Carol Crowther

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.

Expressed anti-HBV primary microRNA shuttles inhibit viral replication efficiently in vitro and in vivo.

The use of RNA interference (RNAi) to inhibit gene expression is potentially applicable in the treatment of viral infections such as hepatitis B virus (HBV) persistence. Although efficient HBV gene silencing by short hairpin RNA (shRNA) expressed from RNA polymerase (Pol) III promoters has been reported, constitutive high-level transcription may cause harmful side effects. Here, we report an approach that allows the use of a Pol II promoter to improve transcription regulation of expressed RNAi effecters. Pol II [cytomegalovirus (CMV)] or Pol III (U6) promoter cassettes that transcribe anti-HBV primary microRNA (pri-miR)-122 and pri-miR-31 shuttles were generated. In cultured cells both types of pri-miR-like sequences effected knockdown of markers of viral replication (>80%) and were processed to form intended 21-nucleotide guides. The concentration of CMV-expressed miRs was approximately 85-fold lower than the U6 shRNA-derived guide RNA. When cells were co-transfected with pri-miR expression cassettes, attenuation of independent RNAi-mediated gene silencing was not observed, which is in contrast to the action of U6 shRNA expression cassettes. The efficacy of the anti-HBV pri-miR shuttles in vivo was verified using the murine hydrodynamic injection model. Employing Pol II-expressed pri-miR mimics may be useful in the treatment of HBV infection, and potentially also for generic application in RNAi-based therapy.

Therapeutic potential of adenoviral vectors for delivery of expressed RNAi activators.

Importance of the field: Harnessing RNA interference (RNAi) to silence pathology-causing genes has shown promise as a mode of therapy. The sustained gene inhibition that may be achieved with expressed sequences is potentially useful for treatment of chronic viral infections, but efficient and safe delivery of these sequences remains a challenge. It is generally recognized that there is no ideal vector for all therapeutic RNAi applications, but recombinant adenovirus vectors are well suited to hepatic delivery of expressed RNAi activators. Areas covered in this review: Adenoviruses are hepatotropic after systemic administration, and this is useful for delivering expressed RNAi activators that silence pathology-causing genes in the liver. However, drawbacks of adenoviruses are toxicity and diminished efficacy, which result from induction of innate and adaptive immune responses. In this review, the advantages and hurdles facing therapeutic application of adenoviral vectors for liver delivery of RNAi effectors are covered. What the reader will gain: Insights into adenovirus vectorology and the methods that have been used to make these vectors safer for advancing clinical application of RNAi-based therapy. Take home message: Adenoviruses are very powerful hepatotropic vectors. To make adenoviruses more effective for clinical use, polymer conjugation and deletion of viral vector sequences have been used successfully. However, further modifications to attenuate immunostimulation as well as improvements in large-scale production are necessary before the therapeutic potential of adenovirus-mediated delivery of RNAi activators is realized.

A valine to phenylalanine mutation in the precore region of hepatitis B virus causes intracellular retention and impaired secretion of HBe‐antigen

Aim:  Hepatitis B virus (HBV) e antigen (HBeAg) is translated from precore mRNA as a precore/core protein, which is post‐translationally modified to give rise to the protein that is secreted into the serum. The G1862T mutation in HBV occurs in the bulge of the encapsidation signal within the pregenomic RNA. When the precore mRNA is translated, this mutation results in a valine to phenylalanine substitution at the −3 position to the signal peptide cleavage site at the amino end of the precursor protein. The aim of this study was to determine whether this mutation could affect HBV replication and/or HBeAg expression.

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.

Inhibition of Hepatitis B Virus Replication by Helper Dependent Adenoviral Vectors Expressing Artificial Anti-HBV Pri-miRs from a Liver-Specific Promoter

