Talen-mediated homology directed insertion of anti-viral sequences to inhibit hepatitis B viral gene expression and replication

Dreyer, Timothy James
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More than 350 million people are chronic hepatitis B virus (HBV) carriers. Viral covalently closed circular DNA (cccDNA) persists as a replication intermediate and can remain dormant. Current HBV therapeutics do not eradicate viral cccDNA reservoirs. Transcription activator-like effector nucleases (TALENs) target and cleave specific DNA sequences, and have shown promise as antiviral agents. Here we propose TALEN-mediated homology directed disruption and silencing of the cccDNA. The designer TALENs introduce double stranded breaks (DSBs) at the HBV cccDNA core and surface ORFs, which activate the non-homologous end joining (NHEJ) and homology directed repair (HDR) cellular repair pathways. We utilise the HDR process to introduce specific mutations at the TALEN cleavage sites. Here we facilitate integration of a HBV-targeting artificial primary micro RNA (pri-miR) mimic into the HBV genome by co-introducing TALENs and donor template strands that contain a pri-miR cassette flanked by sequences that are homologous to the TALEN target sites. Integration of the donor sequences was evaluated by PCR and disruption of HBV replication was evaluated using ELISA. HBsAg knockdown when targeting the surface and core targets was 94% and 63% respectively, a significant improvement on use of TALENs alone. PCR analysis and sequencing confirmed successful integration of donor sequences into the HBV core and surface target sites, verifying that HBsAg knockdown is as a result of sequence insertion and possible pri-miR-mediated silencing of HBx. In conclusion, integration of an artificial DNA sequence at specific HBV target sites was demonstrated and the synergistic activity of TALENs and donor template strands shows promising anti-HBV abilities. Results of this study provide the means to improve targeted disruption of HBV DNA by TALEN constructs. Moreover, the potential for combining different anti-HBV gene therapies to result in better viral suppression is demonstrated