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v
Professor Massimo Levrero
Sapienza University of Rome, Italy
18 th Annual Resistance and
Antiviral Therapy Meeting
Thursday 18 September 2014, Royal College of Physicians, London
Massimo LevreroDipartimento di Medicina Interna e Specialita’ Mediche - DMISM
IIT - Center for Life-Nanoscience
Sapienza Universita’ di Roma
Can we eradicate chronic hepatitis B infection?
Dipartimento dimedicina interna e specialitÀ mediche
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Disclosures
● BMS: Advisory Board and invited speaker; investigator
● Gilead: Advisory Board and invited speaker; investigator
● Janssen: Advisory Board and invited speaker, investigator
● MSD: Advisory Board and invited speaker; investigator
● Roche: Consultancy; nvited speaker; investigator
● Tekmira: Advisory Board
Outline
1. Concepts on HBV cure
2. HBV cccDNA as a minichromosome: HBV replication is
controlled by epigenetic mechanisms
1. modulation of cccDNA function by epigenetic drugs
2. anti-capsid drugs also target cccDNA
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3
H
Host cell
cccDNA
Host DNA
Integrated DNA
Nucleus
Long-term suppression
of viral replication
(DAA)
Adapted from 1. Soriano V, et al. J Antimicrob Chemother 2008;62:1-4. 2. Locarnini S and Zoulim F. Antiviral Therapy
2010;15 (suppl 3):3-14. 3. Sarrazin C and Zeuzem S. Gastroenterology 2010;138:447-462.
HBV the concept of « cure »
Viral Suppression
Functional cure (very few)
Eradication
�. anti-HBV therapies achieve ~ 100 % long term on
treatment suppression but only 20 to 15 % off treatment
control with IFN (12 months) or NUCs (3 to 5 years) off
treatment sustained viral suppression .. adherence
Suppression
of viral replication
Immune control
(IFNα)
• High rates of regression or
halting of fibrosis/cirrhosis with
TDF/ETV
• Established safety profile up to
Year 7 of treatment
• Resistance with some
antivirals (LAM, ADV)
• Risk of HCC persists
• Persistence of cccDNA (even
after therapy or spontaneous
recovery)
- Viral reactivation
- Hepatocarcinogenesis
• Integrated viral genome
- Hepatocarcinogenesis
HBV1,2
HBV challenges
• Treat more effectively: HBV and HCC
• Develop/use tools to Predict Treatment Outcomes– qHBsAg
– qHBeAg
• Aim for cure
– Functional cure
• off-therapy persistent HBV suppression
– cccDNA eradication
• surrogate endpoint HBsAg loss and anti-HBs seroconversion
• Strategies
– Immune system
– Viral targets
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HBV life cicle
from Nassal et al, Virus Research 2008
• HBV hepatic “latent” reservoires (non integrated functionally
competent HBV genomes) is now established – OBI and more
• extra-hepatic reservoires described & but never formally
established &. cccDNA presence unproven
cccDNA half-life: supposedly long, not established
Antivirals do not directly target cccDNA
Modified from Nassal et al, Virus Research 2008
?
