Expression of shRNA from a tissue-specific pol II promoter is an effective and safe RNAi therapeutic
Expression of shRNA from a tissue-specific pol II promoter is an effective and safe RNAi therapeutic
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DOI：
10.1038/mt.2008.144
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发表时间：
2008-09-01
影响因子：
12.4
通讯作者：
Kay, Mark A.
中科院分区：
文献类型：
--
作者：
Giering, Jeffery C.;Grimm, Dirk;Kay, Mark A.
已经观察到一些短发夹RNA（shorth-hairpin RNA，shRNA）的过表达可以诱导急性细胞毒性。这引起了人们对使用RNA干扰（RNAi）技术作为潜在治疗工具的安全性的担忧。我们已经试图通过开发用于来自肝脏来源的聚合酶（pol）II启动子的shRNA的组织特异性表达的单顺反子载体来解决表达控制的这一挑战。这种新的构建体在体外肝癌细胞和体内小鼠肝脏中有效地诱导靶沉默。为了证明这种方法的治疗潜力和提高的安全性，我们选择了一种靶向B肝炎病毒（HBV）包膜表面抗原（sAg）的shRNA，当从常用的U6启动子表达时，它是毒性最大的。将其作为双链DNA包装到腺相关病毒（AAV）假型8中，并以高颗粒剂量（1 × 10（12））将其递送至HBV转基因小鼠，导致血清sAg稳定降低至起始水平的85%，而没有任何伴随的肝损伤迹象。随着耐受性的提高，肝脏特异性pol II shRNA表达在注射后持续了一年以上。我们的结论是，这种pol II shRNA表达系统与一个有效的传递载体相结合，代表了一种有效的替代方案，无论是基于U6的战略或系统，通过使用额外的元素，实现组织特异性。
It has been observed that overexpression of some short-hairpin RNAs (shRNAs) can induce acute cytotoxicity. This has raised concerns about the safety of using RNA interference (RNAi) technology as a potential therapeutic tool. We have sought to address this challenge of expression control by developing a mono-cistronic vector for the tissue-specific expression of an shRNA from a liver-derived polymerase (pol) II promoter. This new construct efficiently induces target silencing in hepatoma cells in vitro and in mouse livers in vivo. In order to demonstrate the therapeutic potential and improved safety of this approach, we selected an shRNA targeting the envelope surface antigen (sAg) of hepatitis B virus (HBV), which is among the most toxic when expressed from the commonly used U6 promoter. Packaging it as a double-stranded DNA into an adeno-associated virus (AAV) pseudotype 8 and delivering it at a high particle dose (1 x 10(12)) to HBV transgenic mice resulted in the stable reduction of serum sAg to 85% of starting levels, without any concomitant sign of liver damage. With this improved tolerability, the liver-specific pol II shRNA expression persisted for more than one year after the injection. We conclude that this pol II shRNA expression system combined with a potent delivery vector represents an effective alternative to either U6-based strategies or systems that achieve tissue specificity through the use of additional elements.