Research on applying RNA interference (RNAi) to counter HBV replication has led to identification of potential therapeutic sequences. However, before clinical application liver-specific expression and efficient delivery of these sequences remain an important objective. We recently reported short-term inhibition of HBV replication in vivo by using helper dependent adenoviral vectors (HD Ads) expressing anti-HBV sequences from a constitutively active cytomegalovirus (CMV) promoter. To develop the use of liver-specific transcription regulatory elements we investigated the utility of the murine transthyretin (MTTR) promoter for expression of anti-HBV primary microRNAs (pri-miRs). HD Ads containing MTTR promoter effected superior expression of anti-HBV pri-miRs in mice compared to HD Ads containing the CMV promoter. MTTR-containing HD Ads resulted in HBV replication knockdown of up to 94% in mice. HD Ads expressing trimeric anti-HBV pri-miRs silenced HBV replication for 5 weeks. We previously showed that the product of the codelivered lacZ gene induces an immune response, and the duration of HBV silencing in vivo is likely to be attenuated by this effect. Nevertheless, expression of anti-HBV pri-miRs from MTTR promoter is well suited to countering HBV replication and development of HD Ads through attenuation of their immunostimulatory effects should advance their clinical utility.

Inhibition of HBV replication in vivo using helper-dependent adenovirus vectors to deliver antiviral RNA interference expression cassettes.

BACKGROUND HBV is hyperendemic to southern Africa and parts of Asia, but licensed antivirals have little effect on limiting life-threatening complications of the infection. Although RNA interference (RNAi)-based gene silencing has shown therapeutic potential, difficulties with delivery of anti-HBV RNAi effectors remain an obstacle to their clinical use. To address concerns about the transient nature of transgene expression and toxicity resulting from immunostimulation by recombinant adenovirus vectors (Ads), utility of RNAi-activating anti-HBV helper-dependent (HD) Ads were assessed in this study. METHODS Following intravenous administration of 5×10(9) unmodified or pegylated HD Ad infectious particles to HBV transgenic mice, HBV viral loads and serum HBV surface antigen levels were monitored for 12 weeks. Immunostimulation of HD Ads was assessed by measuring inflammatory cytokines, hepatic function and immune response to the co-delivered LacZ reporter gene. RESULTS Unmodified and pegylated HD Ads transduced 80-90% of hepatocytes and expressed short hairpin RNAs (shRNAs) were processed to generate intended HBV-targeting guides. Markers of HBV replication were decreased by approximately 95% and silencing was sustained for 8 weeks. Unmodified HD Ads induced release of proinflammatory cytokines and there was evidence of an adaptive immune response to β-galactosidase. However the HD Ad-induced innate immune response was minimal in preparations that were enriched with infectious particles. CONCLUSIONS HD Ads have potential utility for delivery of therapeutic HBV-silencing sequences and alterations of these vectors to attenuate their immune responses may further improve their efficacy.

EFFICIENT INHIBITION OF HEPATITIS B VIRUS REPLICATION IN VIVO USING PEG-MODIFIED ADENOVIRUS VECTORS

Achieving safe delivery of anti-hepatitis B virus (HBV) RNA interference (RNAi) effectors is an important objective of this gene-silencing technology. Adenoviruses (Ads) have a natural tropism for the liver after systemic administration, and are useful for delivery of expressed anti-HBV RNAi sequences. However, a drawback of Ad vectors is diminished efficacy and toxicity that results from stimulation of innate and adaptive immunity. To attenuate these effects we used monomethoxy polyethylene glycol-succinimidyl propionate (mPEG-SPA) to modify first-generation vectors that express an anti-HBV RNAi effector. Efficient hepatocyte transduction and knockdown of HBV replication were achieved after intravenous administration of 5 x 10(9) PEGylated or native recombinant Ads to HBV transgenic mice. After the first injection, circulating HBV viral particle equivalents (VPEs) remained low for 3 weeks and began to increase after 5 weeks. A second dose of PEGylated anti-HBV Ad caused a less sustained decrease in circulating VPEs, but no silencing after a second dose was observed in animals treated with unmodified vector. Release of inflammatory cytokines, including monocyte chemoattractant protein-1 (MCP-1), interferon-gamma, interleukin-6, and tumor necrosis factor-alpha, was elevated in animals receiving unmodified vectors. However, only a modest increase in MCP-1 was observed in mice that received a second dose of PEG Ads. Also, polymer-conjugated vectors induced a weaker adaptive immune response and were less hepatotoxic than their unmodified counterparts. Collectively, these observations show that PEG modification of Ads expressing RNAi effectors improves their potential for therapeutic application against HBV infection.

