Abstract
Purpose :
Suprachoroidal space (SCS) injection of adeno-associated virus (AAV) provides widespread transgene expression in rat and monkeys (Ding et al. 2019). Here, we developed a technique to perform SCS injections in mice and tested various AAVs to determine whether similar results could be achieved in mice.
Methods :
Adult C57BL/6 mice were injected with 3 uL of AAV9 (n=12 eyes) or AAV8 (n=14) CAG-GFP (1E13 vg/mL). For SCS injections, a 30-gauge needle was used to create a shallow incision in the sclera. A 33-gauge needle with 20-degree bevel attached to a 10 uL Hamilton syringe was advanced under the sclera. Correct localization into the SCS was confirmed by evaluating the bevel, which is observable under the transparent sclera but not the pigmented choroid. Once the bevel was within the SCS, the solution was slowly injected. Two weeks post-injection, fundus autofluorescence (FAF) imaging was performed, mice were sacrificed, and eyes were fixed and prepared for flatmounts (RPE/sclera, retina, cornea) or cryosections. Sections and flatmounts were stained with anti-GFP antibody. HALO software was used to quantify the total area of anti-GFP signal in RPE and retinal flatmounts.
Results :
FAF imaging showed widespread GFP signal in the eye with AAV8 & AAV9 injection. Similarly, RPE and retinal flatmounts showed extensive GFP expression with both viral vectors. Cryosections showed that GFP signal in the retina was mostly found in the photoreceptor cell layer. Quantification of the total area of anti-GFP signal in RPE flatmounts revealed the greatest coverage in AAV9 group (59% ± 25%), followed closely by AAV8 (49% ± 18%). In the retina, AAV8 & AAV9 provided equal area of GFP expression (55% ± 27% and 56% ± 28% coverage, respectively). Lastly, GFP signal was also found in flatmounts of sclera and cornea (stromal layer and endothelial cells) with both vectors.
Conclusions :
SCS injection of AAV8 and AAV9 provided widespread transgene expression. Several ocular tissues were transduced, including the sclera, RPE, retina, and cornea. The ability to perform SCS injections in mice allows for the exploration of this delivery method in relevant mouse models of ocular disease.
This is a 2021 ARVO Annual Meeting abstract.