Author: Alison Jayne Clarke
Clarke, Alison Jayne, 2013 Gene Therapy of the Sheep Cornea for the Prolongation of Corneal Graft Survival, Flinders University, School of Medicine
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Although corneal transplants enjoy good short-term survival, their long-term survival is poor. The eye has long been heralded as an immune-privileged site, however this privilege is in a constant state of balance and, if tipped too far by inflammatory forces, corneal transplants will undergo irreversible rejection. This is the major cause of graft failure. Gene therapy has shown potential in experimental transplantation, to reduce the rejection response. Previous studies in our laboratory have shown prolongation of sheep corneal graft survival, one such using an adenoviral vector expressing the interleukin-10 (IL-10) therapeutic transgene under the control of a cytomegalovirus promoter (CMV), and another using the lentiviral vector expressing the same transgene but under the control of the Simian virus type 40 early promoter (SV40). The aim of this study was to investigate gene therapy with a cocktail of vectors designed to induce long-term transplant survival in a sheep model of corneal transplantation. In a direct comparison of internal promoters in a lentiviral vector, gene expression induced by a CMV promoter and the SV40 promoter and was measured from transduced sheep corneas in vitro. The CMV promoter induced significantly higher transgene expression than the SV40 promoter at both the mRNA and protein level (p= 0.006, p≤ 0.001, respectively). Thus the lentivirus vector with the CMV promoter and transgene interleukin-10 was then tested in vivo in an outbred sheep model of orthotopic, penetrating corneal transplantation with high risk of rejection. This single gene therapy applied to the donor cornea significantly prolonged corneal graft survival, with treated grafts surviving a median of 26 days compared with 21 days for the control allografts (p= 0.043). The polycation protamine sulphate was investigated as a possible non-toxic virus transduction enhancer to improve gene expression from the lentiviral vector. It was found to increase transgene expression 14-fold in vitro (p≤ 0.001), however was deemed not to be successful enough to warrant pursuing in vivo. Therapeutic transgenes IL-10, indoleamine 2,3-dioxygenase, endostatin::kringle5 fusion gene (EK5), soluble fms-like tyrosine kinase 1 (sFlt-1), and Bcl-2 family protein, Bcl-xL, had previously been shown to prolong corneal graft survival in animal models, or to reduce corneal neovascularisation. Individual lentiviral vectors expressing these transgenes, each with CMV promoters, were constructed, viruses prepared and tested in vitro for gene expression by qRT-PCR analysis of mRNA and protein expression from sheep corneal endothelial cells. Individual activity assays were performed in cell culture to confirm biologic function of the transgenes. One individual adenoviral vector expressing IL-10 was previously prepared and tested in the laboratory. A cocktail of lentiviral and adenoviral vectors was investigated, initially in vitro to test for vector interference, and finally in vivo for prolongation of corneal graft survival. In vitro cocktail results of transgene expression showed no vector interference occurred when the viruses were used together in a transduction combination. In vivo cocktail therapy with an adenoviral vector expressing IL-10 and two lentiviral vectors expressing EK5 and Bcl-xL did not significantly prolong corneal graft survival, with cocktail therapy-treated and mock-vector treated allografts both having a median survival of 22 days (p= 0.68). The cocktail approach was chosen to utilise the best features of both viral vectors; the adenovirus to give early and strong gene expression, and the lentivirus to give long-term gene expression. However, with a good pre-clinical model, proven therapeutic genes and a combination of useful vectors, prolongation of graft survival could not be achieved. Gene therapy of the cornea for allograft prolongation has some obstacles to overcome before it can reach its full potential.
Keywords: gene therapy,cornea,transplantation,IL-10,sFlt-1,EK5,Bcl-xL,IDO,animal model,lentivirus,adenovirus
Subject: Ophthalmology thesis, Medicine thesis
Thesis type: Doctor of Philosophy
School: School of Medicine
Supervisor: Professor Keryn Williams