Reduction of the Plasmid Vector Backbone Length Enhances Reporter Gene Expression

Gene therapy has been researched using both viral and non-viral delivery methods. While viral vectors display high gene expression levels, there are safety concerns. These non-viral delivery methods, lipofectamine and gene electrotransfer, help mitigate these safety concerns. However, it does not yield high gene expression. To try to remedy this, plasmid DNA with a decreased backbone length was delivered utilizing lipofectamine and gene electrotransfer (GET). The pDNA compared was plasmid with a traditional backbone length, gWiz™, and Nanoplasmid™, which has a decreased backbone length. The DNA encodes firefly luciferase (Luc). The DNA was tagged with c-myc (myc) and DYKDDDDK (DDK). Gene expression was analyzed in rat tenocytes, mouse melanoma cells, and gene delivery to the skin and ventricular myocardium in rats. Decreasing the plasmid vector backbone size yielded an increase in gene expression by 10-fold in rat tenocytes in vitro and rat myocardium in vivo. This increase in gene expression was observed with the use of lipofectamine as well as GET. There was a more conservative improvement in gene delivery to the skin. Further, with the pulsing parameters in conjunction with the Nanoplasmid™, the muscle layer was successfully transfected. Previous studies found that gene delivery to the skin using GET was limited to the epidermis. In vitro experiments showed a higher efficiency using Nanoplasmid™ in both equimolar and equal mass testing. In vivo studies were conducted using equal masses of the pDNA. Decreasing the plasmid vector backbone may help non-viral gene transfer methods to reach therapeutic gene expression levels