Poly-L-glutamate, an anionic polymer, enhances transgene expression for plasmids delivered by intramuscular injection with in vivo electroporation

Intramuscular (i.m.) injection of plasmids followed by electro-permeabilization is an efficient process to deliver genes into skeletal myofibers that permits proteins to be produced and secreted at therapeutically relevant levels. To further improve skeletal muscle as a bioreactor, we identified a formulation that elevates transgene expression in myofibers after i.m. injection and electroporation. With secreted placental alkaline phosphate (SEAP) as reporter gene, plasmid formulated with poly-L-glutamate produced two- to eight-fold higher levels of SEAP in mouse serum than plasmid in saline. Various concentrations and molecular weights of poly-L-glutamate were similarly effective, but 6 mg/ml of 15-50 kDa poly-L-glutamate consistently yielded the highest expression levels. The poly-L-glutamate formulation was effective in two different muscle groups in mice at various plasmid doses for several transgenes, including an erythropoietin (EPO) gene, for which expression was elevated four-to 12-fold in comparison to animals that received EPO plasmid in saline. Transgene expression was localized to myofibers. Poly-L-glutamate may improve transgene expression in part by increasing plasmid retention in skeletal muscle. Poly-L-glutamate did not enhance gene transfer in the absence of electroporation. Therefore, the polymer is a novel formulation that specifically enhances the transfer and expression of genes delivered with electroporation.