• 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.

      Cowen, Rachel L.; Williams, Judith C.; Emery, Steve; Blakey, David; Darling, John L.; Lowenstein, Pedro R.; Castro, Maria G. (nature.com, 2002)
      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.
    • Adenovirus-mediated expression of HSV1-TK or Fas ligand induces cell death in primary human glioma-derived cell cultures that are resistant to the chemotherapeutic agent CCNU.

      Maleniak, Tricia C.; Darling, John L.; Lowenstein, Pedro R.; Castro, Maria G. (Nature Publishing Group, 2001)
      Due to minimal treatment success with surgery, radiotherapy, and chemotherapy, the aim of this study was to test the therapeutic potential of gene therapy for the treatment of glioblastoma multiforme (GBM). We have quantitatively analyzed two gene therapy approaches using short-term human glioma cell cultures derived from surgical biopsies (designated IN859, IN1612, IN2045, IN1760, and IN1265) and compared the results of gene therapy with the chemosensitivity of the same cells. All of the glioma cell cultures tested were susceptible to recombinant adenovirus (RAd)-mediated infection. Expression of herpes simplex virus type 1-thymidine kinase (RAd128), followed by ganciclovir treatment, induced apoptosis in all of the glioma cell cultures studied, including three that are resistant to the chemotherapeutic drug 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU). Expression of murine Fas ligand (RAdhCMV-mFasL) also induced cell death in four of the five cell cultures studied. One cell culture that was resistant to CCNU was also resistant to apoptosis induced by mFasL expression. These results suggest that sensitivity to chemotherapeutic agents does not necessarily correlate with the sensitivity to gene therapy treatments. RAds expressing therapeutic gene products in human glioma cell cultures are able to induce apoptosis even in some cells that are resistant to a commonly used chemotherapeutic agent. Therefore, RAd-mediated gene transfer could be a good candidate to further develop gene therapy for the treatment of GBM.