title: Use of replication-conditional adenovirus as a helper system to enhance delivery of P450 prodrug-activation genes for cancer therapy.
abstract: 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.
authors.affiliation: Division of Cell and Molecular Biology, Department of Biology, Boston University, Boston, Massachusetts 02215, USA.
authors.forename: Youssef
authors.initials: Y
authors.lastname: Jounaidi
authors.forename: David J
authors.initials: DJ
authors.lastname: Waxman
mesh_terms: D000256:Adenoviridae / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival; D065702:Cytochrome P-450 CYP2B6; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007668:Kidney; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000378:metabolism; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D014779:Virus Replication
chemical_list: D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D010089:Oxidoreductases, N-Demethylating; D009251:NADPH-Ferrihemoprotein Reductase