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Current gene transfer vectors are extremely limited for selective vascular cell delivery due to their promiscuous tropism and low efficiency of gene delivery to the vasculature. We have sought to improve the efficiency of gene transfer to vascular endothelial cells using phage display. Using bio-panning on whole cells, we have isolated a panel of 60 7-mer peptides that have the ability to bind endothelial cells but not to non-endothelial cells including vascular smooth muscle cells and hepatocytes. One candidate peptide was cloned upstream of a single chain antibody (scFv) generated against the knob domain of adenovirus type 5, expressed in bacteria and purified. While the scFv alone inhibited adenoviral fiber-dependent infection of all cell types tested (to >95% in hepatocytes), the scFv-peptide fusion mediated selective infection into endothelial cells without infection into non-endothelial cells types. Furthermore, the level of infection achieved in endothelial cells was 15 fold higher than that achieved with fiber-mediated gene transfer alone. We have additionally isolated 15 individual peptides that have the ability to target the endothelial leptin-like oxidised LDL receptor (LOX-1), a receptor highly expressed in endothelial cells in hypertensive models and in atherosclerotic lesions, by phage bio-panning on cells ectopically expressing the LOX-1 receptor. Candidate peptides mediated significantly higher binding to LOX-1 expressing cells compared to LOX-1 negative cells. Their ability to re-target adenoviral gene transfer is being tested. Our results demonstrate that small, novel peptides isolated by phage display have the ability to retarget gene transfer selectively and efficiently to vascular endothelial cells. This has important implications for targeting gene transfer to endothelial cells for molecular and therapeutic protocols in hypertension.
Variables Pertinent to the Efficiency of Adeno-Associated Virus (AAV) Vectors Mediated Gene Transfer to Human Vascular Endothelial Cells.
