Immunotherapy is emerging as an innovative and promising approach to overcome the immune-suppressive tumor pathways and to stimulate the immune system for destroying cancer cells. Cancer immunotherapy can be implemented through the development of vaccines encoding for neoantigens, uniquely expressed in tumor cells. Moreover, studies demonstrated that other compounds, such as immune checkpoint inhibitors and cytokines, can increase the efficacy of cancer vaccines. The aim of this project is to develop two immune stimulating self-amplifying mRNAs (saRNAs) to be used in combination for the treatment of colorectal cancer (CRC), which is a very common type of cancer and still a significant death cause. The first saRNA is a vaccine that encodes specific neoantigens of either CT26 or MC-38 CRC Cells. The second saRNA, called ImStim, will produce the cytokine IL-12 and a nanobody that blocks PD-L1. The IL-12 and anti-PD-L1 will be fused to a tumor targeting nanobody to reduce side effects. For an efficient in vivo delivery, the saRNA vaccine as well as the ImStim saRNA will be formulated in lipid nanoparticles. Moreover, since levels of IL-12 should be controlled to avoid toxicity, its expression will be controlled by an ON/OFF switch, recently developed by our team. The saRNA vaccines and ImStim saRNA will be first validation in healthy mice. Subsequently, their efficacy will be investigated in CRC orthotopic mouse models (CT26 and MC38), both at early- and late-stage.