Induction of rapid and highly efficient expression of the human ND4 complex I subunit in the mouse visual system by self-complementary adeno-associated virus.
Induction of rapid and highly efficient expression of the human ND4 complex I subunit in the mouse visual system by self-complementary adeno-associated virus.
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DOI：
10.1001/archophthalmol.2010.135
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发表时间：
2010-07
影响因子：
--
通讯作者：
Guy, John
中科院分区：
文献类型：
--
作者：
Koilkonda, Rajeshwari D.;Chou, Tsung-Han;Porciatti, Vittorio;Hauswirth, William W.;Guy, John
目的：利用自补充腺相关病毒(ScAAV)载体眼部基因治疗Leber遗传性视神经病变(LHON)患者的急性视力丧失，以证明在脊椎动物视网膜中异位表达正常人类ND4亚单位复合体I的高效和快速。将核编码的人ND4亚基与三磷酸腺苷合成酶(ATPc)亚基C亚基的P1亚型和FLAG表位融合，包装在scAAV2衣壳或单链AAV2衣壳中。这些构建物被注射到小鼠的玻璃体腔中。对侧眼注射scAAV-绿色荧光蛋白(GFP)。1周后检测视网膜电图及人ND4亚单位和绿色荧光蛋白的基因表达。定量分析注射ND4FLAG的眼与Thy1.2标记的视网膜神经节细胞(RGCs)。在接种scAAV-ND4FLAG、ssAAV-ND4FLAG和GFP的眼睛中，图形视网膜电波幅值保持正常。共聚焦显微镜下可见典型的scAAV-ND4FLAG核周线粒体表达，几乎在整个视网膜扁平上。相反，scAAV-GFP呈胞浆和胞核表达。相对于Thy1.2阳性的视网膜节细胞，scAAV-ND4FLAG阳性视网膜节细胞的定量结果为91%，而ssAAV-ND4FLAG阳性视网膜节细胞的定量结果为51%。治疗LHON引起的急性视力丧失可能是通过scAAV载体传递的正常人类ND4亚单位复合体I基因，该基因在大多数LHON病例中发生突变。与大多数视网膜退行性变导致多年缓慢进行性视力丧失不同，由于线粒体DNA突变导致的LHON会导致中风、双侧严重且通常不可逆转的视力丧失。为了抢救LHON的急性视力损失，需要一个高效、快速的基因表达系统。
To demonstrate the high efficiency and rapidity of allotopic expression of a normal human ND4 subunit of complex I in the vertebrate retina using a self-complementary adeno-associated virus (scAAV) vector for ocular gene delivery to treat acute visual loss in Leber hereditary optic neuropathy (LHON). The nuclear-encoded human ND4 subunit fused to the P1 isoform of subunit C of adenosine tri-phosphate synthase (ATPc) mitochondrial targeting sequence and FLAG epitope was packaged in scAAV2 capsids or single-stranded (ss) AAV2 capsids. These constructs were injected into the vitreous cavities of mice. The contralateral eyes were injected with scAAV–green fluorescent protein (GFP). One week later, pattern electroretinograms and gene expression of the human ND4 subunit and GFP were evaluated. Quantitative analysis of ND4FLAG-injected eyes was assessed relative to Thy1.2-labeled retinal ganglion cells (RGCs). Pattern electroretinogram amplitudes remained normal in eyes inoculated with scAAV-ND4FLAG, ssAAV-ND4FLAG, and GFP. Confocal microscopy revealed the typical perinuclear mitochondrial expression of scAAV-ND4FLAG in almost the entire retinal flat mount. In contrast, scAAV-GFP expression was cytoplasmic and nuclear. Relative to Thy1.2-positive RGCs, quantification of scAAV-ND4FLAG–positive RGCs was 91% and that of ssAAV-ND4FLAG–positive RGCs was 51%. Treatment of acute visual loss due to LHON may be possible with a normal human ND4 subunit gene of complex I, mutated in most cases of LHON, when delivered by an scAAV vector. Unlike most retinal degenerations that result in slowly progressive loss of vision over many years, LHON due to mutated mitochondrial DNA results in apoplectic, bilateral severe and usually irreversible visual loss. For rescue of acute visual loss in LHON, a highly efficient and rapid gene expression system is required.
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影响因子：
4.2
作者：
Keeney, Paula M.;Quigley, Caitlin K.;Bennett, James P., Jr.
通讯作者：
Bennett, James P., Jr.
影响因子：
5.1
作者：
McCarty, DM;Monahan, PE;Samulski, RJ
通讯作者：
Samulski, RJ
影响因子：
5.1
作者：
McCarty, DM;Fu, H;Samulski, RJ
通讯作者：
Samulski, RJ
影响因子：
4.2
作者：
Hauswirth, William W.;Aleman, Tomas S.;Jacobson, Samuel G.
通讯作者：
Jacobson, Samuel G.
影响因子：
4.4
作者：
Guy, John;Qi, Xiaoping;Hauswirth, William W.
通讯作者：
Hauswirth, William W.