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Reductions in cccDNA under NUC and ARC-520 therapy
in chimpanzees with chronic hepatitis B virus infection
implicate integrated DNA in maintaining circulating
HBsAg
Christine I. Wooddell1, Deborah Chavez2, Jason E. Goetzmann3,
Bernadette Guerra2, Ryan M. Peterson1, Helen Lee2,
Julia O. Hegge1, Robert Gish4, Stephen Locarnini5, Christopher
R. Anzalone1, Robert E. Lanford2, David L. Lewis1; 1Arrowhead
Madison, Arrowhead Research Corporation, Madison, WI; 2Texas
Biomedical Research Institute, San Antonio, TX; 3New Iberia
Research Center, University of Louisiana at Lafayette, New Iberia,
LA; 4Department of Medicine, Stanford University Medical Center,
San Diego, CA; 5Victorian Infectious Diseases Reference Laboratory,
Melbourne, VIC, Australia
Background: RNAi therapeutic ARC-520 designed to target
all cccDNA-derived transcripts reduces viral antigenemia for
>1 month after single doses in HBV patients. Here we report
the effect of multiple ARC-520 doses on hepatic HBV DNA
and RNA in HBV chimps. Methods: 9 chimps (5 M, 4 F; 9-37
yrs) received 6-11 monthly injections of ARC-520 concurrent
with NUC therapy. 5 were HBeAg-positive (HBeAg+), baseline
DNA 8-9 log10 IU/mL serum, and 4 were HBeAg-negative
(HBeAg-), ≤3 log10 IU/mL. Chimps received NUCs for
8-24 weeks prior to ARC-520 dosing. Liver biopsies from 8
chimps were taken at baseline, completion of NUC lead-in
and on study. HBV DNA, +/- plasmid-safe DNase digestion to
enrich cccDNA, was measured by qPCR. Pre-core/core RNA
(C probe) and total HBV RNA (Total probe) were measured
by RT-qPCR. The Guide for the Care and Use of Laboratory
Animals was strictly adhered to. Results: During NUC lead-in,
total liver HBV DNA decreased 1.1-2.5 log10 in HBeAg+ but
not appreciably in HBeAg- chimps. cccDNA in HBeAg+ chimps
decreased 0.7 ± 0.6 log10. Following addition of ARC-520 in
HBeAg+, total liver DNA decreased from baseline by 1.5–2.9
log10 and cccDNA by 1.4 ± 0.7 log10, the degree of reduction
generally correlating with duration of treatment. Neither
total HBV DNA nor cccDNA levels changed remarkably in
HBeAg- during the study, which at baseline had 2-4 orders of
magnitude less cccDNA than HBeAg+ chimps. HBeAg- chimps
had 50-fold more DNase-sensitive HBV DNA, possibly indicating
the majority is integrated DNA rather than cccDNA.
HBV RNA was not reduced by NUCs, but with addition of
ARC-520 RNA reductions tracked qHBsAg reductions. In
HBeAg+, Total probe detected 1-2x as many transcripts as
the C probe, suggesting similar levels of core/pre-core and
S transcripts. In HBeAg-, the Total probe detected 37x more
transcripts than the C probe, supporting a greater proportion of
HBsAg transcripts being produced from integrated HBV DNA
in HBeAg- chimps. Integration between DR1 and DR2 would
result in HBV RNA lacking ARC-520 target sites, consistent
with greater HBsAg reduction in HBeAg+ (1.7 ± 0.5 log10)
than HBeAg- chimps (0.7 ± 0.1 log10). Administration of siRNA
targeted to integrant-produced transcripts resulted in HBsAg
reductions up to 2.3 log10 beyond those produced by ARC-520
in HBeAg-. Conclusions: 1) ARC-520 reduced total liver DNA
and cccDNA beyond levels achieved in HBeAg+ with NUCs
during lead-in; 2) ARC-520 but not NUCs reduced HBV RNA
and antigens; 3) integrated HBV DNA may be important in
maintaining HBsAg in chronic HBV, especially in HBeAg-. This
finding, if confirmed, has important implications for development
of new HBV therapies.
Disclosures:
Christine I. Wooddell - Employment: Arrowhead Research Corporation
Ryan M. Peterson - Employment: Arrowhead Research Corporation
Julia O. Hegge - Employment: Arrowhead Research Corp
Robert Gish - Advisory Committees or Review Panels: Gilead, AbbVie, Arrowhead;
Consulting: Eiger, Isis, Genentech; Speaking and Teaching: Gilead, Abb-
Vie; Stock Shareholder: Arrowhead
Stephen Locarnini - Consulting: Gilead, Arrowhead; Employment: Melbourne
Health
Robert E. Lanford - Grant/Research Support: Arrowhead Research
David L. Lewis - Employment: Arrowhead Research Corporation
The following authors have nothing to disclose: Deborah Chavez, Jason E. Goetzmann,
Bernadette Guerra, Helen Lee, Christopher R. Anzal |
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楼主: disan
发表于 2015-10-3 13:12
