RESUMEN
High-titer, purified herpes simplex virus thymidine kinase (HSV-TK) retroviral particles, followed with intraperitoneal ganciclovir (GCV) were tested in Fischer rats bearing 1-week established 9L gliosarcomas. 9L cells were infused intracranially through a cannula on day 0, given intracranial infusions of HSV-TK retroviral particles on days 7-12, and given 5 or 10 daily doses of intraperitoneal GCV starting at day 14. Tumor volumetric studies performed on rat brains at day 26 after tumor infusion revealed significant differences in experimental groups receiving HSV-TK retroviral particles plus 10-day GCV or the 100% transduced 9L-TK group receiving GCV versus control groups treated with either buffer, HSV-TK vector, or either 5- or 10-day regimens of GCV alone. The duration of GCV administration and the volume of tumor burden influenced efficacy. Adjuvant dexamethasone did not significantly affect efficacy. Experiments in which 9L rechallenge of animals treated with 9L-TK/GCV or 9L tumors treated with HSV-TK vector/GCV indicated that a host immune response was involved in rejecting the rechallenge tumor. Outcome was dependent upon the site and size of the rechallenge inoculum. In vitro, bystander effects were significant but were especially marked when cell-to-cell contact was maintained. Cumulatively, the data indicate that both the bystander effect and the host immune response are responsible for the efficacy observed in the suicide gene therapy paradigm using purified HSV-TK retroviral particles and GCV to treat smaller tumor burden in rats with 9L gliosarcoma.
Asunto(s)
Neoplasias Encefálicas/terapia , Terapia Genética , Glioblastoma/terapia , Retroviridae/genética , Timidina Quinasa/genética , Animales , Formación de Anticuerpos/efectos de los fármacos , Antineoplásicos/uso terapéutico , Antivirales/uso terapéutico , Astrocitoma/mortalidad , Astrocitoma/terapia , Neoplasias Encefálicas/mortalidad , Dexametasona/uso terapéutico , Ganciclovir/uso terapéutico , Glioblastoma/mortalidad , Herpesvirus Humano 1/enzimología , Masculino , Trasplante de Neoplasias , Ratas , Ratas Endogámicas F344 , Esteroides/administración & dosificación , Esteroides/uso terapéutico , Timidina Quinasa/aislamiento & purificación , Timidina Quinasa/metabolismo , Células Tumorales Cultivadas , ViriónRESUMEN
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.
Asunto(s)
Terapia Genética , Vectores Genéticos , Neoplasias Experimentales/terapia , Retroviridae/genética , Simplexvirus/genética , Transducción Genética , Animales , Femenino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Timidina Quinasa/genética , VolumetríaRESUMEN
Replication-defective, highly purified retroviral vectors (Retrovector), at titers of 10(8) colony forming units/mL, were prepared that conferred either beta-galactosidase or herpes simplex thymidine kinase (HSV-TK) activity. 9L gliosarcoma cells, transduced efficiently in vitro, were highly sensitive to ganciclovir (GCV). The mean frequency of in situ transduction, measured by flow cytometry of single-cell tumor suspensions isolated from rat brains, was 3.2 +/- 0.6%; similar assessments were made by staining of beta-galactosidase or by immunohistochemistry with anti-HSV-TK. In vitro HSV-TK-transduced and G418-selected 9L-TK gliosarcoma tumors treated with GCV were eradicated in approximately 53% of the animals (10/19) at day 26, however, 89% (17/19) histologically showed < 1% tumor volume. Histologic evaluation at day 26 of animals with established 9L tumors treated with intralesional injection of HSV-TK vector followed by GCV treatment showed that 29% (4/14) had no tumor; 50% (7/14) had < 1% tumor volume. Regression of tumors proceeded over the time since the complete rate was increased at day 60. Neither HSV-TK vector particles nor GCV alone altered the histological profile of 9L tumors, but substantial numbers of CD4+ and CD8+ lymphocytes infiltrated the tumors of animals treated with both. In cured animals, the former tumor bed contained cell debris, immune cells, and fibroblasts and was without damage to adjacent brain. The efficacy of suicide gene therapy for rat gliosarcoma using highly purified virion vectors approaches that of packaging cell lines.