Penn Center for Molecular Discovery, Department of Chemical and Biomolecular Engineering, Institute for Medicine and Engineering, Philadelphia, Pennsylvania, USA.
Mol Ther. 2011 Jun;19(6):1152-60. doi: 10.1038/mt.2011.4. Epub 2011 Feb 8.
Intracellular barriers to adeno-associated virus (AAV) transduction may limit gene delivery. We screened a short interfering RNA (siRNA) library targeting 5,520 genes to help identify pathways that modulate AAV transduction of human endothelium. In replicate screening, 50 pools (three siRNAs per gene) resulted in greater than eightfold reporter gene expression enhancement. Single siRNA confirmation tests demonstrated that at least one siRNA from each of the top 10 pools provided greater than twofold enhancement. Several siRNAs when used together resulted in additive effects and two of the most potent siRNA sequences were enhancers in cultured airway epithelium. However, enhanced transduction was not correlated with mRNA knockdown by quantitative real time PCR, indicating an off-target mechanism. In fact, four of the five most potent siRNAs contained a consensus hexamer region 5'-UGUUUC-3' at positions 2-7 of the antisense strand. The point mutation U4A within this region (but not mutations at positions 1 or 14) disrupted transduction enhancement, indicating a microRNA (miRNA)-like mechanism. Transcription profiling indicated that the hexamer also resulted in perturbation of the interferon pathway via reduced interferon-induced protein 44-like (IFI44L), interferon-inducible myxovirus resistance 1 (MX1), and interferon-induced protein with tetratricopeptide repeats (IFIT5) mRNAs. Direct interferon (α, β, and ω) receptor 2 (IFNAR2) knockdown resulted in greater than twofold transduction enhancement. In addition to providing insight into AAV biology and enhanced transduction, the results demonstrate certain beneficial siRNA off-target effects.
细胞内的腺相关病毒（AAV）转导障碍可能会限制基因传递。我们筛选了一个针对 5520 个基因的小干扰 RNA（siRNA）文库，以帮助鉴定调节人内皮细胞中 AAV 转导的途径。在重复筛选中，50 个池（每个基因 3 个 siRNA）使报告基因表达增强了 8 倍以上。单 siRNA 确认测试表明，前 10 个池中的每个 siRNA 至少有一个提供了 2 倍以上的增强。一些 siRNA 一起使用时会产生相加效应，两种最有效的 siRNA 序列在培养的气道上皮细胞中也是增强剂。然而，增强的转导与定量实时 PCR 测定的 mRNA 敲低无关，表明存在非靶向机制。事实上，在最有效的五个 siRNA 中有四个包含一个在反义链的 2-7 位上的 5'-UGUUUC-3'的一致六聚体区域。该区域内的点突变 U4A（但 1 位或 14 位的突变除外）破坏了转导增强，表明存在 microRNA（miRNA）样机制。转录谱分析表明，六聚体还通过降低干扰素诱导蛋白 44 样（IFI44L）、干扰素诱导的粘液病毒抗性 1（MX1）和干扰素诱导的四肽重复蛋白（IFIT5）mRNA，导致干扰素途径受到干扰。直接干扰（α、β 和 ω）受体 2（IFNAR2）敲低导致转导增强超过 2 倍。除了提供对 AAV 生物学和增强转导的深入了解外，这些结果还证明了某些有益的 siRNA 非靶向效应。
