Abstract:
Chronic infection with hepatitis B virus (HBV) remains an important global health problem.
Currently licensed therapies have modest curative efficacy, which is as a result of their
transient effects and limited action on the viral replication intermediate comprising
covalently closed circular DNA (cccDNA). Gene editing with artificial HBV-specific
endonucleases and use of artificial activators of the RNA interference pathway have shown
anti-HBV therapeutic promise. Although results from these gene therapies are encouraging,
maximizing durable antiviral effects is important. To address this goal, a strategy that entails
combining gene editing with homology-directed DNA recombination (HDR), to introduce
HBV-silencing artificial primary microRNAs (pri-miRs) into HBV DNA targets, is reported
here. Previously described transcription activator-like effector nucleases (TALENs) that
target the core and surface sequences of HBV were used to introduce double stranded
breaks in the viral DNA. Simultaneous administration of donor sequences encoding artificial
promoterless anti-HBV pri-miRs, with flanking arms that were homologous to sequences
adjoining the TALENs' targets, augmented antiviral efficacy. Analysis showed targeted
integration and the length of the flanking homologous arms of donor DNA had a minimal
effect on antiviral efficiency. These results support the notion that gene editing and
silencing may be combined to effect improved inhibition of HBV gene expression.
