RNA interference (RNAi) is a universal and evolutionarily conserved phenomenon of post-transcriptional gene silencing by means of sequence-specific mRNA degradation, triggered by small double-stranded RNAs. Because this mechanism can be efficiently induced in vivo by expressing target-complementary short hairpin RNA (shRNA) from non-viral and viral vectors, RNAi is attractive for functional genomics and human therapeutics. Here we systematically investigate the long-term effects of sustained high-level shRNA expression in livers of adult mice. Robust shRNA expression in all the hepatocytes after intravenous infusion was achieved with an optimized shRNA delivery vector based on duplex-DNA-containing adeno-associated virus type 8 (AAV8). An evaluation of 49 distinct AAV/shRNA vectors, unique in length and sequence and directed against six targets, showed that 36 resulted in dose-dependent liver injury, with 23 ultimately causing death. Morbidity was associated with the downregulation of liver-derived microRNAs (miRNAs), indicating possible competition of the latter with shRNAs for limiting cellular factors required for the processing of various small RNAs. In vitro and in vivo shRNA transfection studies implied that one such factor, shared by the shRNA/miRNA pathways and readily saturated, is the nuclear karyopherin exportin-5. Our findings have fundamental consequences for future RNAi-based strategies in animals and humans, because controlling intracellular shRNA expression levels will be imperative. However, the risk of oversaturating endogenous small RNA pathways can be minimized by optimizing shRNA dose and sequence, as exemplified here by our report of persistent and therapeutic RNAi against human hepatitis B virus in vivo.

Hepatitis B virus (HBV) infection substantially increases the risk of chronic liver disease and hepatocellular carcinoma in humans. RNA interference (RNAi) of virus-specific genes has emerged as a potential antiviral mechanism. Here we show that RNAi can be applied to inhibit production of HBV replicative intermediates in cell culture and in immunocompetent and immunodeficient mice transfected with an HBV plasmid. Cotransfection with plasmids expressing short hairpin RNAs (shRNAs) homologous to HBV mRNAs induced an RNAi response. Northern and Southern analyses of mouse liver RNA and DNA showed substantially reduced levels of HBV RNAs and replicated HBV genomes upon RNAi treatment. Secreted HBV surface antigen (HBsAg) was reduced by 94.2% in cell culture and 84.5% in mouse serum, whereas immunohistochemical detection of HBV core antigen (HBcAg) revealed >99% reduction in stained hepatocytes upon RNAi treatment. Thus, RNAi effectively inhibited replication initiation in cultured cells and mammalian liver, showing that such an approach could be useful in the treatment of viral diseases.

A virus given the name ground squirrel hepatitis virus (or GSHV), with many of the unique characteristics of human hepatitis B virus (HBV), has been found in Beechey ground squirrels in northern California. (2), the viral DNA (3), a DNA polymerase activity (4, 5), and probably hepatitis B e antigen (HBeAg) in a cryptic form (6). No crossreaction has been found between HBsAg and a large number of antigens of other viruses that have been tested (7). HBcAg and HBeAg were also considered unique to HBV.The viral DNA is circular and has a contour length consistent with that of double-stranded DNA of approximately 3200 base pairs (bp) (3). However, as isolated from virions, the DNA was not completely double stranded. Between 15% and 50% of the length of different molecules was single stranded (8-10). A DNA polymerase activity in the viral core catalyzed a repair reaction that converted the DNA to fully double-stranded circular molecules of approximately 3200 bp (8-10).Persistent infection with HBV is common, continues for years (11), and is accompanied by continuous circulation in the blood of high concentrations of incomplete viral forms consisting of small spherical HBsAg particles 16-25 nm in diameter and long filamentous HBsAg containing structures approximately 22 nm in width and up to 500 nm in length (1, 2, 12) as well as complete virions usually in much lower concentrations. Persistent infection in humans is associated with chronic hepatitis (13) Gerin, personal communication).Here we describe a virus found in ground squirrels in one region of northern California that is similar to HBV and WHV and may represent a third member of this class of viruses.MATERIALS AND METHODS Sera. Sera were obtained from all animals by cardiac puncture. Apparently healthy Beechey ground squirrels (Spermophilus beecheyi) were obtained from Point Lobos, CA (location A), a region adjacent to the Stanford University campus (location B), and a region near Santa Barbara, CA (location C). Sera containing high concentrations of HBV were obtained by plasmapheresis of a persistently infected patient after informed consent was obtained.Assay for Virion DNA Polymerase Activity. Particle-associated DNA polymerase levels in sera were determined by a modification of the method previously described (19). Serum samples were centrifuged in an Eppendorf centrifuge at 10,000 rpm for 10 min to remove precipitated protein and other debris; 200 Ml of each supernatant was layered over 500 ,ul of 30% sucrose (wt/vol) containing 10 mM Tris-HCI, 0.1 M NaCI, 5 mM EDTA, 0.1% 2-mercaptoethanol, and 1 mg of centrifuged bovine serum albumin per ml in 1-ml polycarbonate tubes and particles were pelleted in a Spinco type 25 rotor for 16 hr at 25,000 rpm and 4°C. Supernatants were removed by suction and discarded, and inner walls of the tubes were wiped dry. The pellets were resuspended in 50 ,l of 1.5% Nonidet P-40/0.1% 2-mercaptoethanol/10 mM Tris-HCI, pH 7.5/0.1 M NaCl. Then 25 ,ul of 0.2 M Tris-HCl (pH 7.5), 80 The publication costs of this article...

