Novel gene therapeutic approaches to prolong corneal allograft survival

Cornea transplantation (penetrating keratoplasty) is the most frequent form of allograft transplantation in humans with a 90 % success rate in the first 2 years, however, the rate of survival decreases over time and drops to approximately 62% after 10 years. The predictions for 'high risk' graft survival are even poorer. Despite advances in microsurgery and immunosuppressive treatment protocols a significant number of corneal grafts still undergo immune-mediated allograft rejection. Therefore, alternative approaches are needed to prevent corneal transplant rejection such as genetic modification of donor corneas. Gene therapy has been widely applied in preclinical studies to manipulate the corneal graft rejection process. The main goal of corneal gene therapy is to achieve long-term allograft survival without the need for other treatments or only restricted to short periods of time thereby reducing undesired side effects. Herein, two distinctive strategies in a preclinical model of rat cornea transplantation to prevent allograft rejection were investigated using lentiviral (LV) vectors as vehicles for therapeutic gene delivery and overexpression in the corneal allograft. The first part of this work was based on known anti-apoptotic properties of nerve growth factor (NGF). As previously reported, adenovirus (Ad)-mediated NGF overexpression reduces apoptosis in corneal endothelial cells and prolongs allograft survival in an in vivo rat model, however, the mechanisms were not fully understood and the use of adenoviral vectors raised concern. The aim of this part of the project was to develop an anti-apoptotic strategy using alternative, low immunogenic LV vectors expressing NGF (LV.NGF) for rat cornea transplantation and investigation of the underlying mechanisms of cytoprotection. It was shown that LV.NGF transduced corneal endothelial cells secreted biologically active NGF. In addition, supernatants from LV.NGF transduced immortalized human corneal endothelial cells (HCEC) significantly reduced thapsigargin (TG)-induced apoptosis in control PC12 cells, however, no protective effect of either recombinant NGF or supernatant containing NGF could be observed in HCEC. Subsequently, a protocol for LV-mediated transduction of ex vivo cultured corneas was established. However, in contrast to previous results showing that Ad.NGF gene transfer in cultured corneas resulted in significant prolongation of allograft survival, donor corneas transduced with LV.NGF did not show any protective effect in vivo. Different expression levels of the therapeutic gene after Ad- or LV gene transfer may account for these results. The second part of this project was to investigate the role of LV-mediated overexpression of Programmed Death Ligand-1 (PD-L1) on rat corneal allograft survival. In contrast to LV.NGF treatment, allogeneic LV.PD-L1 transduced corneas showed a high percentage (83%) of graft survival. This striking result was associated with a reduction of natural killer T (NKT) cell and cytotoxic CD8+ T cell infiltration to the graft as measured by flow cytometry. Graft opacity, as an indicator of cell infiltration, was present but was significantly reduced in PD-L1 transduced corneas compared to control or EGFP expressing corneas. Herein, the data provide evidence that therapeutic applications of LV-based cornea gene therapy might be a valuable clinical approach for treatment of corneal endothelium failure and/or graft rejection.

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University of Galway Theses (PhD Theses)