Despite efficient uptake in hepatocytes of RO7062931 mirroring other GalNAc-ASOs, the HBsAg response is strikingly different:1. A negligible HBsAg response accompanies the 0.5mg/kg dose. Significant protein reductions (~35%) are observed with similarly dosed GalNAc-ASOs within 15 days. [2] 2. The mean HBsAg reduction after 15 days of RO7062931 dosing is weak (~0.2 log 10 IU/mL), even at 3 mg/kg, and highly variable (HBsAg nadir of −0.17 to −0.85 log 10 IU/mL). Efficient target engagement by RO7062931 should lead to about 1 log 10 IU/mL HBsAg reduction in all patients at 15 days.Although RO7062931 is designed to target HBV mRNA cleavage from covalently closed circular DNA or integrated HBV DNA, the inactivation of antisense or RNA interference by single-point mutations in the target site are well known. HBV quasi-species in humans are uniquely prolific and can rapidly evolve during therapy. Pre-existing or rapidly evolving escape mutants are a very likely explanation for a weak HBsAg response to RO7062931, which is actually inconsistent with target engagement. Off-target effects with ASOs from immunostimulation (through toll-like receptor 9 activation) or the formation of oligonucleotide aptamers are also well described in the clinic. The aptamer issue is potentially more problematic in RO7062931 due to the presence of locked nucleic acids, which significantly alter oligonucleotide structure. Such potential off-target effects driving the HBsAg response to RO7062931 cannot be ruled out.