Project Summary/Abstract Vaccination is a potent regulator of the immune system and much research is being conducted to understand how antigen-specific stimulation can be used to induce and redirect anti-tumor immunity. Our team develops a cancer immunotherapy program using novel AAV vectors to deliver antigens both directly and by cross- presentation to antigen presenting cells. Optimization of naturally occurring AAV serotypes and novel methods of AAV production are necessary to warrant wide use of these vectors for cancer immunotherapy. The main aims of this proposal is to determine the safety and efficacy of our novel AAV-based vaccines as part of preclinical studies by using companion dogs with oral melanoma. In recent years, we have undertaken studies to gain a better understanding of the underlying molecular mechanisms of AAV and host immune system interaction via the high efficiency transduction of DCs. We have made the following scientific achievements, which form the basis of the current proposal: • Identified and mutagenized critical surface-exposed serine and threonine residues on the AAV capsid that are involved in intracellular trafficking of virus in the host cells. • Demonstrated that rational modifications in the AAV expression cassette results in antigen processing by dendritic cells (DCs), leading to a stronger and prolonged antigen-specific immune response. • Developed the next generation of highly efficient AAV-vectors that expressed a tumor-associated antigens (premelanosome protein gp100 (also known as Pmel), tyrosinase (Tyr), tyrosinase-related protein 1(TRP1), and dopachrome tautomerase (TRP2). • Tested these novel AAV vectors for their ability to stimulate a specific T-cell clone proliferation and protective immune response in a small animal model. In this proposal, we will test ability of our recently developed optimized AAV capsid and expression cassette, whose unique properties enhance a cellular/humoral immune response toward vector encoded tumor antigens. Successful completion of the current proposal will result in: • Production of high titers of a therapeutic AAV-based vaccine. • Identification of an optimal AAV-based dose for vaccination that can be used to slow/eliminate the progression of melanoma cancer in a large animal model such as companion dog-patients with spontaneous cancer. • Mechanism of the novel cancer vaccine function in the most appropriate animal model. The optimized AAV vector can be potentially used as a vaccine platform for the treatment of cancers in canine and humans.