AAV 44.9- a novel capsid that efficiently transduces photoreceptors and retinal pigment epithelium

Abstract

Purpose : Adeno-associated virus (AAV) has become the vector of choice for targeting therapeutic genes to the retina. Both naturally occurring and synthetic AAVs have been identified that display retinal tropism. Recently, a novel AAV capsid, 44.9, was isolated as a contaminant of a laboratory stock of adenovirus. The purpose of our study was to evaluate its transduction of ocular cell lines and mouse retina following intravitreal (Ivt) or subretinal (SR) injection with emphasis on photoreceptor transduction efficiency.

Methods : A self-complementary AAV construct containing the smCBA promoter driving an mCherry reporter was packaged into AAV44.9, AAV5 or AAV8(Y733F). ARPE19 (human RPE) and 661W (mouse cone) cell lines were infected and transduction evaluated by flow cytometry. Vectors were also Ivt and SR injected into Nrl-GFP mice that constitutively express GFP in rods. Transduction was qualitatively evaluated by fluorescent fundoscopy. Transduction of rods and non-rod cells (all other neural retina) was quantified by flow cytometry and vector tropism was determined by microscopy of retinal sections. Next, we evaluated whether addition of a tyrosine to phenylalanine (Y-F) mutation at a position shown to improve transduction efficiencies of other AAV capsids enhanced AAV44.9. Finally, we asked whether AAV44.9 could effectively transduce cones by SR injecting WT mice with AAV44.9 containing the cone preferential IRBP/GNAT2 promoter and a GFP reporter.

Results : Results: AAV44.9 vectors packaged with high efficiency. Transduction of ocular cell lines was achievable, however efficiency was low. Ivt injection resulted in limited retinal transduction, similar to that observed for AAV5 and AAV8(Y733F). Subretinal injection resulted in extensive transduction that was primarily restricted to photoreceptors and RPE. In Nrl-GFP mice SR injected with AAV44.9, the percent of transduced rods (GFP and mCherry positive cells) was higher (63.5%) than in mice injected with benchmark vectors AAV5 (40.8%) and AAV8(Y733F) (53.9%). Surprisingly, the addition of the Y733F mutation to AAV44.9 did not improve transduction efficiency in vitro or in vivo. Analysis of cone transduction is underway.

Conclusions : AAV44.9 is a promising candidate for photoreceptor- targeted gene therapies. It remains to be seen whether additional improvements through rational design are achievable.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.