1 yr of monotherapy with nucleos(t)ide analogues (ADV, LAM, ETV)
reduced median intrahepatic cccDNA amounts by 1 log
Zoulim,Petersen,Locarnini, Gastroenterology 2004,
Wong, Antivir Ther 2006, Sung, Gastroenterology 2005
No data on IFN-a monotherapy
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Persistence of cccDNA
Belloni, Levrero, Gaeta HBV meeting 2010
B2B1 B3 B4 C2C1 N1 N2
pg
RN
Acp
/ng
cD
NA
0
0.02
0.04
0.06
0.08
0.10
0
0.25
0.50
0.75
1.0
cccD
NA
co
pie
s/c
ell
1149.4neg>250
HBsAg
Persistence of cccDNA in 3 out of 4 patients with long
term HBV suppression under lamivudine
In 2 out 3 patients cccDNA is inactive (no pgRNA)
• Detected in the liver of NUCs long-term suppressed patients after HBsAg to anti-HBs
seroconversion [Maynard, 2005; Belloni unpublished]
• Detected in the liver of HBsAg negative patients (occult HBV infection)
[Werle-Lapostolle, 2004; Pollicino unpublished]
• Present in 30 /30 patients with occult HBV infection and HCC [Pollicino, 2004]
liver tissue
Huh7 or HepG2 cells
transient transfection of
linear full-length HBV monomers
HBV
cccDNA as a minichromosome: the cccDNA ChIP assays
Pollicino et al. Gastroenteroplogy 2006
Levrero et al. J Hepatol, 2009
Belloni, PNAS 2009
Belloni, JCI 2012
A methodology to study cccDNA function
[ HBV minichromosome structure ]
[ modifications of cccDNA bound histones ]
[ binding of TF and coregulators ]
in vitro,
in animal models
ex vivo (liver samples/biopsies)
PCR or real time PCR
with cccDNA
specific primers
crosslink
sonicate
reverse crosslink
purify DNA
Reference
input DNA
immunoprecipitate
with specific antibodies
Massive Parallel
Sequencing
(ChIP-Seq)
1) cccDNA chromatin Immuno
Precipitation assay (ChIP)
2) PCR-based method that allows
cccDNA quantitation
3) transient transfection of linear
HBV full-length genomes into HuH7
hepatoma cells
Bock, T. et al 1994.
Bock, T. et al 2001.
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Studying cccDNA back in 2004
from Nassal et al, Virus Research 2008
- No reliable infection system
- HBV transgenic mice: cccDNA (-)
- HBV stable cell lines (2.2.15) cccDNA +/-
- HBV Transfection:
• 1.2-1.3 HBV constructs: cccDNA +/- or (-)
• 1.0 HBV linear genome: cccDNA ++
1.0 HBV1.2-1.3 HBV or
HBV DNA transfection
cccDNA
formation
cccDNA
transcription
Epigenetic marks
of open and condensed chromatin
Arrowsmith CH et al., Nat Rev Drug Discov. 2012
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HBV cccDNA ChIP assay
• HBV replication parallels the acetylation status of HBV
cccDNA-bound H3 and H4 histones in HBV replicating cells
and in vivo in patients
• Transcription factors and transcriptional coactivators and
repressors are recruited onto cccDNA
– Stat1/2, Stat3, HNF1a, HNF4a, p65, YY1, FXR
– HATs (p300, PCAF, CBP)
– HDACs (HDAC1, hSirt1)
– HMTs (Ezh2); PRMTs (PMRT1; PMRT5) DNA-MTs (DNMT3a)
• Viral proteins bind to and modulate cccDNA functions
– HBx (increases/required for transcription; increase p300 and
prevents HDAC1 binding)
– HBc
Pollicino, Gastroenterology 2006; Belloni, PNAS 2009; Levrero, J Hepatol 2009; Belloni, JCI 2012
PCAF
Sirt1 Sirt1
HDAC1HDAC1
HistonesTFTF
TF TF
PCAF p300
TF TF
HistonesTF
PCAFp300
TF
HBx
TF TF
PCAF p300
TF
HBx
TF
Active cccDNA
Inactive cccDNA
Acetylation of cccDNA-Bound H3 and H4 Correlates to
HBV Viremia Levels in Chronic Hepatitis B Patients
ChIP of liver nuclear extracts from
10 HBsAg-posi tive CH pts
using specific antibodies
to AcH3, AcH4, HDAc1 or control IgG
A. B.
Serum HBV DNA quantification in HBsAg-positive pts with
- active (AcH3 - AcH4 positive/HDAc1 negative, 4 cases)
(AcH3-AcH4 positive/HDAc1 positive, 2 cases)
- suppressed (AcH3-AcH4 negative/ HDAc1positive, 4 cases)
HBV replication. P value: Wilcoxon rank sum test.
