PROJECT SUMMARY Orlance has developed a next-generation Gene Gun (MACH-1 GG) that efficiently delivers DNA and RNA into epidermal cells, leading to robust immune responses. Sequencing of tumors from individual patients has led to the identification of personalized neoantigens that could be targeted with cancer vaccines. However, technologies that can effectively deliver these cancer neoantigens and promote the induction of localized tumor specific T cell responses are still needed. Nucleic acid (NA; DNA and RNA) vaccines administered using specific formulations or delivery technologies that achieve intracellular delivery offer considerable promise to achieve this goal. These include electroporation (EP) or jet injection for IM delivery of DNA or lipid nanoparticles (LNPs) for IM delivery of RNA. These delivery modalities, however, have different drawbacks including a requirement for high doses (1-5 mg of DNA), ultra-cold storage due to limited stability (RNA/LNPs), reactogenicity or pain post-administration, and a limited ability to target immune responses to specific tissues. The GG entails the delivery of room temperature stable lyophilized DNA or RNA vaccines on gold microparticles. It achieves painless and direct intracellular delivery into skin cells with very low doses (1-4 µg) that results in systemic, mucosal and localized skin immune responses that could provide a benefit for treatment of cancers and, in particular, melanoma. The MACH-1 GG is based on a previous successful GG that induced strong antibody and T cell responses in phase I human clinical trials. The MACH-1 provides significant improvements over this earlier device. Here, we will investigate the feasibility of using MACH-1 to deliver DNA or RNA cancer vaccines in mice and test the hypothesis that MACH-1 will offer advantages in immunogenicity and efficacy over other DNA/RNA delivery technologies for melanoma. We will first determine if co-delivering a novel set of genetic adjuvants will increase the ability of MACH-1 DNA and RNA vaccines to induce melanoma-specific T cell responses. Next, we will determine if combining DNA and RNA in the same dose or in a prime-boost regimen offers synergistic effects. We will then compare MACH-1 delivery of DNA and/or RNA melanoma vaccines to DNA delivery by EP or RNA delivery by LNPs for immunogenicity and protective efficacy in mice. This work will be accomplished in two Aims: Aim 1: Investigate the impact of genetic adjuvants on the immunogenicity and efficacy of GG delivered DNA and RNA melanoma vaccines. Aim 2: Determine if combining the optimized adjuvanted DNA and RNA vaccines in the same dose or in a prime-boost regimen enhances immunogenicity and efficacy compared to EP delivery of DNA and LNP delivery of RNA in a mouse model of melanoma. Successful completion of these Aims will establish MACH-1 as an effective device to deliver cancer vaccines.

PROJECT NARRATIVE Orlance’s Gene Gun uniquely enables needle-free delivery of DNA and RNA vaccines into the epidermis of the skin. We hypothesize that the ability of the gene gun to directly deliver DNA and RNA into cells in the epidermis will make it more effective than conventional intramuscular delivery of DNA and RNA vaccines in targeting tumor cells localized in the skin. Here, we will optimize gene gun delivery of DNA or RNA to identify a regimen that induces strong tumor specific immune responses and protection from melanoma in a mouse model.