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Title: Development of novel non-viral vectors for use in the immunomodulation of corneal transplantation
Author: Seow, Wei Yang
ISNI:       0000 0004 2713 3241
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2011
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About 25% of corneal grafts will fail within five years (mainly due to T-cell-mediated rejection) and the ex-vivo delivery of immunomodulatory genes to the cornea is one attractive method to improve survival rates. Early efforts focused on the use of viral vectors, whose toxicity and immunogenicity remain formidable barriers. Here, we developed an arginine-rich oligopeptide featuring a novel tri-block design. Each block served a specific purpose – DNA binding, endosomal escape and cell membrane penetration – and was systematically optimised. The peptides could effectively bind and protect DNA from physical and enzymatic degradation. Since the corneal endothelium is an important target for therapy, mouse corneal endothelial cells (MCEC) were chosen for initial in-vitro testing. The peptides depended heavily on cell surface heparan sulfate and utilised multiple endocytosis pathways to gain entry into MCEC. Importantly, they mediated indolamine 2,3-dioxygenase (IDO) mRNA and protein expression efficiently in both MCEC and murine corneas. IDO catalyses the biodegradation of tryptophan which T-lymphocytes sensitively require for growth. Hence, IDO over-expression in the cornea can prolong allograft survival by locally suppressing T-cell activity. We demonstrated that the IDO expressed was biologically active and could suppress the growth of CD4 T-cells in a proliferation assay. Immunohistochemistry further determined that IDO expression was localised to the corneal endothelium and epithelium. Corneal transplantations were then performed using a fully MHC-mismatched mouse model. However, the survival of IDO-transfected grafts was only marginally prolonged relative to GFP-transfected corneas and was shortened compared to untreated corneas. Toxicity, as evidenced by propidium iodide staining, was suspected to shorten graft survival following peptide-mediated transfection. Efforts to modulate toxicity by reducing the transfection period in the presence of chloroquine also failed to prolong graft survival. Future work should therefore focus on reducing toxicity while improving the transfection efficiency of this peptide carrier.
Supervisor: George, Andrew ; Yang, Yi-Yan Sponsor: A*STAR Graduate Academy
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral