.oriP.luc, or when EBV-negative CNE-2Z cells were exposed to adv.oriPncFasL. Casp-8 and casp-3 activities were induced 4 and 10-fold respectively, in a dose-dependent manner (0-25 pfu/cell of adv.oriPncFasL). The addition of the Z-LETD (10 uM) completely abolished casp8 activity; casp3 activity was reduced close to basal levels, indicating that our therapeutic strategy is inducing apoptosis through the predicted Fas death receptor pathway. Conclusion We have successfully developed a novel adenoviral vector that incorporates EBV-specific gene expression of a mutant FasL, which achieves significant cytotoxicity, mediated primarily by apoptosis, through the Fas pathway. This strategy will be next evaluated in an in vivo system, to determine its full therapeutic potential and safety.
KEYWORDS: cancer gene therapy, nasopharyngeal cancer, FasL, apoptosis