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).

Perhaps the 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.

References:

Davis ME (2002) Non-viral gene delivery systems. Curr Opin Biotechnol 13, 128-31.

Kurisawa M, Yokoyama M, Okano T (2000a) Gene expression control by temperature with thermo-responsive polymeric gene carriers. J Control Release 69, 127-37.

Kurisawa M, 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 345, 227-32.

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 4, 138-45.

Vectors, 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!