Surgical removal of internal limiting membrane and layering of AAV vector on the retina under air enhances gene transfection in a nonhuman primate

PURPOSE. To determine if the surgical removal of the internal limiting membrane (ILM) in nonhuman primates (NHPs) will result in safe and effective transfection of adeno-associated viral (AAV2) vectors using green fluorescent protein (GFP) as a reporter. METHODS. Six Macaca fascicularis NHP eyes underwent vitrectomy, ILM peel with layering of 1.7 × 10¹³genome copies per milliliter of AAV2-GFP under air. Four control eyes underwent only vitrectomy and pooling under air. The intensity and area transfected was quantified in vivo with fundus autofluorescence (FAF) imaging. NHPs were euthanized 16 weeks postsurgery and immunohistochemical analysis assessed GFP expression at the cellular level. RESULTS. There was a larger area of fluorescence in ILM peeled eyes then in non-ILM peeled eyes (50.7 [33.1–58.4] pixel²versus 5.1 [0.6–7.6] pixel², P < 0.01). The intensity of fluorescence was also higher in ILM peeled eyes (10.3 [2.2–18.5] vs. 1.9 [0.6–4.4], P = 0.05). Non-ILM peeled eyes displayed fluorescence confined to the foveal center. Histological sections showed colocalization in the Müller cell layer, ganglion cell layer, and photoreceptor cell layer in the ILM peeled eyes. In non-ILM peeled eyes GFP expression was only in the ganglion cell layer in three eyes and was confined to the immediate vicinity of the fovea. CONCLUSIONS. ILM appears to be the predominate barrier to AAV transfection. An efficacious and safe method of AAV2 gene delivery, taking into account the potential need for repeat treatments, appears to be the surgical removal of ILM and layering of AAV under air.v.