| Summary: | Persistent hepatitis B virus (HBV) infection remains a serious medical problem worldwide, with an estimated global burden of 257 million carriers. Prophylactic and therapeutic interventions, in the form of a vaccine, immunomodulators, and nucleotide and nucleoside analogs, are available. Vaccination, however, offers no therapeutic benefit to chronic sufferers and has had a limited impact on infection rates. Although immunomodulators and nucleotide and nucleoside analogs have been licensed for treatment of chronic HBV, cure rates remain low. Transcription activator-like effector nucleases (TALENs) designed to bind and cleave viral DNA offer a novel therapeutic approach. Importantly, TALENs can target covalently closed circular DNA (cccDNA) directly with the potential of permanently disabling this important viral replicative intermediate. Potential off-target cleavage by engineered nucleases leading to toxicity presents a limitation of this technology. To address this, in the context of HBV gene therapy, existing TALENs targeting the viral <i>core</i> and <i>surface</i> open reading frames were modified with second- and third-generation <i>Fok</i>I nuclease domains. As obligate heterodimers these TALENs prevent target cleavage as a result of <i>Fok</i>I homodimerization. Second-generation obligate heterodimeric TALENs were as effective at silencing viral gene expression as first-generation counterparts and demonstrated an improved specificity in a mouse model of HBV replication.
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