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Inhibition of HBV replication by RIG-I stimulation with 5`-triphosphated siRNA in vitro and in vivo.

Köln, Universität zu Köln, Mathematisch-Naturwissenschaftliche Fakultät, Diss., 2009, 171 S.
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For a proportion of patients, HBV infection results in chronic disease with severe consequences like liver cirrhosis or hepatocellular carcinoma. Approved therapies of chronic hepatitis B include administration of antivirally active IFN-alpha and inhibitors of viral reverse transcription. These drugs control replication, but HBV cccDNA, the episomal transcription template, persists in most cases. Hence, lifelong therapy is required, accompanied by severe side effects and development of drug resistant mutants. An alternative and probably safe immunotherapeutic approach for clearance of HBV may be achieved by the stimulation of pattern-recognition receptors inducing an endogenous type-I IFN response. In this study the cytosolic helicase RIG-I was triggered by in vitro transcribed 5�-triphosphated (3p-) dsRNA. Stimulation induced an RIG-dependent antiviral type-I IFN response and controlled HBV replication in vitro in stable HBV replicating cell lines as well as in HBV infected primary human hepatocytes. In vivo, virus replication in HBV transgenic animals was transiently controlled when Rig I ligands were complexed and i.v. injected. To enhance and prolong the antiviral effect, siRNAs targeting overlapping open reading frames of the HBV genome at the 3´-end of multiple HBV-RNAs were designed and investigated whether they can be in vitro transcribed and act as RIG-I ligands, and thus combine the immune stimulatory potential with HBV specific gene silencing. 3p-siRNAs were transfected into HBV replicating cells, induced INF-I and IFN-stimulated genes. HBV replication markers were significantly reduced and the antiviral effect of 3p-siRNA was superior to siRNA or 3p-RNA alone. Stronger effects of 3p-siRNAs on HBV replication were confirmed in HBV infected primary human hepatocytes, as well as in vivo. Nevertheless, the gene silencing effect of 3p-siRNA seemed to be limited in the mouse model due to insufficient targeting of hepatocytes, the HBV host cells. Application of cholesterol coupled siRNA improved hepatocyte specific targeting. Furthermore, we showed that 3p-siRNA besides a direct antiviral effect additionally induced infiltration of cytotoxic CD8+ T cells to the liver, potentially leading to elimination of infected hepatocytes. The results of this study demonstrate that HBV-specific 5`-triphosphated siRNAs efficiently block HBV replication in vitro and in vivo. Combination of endogenous type-I IFN induction by stimulation of RIG-I with HBV sequence-specific gene silencing by RNAi in one single molecule could be a promising alternative to the actual standard therapeutic approaches against chronic HBV infection.
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Publikationstyp Sonstiges: Hochschulschrift
Typ der Hochschulschrift Dissertationsschrift
Quellenangaben Band: , Heft: , Seiten: 171 S. Artikelnummer: , Supplement: ,
Hochschule Universität zu Köln
Hochschulort Köln
Fakultät Mathematisch-Naturwissenschaftliche Fakultät