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biopharmaceuticals biochemistry and biotecnology - Walsh G.

Walsh G. biopharmaceuticals biochemistry and biotecnology - John Wiley & Sons, 2003. - 572 p.
ISBN 0-470-84327-6
Download (direct link): biochemistryandbiotechnology2003.pdf
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While 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 (both blood cells and vascular endothelial cells) and serum proteins. Also, following i.v. injection, such DNA complexes tend in practice 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 or 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 11.9). A proportion of endocytosed plasmid DNA escapes from the endosome by entering the cytoplasm (thereby escaping liposomal destruction (Figure 11.9). 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.
Once in the cytoplasm, a proportion of plasmid molecules are probably 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.
Figure 11.9. 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
Manufacture of plasmid DNA
Plasmid DNA is routinely extracted and purified from various microbial cells at the research level. However, industrial-scale manufacture to the exacting standards of purity demanded of pharmaceutical products is a pursuit still in its infancy, and little has been published on the subject. The overall generalized approach used to produce plasmid DNA for the purposes of gene therapy trials is presented in Figure 11.10. Prior to its manufacture, researchers would have constructed an appropriate vector housing the therapeutic gene and introduced it into a producer microorganism, such as E. coli. Routine large-scale plasmid manufacture then entails culture of a batch of producer microorganisms by fermentation, followed by plasmid extraction and purification. In this regard, the overall approach used resembles the approaches taken in the large-scale manufacture of recombinant therapeutic proteins, as described in Chapter 3.
Industrial-scale microbial fermentation (upstream processing) has also been described in Chapter 3, to which the reader is referred. Fermentation promotes microbial cell replication and thus the biosynthesis of large quantities of plasmid. Subsequent to fermentation, the microbial cells are harvested (collected) by either centrifugation or microfiltration. Following resuspension in a low volume of buffer, the cells must be disrupted in order to release the plasmids therein. This appears to be most commonly achieved by the addition of a lysis reagent, consisting of NaOH and SDS (sodium dodecyl sulphate). The combination of high pH and detergent action disrupts the microbial cell wall and membranes, with consequent release of the intracellular contents. In addition to the desired plasmid DNA, this crude mixture will also contain various impurities that must be removed by subsequent downstream processing steps. Notable impurities include:
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