Publications

The complexes formed between plasmid DNA and polycationic peptides such as those based on poly(L-lysine) form the basis of numerous non-viral gene delivery vectors. In this paper, we describe several distinct methodologies for monitoring the formation of such nanoparticles formed by electrostatic interation between plasmid DNA and polycations such as those based on poly(L-lysine). Effects of N:P (charge) ratio and polycation molecular weight are also examined with respect to complex stability to dilution and physiological levels of salt (150mM), ultimately drawing the conclusion that polyelectrolyte complexes for targeted application in vivo should be based on high molecular weight polycations, or should be stabilised to prevent their dissociation under physiological salt concentrations. Click the link above to view this paper.

This paper addresses one of the most significant quandries of systemic application of gene therapy vectors, namely how to achieve systemic inertness of the applied vectors (ie procluding interactions with the bodies immune system and blood serum factors) whilst providing a high degree of intracellular activity following uptake into the target cells. By using a combination of pHPMA, a amino-reactive hydrophilic polymer that can (at least theorectically) be used to surface modify any gene therapy vectors with available surface amino groups, increasing their solubility whilst decreasing their interaction with blood and immune components, with a high molecular weight poly(L-lysine) designed to incorporate regular disulphide bonds (that undergo reduction at intracellular gluatathione levels), we have created nanoparticles that show excellent stability in the extracellular compartment coupled with high levels of intracellular activity. Furthermore, cell specific targeting ligands (such as bFGF) can be incorporated into the vector (via spare amino-reactive ester groups on the polymer) to increase cell association and specificity. Click the link above to view this paper.

In this paper we describe how small (7-mer) oligopeptides were iterated using phage display technology that bind to bFGF receptors. The receptors for bFGF have been shown to be upregulated in numerous tumours in vivo, presumably to help stimulate angiogensis. Therefore, the possibility of using bFGF to target gene delivery vectors (or chemotherapeutic drugs) to the the tumour vasculature is an appealing one. However, the likelihood of being able to use bFGF systemically is limited by the large, hydrophobic nature of the molecule, its difficulty (and cost) to mass produce, its potential angiogenic effects, and potential interaction with the bodies natural vasculature, presumably lowering the dose limiting toxicity. Therefore, we reasoned that small, oligopeptides that bind to the same receptor (both on cells in vitro, and resected tumours ex vivo), could easily be incorporated into either viral, or non-viral gene delivery vectors mediating selective delvery of their DNA payload to the bFGF recptor expressing cells of the tumour vasculature. Importantly, whilst the peptides were shown to bind to bFGF receptors, they did not stimulate the strong mitogenic effect observed using bFGF. Click the link above to veiw this paper.

Translocation of exogenous DNA from the cytosolic compartment to the nucleus of non-dividing cells is considered to be the greatest rate limiting step in successful gene delivery. In the absence of nuclear translocation of the exogenous DNA, transcription and translation cannot occur, and phenotypic correction of the diseased cell will not be achieved. An alluring alternative to the use of DNA to mediate cellular correction is to bypass the requirement for cytoplasmic to nuclear translocation by delivering mRNA, that can readily undergo translation in the cytosol. In this paper we demonstrate the feasibility of this approach using reporter mRNA. This "cytoplasmic expression system" provides a short term burst of expression but could feasibly be used to set up an autonomous cytoplasmic expression system such as those based upon T7 polymerase. Click on the link above to view this paper.

In this paper we address one of the fundamental difficulties in developing targeted gene delivery vectors for in vivo application, namely that cells cultured in vitro show loss of polarisation (compared to in vivo) and deregualtion of cellular receptors (compared to in vivo). Frequently, vector targeting strategies that are shown to be efficient in vitro are twarted by lack of in vivo efficacy, attributable to the significant changes in cellular phenotype exhibited in the two very different environments. In order to achieve targeted gene delivery in vivo, we have utilised phage display technology to identify receptor binding oligopeptides that are expressed on polarised cells in vivo. To achieve this, receptor binding oligopeptides were recovered by biopanning against the endothelial cells of human umbilical veins. The resulting peptides bound not only to the polarised cells ex vivo, but also to cultured HUVECs in culture, but showed little or no affinity for other cultured cell lines indicating selectivity for the vascular endothelial cells. To view this paper, click on the link above.

In this article, the current status in the field of non-viral gene therapy is reviewed. Initially focussing upon the multitude of different approaches applied in the field (lipoplexes, polyplexes, ultrasound etc), the review then goes on to discuss the numerous pitfalls en-route to achieving targeted gene delivery in vivo, both at the extracellular and intracellular levels. To view this article, click the link above.