Non-Viral Methods of DNA Transfer
Viral vectors all induce an immunological response to some degree & may have
safety risks (such as insertional mutagenesis & toxicity problems). Furthermore
their capacity is limited & large scale production may be difficult to achieve.
Non-viral methods of DNA transfer require only a small number of proteins, have
a virtually infinite capacity, have no infectious or mutagenic capability &
large scale production is possible using pharmaceutical techniques. There are
three methods of non-viral DNA transfer. namely:
- naked DNA
- liposomes
- molecular conjugates
Naked DNA (in the form of a plasmid) can be directly injected into muscle cells (Wolff et al, 1990) or attached to gold particles that are bombarded into the tissue (Cheng et al, 1993). Though not very efficient, this can result in prolonged low level expression in vivo. The simplicity of this method, & sustained expression has led to the development of DNA vaccines. Compared to conventional attenuated & protein based vaccines, they are unaffected by pre-existing immunity e.g. due to maternal antibodies, relatively cheap, & can deliver a number of pathogen antigens on a single plasmid (Manickan et al, 1997). DNA vaccines are being developed for those pathogens where there is no existing vaccine e.g. HIV (Lekutis et al, 1997) or the current vaccine not fully effective e.g. influenza (Macklin et al, 1998). By using a highly conserved gene Ulmer et al (1993) were able to immunise mice against two serologically distinct influenza virus strains. In most cases however, DNA vaccines have not been shown to be better than the existing vaccines (Macklin et al, 1998). The actual type of immune response can be controlled by cotransformation of a gene coding for the appropriate cytokine (Xiang & Ertl, 1995) & this method may prove useful in redirecting inappropriate immune responses (Manickan et al, 1997). Other uses for naked DNA include cancer immunopotentiation (discussed below, Corr et al, 1997), repair of pancreatic insulin function (Goldfine et al, 1997), & stimulation of collateral blood vessel development (Takeshita et al, 1997). Expression of the gene product in muscle tissue can be improved by the coadministration of collagenase, papaverine & ischaemic conditions (Budker et al, 1998). The use of a muscle specific promoter (Skarli et al, 1998) & other intragene regulatory sequences, such as the poly A & transcription termination sequence (Hartikka et al, 1996) will also improve transgene expression.
Liposomes are lipid bilayers entrapping a fraction of aqueous fluid. DNA will spontaneously associate to the external surface of cationic liposomes (by virtue of its charge) & these liposomes will interact with the cell membrane (Felgner et al, 1994). In vitro up to 90% of certain cell lines may be transfected. By including a small amount of an anionic lipid in an otherwise cationic liposome the DNA can be incorporated into the internal surface of the liposome, thus protecting it from enzymatic degradation. (Crespo et al, 1996, cited Alio, 1997). To facilitate uptake into the cell as endosomes, targeting proteins have been included in liposomes, e.g. anti-MHC antibody (Wang & Huang, 1987) transferrin (Stavridis et al, 1986), & the Sendai virus or its F protein (Dzau et al, 1996). The Sendai virus additionally allows the plasmid DNA to escape from the endosome into the cytoplasm, thus avoiding degradation. The inclusion of a DNA binding protein (28 kDa high mobility group 1 protein) enhances transcription by bringing the plasmid into the nucleus (Dzau et al, 1997). Further proposed improvements include incorporating the Epstein-Barr virus Ori p & EBNA1 genes in the plasmid (as described above for HSV-1 amplicons) to maintain the plasmid as an episomal element (Alio, 1997).
Molecular conjugates consist of protein or synthetic ligands to which a DNA binding agent has been attached. Delivery to the cell can be improved by using similar techniques to those for liposomes. Targeting proteins include asialoglycoprotein (Wagner et al, 1991), transferrin (Wu et al, 1989), polymeric IgA (Ferkol et al, 1993) & adenovirus (Madon & Blum, 1996). Transgene expression tends to be transient & is limited by endosome/lysosomal degradation.
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