hepatitisb: new treatment targetsand possible cure · chimpanzees with chronic hepatitis b virus...
TRANSCRIPT
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Hepatitis B: New TreatmentTargets and Possible Cure
Fabien Zoulim
Hepatology Department, Hospices Civils de Lyon
INSERM U1052, Cancer Research Center of Lyon
Lyon University, France
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Why a need for new antiviral targets for hepatitis B ?
Current antivirals achieve viral suppression in the majority of patients (in western countries)
Issues with antiviral drug resistance in developing countries (use of low barrier to resistance antivirals)
The cure rate (cccDNA / HBsAg loss) remains very low
Life-long therapy is needed in the majority of cases
Treatment with finite duration if:
� cccDNA control or loss
� HBsAg loss
HBsAg clearance is associated with a lower risk of HCC development
Zoulim, Antiviral Research 2012
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Definition of HBV cure
Virologic definition
- Functional cure
- Situation where antiviral therapy could be stopped
with a minimal risk of viral reactivation
- HBsAg loss with anti-HBsAb seroconversion
- cccDNA inactivation and/or control by host
mechanisms
- Complete cure
- HBsAg clearance and cccDNA eradication
Clinical definition
- Functional cure associated with a regression in the
risk of progression of fibrosis and HCCZeisel, Lucifora et al, Gut 2015
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Results of current antivirals
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Zoulim & Locarnini, Gastroenterology 2009; Zoulim Antiviral Research 2012; Mico et al J Hepatol 2013; Lucifora et al Science 2014
NK
cells
Innate responses
cells
CD8
+
cells
B
cells
cells
CD4
+
cells
Adaptive immune
responses
Nucleos(t)ide analogues
Interferon
alphaNTCP
Mode of action of antivirals for CHB
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Current treatments: virus suppression and sustained disease control
Entecavir1,2 Tenofovir3 PEG-IFN α-2a4,5
HBeAg positive n = 354 n = 176 n = 271
HBV DNA undetectable 67% 76% 25%a
HBeAg seroconversion 21% 21% 27%
ALT normalisation 68% 68% 39%
HBsAg loss 2% 3.2% 2.9%b
HBeAg negative n = 325 n = 250 n = 177
HBV DNA undetectable 90% 93% 63%a
ALT normalisation 78% 76% 38%
HBsAg loss 0.3% 0% 0.6%b
1. Chang T-T, et al. N Engl J Med 2006;354:1001–10.
2. Lai C-L, et al. N Engl J Med 2006;354:1011–20.
3. Marcellin P, et al. N Engl J Med 2008;359:2442–55.
4. Lau GKK, et al. N Engl J Med 2005;352:2682–95.
5. Marcellin P, et al. N Engl J Med 2004;351:1206–17.
Results at 48 weeks a HBV DNA < 400 copies/mL; b At 72 weeks
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Persistence of intrahepatic viral DNA synthesis during Tenofovir therapy
(HIV-HBV cohort)
Boyd et al, in revision
New round of infection and/or replenishment of the cccDNA pool occur
despite « viral suppression »
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NUC Therapy
HB
V D
NA
ch
an
ge f
rom
baselin
e (
log
10
c/m
L)
0.0
-1.0
-2.0
-3.0
-4.0
+1.0
Time
Long-term therapy is required to maintain viral suppression
HBsAg
HBVDNA
cccDNA
SERUM
LIVER
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New treatment concepts for a functional cure of HBV infection
Antiviral
s
Therapy0.0
-1.0
-2.0
-3.0
-4.0
+1.0
Time
HBsAg
HBVDNA
cccDNA
Immune
restoration
SERUM
LIVER Decay or epigenetic control
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0 4 8 12 16 20 24
0
2
4
6
8
10
Stopping TDF therapy after long-term viral suppressionHigh rates of viral replapse & ALT elevations3 patients with HBsAg loss out of 41
24-week TFFU Completers (n=41)
HBeAg positive (n=4)
HB
V D
NA
(L
og10
IU/m
L)
HBeAg negative (n=37)
Follow-up Week
Buti et al AASLD 2015
