SELF-AMPLIFYING RNA VECTORS ENCODING FOR SURVIVIN AS ANTI-TUMORAL VACCINE CANDIDATES

Tumor associated antigens (TAAs) can be the target for cancer therapies. In particular, vaccination represents an attractive strategy to elicit specific immune responses against TAAs, harnessing the immune system to fight against tumor. Adenovirus and poxvirus derived-viral vectors have been widely used in infectious diseases and as cancer treatment in pre-clinical and clinical trials showing encouraging results in prolonging survival and tumor protection. Nevertheless, viral vectors present various limitations such as a possible pre-existing immunity against the vector, and the induction of neutralizing antibody responses against the vector itself, therefore multiple vaccinations with the same vector become inefficient. The aim of this thesis is to investigate an alternative strategy to overcome viral vector limitations; we focus our attention on alphavirus derived self-amplifying (SA) RNA vectors, when carry non-structural genes to encode the RNA replication machinery, while the structural genes are replaced with the transgene of interest. In the final configuration (Novartis patent) SA-RNA will be delivered by specially designed totally non immunogenic liposomes. SA-RNA presents different advantages: it derives from a totally chemical procedure (no cell cultures involved), it induces high gene expression, it is transiently expressed only in the cytoplasm, it does not induce an immune response against vector itself and it is fast (and cheap) to manufacture. To validate this new technology, Survivin has been chosen as a suitable benchmark TAA because it is over-expressed in virtually every human cancer, and it presents multiple functions involved in tumor maintenance and progression. SA-RNA encoding Survivin has been tested in vitro in terms of ability to replicate inside the cells and to produce Survivin protein. To test SA-RNA in in vivo experiments viral replicon particles (VRPs) have been used as delivery strategy, because the liposomes are not available as yet. VRPs do not present pre-existing immunity against the vector and they are poorly immunogenic, allowing repetitive vaccinations. VRPs encoding Survivin have been tested in vivo in wild-type mice, to evaluate a possible Survivin-specific immune response. The results show that there is a weak immune response against Survivin, in line with what described in the literature, and the experiments suggest that the best immunization schedule is 3 doses 2 weeks apart. In the last part of the project, the efficacy of VRPs encoding Survivin has been tested in two mouse cancer models: pancreatic cancer and malignant mesothelioma. In pancreatic cancer model, a trend in survival increase is observed in treated mice. In malignant mesothelioma model, only an initial difference in survival has been shown, nevertheless, VRPs encoding Survivin induce a biological effect in term of reduced tumor dissemination and increased intra-tumoral cell death in treated mice. From these results and similar results obtained with other vectors, it is clear that Survivin vaccination has a documentable biological effect on tumor growth, but that is not by itself promoting any significant reduction of tumor mortality. However, the well documented increase in intratumoral cell death (see our TUNEL results) and in hemorragic necrosis (Bertino et al., 2012) suggest to experiment anti-Survivin immunotherapy in association with other anti-cancer treatment, such as chemotherapy, inflammatory cytokines, as interferon (IFN-α) and interluekin-2 (IL-2), or checkpoint inhibitors. The next step of the project is to understand the mechanism that causes intratumoral cell death and hemorragic necrosis. In general, SA-RNA platform, integrated with the liposome delivery system, will be suitable for many vaccines, including the peptide collections involved in the personalized therapy schemes, now under active investigation.