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.

Growth environment influences B16.F10 mouse melanoma cell response to gene electrotransfer.

We developed and characterized a 3D collagen hydrogel model for B16.F10 melanoma tumors. Cells in this 3D environment exhibited lower proliferation than cells in the conventional 2D culture environment. Interestingly, the basal expression levels of several genes varied when compared to conventionally grown cells. In each growth environment, a significant number of melanoma cells were transfected by plasmid electroporation (electrotransfer), although expression could only be ascertained on the surface of the 3D constructs. Cellular responses to plasmid entry as demonstrated by pro-inflammatory cytokine and chemokine upregulation varied based on the growth environment, as did the mRNA levels of several putative DNA-specific pattern recognition receptors (DNA sensors). Unexpectedly, when plasmid DNA was delivered while cells where attached in the 2D or 3D environments, the mRNAs of the DNA sensor p204 and the inflammatory mediator TNFα were regulated in cells receiving pulses only. However, we were unable to confirm coordinate upregulation of TNFα and p204 proteins. This study confirms that cell responses differ significantly based on their environment, and demonstrates the difficulty of extending experimental observations between cell environments.