Research ArticleInfectious Disease

RNAi-based treatment of chronically infected patients and chimpanzees reveals that integrated hepatitis B virus DNA is a source of HBsAg

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Science Translational Medicine  27 Sep 2017:
Vol. 9, Issue 409, eaan0241
DOI: 10.1126/scitranslmed.aan0241
  • Fig. 1. Serum HBsAg, HBcrAg, and HBeAg reduction in human patients treated with a single dose of ARC-520.

    CHB patients were given a single intravenous dose of ARC-520 (1 to 4 mg/kg) on a background of daily oral NUCs. HBsAg (A) or HBcrAg (C) reduction in CHB patients who were NUC-experienced HBeAg-negative and received single doses (1 to 4 mg/kg) (cohorts 1 to 4, n = 6). (B) HBsAg reduction in CHB patients who were NUC-experienced HBeAg-negative (cohort 4, n = 6) or NUC-experienced HBeAg-positive (cohort 5, n = 6) who received a single dose (4 mg/kg). (D) HBcrAg and HBeAg reduction in CHB patients who were NUC-experienced HBeAg-positive and received a single dose (4 mg/kg) (cohort 5, n = 6). (E) HBsAg reduction for individual CHB patients who were NUC-naïve HBeAg-positive and received a single dose (4 mg/kg) in cohort 7 (n = 6). (F) HBsAg, HBeAg, and HBcrAg reductions in CHB patients who were NUC-naïve HBeAg-positive and received a single dose (4 mg/kg) (cohort 7, n = 5). (G) HBsAg reduction in CHB patients who were HBeAg-negative and NUC-naïve (cohort 7, n = 6) or NUC-experienced (cohort 4, n = 6). PBO, patients on NUC therapy given placebo injection. Error bars show SEM.

  • Fig. 2. Response to repeat dosing of chimpanzees with ARC-520.

    (A) After a prestudy evaluation, nine chimpanzees began daily oral NUC dosing. After a variable NUC lead-in period to reduce viremia, NUC treatment continued concomitant with Q4W dosing of ARC-520 that began on day 1. Dosing days are indicated by vertical dashed lines. Blood samples were collected periodically throughout the study, and serum HBV DNA (B), HBeAg (C), and HBsAg (D and E) were measured and are shown for individual animals. (F) Mean log10 change in HBsAg for the 8 weeks preceding the first dose of ARC-520 and during ARC-520 treatment of four HBeAg-positive (●) and four HBeAg-negative (□) and in the HBeAg transitional chimpanzee (▲). BLD, below the limit of detection. *, chimpanzee 89A008 transitioned from HBeAg-positive to HBeAg-negative during the NUC lead-in period. Boxes in (B) and (C) indicate the first day of daily ETV dosing.

  • Fig. 3. Serum HBsAg and liver HBV mRNA reduction in chimpanzees dosed with ARC-520.

    After prestudy evaluation, nine chimpanzees began NUC dosing for a lead-in period of 8 to 24 weeks. Q4W dosing with ARC-520 began on day 1. Liver mRNA from biopsies of HBeAg-positive chimpanzees (A2A004, A3A006, and A4A014) and HBeAg-negative chimpanzees (88A010, 95A008, and 95A010) was evaluated for HBV gene expression by RT-qPCR with probe sets in core to measure the amount of precore mRNA/pgRNA and in X to measure total HBV mRNA. (A) Mean total HBV mRNA amounts were compared between HBeAg-positive and HBeAg-negative chimpanzees before study, after the NUC lead-in (day 1), and 1 week after the second ARC-520 injection (day 36). (B) The numbers of total HBV transcripts and precore/pgRNA transcripts on day 1 are compared for the HBeAg-positive and HBeAg-negative chimpanzees. Error bars show SEM.

  • Fig. 4. Liver HBV mRNA paired-end sequencing reads in HBeAg-positive and HBeAg-negative chimpanzees.

