Targeted transgene expression in Muller glia of normal and diseased retinas using lentiviral vectors
Targeted transgene expression in Muller glia of normal and diseased retinas using lentiviral vectors
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DOI：
10.1167/iovs.05-1570
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发表时间：
2007-04-01
影响因子：
4.4
通讯作者：
Flannery, John G.
中科院分区：
文献类型：
--
作者：
Greenberg, Kenneth P.;Geller, Scott F.;Flannery, John G.
目的。米勒胶质细胞在视网膜稳态和功能中发挥着至关重要的作用。通过病毒基因传递对 Muller 细胞进行基因修饰对于研究其正常生理学和在视网膜疾病状态中的作用具有重要意义。然而，在递送基因转移载体后，Muller 细胞中稳定且有效的转基因表达仍然难以捉摸。转录和转导靶向方法被用来设计重组的基于 HIV-1 的慢病毒 (IV) 载体，能够在健康和患病的啮齿动物视网膜中高效、持续地表达 Muller 细胞转基因。方法。含有神经胶质细胞特异性启动子（CD44、神经胶质纤维酸性蛋白和波形蛋白）和增强型绿色荧光蛋白（eGFP）cDNA的表达盒被克隆到IV骨架中，这些骨架被包装到展示水泡性口炎病毒（VSV）或罗斯河病毒（RRV）包膜表面糖蛋白的传染性载体颗粒中。通过玻璃体内和视网膜下方法将载体注射到1至180天的野生型Sprague-Dawley (SD)和视网膜退化S334Ter(+/-)转基因大鼠中。采用体内荧光眼底成像和免疫荧光共聚焦显微镜比较表达效率、细胞类型特异性和时间表达特征。结果。病毒假型、调控启动子和手术递送位点的选择均对 Muller 细胞中 eGFP 转基因表达水平产生可测量的影响。通过视网膜下注射含有 CD44 启动子的 VSV-G 假型 IV 载体，SD 视网膜中的表达水平最高。使用这些载体，在单次视网膜下注射后，在 Muller 胶质细胞中观察到 eGFP 持续表达超过 6 个月，覆盖了 25% 至 30% 的视网膜表面积。免疫组织化学（α-谷氨酰胺合成酶）显示大约 95% 的 Muller 细胞在注射部位附近区域被转导。根据视网膜电图 (ERG) 评估，这些病毒载体的递送和随后的 Muller 细胞 eGFP 表达对视觉功能没有负面影响。结论。含有神经胶质细胞特异性启动子的假型 IV 载体可有效转导并指导视网膜 Muller 神经胶质细胞的持续转基因表达。这种类型的载体将可用于视网膜疾病的实验治疗以及视网膜的生理和发育研究。
Purpose. Muller glia play crucial roles in retinal homeostasis and function. Genetic modification of Muller cells by viral gene delivery would be valuable for studies of their normal physiology and roles in retinal disease states. However, stable and efficient transgene expression in Muller cells after delivery of gene transfer vectors has remained elusive. Transcriptional and transductional targeting approaches were used to engineer recombinant HIV-1-based lentiviral (IV) vectors capable of highly efficient and sustained Muller cell transgene expression in healthy and diseased rodent retinas.METHODS. Expression cassettes containing glia-specific promoters (CD44, glial fibrillary acidic protein, and vimentin) and an enhanced green fluorescent protein (eGFP) cDNA were cloned into IV backbones, which were packaged into infectious vector particles displaying either the vesicular stomatitis virus (VSV) or Ross River virus (RRV) envelope surface glycoproteins. Vectors were injected by intravitreal and subretinal approaches in wild type Sprague-Dawley (SD) and retinal degenerate S334Ter(+/-) transgenic rats aged 1 to 180 days. In vivo fluorescent fundus imaging and immunofluorescent confocal microscopy were used for comparison of expression efficiency, cell type specificity, and temporal expression characteristics.RESULTS. The choice of viral pseudotype, regulatory promoter, and surgical delivery site each had a measurable effect on the level of eGFP transgene expression in Muller cells. The highest expression levels in SD retinas were attained with subretinal injection of VSV-G pseudotyped IV vectors containing the CD44 promoter. With these vectors, persistent eGFP expression in Muller glia was observed for more than 6 months, covering 25% to 30% of the retinal surface area after a single subretinal injection. Imunohistochemistry (alpha-glutamine synthetase) revealed that approximately 95% of the Muller cells were transduced in the region near the injection site. Delivery of these viral vectors and subsequent Muller cell eGFP expression had no negative impact on visual function, as assessed by electroretinography (ERG).CONCLUSIONS. Pseudotyped IV vectors containing glia-specific promoters efficiently transduce and direct sustained transgene expression in retinal Muller glia. Vectors of this type will be useful for experimental treatment of retinal disease, as well as for physiological and developmental investigations of the retina.