Self-complementary AAV5 vector facilitates quicker transgene expression in photoreceptor and retinal pigment epithelial cells of normal mouse.
Self-complementary AAV5 vector facilitates quicker transgene expression in photoreceptor and retinal pigment epithelial cells of normal mouse.
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DOI：
10.1016/j.exer.2010.01.011
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发表时间：
2010-05
影响因子：
3.4
通讯作者：
Pang, Ji-Jing
中科院分区：
文献类型：
--
作者：
Kong, Fansheng;Li, Wensheng;Li, Xia;Zheng, Qinxiang;Dai, Xufeng;Zhou, Xiangtian;Boye, Sanford L.;Hauswirth, William W.;Qu, Jia;Pang, Ji-Jing
关键词：
为了阐明自补型(Sc)AAV5载体在正常视网膜中的转导效率和细胞类型特异性是否与标准的单链AAV5载体相似，将1微升的scAAV5-smCBA-GFP载体(1x1012基因组含颗粒/ml)和AAV5-smCBA-GFP载体(1x1012基因组含颗粒/ml)分别注射到成年C57BL/6J小鼠的右眼和左眼(均经角膜)。分别于注射后第1、2、5、7、10、14、21、28和35天摘除眼球，取视网膜色素上皮(RPE)、神经视网膜和眼球切片，荧光显微镜下观察绿色荧光蛋白(GFP)的表达。经角膜下注射scAAV5-smCBA-GFP载体后，GFP在PID1附近的RPE和PID2~5之间的神经视网膜整体中首次检测到GFP的表达，在PID10~14之间和P14~P21之间的RPE整体中GFP表达达到峰值并稳定，绿色荧光强而均匀，覆盖整个视网膜整体。冰冻切片支持以下结果：GFP在视网膜色素上皮(RPE)中的表达最早出现在PID1-2附近，并很快扩散到光感受器(PR)细胞；在PID7，GFP的中等表达主要出现在PR和RPE层；在PID14-21之间，GFP在RPE和PR细胞中有较强且均匀的表达。在视网膜下注射AAV5-smCBA-GFP后，GFP的表达首先在PID5-7左右的RPE整细胞和PID7-10左右的神经视网膜整细胞中检测到；在RPE全细胞中，ssAAV5介导的GFP表达在PID21左右达到峰值，在神经视网膜整细胞中在PID28左右达到峰值；AAV5处理眼的切片也支持从全细胞中获得的结果：GFP首先在RPE中检测到，然后扩散到PR细胞。在rPE中，scAAV5介导的GFP表达峰值与AAV5介导的相似。但在PR细胞中，scAAV5介导的峰值GFP表达强于AAV5。经角膜玻璃体内注射scAAV5-GFP或AAV5-GFP后，所有视网膜细胞(RPE细胞、神经视网膜细胞和视网膜切片)均未检测到GFP荧光。经角膜玻璃体内注射后，scAAV5和AAV5均不能转导视网膜细胞。本研究中使用的scAAV5载体比匹配的AAV5载体更早开始转基因表达，并且在视网膜下注射后在PR细胞中有更强的转基因表达。我们的数据证实了先前的报道，即单链AAV载体比标准单链AAV载体具有更早的发病时间。在解决某些RPE和/或PR细胞相关的视网膜营养不良模型时，scAAV5载体可能比标准的单链AAV载体更有用，特别是对于需要快速和强劲的转基因表达以防止PR细胞早期退化的人视网膜色素变性的小鼠模型。
To clarify whether transduction efficiency and cell type specificity of self-complementary (sc) AAV5 vectors are similar to those of standard, single stranded AAV5 vectors in normal retina, one micro liter of scAAV5-smCBA-GFP vector (1X1012 genome containing particles/ml) and AAV5-smCBA-GFP vector (1X1012 genome containing particles/ml) were subretinally or intravitreally (in both cases through the cornea) injected into the right and left eyes of adult C57BL/6J mice, respectively. On post-injection day (PID) 1, 2, 5, 7, 10, 14, 21, 28 and 35, eyes were enucleated; retinal pigment epithelium (RPE) wholemounts, neuroretinal wholemounts and eyecup sections were prepared to evaluate green fluorescent protein (GFP) expression by fluorescent microscopy. GFP expression following trans-cornea subretinal injection of scAAV5-smCBA-GFP vector was first detected in RPE wholemounts around PID 1 and in neuroretinal wholemounts between PID 2 and 5; GFP expression peaked and stabilized between PID 10-14 in RPE wholemounts and between P14 and P21 in neuroretinal wholemounts with strong, homogeneous green fluorescence covering the entire wholemounts. The frozen sections supported the following findings from the wholemounts: GFP expression appeared first in RPE around PID 1-2 and soon spread to photoreceptors (PR) cells; by PID 7, moderate GFP expression was found mainly in PR and RPE layers; between PID 14 and 21, strong and homogenous GFP expression was observed in RPE and PR cells. GFP expression following subretinal injection of AAV5-smCBA-GFP was first detected in RPE wholemounts around PID 5-7 and in neuroretinal wholemounts around PID 7-10; ssAAV5 mediated GFP expression peaked at PID 21 in RPE wholemounts and around PID 28 in neuroretinal wholemounts; sections from AAV5 treated eyes also supported findings obtained from wholemounts: GFP expression was first detected in RPE and then spread to the PR cells. Peak GFP expression in RPE mediated by scAAV5 was similar to that mediated by AAV5. However, peak GFP expression mediated by scAAV5 in PR cells was stronger than that mediated by AAV5. No GFP fluorescence was detected in any retinal cells (RPE wholemounts, neuroretinal wholemounts and retinal sections) after trans-cornea intravitreal delivery of either scAAV5-GFP or AAV5-GFP. Neither scAAV5 nor AAV5 can transduce retinal cells following trans-cornea intravitreal injection. The scAAV5 vector used in this study directs an earlier onset of transgene expression than the matched AAV5 vector, and has stronger transgene expression in PR cells following subretinal injection. Our data confirm the previous reports that scAAV vectors have an earlier onset than the standard, single strand AAV vectors. scAAV5 vectors may be more useful than standard, single stranded AAV vector when addressing certain RPE and/or PR cell-related models of retinal dystrophy, particularly for mouse models of human retinitis pigmentosa that require rapid and robust transgene expression to prevent early degeneration in PR cells.
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