Gene electrotransfer of IL-2 and IL-12 plasmids effectively eradicated murine B16.F10 melanoma

Gene therapy has become an important approach for treating cancer, and electroporation represents a technology for introducing therapeutic genes into a cell. An example of cancer gene therapy relying on gene electrotransfer is the use of immunomodulatory cytokines, such as interleukin 2 (IL-2) and 12 (IL-12), which directly stimulate immune cells at the tumour site. The aim of our study was to determine the effects of gene electrotransfer with two plasmids encoding IL-2 and IL-12 in vitro and in vivo. Two different pulse protocols, known as EP1 (600 V/cm, 5 ms, 1 Hz, 8 pulses) and EP2 (1300 V/cm, 100 µs, 1 Hz, 8 pulses), were assessed in vitro for application in subsequent in vivo experiments. In the in vivo experiment, gene electrotransfer of pIL-2 and pIL-12 using the EP1 protocol was performed in B16.F10 murine melanoma. Combined treatment of tumours using pIL2 and pIL12 induced significant tumour growth delay and 71% complete tumour regression. Furthermore, in tumours coexpressing IL-2 and IL-12, increased accumulation of dendritic cells and M1 macrophages was obtained along with the activation of proinflammatory signals, resulting in CD4 + and CD8 + T-lymphocyte recruitment and immune memory development in the mice. In conclusion, we demonstrated high antitumour efficacy of combined IL-2 and IL-12 gene electrotransfer protocols in low-immunogenicity murine B16.F10 melanoma

\u3ci\u3eIn Vitro and In Vivo\u3c/i\u3e Correlation of Skin and Cellular Responses to Nucleic Acid Delivery

Skin, the largest organ in the body, provides a passive physical barrier against infection and contains elements of the innate and adaptive immune systems. Skin consists of various cells, including keratinocytes, fibroblasts, endothelial cells and immune cells. This diversity of cell types could be important to gene therapies because DNA transfection could elicit different responses in different cell types. Previously, we observed the upregulation and activation of cytosolic DNA sensing pathways in several non-tumor and tumor cell types as well in tumors after the electroporation (electrotransfer) of plasmid DNA (pDNA). Based on this research and the innate immunogenicity of skin, we correlated the effects of pDNA electrotransfer to fibroblasts and keratinocytes to mouse skin using reverse transcription real-time PCR (RT-qPCR) and several types of protein quantification. After pDNA electrotransfer, the mRNAs of the putative DNA sensors DEAD (AspGlu-Ala-Asp) box polypeptide 60 (Ddx60), absent in melanoma 2 (Aim2), Z-DNA binding protein 1 (Zbp1), interferon activated gene 202 (Ifi202), and interferon-inducible protein 204 (Ifi204) were upregulated in keratinocytes, while Ddx60, Zbp1 and Ifi204 were upregulated in fibroblasts. Increased levels of the mRNAs and proteins of several cytokines and chemokines were detected and varied based on cell type. Mouse skin experiments in vivo confirmed our in vitro results with increased expression of putative DNA sensor mRNAs and of the mRNAs and proteins of several cytokines and chemokines. Finally, with immunofluorescent staining, we demonstrated that skin keratinocytes, fibroblasts and macrophages contribute to the immune response observed after pDNA electrotransfer