AL
T,
Mu
ltip
le o
f U
LN
Follow-up Week
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Characterisation of the immune profile in Chronic Hepatitis B, with CyTOF, to identify biomarkers of
immune control following NUC therapy discontinuationExpanded global CD4+ T cell memory populations in ‘non-flare’ vs flare patients
CD4+ T cells CD127+ (IL7R) CD4+ T cells
Non flare
Flare
CD
4+
T c
ells
(%)
0
20
Naive
*80
40
60
TCM TEM TEMRA
*
CD
127 +
CD
4+
T c
ells
(%)
20
40
Naive
*100
60
80
TCM TEM TEMRA
HBV polymerase specific T cell responses on NUC therapy are predictive of hepatic flares with
NUC discontinuationOn NUC therapy NUC discontinuation
0
100
X
300
200
Core Env Pol
*250
150
50
*** *
SF
C/1
05
cells
0
100
X
1000
200
300
Core Env Pol
** *
� Patients with detectable frequencies of circulating HBV-specific T cells controlled
HBV replication / no hepatic flare following NUC discontinuation
SF
C/1
05
cells
Gill US, et al. AASLD 2015, San Francisco. #167
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Improvement of existing drugsExample of TAF for tenofovir
N
N
N
N
NH2
OP
O
OO
O
O
O
OO
O
N
N
N
N
NH2
OP
O
HOOH
N
N
N
N
NH2
OP
O
NH O
O
O
Tenofovir
Disoproxil FumarateTenofovir
Tenofovir
Alafenamide
LYMPHOID CELLS/
HEPATOCYTESPLASMAGUT
TFV
TFV
TFV-MP
TFV-DP
TDF/TFVTDF
TFV TDF TAF
TAF
Cathepsin A
CES1
TAF TAF
♦ Improved stability in
plasma:
– Enhanced delivery of
active form (TFV-DP) to
hepatocytes
– Lower doses are used;
systemic exposures of
TFV reduced
Agarwal K et al. AASLD 2013, Poster # 973
Murakami E et al. HepDART 2013, Abstract 104CES1 = carboxylesterase 1; DP= di-phosphate; MP= mono-phosphate.
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Phase 1B results: HBV DNA kinetics on 28 days
♦No differences in viral declines over range of TAF 8 mg to 120 mg
♦Viral suppression over 4 weeks with TAF was similar to TDF
Study Day
TAF 8 mg (n= 10)
TAF 40 mg (n=10)
TAF 120 mg (n=11)
TDF 300 mg (n=10)
TAF 25 mg (n=10)
Study Day
Me
an
HB
V D
NA
(L
og10
IU/m
L)
Agarwal K et al. AASLD 2013, Poster # 973
GS-US-320-0101 - Clinicaltrials.gov NCT01671787
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New targets for HBV therapy
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Vaccine therapy
Check-point
inhibitors
TLR agonistsBlockade
of immune-
suppressive
cytokines
Chimeric antigen
Receptors (CAR)
Antiviral cytokines
Entry inhibitors
Core modulators
Targeting cccDNA
Polymerase
inhibitors
RNA
interference
Egress Inhibitors
Core modulators
Targeting
HBx
Testoni and Zoulim, Hepatology 2015
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Li et al, elife 2012
Urban et al, Gastroenterology 2014
Model for HBV entry in hepatocytes and
development of entry inhibitors
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Myrcludex B, a peptidic inhibitor of
NTCP-mediated entry of HBV and HDV
• Myrcludex B is an HBV preS-derived lipopetide binding sodium-taurocholate co-transporting polypeptide
(NTCP).
• Myrcludex B inhibits HBV and HDV receptor function of NTCP in vitro and in animal models (IC50∼ 80 pM).
• Myrcludex B inhibits NTCP-mediated bile salt uptake into hepatocytes (IC50∼ 100 nM).
• Myrcludex B specifically targets liver hepatocytes after subcutanous administration.
• Myrcludex B showed safety in Phase I clinical trials.
⇒ (A) Proof of safety and efficacy in chronically HBV infected individuals.
⇒ (B) Proof of safety and efficacy in chronically HBV/HDV co-infected individuals.
AASLD 2014 Boston, SIG HBV, Nov. 10thS Urban Heidelberg U & MyrGmbH
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HBV Serum DNA-levels decline
during Myrcludex B treatment
0,5 mg/d 1 mg/d 2 mg/d
5 mg/d 10 mg/d ETV 0,5 mg/d
⇒ HBV DNA levels decline significantly during Myrcludex B treatment in all groups.
