重组腺相关病毒2、6、8、9型玻璃体腔注射在小鼠视网膜中的趋向性表达

Tropisms of rAAV2,6,8,9 serotypes for intravitreal injection in the mouse retina

目的比较腺相关病毒(AAV)不同血清型对小鼠视网膜层的趋向性。方法构建pFastBacDual-inCap衣壳质粒和pFastBacDual-ITR-CMV-EGFP基因组质粒;包装重组腺相关病毒2型(rAAV2)、rAAV6、rAAV8、rAAV9;以感染复数(MOI)2000的比例感染HEK293T细胞,24 h后观察荧光表达;应用随机数字表法将C57BL/6小鼠分为rAAV2、rAAV6、rAAV8、rAAV9组和对照组,每组5只,分别双眼玻璃体腔注射rAAV2、rAAV6、rAAV8、rAAV9和磷酸盐缓冲液(PBS)各1μl,注射后2周,制作眼球冰冻切片以及视网膜铺片,分别应用倒置荧光显微镜和激光扫描共焦显微镜观察rAAV不同血清型增强型绿色荧光蛋白(EGFP)荧光强度和感染部位。应用倒置荧光显微镜观察玻璃体腔注射rAAV2后2周、1个月、3个月EGFP荧光强度变化。结果重组病毒对HEK293T的感染效率从高到低依次为rAAV2、rAAV6、rAAV8和rAAV9,转导效率分别为39.5%、18.4%、8.7%和4.6%;在小鼠视网膜中,rAAV2和rAAV6在神经节细胞表达水平较高,rAAV8和rAAV9在视网膜色素上皮(RPE)和光感受器细胞中表达水平较高;rAAV2介导EGFP可于注射后2周、1个月、3个月内在视网膜稳定表达。结论rAAV不同血清型对视网膜RPE细胞和神经节细胞具有高趋向性和高效表达,rAAV2玻璃体腔注射后转导效率较高,且在注射后3个月内稳定表达。

Objective To compare the tropism of different adeno-associated virus(AAV)serotypes in retinal cells.Methods The plasmids pFastBacDual-inCap and pFastBacDual-ITR-CMV-EGFP were constructed for AAV packaging with the baculovirus expression system.Recombinant adeno-associated virus type 2(rAAV2),6,8 and 9 serotypes were packaged,and the infectivity of rAAV was evaluated by infecting HEK293T cells at multiplicity of infection(MOI)2000.Twenty-five C57BL/6 mice were divided into five groups,with five mice per group.In the three experimental groups,both eyes of each mouse were injected 1μl rAAV intravitreally,and 1μl phosphate buffered saline(PBS)for the eyes of the control group.Two weeks after injection,the retinal tissues were collected for preparing flat mounts and cryosections.Enhanced green fluorescent protein(EGFP)gene expression was observed via fluorescence microscopy and laser scanning confocal microscopy.The study protocol was approved by the Ethics Committee of Suzhou Institute of Biomedical Engineering and Technology.Results The infection efficiency of the recombinant virus to HEK293T cells was rAAV2>rAAV6>rAAV8>rAAV9,and the transduction efficiency was 39.5%,18.4%,8.7%and 4.6%,respectively.In mouse retinal transduction,rAAV2 and rAAV6 were highly expressed in the ganglion cells,and rAAV8 and rAAV9 were highly expressed in the retinal pigment epithelium(RPE)and photoreceptor cells.rAAV2-mediated EGFP expression in retinas was stable within three months after injection.Conclusions Different rAAV serotypes have varying tropism and transduction efficiencies in retinal cells through intravitreal injection,rAAV2 has a high transduction efficiency and it can be stably expressed in retinas within three months after injection.