    The HBV mRNA and HBV protein open reading frames are positioned relative to the coordinates of the HBV genome. The mRNA-seq read histograms are shown for HBeAg-positive chimpanzees 4x0139, A2A004, A3A006, and A4A014; for HBeAg transitional chimpanzee 89A008; and for HBeAg-negative chimpanzees 88A010, 95A010, and 95A008. Locations of the DR1 sequence (red line), HBV PAS (brown dashed line), and binding sites for the siRNAs in ARC-520 (siHBV-74 and siHBV-77) are indicated.

  • Fig. 5. Mapping of HBV S transcripts from HBeAg-positive and HBeAg-negative chimpanzees.

    Total RNA isolated from the liver biopsies after the NUC lead-in and before ARC-520 dosing on day 1 from HBeAg-positive chimpanzee A2A004 and HBeAg-negative chimpanzee 88A010 was reverse-transcribed, size-selected, and sequenced with SMRT sequencing. (A) Full-length nonconcatemer reads were aligned to each chimpanzee’s consensus HBV DNA sequence. Green lines represent HBV-containing transcripts. Dark green represents sequences aligning to HBV, and light green represents those not aligning to HBV. The HBV coordinates are shown with elements DR2, DR1, and the HBV PAS. (B) Cumulative fraction of HBV-chimpanzee breakpoints was plotted against the HBV coordinate.

  • Fig. 6. Treatment of HBeAg-negative chimpanzees with siRNA targeted outside the DR1-DR2 region.

    After a NUC lead-in, all four HBeAg-negative chimpanzees were given seven Q4W doses of ARC-520. Chimpanzees 4x0506 and 95A008 were then given an additional three doses of ARC-520 (4 mg/kg), whereas 95A010 and 88A010 were given three doses of siHBV-75 (4 mg/kg) plus ARC-EX1 (4 mg/kg) delivery reagent. HBsAg in serum was measured.

  • Table 1. Characterization of HBV transcripts and PAS sequences in HBeAg-negative and HBeAg-positive chimpanzees.

    Total liver RNA collected after the NUC lead-in and before ARC-520 dosing (day 1) from HBeAg-negative chimpanzee 88A010 and HBeAg-positive chimpanzee A2A004 was sequenced with the SMRT approach. All mRNA sequences that could be at least partially aligned with the HBV genome were analyzed to determine the number that contained entirely HBV sequence, HBV-chimpanzee fusion sequence, HBV-HBV fusion sequence, or HBV fused to an unknown sequence.

    Characterization of HBV transcripts
    HBeAg-negative 88A010HBeAg-positive A2A004
    HBV transcriptsNumber of transcriptsPercentageNumber of transcriptsPercentage
    HBV nonfusion12822.7246690.5
    HBV-chimpanzee375*66.4351.3
    HBV-HBV5710.12188.0
    HBV-other50.970.3
    Total5651002726100
    Analysis of PAS sequences in nonfusion HBV transcripts
    HBeAg-negative 88A010HBeAg-positive A2A004
    PAS sequenceNumber of transcriptsPercentageNumber of transcriptsPercentage
    TATAAA8062.5239697.2
    CATAAA4232.87§0.3
    Other10.8180.7
    None detected53.9451.8
    Total1281002466100

    *Thirteen reads with reverse orientation were excluded from further analysis.

    †Two reads with reverse orientation were excluded from further analysis, and 12 transcripts with poly(A) or poly(T) appear to have been misidentified as fusion transcripts.

    ‡One hundred sixty-six transcripts contain gaps in HBV sequence consistent with splicing (39).

    §Two of seven could be mutants or sequencing errors (TATAAA expected based on the HBV coordinate).

    Supplementary Materials

    • www.sciencetranslationalmedicine.org/cgi/content/full/9/409/eaan0241/DC1

      Materials and Methods

      Fig. S1. HBV sequence elements near DR1 and DR2.

      Fig. S2. Serum HBsAg, HBeAg, and HBcrAg reduction in human patients treated with two doses of ARC-520 2 weeks apart.

