Date of Degree
PhD (Doctor of Philosophy)
Kevin G. Rice
The 21st century has been called the age of genomic medicine, yet gene therapy for medicinal use remains a theory. One reason that there are no safe and effective treatments for human disease is the lack of a vehicle capable of delivering genetic material to a specific target. In nature we observe gene pathology by viral vectors, which deliver their own genetic material to specific host cells efficient at spreading the viral blueprint throughout the organism.
The aim of my research into gene therapy has been to develop a synthetic vector with the delivery capability of viral vectors found in nature. This includes the ability to protect genetic cargo from modification and degradation in vivo, target to a desired cell type within a specific tissue, facilitating absorption into the cell, and delivery to the nucleus, where expression of genetic material occurs.
The goal of this thesis project was to synthesize a novel vector which would selectively target the dendritic cell SIGN receptor, mirroring the method of pathogens such as HIV, which target this receptor and subsequently the immune system, resulting in chronic infection.
The vector we designed contains two major components, the high mannose N-glycan Man9GlcNAc2Asn, and a peptide composed of nine amino acids: four lysine spacing residues, four lysines derivatized with acridine on the epsilon amine of their side chains, and a cysteine for conjugation to the glycan. This compound, the Man9-AcrLys Glycopeptide, was engineered to intercalate into plasmid DNA via the acridine functional groups and to bind the DC-SIGN receptor through the glycan's mannose residues.
The vehicle was tested in vitro in CHO cells bearing a recombinant DC-SIGN receptor in the context of luciferase reporter gene delivery. We found that under equal treatment conditions, DC-SIGN (+) CHO cells expressed more luciferase and were 100-fold more luminescent than control DC-SIGN (-) CHO cells.
My delivery method was further analyzed in a cell-sorting FACS experiment. I covalently labeled pGL3 reporter plasmid with a fluorophore, and transfected the CHO cells under typical transfection conditions. The experimental results confirmed preferential DC-SIGN mediated gene delivery.
Acridine, DC-SIGN, Dendritic Cell, N-glycan, non-viral gene delivery, Soybean Agglutinin
Copyright 2009 B. Kevin Anderson