Dendritic cell (DC) cancer vaccines have become an established treatment modality for patients with a wide range of cancers. However, the therapeutic potential of this immunotherapy is often limited by negative feedback mechanisms that normally prevent sustained activation of the immune system, thereby reducing the risk of autoimmunity. To overcome these challenges, a new generation of DC vaccines have been developed using RNA interference (RNAi) technology to inhibit the expression of immunosuppressive factors, such as indoleamine 2,3-dioxygenase 1 (IDO1). The developed vaccines effectively stimulated T cells and demonstrated anti-tumor activity in cancer patients. This chapter describes the design and enzymatic synthesis of siRNA targeting IDO, along with its incorporation into DC vaccines under Good Manufacturing Practice.