Establishment of Parameters for Optimal Transduction Efficiency and Antitumor Effects with Purified High-Titer HSV-TK Retroviral Vector in Established Solid Tumors

Abstract
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 nonmurine 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 108 cfu/ml. Purified, high-titer retroviral preparations were injected direcly 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. Retroviral gene therapy is a potentially useful method for delivery of the thymidine kinase (TK) gene into solid tumors, allowing for selective killing of transduced cells upon administration of the prodrug ganciclovir (GCV). Effective elimination of tumor cells in an animal model depends upon several clinically relevant parameters such as retroviral vector titer, schedule and route of administration, transduction efficiency, and thorough in vitro screening of candidate cell lines for transducibility and sensitivity to GCV prior to initiation of animal studies. Purified, high-titer retroviral vector delivered as a series of injections inside of and surrounding an established mouse tumor caused transduction of approximately 20% of tumor cells, and when followed by systemic GCV delivery, caused significant reduction in tumor volume and some complete cures.