O'Donnell, M.W.; (2005) Lipidic peptide dendrimers: Potential gene delivery agents for the treatment of haemophilia B. Doctoral thesis , University of London.

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Abstract

Haemophilia B is an X-linked recessive bleeding diathesis caused by a deficiency of biologically active factor EX (FIX). Correction of this phenotype is possible by introducing a normal copy of the gene into cells capable of producing functional FIX. Engineered virus or synthetic molecules have been utilised as carriers to perform this task with varying levels of success. Lipidic peptide dendrimers (LPD) have an in vitro capacity for gene transfer, are able to transfect a number of cell lines and exhibit nMnimal cytotoxicity. This thesis reports the characteristics and performance of LPD as a means of delivering a FIX gene construct. LPD/DNA complexes are capable of delivering a human FIX gene to cultured cells and produce a functional protein. The complexes showed improved gene transfer to cultured liver cells when a co-lipid displaying a ligand specific to the cell-type was incorporated into the complexes. In vivo experiments involved delivery of the complexes to mice by way of oral and intra-venous administration. Liquid scintillation counting and PCR studies indicated the presence of both dendrimer and plasmid DNA respectively in the liver of treated mice, but not in tissues of the reproductive or central nervous system. Factor IX protein was not detected in murine tissues by immunohistochemistry or in the plasma by ELISA. Gastro-intestinal tract tissue indicated activity of a reporter gene from glucose-modified complexes delivered orally. Immune responses to the complexes were investigated by means of complement activation and cytokine gene expression assays. Cytokines IL-lp, TNFa and IL-6 were investigated using RT-PCR to quantify localised and systemic inflammatory responses. These tests showed a general increase in the expression of inflammatory cytokines, particularly in the liver of animals to which the complexes were administered. All LPD/DNA complexes were found to activate complement to different degrees and relative to the charge density of the dendrimer used.

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