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.