Pollicino et al., Gastroenterology, 2006
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spontaneously
iatrogenic
immunosuppression
iatrogenic
immunosuppression
tolerance chronic hepatitis inactive carrier pre-core mt occult HBV
0,001
0,01
0,1
1
10
100
1000
DNA
cccDNA status in HBV patients
Histones
PCAFp300 PCAF
p300
High Replication Low Replication
Sirt1
Sirt1HDAC1
HDAC1
Histones
Ezh2
Sirt1
Sirt1HDAC1
HDAC1
Histones
HP1MeCP2
Suv39
Occult HBV
Input 1 2 3 4 5 6 IgG
ChIP
Low-replicative to latent infectionEpigenetic control
Pollicino et al., Gastroenterology 2006;
Belloni et al., HBV meeting 2006
Pollicino, unpublished
cccDNA ChIP assay in vitro and in vivo
HBV replication models
HepG2,
HepaRG
PHH
wt HBV
cccDNA
Bac-HBV-1.1
uPA/SCID mice
in vivo
HBV infection
Human Hepatocytes in uPA mice[Belloni, 2012; Belloni & Allweiss, unpublished]
• cccDNA detected
• H3 and AcH3 ChIP performed
• infection efficiency: cccDNA levels required 0.5-1 cp/cell
Bac-HBV transduction of HepaRG cells[Lucifora, 2008]
• cccDNA formed from nucleocapsid recycling
• AcH3/H4 ChIP positive
HBV infection of PHH or HepaRG cells[Lucifora, 2011; Sonnabend & Belloni, unpublished]
• cccDNA detected
• H3 and AcH3 ChIP performed
• infection efficiency as limitation
0.2 cccDNA/cell = cccDNA ChIP LOD
0.5-1.0 cccDNA/cell = multiple parameters cccDNA ChIP
wt HBV
cccDNANTCP-HepG2
HBV infection of NTCP-HepG2 cells[Sonnabend & Belloni, unpublished]
• cccDNA detected
• H3 and AcH3 ChIP performed
Liver biopsies
Human Hepatocytes[Pollicino, 2006; Belloni, Pollicino, Guerrieri, unpublished]
• cccDNA detected
• H3, AcH3, HDACs, HMTs ChIP performed
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cccDNA
formation
cccDNA Y Y Y N N N Y/N
cccDNA
chromatin
assembly
cccDNA
ChIPY/N * Y/N* Y/N* N Y N Y
cccDNA
chromatin
function
cccDNA
ChIPY/N* Y/N* Y/N* Y/N* Y Y/N Y
cccDNA
stability
cccDNA Y/N Y/N Y Y/N Y/N Y N
cccDNA
transcription
pgRNA Y Y Y Y Y Y/N Y/N
Core particles
recyclingY Y Y/N Y/N N Y Y
HBV
replication
Cp
HBV-
DNA
Y Y Y Y Y Y Y
Chronic
infectionN Y/N Y Y N Y/N Y/N
HepaRG
PHH
[infection]
NTCP
HepG2
HepaRG
[infection]
uPA/SCID
Mice
[infection]
HBV
isolated
hepatocytes
uPA mice
[ex vivo]
HepG2
HBV 1.0
[transfection]
HepG2
HBV H1.3
[stable]
HepG2
AD38
[inducible]
* inferred; direct study limited by cccDNA levels
HBV models for cccDNA
HBV cure
• Aim for cure
– Functional cure
Off-therapy persistent HBV suppression
– cccDNA eradication
• Strategies
- Immune system
- Viral targets
cccDNA degradation
cccDNA silencing
cccDNA dilution
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Adapted from Nassal M et al. Virus Res 2008;134:235–249;
Grimm D et al. Hepatol Int 2011;5:644–653.
cccDNA formation
inhibitors
Nucleocapsid assembly
inhibitors
cccDNA transcription
Inhibitors
Emerging antiviral approaches
• cccDNA - silencing
- degradation
• pgRNA - siRNAs
• sub-genomic RNAs - siRNAs
• RNAse H inhibitors
• “capsid”- Core protein Assembly
Modulators (CpAMs)
• Entry inhibitors (HBV and HDV)
• Prenylation inhibition (HDV)
• HBsAg release inhibitors
• Cyclophilin inhibitors
• TLR agonists
• Therapeutic vaccination
anti-HBV siRNAs
cccDNA
destabilization/degradation
Adapted from Petersen J et al. Hepatol Rev 2007;4:9–13 and Levrero M et al. J Hepatol 2009;51:581–592.
