Screening of Adeno-associated virus (AAV) Serotypes for Rabbit Retina delivery

Abstract

Purpose : The choice of AAV serotype and route of administration are critical determinants of the efficacy and safety of ocular gene therapy and need to be well optimized in preclinical models. Rabbits are classic animal model for ocular diseases. Currently there is lack of evidence on the tropism of AAV serotypes in rabbit retina. Here we assessed the effects of AAV serotypes and the administration routes on the retinal tropism and transfection efficiency in rabbit retina.

Methods : We screened 6 AAV serotypes: AAV2,AAV5,AAV8, AAV9, AAV44.9(E531D) and AAV2(TrpYF) in rabbit eyes. New Zealand White rabbits at 2-6 months (n=3 eyes/group) were injected with 1E11 vg/eye in 100 μL PBS in intravitreal injection (IVTI) or with 5E10 vg/eye in 50 μL PBS in subretinal injection (SRI). GFP expression was evaluated by fundus fluorescence imaging weekly. Injected rabbits were sacrificed at 4 weeks post injection(psi) and the localization of the GFP expression were determined by immunofluorescent staining in the fixed eye balls. All the animal experiments were conducted under a protocol approved by the UM IACUC.

Results : At 4 weeks psi, AAV2 showed tropism in retinal epithelium cells, while AAV5 and 9 transfect more in the photoreceptor layers. In SRI, AAV44.9(E531D) showed photoreceptor tropism and highly efficient transfection better than all the other serotypes tested, and the GFP expression region expanded outside of the injection bleb and in some cases, reached the peripheral and center retina in a single injection. In IVT injections, AAV2(TrpFY) showed tropism to inner retinal layers with efficiency outperformed all the other serotypes tested.

Conclusions : All the 6 AAV serotypes studied efficiently transduced rabbit retinal cells with different transfection efficiency and tropism depend both on serotype and injection route. Appropriate AAV serotype and injection method should be selected based on target cells and treatment goals.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

 

Figure 1. AAV serotype screening in rabbit retina. A),B),E),F). Multiple fundus fluorescnce Images were stitched to show retinal montage. GFP expression was detected in live rabbits at 4 weeks post injection(psi). C),D),G),H). Cross sections of the fixed eyeball were detected by immunofluorescence staining using an anti-GFP antibody (green). Nuclei were counterstained with DAPI (blue). ONL: Outer nuclear layer. INL: Inner nuclear layer.