      Fig. S3. Reduction of HBV serum DNA in CHB patients treated with a single dose of ARC-520 on a background of NUCs.

      Fig. S4. Quantitation of liver HBV DNA in chimpanzees.

      Fig. S5. Mapping of HBV DNA integration sites.

      Fig. S6. Massive paired-end sequencing of chimpanzee liver mRNA and alignment to HBV reference genome.

      Fig. S7. Immunohistochemical staining of HBsAg in livers of HBeAg-positive and HBeAg-negative chimpanzees.

      Table S1. Individual CHB patient demographics at screening and maximum HBsAg reduction from baseline in clinical trial Heparc-2001.

      Table S2. Mean CHB patient demographics at screening and maximum HBsAg reduction from baseline in clinical trial Heparc-2001.

      Table S3. Treatment-emergent adverse events in cohorts 1 to 7 of the Heparc-2001 study.

      Table S4. Characteristics of chimpanzees chronically infected with HBV and treatment regimens.

      Table S5. mRNA-seq data obtained from liver biopsies of chimpanzees chronically infected with HBV.

      Table S6. PASs in HBV-chimpanzee fusion transcripts of HBeAg-negative chimpanzee 88A010.

      Table S7. HBV genome characteristics determined from liver DNA sequencing of study chimpanzees.

      Table S8. Primary data.

    • Supplementary Material for:

      RNAi-based treatment of chronically infected patients and chimpanzees reveals that integrated hepatitis B virus DNA is a source of HBsAg

      Christine I. Wooddell,* Man-Fung Yuen, Henry Lik-Yuen Chan, Robert G. Gish, Stephen A. Locarnini, Deborah Chavez, Carlo Ferrari, Bruce D. Given, James Hamilton, Steven B. Kanner, Ching-Lung Lai, Johnson Y. N. Lau, Thomas Schluep, Zhao Xu, Robert E. Lanford, David L. Lewis

      *Corresponding author. Email: cwooddell{at}arrowheadpharma.com

      Published 27 September 2017, Sci. Transl. Med. 9, eaan0241 (2017)
      DOI: 10.1126/scitranslmed.aan0241

      This PDF file includes:

      • Materials and Methods
      • Fig. S1. HBV sequence elements near DR1 and DR2.
      • Fig. S2. Serum HBsAg, HBeAg, and HBcrAg reduction in human patients treated with two doses of ARC-520 2 weeks apart.
      • Fig. S3. Reduction of HBV serum DNA in CHB patients treated with a single dose of ARC-520 on a background of NUCs.
      • Fig. S4. Quantitation of liver HBV DNA in chimpanzees.
      • Fig. S5. Mapping of HBV DNA integration sites.
      • Fig. S6. Massive paired-end sequencing of chimpanzee liver mRNA and alignment to HBV reference genome.
      • Fig. S7. Immunohistochemical staining of HBsAg in livers of HBeAg-positive and HBeAg-negative chimpanzees.
      • Table S1. Individual CHB patient demographics at screening and maximum HBsAg reduction from baseline in clinical trial Heparc-2001.
      • Table S2. Mean CHB patient demographics at screening and maximum HBsAg reduction from baseline in clinical trial Heparc-2001.
      • Table S3. Treatment-emergent adverse events in cohorts 1 to 7 of the Heparc-2001 study.
      • Table S4. Characteristics of chimpanzees chronically infected with HBV and treatment regimens.
      • Table S5. mRNA-seq data obtained from liver biopsies of chimpanzees chronically infected with HBV.
      • Table S6. PASs in HBV-chimpanzee fusion transcripts of HBeAg-negative chimpanzee 88A010.
      • Table S7. HBV genome characteristics determined from liver DNA sequencing of study chimpanzees.

      [Download PDF]

      Other Supplementary Material for this manuscript includes the following:

      • Table S8 (Microsoft Excel format). Primary data.

      [Download Table S8]

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