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Use of Replication-Conditional Adenovirus as a Helper System to Enhance Delivery of P450 Prodrug-Activation Genes for Cancer Therapy

Division of Cell and Molecular Biology, Department of Biology, Boston University, Boston, Massachusetts

Cytochrome P450 (CYP) gene transfer sensitizes tumor xenografts to anticancer prodrugs such as cyclophosphamide (CPA) without a detectable increase in host toxicity. Optimal prodrug activation is achieved when a suitable P450 gene (e.g., human CYP2B6) is delivered in combination with NADPH-cytochrome P450 reductase (P450R), which encodes the flavoenzyme P450 reductase. We sought to improve this gene therapy by coordinated delivery and expression of P450 and P450R on a single bicistronic vector using an internal ribosomal entry site (IRES) sequence. Retrovirus encoding a CYP2B6-IRES-P450R expression cassette was shown to induce strong P450-dependent CPA cytotoxicity in a population of infected 9L gliosarcoma cells. Adeno-P450, a replication-defective, E1/E3 region-deleted adenovirus engineered to express CYP2B6-IRES-P450R, induced intracellular CPA 4-hydroxylation, and CPA cytotoxicity, in a broad range of human cancer cell lines. However, limited Adeno-P450 gene transfer and CPA chemosensitization was seen with certain human tumor cells, notably PC-3 prostate and HT-29 colon cancer cells. Remarkable improvements could be obtained by coinfecting the tumor cells with Adeno-P450 in combination with Onyx-017, an E1b-55k gene-deleted adenovirus that selectively replicates in p53 pathway-deficient cells. Substantial increases in gene expression were observed during the early stages of viral infection, reflecting an apparent coamplification of the Adeno-P450 genome, followed by enhanced viral spread at later stages, as demonstrated in cultured tumor cells, and in A549 and PC-3 solid tumor xenografts grown in scid mice. This combination of the replication-defective Adeno-P450 with a replication-conditional and tumor cell-targeted helper adenovirus dramatically improved the low gene transfer observed with some human tumor cell lines and correspondingly increased tumor cell-catalyzed CPA 4-hydroxylation, CPA cytotoxicity, and in vivo antitumor activity in a PC-3 tumor xenograft model. The use of tumor-selective, replicating adenovirus to promote the spread of replication-defective gene therapy vectors, such as Adeno-P450, substantially increases the therapeutic potential of adenoviral delivery systems, and should lead to increased activity and enhanced tumor selectivity of cytochrome P450 and other gene-directed enzyme prodrug therapies.

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