Surgical Removal of Internal Limiting Membrane and Layering of AAV Vector on the Retina Under Air Enhances Gene Transfection in a Nonhuman Primate
Abstract
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. Six Macaca fascicularis NHP eyes underwent vitrectomy, ILM peel with layering of 1.7 × 1013 genome 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. There was a larger area of fluorescence in ILM peeled eyes then in non-ILM peeled eyes (50.7 [33.1-58.4] pixel2 versus 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 e...Continue Reading
References
Citations
Related Concepts
Related Feeds
CREs: Gene & Cell Therapy
Gene and cell therapy advances have shown promising outcomes for several diseases. The role of cis-regulatory elements (CREs) is crucial in the design of gene therapy vectors. Here is the latest research on CREs in gene and cell therapy.