Felix H. Salazar

Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways

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.

Inhibition of hepatitis B virus in mice by RNA interference

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.

In vivo antiviral efficacy of prenylation inhibitors against hepatitis delta virus

Hepatitis delta virus (HDV) can dramatically worsen liver disease in patients coinfected with hepatitis B virus (HBV). No effective medical therapy exists for HDV. The HDV envelope requires HBV surface antigen proteins provided by HBV. Once inside a cell, however, HDV can replicate its genome in the absence of any HBV gene products. In vitro, HDV virion assembly is critically dependent on prenyl lipid modification, or prenylation, of its nucleocapsid-like protein large delta antigen. To overcome limitations of current animal models and to test the hypothesis that pharmacologic prenylation inhibition can prevent the production of HDV virions in vivo, we established a convenient mouse-based model of HDV infection capable of yielding viremia. Such mice were then treated with the prenylation inhibitors FTI-277 and FTI-2153. Both agents were highly effective at clearing HDV viremia. As expected, HDV inhibition exhibited duration-of-treatment dependence. These results provide the first preclinical data supporting the in vivo efficacy of prenylation inhibition as a novel antiviral therapy with potential application to HDV and a wide variety of other viruses.

Controlling HBV Replication in Vivo by Intravenous Administration of Triggered PEGylated siRNA-Nanoparticles

Harnessing RNA interference (RNAi) to inhibit hepatitis B virus (HBV) gene expression has promising application to therapy. Here we describe a new hepatotropic nontoxic lipid-based vector system that is used to deliver chemically unmodified small interfering RNA (siRNA) sequences to the liver. Anti HBV formulations were generated by condensation of siRNA (A component) with cationic liposomes (B component) to form AB core particles. These core particles incorporate an aminoxy cholesteryl lipid for convenient surface postcoupling of polyethylene glycol (PEG; C component, stealth/biocompatibility polymer) to give triggered PEGylated siRNA-nanoparticles (also known as siRNA-ABC nanoparticles) with uniform small sizes of 80-100 nm in diameter. The oxime linkage that results from PEG coupling is pH sensitive and was included to facilitate acidic pH-triggered release of nucleic acids from endosomes. Nanoparticle-mediated siRNA delivery results in HBV replication knockdown in cell culture and in murine hydrodynamic injection models in vivo. Furthermore repeated systemic administration of triggered PEGylated siRNA-nanoparticles to HBV transgenic mice results in the suppression of markers of HBV replication by up to 3-fold relative to controls over a 28 day period. This compares favorably to silencing effects seen during lamivudine treatment. Collectively these observations indicate that our PEGylated siRNA-nanoparticles may have valuable applications in RNAi-based HBV therapy.

Potent efficacy of entecavir (BMS-200475) in a Duck model of hepatitis B virus replication

ABSTRACT The ability of entecavir (ETV) to inhibit Duck hepatitis B virus (DHBV) infection in duck hepatocytes and ducklings was examined using lamivudine (3TC) as a comparator drug. ETV exhibited antiviral activity (50% effective concentration [EC50], 0.13 nM) in DHBV-infected duck hepatocytes that was >1,000-fold more potent than that of 3TC (EC50, 138 nM). A 21-day treatment of ducklings with 1 mg of ETV per kg of body weight per day by oral gavage resulted in a mean reduction of log10 3.1 in serum DHBV DNA levels. Daily treatment with 0.1 mg of ETV/kg was nearly as effective, achieving an average viral DNA level decrease of log10 2.1. Reducing the daily dose of ETV to only 0.01 mg/kg resulted in an average viral DNA level decrease of log10 0.97. Daily treatment with 25 mg of 3TC/kg resulted in an average viral DNA level decrease of log10 0.66, compared to the log10 0.20 drop seen for ducklings given the vehicle alone. ETV was also more effective in decreasing the DHBV DNA levels in duck livers after 21 days of treatment, causing average drops of log10 1.41, log10 0.76, and log10 0.26 for dose levels of 1.0, 0.1, and 0.01 mg/kg, respectively, compared to a decrease of log10 0.06 for 3TC at a dose level of 25 mg/kg. Levels of viral covalently closed circular DNA in the treatment group receiving 1 mg of ETV/kg were reduced compared to those in the vehicle-treated group. ETV and 3TC were both well tolerated in all treated animals. These results show that ETV is a highly potent and effective antiviral in the DHBV duck model.

Exploring gene-deleted adenoviral vectors for delivery of short hairpin RNAs and reduction of hepatitis B virus infection in mice

RNA interference based therapeutic approaches hold promise for the treatment of patients chronically infected with hepatitis B virus (HBV). To conquer HBV infection, long‐term suppression of target transcripts in all hepatocytes without toxic effects may be required. The present study explored gene‐deleted adenoviral vectors (GD‐AdV) lacking all viral coding sequences for delivery of the previously described short hairpin RNA (shRNA) HBVU6no.2, which was demonstrated to result in post‐transcriptional knock‐down of HBV transcripts.

