Intravitreal delivery of AAV2 transduces porcine retinal ganglion cells.

Abstract

Purpose : Adeno-associated virus serotype 2 (AAV2) is a promising gene therapy platform for glaucoma and other retinal ganglion cell (RGC) degenerative diseases. Although pro-survival and pro-regenerative AAV2 gene therapies have been identified in mouse, advancement towards human therapeutics has been limited due to a lack of data in a large animal model. Here we test transduction of porcine RGCs with AAV2 in vivo while evaluating vector promoters and optimizing injection technique.

Methods : Yucatan minipigs (5.5-6 months old) were intravitreally co-injected with AAV2-UBC-eGFP and AAV2-CAG-TdTomato to compare promoter strength. In order to optimize vector delivery, three injection strategies were evaluated: (1) 1-point injection at the temporal aspect of the eye 4 mm posterior to the limbus, (2) 2-point injection at the temporal and nasal aspects, and (3) 4-point injection at the temporal, nasal, superior, and inferior aspects. Four weeks after injection, retinas were harvested, stained by immunofluorescence for Brn3a (RGC marker), flat-mounted, and imaged by confocal microscopy. Fluorescent cells were quantified on ImageJ, and transduction efficiency was determined by calculating the percentage of Brn3a+ cells that were eGFP+ or TdTomato+. All research was conducted in compliance with IACUC approval and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Results : Both UBC and CAG promoters drove high fluorophore expression in RGCs (75% and 74% transduction efficiency, respectively). The 2-point injection strategy resulted in high transduction efficiency across the retina, whereas 1-point injection resulted in high transduction in the temporal retina but moderate or low transduction elsewhere, and 4-point injection resulted in minimal transduction across the retina.

Conclusions : AAV2 is a viable technology for gene delivery in porcine RGCs in vivo. Both UBC and CAG promoters strongly promote transduced gene expression in porcine RGCs, and a 2-point injection method yielded optimal delivery efficiency.

This is a 2021 ARVO Annual Meeting abstract.

 

AAV2-mediated expression in porcine RGCs. Four weeks after 2-point delivery of AAV2-UBC-eGFP and AAV2-CAG-TdTomato, retinas were harvested and stained for Brn3a by immunofluorescence. Both UBC and CAG promoters drove robust expression in RGCs of the temporal retina. Scale bar, 100 um.