Program in Integrative Cardiac Metabolism, Center for Cardiovascular Research and Department of Physiology and Biophysics, University of Illinois at Chicago, College of Medicine, Chicago, IL 60612, USA.
Curr Gene Ther. 2012 Dec;12(6):454-62. doi: 10.2174/156652312803519760.
This study examines the feasibility of using the adenoviral delivery of DNA for a non-native microRNA to suppress expression of a target protein (cytosolic NADP(+)-dependent malic-enzyme 1, ME1) in whole heart in vivo, via an isolated-heart coronary perfusion approach. Complementary DNA constructs for ME1 microRNA were inserted into adenoviral vectors. Viral gene transfer to neonatal rat cardiomyocytes yielded 65% suppression of ME1 protein. This viral package was delivered to rat hearts in vivo (Adv.miR_ME1, 10(13) vp/ml PBS) via coronary perfusion, using a cardiac-specific isolation technique. ME1 mRNA was reduced by 73% at 2-6 days post-surgery in heart receiving the Adv.miR_ME1. Importantly, ME1 protein was reduced by 66% (p < 0.0002) at 5-6 days relative to sham-operated control hearts. Non-target protein expression for GAPDH, calsequestrin, and mitochondrial malic enzyme, ME3, were all unchanged. The non-target isoform, ME2, was unchanged at 2-5 days and reduced at day 6. This new approach demonstrates for the first time significant and acute silencing of target RNA translation and protein content in whole heart, in vivo, via non-native microRNA expression.
本研究通过离体心脏冠状动脉灌流的方法，考察了利用腺病毒载体将非天然 microRNA 的 DNA 递送至整体心脏内以抑制靶蛋白（胞质 NADP(+)-依赖性苹果酸酶 1，ME1）表达的可行性。将 ME1 microRNA 的 cDNA 构建体插入腺病毒载体中。腺病毒载体转染新生大鼠心肌细胞，使 ME1 蛋白表达抑制 65%。采用心脏特异性分离技术，通过冠状动脉灌流将这种病毒制剂（Adv.miR_ME1，10(13) vp/ml PBS）递送至大鼠体内。接受 Adv.miR_ME1 的心脏在术后 2-6 天 ME1 mRNA 减少 73%。重要的是，与假手术对照心脏相比，5-6 天 ME1 蛋白减少 66%（p < 0.0002）。非靶蛋白 GAPDH、钙结合蛋白和线粒体苹果酸酶 ME3 的表达均无变化。非靶标同工酶 ME2 在 2-5 天无变化，6 天减少。该新方法首次证明，通过非天然 microRNA 表达，可在整体心脏内实现靶 RNA 翻译和蛋白含量的显著急性沉默。
