In vitro and in vivo 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.

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.

Gene electrotransfer of proinflammatory chemokines CCL5 and CCL17 as a novel approach of modifying cytokine expression profile in the tumor microenvironment.

The effectiveness of immunotherapy highly correlates with the degree and the type of infiltrated immune cells in the tumor tissue. Treatments based on modifying the immune cell infiltrate of the tumor microenvironment are thus gaining momentum. Therefore, the aim of our study was to investigate the effects of gene therapy with two proinflammatory chemokines CCL5 and CCL17 on inflammatory cytokine expression profile and immune cell infiltrate in two murine breast tumor models, 4T1 and E0771, and two murine colon tumor models, CT26 and MC38. In vitro, lipofection of plasmid DNA encoding CCL5 or CCL17 resulted in changes in the cytokine expression profile similar to control plasmid DNA, implying that the main driver of these changes was the entry of foreign DNA into the cell's cytosol. In vivo, gene electrotransfer resulted in high expression levels of both Ccl5 and Ccl17 transgenes in the 4T1 and CT26 tumor models. Besides a minor increase in the survival of the treated mice, the therapy also resulted in increased expression of Cxcl9 and Ifnγ, potent activators of the immune system, in CT26 tumors. However, this was not recapitulated in changes of TME, implying that a further refinement of the dosing schedule is needed.