ANTISENSE GENE THERAPY DIRECTED AGAINST EBNA-1 IN EBVASSOCIATED B CELL LYMPHOMAS RESULTS IN POTENTIATION OF CHEMOTHERAPY IN VITRO AND DECREASED TUMOR VIRULENCE IN SCID MICE

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Background: With prolonged survival of late-stage AIDS patients, AIDS-related lymphomas (ARL) are occurring with an increased frequency, resulting in approximately 3000-9000 new cases per year. Overall prognosis remains exceedingly poor. EBV is highly associated with ARL (30-70% of systemic lymphomas and up to 100% of CNS lymphomas) and likely plays a significant role in pathogenesis. We investigated the effects of ablating EBNA-1, an EBV transforming gene, using antisense oligodeoxynucleotides (oligos). Methods: Antisense (AS) oligos corresponded to codons 16-30 of theEBNA-I open reading frame. Scrambled AS (SAS) control was created using the same nucleotides in random order. Chemotherapeutic agents included adriamycin, etoposide (VP-16), and taxol. Cell lines included human B cells transformed with EBV strain B95-8, EBV negative B cell lymphomas, and EBV negative, non-B cell tumors. Cellular proliferation was determined by tritiated thymidine uptake as a measure of cytotoxicity. Apoptosis was measured by JAM assay or fluorescent staining. In vivo studies utilized SCID mice inoculated by intraperitoneal injection with B cell tumors. Results: The addition of as little as 3 µM AS resulted in a significant reduction of the LD50 of chemotherapeutic agents (equivalent to 0.007 µg/ml of VP-16 in the presence of the LD50 of adriamycin, for example). Although both AS and SAS potentiated the effect of chemotherapy on all tumor cell lines tested, AS potentiated the cytotoxicity of chemotherapy significantly more than SAS in EBV positive cell lines, but not in EBV negative cell lines (p<0.005,t-test). Apoptosis due to chemotherapy also appeared to be enhanced by prior AS treatment in EBV positive cell lines but not in EBV negative cell lines. In SCID mice inoculated with EBV positive human B cells, prior in vitro AS treatment of tumors (without chemo) enhanced survival by two-fold (median 102 days vs 56 days for saline and 42 days for SAS). Experiments using chemo plus oligos in SCID mice are underway. Conclusions: Ablation of EBNA-1 by AS potentiates the cytotoxicity of chemotherapy in an in vitro model of ARL. Non-specific cytotoxicity due to SAS was also seen. Enhanced cytotoxicity may be related to increased apoptosis as a result of EBNA-1 ablation and/or other downstream effects. Ex vivo EBNA-1 ablation by AS also increased survival in vivo in SCID mice inoculated with these tumors. These studies provide evidence for a significant role played by EBV in ARL and may lead to therapies for ARL based on EBV gene ablation.