On the basis of the significant roles of TGFβ and Smad proteins in regulating collagen expression, there have been several studies attempting to block the TGFβ/Smad signal and suppress organ fibrosis by using the adenoviral gene transfer techniques. One of them blocked the TGFβ signalling by expressing a dominant‐negative TGFβ type II receptor, and prevented liver fibrosis and dysfunction in dimethylnitrosamine‐treated rats.69
It also prevented the progression of pre‐existing liver fibrosis and enhanced hepatocyte regeneration.70
Similarly, a soluble form of TGFβ type II receptor consisting of only the extracellular domain suppressed experimental liver fibrosis induced by common bile duct ligation71
or dimethylnitrosamine administration.72
Others injected a recombinant adenovirus carrying inhibitory Smad7 cDNA into rats and succeeded in treating liver fibrosis induced by common bile duct ligation.73
All of these adenoviral gene treatments have a great potential in treating liver fibrosis in humans. However, there are several concerns arising before their clinical application. Firstly, it should be seriously considered that blocking of the TGFβ/Smad signal may result in the promotion of carcinogenesis. As already described, TGFβ has an antiproliferative effect on most epithelial types of cells including parenchymal hepatocytes, and Smad4 was originally identified as the product of a tumour suppressor gene. It is especially important in the case of advanced liver fibrosis in humans, as most hepatocellular carcinomas originate from the underlying cirrhotic liver tissue. Owing to its critical roles in immune suppression, generalised blockade of TGFβ activity may also lead to excessive immune reactions and extensive inflammation. In relation to the first problem, the second concern is the limitation in tissue specificity and cell type specificity of gene delivery. All of the above experiments used strong enhancer/promoter elements such as the one derived from cytomegalovirus. These strong expression units exhibit no tissue specificity, which may cause severe adverse effects in the non‐target organs. Considering that collagens play critical parts in the maintenance of organ architecture and tissue integrity as well as in various physiological processes, it is more logical to use a tissue‐specific enhancer/promoter sequence for the fibrotic tissue‐specific regulation of collagen metabolism.
Several promoter sequences have been tested for their ability to mediate cell‐specific expression. One of them, the promoter of tissue inhibitor of metalloproteinase 1 gene, which is activated during hepatic fibrogenesis, induced cell death in culture activated HSC by expressing the herpes simplex virus thymidine kinase.74
We have recently shown in vivo the cell type‐specific gene expression by using a recombinant adenovirus carrying the −17 kilobase tissue‐specific COL1A2
Under the control of this COL1A2
enhancer, enhanced green fluorescent protein was expressed only in CCl4
‐treated liver but not in untreated normal liver. It was expressed mainly in activated HSC in the fibrotic liver, but not in any other organs including kidney, lung and skin (fig 5). Furthermore, adenovirus‐mediated overexpression of YB‐1 driven by the COL1A2
enhancer inhibited COL1A2
promoter activation after CCl4
injections, and subsequently suppressed the progression of liver fibrosis.75
These results validate a new concept of the treatment for liver fibrosis by suppressing excessive collagen deposition in fibrotic tissue without affecting non‐target normal organs.
Figure 5Cell type‐specific gene expression in the fibrotic liver tissue by using a tissue‐specific ×2(I) collagen gene enhancer sequence.75 When using the CAG expression unit as a control, enhanced green fluorescent protein (more ...)
On the other hand, the adenoviral system still has several limitations and problems, despite the cell type‐specific gene expression described above. Firstly, adenovirus is immunogenic and rapidly induces neutralising antibodies. Thus, it is not suitable for repeated injections in vivo. Secondly, the virus has dose‐related cytotoxicity, which raises safety concerns on clinical application. From this point of view, development of better delivery systems is needed to treat chronic diseases such as organ fibrosis more effectively. Another attempt for cell type‐specific fibrosis treatment uses modified human albumin as a selective carrier of antifibrotic reagents. For example, mannose‐6‐phosphate76
and a PDGF receptor‐recognising macromolecule77
have been tested for their binding capacity, specifically to activated HSC.