As the first demonstrations of a therapeutic application of siRNA in vivo were done in mouse models of acute liver failure (ALF), the next paragraphs will give a brief introduction into this disease, followed by a discussion of the therapeutic use of siRNA to suppress apoptosis in this model.
ALF is defined as a dramatic clinical syndrome in which a previously normal liver fails within days or weeks. Three subgroups of ALF can be distinguished, hyperacute, acute, and subacute liver failure. Despite the frequent occurrence of cerebral edema and renal failure in patients with hyperacute liver failure, prognosis without transplantation is relatively good. Survival rates in patients with acute and subacute liver failure, however, are at best 15% (20,21). The etiology of ALF shows marked worldwide variation: in underdeveloped countries viral causes predominate, whereas drug-induced hepatotoxicity and seronegative hepatitis predominate in most countries of the Western world (22). To this day, the management of these varying clinical scenarios is essentially supportive. It aims to identify and remove the insult that led to destruction of the liver, whereas preventing associated complications, such as acute renal and respiratory failure, bleeding diatheses, severe sepsis, cerebral edema, and encephalopathy. Overall mortality in patients with severe ALF remains high, ranging from 40 to 80%. Although liver support devices or hepatocyte transplantation may in time have a place in treatment, currently liver transplantation remains the only therapeutic option that has been shown to significantly improve the outcome of patients with ALF. Because of limitations of donor organs and the requirement of lifelong and life-limiting immunosuppression, liver transplantation should be only performed in patients who are unlikely to recover from ALF. In patients who recover from ALF with medical support, the liver almost always returns to normal, both structurally and functionally. Consequently, prevention of destruction of liver cells in the time-course of ALF and support of liver regeneration are the most important goals in management of ALF by molecular therapies in the future.
Several molecular mechanisms can initiate liver cell injury and can further aggravate ongoing damage processes (23). Mitochondria are the prominent targets for hepatotoxicity of many drugs, leading to impairment of energy metabolism and intracellular oxidative stress. Once hepatocellular function is impaired, accumulation of hydrophobic bile acids causes additional cyto-toxicity. Although drug-induced hepatotoxicity appears to be mediated by both apoptosis and necrosis, viral infection predominantly induces cell death of hepatocytes by apoptosis. In contrast to necrosis, apoptosis is a highly conserved physiological process important in normal development and tissue homeostasis of multicellular organisms. Apoptosis occurs by two pathways: a death receptor pathway and a mitochondrial pathway. Signals released from the cytoplasm and/or from the cell membrane activate a well-characterized cascade of caspases (cysteine aspartase), which execute apoptotic cell death (24-27). Receptor-mediated apoptosis, as triggered by the tumor necrosis factor-R, Fas-, or TNF-related apoptosis-inducing ligand (TRAIL)-receptor 1, has been reported to be involved in the pathogenesis of different liver diseases like viral hepatitis, ALF, autoimmune hepatitis, ischemia-reperfusion injury, nonalcoholic steatohepatitis, and toxic liver damage like Wilson's disease or bile acid-induced hepatotoxicity (28-33). Therefore, the apoptotic pathway provides attractive targets for molecular therapy to prevent further liver damage and provides a condition for successful liver regeneration in ALF.
Recently, Song et al. (16) and our group (34) reported the therapeutic use of siRNA in mouse models of ALF. Song et al. used siRNA duplexes targeting the Fas (CD95) receptor. Three consecutive applications of Fas-siRNA led to an uptake of siRNA in more than 80% of all hepatocytes resulting in an 8- to 10-fold downregulation of Fas mRNA expression in the liver. To note, comparable downregulation of Fas had been shown before using ASO (35), however, this required treating the mice with an approx 14-fold higher amount of anti-Fas ASO (6 mg/kg body weight for 12 consecutive days). In accordance with the results from Zhang et al. (35), inhibition of Fas expression by siRNA protected the hepatocytes against treatment with the Fas-activating antibody Jo-2
and resulted in significantly increased survival. Remarkably, Fas-siRNA also conferred protection against ConA-mediated acute liver damage, whereas Fasantisense did not.
As it is well established that in addition to FasL, tumor necrosis factor a and TRAIL are also involved in the pathogenesis of viral hepatitis (36,37), we reasoned that an essential early downstream mediator of all death receptors would be the most suitable target to achieve the best therapeutic effects in preclinical animal models of viral hepatitis and ALF. To test this, we directed siRNA against caspase-8, which is a key downstream effector in receptor-mediated apoptosis. A single dose of 0.45-0.6 nmol/g body weight of caspase-8-siRNA resulted in very effective inhibition of caspase-8 expression in the liver, thus leading to protection against Jo-2-mediated liver damage or liver damage induced by an adenovirus overexpressing Fas ligand (Ad-FasL). With regard to potential clinical applications, it is noteworthy that caspase-8-siRNA not only prevented acute liver damage but was also highly effective when delivered into an ongoing ALF. Furthermore, it is of particular interest that in our study the therapeutic efficiency of caspase-8-siRNA was shown in acute viral hepatitis that was triggered by wild-type adenovirus, which better resembles the multiple molecular events in human acute hepatitis.
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