In vivo gene therapy: Using a recombinant vaccinia virus as a cytokine gene delivery system to localized murine tumors

Gene therapy of cancer is emerging as a new approach for immunotherapy. Genetically modified tumor cells which secrete different cytokines have been shown to elicit protective anti-tumor effects in experimental animals. However, in vitro modification of tumor cells or immune effector cells from individual patients is not always clinically feasible. Thus, the focus of this study was to investigate the potential of using recombinant vaccinia virus for intratumoral cytokine gene transfer based on the following criteria: (1) in vivo infectability, (2) the ability to introduce encoded gene products into tumor cells and (3) the expression of encoded gene product in the presence of pre-existing anti-vaccinia immunity. The first objective was to determine if encoded gene transfer could be demonstrated using vaccinia recombinants H1-VAC and NP-VAC encoding influenza hemagglutinin and nucleoprotein, respectively, as reporter constructs. Using a murine bladder tumor model, intravesical instillation of H1-VAC was shown to induce systemic anti-vaccinia immunity as defined by vaccinia specific CTLs. Vaccinia recombinants were demonstrated to efficiently mediate local gene transfer of encoded reporter genes into murine bladder tumors in vitro and established tumors in vivo. Of significant importance, intratumoral gene transfer using vaccinia recombinants was achieved in mice preimmunized to vaccinia. Thus, vaccinia virus recombinants were demonstrated to effectively introduce encoded gene products locally into tumors in situ, even in the presence of pre-existing immunity to vaccinia, supporting its use for in vivo gene transfer. The second objective was aimed at evaluating if a cytokine producing vaccinia recombinant could efficiently mediate cytokine gene transfer into tumors in situ. A recombinant vaccinia virus encoding the murine granulocyte macrophage-colony stimulating factor gene (GM-CSF; referred to as VV-GM) was constructed under the control of the vaccinia early/late P$\sb{7.5}$ promoter. Significant amounts of biologically active cytokine were detected in supernatants from VV-GM infected tumor cells as determined by bioassay and ELISA. Using a murine melanoma tumor model, systemic immunity to vaccinia virus was shown not to inhibit infection/transfection of VV-GM in vivo. Using specially designed vaccinia specific RT-PCR primers, kinetic studies showed that virally encoded GM-CSF mRNA was detected in tumors transfected with VV-GM up to 72 hrs and 24 hrs in naive and virus immune mice, respectively. The intratumoral transfection of VV-GM was shown to recruit host effector cells to the local tumor site and inhibit established B16. F10 melanoma tumor growth. In sum, these findings demonstrated the feasibility of using recombinant vaccinia virus as a cytokine delivery system to localized tumors for in vivo gene therapy