Real-time, noninvasive in vivo assessment of adeno-associated virus-mediated retinal transduction.
Real-time, noninvasive in vivo assessment of adeno-associated virus-mediated retinal transduction.
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DOI：
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发表时间：
1997-12
影响因子：
4.4
通讯作者：
Jean Bennett;Dongsheng Duan;John F. Engelhardt;A. Maguire
中科院分区：
文献类型：
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作者：
Jean Bennett;Dongsheng Duan;John F. Engelhardt;A. Maguire
目的评价重组腺相关病毒（rAAV）介导的视网膜转导在成年免疫活性小鼠体内的效率、细胞特异性、稳定性和毒性。评估绿色荧光蛋白（GFP）用于实时、无创监测体内视网膜转基因表达的有效性。在视网膜下注射携带GFP互补DNA（cDNA）的重组腺相关病毒后11周，在小鼠队列中进行眼部GFP表达的评估。使用激发屏障滤光片，通过检眼镜直接观察荧光进行体内检查。视网膜组织的组织学分析用于鉴定转导的细胞并评估炎症。结果视网膜GFP表达可以在体内实时监测，非侵入性成像。重组AAV有效地转导神经视网膜和视网膜色素上皮（RPE）的多种细胞。直到感染后1周才观察到转基因表达。GFP表达细胞的数量在3周内增加，并且表达光感受器和RPE的细胞持续至少11周（实验终止）。没有炎症反应的临床或组织学证据。结论rAAV介导的视网膜基因转移是一种稳定、有效的基因转移方法，且无临床或组织学毒性和免疫反应。绿色荧光蛋白允许作为时间函数的视网膜转基因表达的程度和位置的非侵入性评估，并且有望单独使用和作为实验性或治疗性递送到视网膜的其他转基因的标签。
PURPOSE To evaluate the efficiency, cell specificity, stability, and toxicity of recombinant adeno-associated virus (rAAV)-mediated retinal transduction in vivo in the adult immunocompetent mouse. To assess the usefulness of green fluorescent protein (GFP) for real-time, noninvasive monitoring of retinal transgene expression in vivo. METHODS Assessment of ocular GFP expression was performed in cohorts of mice for 11 weeks after subretinal injection of a recombinant adeno-associated virus carrying the complementary DNA (cDNA) for GFP. Examinations were performed in vivo by direct observation of fluorescence by ophthalmoscopy, using excitation-barrier filters. Histologic analyses of retinal tissue were used to identify transduced cells and to assess inflammation. RESULTS Retinal GFP expression can be monitored in vivo using real-time, noninvasive imaging. Recombinant AAV efficiently transduces a variety of cells of the neural retina and of the retinal pigment epithelium (RPE). Transgene expression was not observed until 1 week after infection. The number of GFP-expressing cells increased over 3 weeks, and expressing photoreceptors and RPE, cells persisted at least through 11 weeks (the termination of the experiment). There was no clinical or histologic evidence of inflammatory response. CONCLUSIONS Retinal gene transfer mediated by rAAV is stable and efficient and is associated with no clinically or histologically detectable toxicity or immune reaction. Green fluorescent protein allows noninvasive assessment of the extent and location of retinal transgene expression as a function of time and promises to be useful alone and as a tag for other transgenes delivered experimentally or therapeutically to the retina.