Disruption of HBV gene expression using TALEN encoding Adeno- associated viral vectors

Abstract

Hepatitis B Virus (HBV) infection is a global health problem with around 254 million people chronically infected world-wide. HBV infection results in both acute and chronic liver disease, with the latter resulting in hepatocellular carcinoma. Despite the availability of a vaccine and several approved drugs, the number of infections is still increasing. Upon infection with HBV, the viral genome is transported to the nucleus of an infected cell where it is repaired to covalently closed circular DNA (cccDNA). cccDNA serves as a template for the transcription of viral RNA and translation of viral proteins. cccDNA can remain as a viral reservoir within the nucleus of an infected cell, which allows the virus to persist in the host even during periods of low viral activity. Currently approved drugs are incapable of targeting cccDNA, which emphasises the need for a more suitable therapy for HBV. The HBV genome contains four overlapping open reading frames (ORFs) surface, core, polymerase, and X. Gene editing technologies such as those using transcription activator-like effector nucleases (TALENs) designed to target these four open reading frames have demonstrated good efficacy in vitro and in vivo. TALENs use two monomers to introduce double stranded DNA breaks within the viral DNA. Using host machinery, the double stranded break is repaired by error prone non- homologous end joining, which introduces mutations within the genome. Progression of these anti-HBV TALENs to clinical application has been delayed due to the lack of a suitable delivery vehicle. This study assessed the efficacy of Adeno-associated viruses (AAVs) in the delivery of anti-HBV TALENs. AAV serotype 2 (AAV 2), AAV serotype 8 (AAV 8) and an artificial AAV (Anc 80) encoding a single monomer of surface or core targeting TALENs were successfully produced. Using immunofluorescent detection, all TALEN monomers were detected in AAV 2 and Anc 80 infected cells. Anti-HBV effects were assessed in vitro using the transient plasmid transfection, HBV infection and AAV infection (AAV-HBV) models of HBV replication, with the latter demonstrating the most efficient anti-HBV activity. In the AAV-HBV model, ~ 90 % reduction of HBsAg levels were obtained when using AAV 2 or Anc 80 encoding anti-surface TALENs and, ~ 60 % reduction in HBsAg levels when using AAV 2 or Anc 80 encoding anti-core TALENs. This data was supported by reduction in HBV transcripts. No vector toxicity was viii observed in vitro using an MTT assay. In an AAV-HBV mouse model, a significant reduction in HBsAg, HBcAg and HBV transcript levels were noted with Anc 80 encoding surface and core targeting anti-HBV TALENs without adverse effects. A significant reduction in HBcAg and HBV transcript levels were noted with AAV 8 encoding surface and core targeting anti-HBV TALENs, while AAV 8 surface targeting anti-HBV TALENs also resulted in a significant reduction in HBsAg levels. AAVs have demonstrated great potential as gene delivery vehicles but a major disadvantage is that 30-60 % of the population have developed neutralising antibodies (nAbs) against naturally occurring AAV serotypes (e.g AAV 8 and AAV 2) prompting an immune response and subsequent clearance before a therapeutic benefit can be observed. Various strategies have been investigated to evade the immune system, and one such strategy let to the discovery of Anc 80 the predicted ancestor of AAV 1,2,8 or 9, but is more closely related to AAV 8. In this study, Anc 80 was further characterised for its ability to mediate transgene expression in the presence of AAV 2 and AAV 8 specific antibodies (Abs). Data from a luciferase expression- based in vitro neutralization assays using mouse or human derived Abs and in vivo neutralization assay using mice demonstrated that Anc 80 is partially neutralised by AAV 2 and/or AAV 8 specific Abs. Whether this residual transgene expression is sufficient to suppress HBV gene expression was assessed using AAV-HBV mouse model and AAVs expressing HBV targeting primary microRNA (apri-miRNA). In the presence of AAV specific Abs, a significant reduction in HBsAg levels were observed. However, this was not specific to anti-HBV apri- miRNA expressing vectors, but most probably as a result of AAV-HBV transduced cell clearance by cross reacting AAV/Anc 80 specific antibodies. This study highlighted AAV as a good delivery vehicle for anti-HBV TALENs without any safety or efficacy concerns. The viral vector Anc 80 adds to the AAV toolbox for designing novel HBV targeted gene therapeutics. However, the appeal of Anc 80 being able to evade pre-existing immunity to naturally occurring AAVs is not supported in this study.