Miniature pig model to investigate retinal microglial cells after subretinal injection of rAAV

Abstract

Purpose : Retinal gene therapy by subretinal injection of recombinant Adeno-Associated Viral vectors (rAAV) is effective in patients but still faces adverse effects related to inflammatory reactions that compromise the safety and long-term efficacy of the treatment. Modelling of retinal immune cell activation is needed to develop and evaluate less inflammatory strategies in patients. As there is little data available on the reactivity of retinal microglial cells to rAAV delivery, the aim of this study is to assess the transduction and the recruitment of these mononuclear phagocytes after subretinal injection of rAAV in pigs.

Methods : Subretinal injection of rAAV was performed in the eyes of 2 Yucatan miniature pigs (6-month-old, 20kg) using either rAAV8-CAG-GFP or rAAV6-CAG-GFP (150µl, 5e11 vg). To replicate the patient's conventional preventive anti-inflammatory treatment, subconjunctival injection of 0.5ml of long-acting corticosteroid (methylprednisolone, 40mg/ml) was performed at the end of the procedure in each eye. In vivo and post-mortem retinal imaging were done 4 weeks after injection. Iba1+ microglial cells were counted on confocal slices of flat-mount retinas from the right eyes of the pigs in 6 random squares of 500µm side length in the injected zone (IZ), peripheral non-injected zones (NIZ) and macular NIZ.

Results : After surgery, there was no signs of inflammation or toxicity. Optical coherence tomography (OCT) did not show any structural difference between IZ and NIZ. Retinotomies were barely noticeable in vivo whereas local inflammation was evident on flat-mount retinas. Green fluorescence was strong in IZ and absent in NIZ. Microglial Iba1+ cells were detected but none of them showed green fluorescence. They were significantly more numerous in IZ than in NIZ in rAAV8-injected pig (mean 23.5 cells/square, +/-sd 6.3 in IZ vs 0.3+/-0.5 in peripheral NIZ and 0.4+/-0.8 in macular NIZ, p=0.003) and in rAAV8-injected pig# (mean 61.3 +/-35.0 in IZ vs 5.8+/-3.8 in peripheral NIZ and 0 in macular NIZ, p=0.005).

Conclusions : After subretinal injection of rAAV, microglial cells were not transduced. However, they were massively recruited in the IZ. This inflammatory reaction was not clinically detectable. It highlights the lack of sensitivity of clinical examination and OCT to detect retinal inflammation and the need to further study the impact of microglial cells activity in rAAV gene therapy studies.

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