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• their low/non-immunogenicity;
• non-occurrence of integration of the therapeutic gene into the host chromosome (this
eliminates the potential to disrupt essential host genes or to activate host oncogenes.
The initial approach adopted entailed administration of ‘naked’ plasmid DNA housing the gene of interest. This avenue of research was first opened in 1990, when it was shown that naked plasmid DNA was expressed in mice muscle cells subsequent to its i.m. injection. The plasmid DNA concerned housed the b-galactosidase gene as a reporter. Subsequent expression of b-galactosidase activity could persist for anything from a few months to the remainder of the animal’s life. The transfection rate recorded was low (1-2% of muscle fibres assimilated the DNA) and the DNA was not integrated into the host cell’s chromosomes.
Up until this point, it was assumed that naked DNA injected into animals would not be spontaneously taken up and expressed in host cells. This finding vindicated the cautious approach taken by the FDA and other regulatory authorities with regard to the presence of free DNA in biopharmaceutical products (Chapter 3).
Scientists have also since demonstrated that DNA (coated on microscopic gold beads) propelled into the epidermis of test animals with a ‘gene gun’ is expressed in the animal’s skin cells. Furthermore, the introduction in this fashion of DNA coding for human influenza viral antigens resulted in effective immunization of the animal against influenza. Similar results, using other pathogen models, have also now been generated. It is assumed that expressed antigen is secreted by the cell and, in this way, is exposed to immune surveillance. Further research has illustrated that systematic administration via i.v. injection rarely achieves meaningful cell transfection. This is most likely due to the high nuclease levels present in serum. In contrast, free nuclease activity in muscle tissue is extremely low.
Modern non-viral-based systems generally entail complexing/packaging the gene of interest (present, along with appropriate promoters, etc., in a circular plasmid) with additional molecules, particularly various lipids or some polypeptides. These generally display a positive charge and hence interact with the negatively-charged DNA molecules. The function of such carrier molecules is to stabilize the DNA, protect it, e.g. from serum nucleases, and ideally to modulate interaction with the biological system, e.g. help target the DNA to particular cell types — or away from other cell types.
NUCLEIC ACID THERAPEUTICS 477
Pol ó lysine
Figure 11.7. Structure of some cationic lipids and polylysine
The most commonly used polymers are the cationic lipids and polylysine chains (Figure 11.7). Cationic lipids can aggregate in aqueous-based systems to form vesicles/liposomes, which in turn will interact spontaneously with DNA (Figure 11.8). Initially, the negatively-charged plasmid DNA probably acts as a bridge between adjacent vesicles. Further DNA-vesicle interactions quickly generate a complex 3-D lattice-like system composed of flattened vesicles (some of which probably rupture) interspersed with plasmid DNA. The lipid component of such ‘lipoplexes’ should therefore provide a measure of physical protection to the therapeutic gene.
Gene therapy results to date using this approach have been mixed. The process of lipoplex formation is not easily controlled and hence different batches made under seemingly identical conditions may not be structurally identical. Furthermore, in vitro test results using such lipoplexes can correlate very poorly with subsequent in vivo performance. Clearly, more research is required to underpin the rational use of lipoplexes for gene therapy purposes. The same is true
Figure 11.8. Initial interaction of plasmid DNA with cationic (positively charged) vesicles. Refer to text for further details
for other polymer-based synthetic gene delivery systems, the most significant of which is the polylysine-based system. Polylysine molecules, due to their positive charge (Figure 11.7) can also form electrostatic complexes with DNA. However, the stability of such ‘polyplexes’ in biological fluids can be problematic. Furthermore, polyplexes tend to be rapidly removed from the circulation, prompting a low plasma half-life. These difficulties can be alleviated in part by the attachment of polyethylene glycol (PEG) molecules. PEG attachment is also used to increase the serum half-life of various therapeutic proteins, such as some interferons (Chapter 4).
No matter what their composition, such synthetic gene delivery systems also meet various biological barriers to efficient cellular gene delivery. Viral vector-based systems are far less prone to such problems, as the viral carrier has evolved in nature to overcome such obstacles. Obstacles relate to:
• blood-related issues;
• biodistribution profile;
• cellular targeting;
• cellular entry and nuclear delivery.