Efficiency of Adenoviral Gene Transfer to Outflow Cells in Perfused Human Anterior Segments

Abstract

Abstract: : Purpose: Adenoviral transfer of genes to trabecular meshwork (TM) and Schlemm's canal (SC) cells in cultured human anterior segments is now a common tool for studying outflow biology. In the present study we investigated the efficacy of two different methods of delivering adenovirus to TM and SC cells. Methods: Replication-deficient adenoviruses having coding sequence for either beta-galactosidase (ß-gal) or aquaporin-1 (AQP1) under the control of the CMV promoter were used in experiments. Efficiency of gene transfer was first verified by infecting and assaying cultured human TM and SC cells grown on permeable filters for ß-gal activity and water permeability. Next, ostensibly normal paired human eyes were prepared using standard techniques and perfused for 2-5 days to measure baseline facilities. Eyes were then infected by one of two methods: (1) standard trans-corneal puncture using a 30G needle and tuberculin syringe, followed by injection of 100 µl of virus containing approximately 10⁶ infective viral particles/ml (total dose 10⁵ viral particles); or (2) injection of the virus into a 1 mm diameter silastic segment of supply tubing immediately upstream of the perfusion dish. Five days after viral injection, eyes were harvested, fixed and wedges from each of four quadrants were examined histologically. Sections were assayed for ß-gal activity, immunolabelled for AQP1 expression and/or stained with toludine blue. Results: Eyes receiving 10⁵ viral particles by trans-corneal injection showed variable levels of ß-gal activity, inconsistent levels of AQP1 expression, and highly variable TM cellular morphology, ranging from excellent preservation to cellular lysis. Eyes receiving an equivalent viral dose by method (2) showed higher transfer efficiency, as judged by almost complete TM cell loss (indicative of viral toxicity) and intense extracellular ß-gal activity from the residual cytoplasm. At lower doses (1/3 to 1/10 of that used in method (1)) ß-gal activity was comparable to that seen in method (1), while TM cell morphology was, on average, better. Conclusions: In our hands, trans-corneal injection of virus led to inconsistent results and generally lower transfection efficiency than with method (2). This may be due to unavoidable small leaks during needle withdrawal from the cornea, relative flow stasis near the cornea, and/or viral binding by corneal endothelium. We recommend viral injection into the supply tubing.

Keywords: outflow: trabecular meshwork • gene transfer/gene therapy • adenovirus