Gene Therapy for Chronic HBV—Can We Eliminate cccDNA?

Bloom, Kristie; Maepa, Mohube Betty; Ely, Abdullah; Arbuthnot, Patrick

As with ZFNs, TALENs are dimeric engineered nucleases that comprise a DNA-binding protein fused to an endonuclease domain. The transcriptional activator-like effector (TALE) is derived from the Xanthomonas bacteria where individual repeat domains comprising 33-35 amino acids recognize a single base pair [41, 42] . The nucleotide binding specificity of these repeats is predetermined by repeat-variable di-residues (RVDs) located at amino acid positions 12 and 13 [43, 44] . Linking multiple repeats in a defined order generates engineered TALEs with highly specific DNA binding properties. Unlike with ZFNs, nucleotide binding affinity of each monomer comprising the DNA binding domain is not influenced by a neighboring unit. Our group first described antiviral efficacy of engineered TALENs on HBV cccDNA in cultured cells and inhibition of viral replication in a murine model [45] . TALEN dimers designed to bind within the surface (S) and core (C) ORFs showed optimal cleavage activity in the HepG2.2.15 cell line without measurable cytotoxicity. Importantly, cccDNA targeted disruption frequencies of 35% and 12% were achieved with the S and C TALEN pairs respectively. Using the murine hydrodynamic injection (HDI) model, co-administration of HBV replication-competent plasmids with TALEN-encoding sequences demonstrated in vivo antiviral efficacy of the nucleases and there was no evidence of liver toxicity. A significant and substantial reduction in serum concentrations of HBsAg and circulating viral particle equivalents was observed in TALEN-treated mice, and targeted mutagenesis of up to 87% was achieved. Deep sequencing verified large deletions in viral DNA, which were likely to inactivate HBV replication. A subsequent study by Chen et al. confirmed the cccDNA-specific antiviral potential of TALENs designed to target conserved regions within the polymerase (RNaseH sequence) and C ORFs [46] . This was achieved in liver-derived Huh7 cells transfected with linear viral sequences that generate cccDNA and recapitulate HBV replication [11, 12] . A reduction in viral protein expression was observed across genotypes A, B, C, and D, emphasizing the applicability of anti-HBV TALENs to a variety of viral isolates. Moreover, a synergistic effect was shown when combining IFN-α with core TALENs, to result in an approximately 60% reduction in copies of cccDNA. In another study, co-transfection of linear donor sequences encoding trimeric gene silencers significantly augmented anti-HBV efficacy of S or C TALEN pairs in HepG2.2.15 cells [47] . This approach exploited the homology directed repair pathway to introduce the artificial primary microRNA-encoding sequences directly into viral DNAs. In doing so, the viral genome may be permanently disrupted and after homologous recombination, HBV DNA transcribes an antiviral sequence from its own rearranged genome.