Silencing cccDNA
“functional cure” – off therapy control
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IFNα targets the HBV cccDNA
Interferon-α (IFNα) inhibits HBV transcription and
replication in vitro and in vivo by favoring the long
term recruitment to the nuclear cccDNA mini-
chromosome of the class III HDAC hSirt1 and of
the PRC2 repressive complex, including the
transcriptional co-repressors HDAC1 and Ezh2
RBAP48
Suz12
Ezh1
Me Me
YY1
PRC2 complex
EED
Ezh2
Ac
Ac
IFNa (1000 UI/ml)
NT
Belloni et al., JCI 2012
Effects of IFNα treatment on HBV
replication and transcription confirmed
in humanized uPA/SCID mice
The HBV ISRE mediates IFNα transcriptional
repression
Pollicino T et al. Gastroenterology 2006;130:823–837; Adapted from Levrero M et al. J Hepatol 2009;51:581–592;Belloni L et al. J Clin Invest 2012;122:529–537.
Histone acetylation/methylation
affects the regulation of gene expressionIFNα treatment is accompanied by a
decrease in the acetylation of cccDNA
bound H4 histones in vitro
PCAFPCAF
p300p300
Sirt1Ezh2YY1HDAC1
acetylated histones
deacetylated histones
+ IFNα
HBx mt
Transcription of the HBV cccDNA mini-
chromosome can be regulated epigenetically
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IFNα targets the HBV cccDNA
Our results indicate that IFNα induces a
condition of “active epigenetic control” of
HBV cccDNA transcription that likely
contributes to the persistent, yet reversible,
“off therapy” inhibition of HBV replication (30-
35% of HBeAg+ patients and 20-25% of
HBeAg- patients) and suggest that HBV
cccDNA function can be modulated by
epigenetic drugs
RBAP48
Suz12
Ezh1
Me Me
YY1
PRC2 complex
EED
Ezh2
Ac
Ac
HBV-specific CD8
CMV-specific CD8
Micco L et al J. Hepatol. 2012Belloni et al., JCI 2012
HepAD38 cells
HBV stable clone
Ladner 1997cccDNA
- Tet
days0 36
Cp-HBV-DNA
pgRNA
Drug
Ezh2JMD3
MC3119
Me Me
Ezh2
Ac
Ac
Palumbo GA et al. AASLD 2013 Abs 928
Ladner SK et al. Antimicrob Agents and Chemo1997;41:1715-20.
Modulation of Ezh2 histone methyltransferase
activity mimics IFNα-induced repression of
cccDNA transcription
0
0.2
0.4
0.6
0.8
1
1.2
2^
-(CtpgRNA
-Ctb
eta
ctin
a)
pgRNAChIP
α-Me3K27H3
Fold
in
du
ctio
n
0
1
2
3
4
0
0.2
0.4
0.6
0.8
1
1.2
HB
V D
NA
co
pie
s/ce
ll
Cp HBV-DNA
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• Modulation of Ezh2 activity by MC3119 mimics IFNa-induced
transcriptional repression of the cccDNA.
