812. Cellular Delivery of Gene Therapy Vectors

Abstract

To achieve targeted, truly systemic therapeutic gene delivery to tumors, we exploited the observation that infection by a retrovirus proceeds initially through a non-specific adsorption stage prior to envelope-receptor recognition/internalization. Antigen specific, primary murine OT-1 T cells can be incubated in vitro with retroviral stocks which adhere to the cell surface. In vitro, virus can be handed off to target cells upon co-culture with the T cells, dependent upon cell contact, envelope/receptor interactions and local heparanase expression as provided by T cell activation upon encounter of antigen (ovalbumin) by the OT-1 T cells. Retroviral particles, pre-adsorbed to the surface of OT-1 T cells in vitro, were available for hand off in vivo to B16ova tumors distant from the site of T cell injection at high enough concentrations for therapeutic levels of gene transfer to occur. We have now delivered a variety of therapeutic genes to tumors, including HSVtk, IL-12 and the chemokine CCL-21. Pre- adsorption of vectors encoding each of these genes has led to therapeutic benefits in terms of direct local cell killing (HSVtk in combination with lymphodepletion), generation of potent immunizing activity (IL-12) and significant enhancement of the efficacy of subsequent doses of adoptively transferred, unmodified T cells (CCL-21). We have also characterized a second pathway by which viral particles expressing no envelope (BALD particles) can be handed off from T cells dependent upon T cell activation. Release of viral particles from the T cell surface coincides with the penetration of perforin-containing cytotoxic vesicles from the antigen activated T cells. The endosomolytic properties of perforin promote release of these viral particles from the endosome and, in situations where direct T cell killing does not occur, the virus goes onto to generate a productive infection. Thus, T cell mediated delivery of MLV core particles can be used to deliver therapeutic retroviral vectors in vivo using targeting at the level of T cell activation, thereby dispensing with the need to target viral envelopes. We have also investigated the possibilities of using antigen specific T cells to deliver replication competent viruses to tumors both in vitro and in vivo. In particular, we have demonstrated that replication competent Vesicular Stomatitis virus expressing GFP can be used to load OT-1 T cells. The virus infects these T cells only at very low levels and does not kill them. However, the T cells produce VSV particles at appreciable levels 48-72 hours post loading suggesting that they may be able to carry the viruses to tumors in vivo and lead to productive infection following intratumoral T cell accumulation. Therefore, the protection, concentration, and targeted delivery of viruses by T cell carriers offers great potential for the delivery of vector stocks into the circulation of patients. By careful selection of the virus, and therapeutic gene that is used, significant synergy can be achieved over the use of virotherapy, or adoptive T cell therapy, alone.