Transscleral Delivery of Proteins by Periocular Gene Transfer

Abstract

Abstract: : Purpose: The aim of this study was to express proteins of various sizes in the periorbital space of mice by gene transfer and evaluate the ability of each of the proteins to enter the eye. Methods: Adult C57BL/6 mice were given a periocular injection of E1-, E3-, E4-deleted adenoviral vector (Ad) encoding transforming growth factor-ß1 (TGF-ß1, a 25 kDa protein), pigment epithelium-derived factor (PEDF, a 50 kDa protein), or the soluble extracellular domain of VEGF receptor-1 (sFlt-1, a 78 kDa protein). Mice were euthanized 24 hours after injection and sclera, choroid, and retina were dissected and frozen. Levels of the appropriate protein were measured in each of the tissues by ELISA. Results: Compared to uninjected mice (n = 5), mice given periocular injection of Ad.TGF-ß1 (n =5) had significant elevation of TGF-ß1 (pg TGF-ß1/µg total protein) in sclera (0.22 vs 0.13; p=0.02) and choroid (0.1 vs 0.05; p=0.04), but not the retina, giving a sclera:choroid:retina (scr) ratio of 1:0.45:undetectable. Compared to uninjected mice (n = 5), mice given a periocular injection of Ad.PEDF (n =5) had increased PEDF in sclera (51.56 vs 2.89; p=0.008); choroid (21.67 vs 1.78; p=0.011), and retina (6.86 vs 3.39; p=0.002) giving a 1:0.42:0.32 scr ratio. Compared to uninjected mice (n =5), mice given a periocular injection of Ad.sFlt-1 had increased levels of sFlt-1 in the sclera (7.21 vs 0.75; p=0.0005); choroid (4.27vs. 0.46;p=4.83x10^-5), and retina (0.20vs.0.01; p=0.035) giving a 1:0.6:0.05 scr ratio. Conclusions: These data suggest that the sclera provides a modest barrier for entry of proteins into the choroid from the periocular space, while there is a strong barrier between the choroid and the retina (choroidal blood flow and the RPE are likely to be components of the barrier). Both barriers become more stringent with increasing molecular weight, but even a 78 kDa protein can gain entry into the retina from the periocular space. Due to the steep gradient between choroids and retina even small proteins, like TGF-ß1, must be expressed at high levels in the periocular space in order to achieve detectable levels in the retina.

Keywords: gene transfer/gene therapy • growth factors/growth factor receptors • adenovirus