Preventing Posterior Capsule Opacification Using a Recombinant Adenovirus-Encoded p21WAF1/CIP1 Transgene

Abstract

Abstract: : Purpose: Posterior capsule opacification (PCO) is a common complication following contemporary cataract surgery due to lens epithelial cell (LEC) proliferation. A recombinant adenovirus vector expressing the cell cycle inhibitor p21^WAF1/CIP1 (rAd-p21) can inhibit ocular fibroblast proliferation. The purpose of this study was to: 1) establish a rabbit model of PCO; 2) test adenoviral transduction of LEC in vitro and in vivo; and 3) assess the effect of rAd-p21 on LEC proliferation. Methods: Twenty-one rabbits underwent unilateral phacoemulsification. PCO was scored by slit lamp, and assessed weekly during the first month and at the end of the second month. To investigate adenoviral transduction of LECs in vivo, 100µl rAd-LacZ reporter [8.1 x 10¹⁰ particles (p)] was mixed with 100µl of Healon® and injected into the empty capsular bag of four rabbits for 5 min post-phacoemulsification. The mixture was then removed. Three days post injection, whole eyes were X-gal stained and processed to slides for histological analyses. In vitro, primary rabbit and human LECs were treated with increasing doses of a reporter construct expressing the green fluorescent protein, rAd-GFP (1 x 10⁷-10⁹ p/mL) for 48h. Percentage of cells expressing GFP was determined by FACS analysis. To determine the anti-proliferative effects of rAd-p21, LECs were treated with increasing doses of rAd-p21 (1 x 10⁷ -10⁹ p/mL) for 48h, at which time cells were pulse labeled with bromodeoxyuridine (BrdU). Percentage of BrdU positive cells was determined by FACS analysis. Results: PCO formation occurred within 1-2 weeks post surgery, increasing in severity at 3-4 weeks. Thereafter, PCO scores stabilized up to 2 months post-operatively. Lens-capsular-bag injection of rAd-LacZ demonstrated positive LacZ expression in LECs, the target cell in PCO. A dose-dependent expression of GFP was observed in rabbit (3-81%) and human (3-100%) LECs. A dose-dependent and gene specific inhibition of BrdU incorporation was observed in rAd-p21 treated human and rabbit LEC compared to controls. Percentage of cells incorporating BrdU was reduced from 38% to 2% and from 54% to 18%, for human and rabbit respectively. Conclusions: Adenovirus is an efficient vector for gene transfer into target LECs in vitro and in vivo, resulting in the desired anti-proliferative effect of rAd-p21 in vitro. Efficient PCO formation makes the rabbit model suitable for studies of PCO prevention by rAd-p21.

Keywords: gene transfer/gene therapy • posterior capsular opacification (PCO) • animal model