Nonviral vectors

Although viral-mediated gene delivery systems currently predominate, a substantial number of current clinical trials use non-viral-based methods of gene delivery. General advantages quoted with respect to non-viral delivery systems include:

• 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 P-galactosidase gene as a reporter. Subsequent expression of P-galactosi-dase 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 per cent 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 biophar-maceutical products (Chapter 7).

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 from, for example, 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).

The most commonly used polymers are the cationic lipids and polylysine chains (Figure 14.8). Cationic lipids can aggregate in aqueous-based systems to form vesicles/liposomes, which in turn ch3

CTAB DOTMA

DMRI

, O-C18H35

Polylysine

Figure 14.8 Structure of some cationic lipids and polylysine

n will interact spontaneously with DNA (Figure 14.9). Initially, the negatively charged plasmid DNA probably acts as a bridge between adjacent vesicles. Further DNA/vesicle interactions quickly generate a complex three-dimensional 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; 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 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 14.8), 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 circulation, prompting a low plasma half-life. These difficulties can be alleviated in part by the attachment of PEG molecules. PEG attachment is also used to increase the serum half-life of various therapeutic proteins, such as some interferons (Chapter 8).

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.

Whereas lipoplexes/polyplexes generally protect the plasmid from serum nucleases, the overall positive charge characteristic of these structures leads to their non-specific interactions with cells

Figure 14.9 Initial interaction of plasmid DNA with further details cationic (positively charged) vesicles. Refer to text for

Figure 14.9 Initial interaction of plasmid DNA with further details cationic (positively charged) vesicles. Refer to text for

(both blood cells and vascular endothelial cells) and serum proteins. Also, following i.v. injection, such DNA complexes in practice tend to accumulate in the lung and liver. Targeting of DNA complexes to specific cell types also poses a considerable (largely unmet) technical challenge. Approaches, such as the incorporation of antibodies directed against specific cell surface antigens may provide a future avenue of achieving such cell-selective targeting. However, it is currently believed that ionic interactions constitute a predominant binding force between the positively charged lipoplexes/polyplexes and the negatively charged eukaryotic cell surface. Such electrostatic interactions may even override more biospecific interactions characteristic of antibody- or receptor-based systems. Currently, probably the most effective means of delivering such vectors to target tissue/cells is to inject them into/beside the target area.

However targeted to the appropriate cell surface, if it is to be clinically effective, the therapeutic plasmid must enter the cell and reach the nucleus intact. Cellular entry is generally achieved via endocytosis (Figure 14.10). A proportion of endocytosed plasmid DNA escapes from the endosome by entering the cytoplasm, thereby escaping liposomal destruction (Figure 14.10). The molecular mechanism by which escape is accomplished is, at best, only partially understood. Anionic lipid constituents of lipoplexes, for example, may fuse directly with the endosomal membranes, facilitating direct expulsion of at least a portion of the plasmid DNA into the cytoplasm. Generally, the DNA is released in free form (i.e. uncomplexed to any lipid).

Some attempts have been made to rationally increase the efficiency of endosomal escape. One such avenue entails the incorporation of selected hydrophobic (viral) peptides into the gene delivery systems. Many viruses naturally enter animal cells via receptor-mediated endocytosis. These viruses have evolved efficient means of endosomal escape, usually relying upon membrane-disrupting peptides derived from the viral coat proteins.

Endosome

Endosome

Figure 14.10 Overview of cellular entry of (non-viral) gene delivery systems, with subsequent plasmid relocation to the nucleus. The delivery systems (e.g. lipoplexes and polyplexes) initially enter the cell via endocytosis (the invagination of a small section of plasma membrane to form small membrane-bound vesicles termed endosomes). Endosomes subsequently fuse with golgi-derived vesicles, forming lysosomes. Golgi-derived hydrolytic lysosomal enzymes then degrade the lysosomal contents. A proportion of the plasmid DNA must escape lysosomal destruction via entry into the cytoplasm. Some plasmids subsequently enter the nucleus. Refer to text for further details

Figure 14.10 Overview of cellular entry of (non-viral) gene delivery systems, with subsequent plasmid relocation to the nucleus. The delivery systems (e.g. lipoplexes and polyplexes) initially enter the cell via endocytosis (the invagination of a small section of plasma membrane to form small membrane-bound vesicles termed endosomes). Endosomes subsequently fuse with golgi-derived vesicles, forming lysosomes. Golgi-derived hydrolytic lysosomal enzymes then degrade the lysosomal contents. A proportion of the plasmid DNA must escape lysosomal destruction via entry into the cytoplasm. Some plasmids subsequently enter the nucleus. Refer to text for further details

Once in the cytoplasm, a proportion of plasmid molecules are likely degraded by cytoplasmic nucleases, effectively further reducing transfection efficiencies. There are two potential routes by which plasmid DNA could reach the nucleus:

• direct nuclear entry as a consequence of nuclear membrane breakdown associated with mitosis;

• transport through nuclear pores, which may occur via passive diffusion or specific energy-requiring transport processes.

Overall, it is estimated that only one in 104-105 plasmids taken up by endocytosis will enter the nucleus intact and be successfully expressed.

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