Biomedical Engineering Reference
In-Depth Information
contaminants, as well as RNA. However, genomic DNA and endotoxins generally co-purify with
the plasmid DNA. Additional chromatographic approaches based upon reverse-phase and affi nity
systems have been developed at laboratory scale at least.
A signifi cant feature of plasmid purifi cation employing capture chromatography (i.e. involving
plasmids binding to the chromatographic beads) is the low plasmid-binding capacities observed.
The pore size of commercially available capture chromatographic media is insuffi ciently large to
allow entry of plasmids, restricting binding to the bead surface. Binding capacities can, therefore,
be 100-fold or more lower than those observed when the same media are used to purify (much
smaller) therapeutic proteins (Chapter 6).
Purifi ed plasmids may then be analysed using various analytical techniques. Freedom from con-
taminating nucleic acid/proteins can be assessed electrophoretically. Endotoxin and sterility tests
would also be routinely undertaken. The purifi ed plasmid DNA must next be formulated to yield
the fi nal non-viral delivery system. Formulation studies relating to such systems remain an area re-
quiring further investigation. Most work reported to date relates to formulating/stabilizing lipoplex-
based gene delivery systems. Aqueous suspensions of these (and other) non-viral-based systems tend
to aggregate quickly (in a matter of minutes to hours). In order to circumvent this problem, the fi nal
delivery systems were often actually formulated at the patient bedside in earlier clinical trials.
Research aimed at identifying appropriate stabilizing excipients/formulation formats is ongoing.
Simple freezing is an option, particularly as frozen formulations would be immune to agitation-in-
duced aggregation. However, the process of freezing, particularly slow freezing, in itself induces ag-
gregation. This can be minimized by fl ash freezing (e.g. by immersion in liquid nitrogen), although
this approach may not prove practicable at an industrial scale. The addition of cryoprotectants may
help minimize this problem, and initial studies indicate that various sugars (e.g. glucose, sucrose
and trehalose) show some potential in this regard. Another avenue under investigation relates to the
generation of a fi nal freeze-dried product. Again, issues such as the (relatively) slow freezing proc-
ess characteristic of industrial-scale freeze-driers complicate attaining this goal in practice.
14.4 Gene therapy and genetic disease
Well over 4000 genetic diseases have been characterized to date. Many of these are caused by lack of
production of a single gene product or are due to the production of a mutated gene product incapable
of carrying out its natural function. Gene therapy represents a seemingly straightforward therapeutic
option that could correct such genetic-based diseases. This would be achieved simply by facilitating
insertion of a 'healthy' copy of the gene in question into appropriate cells of the sufferer.
Although simple in concept, the application of gene therapy to treat/cure genetic diseases has,
thus far, made little impact in practice. The slow progress in this regard is likely due to a number
of factors. These include:
•
The number of genetic diseases for which the actual gene responsible has been identifi ed and
studied are relatively modest, although completion of the human genome project should rapidly
accelerate identifi cation of such genes.
•
As discussed previously, none of the fi rst-generation gene-delivering vectors have proven fully
satisfactory.
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