Immunotoxicology and tissue response profiles of electro gene transfer (EGT) in skeletal muscle by microarray analysis

Electro gene transfer (EGT) is a common method for delivery of DNA into skeletal muscle to produce local effects, secretion of molecules or vaccination. Despite wide use of EGT, little is known about safety aspects associated with the electric field or the gene expression changes associated with immunological responses. Our goal was to perform a safety assessment of EGT using microarrays to analyse gene expression changes, in conjunction with histological analysis. The microarray data was used to create a set of marker genes that will be beneficial for comparing the safety of other EGT protocols and potentially other gene therapy approaches in muscle. We compared gene expression profiles in mouse muscle from non-electrotransferred controls, saline electro-transferred muscle and muscle treated with non-expressing plasmid in order to delineate the contribution of each process. We first demonstrate that the number and magnitude of gene expression changes in saline-EGT muscle were modest, in accordance with the limited infiltration and structural changes observed histologically. The majority of up-regulated genes were attributed to a muscle regeneration, largely in association with needle track damage. In contrast, EGT of a non-expressing plasmid produced large increases in the number and degree of gene expression in accordance with greater evidence of muscle injury and remodelling with substantial infiltration observed histologically. Importantly, many genes involved in the innate immune system recognition of nucleic acids were up-regulated suggesting that the innate immune system is responsible for triggering inflammation and that several new, but poorly characterised pathways may be involved. Overall, our results indicate that the EGT procedure itself is safe, although the presence of plasmid DNA is capable of exaggerating regenerative and innate immune responses which will provide insights into vaccine action and potential safety concerns for human trials using EGT.