Hepatitis B virus (HBV) is hyperendemic to southern Africa and parts of Asia where it is a major cause of serious liver disease. Licensed antivirals for chronically infected individuals are only partially effective and approximately one million deaths occur annually as a result ofpersistent infection with the virus. Although RNA interference (RNAi) based gene silencingof HBV has been successfully demonstrated, difficulties with delivery of anti-HBV RNAieffectors remains an obstacle to their clinical use. Recombinant adenoviruses (Ads), amongst the most efficient hepatotropic gene vectors following systemic administration, have been successfully used to deliver expressed anti-HBV RNAi sequences. However, a drawback of Ad vectors is diminished efficacy and toxicity that results from stimulation of innate andadaptive immunity.To attenuate these effects we used polyethylene glycol (PEG) to modify first generation recombinant Ad (FG Ad) vectors that express an anti-HBV short hairpin (shRNA) sequence. Efficient hepatocyte transduction occurred and expressed shRNAs were processed to generate intended HBV-targeting guides. Inhibition of HBV replication was achieved after intravenous administration of PEGylated or native recombinant first generation Ads (FG Ads) to HBV transgenic mice. 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 was elevated in animals receiving unmodified vectors and only a modest increase in monocyte chemotactic protein-1 (MCP-1) was observed in mice that received a second dose of PEG Abstract Ads. Also, polymer-conjugated vectors induced a weaker adaptive immune response and were less hepatotoxic than their unmodified counterparts. To address concerns about the transient nature of transgene expression by FG Ads resulting from immunostimulation, third generation helper-dependent (HD Ad) were utilised to delivered anti-HBV RNAi effectors. Seven days after intravenous administration of infectious HD Ads to HBV transgenic mice, 80-90% of hepatocytes were transduced and markers of HBV replication were decreased by approximately 95% which was sustained for 8 weeks. HD Ad-induced release of proinflammatory cytokines was minimal in preparations that were enriched with infectious particles. PEGylated HD Ad vectors caused similar anti- HBV effects and may be useful to evade interaction with vector-sequestrating receptors and further attenuate immunostimulation. Collectively these observations indicate that PEG modification of Ads and the use of HD Ads may have utility for delivery of therapeutic HBVsilencing sequences. Future work will focus on improving strategies to avoid immune detection and utilisation of HD Ad vectors for other HBV targeting sequences.

Hepatitis B virus (HBV) is a major global public health burden, with over 350 million people chronically infected. This results in approximately 600,000 liver cancer-related deaths annually. Chronic HBV infections are normally managed with long-term anti-HBV therapeutics, such as reverse transcription inhibitors, which target post-transcriptional viral processes without affecting the cccDNA. Treatment failure however is largely as a result of the stability of this episomal viral DNA. The cccDNA minichromosome serves as a reservoir of HBV DNA and is capable of re-establishing viral replication following withdrawal of treatment. Designer nucleases, like transcription activator-like effector nucleases (TALENs), have recently been used to create double stranded breaks (DSBs) at target-specific endogenous DNA loci. These nucleases are designed as pairs, which upon dimerisation cleave double-stranded DNA. Subsequent activation of the cellular non-homologous end-joining (NHEJ) pathway often results in targeted mutagenesis at the DSB site. As TALENs may be designed to bind to any DNA sequence, they are commonly used as genetic engineering agents. Inactivation of HBV cccDNA, using these engineered TALENs, presents a unique approach to disabling viral replication permanently. To investigate this, a panel of TALENs targeting the core (C), surface (S) and two different polymerase (P1 and P2) regions of HBV cccDNA were generated using a Golden gate modular assembly approach. TALENs were initially tested in two liver-derived cell lines. Firstly as transient co-transfections in Huh7 cells using a HBV replication competent plasmid, followed by long term investigations in HepG2.2.15 cells which model HBV replication in vitro. Inactivation of HBV was determined by measuring markers of viral replication, whilst TALEN-mediated targeted disruption was verified by T7 endonuclease 1 (T7E1) or CELI endonuclease assays and sequencing. In vitro, the S TALEN inhibited HBsAg secretion by 80% in Huh7 cells and 60% in HepG2.2.15 cells. Furthermore, S TALEN-mediated targeted disruption occurred in 35-47% of cccDNA copies, whilst the C TALEN resulted in 11% targeted disruption of cccDNA in without inhibition of HBsAg expression. The P2 TALEN showed no anti-HBV efficacy, however the P1 TALEN inhibited HBsAg expression by up to 60% without any evidence of site-directed cleavage. As this TALEN binding site spans the HBV Enhancer I sequence, knock-down of HBsAg expression is most likely to occur as a result of transient transcriptional repression. To confirm whether permanent repression of HBV transcription could be achieved, a KRAB-based transcription activator-like repressor (rTALE) targeting the HBV pre-S2 promoter was generated. Using an in vitro reporter gene assay, the pre-S2 rTALE inhibited luciferase expression by up to 90%. However this was only achieved using high molar concentrations of the repressor, suggesting multiple rTALEs may improve HBV transcriptional repression. As the S and C TALENs displayed significant anti-HBV efficiency in vitro, they were tested in a murine hydrodynamic injection model of HBV replication. In vivo, the S TALEN inhibited HBsAg secretion by 95% and induced disruption in 77–87% of HBV DNA targets. In addition the C TALEN inhibited HBcAg expression and induced disruption in 78-93% of HBV DNA targets. Additionally, serological analysis showed a reduction in circulating virions and no apparent liver toxicity, as determined by real-time PCR (qPCR) and aspartate transaminase (AST)/ alanine aminotransferase (ALT) liver function tests respectively. Deep sequencing at the S and C TALEN binding sites showed targeted mutagenesis of HBV DNA in samples extracted from murine hepatocytes transfected with TALENs, however wild-type sequences were exclusively detected in mice that had not been treated with anti-HBV TALENs. Furthermore, frameshift deletions were predominantly detected indicating major disruptions in the HBV surface and core sequences. These results indicate that TALENs designed to disable and silence HBV cccDNA are effective both in vitro and in vivo and as such provide a promising therapeutic approach to treat this serious infection.