⇒ Pronounced effects by > 1log in 6/8 patients were observed in the 10 mg dosing group.
⇒ 7/40 showed > 1log HBV reduction in lower dosing groups.
AASLD 2014 Boston, SIG HBV, Nov. 10thS Urban Heidelberg U & MyrGmbH
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Lucifora et al, Science 2014
Zoulim, et al, Clin
Gastroenterol Hepatol 2013
Belloni et al, JCI 2012
Koeniger etal, PNAS 2014
Tropberger et al, PNAS 2015
Hepatocyte turn-over
cccDNA silencing
cccDNA
degradation
cccDNA
formation
Targeting cccDNA
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Lucifora et al, Science 2014; Shlomai & Rice, Science 2014
Model for cccDNA degradation
IFNalpha /Lymphotoxin beta can induce APOBEC3A/B dependent
degradation of HBV cccDNA
Similar observation with IFNγ and TNFα – Xia et al, Gastroenterology 2015
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Epigenetics of covalently closed circular (ccc)DNA
Regulation by viral proteins (HBc and HBx)
Michael Nassal Gut doi:10.1136/gutjnl-2015-309809
Pollicino et al. Gastroenteroplogy 2006
Levrero et al. J Hepatol, 2009
Lucifora et al, J Hepatol 2012
Belloni et al, PNAS 20O9
Belloni et al, J Clin Invest 2012
Strubin et al, HBV conference 2015
Silencing
Interferon alpha,
Capsid inhibitors,
Epigenome modifyers
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Challenges in targeting cccDNA
• cccDNA formation: involves nuclear enzyme / DNA repair machinery
• cccDNA degradation: is the whole pool of cccDNA susceptible to
degradation ? will all infected cells be susceptible ?
• cccDNA damage: CRISPR/cas9 technologies and others. Issues
with delivery ?
• cccDNA silencing: targeting virus-specific mechanisms to avoid
safety issues
• Hepatocyte turn-over: may trigger the clonal selection of
hepatocytes in the context of an oncogenic virus
• Small molecules needed !
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Targeting the HBV capsid
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Targeting HBV nucleocapsids
Deres et al, Science 2003
Klumpp et al, PNAS 2015
Heteroaryldihydropyrimidines
Destabilization of nucleocapsids
Phenylpropenamide derivatives
Prevent pgRNA encapsidation
Antimicrob Agents Chemother. 2002.
Novel classes of capsid inhibitors based on the 3D structure of HBc
Novira, Assembly Biosciences, Janssen, Roche, and others
Phase 1 studies with Novira completed
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Effective reduction of serum HBV DNA by NVR 3-778(Novira)
All treatments reduced HBV DNA
ETV and NVR 3-778
monotherapies show similar
antiviral activity (p>0.05)
PEG-IFN + NVR 3-778
combination provides
highest antiviral efficacy
5/5 mice achieve serum HBV
DNA BLQ
25
Lam A, et al. AASLD 2015, San Francisco. #33
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Phase 1b clinical trial: NVR 3-778 reduces serum HBV DNA and RNA (Novira)
Serum HBV DNA: mean 1.7 log reduction (600 mg BID)
Serum HBV RNA: mean 0.86 log reduction (600 mg BID)
26
Cohort I: 600 mg BID
mean serum HBV RNA change from baseline
Yuen M-F, et al. AASLD 2015, San Francisco. #LB-10
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mRNA
mRNAdegradation
dsRNAdicer
Cleavage
Strand separation
Complementary pairing
Cleavage
(A)n
(A)n
Natural Process of RNAi
Synthetic siRNA
Targeted Gene
Silencing
RISC
RNA Interference
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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
Wooddell CI, et al. AASLD 2015, San Francisco.
