Efficiency and Safety of AAV-Mediated Gene Delivery of the Human ND4 Complex I Subunit in the Mouse Visual System
Efficiency and Safety of AAV-Mediated Gene Delivery of the Human ND4 Complex I Subunit in the Mouse Visual System
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DOI：
10.1167/iovs.08-3214
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发表时间：
2009-09-01
影响因子：
4.4
通讯作者：
Hauswirth, William W.
中科院分区：
文献类型：
--
作者：
Guy, John;Qi, Xiaoping;Hauswirth, William W.
目的.目的评价腺相关病毒（AAV）介导的人ND 4复合物I亚基基因转染小鼠视觉系统的有效性和安全性。将与ATPc线粒体靶向序列和FLAG表位融合的核编码的人ND 4亚基包装在注射到小鼠右眼中的AAV 2衣壳中。将AAV-GFP注射到左眼中。一个月后，模式视网膜电图（PERG），ATP合成率，基因表达，并纳入人ND 4亚基到小鼠复合物I进行了评价。与注射绿色荧光蛋白（GFP）的眼睛相比，对注射ND 4 FLAG的眼睛进行定量分析。在ND 4FLAG和GFP接种的眼睛中，ATP合成率和PERG振幅相似。接受ND 4FLAG的眼睛的PERG潜伏期较短。免疫沉淀的鼠复合物I得到预期的52-kDa的经处理的人ND 4FLAG条带。共聚焦显微镜显示FLAG的核周表达共定位与大肠杆菌特异性荧光染料。透射电子显微镜显示线粒体内的FLAG免疫金。与Thy1.2阳性视网膜神经节细胞（RGC）相比，FLAG阳性RGC的定量为38%，GFP阳性RGC的定量为65%。AAV-ND 4FLAG中Thy1.2阳性RGC计数与注射AAV-GFP的对照眼中的计数相似。人ND 4经适当处理后，玻璃体内注射后，输入到RGCs的线粒体和小鼠视神经轴突中。虽然它有大约三分之二的GFP的效率，正常人ND 4在鼠线粒体中的表达并没有诱导RGC的损失，ATP合成，或PERG振幅，这表明异位ND 4可能是安全的治疗Leber遗传性视神经病变的患者。（Invest Ophthalmol维斯科学。2009; 50：4205-4214）DOI：10.1167/iovs.08-3214
PURPOSE. To evaluate the efficiency and safety of AAV-mediated gene delivery of a normal human ND4 complex I subunit in the mouse visual system.METHODS. A nuclear encoded human ND4 subunit fused to the ATPc mitochondrial targeting sequence and FLAG epitope were packaged in AAV2 capsids that were injected into the right eyes of mice. AAV-GFP was injected into the left eyes. One month later, pattern electroretinography (PERG), rate of ATP synthesis, gene expression, and incorporation of the human ND4 subunit into the murine complex I were evaluated. Quantitative analysis of ND4FLAG-injected eyes was assessed compared with green fluorescent protein (GFP)-injected eyes.RESULTS. Rates of ATP synthesis and PERG amplitudes were similar in ND4FLAG- and GFP-inoculated eyes. PERG latency was shorter in eyes that received ND4FLAG. Immunoprecipitated murine complex I gave the expected 52-kDa band of processed human ND4FLAG. Confocal microscopy revealed perinuclear expression of FLAG colocalized with mitochondria-specific fluorescent dye. Transmission electron microscopy revealed FLAG immunogold within mitochondria. Compared with Thy1.2-positive retinal ganglion cells (RGCs), quantification was 38% for FLAG-positive RGCs and 65% for GFP-positive RGCs. Thy1.2 positive-RGC counts in AAV-ND4FLAG were similar to counts in control eyes injected with AAV-GFP.CONCLUSIONS. Human ND4 was properly processed and imported into the mitochondria of RGCs and axons of mouse optic nerve after intravitreal injection. Although it had approximately two-thirds the efficiency of GFP, the expression of normal human ND4 in murine mitochondria did not induce the loss of RGCs, ATP synthesis, or PERG amplitude, suggesting that allotopic ND4 may be safe for the treatment of patients with Leber hereditary optic neuropathy. (Invest Ophthalmol Vis Sci. 2009; 50: 4205-4214) DOI: 10.1167/iovs.08-3214