Gene Therapy for Chronic HBV—Can We Eliminate cccDNA?

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

Zinc fingers (ZFs) are abundant multifunctional mammalian proteins that occur naturally as transcription factors. By exploiting the specific targeting of nucleotide triplets of single ZFs, these proteins may be engineered to form arrays with defined DNA binding properties [36] . Addition of a FokI effector domain at the C-terminus of a ZF yields an engineered nuclease that cuts one strand of a DNA duplex. ZFN dimers, with cognates on complementary strands of the DNA duplex, may thus be used to create double stranded breaks [37] . Anti-HBV ZFNs were first described in a proof-of-concept experiment where 36% of plasmid-derived viral sequences were disrupted in a cell culture model of viral replication [38] . More recently Weber et al. reported on delivery of self-complementary adeno-associated viral vectors (scAAVs) encoding ZFNs targeting the polymerase/X (1), polymerase/core (2) and polymerase (3) viral open reading frames (ORFs) [39] . Using inducible liver-derived HepAD38 cells to mimic natural HBV infection [40] , ZFN pairs 1, 2 and 3 cleaved 9.8, 34, and 28% of the viral targets respectively. These results were confirmed using single molecule real time sequencing which additionally identified potential off targets, albeit at low frequencies. Interestingly ZFN pair 2 resulted in the highest detectable targeted disruption, yet it was also found to be cytotoxic. Only ZFN pair 3 showed antiviral efficacy but cleavage of cccDNA could not be verified.