Biomedical Engineering Reference
In-Depth Information
The choice of target cells is another point worthy of discussion. In some instances, this choice is
predetermined, e.g. treatment of the genetic condition, familial hypercholesterolaemia, would require
insertion of the gene coding for the low-density lipoprotein receptor specifi cally in hepatocytes.
In other cases, however, some scope may be available to choose a target cell population. Even
in the case of redressing some genetic diseases, it may not be necessary to correct genetically the
exact population of cells affected. For example, a hallmark of several of the best characterized ge-
netic diseases is the exceedingly low production of a circulatory gene product. Examples include
clotting factors VIII and IX, a lack of which leads to haemophilia. It may be possible to correct
such defects by introducing the appropriate gene into any recipient cell capable of exporting the
gene product into the blood. Is such cases, choosing a target cell could be made upon practical
considerations, such as their ease of isolation and culture, their capacity to express (and excrete)
the protein product, and their half lives in vivo .
Several cell types, including keratinocytes, myoblasts and fi broblasts, have been studied in this
regard. It has been shown, for example, that myoblasts, into which the factor IX gene and the
growth hormone gene have been introduced, could express their protein products and secrete them
into the circulation.
14.3 Vectors used in gene therapy
A list of the various vectors capable of introducing genes into recipient cells has been provided in
Table 14.2. These vectors are conveniently categorized as being viral-based or non-viral-based sys-
tems. The main vector systems developed, thus far, are discussed in somewhat more detail below.
14.3.1 Retroviralvectors
Some 24 per cent of all gene therapy clinical trials undertaken to date have employed retroviral
vectors as gene delivery systems. Retroviruses are enveloped viruses. Their genome consists of
ssRNA of approximately 5-8 kb. Upon entry into sensitive cells, the viral RNA is reverse tran-
scribed and eventually yields double-stranded DNA. This subsequently integrates into the host cell
genome (Box 14.1). The basic retroviral genome contains a minimum of three structural genes:
Box 14.1
The retroviral life cycle
The retroviral life cycle (Figure 14.B1) begins with the entry of the enveloped virus into the cell.
The viral reverse transcriptase enzyme then copies the viral RNA genome into a single (minus)
DNA strand and, using this as a template, generates double-stranded DNA. The double-stranded
DNA is then randomly integrated into the host cell genome (the proviral DNA). Transcription
of the proviral genes host cell's transcription machinery yields mRNA that directs synthesis of
mature virion particles. The viral particles bud out from the cell's plasma membrane, picking
up a membrane-derived outer coat as they do so.
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