444. ADP-Overexpressing Adenovirus Elicits Enhanced Cytopathic Effect by Induction of Apoptosis
Authors: Zeng B. Zhu, Sharmila K. Makhija, Baogen Lu, Minghui Wang, Angel A. Rivera, Gene P. Siegal, Ronald D. Alvarez, David T. Curiel
Journal: Molecular Therapy (2004)

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Conditionally replicating adenoviruses (CRAds) represent a promising new modality for the treatment of cancer. A key contribution in this regard was the introduction of tumor-selective viral replication for amplification of the initial inoculums. Specifically, following cellular infection, the virus replicates selectively in the infected tumor cells and kills the cells by cytolysis. Next, the progeny virions infect surrounding target cells, replicate and eradicate the infected tumor cells, leaving normal cells unaffected. Similar to adenoviral vectors, however, CRAd agents are limited in their use in cancer gene therapy for two main reasons: they have low infection efficacy due to paucity of expression of the primary adenovirus receptor, coxsackie-adenovirus receptor (CAR), and they lack specificity, thus infecting normal cells, which leads to the toxicity. In this study, we propose an improved CRAd agent to overcome these two obstacles. We constructed a novel CRAd agent, CRAd-Survivin, which overcomes the limitations described above and has the following characteristics: 1) The E1 gene of the adenovirus, which responses to adenoviral replication, is driven by a novel tumor specific promoter (tsp), the survivin promoter; 2) The vector maintains the E3 region containing the sequence coding the “death domain” to aid the cytocidal effect; 3) A modified RGD sequence (RGD4C) is incorporated into the HI loop of the fiber, which increases the infectivity in target cells; 4) We constructed the CRAd-Surivin by inserting a SV40 poly-A signal sequence between the LITR and tsp. This modification halts the transcription signal from LITR-Enh via the inserted SV40 poly-A signal. Five different cancer cell lines, including pancreatic cancer (PC-3), breast cancer (MDA-MB-361), melanoma (Mel-28), ovarian cancer (OV4), and glioma (D65), were infected with this improved CRAd agent. All cells were plated in a 24 well plate, and infected with 0, 0.1, 1.0, or 10 pfu/cell of CRAd-Survivin or a negative control, Ad-Survivin, which is a non-replicating agent. The cytocidal effect of the CRAd agent was detected by crystal violet stain, and the cell viability of tumor was measured by exclusion of Trypan Blue stain.

The viabilities of D65 and MDA-MB-361 cells were 3.3% and 1.0%, respectively, 10 days post-infection at 0.1 pfu/cell of CRAd-Survivin. In contrast, the viabilities were 97% and 80% 10 days post-infection at 0.1 pfu/cell of Ad-Survivin, the non-replicative control. The viabilities of PC-3, Mel-28, and OV4 were 70.0%, 30%, and 95%, respectively, under the same experimental conditions. Further, strong cytocidal effects were observed in different glioma cell lines, including U118, U373, U87, and M59. These preliminary data indicate that an infectivity enhanced, tumor-specific CRAd agent, CRAd-Survivin, may be an excellent candidate for treatment of glioma and breast cancer by oncolysis.