Engineered AAV capsid variants allow efficient in vivo and ex vivo gene transfer into the porcine retina

Abstract

Purpose : The pig gains more and more attention as an important model for inherited as well as acquired cone related retinal dystrophies. Vector-based systems for the gene transfer into the porcine retina are needed and vectors based on adeno-associated viruses (AAV) have shown some efficiency following subretinal delivery. However, intravitreal delivery would circumvent some of the limitations associated with subretinal delivery and allow pan-retinal transduction of target cells. Here we assess the capacity of newly developed AAV capsids based on directed evolution to transduce photoreceptor cells in the porcine retina following subretinal and intravitreal delivery in vivo as well as on organotypic retina explants cultures.

Methods : Twelve Libechov minipigs were used in the in vivo studies and divided into 4 groups, receiving either AAV2.GL, AAV2.NN, or AAV2-7m8 containing the GFP gene under the control of the CMV promoter, and vehicle control either subretinally or intravitreally. All eyes received 1x10¹¹ vg/ml in 50 µl volume. For ex vivo experiments, eyes from a local slaughterhouse were processed and explants cultured on semipermeable transwells. Each explant received 5x10¹⁰ vg in 20 µl onto the inner limiting membrane and was cultivated for 7 days prior to analysis. All eyes were processed for immunohistochemistry and stained with antibodies against GFP, GFAP, PNA and PKCα.

Results : Subretinal delivery resulted in a very strong GFP signal mostly in photoreceptor cells with the strongest signal observed for the AAV2.NN vector. Intravitreal delivery showed GFP positive cells spread throughout the entire retina and in all retinal layers, showing a strong signal with all three vectors tested, particularly in photoreceptor and Müller cells. Ex vivo gene transfer resulted in strong photoreceptor and Müller cell signal using all three vectors.

Conclusions : AAV capsids based on directed evolution of AAV2 enable efficient gene transfer through the inner limiting membrane, a barrier so far hindering efficient pan-retinal gene therapy for retinal diseases. Efficient gene transfer into the pig retina is a prerequisite for testing new strategies in this relevant large animal model.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.