Tumor gene therapy using Semliki forest virus replicons

Abstract: A major challenge in cancer research is to design effective vaccines that can break immunological tolerance to tumor antigens. Over the past ten years several immunotherapeutic strategies aimed at modulating the patient's immune response to fight cancer have been tested in clinical trials. Recently, there has been an increase in attention in the use of recombinant viruses as vectors for the in vivo delivery of genes with immunotherapeutic properties. One such vector are replicons based on Semliki Forest virus (rSFV), which have shown efficacy as recombinant vaccines in a variety of animal models. In this thesis, the potential of rSFV replicons as recombinant vaccines for the treatment of tumors was investigated. The well-characterized murine P815 mastocytoma was chosen as a tumor model because of its resemblance to human tumors expressing shared tumor-associated antigens (TAA). Recombinant SFV particles were constructed encoding P815A, a weakly immunogenic TAA expressed by P815 tumor cells. Mice vaccinated with these particles generated strong cytotoxic T cell responses and were protected against a lethal challenge with the P815 tumor. The therapeutic efficacy of these vectors was investigated in mice bearing established P815 tumors. Injection of rSFV encoding P1A or the immunostimulatory cytokine IL12, resulted in delayed tumor progression and in several mice complete tumor eradication. The efficacy of the treatment was dependent on the size of the treated tumor and was only achieved by local intratumoral injection of the virus. In accordance with these findings, expression of a rSFV-RNA was localized exclusively in the tumor and the local draining lymph node. Further studies were conducted to address biosafety issues related. to the in vivo distribution and persistence of rSFV particles after injection by different routes. RT-PCR analysis demonstrated that, after i.v. injection, rSFV-RNA distributed to a variety of different tissues, whereas it was confined locally after i.m. and s.c. injections. Furthermore, the expression of vector RNA was transient, and no viral RNA could be detected ten days after inoculation. In addition, all tested routes of immunization generated significant levels of antigen-specific CTL responses, and tetramer analysis showed that the distribution of antigen-specific CD8+T cells correlated with the in vivo expression pattern of rSFV, with preferential homing of antigen-specific CTLs to the local lymph node after s.c. injection. The studies presented in this thesis propose an alternative immunotherapeutic strategy to treat cancer, based on the use of rSFV-replicon vectors for the in vivo expression of tumor antigens and cytokine genes.