Efficient Silencing of Hepatitis B Virus by Helper-dependent Adenovirus Vector-mediated Delivery of Artificial Antiviral Primary Micro RNAs

Hepatitis B virus (HBV) infection is endemic to southern Africa and parts of Asia where approximately 350 million individuals are chronically infected. Persistent infection increases risk for the serious complications of cirrhosis and hepatocellular carcinoma. Licensed HBV treatments rarely eradicate the virus, which makes developing new strategies for the treatment of chronic HBV a priority. Pol II-transcribed mono- and trimeric primary micro RNAs (primiRNAs) have previously been used to activate RNA interference (RNAi) and inhibit HBV gene expression, indicating that this approach holds promise for HBV therapy. Nevertheless, achieving safe and efficient delivery of anti-HBV RNAi expression cassettes remains an important objective before therapeutic application of this gene silencing technology is realized. Recombinant adenoviruses (Ads) are amongst the most efficient hepatotropic gene delivery vehicles, but a drawback of their use is transient transgene expression and toxicity that results from induction of host immune responses. To diminish immunostimulation of anti-HBV RNAi-activating vectors, helper-dependent (HD) Ads with all viral proteinencoding sequences removed from their genomes, were generated. A CMV Pol II promoter element was used to transcribe antiviral pri-miRNAs that target HBV. Processing of the anti-HBV pri-miRNA RNAi activators occurred according to intended design. Assessment in cultured cells and in a HBV transgenic model of the infection demonstrated that HD Ads delivered the silencing sequences efficiently and replication of the virus was inhibited without causing overt toxic effects. Collectively these data augur well for clinical use of HD Ads to deliver Pol II HBV-silencing cassettes to counter the persistent infection.

483. Epigenetic Silencing of Hepatitis B cccDNA In Vitro and In Vivo Using AAV-Delivered Engineered Repressor Transcription Activator-Like Effector

Hepatitis B virus (HBV) infection is hyper-endemic (>8 % chronic carriers) to parts of Asia and sub-Saharan Africa. Persistent HBV infection predisposes to liver diseases such as cirrhosis and hepatocellular carcinoma. Current anti-HBV therapies face several limitations. RNA interference-based therapies lack the robustness and specificity required for therapeutic effect while interferon-α and nucleotide/side analogs function post-transcriptionally and thus allow for the persistence of the covalently closed circular (cccDNA). cccDNA may persist indefinitely and enables re-initiation of HBV replication after withdrawal of treatment. Disabling the cccDNA is essential for the successful treatment of chronic HBV. Our group previously described effective anti-HBV transcription activator-like effector (TALE) nucleases (TALENs). Although potentially useful to counter HBV replication, one drawback is that viral sequences integrated into the host genome may be susceptible to digestion by the TALENs. Transcriptional silencing, rather than cleavage, of cccDNA may therefore be preferable to avoid causing undesirable mutations in the host. To this end, TALE binding domains designed to target the viral preS2 promoter and the basic core promoter/enhancer II regions were fused to a Kruppel-associated box repressor domain to generate repressor-TALEs (rTALEs). These rTALEs were shown to inhibit viral replication in vitro and in vivo without inducing significant toxicity. In an in vivo murine model using hydrodynamic transfection with an HBV replication-competent plasmid, a reduction in secreted HBV surface antigen (HBsAg) of 97% and 98% was seen at day 3 and 93% and 96% at day 5 for P1L and P1R respectively. To develop this approach as a feasible therapy, rTALE-encoding sequences were incorporated into recombinant adeno-associated viruses (rAAVs) and assessed in the HepG2.hNTCP-C4 cell line. These cells overexpress the HBV receptor, human sodium taurocholate co-transporting peptide (hNTCP). The HepG2.NTCP-C4 cell line is infectable with HBV and viral gene expression is dependent on formation of cccDNA. Measurement of viral markers of replication may thus be used as an indicator of inhibitory effects on cccDNA. The anti-HBV efficacy of the rTALEs and epigenetic modification of the targeted HBV DNA was characterized. Chromatin immunoprecipitation assays determined the binding of the rTALEs to the cccDNA and induction of epigenetic markers of viral gene suppression. Mobility shift assays confirmed specificity of rTALE binding. In vivo efficacy of rAAV-delivered rTALEs was evaluated in transgenic HBV mice. Our study provides valuable information on the potential therapeutic utility of rTALEs and demonstrates the feasibility of the approach for treatment of HBV.