Investigating Lentivirus-Mediated Gene Transfer of Nerve Growth Factor in Corneal Endothelial Cells and Analysis of Its Protective Effects

Abstract

Purpose: : We have recently shown that adenovirus-mediated nerve growth factor (NGF) over-expression reduces apoptosis in corneal endothelial cells in an in vivo model, however, the mechanisms were not fully understood. Herein we investigated the optimal conditions for lentiviral gene transfer and the anti-apoptotic effects of NGF on corneal endothelial cells in more detail.

Methods: : Lentiviral vectors (LV) encoding for NGF or EGFP as a control under both viral (CMV) and cellular (EF-1α) regulatory elements were constructed. Gene over-expression was measured in HeLa cells, immortalized Human Corneal Endothelial Cells (iHCEC) and rat corneas (RC) by flow cytometry or specific ELISA, respectively. To verify NGF activity, supernatants from transduced cells were added to NGF-responsive indicator rat PC12 cells and neurite outgrowth was monitored. Apoptosis on iHCEC and PC12 was investigated by flow cytometry (appearance of sub-diploid DNA peak) and Western blot for the presence of cleaved caspase-3.

Results: : LV_EF-1α-EGFP and LV_CMV-EGFP vectors were generated and titrated on HeLa cells (1.5*10⁷ TU/ml and 2.0*10⁶ TU/ml, respectively). RC were incubated ex vivo with LV_CMV-EGFP (2*10⁴ TU/cornea) for 1h, 4h and 20h. 5 days after transduction, RC were dissected and EGFP expression was analyzed by flow cytometry. We observed increased levels of EGFP expression (1,95%; 2,68%; 5.5% of EGFP positive cells after 1h, 4h, 20h of incubation, respectively). Moreover, we could show that both HeLa cells as well as RC can be efficiently transduced with LV_EF-1α-NGF and LV_CMV-NGF resulting in high levels of therapeutic gene expression (for HeLa cells: 3,73±0,84 ng/ml and 2,37±0,48 ng/ml 5 days after transduction, respectively). Secreted NGF from HeLa cells and RC transduced with both LV constructs was able to induce neurite outgrowth in rat PC12 cells indicating functional activity. In addition, supernatants from NGF transduced cells significantly reduce thapsigargin-induced apoptosis in PC12 cells by approximately 38%, however, in iHCEC no protective effect of recombinant NGF or supernatants from LV-NGF transduced cells could be observed.

Conclusions: : LV are efficient tools for ex vivo genetic modification of iHCEC and RC. LV-NGF over-expression is a promising approach for the prevention of corneal endothelial cell apoptosis, however further studies have to be performed in RC.

Keywords: gene transfer/gene therapy • cornea: endothelium • apoptosis/cell death