Tropisms of rAAV2, 6, 8, 9 serotypes for intravitreal injection in the mouse retina
10.3760/cma.j.cn115989-20200326-00210
- VernacularTitle: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
- Author:
Taiming LI
1
;
Zheng WEI
;
Xiaomei LIU
;
Jiahong SHAO
;
Yun YUAN
;
Jinjing CAO
;
Feifei WANG
;
Dongxin WANG
;
Wensheng LI
;
Chun ZHANG
Author Information
1. 中国药科大学生命科学与技术学院，南京 210009
- From:
Chinese Journal of Experimental Ophthalmology
2020;38(5):381-387
- CountryChina
- Language:Chinese
-
Abstract:
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 1 μl rAAV intravitreally, and 1 μl 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.
