Inactivating hepatitis B virus replication using obligate heterodimeric TALEN- encoding mRNA

Abstract

The hepatitis B virus (HBV) poses a significant global health challenge, as chronic infection can lead to cirrhosis and hepatocellular carcinoma, resulting in over 1 million deaths annually. While vaccines exist, they have limitations, particularly in addressing chronic infections or non- responders. Current treatments, including nucleoside/nucleotide analogues and pegylated interferon-α, are inadequate for curing chronic HBV infections because they cannot eliminate covalently closed circular DNA (cccDNA) from infected cell nuclei. cccDNA persists as a viral gene transcription template, sustaining chronic infection and allowing viral reactivation upon treatment cessation. Obligate heterodimeric transcription activator-like effector nucleases (TALENs) have emerged as a promising tool for targeting HBV. These TALENs feature a DNA- binding domain and a second-generation FokI nuclease domain that enable precise DNA cleavage exclusively when heterodimeric TALENs are in close proximity on opposite DNA strands, preventing homodimeric cleavage and minimising off-target effects. This cleavage activates non- homologous end-joining DNA repair, inducing mutations at double-strand break sites. To assess the clinical potential of TALENs targeting HBV core and surface open reading frames, TALEN- encoding mRNA was synthesised by in vitro transcription using T7 RNA polymerase and three distinct cap analogues: anti-reverse cap, post-transcriptional Vaccinia cap, and CleanCap® AG. Immunocytochemistry was first performed in liver-derived cells to detect the haemagglutinin tag located within the TALEN construct. This experiment confirmed that optimal expression of Core and Surface TALENs was achieved using CleanCap® AG. TALEN efficacy was then tested in three in vitro models: Huh7 cells with HBV replication-competent plasmid, HepG2-hNTCP cells infected with HBV, and Huh7 cells transduced with adeno-associated virus type 2 (AAV2) containing HBV genome sequences from subgenotype D3. Surface TALENs reduced HBsAg Page | vii levels by 40%, 30%, and 90% across the models, with no observed cytotoxicity. In vivo studies using lipid nanoparticle-encapsulated Core and Surface TALEN-encoding mRNA in a transgenic HBV murine model showed a 63% reduction in HBsAg and over 99% reduction in viral particle equivalents, with no liver toxicity or significant inflammatory response. While Sanger sequencing did not detect targeted mutations with sufficient sensitivity, next-generation sequencing could offer more detailed insights as observed data indicate a significant difference in viral fitness between mock and TALEN-treated mice. This study represents the first successful production of obligate heterodimeric TALENs as mRNA. It highlights their potential as a therapeutic approach for chronic HBV infection by targeting and permanently inactivating HBV production.