PURPOSE. To investigate the efficacy of two different methods of adenoviral transfer of genes to trabecular meshwork (TM) and Schlemm's canal (SC) cells in cultured human anterior segments, using both experimental and numerical analyses. METHODS. Replication-deficient adenoviruses having coding sequence for β-galactosidase β-gal) under the control of the cytomegalovirus promoter were used. Efficiency of gene transfer over time was verified by infecting cultured human TM cells and assaying for β-gal activity. Next, ostensibly normal paired human eyes were prepared by standard techniques and perfused for 2 to 5 days to measure baseline facilities. Eyes were then infected by one of two methods: standard transcomeal puncture, or injection into a 1 mm diameter silastic segment of supply tubing immediately upstream of the perfusion dish. In both cases, the nominal total dose was 2 × 108 viral particles. Five days after viral injection, eyes were harvested and fixed, and wedges from each of four quadrants were examined histologically. Sections were assayed for β-gal activity and/or stained with toluidine blue. In a parallel study, flow and viral transport within perfused anterior segments were numerically simulated for conditions that approximated those used experimentally. RESULTS. Eyes receiving viral particles by transcomeal injection showed variable levels of β-gal activity and highly variable TM cellular morphology, ranging from excellent preservation to cellular lysis. Eyes receiving an equivalent viral dose via the supply tubing showed higher transfer efficiency, as judged by almost complete TM cell loss (indicative of viral toxicity) and intense extracellular β-gal activity from the residual cytoplasm. At lower doses (1/3 to 1/1000 of that used in transcomeal injection) β-gal activity was still present, while TM cell morphology was good at the lower viral doses. Computer modeling showed that the region beneath the cornea was nearly stagnant, and consequently virus introduced into this region by transcorneal injection was delivered very slowly to the TM. This caused the effective delivered viral dose to be low and sensitively dependent on the volume and shape of the transcorneally injected virus bolus. CONCLUSIONS. Injection of adenovirus into supply tubing led to consistent delivery of reporter gene and approximately 300-fold greater efficiency of gene transfer compared to the transcomeal injection method, and is therefore the preferred method for introducing viral particles into perfused anterior segments. These findings were consistent with computer modeling of flow and mass transport in perfused anterior segments. Although these quantitative results are specific to adenovirus, this general trend should hold for a wide range of perfused compounds.
ASJC Scopus subject areas
- Sensory Systems
- Cellular and Molecular Neuroscience