Recombinant Adeno-associated Virus 2-mediated-transfection Of An Enhanced Green Fluorescent Protein Gene Into Rabbit Lens Epithelial Cells In Vivo

Objective To investigate the expression of enhanced green fluorescent protein (EGFP) in rabbit lens epithelial cells transfected with recombinant adeno-associated virus 2 (rAAV2)carrying the EGFP gene, and to determine whether it is feasible to use rAAV 2 as a vector in the gene therapy of posterior capsule opacification(PCO).Methods1. 36 healthy rabbits(36 eyes) were randomly divided into 4 groups: groupA, groupB, groupC and group D. The right eye of each rabbit received phacoemulsification. At the end of the phacoemulsification procedure, we injected 0.1ml of rAAV-EGFP suspension (0.1ml containing 10⁸, 10⁹, 10¹⁰ viral particles) into the capsular bags of the rabbits of groupB, groupC and groupD. And the rabbits of group A receiving 0.1ml of balenced salt solution(BSS) were used as control.2. In the next four weeks after operation, every right eye undertook the slit-lamp examination and the corneal endothelium microscope examination. On the first , second and forth week after the surgery, the expression of EGFP and the effect of rAAV 2 vector on lens epithelial cells, coreal and iris were detected, and the transfection efficiency was calculated.Results1. Slit-lamp examination: On the first day after the surgery, varied degree of inflammatory reaction occurred on all eyes, relieved gradually after one week, and disappeared after two weeks.2. Corneal endothelium microscope examination: There was no statistical difference among each group on corneal endothelial cell density, mean cell area and coeffient of variation.3. Expression and location of EGFP: The intensity of green fluorescence in the residual anterior capsule and the equator of lens was higher than that in the centre of the posterior capsule . EGFP- expressing cells in the residual anterior capsule and the equator of lens were much more than that in the centre of the posterior capsule.4. The transduction efficiency of rAAV2-mediated EGFP: One week after phacoemulsification , green fluorescence was observed under fluorescence microscope in the casular bags of GroupB,GroupC and GroupD. The transduction efficiency of GroupD reached 94.49% on the fourth week after the surgery. The transduction efficiency of each group had statistical difference at different time.5. Histological examination: The histological damage to lens epithelial cells, cornea and iris in every group was not obvious.Conclusion rAAV2-EGFP can be transducted into lens epithelial cells in vivo stably and efficiently. rAAV2 has no histological damage to the anterior segment of rabbit eye. rAAV2 is suitable as a vector in the gene therapy of PCO.