Studies on the Development of Targeted Gene Delivery Systems for Reversal of Atherosclerosis and obesity

Abstract

Endothelial dysfunction combined with inflammation leads to atherosclerosis. Endothelium-specific delivery of therapeutic agents at the cellular level—specifically in vivo—is still a difficult task for proper management of atherosclerosis. We designed a redox-sensitive poly(oligo-L-arginine) (rsPOLA) playing dual roles as an endothelium α-2 adrenoceptors(α-2ARs)-targeted gene carrier and as a substrate for endothelial nitric oxide synthase (eNOS). Overexpression of α-2ARs on atherosclerotic endothelial cells was confirmed and the eNOS/rsPOLA nanoplexes following systemic injection demonstrated to 1) enhance eNOS gene delivery into endothelial cells via α-2ARs/L-arginine specific binding, 2) increase intracellular level of nitric oxide, 3) suppress inflammatory response in endothelium and finally 4) reduce atherosclerotic plaque in a Ldlr-/- atherosclerotic mouse model. Among the tested nanoplexes [eNOS/rsPOLA, eNOS/(poly(oligo-D-arginine), rsPODA) and eNOS/(racemic mixture, rsRM)], eNOS/rsPOLA reduced atherosclerotic inflammation most effectively as we hypothesized. Current treatment strategy provides strong potential for further development of a gene therapeutic system to ameliorate inflammation and progressive atherosclerotic plaques. Obesity is a progressively prevailing in developed societies. In spite of all major progress in the understanding of molecular mechanisms of obesity, currently available commercial anti-obesity drugs have been shown very limited efficacy with severe side effects. Anti-obesity drug development industry is thus concentrating on targeting adipocytes that store excess fat. In this study we used a peptide motif (CKGGRAKDC) that homes to adipocytes, thus named as adipocyte-targeting sequence (ATS). An adipocyte-targeting fusion-oligopeptide gene carrier was developed by conjugating ATS and 9-arginine (ATS-9R). This peptide binds with prohibitin, a vascular marker of adipose tissue. We also constructed a single guide RNA (sgRNA) and complexed it with deactivated Cas9 protein (dCas) to develop a complex to knock-down fatty-acid-binding protein 4 (FABP4) via CRISPR interference technique. Transfection of dCas9+sgFABP4 through ATS-9R caused FABP4 knock down in pre adipocytes and mature adipocytes (>40%). The dCas9+sgFABP4/ATS-9R oligoplexes could be a safe therapeutic approach to regress and treat obesity as well as obesity-induced metabolic syndromes.