Kleinman Mark E, Yamada Kiyoshi, Takeda Atsunobu, Chandrasekaran Vasu, Nozaki Miho, Baffi Judit Z, Albuquerque Romulo J C, Yamasaki Satoshi, Itaya Masahiro, Pan Yuzhen, Appukuttan Binoy, Gibbs Daniel, Yang Zhenglin, Karikó Katalin, Ambati Balamurali K, Wilgus Traci A, DiPietro Luisa A, Sakurai Eiji, Zhang Kang, Smith Justine R, Taylor Ethan W, Ambati Jayakrishna
Department of Ophthalmology, University of Kentucky, Lexington, Kentucky 40506, USA.
Nature. 2008 Apr 3;452(7187):591-7. doi: 10.1038/nature06765. Epub 2008 Mar 26.
Clinical trials of small interfering RNA (siRNA) targeting vascular endothelial growth factor-A (VEGFA) or its receptor VEGFR1 (also called FLT1), in patients with blinding choroidal neovascularization (CNV) from age-related macular degeneration, are premised on gene silencing by means of intracellular RNA interference (RNAi). We show instead that CNV inhibition is a siRNA-class effect: 21-nucleotide or longer siRNAs targeting non-mammalian genes, non-expressed genes, non-genomic sequences, pro- and anti-angiogenic genes, and RNAi-incompetent siRNAs all suppressed CNV in mice comparably to siRNAs targeting Vegfa or Vegfr1 without off-target RNAi or interferon-alpha/beta activation. Non-targeted (against non-mammalian genes) and targeted (against Vegfa or Vegfr1) siRNA suppressed CNV via cell-surface toll-like receptor 3 (TLR3), its adaptor TRIF, and induction of interferon-gamma and interleukin-12. Non-targeted siRNA suppressed dermal neovascularization in mice as effectively as Vegfa siRNA. siRNA-induced inhibition of neovascularization required a minimum length of 21 nucleotides, a bridging necessity in a modelled 2:1 TLR3-RNA complex. Choroidal endothelial cells from people expressing the TLR3 coding variant 412FF were refractory to extracellular siRNA-induced cytotoxicity, facilitating individualized pharmacogenetic therapy. Multiple human endothelial cell types expressed surface TLR3, indicating that generic siRNAs might treat angiogenic disorders that affect 8% of the world's population, and that siRNAs might induce unanticipated vascular or immune effects.
针对年龄相关性黄斑变性导致的致盲性脉络膜新生血管（CNV）患者，开展的靶向血管内皮生长因子-A（VEGFA）或其受体VEGFR1（也称为FLT1）的小干扰RNA（siRNA）临床试验，是以细胞内RNA干扰（RNAi）实现基因沉默为前提的。然而，我们发现CNV抑制是一种siRNA类效应：与靶向Vegfa或Vegfr1的siRNA相比，靶向非哺乳动物基因、非表达基因、非基因组序列、促血管生成和抗血管生成基因以及无RNAi活性的siRNA（长度为21个核苷酸或更长），在小鼠中均能同等程度地抑制CNV，且不存在脱靶RNAi或干扰素-α/β激活现象。非靶向（针对非哺乳动物基因）和靶向（针对Vegfa或Vegfr1）的siRNA通过细胞表面的Toll样受体3（TLR3）、其接头蛋白TRIF以及干扰素-γ和白细胞介素-12的诱导来抑制CNV。非靶向siRNA抑制小鼠皮肤新生血管生成的效果与Vegfa siRNA相同。siRNA诱导的新生血管生成抑制需要最短21个核苷酸的长度，这是模拟的2:1 TLR3-RNA复合物中的一个必要连接条件。表达TLR3编码变体412FF的人的脉络膜内皮细胞对细胞外siRNA诱导的细胞毒性具有抗性，这有利于个体化药物遗传学治疗。多种人类内皮细胞类型均表达表面TLR3，这表明通用siRNAs可能治疗影响全球8%人口的血管生成性疾病，并且siRNAs可能会诱导意想不到的血管或免疫效应。