Persistence of hepatocytes transplanted into the same or related species has been established. The long-term engraftment of human hepatocytes into rodents would be useful for the study of human viral hepatitis, where it might allow the species, technical and size limitations of the current animal models to be overcome. Although transgenic mice expressing the hepatitis B virus (HBV) genome produce infectious virus in their serum, the viral life cycle is not complete, in that the early stages of viral binding and entry into hepatocytes and production of an episomal transcriptional DNA template do not occur. As for hepatitis delta virus (HDV), another cause of liver disease, no effective therapy exists to eradicate infection, and it remains resistant even to recent regimens that have considerably changed the treatment of HBV (ref. 13). Here, we demonstrate long-term engraftment of primary human hepatocytes transplanted in a matrix under the kidney capsule of mice with administration of an agonistic antibody against c-Met. These mice were susceptible to HBV infection and completion of the viral life cycle. In addition, we demonstrate super-infection of the HBV-infected mice with HDV. Our results describe a new xenotransplant model that allows study of multiple aspects of human hepatitis viral infections, and may enhance studies of human liver diseases.

A unique series of simple "unnatural" nucleosides has been discovered to inhibit hepatitis B virus (HBV) replication. Through structure-activity analysis it was found that the 3-OH group of the ␤-L-2-deoxyribose of the ␤-L-2-deoxynucleoside confers specific antihepadnavirus activity. The unsubstituted nucleosides ␤-L-2-deoxycytidine, ␤-L-thymidine, and ␤-L-2-deoxyadenosine had the most potent, selective, and specific antiviral activity against HBV replication. Human DNA polymerases (␣, ␤, and ␥) and mitochondrial function were not affected. In the woodchuck model of chronic HBV infection, viral load was reduced by as much as 10 8 genome equivalents/ml of serum and there was no drug-related toxicity. In addition, the decline in woodchuck hepatitis virus surface antigen paralleled the decrease in viral load. These investigational drugs, used alone or in combination, are expected to offer new therapeutic options for patients with chronic HBV infection.Infection with hepatitis B virus (HBV) is a major world health problem, affecting 5% of the population. More than 2 billion people have been infected with the virus, and 350 million of them are chronic carriers at risk of death from cirrhosis and liver cancer (49).Several strategies have been evaluated for the treatment of chronic HBV infection with the goal of eliminating persistent viral replication and preventing progression to chronic active hepatitis and liver failure. Currently, the only approved treatment options are alpha interferon (IFN) and lamivudine (␤-L-2Ј,3Ј-dideoxy-3Ј-thiacytidine [3TC]). Unfortunately, the rate of response to IFN is low, and drug-associated side effects are significant (24,55). Individuals who are immunosuppressed (e.g., transplant recipients or those coinfected with the human immunodeficiency virus [HIV]) rarely respond to IFN therapy (13). Lamivudine is a well-known example of the class of ␤-Lnucleoside analogs that has recently drawn attention as antiviral and anticancer agents (52). As with IFN, however, a complete antiviral response, as assessed by HBe seroconversion, is seen in only a minority of patients after 1 year of treatment (27). In addition, cessation of lamivudine therapy or development of viral resistance may lead to a marked rebound in viral replication which can be life threatening (hepatitis flare) in HIV-HBV-coinfected patients (2, 30). Lamivudine resistance is now recognized in 16 to 32% of HBV-infected patients after 1 year of treatment and in as many as 58% after 2 to 3 years (14,27,30).Since the Food and Drug Administration approved lamivudine for the treatment of HIV infection in the United States in 1996 and for HBV in 1998, intensive studies on "unnatural" L-nucleosides as agents against HIV, HBV, and herpesviruses (including Epstein-Barr virus [EBV]) and as anticancer agents have been conducted (23). Now, through an extensive structure-activity analysis, we have found that the 3Ј-OH group of the ␤-L-2Ј-deoxyribose of the ␤-L-2Ј-deoxynucleoside series confers unique specificity for anti-HBV activity. In this ...