• PERSPECTIVE:
- explore sequential treatments as a model for IFN sparing regimens
- increase the subset of IFN SVRs
Make active carriers „true“ inactive
and, eventually, over time „occult“
carriers by „locking“ the cccDNA
Proof of concept of modulation of cccDNA function
by “epigenetic” compounds
Palumbo GA et al. AASLD 2013 Abs 928
Proof of concept of modulation of cccDNA function
by “epigenetic” compounds
TFn1TFn2
Ac AcAc
Ac
• Inhibitors of PCAF/p300 and activators of
hSirt1/2 display the most significant
effects on cccDNA transcription and HBV
replication
• Inhibition of cccDNA bound HATs activity
leads to detachment of (PCAF) and p300
• The hSirt1/2 agonist induce a shift in
hSirt1/ hSirt2 occupancy and global
cccDNA-bound H4 deacetylation
• These results provide a proof of concept
that small molecules / drugs that affect
cccDNA bound chromatin modifying
enzymes can modulate HBV transcription
and replication
• PERSPECTIVE
• - possible synergisms ??
TFn1TFn2
Ac Ac
Ac
Ac
Active cccDNA
Suppressed cccDNA
Palumbo GA et al. AASLD 2013 Abs 928
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HBc protein / capsid
HBV capsid
(120 HBc dimers)
Bock, 2001
HBc dimer
Input
aHBc
IgG
wt
mock
aHBc
5
10
15
20
Arb
itra
ry U
nit
s
Belloni 2009
� HBc binds the cccDNA and regulates its function
HBc protein / capsid
HBV capsid
(120 HBc dimers)
Bock, 2001
HBc dimer
Input
aHBc
IgG
wt
mock
aHBc
5
10
15
20
Arb
itra
ry U
nit
s
Belloni 2009
ChIP aHBc
Lupacchini (unpublished)
� HBc binds the cccDNA and regulates its function
� HBc binds to cellular promoters and regulates gene expression
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HBV capsid
(120 HBc dimers)
HBc dimer
HAP12
� The Hap12 Core Protein Assembly Modulator (CpAM)
• inhibit HBV replication
CpAMs
HBV capsid
(120 HBc dimers)
HBc dimer
HAP12
� The Hap12 Core Protein Assembly Modulator (CpAM)
• inhibit HBV replication
• target the cccDNA
NT
1µM HAP
Palumbo (unpublished)
CpAMs
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HBV capsid
(120 HBc dimers)
HBc dimer
HAP12
� The Hap12 Core Protein Assembly Modulator (CpAM)
• inhibit HBV replication
• target the cccDNA
• affect HBc occupancy on both the cccDNA and cellular promoters
NT
1µM HAP
Palumbo (unpublished)
CpAMs
HEPATITIS B CORE (HBC) PROTEIN IS A KEY AND VERY EARLY
NEGATIVE REGULATOR OF THE INTERFERON RESPONSE
HBc affects the epigenetic state of the host genome to control innate immunity- very early inhibition of the hepatocyte IFN response by core protein through the recruitment of
epigenome-modifying enzymes (Ezh2 and G9 HMTs) that leads to repressive marks (H3K27me3
and H3K9me3)
Gruffaz M, Testoni B et al AASLD 2013 Abs 136
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HBV capsid
(120 HBc dimers)
HBc dimer
HAP12
� The Hap12 Core Protein Assembly Modulator (CpAM)
• inhibit HBV replication
• target the cccDNA
• affect HBc occupancy on both the cccDNA and cellular promoters
NT
1µM HAP
Palumbo (unpublished)
CpAMs
More assembly
Products are normal.ish
more free dimers
More assembly
Larger products
Different CpAMs – Different Results ?
Adapted from Zlotnick, ICAR 2014
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CpAMs Conclusions
● HAP12 “early treatment” inhibits cccDNA accumulation and
HBV replication
[direct effect on cccDNA or NUC-like effect on capsid recycling ?]
● HAP12 treatment affects significantly an existing cccDNA pool
[experiments in HepG2-NTCP stable infection needed]
● HAP12 treatment apparently does not change HBc nuclear
levels
[how it works ?]
● HAP12 treatment effect on cccDNA transcription “possible”
but not “proven”
Lucifora et al. Science 343, 1221-8, 2014
- Interferon-α and lymphotoxin-β-receptor
activation up-regulated APOBEC3A and 3B
cytidine-deaminases, respectively, in HBV-
infected cells, primary hepatocytes and
human liver-needle biopsies.
