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Aim: To test the hypothesis that enhancement of the activity of heme oxygenase can interfere with processes of fibrogenesis associated with recurrent liver injury, we investigated the therapeutic potential of over-expression of heme oxygense-1 in a CCl(4)-induced micronodular cirrhosis model., Methods: Recombinant adeno-associated viruses carrying rat HO-1 or GFP gene were generated. 1x 10(12) vg of adeno-associated viruses were administered through portal injection at the time of the induction of liver fibrosis., Results: Conditioning the rat liver with over-expression of HO-1 by rAAV/HO-1 significantly increased the HO enzymatic activities in a stable manner. The development of micronodular cirrhosis was significantly inhibited in rAAV/HO-1-transduced animals as compared to controls. Portal hypertension was markedly diminished in rAAV/HO-1-transduced animals as compared to controls, whereas there are no significant changes in systolic blood pressure. This finding was accompanied with improved liver biochemistry, less infiltrating macrophages and less activated hepatic stellate cells (HSCs) in rAAV/HO-1-transduced livers., Conclusion: Enhancement of HO activity in the livers suppresses the development of cirrhosis.

Liver fibrosis is the consequence of activation of hepatic stellate cells mediated by persistent or recurrent liver injury, where oxidative stress or inflammatory response resulting from immune cells and cytokines are involved. Targeting of hepatic stellate cells could be an important strategy for the therapy of liver fibrosis. In this study, we showed a tropism of recombinant adeno-associated virus (rAAV, serotype 2) with high efficiency in transduction of a homeostatic gene, heme oxygenase-1 (HO-1), to activated stellate cells. The binding of rAAVs to stellate cells increased significantly after serum-stimulated activation compared with quiescent status. Portal injection of rAAVs to normal or carbon tetrachloride (CCl(4))-induced liver fibrosis showed a distinct distribution of rAAV binding. The majority of injected rAAVs bound to the cells in fibrotic areas that were associated with higher expression levels of fibroblast growth factor receptor-1alpha at 2 hours after administration. Isolation of different types of cells from CCl(4)-induced fibrotic livers showed predominant expression of transgene in stellate cells after rAAV/HO-1 administration on day 3 and remained stable for 12 weeks. In addition, HO-1-transduced stellate cells showed reduced transcript levels of type 1 collagen and impaired proliferative ability compared with controls. With this approach, the severity of established micronodular cirrhosis was markedly reduced. In conclusion, these findings suggest a new approach for the treatment of liver fibrosis using adeno-associated virus-mediated gene transfer.

Background: Allograft deterioration is the major obstacle to organ transplantation as a long-term treatment of end-stage heart failure. In this study, we transduced the antioxidant gene, heme oxygenase-1 (HO-1), to heart grafts using a recombinant adeno-associated viral vector (rAAV) in a rat heart transplantation model and investigated its potentiality in prevention of chronic graft deterioration., Methods and Results: rAAV/HO-1 was administered to heart grafts through the coronary arteries during cold preservation. We investigated the expression patterns and activities of transgene, graft survival, graft histomorphology, and relevance of HO-1 expression on graft survival and chronic graft deterioration by itself. Long-term allograft survival can be achieved by rAAV/HO-1-mediated stable transgene expression. The development of graft arteriosclerosis and interstitial fibrosis was prevented in rAAV/HO-1-transduced allografts on day 100. rAAV/HO-1-mediated long-term graft protection was accompanied by remarkable downregulation of the intragraft mRNA level of macrophage migration inhibitory factor, tumor necrosis factor-alpha, and transforming growth factor-beta1. Blockage of HO activities by zinc protoporphyrin IX at different posttransplant phases showed that the stable expression of HO-1 is a prerequisite for both survival of grafts and prevention of graft arteriosclerosis., Conclusions: rAAV/HO-1 gene transfer represents a novel therapeutic approach to prevent chronic allograft deterioration in clinical heart transplantation.

Recombinant adeno-associated virus vector (rAAV) is an effective and safe gene-delivery tool. However, its application in solid-organ transplantation has not been addressed. The present study is designed to introduce human cytotoxic T-lymphocyte-associated antigen 4 immunoglobulin G (hCTLA4Ig) by rAAV (rAAV-hCTLA4Ig) into rat liver grafts to analyze the effects of virus titer, exposure time, and route of administration on transgene expression and possible side effects caused by the gene-delivery approach. Different rAAV-hCTLA4Ig titers were introduced into liver grafts through back-table portal vein perfusion and preserved for a certain time. rAAV-hCTLA4Ig also was administered by intravenous and intramuscular injection. Transgene expression in grafts and plasma was detected by immunohistochemistry and enzyme-linked immunosorbent assay. Intragraft cytokine level was detected by reverse-transcriptase polymerase chain reaction. Anti-hCTLA4Ig antibodies in plasma were detected by flow cytometry. A higher virus titer (1 x 10(12) viral genomes/animal) introduced through back-table portal vein perfusion and a longer preservation time (3 hours) achieved a greater level of transgene expression until day 180. Back-table portal vein perfusion induced a greater level of hCTLA4 expression in plasma than intramuscular or intravenous injection. Increased interleukin-2 and interferon-gamma messenger RNA levels were detected in grafts with rAAV-hCTLA4Ig gene transfer compared with those without virus delivery, but the response was minor. Such a cellular immune response could be suppressed by low-dose FK506 administration during the first 3 postoperative days. Anti-hCTLA4Ig antibodies could be detected in long-term surviving animals, but the extent of humoral response was not severe. This study shows that rAAV can be an effective and safe vector for gene delivery in liver transplantation.