The development of synthetic gene delivery systems

Abstract: This thesis describes a new platform technology. The technique is referred to as Bioplex (biological complex) in analogy with the nomenclature for other transfection reagents, since we attach biological functions to a nucleic acid complex via sequence-specific hybridisation. The technology is based on the use of two particular aspects of peptide nucleic acids, PNA, (or analogues thereof, namely the high sequence specificity, making these molecules most suitable as genetic anchors, and the possibility of making a continuous peptide synthesis, allowing PNA and a peptide moiety to become adjacent without involving cumbersome linking chemistry and purification of the conjugated peptides. We use the anchor property (trans) as a means of incorporating, in a sequencespecific fashion, various functional elements in cis relative to the PNA. This enables modulation of nucleic acids, including multifunctional approaches that permit improved gene delivery. PNA is a synthetic compound based on the linkage of purines or pyrimidines to a neutral pseudo-peptide backbone instead of the charged pentosephosphate moiety. The neutral charge contributes to an increased Tm value for PNA-DNA/ RNA interactions versus "homotypic" DNA (DNA-DNA) or RNA (RNA-RNA), or RNA-DNA duplex formation. Under certain circumstances, these properties will permit "strand-invasion" at room temperature, i.e. allow a PNA molecule to invade a DNA duplex, a phenomenon dependent on the inherent DNA breathing activity. Thus, this thesis describes how to link peptide functions directly to DNA via hybridisation instead of covalent linkage or via non-specific charge-interactions. This technique is applicable both in vitro and in vivo, and avoids the potential hazards of vectors based on viruses and other microorganisms. In the first article we show that PNA can be used as an anchor for peptide functions and that it is possible to confer peptide functions both to oligonucleotides and plasmids in vitro (I). We subsequently investigated the in vivo activity of PNA-NLS molecules hybridised to fluorescent oligonucleotides. We introduced the denomination Bioplex to describe the category of transfection complexes where a function has been linked directly to a nucleic acid (II). The third work delves into the use of Bioplex mediated transfection without helper reagents such as polyethyleneimine. The intracellular localisation of the PNA-NLS hybridised nucleic acid was also investigated by deconvolution microscopy (III). Anchoring 1 or 4 RGD-PNA peptides to a fluorescently labelled oligonucleotide carrier and comparing the cellular fluorescence after transfection showed the synergistic effect of multiple ligands. By adding a PNA-NLS molecule to the DNA/(PNA-RGDx4) complex and using a lysosome disruptive reagent we showed the synergistic potentiation of using multivalent receptor targeting, endosome escape and subsequent nuclear translocation in combination (IV). Plasmid DNA is the main target for clinical Bioplex application. We investigated the parameters necessary to achieve PNA-anchor hybridisation and PNA-NLS mediated transfection efficacy. The reporter construct used is a 6.7 kb plasmid containing a GFP-Luciferase fusion gene. This allowed us to compare the read-out of two separate assay systems thus avoiding potential inherent problems in either one of them (V).