- HBV-core protein mediates the interaction
with nuclear cccDNA resulting in cytidine-
deamination, apurinic/apyrimidinic site
formation and finally cccDNA degradation
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Lucifora et al. Science 343, 1221-8, 2014
- Interferon-α and lymphotoxin-β-receptor
activation up-regulated APOBEC3A and 3B
cytidine-deaminases, respectively, in HBV-
infected cells, primary hepatocytes and
human liver-needle biopsies.
- HBV-core protein mediates the interaction
with nuclear cccDNA resulting in cytidine-
deamination, apurinic/apyrimidinic site
formation and finally cccDNA degradation
Targeted gene disruption strategies
Recognized challenge: delivery vehicle
� Zinc Finger
� Talen
� CRISP / Cas
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Targeted gene disruption strategies
Recognized challenge: delivery vehicle
� Zinc Finger
� Talen
� CRISP / Cas
� TALENs treatment
reduces cccDNA level in
vitro and and HBV pgRNA
in vivo
Chen, Mol Therapies, 2014
� Inhibition of HBV replicazion with ZNF in Hep AD38 cells Weber, Plos1, 2014
Conclusions
● Still striving for finite therapy (“functional cure”)
�. “where the old and the new ways meet”;
● In theory, the ideal goal of antiviral therapy for CHB
would be complete HBV elimination including complete
eradication of cccDNA from infected hepatocytes
.� “a new beginning” � and keep open mind and acombinatorial attitude (HAPs+NUCs; HAPs+Mycludex �.)
● Lack of adequate in vitro replication models to study
cccDNA
�. “one model does not fit all �. yet”
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Future directions: target & drug discovery to cure HBV infection
Immune modulation
• Toll-like receptors
agonists, Gilead,
Roche
• Anti-PD-1 mAb,
BMS, Merck
• Vaccine therapy
Transgene, Gilead,
Roche Innovio,
Medimmune, ITS
Development stage: preclinical, clinical
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, Assemblyparm, Gilead, Janssen
Polymerase inhibitors
• Nucleoside
analogues, e.g.
Gilead, BMS
• Non-nucleoside,
e.g. LB80380
Inhibitors of HBsAg
release, ReplicorRNA interference,
Arrowhead, Tekmira,
Alnylam, GSK
What Might HBV Cure Will Look Like?
Potent NA
cccDNA
Inhibitor
Immune
Activator
HBV
Antigen
Inhibitor
⊕
⊕
⊕
to prevent viral spread and cccDNA re-amplification
safe and selective agent to reduce or silence cccDNA
agent(s) to activate specific antiviral immune
responses or relieve repression/exhaustion of the
immune system
agent(s) to block/inhibit the HBV life-cycle [entry,
cell-spread, capsid assembly, HBx, HBeAg, HBsAg]
Modified from S. Locarnini 6.2014
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Laura Belloni Natalia Pediconi
Francesca Guerrieri Cecilia Scisciani
Aurora Palumbo Ludovica Calvo
Letizia Cimino Barbara Testoni
Rossana DeIaco Valeria Schinzari
Massimo Levrero
Collaborations:
Dept. of Internal Medicine - University of Messina
Giovanni Raimondo
Teresa Pollicino
INSERM U761 -Lyon
Fabien Zoulim
Barbara Testoni
University Medical Hospital Hamburg
Jorg Petersen
Maura Dandri
TUM - Helmholtz Zentrum München
Ulrike Protzer
Julie Lucifora
Laboratory of Gene Expression
With the support of Fondazione
Andrea
Cesalpino
Anna TramontanoAndrea Sbardellati,
Daniel D’Andrea
Francesco Cicconardi
Collaborations at Sapienza:
Dept of Molecular & Cellular Biochemistry
Indiana University
Adam Zlotnick
Antonello MaiDaniela Secci
Dante Rotili
Sergio Valente
Dept of Molecular Virology – Heidelberg Univ Hospital
Jessika Sonnabend
Stephan Urban
Assembly Pharma
Uri Lopatin