#32
Total Liver HBV DNA cccDNA
Pre-
study
After NUC
lead-in
Pre-
study
After NUC
lead-in
108
107
106
105
104
103
102
HBeAg
+
HBeAg-
LLOQ
Liv
er
HB
V D
NA
(G
E/µ
g)
� ARC-520 reduced total liver DNA and
cccDNA beyond levels achieved in
HBeAg-pos NUC treatment during lead-in
� ARC-520 and NOT NUC reduced
intrahepatic HBV RNA and antigens
109
108
107
106
105
104
HBeAg+ HBeAg-
HB
V m
RN
A in liv
er
(copy/µ
g)
Total HBV RNAPre-core/Core RNA1000
Weig
hte
d R
NA
reads
HBeAg+ HBeAg-
100
10
1
Total HBV RNAPre-core/Core RNA
50%
5%
49%
5%
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ARC-520 produces deep and durable knockdown of viral antigens and DNA in a phase II study in patients with chronic hepatitis B
Yuen M-F, et al. AASLD 2015, San Francisco. #LB-9
Small dose-related reduction in HBsAg
Maximum effective dose not reached
HBV DNA results pending in ETV naïve patients
-1.4
-1.2
-1
-0.8
-0.6
-0.4
-0.2
0
0.2
-10.00 10.00 30.00 50.00 70.00 90.00
Log
Re
du
ctio
n i
n v
ira
l a
nti
ge
n
Days
HBsAg PBO HBsAg ARC-520 HBeAg PBO
HBeAg ARC-520 HBcrAg PBO HBcrAg ARC-520
HBsAg reduction in ETV naïve
patients with a single 4 mg dose
(cohort 7)
HBV antigen reduction in ETV
experienced HBeAg-positive
patients with a single 4 mg dose
(cohort 5)
Direct antiviral effect lasted up to 57 days after
a single dose of ARC-520, delayed response
duration >85 days
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Restoration of antiviral immunity
Bertoletti A, Gehring AJ (2013) Immune Therapeutic Strategies in Chronic Hepatitis B Virus Infection: Virus or Inflammation
Control?. PLoS Pathog 9(12): e1003784. doi:10.1371/journal.ppat.1003784
http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003784
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Antiviral activity of a TLR7 agonist
in HBV infected chimpanzees
Lanford et al, Gastroenterology 2013
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The Oral Toll-Like Receptor-7 Agonist GS-9620 in
Patients with Chronic Hepatitis B Virus Infection
Gane et al, Journal of Hepatology, 2015
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Effective T-cells control virus Exhausted T-cells lose control of
virus
CD8 T cells
Infected hepatocytes Infected hepatocytes
INF-γ
TNF-α
IL-2
Granzyme
Perforin
Can an effective antiviral T-cell response be recovered?
Patients who have resolved HBV Patients with chronic HBV
Restoration of defective T-cell immune control
Figure adapted from Nebbia G, et al. Q J Med 2012;105:109–13 and Freeman G, et.al. J Exp Med 2006;203(10):2223–7.
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Effective T-cells control virus Exhausted T-cells lose control of
virus
CD8 T cells
Infected hepatocytes Infected hepatocytes
INF-γ
TNF-α
IL-2
Granzyme
Perforin
Prolonged nucleotide therapy in a subset of patients1
Patients who have resolved HBV Patients with chronic HBV
Restoration of defective T-cell immune control
1. Boni C, et al. Gastroenterology 2012;143:963–73
Figure adapted from Nebbia G, et al. Q J Med 2012;105:109–13 and Freeman G, et.al. J Exp Med 2006;203(10):2223–7.
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Effective T-cells control virus Exhausted T-cells lose control of
virus
CD8 T cells
Infected hepatocytes Infected hepatocytes
INF-γ
TNF-α
IL-2
Granzyme
Perforin
Specific immunomodulation of existing T-cells e.g. PD-1 blockade1,2
Patients who have resolved HBV Patients with chronic HBV
Restoration of defective T-cell immune control
1. Fisicaro P, et al. Gastroenterology 2010;138:682–93. 2. Fisicaro P, et al. Gastroenterology 2012;143:1576–85
Figure adapted from Nebbia G, et al. Q J Med 2012;105:109–13 and Freeman G, et.al. J Exp Med 2006;203(10):2223–7.
