Abstract
Purpose :
Glaucoma leads to loss of retinal ganglion cells and optic nerve degeneration, and blindness. Current medications are only effective in managing the condition and they are critically undermined by patient non-compliance. In this study, we evaluate the tropism of nine recombinant adeno-associated viral vector (rAAV) serotypes in the anterior chamber as part of the development of gene therapy for glaucoma, which will likely require genetic modification of cells in the aqueous outflow pathway or cornea.
Methods :
C57BL/6J mice (48 eyes) were bilaterally intracamerally injected with 2 μL of either rAAV2/1, 2/2, 2/2[MAX], 2/2[7m8], 2/5, 2/6, 2/7, 2/8 or 2/9 vector packaging a GFP reporter gene driven by a ubiquitously expressing chicken beta-actin promotor at concentrations greater than or equal to 2.21x1012 viral genomes/ml. 8-weeks post-injection, animals were imaged using confocal scanning laser ophthalmoscopy (cSLO) to determine transduction efficiency. After in vivo cSLOimaging, animals were euthanized, and eyes were enucleated for histological analysis. Flat mounts were stained for ZO-1 to determine endothelial cell transduction, and cryosections to determine tissue transduction for each serotype. 14-15 organ units per mouse harvested for biodistribution analysis.
Results :
Histology and cSLO imaging revealed that rAAV2/2 showed the least amount of transduction with sparse transduction in the iris and corneal endothelium (CE). 2/2[MAX] and 2/2[7m8] primarily transduced the iris predominantly around the pupil. rAAV2/1, 2/5, 2/6, 2/7, 2/8, and 2/9 successfully transduced the cornea. Flat mount from the cornea showed that rAAV2/2, 2/2[MAX], 2/2[7m8] sparsely transduced the CE. rAAV2/1, 2/7, 2/8, and 2/9 transduced mostly stroma, and some transduction in the CE. rAAV2/5 and 2/6 were the most successful in transducing the CE with rAAV2/5 showing the strongest GFP expression. The biodistribution of rAAV in different organs is currently being investigated.
Conclusions :
In this study, we evaluated the tropism of rAAV serotypes within the anterior chamber of C57BL/6J mice. As is the case following rAAV delivery via more traditional injection routes, such as intravitreal or subretinal, tropism varied greatly following delivery to the anterior chamber, with rAAV2-based serotypes prioritizing transduction of the angle and iris, and expression of rAAV2/5 effectively limited to the CE.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.