Biomedical Engineering Reference
In-Depth Information
Once in the cytoplasm, a proportion of plasmid molecules are likely degraded by cytoplasmic
nucleases, effectively further reducing transfection effi ciencies. 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 specifi c energy-
requiring transport processes.
Overall, it is estimated that only one in 10
4
-10
5
plasmids taken up by endocytosis will enter the
nucleus intact and be successfully expressed.
14.3.5 Manufacture of plasmid DNA
The overall generalized approach used to produce plasmid DNA for the purposes of gene therapy
trials is presented in Figure 14.11. Prior to its manufacture, researchers would have constructed
an appropriate vector housing the therapeutic gene and introduced it into a producer microorgan-
ism, usually
E. coli
. Routine large-scale plasmid manufacture then entails culture of a batch of
producer microorganisms by fermentation, followed by plasmid extraction and purifi cation. In this
regard, the overall approach used resembles the approaches taken in the large-scale manufacture
of recombinant therapeutic proteins, as described in Chapters 5 and 6.
Industrial-scale microbial fermentation (upstream processing) has also been described in Chapter 5, 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 microfi ltration. 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. The combination of high pH and deter-
gent action disrupts the microbial cell wall/membranes with consequent release of the intracellular con-
tents. In addition to the desired plasmid DNA, this crude mixture will also contain various impurities,
which must be removed by subsequent downstream processing steps. Notable impurities include:
•
cell wall debris and some intact cells;
•
proteins;
•
genomic DNA;
•
RNA;
•
low molecular mass metabolites;
•
endotoxin.
After lysis is complete, the next step can entail the addition of a high-salt neutralization solu-
tion, such as potassium acetate. This promotes formation of aggregates of genomic DNA and
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