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Xu Cao, Heather Kayew Mak, Jasmine Sum-Yee Yung, Heidi Ng, Christopher Kai-Shun Leung; Transfection efficiency of hybrid adeno-associated virus (AAV) vectors in mouse retinal ganglion cells (RGCs). Invest. Ophthalmol. Vis. Sci. 2017;58(8):4875.
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© ARVO (1962-2015); The Authors (2016-present)
Adeno-associated virus (AAV) is a replication-deficient virus that can infect RGCs with low immune responses, making it a good candidate for gene therapy. Although capsid protein is a major determinant of cellular tropism and a variety of hybrid AAV vectors have been engineered with capsid protein of AAV serotypes 1-9, little is known about the transfection efficiency of these AAV serotypes in RGCs via intravitreal administration. Here, we examined the efficiency of seven AAV serotypes in the mouse retina after intravitreal administration.
Each eye of 13 4-week-old C57BL/6 mice eyes received intravitreal injection of one of the following GFP-expressing AAV serotypes: AAV2/1, AAV2/2, AAV2/4, AAV2/5, AAV2/6, AAV2/8 and AAV2/9. The retinas were imaged non-invasively using a confocal scanning laser ophthalmoscope (CSLO) at day 3, week 1, 2, 3, 4 and 8 following intravitreal injection. The retinas were then isolated for immunohistochemistry. Transfection efficiency was calculated with reference to the proportion of the number of GFP and TUJ1 positive cells to the number of TUJ1 positive cells in the ganglion cell layer.
With in vivo CSLO imaging, florescent signals were first detected 3 days after intravitreal injection and the signals peaked after 1 week (Fig.1). AAV2/1, AAV2/2, AAV2/4 and AAV2/6 transfected RGCs with transfection efficiency of 0.22± 0.11%, 14.33±0.76%, 0.02±0.02 %, and 0.08±0.05 %, respectively. AAV2/2 showed the highest transfection efficiency and the transfection was evenly distributed in the retina. AAV2/5, AAV2/8 and AAV2/9, however, did not transfect RGCs and the GFP signals were primarily localized to the injection site.
Intravitreal administration of AAV2/2 had the greatest efficiency for transfecting RGCs compared with other AAV serotypes. Long term expression was observed up to 2 months of follow-up. Our data supports intravitreal administration of AAV2/2 can provide a safe and effective approach for gene delivery to RGCs.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
Figure 1. In vivo CSLO imaging of a mouse retina at day 3, week 1, 2, 3 and 4 following intravitreal injection of AA2/2 (left to right).
Figure 2. Immunohistochemical staining of a retinal cross-section (left to right) showing GFP (green), TUJI (red), DAPI (blue) fluorescent signals and the brightfield and the merged images.
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