495. Activation of DNA Pattern Recognition Receptors After Plasmid Electrotransfer in Melanoma Cells and Tumors

Abstract

In vivo electroporation or electrotransfer, the application of controlled electric pulses, enhances delivery of plasmid DNA to a wide variety of healthy tissues as well as many tumor types. Electrotransfer of pDNA encoding therapeutic genes substantially increases gene expression, enhancing subsequent therapeutic effects. Delivery of therapeutic plasmid DNA has reached clinical trials in the US and in Europe, primarily for cancer therapies and infectious disease vaccines. In several preclinical tumor models, delayed tumor growth, increased survival time, and even complete tumor regression can occur with intratumoral electroporation, also known as electrotransfer, of DNA oligonucleotides or plasmid DNA devoid of a therapeutic gene (empty vector). In B16.F10 mouse melanomas, these effects are preceded by significant elevation of several proinflammatory cytokines and chemokines including IFNβ, implicating the binding and activation of intracellular DNA-specific pattern recognition receptors in response to DNA electrotransfer. The purpose of this study was to investigate whether melanoma tumors and cells express cytosolic DNA sensors and whether these sensors respond to pDNA electrotransfer. Histologically, tumor necrosis independent of caspase-3 was observed. Although the mRNAs for several DNA sensors were detected in tumors, none was significantly upregulated. In B16.F10 cells in culture, IFNβ mRNA and protein levels were significantly upregulated after pDNA electrotransfer. The mRNAs for several DNA sensors were present in these cells and DAI, DDX60, and p204 mRNAs were significantly upregulated after pDNA electrotransfer. DDX60 protein levels were coordinately upregulated. Mirroring the observation of tumor necrosis, cells underwent a significant pDNA concentration-dependent decrease in proliferation and survival. Taken together, increased IFNβ and DNA sensor expression accompanied by cell death and tumor necrosis indicate that pDNA electrotransfer activates intracellular DNA sensors in B16.F10 cells and tumors, producing both in vitro and in vivo effects. The absence of activation of DNA sensors in vivo could be due to the lower transfection efficiency compared to that in vitro or to dilution by other tumor cell types. Electrotransfer is an efficient means of enhancing plasmid DNA introduction into tissues such as skin, muscle, and tumors for therapeutic application. Localized inflammation and induced cell death may contribute to cancer gene therapies but may impede gene therapies for which these effects are not desirable.