Abstract
Purpose :
Subretinal injection under the fovea increases risk of macular hole formation, loss of foveal cones and visual acuity decline. We previously identified novel AAV capsids that exhibit enhanced lateral spread and potency after subretinal injection in mice. Here, we evaluated whether these capsids would promote transduction of foveal cones following extrafoveal subretinal injection (i.e. fovea remains attached during and after surgery).
Methods :
Seven cynomolgus macaques were injected subretinally with novel AAV vectors containing either CBA or hGRK1 promoters driving GFP at a concentration of 1e12 vg/ml. Vectors included 1) an AAV2-based capsid containing 4 proteosomal avoidance (Y-F) mutations and canonical HSPG binding residue substitutions- AAV2(4pMutΔHS), 2) the novel naturally occurring capsid AAV44.9, and 3) a rationally designed version of the latter AAV44.9(E531D). AAV5 was used as a control. In 8 eyes, two 60µL injections were performed (one intentionally subfoveal, one peripheral). In 6 eyes, three 30µL perifoveal injections were performed. Optical coherence tomography (OCT) and confocal scanning laser ophthalmoscopy (cSLO) were performed at regular intervals. At 6 weeks post-injection, retinal cryosections were prepared and stained with a cone arrestin antibody. Rod and cone transduction was quantified within and outside the injection blebs.
Results :
Intentional subfoveal delivery of all vectors drove highly efficient GFP expression within foveal photoreceptors. Loss of ellipsoid zone and foveal bulge on OCT were apparent within these eyes, with partial or complete recovery through week 6. Extrafoveal injection produced no OCT changes within the fovea of any eyes. GFP fluorescence extending beyond subretinal bleb margins was apparent in cSLO images at 2 weeks post-injection with novel vectors, and spread continued until study termination. This was not the case for AAV5. GFP intensity increased through week 6 for all vectors . Extrafoveal injection of novel vectors led to transduction of up to 98% of foveal cones.
Conclusions :
AAV44.9 and AAV2(4pMutΔHS) are highly efficient at transducing macaque photoreceptors following subretinal injection. Notably, both transduce foveal cone photoreceptors without the requirement of foveal detachment. This may be vital to the prevention of visual acuity decline in diseased retinas undergoing gene therapy.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.