Intravitreal Delivery of rAAV2 Vectors to the 13-Lined Ground Squirrel Retina

Abstract

Purpose : To deliver rAAV2 vectors to 13-lined ground squirrel (13-LGS) cone photoreceptors via intravitreal injection and assess artefacts arising from these injections that may impact subsequent longitudinal imaging studies.

Methods : Newborn (P1; n=43) and adolescent (P70-163; n=11) 13-LGS received intravitreal injections of a capsid mutant rAAV2/2 vector containing the [7m8] peptide insertion (R588_Q589insLALGETTRPA) and five point mutations (Y272F, Y444F, T491V, Y500F, Y730F) termed herein, rAAV2[MAX], packaging an mCherry reporter gene under control of either a ubiquitously expressing CBA promoter or a photoreceptor specific GRK1 promoter. Cornea/lens damage was examined using a Bioptigen Envisu R2200 OCT. Retinal transduction of mCherry was assessed in vivo using a Heidelberg Spectralis confocal SLO, and ex vivo with immunocytochemistry. Cone transduction of mCherry was quantified with flow cytometry using dissociated 13-LGS retina incubated with antibodies to M- and S-opsin.

Results : Iris synechia was found in 65% of newborn-injected eyes, but only 1 (4%) of the adolescent-injected eyes. The outcome of iris synechia in newborn-injected 13-LGS was independent of injection substance (p = 0.29; Chi Square test), suggesting it was due to the injection procedure. Conversely, 27% of adolescent-injected eyes had hyper-reflective puncta in anterior and/or posterior chambers, but this was not seen in any newborn-injected eyes. This presumed inflammatory response was specific to rAAV2[MAX] injected eyes with the CBA promoter. Successful but variable cone transduction was seen using flow cytometry regardless of injection age or promoter used. From 0.03% to 15.1% of opsin positive cells also expressed mCherry. Newborn injections of rAAV2[MAX]-CBA resulted in strong lens expression of mCherry, and cone transduction was minimal in these animals. Preliminary flow cytometry results suggest that 13-LGS cones are transduced most efficiently with rAAV2[MAX]-CBA administered during adolescence.

Conclusions : Intravitreal injections of rAAV2[MAX] with either CBA or GRK1 promoters can effectively target cone photoreceptors in the 13-LGS. Injecting these vectors in newborn animals often results in mild iris synechia and lens damage, which in most cases does not inhibit retinal imaging with OCT. Injecting these vectors in adolescent animals can result in inflammation, which in only one instance inhibited OCT retinal imaging.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.