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John Neidhardt, Carsten Roeger, Jasmin Segelken, Romain Da Costa; Retina-wide AAV-transduction in mice: A novel method combining vitrectomy and intravitreal injection. Invest. Ophthalmol. Vis. Sci. 2016;57(12):755.
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© ARVO (1962-2015); The Authors (2016-present)
Injections of adeno-associated viruses (AAV) into the retina have been extensively used for various approaches in gene therapy or basic science. However, standard retinal injection techniques show limited transduction rates - typically about 20-30% of retinal cells can be transduced in mice. We developed a novel method that allows retina-wide AAV-transduction.
Recombinant AAV2/8 particles with a viral titer of 1*1012 to 1*1013 (VG/ml) were produced applying standard techniques. The AAV encoded GFP was driven by a CMV promotor. Adult C57Bl/6J mice were vitrectomized followed by an AAV-injection of 1.5 microliter into the vitreous. GFP expression was visualized after 1-4 months post-injection by fluorescence-based fundus imaging. ERG measurements were performed. To fine-map GFP-positive cell in retinal layers, immunohistochemistry on retinal flatmounts was combined with laser scanning microscopy using antibody markers for photoreceptors (blue cone opsin) and retinal ganglion cells (Brn3a).
We describe a novel intravitreal injection procedure that leads to transduction of more than 80% of the retina, an efficacy that exceeds that of standard retinal injections. Over 90 mice have been evaluated. An aspiration of vitreous tissue (vitrectomy) prior to a single intravitreal injection of a GFP-expressing AAV2/8 resulted in an almost complete transduction of the retina. Fluorescence laser scanning microscopy showed GFP signals in all retinal layers indicating that the retina was fully penetrated by the AAV. In addition, the optic nerve and nerve fiber layers were GFP positive. ERG measurements and analyses of retinal flatmounts suggested that the novel method does not lead to obvious side effects on the morphological and physiological level.
We developed a novel method to transduce the vast majority of retinal cells with a single AAV injection. Our results will have implications on basic science applications and on virus-based gene therapies in mouse models. The new method may further provide the basis to advance AAV applications to the human retina.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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