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

Authors: Li Taiming,  Wei Zheng,  Liu Xiaomei,  Shao Jiahong,  Yuan Yun,  Cao Jinjing,  Wang Feifei,  Wang Dongxin,  Li Wensheng,  Zhang Chun
DOI: 10.3760/cma.j.cn115989-20200326-00210
Published 2020-05-10
Cite as Chin J Exp Ophthalmol, 2020,38(05): 381-387.

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.

Key words:

Adeno-associated viruses; Gene therapy; Intravitreal injection; Retina; Retinal diseases

Contributor Information

Li Taiming
School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China
Wei Zheng
School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China
Liu Xiaomei
Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou 215163, China
Shao Jiahong
Suzhou Gene Medicine Co.Ltd, Suzhou 215002, China
Yuan Yun
Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou 215163, China
Cao Jinjing
Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou 215163, China
Wang Feifei
Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou 215163, China
Wang Dongxin
Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou 215163, China
Li Wensheng
Aier School of Ophthalmology, Central South University, Changsha 410006, China
Zhang Chun
Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou 215163, China
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Updated: June 24, 2020 — 11:18 am