Inhibition of hepatitis B virus replication in vivo using lipoplexes containing altritol-modified antiviral siRNAs

Chronic infection with the hepatitis B virus (HBV) occurs in approximately 6% of the worlds population and carriers of the virus are at risk for complicating hepatocellular carcinoma. Current treatment options have limited efficacy and chronic HBV infection is likely to remain a significant global medical problem for many years to come. Silencing HBV gene expression by harnessing RNA interference (RNAi) presents an attractive option for development of novel and effective anti HBV agents. However, despite significant and rapid progress, further refinement of existing technologies is necessary before clinical application of RNAi-based HBV therapies is realised. Limiting off target effects, improvement of delivery efficiency, dose regulation, and preventing reactivation of viral replication are some of the hurdles that need to be overcome. To address this we assessed the usefulness of the recently described class of altritol-containing synthetic siRNAs (ANA siRNAs), which were administered as lipoplexes and tested in vivo in a stringent HBV transgenic mouse model. Our observations show that ANA siRNAs are capable of silencing of HBV replication in vivo. Importantly, non specific immunostimulation was observed with unmodified siRNAs, and this undesirable effect was significantly attenuated by ANA modification. Inhibition of HBV replication of approximately 50% was achieved without evidence for induction of toxicity. These results augur well for future application of ANA siRNA therapeutic lipoplexes.

441. Short Hairpin RNA Interference Expressed from a Gene-Deleted Adenoviral Vector Results in Reduction of Hepatits B Surface Antigen Levels In Vitro and in a Small Animal Model for Hepatitis B Infection

Top of pageAbstract There is substantial need for new treatment strategies for chronically hepatitis B virus (HBV) infected patients. This study explores the rational design of novel adenoviral gene therapy vectors for efficient delivery of RNA interference (RNAi) inhibitor molecules against HBV. Over the recent years various viral and non-viral vectors have been studied for delivery of short hairpin RNAi (shRNAi) expression cassettes but the in vitro and in vivo efficacy of significantly safer gene-deleted adenoviral vectors (HD vectors) for delivery of shRNAi remains to be tested. To show in vivo efficacy regular FVB mice (n=3 per group) were co-injected with a HD vector with a shRNAi expression cassette against luciferase and a second adenoviral vector expressing luciferase. We found up to 96% reduction of luciferase expression seven days post-injection. Our laboratory previously demonstrated that a 25-mer shRNAi against HBV (HBVU6no.2) driven by a polymerase III promoter was sufficient to inhibit HBV in a non-viral gene therapy approach in vivo (McCaffrey et al., Nat Biotechnol 21:639-644, 2003) but was lethal in the context of a novel self-complementery adeno-associated viral vector (AAV) in HBV transgenic and normal mice (Grimm et al., 2004). In this study we created a gene-deleted adenoviral vector with the same shRNAi expression cassette (FTC/HBVU6no.2). In cells stably transfected with the HBV genome we observed up to 60% reduction of secreted surface antigen (sAg) levels, a marker for progression of HBV infection. In a further step we transfused a plasmid containing the HBV-genome into mouse liver of C57Bl/6 mice (n=4 per group) by high pressure tail vein injection followed by injection of 5|[times]|10e9 transducing units of the HD vector FTC/HBVU6no.2. This viral dose was sufficient to transduce 100% of all hepatocytes. We observed a 70% drop in serum sAg levels. Control mice which received only the HBV containing plasmid or a HD vector with a beta-galactosidase (|[beta]|-Gal) expression cassette showed a 29% and a 73% drop in serum sAg levels, respectively. HBV transgenic mice (n=4 per group) were either injected with 5|[times]|10e9 transducing units of the HD vector FTC/HBVU6no.2 or a |[beta]|-Gal expressing HD vector. After a transient increase in sAg levels in both groups, we observed an up to 45% and 73% reduction in serum sAg levels compared to the vehicle control group, respectively. Limited toxicity studies were performed and highest liver enzyme levels were measured 20 days post-injection which returned to normal levels 40 days post-injection for the FTC/HBVU6no.2 but not the |[beta]|-Gal group. We speculate that an immune response against the transgene |[beta]|-Gal is responsible for the significant drop in serum sAg levels and the sustained increased liver enzyme levels. In summary, this study demonstrates that gene-deleted adenoviral vectors can be used to deliver small inhibitor molecules in vivo and with further improvements these vectors will play an important role in gene therapy approaches based on shRNAi.