Intravitreal AAV delivery causes long-term subclinical microglia activation in mice

Abstract

Purpose : Recombinant adeno-associated virus (rAAV) vectors have emerged as an important delivery platform for the treatment of ocular disease. Intravitreal (IVT) administration of rAAV is recognised to elicit acute intraocular inflammation with a main adaptive component, but unexplained late-onset inflammation has also been observed in some patients. We hypothesise that IVT injection of rAAV induces long-term activation of retinal microglia (MG), leading to dysregulation of retinal homeostasis.

Methods : Cx3cr1^CreER:R26-tdTomato (C57BL/6J) heterozygous mice (M/F; 10-12 wk.; n = 4-8) received IVT injection of 1E10 gc/eye rAAV2 (eGFP or null constructs). Clinical imaging (fundus/OCT/fluorescence) was used for longitudinal monitoring of rAAV-mediated inflammation. At selected timepoints (Day 12, 28, 42 & 83) post-injection, individual retinae were processed and tdTomato⁺ MG and CD45⁺ infiltrating cells phenotyped by flow cytometry (FACS). To understand AAV-mediated changes in transcription, 600 tdTomato⁺ MG were FACS-sorted from single retinae and transcriptomes generated from naïve and injected eyes (null) on D12 and D28.

Results : IVT rAAV2 administration induces inflammation peaking at D10-12, characterised by infiltration of non-MG CD45⁺ cells (q < 0.0001) with a main CD3⁺ component, accompanied by a 205±121% increase in and perivascular accumulation of the MG. FACS demonstrates MG activation, indicated by reduced expression of homeostatic P2RY₁₂ (q < 0.0001) and increased levels of CD44 & BST2 (q < 0.0001) activation markers, with only partial recovery by D28. RNA-Seq analysis of MG activation (e.g. Ccl5, Cd44, Bst2) and homeostasis (e.g. P2ry12, Gpr34, Hpgd) marker transcripts confirms the plasticity of the response, matching observed changes in clinical inflammation and FACS. Despite trends toward resolution by D42, FACS shows non-MG CD45⁺ count and MG activation altered through D82, as imaging reveals inflammation (optic disc enlargement or perivascular MG accumulation) of variable severity, with limited vitreous infiltration, in 25% (12/48) of eyes at D70-80. Late inflammation was observed in both null vector eyes (4/12) and eGFP vector eyes (8/12).

Conclusions : IVT administration of rAAV leads to long-term activation of retinal MG, concurrent with late-onset inflammation. Our research aims to further characterise early and late-stage molecular changes in MG and identify pathways for therapeutic intervention.

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