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Triple combination therapy of ETV treatment, DNA
vaccination and in vivo PD-L1 blockade in WHV infected
woodchucks
Liu J, Zhang E, Ma Z, Wu W, et al. (2014) Enhancing Virus-Specific Immunity In Vivo by Combining Therapeutic Vaccination and
PD-L1 Blockade in Chronic Hepadnaviral Infection. PLoS Pathog 10(1): e1003856. doi:10.1371/journal.ppat.1003856
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In vivo PD-L1 blockade synergizes with therapeutic
vaccination to control WHV replication
Liu J, Zhang E, Ma Z, Wu W, et al. (2014) Enhancing Virus-Specific Immunity In Vivo by Combining Therapeutic Vaccination and
PD-L1 Blockade in Chronic Hepadnaviral Infection. PLoS Pathog 10(1): e1003856. doi:10.1371/journal.ppat.1003856
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Antiviral
s
Therapy
HB
V D
NA
ch
an
ge
fro
m b
as
elin
e (
log
10
c/m
L)
0.0
-1.0
-2.0
-3.0
-4.0
+1.0
Time
New treatment concepts for a functional cure of HBV infection
HBsAg
HBVDNA
cccDNA
Immune
restoration
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Target & drug discovery to cure HBV infection
Immune modulation
• Toll-like receptors
agonists, Gilead,
Roche
• PD1 blockade,
BMS, Merck etc.
• Vaccine therapy
Transgene, Gilead,
Roche Innovio,
Medimmune, ITS
Zoulim F, et al. Antiviral Res 2012;96(2):256–9; HBF Drug Watch, Available at:
http://www.hepb.org/professionals/hbf_drug_watch.htm.
HBx
Endosome
rcDNA
cccDNA
Polymerase
pgRNA
Core
Surface
proteins
Entry inhibitors
• Lipopeptides, e.g.
Myrcludex-B
Targeting
cccDNA
Inhibition of nucleocapsid
assembly, Novira, AssemblyBiosc,
Gilead, Janssen, Roche
Polymerase
inhibitors
• Nucleoside
analogues, e.g.
Gilead, BMS
• Non-nucleoside,
e.g. LB80380
• RNAseH inhibitors
Targeting HBsAg
Mab, Gilead
Release, Replicor
RNA interference,
Arrowhead, Arbutus,
Alnylam, GSK
Cyclophilin
inhibitors
Arbutus
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Can we cure the liver disease ?
• Mechanisms of HBV-induced HCC involve several
factors: inflammation, fibrosis, chromosomal integration,
HBx etc.
• Improvement of liver inflammation & fibrosis with long-
term NUC
• HCC not always seen on a background of cirrhosis
• Clonal expansion of hepatocytes not supporting HBV
replication occurs even before cirrhosis
• NUCs decrease, but don’t eliminate, the risk of HCC
• Early treatment intervention & better antivirals needed
Zoulim & Mason, Gut 2012; 61 : 333-336
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Acknowledgements
Hepatology Unit INSERM U1052 Collaborations
David Durantel
Barbara Testoni
Julie Lucifora
Malika Ait-Goughoulte
Souphalone Luangsay
Marion Gruffaz
Nathalie Isorce
Fanny Lebossé
Maelenn Fournier
Maud Michelet
Judith FresquetLabEx
C. Caux, Lyon CRCL
FL. Cosset, Lyon CIRI
K. Lacombe, Paris
M. Levrero, Rome/Lyon
JP Quivy, Institut Curie
IHU
Maelle Locatelli
Valentina d’Arienza
Pascal Jalaguier
Thomas Lahlali
Dulce Alafaiate
Lucyna Cova
Romain Parent
Anna Salvetti
Birke Bartosch
Eve Pecheur
Boyan Grigorov
Christophe Combet
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Third ANRS “HBV cure” Workshop HBV pathobiology and target discovery
Scientific coordination: Fabien Zoulim
Tuesday, May 31st, 2016
Union internationale des chemins de fer (UIC) 16, rue Jean Rey - 75015 PARIS
HBV cure 2014: Zeisel, M. B. et al. Towards an HBV cure: state-of-the-art and unresolved
questions-report of the ANRS workshop on HBV cure. Gut, doi:10.1136/gutjnl-2014-308943 (2015).
HBV cure 2015: http://www.anrs-hbvcure2015.com/
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15/12/2015 43
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348 HBeAg(+) and HBeAg(-) CHB patients from phase 3 studies who enrolled in a
long-term rollover study were evaluated for long-term liver histology outcomes51% of
patients had regression of fibrosis, including 71/96 patients with cirrhosis
(Ishak score ≥ 5) at phase 3 study baseline
Regression of fibrosis and cirrhosis during long-term Tenofovir therapy
Marcellin P, et al. Lancet 2013;381:468–75.
