Gene Therapy to Modulate Wound Healing following Trabeculectomy: Transfection of Tenon Fibroblasts with Oligonucleotides

Abstract

Abstract: : Purpose: The process of wound healing following trabeculectomy involves Tenon and episcleral fibroblasts as target cells. This process is one of the most important factors that distinguish between a surgical success or failure. Current clinical concepts mainly involve antifibroblastic substances or, more recently, agents that interfere with specific growth factors. We now entertain the concept and possibility of a gene transfer into the target cells. As a first step, it has to be show that it is in general possible to get specific gene fragments in a relevant number into the fibroblasts. Secondly, it has to be determined which genes or combinations of these will be effective to interfere with the viability of proliferating fibroblasts. As a first gene sequence, we have chosen a p53 antisense oligonucleotide. This gene interferes with the function of p53 to reduce apoptosis and blocks proliferating cells during mitosis. Methods: 3T3 fibroblasts and human Tenon fibroblasts were cultured. A mixture of polycationic lipids (Metafectene) and p53 antisense oligonucleotides was added after 24 hours. At another 24 hours of incubation a cristall-violett-essay was performed to determine cell viability. Controls included no treatment and exposure to mitomycin. Concentrations of Metafectene ranged from 4µl/ml to 80µl/ml, and concentrations of the oligonucleotide ranged from 1µg/ml to 6µg/ml.. Results: Cellular penetration of the liposome-DNA-complex was high as quantified by the fluorescein-label of the oligonucleotide. Cell growth was markedly reduced in 3T3 cells when treated with the highest concentrations (down to 24.1%±0.7%; p<0.0001, t-test as compared to untreated controls. For comparison: mitomycin 20.2%±1.6%). In human Tenon fibroblasts, the reduction of cell growth was less marked and only significant when treated with the highest concentrations (down to 71.1%±1.8%; p<0.001, t-test as compared to untreated controls. For comparison: mitomycin 70.1%±0.3%). Conclusions: The idea presented here is a new approach to possibly interfere with postoperative cell proliferation. This new avenue to directly influence cell function and proliferation may be promising to effectively inhibit undesired subconjunctival scarring in the early post-operative period. More steps will probably be needed before the introduction into clinical practice.

Keywords: apoptosis/cell death • gene transfer/gene therapy • wound healing