Costos de Obra

1.4.7) Role o f epithelial cell permeability in gene transfer

Although severe lung injury may facilitate receptor mediated gene transfer by upregulating and redistributing epithelial receptors, much lower levels o f lung damage may facilitate gene transfer by increasing transepithelial permeability and increasing receptor-ligand interaction. Hypo-osmotic shock can increase permeability o f epithelial

cell layers after only eight minutes exposure to deionised water (Widdicombe et al., 1996). Cell layers become permeable to macromolecules due to increases in cellular permeability, resolving rapidly, and paracellular transepithelial permeability, which is more prolonged (Widdicombe et al., 1996). Hypotonic shock can increase cell membrane permeability by endocytosis (Okada and Rechsteiner, 1982), and transepithelial permeability by disruption o f tight junction complexes (Anderson and Van Itallie, 1995; Gumbiner, 1993). Tight junctions are calcium dependent and intraepithélial permeability can be transiently increased using a number o f calcium chelating agents (Wang et al., 2000b). Treatment o f human bronchial epithelial cell layers with either water, or the calcium chelating agent Ethylene glycol-bis ({3-aminoethyl ether)-N,N,N’,N’-tetraacteic acid (EGTA), can lead to increased transepithelial permeability in less than 60 seconds. Transduction efficiency o f amphotrophic viruses in these cell layers was significantly enhanced following treatment with either water or EGTA, and remained receptor dependent (Wang et ah, 1998). These methods o f increasing transepithelial permeability have also lead to increased gene transfer in vivo (Wang et ah, 2000b), and naked DNA dissolved in water, instilled intratracheally, has been shown to give higher gene expression than when dissolved in isotonic saline (Sawa et ah, 1996). Although deionised water may increase gene expression by increasing transepithelial permeability, it will also affect the physico-chemical properties o f non-viral gene delivery systems.

1.4.8) Effect o f physical characteristics o f non-viral vectors on gene transfer

The size and charge o f non-viral complexes can be affected in different ionic environments. How this affects transfection efficiency is currently poorly understood.

This is partly because in vitro studies evaluating non-viral vectors depend on the cell type and vector being studied with few general rules emerging, although it is accepted that transfection is less efficient in the presence o f serum (Scheule and Cheng, 1998). The surface charge o f cationic liposome - DNA complexes, measured by zeta potential, is dependent on the complex buffer. When DC-Chol/DOPE, a cationic liposome, was complexed with DNA in water, the surface charge was proportional to the lipid: DNA ratio, becoming negative at a lipid: DNA ratio o f 4: 1. Whereas in DMEM, the zeta potential o f the complex was negative immediately on addition o f DNA, and did not alter at any lipid: DNA ratio (Son et al., 2000). Increasing ionic strength, and in particular multivalent anions, facilitates lipid fusion (Monkkonen and Urtti, 1998) and when preformed lipid-DNA complexes are added to cell culture medium their size distribution changes with complex fusion taking place (Jaaskelainen et al., 1994). Lipid aggregation is also seen when cationic liposomes are administered intravenously (Mahato et al., 1998). However, whether these changes are important is uncertain, as there does not appear to be a direct relationship between size, or charge ratio, and in vitro transfection efficiency o f either lipofectin or DOTAP (Stegmann and Legendre, 1997). However, the size and charge characteristics may determine the distribution and duration o f gene expression following intravenous delivery o f liposomes (Mahato et al., 1998). Non-viral lipopolyplex complexes may also aggregate in the presence o f serum (Li et al., 1998). Aggregation is also observed in physiological buffers and in vitro gene expression, using either transferrin conjugated or unmodified PEI complexes, is greater compared with transfection in hypotonic buffers where complex size is smaller and aggregation inhibited (Ogris et al., 1998). There is little data assessing whether physico-chemical

characteristics o f receptor mediated systems affects in vivo transfection, but would appear to suggest that smaller complexes are optimal. Perales and Ferkol have both reported successful targeting o f lOnm and 25nm complexes respectively, and suggesting an optimal ratio o f 1:0.7 DNA to polylysine giving an overall negative charge ratio (Perales et ah, 1994; Ferkol et ah, 1995). Thus, when considering strategies for receptor-mediated gene transfer to the lung, there are a number o f features worthy o f consideration in an attempt to optimise levels o f gene expression whilst minimising the risks o f adverse effects. Clearly, the choice o f receptor ligand is crucial. Having determined this, the overall characteristics o f the complex particularly the charge ratio o f the complex and the size and surface charge o f the complex are factors that may affect levels o f transfection and pulmonary toxicity. By investigating these parameters it may be possible to design a complex that is suitable for the particular needs o f the disease to be treated, in this case pulmonary fibrosis.

1.4.9) Summary

At the present time there is no effective treatment for pulmonary fibrosis. COX-2 is a molecule that may well be important in preventing the abnormal response to lung injury that occurs in this condition. It is an intracellular molecule and therefore can not be administered, either locally or systemically, as a recombinant protein. Gene therapy offers the potential to deliver the gene encoding COX-2 directly to the cells o f the lung, the organ affected in pulmonary fibrosis. The major limitations o f gene therapy relate to problems associated with delivery o f the gene to the cell. Viruses are efficient, but lead to inflammation and immune responses that can be fatal. This limits the number o f times the

gene can be administered, whereas non viral systems have yet to be developed with transfection efficiencies that make them practical for therapeutic purposes. By exploiting receptor mediated cell entry, modification o f non-viral systems may enable more efficient delivery systems, with minimal adverse effects, to be developed.

Section 5