Efficient photoreceptor-targeted gene expression in vivo by recombinant adeno-associated virus
Efficient photoreceptor-targeted gene expression in vivo by recombinant adeno-associated virus
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DOI：
10.1073/pnas.94.13.6916
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发表时间：
1997-06-24
影响因子：
11.1
通讯作者：
Hauswirth, WW
中科院分区：
文献类型：
--
作者：
Flannery, JG;Zolotukhin, S;Hauswirth, WW
我们描述了一种在哺乳动物视网膜光感受器细胞中实现外源基因高效和细胞类型特异性表达的通用方法。重组腺相关病毒(RAAV)载体在注射到视网膜下间隙后将细菌LacZ基因或合成的绿色荧光蛋白基因(GFP)转移到小鼠或大鼠视网膜。用小鼠近端视蛋白启动子(+86~-385)来驱动表达，报告基因产物仅在光感受器中发现，而在任何其他类型的视网膜细胞或邻近的视网膜色素上皮中均未发现。绿色荧光蛋白表达的光感受器通常覆盖视网膜总面积的10-20%。单次注射L后，光感受器直接在注射部位周围区域以近100%的效率被转导，我们估计由于单次视网膜下接种，大约有250万个光感受器被转导。这种水平的基因转移和表达表明了视网膜疾病基因治疗的可行性，分别通过空斑试验和感染中心试验判断，含有GFP的rAAV株基本上没有腺病毒和野生型AAV。因此，高纯度、无辅助病毒的rAAV载体可以实现对终末分化、有丝分裂后的光感受器细胞的高频组织特异性转导。
We describe a general approach for achieving efficient and cell type-specific expression of exogenous genes in photoreceptor cells of the mammalian retina, Recombinant adeno-associated virus (rAAV) vectors were used to transfer the bacterial lacZ gene or a synthetic green fluorescent protein gene (gfp) to mouse or rat retinas after injection into the subretinal space. Using a proximal murine rod opsin promoter (+86 to -385) to drive expression, reporter gene product was found exclusively in photoreceptors, not in any other retinal cell type or in the adjacent retinal pigment epithelium. GFP-expressing photoreceptors typically encompassed 10-20% of the total retinal area after a single 2-mu l injection, Photoreceptors were transduced with nearly 100% efficiency in the region directly surrounding the injection site, We estimate approximately 2.5 million photoreceptors were transduced as a result of the single subretinal inoculation. This level of gene transfer and expression suggests the feasibility of genetic therapy for retinal disease, The gfp-containing rAAV stock was substantially free of both adenovirus and wild-type AAV, as judged by plaque assay and infections center assay, respectively. Thus, highly purified, helper virus-free rAAV vectors can achieve high-frequency tissue-specific transduction of terminally differentiated, postmitotic photoreceptor cells.