Characterization of Hepatitis B Virus Transcripts in Chronically HBV-infected Chimpanzees and Patients Treated with ARC-520 siRNA Demonstrates Transcriptional Silencing of cccDNA

Full-length hepatitis B virus (HBV) transcripts of chimpanzees and patients treated with multidose (MD) HBV siRNA ARC-520 and entecavir (ETV) were characterized by single-molecule real-time (SMRT) sequencing, identifying multiple types of transcripts with the potential to encode HBx, HBsAg, HBeAg, core, and polymerase, as well as transcripts likely to be derived from dimers of dslDNA, and these differed between HBeAg-positive (HBeAg+) and HBeAg-negative (HBeAg-) individuals. HBV transcripts from the last follow-up ~30 months post-ARC-520 treatment were categorized from one HBeAg+ (one of two previously highly viremic patients that became HBeAg- upon treatment and had greatly reduced cccDNA products) and four HBeAg- patients. The previously HBeAg+ patient received a biopsy that revealed that he had 3.4 copies/cell cccDNA (two to three orders of magnitude more cccDNA than HBeAg- chimpanzees) but expressed primarily truncated X and HBsAg from iDNA, like two patients that were HBeAg- at the start of the study and had one copy/cell cccDNA. No HBV transcripts were detected in two other HBeAg- patients that had ~0.3 copies/cell cccDNA, one of which had seroconverted for HBsAg. The paucity of cccDNA-derived transcripts in the presence of high cccDNA demonstrates the transcriptional silencing of HBV following MD siRNA treatment with ETV. Overall design: The initial study with nine chimpanzees aimed to determine the safety, tolerability, and pharmacological effects of ARC-520 siRNA treatment on HBsAg-positive chimpanzees (Pan troglodytes) which are thought to have become infected perinatally [Wooddell et al. Sci Transl Med, 2017]. HBV transcripts were characterized from six of these chimpanzees. The chimpanzees received daily oral entecavir for an 8–20-week lead-in period prior to receiving intravenous ARC-520 (2–4 mg/kg) once every four weeks (Q4W) with continued daily ETV. After 6–10 doses of ARC-520, the siRNA siHBV-75 ± siHBV-74 was tested in the HBeAg- chimpanzees that responded with lower activity to ARC-520. ETV dosing continued 1–2 weeks after the last siRNA dose. Plasma and serum were collected biweekly for the duration of the monitoring period to measure HBsAg, HBeAg, HBV DNA, anti-HBeAg, anti-HBsAg, clinical chemistries and complete blood counts as previously described [Wooddell et al. Sci Transl Med, 2017]. Periodic liver needle biopsies were collected to evaluate HBV DNA and RNA and for histology. All sample collections from the chimpanzees were conducted prior to 14 September 2015, when the U.S. Department of Fish and Wildlife reclassified chimpanzees born in captivity at research centers as endangered. References: 1. Wooddell, C.I., et al., RNAi-based treatment of chronically infected patients and chimpanzees reveals that integrated hepatitis B virus DNA is a source of HBsAg. Sci Transl Med, 2017. 9(409).