Ishak Score
0
20
40
60
80
100
Baseline Year 1 Year 5
6
5
4
3
2
1
0
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Japanese cohorts: Entecavir reduced HCC incidence, compared with controls
PS-matched cohort multivariate cox regression analysis:*
HR 0.37 (95% CI 0.15–0.91) p = 0.030
*Adjusted for age, sex, alcohol, smoking, cirrhosis, HBV genotype, HBeAg status, HBV-DNA, ALT, albumin,
γGTP, total bilirubin and platelet count.
Cum
ula
tive H
CC
rate
s (
%)
Log-rank test: p<0.001
Treatment duration (years)
0
10
20
30
7.2%
13.7%
3.7%
1.2%
0 1 3 5 72 4 6
No. at risk
ETV
Control316
316
316
316
264
277
185
246
101
223
44
200
2
187
2
170
Adapted from Hosaka T, et al. Hepatology 2013 [Epub ahead of print]. doi: 10.1002/hep.26180.
HR, hazard ratio; PS, propensity score
Control
ETV
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HCC occurence in HBsAg loss patients
Simonetti et al, Hepatology 2010
The incidence of HCC after clearance of HBsAg was 36.8 per 100,000
person-year (95% CI 13.5-80.0) which was significantly lower than the rate
in those who remained HBsAg-positive (195.7 cases per 100,000 person-
years of follow-up [95% CI 141.1-264.5; P < 0.001])
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Liver Damage and HBV infection
HCC not always seen on a background of cirrhosis
Liver damage results of immune killing of
hepatocytes
Clonal expansion of hepatocytes not supporting
HBV replication occurs even before cirrhosis
Experimental models show that clonal hepatocyte
repopulation is a major risk factor for HCC
Zoulim & Mason, Gut 2012; 61 : 333-336
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Mechanisms of HBV-related hepatocarcinogenesis
1. Lupberger J, Hildt E. World J Gastroenterol. 2007;13:74–81; 2. But DYK, et al. World J Gastroenterol. 2008;14:1652–6; 3. Neuvert C, et al. J
Hepatol. 2010;52:594–604; 4. Tan YJ. World J Gastroenterol. 2011;17:4853–7; 5. Wong DKH, et al. J Hepatol. 2006;45:553–9;
6. Friedman SL. Gastroenterol. 2008;134:1655–69.
Chronic liver
inflammation,
hepatocyte injury,
proliferating fibroblasts,
fibrosis/cirrhosis
Hepatocellular carcinoma
Chronic HBV Infection
Viral replication
Host DNA mutations
due to high hepatocyte
turnover, cytokine and
growth factor release
Chromosomal
integrationHBV protein X
Chromosomal
instability, altered gene
expression or function
Interference with cellular
transcription and
signaling pathways
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Mason W S et al. Proc. Natl. Acad. Sci. USA 2005; J Virol 2009 & 2010
Analysis of liver tissue for variability in levels of HBV infection - Chimpanzee
Analysis of liver fragments for clonal expansion of cells by serial dilution - woodchucks
Similar observations in HBV infected
chimpanzees and patients
Clonal expansion of hepatocytes not supporting
active viral replication
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� 48 weeks of ADV resulted in significant reductions in :
serum HBV DNA > total intrahepatic HBV DNA > cccDNA
> 14 years of therapy to clear completely viral cccDNA
Werle et al, Gastroenterology 2004
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� 0.8 log10 (84%) decline in cccDNA, not paralleled by a similar decline in the
number of HBcAg+ cells
� Suggests cccDNA depleted primarily by non-cytopathic mechanisms or new
rounds of hepatocyte infection occurred during therapy
Baseline Week 48
Werle et al, Gastroenterology 2004
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Koeniger et al, PNAS 2014
cccDNA formation: identification of TDP2
TDP2: Tyrosyl DNA Phosphodiesterase
Cortes Ledesma et al, Nature 2009
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53
Targeting Hepatitis B Virus With CRISPR/Cas9
Mol Ther Nucleic Acids. 2014; Seeger et al
Induction of deletions in cccDNA
Decreased number of cells expressing viral antigens
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Histone modifications, Pol2 binding and transcriptional profile of cccDNA
Tropberger et al, PNAS 2015
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In vivo proliferation of hepadnavirus‐infected hepatocytes induces loss of cccDNA in mice
Dandri et al, Hepatology 2010