Cascaded bio-responsive delivery of eNOS gene and ZNF gene to collaboratively treat hindlimb ischemia via pro-angiogenesis and anti-inflammation.

Gene therapy is a promising strategy for treating ischemic disease by solving the dual dilemma of ischemia and inflammation. However, its development remains limited by inefficient gene transfection. Hence, we propose a "dual genes + all-adaptive carrier" idea. We have innovatively co-delivered eNOS gene and the ZNF gene encoding its transcription factor to enhance the efficiency of eNOS expression. The overexpressed ZNF protein significantly promotes angiogenesis via regulating the transcription of multiple genes. This implies a potential synergistic effect of eNOS and ZNF genes in anti-ischemic therapy. Additionally, we have designed an all-adaptive gene carrier with cascaded bio-responsive functions based on the characteristic bio-signals of the ischemic site (including extracellular excessive matrix metalloproteinase-2, the endo/lysosomal pH gradient and high cytoplasmic glutathione level). This carrier can sequentially overcome transfection bottlenecks and achieve high transfection. Excitingly, this cascaded bio-responsive delivery strategy remarkably enhanced blood perfusion, accelerated angiogenesis and alleviated inflammation in critical limb ischemia (CLI) mice, which was attributed to the combined effects of pro-angiogenic ZNF expression and synergistically produced eNOS expression. Thereby, we believe that the co-delivery of eNOS and ZNF genes assisted by a cascaded bio-responsive carrier is a powerful strategy to treat CLI.