The degree of AAV-induced retinal inflammation varies based on serotype and route of delivery: intravitreal, subretinal or suprachoroidal.

Abstract

Purpose : Currently, there is a paucity of data describing the variable immune responses to different adeno-associated virus (AAV) serotypes and how these responses differ depending on the route of ocular delivery. The purpose of this study is to histologically assess virus-mediated inflammation of 5 different AAV serotypes following 3 routes of delivery: intravitreal, subretinal or suprachoroidal.

Methods : Two-month-old Sprague-Dawley rats were injected with either AAV1, AAV2, AAV6, AAV8 or AAV9 (each driving eGFP via the constitutive CMV promoter) or sterile buffer via intravitreal, subretinal or suprachoroidal injection (N≥3 eyes per serotype and per route of delivery). For each serotype and route of injection, 10µl of viral particles (5x10¹² vg/ml for a total dose of 5x10¹⁰ vg) was delivered. AAV serotypes were titer-matched to within one-half log of one another. All eyes were examined immediately post-injection to confirm success of injections into the intended space, using a rodent-specific optical coherence tomography instrument. Animals were euthanized at 14-days post-injection and eyes fixed and prepared for standard histology to characterize the degree and distribution of resident microglia and infiltrating macrophages using anti-Iba1 labeling. Labeled cryosections were imaged via confocal microscopy and panoramic images spanning the entire retina composed to quantify the number of Iba1-positive cells in the vitreous, total neural retina or outer nuclear layer (ONL)/subretinal space to develop an overall qualitative grading system for retinal inflammation.

Results : Compared to buffer-injected controls for each route of delivery, AAV2 and AAV6 induced the highest level of macrophage influx across all routes of delivery of the vectors tested, with AAV6 inducing the highest levels of retinal inflammation when delivered suprachoroidally. AAV1-induced inflammation was highest when delivered suprachoroidally, whereas minimal inflammation was seen with intravitreal delivery. AAV8 and AAV9 induced minimal inflammation across all routes of delivery. Importantly, the degree of macrophage recruitment was not correlated with vector-mediated transduction and expression of eGFP.

Conclusions : These data emphasize the importance of considering ocular inflammation when selecting AAV serotypes and ocular delivery routes for the development of gene therapy strategies.

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