Chapter 10 of
Principles of Gene Manipulation (pp 175-176), we discuss
the use of cationic lipids as efficient vehicles for gene delivery
both in vitro and in vivo (a gene delivery vehicle is defined
in the Box below). The advantage of cationic lipids over other lipid-based
transfection agents is that they spontaneously assemble into complexes
with DNA. The complexes have a positive charge allowing them to
interact spontaneously with the negatively charged cell membrane
and this stimulates their uptake by endocytosis. More recently,
however, cationic polymer-based gene delivery vehicles have become
popular. These have the same advantages as cationic lipids but there
is more control over their physical properties because copolymeric
complexes can be produced. The practicalities of such 'lipoplexes'
and 'polyplexes' for gene transfer in vivo have been recently
reviewed (Davis 2002).
most obvious application of in vivo gene transfer using cationic
complexes is human gene therapy. However, for this to be successful,
precise control over transgene expression would be required. Traditional
inducible expression systems, as discussed in the first part of
Chapter 13 in Principles of Gene Manipulation (pp 247-253),
generally rely on the application of chemicals, such as tetracycline,
oestrogen or immunosuppressive drugs. A major problem with these
systems is the difficulty in delivering the inducing agent to particular
tissues and the uneven way in which they are absorbed and eliminated.
It is also possible that large or prolonged doses of such agents
would be toxic and therefore unsuitable for use in humans. Another
way to control gene expression is targeted delivery, which can be
achieved through the use of antibodies or other specific recognition
molecules to facilitate the uptake of cationic complexes by particular
cell types. However, recent developments in polymer technology provide
an alternative solution - controlling the physical and chemical
properties of the gene delivery vehicle itself to regulate transgene
expression (Yokoyama 2002).
The basis of
these developments is that some polymers change their properties
in response to a physical stimulus. For example, Nagasaki et al.
(2000) have described a synthetic polymer whose affinity to DNA
changes upon exposure to UV light. The polymer is based on azobenzene,
and the azo moiety undergoes trans-II-cis isomerism following
UV irradiation causing DNA to be released from the complex. In cultured
cells, exposure to UV light increases transfection efficiency by
up to 50%. Polymeric complexes responsive to UV light have limited
use for gene therapy because it would be difficult to illuminate
internal organs. However, another synthetic complex has been described
that is responsive to heat (Kurisawa et al. 2000a,b, Yokoyama et
al. 2001). This polymer is based on N-isopropylacrylamide (PIPAAm)
and undergoes a phase transition at a lower critical solution temperature
of 32°C. Below 32°C, the polymer is hydrophilic and soluble,
and forms a loose complex with DNA. Above 32°C, the polymer
is hydrophobic and compact, and forms a tight complex by aggregation.
A tight complex is preferable for DNA delivery because it is suitable
for uptake and resistant to nucleases, whereas a loose complex is
better for transcription because it facilitates access by transcription
factors. Therefore, at normal body temperatures the DNA complex
may be taken up efficiently by all cells, but poorly expressed.
Local cooling, e.g. through the application of ice to the body surface
or the use of catheters, can then induce gene expression in specific
tissues or organs. The properties of the polymer can be modified
by increasing the proportion of hydrophobic or hydrophilic chemical
groups, thus lowering or raising the transition temperature, respectively.
Davis ME (2002)
Non-viral gene delivery systems. Curr Opin Biotechnol 13, 128-31.
Yokoyama M, Okano T (2000a) Gene expression control by temperature
with thermo-responsive polymeric gene carriers. J Control Release
Yokoyama M, Okano T (2000b) Transfection efficiency increases by
incorporating hydrophobic monomer units into polymeric gene carriers.
J Control Release 68, 1-8.
Nagasaki T et
al. (2000) Synthesis of a novel water-soluble polyazobenzene dendrimer
and photoregulation of affinity toward DNA. Mol Cryst Liq Cryst
Yokoyama M (2002)
Gene delivery using temperature-responsive polymeric carriers. Drug
Discovery Today 7, 426-32.
Yokoyama M et
al. (2001) Influential factors on temperature-controlled gene expression
using thermo-responsive polymeric gene carriers. J Artifical Org
vehicles and gene transfer
What is the
difference between a vector and a vehicle? Both terms have been
adopted in molecular biology on the basis that they mean 'carrying
something'. A vector is a recombinant DNA or RNA molecule,
usually based on a plasmid or a viral genome, which incorporates
a new piece of DNA or RNA called the transgene. The purpose
of the vector is not to facilitate gene transfer to the host
cell, but to allow the transgene to be cloned, maintained and
if necessary, expressed (e.g. by providing an origin of replication,
a selectable marker, a polylinker and elements to control transcription
and translation). Vectors may also be based on chromosomes or transposons.
In contrast, a vehicle can be anything with which DNA or
RNA is associated or within which it is encapsulated to facilitate
the introduction of the transgene into the host cell. Liposomes
are vehicles containing plasmid vectors. The metal particles used
in bombardment methods are also vehicles. Strictly speaking, a recombinant
viral genome is a vector whereas the capsid that surrounds it is
a vehicle, although this is taking the terminology to extremes!