Enjoji M, Wang F, Nakamuta M, Chan L, Teng B B
Departments of Medicine and Cell Biology, Baylor College of Medicine, Houston, TX 77030, USA.
Hum Gene Ther. 2000 Nov 20;11(17):2415-30. doi: 10.1089/104303400750038516.
In humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. To reduce the levels of apoB mRNA, we used adenovirus-mediated vector to target hammerhead ribozyme at GUA(6679) downward arrow of apoB mRNA (designated AvRB15) in the liver of a dyslipidemic mouse model that is deficient in apoB mRNA editing enzyme and overexpresses human apoB100. In this study, we delivered approximately 4 x 10(11) virus particles of AvRB15 (active ribozyme) or AvRB15-mutant (inactive ribozyme) to the animals. Using Southern blot analysis, we readily detected RB15 DNA in the mouse liver as long as day 35 after injection. This result was correlated with the RNA expression of RB15 by RNase protection assay. Using reverse ligation-mediated polymerase chain reaction, the 3' cleavage product of apoB mRNA was detected, and the exact cleavage site was confirmed by sequencing. Importantly, the levels of human and mouse apoB mRNA decreased approximately 80% after AvRB15 transduction. There was a marked decrease in plasma cholesterol, triglyceride, and human apoB of 42, 51, and 62%, respectively, when compared with the inactive ribozyme-treated group. Moreover, ribozyme cleavage of apoB mRNA generated a truncated protein of the expected size (apoB48.1), which was associated with lipoprotein particles in the very low density, low density, and high density lipoprotein fractions. Taken together, these results indicate that apoB mRNA-specific hammerhead ribozyme can be used as a potential therapeutic agent to modulate apoB gene expression and to treat hyperlipidemia.
在人类中，载脂蛋白B（apoB）的过量产生与早发性冠状动脉疾病呈正相关。为了降低apoB mRNA的水平，我们使用腺病毒介导的载体将锤头状核酶靶向apoB mRNA的GUA(6679)下游（命名为AvRB15），该载体用于缺乏apoB mRNA编辑酶并过表达人apoB100的血脂异常小鼠模型的肝脏。在本研究中，我们向动物递送了约4×10(11)个AvRB15（活性核酶）或AvRB15突变体（无活性核酶）的病毒颗粒。使用Southern印迹分析，我们在注射后长达35天的小鼠肝脏中很容易检测到RB15 DNA。该结果与通过核糖核酸酶保护试验检测的RB15的RNA表达相关。使用反向连接介导的聚合酶链反应，检测到apoB mRNA的3'切割产物，并通过测序确认了确切的切割位点。重要的是，AvRB15转导后人和小鼠apoB mRNA的水平下降了约80%。与无活性核酶处理组相比，血浆胆固醇、甘油三酯和人apoB分别显著降低了42%、51%和62%。此外，apoB mRNA的核酶切割产生了预期大小的截短蛋白（apoB48.1），其与极低密度、低密度和高密度脂蛋白组分中的脂蛋白颗粒相关。综上所述，这些结果表明apoB mRNA特异性锤头状核酶可作为一种潜在的治疗剂来调节apoB基因表达并治疗高脂血症。
