Molecular Studies of IGF-1 Transgene Expression in Transfected Human Retinal Pigment Epithelial Cells Manifesting an Altered Cell Phenotype

Abstract

Abstract: : Purpose: The conceptual theme of our work is that it is possible to modify the phenotype of the retinal pigment epithelium (RPE) by inducing the expression of growth factors using gene therapy. Our goal is to utilize cytokine gene transfer as a therapeutic approach to the treatment of macular degeneration. Previously, we have transfected human insulin-like growth factor-1 cDNA into RPE cells and have shown that IGF-1 transgene expression can effect changes in the RPE phenotype. These studies correlate the observed changes in phenotype with molecular evidence of IGF-1 transgene expression in transduced clones. Methods: An expression vector encoding a promoted hIGF-1 cDNA and a neomycin resistance gene were transfected into human RPE cells. G418 was used to clone transduced cells expressing the neomycin resistance transgene in selective medium. Resistant clones were expanded and screened for changes in the RPE cell phenotype. Transduced clones manifesting changes in the RPE phenotype were screened for IGF-1 transgene expression using RT-PCR and Western analyses. Results: One RPE clone isolated demonstrated a high level of IGF-1 transgene expression in vitro. This clone showed enhanced growth characteristics compared to the recipient RPE cell and was associated with a marked change from an epithelial to fusiform cell morphology during passaging in vitro. Conclusion: Human RPE cells can be transduced and cloned with selectable genetic markers. Stable expression of the IGF-1 transgene in an RPE clone was capable of altering the phenotype of the retinal pigment epithelial cell in vitro.

Keywords: 419 gene transfer/gene therapy • 567 retinal pigment epithelium • 423 growth factors/growth factor receptors