title: Adenovirus vector-mediated delivery of the prodrug-converting enzyme carboxypeptidase G2 in a secreted or GPI-anchored form: High-level expression of this active conditional cytotoxic enzyme at the plasma membrane.
abstract: Carboxypeptidase G2 (CPG2) is a powerful prodrug-converting enzyme. Without a requirement for endogenous enzymes or cofactors, it can directly activate mustard alkylating prodrugs to cytotoxic species, killing both quiescent and dividing cells. This paper provides the first report of its use in the context of a clinically relevant delivery vehicle using adenovirus vectors. To strengthen the efficacy of the prodrug-activating system, the enzyme has been engineered to be secreted or glycosylphosphatidylinositol (GPI) anchored to the extracellular membrane of tumor cells, resulting in an enhanced bystander effect by facilitating diffusion of the active drug through extracellular, rather than intracellular, activation. Using the vectors, we have achieved expression of functional secreted or GPI-anchored CPG2 in a panel of tumor cell lines demonstrating no loss in efficacy as a result of GPI anchor retention. Despite variable transduction efficiencies inherent to these vectors, greater than 50% cell kill was achievable in all of the cell lines tested following only a single exposure to the prodrug ZD2767P. Even in cell lines refractive to infection with the vectors, substantial cell death was recorded, indicative of the enhanced bystander effect generated following extracellular prodrug activation. A direct evaluation of the efficacy of our system has been made against adenoviral delivery of herpes simples virus thymidine kinase plus ganciclovir (GCV), a suicide gene therapy approach already in the clinic. In a short-term human glioma culture (IN1760) resistant to the clinical chemotherapeutic drug CCNU (1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea), thymidine kinase/GCV effected no cell killing compared to 70% cell killing with our system.
authors.affiliation: Molecular Medicine and Gene Therapy Unit, School of Medicine, University of Manchester, UK.
authors.forename: Rachel L
authors.initials: RL
authors.lastname: Cowen
authors.forename: Judith C
authors.initials: JC
authors.lastname: Williams
authors.forename: Steve
authors.initials: S
authors.lastname: Emery
authors.forename: David
authors.initials: D
authors.lastname: Blakey
authors.forename: John L
authors.initials: JL
authors.lastname: Darling
authors.forename: Pedro R
authors.initials: PR
authors.lastname: Lowenstein
authors.forename: Maria G
authors.initials: MG
authors.lastname: Castro
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology*; D002470:Cell Survival; D017931:DNA Primers; D005091:Exons; D005822:Genetic Vectors; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011355:Prodrugs / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D018800:Thy-1 Antigens / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured; D011623:gamma-Glutamyl Hydrolase / Q000235:genetics* / Q000378:metabolism
chemical_list: D017931:DNA Primers; D017261:Glycosylphosphatidylinositols; D011355:Prodrugs; D011993:Recombinant Fusion Proteins; D018800:Thy-1 Antigens; D011623:gamma-Glutamyl Hydrolase