Suicide gene therapy using the herpes simplex thymidine kinase gene and ganciclovir is an attractive strategy for solid tumors. Early animal studies involved intratumoral injection of retroviral producer cells or unprocessed supernatant to generate an antitumor effect. Xenotransplantation of producer cells proved effective in several models, but the crude supernatants from the same cells were of insufficient titer to produce antitumor effects. We have developed new non-murine producer lines that yield replication-defective retroviral vectors encoding thymidine kinase at high titer which are then further purified and processed, resulting in pharmaceutical grade retroviral vectors with titers of up to 10(8) cfu/ml. Purified, high-titer retroviral preparations were injected directly into solid tumors in two syngeneic mouse tumor models. Significant antitumor responses and some cures were observed following systemic ganciclovir therapy. Assays using monoclonal antibodies to measure thymidine kinase protein expression at the single cell level in vitro and in vivo were developed so that therapeutic transgene expression could be quantified. Intralesional delivery resulted in transduction of over 20% of tumor cells in a protocol designed to maximize transduction on the basis of separate analyses of route, dosage, and schedule of vector administration. A consensus strategy evolved in which the combined effects of increased titer and a longer duration of retroviral vector administration interact to maximize transduction efficiency. These results indicate that purified high-titer retroviral vectors have the potential to transfer effective quantities of therapeutic genes into solid tumors in human subjects and highlight some pharmacologic factors that could be valuable in the design of clinical gene therapy protocols.

An important consequence of the suicide gene therapeutic paradigm is the phenomenon of bystander cell killing, the death of adjacent tumor cells not transduced with the thymidine kinase ( TK ) gene from herpes simplex virus ( HSV ) after treatment with the antiviral drug, ganciclovir ( GCV ). Evidence from quantitative in vitro assays of glioma cell lines suggest that both murine and human gliomas are similar in expressing high sensitivity to the bystander effect. In five of six glial tumors examined, the presence of only 5% of HSV -TK -expressing transduced cells in the culture resulted in > 90% tumor cell death / stasis after addition of GCV. Several lines of evidence support gap junction intercellular communication ( GJIC ) as important in the bystander effect. In vitro metabolic assays, performed with GCV in the medium, indicated that more tumor burden was reduced when culture conditions supported cell -cell contact of parental and HSV -TK -transduced cells. Additionally, a double dye transfer assay showed that cell communication through the gap junction is greatest for glioma, less for melanoma, and much less for colorectal carcinoma cell lines. In vitro metabolic assays with mixtures of TK + / TK À homologous tumor cells confirmed that glioma cell lines were more susceptible to bystander killing than melanomas. Assays with chimeric tumor mixtures of TK + / TK À cells showed that the level of the bystander killing obtained was characteristic of the TK À bystander cells. The in vitro findings were confirmed in vivo with GCV -treated homologous and chimeric tumors composed of TK + / TK À cells. Day 21 mean tumor volumes ( MTVs ) indicated the growths obtained were characteristic of the bystander activity reflective of the nontransduced cell population. Furthermore, nontransduced, high -GJIC cells in a chimeric tumor mass appeared to effectively bridge between transduced tumor cells and poorly communicating nontransduced cells. Finally, the importance of a gap junction protein, such as connexin -43, in facilitating the bystander effect was demonstrated with the HT29 low -GJIC cell line. When the TK -nontransduced cell population expressed connexin -43, a better bystander kill was achieved compared to the parental counterpart.

An important consequence of the suicide gene therapeutic paradigm is the phenomenon of bystander cell killing, the death of adjacent tumor cells not transduced with the thymidine kinase ( TK ) gene from herpes simplex virus ( HSV ) after treatment with the antiviral drug, ganciclovir ( GCV ). Evidence from quantitative in vitro assays of glioma cell lines suggest that both murine and human gliomas are similar in expressing high sensitivity to the bystander effect. In five of six glial tumors examined, the presence of only 5% of HSV -TK -expressing transduced cells in the culture resulted in > 90% tumor cell death / stasis after addition of GCV. Several lines of evidence support gap junction intercellular communication ( GJIC ) as important in the bystander effect. In vitro metabolic assays, performed with GCV in the medium, indicated that more tumor burden was reduced when culture conditions supported cell -cell contact of parental and HSV -TK -transduced cells. Additionally, a double dye transfer assay showed that cell communication through the gap junction is greatest for glioma, less for melanoma, and much less for colorectal carcinoma cell lines. In vitro metabolic assays with mixtures of TK + / TK À homologous tumor cells confirmed that glioma cell lines were more susceptible to bystander killing than melanomas. Assays with chimeric tumor mixtures of TK + / TK À cells showed that the level of the bystander killing obtained was characteristic of the TK À bystander cells. The in vitro findings were confirmed in vivo with GCV -treated homologous and chimeric tumors composed of TK + / TK À cells. Day 21 mean tumor volumes ( MTVs ) indicated the growths obtained were characteristic of the bystander activity reflective of the nontransduced cell population. Furthermore, nontransduced, high -GJIC cells in a chimeric tumor mass appeared to effectively bridge between transduced tumor cells and poorly communicating nontransduced cells. Finally, the importance of a gap junction protein, such as connexin -43, in facilitating the bystander effect was demonstrated with the HT29 low -GJIC cell line. When the TK -nontransduced cell population expressed connexin -43, a better bystander kill was achieved compared to the parental counterpart.