Gene delivery of angiogenic growth factors is a promising approach for the treatment of ischemic cardiovascular diseases. However, success of this new therapeutic principle is hindered by the lack of critical understanding as to how disease pathology affects the efficiency of gene delivery and/or the downstream signaling pathways of angiogenesis. Critical limb ischemia occurs in patients with advanced atherosclerosis often exhibiting deficiency in endothelial nitric oxide production. Similar to these patients, segmental femoral artery resection progresses into severe ischemic necrosis in mice deficient in endothelial nitric oxide synthase (ecNOS-KO) as well as in balb/c mice. We used these models to evaluate the influence of severe ischemia on transfection efficiency and duration of transgene expression in the skeletal muscle following plasmid injection in combination with electroporation. Subsequently, we also explored the potential therapeutic effect of the phosphomimetic mutant of ecNOS gene (NOS1177D) using optimized delivery parameters, and found significant benefit both in ecNOS-KO and balb/c mice. Our results indicate that NOS1177D gene delivery to the ischemic skeletal muscle can be efficient to reverse critical limb ischemia in pathological settings, which are refractory to treatments with a single growth factor, such as vascular endothelial growth factor.