Inhibition of choroidal neovascularization by adenovirus-mediated delivery of short hairpin RNAs targeting VEGF as a potential therapy for AMD
Inhibition of choroidal neovascularization by adenovirus-mediated delivery of short hairpin RNAs targeting VEGF as a potential therapy for AMD
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DOI：
10.1167/iovs.05-1610
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发表时间：
2006-08-01
影响因子：
4.4
通讯作者：
Kumar-Singh, Rajendra
中科院分区：
文献类型：
--
作者：
Cashman, Siobhan M.;Bowman, Lisa;Kumar-Singh, Rajendra
目的.脉络膜新生血管（CNV）是年龄相关性黄斑变性（AMD）致盲的主要原因。几条证据表明AMD患者视网膜色素上皮（RPE）中血管内皮生长因子（VEGF）水平升高。目前减弱VEGF或其受体的方法，包括使用小干扰（si）RNA，显示出显著的前景，但仍然具有有限的功效，并且需要重复施用，使用与多种并发症相关的程序。本研究的目的是开发一种方法，用于短发夹（sh）RNA的长期内源性表达，这将显着减弱VEGF，因此作为一种潜在的治疗AMD。开发了从重组腺病毒表达的几种shRNA。在存在过表达VEGF的腺病毒载体的情况下，这些shRNA在人RPE细胞中表达，并评估VEGF衰减的量。随后将表达VEGF的腺病毒载体注射到小鼠的视网膜下腔中，并在表达靶向VEGF的shRNA的腺病毒载体存在下测量CNV的诱导。鉴定了能够在人RPE细胞中沉默VEGF的有效shRNA序列。当从腺病毒骨架表达时，这些shRNA构建体在1：5摩尔比（VEGF与shRNA）下使VEGF沉默94%，在1：0.05摩尔比下使VEGF沉默64%。高表达VEGF的腺病毒载体可在5天内诱导小鼠CNV。将表达VEGF的病毒与靶向VEGF的shRNA共注射到小鼠体内，导致CNV显著减少（84%）。来自腺病毒载体的靶向VEGF的shRNA可有效减弱VEGF并预防CNV。这种方法显示出作为AMD疗法的前景。
PURPOSE. Choroidal neovascularization ( CNV) is the leading cause of blindness in age-related macular degeneration ( AMD). Several lines of evidence implicate increased levels of vascular endothelial growth factor ( VEGF) in retinal pigment epithelium ( RPE) from patients with AMD. Current approaches to attenuate VEGF or its receptors, including the use of small interfering ( si) RNA, show significant promise, but still have limited efficacy and require repeat administrations, using procedures associated with multiple complications. The goal of this study was to develop an approach for long-term endogenous expression of short hairpin ( sh) RNA that would significantly attenuate VEGF and hence act as a potential therapy for AMD.METHODS. Several shRNAs expressed from recombinant adenovirus were developed. These shRNAs were expressed in human RPE cells in the presence of adenovirus vectors overexpressing VEGF, and the amount of VEGF attenuation was evaluated. Adenovirus vectors expressing VEGF were subsequently injected into the subretinal space of mice, and induction of CNV was measured in the presence of adenovirus vectors expressing shRNA targeting VEGF.RESULTS. Potent shRNA sequences were identified that were able to silence VEGF in human RPE cells. When expressed from adenovirus backbones, these shRNA constructs silenced VEGF by 94% at a 1: 5 molar ratio ( VEGF to shRNA) and 64% at a 1: 0.05 molar ratio. Adenovirus vectors expressing high levels of VEGF could induce CNV in mice within 5 days. Co-injection of VEGF-expressing viruses into mice with shRNA targeting VEGF led to a substantial ( 84%) reduction in CNV.CONCLUSIONS. shRNA targeting VEGF from adenovirus vectors allows potent attenuation of VEGF and prevents CNV. This approach shows promise as a therapy for AMD.