Pseudotyping And Culture Conditions Affect Efficiency And Cytotoxicity Of Retroviral Gene Transfer To Human Corneal Endothelial Cells

Abstract

Purpose: : The lack of a regenerative capacity of the corneal endothelium and the potential risk of endothelial decompensation as a response to immune reactions after keratoplasty made several research groups investigate the genetic manipulation of corneal endothelial cells. We sought to evaluate retroviral vectors as a tool to transduce normal human corneal endothelial cells (HCEC) and to optimize transduction to increase gene transfer efficiency.

Methods: : EGFP encoding retroviral vectors based on HIV-1 (human immunodeficiency virus type 1) or MLV (murine leukemia virus), pseudotyped with either VSV-G (vesicular stomatitis virus glycoprotein) or a modified FV Env (foamy virus envelope protein), and PFV (prototype foamy virus) were produced using the 3-plasmid-system in 293T cells. Transduction of HCEC was performed in four culture media that were previously described for specific cultivation of HCECs or organ culture of donor corneas, namely enriched HCEC growth medium F99_HCEC, its unsupplemented basal medium F99, MEM + 2 % FCS (MEM), and Human Endothelial-SFM (SFM). Transduction efficiency was evaluated by marker gene transfer assay. Transgene expression during subcultivation was monitored by fluorescence microscopy. Cytotoxic effects of virus infection were evaluated by resazurin conversion assay.

Results: : PFV and HIV-1 based vectors showed superior transduction efficiency compared to MLV based vectors. Pseudotyping with a modified FV Env further increased transduction efficiency compared to VSV-G. Titers ranged between 3.0x10⁵ (MLV/VSV-G) to 1.95x10⁷ (HIV-1/FV). Transgene expression remained stable throughout subcultivation over 9 passages. In medium SFM, transduction efficiency of PFV, HIV-1/FV and MLV-based vectors was markedly reduced. When cultured in F99-based media, HCEC viability was reduced by retroviral transduction compared to uninfected or mock infected controls, but remained unaffected in SFM and was even increased in MEM.

Conclusions: : Retroviral vectors can efficiently be used to transduce primary HCECs in vitro and to achieve stable, long-term transgene expression. HIV-1 based vectors show higher transduction efficiency than MLV-based vectors, this being further increased by FV Env pseudotyping. However, retroviral transduction efficiency is dependent upon culture conditions and impairs metabolic activity and viability of HCECs in vitro.

Keywords: cornea: basic science • cornea: endothelium • gene transfer/gene therapy