HBV Cure?

The impact ofHBV DNAIntegration?

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Integration of HBV DNA is a key mechanismof HBV persistence

Expression of HBsAg from integrated HBV DNAsequences has been well demonstrated:

. Conservation of HBsAg-encodingsequences in most HBV DNA integrants

. Expression of HBsAg from cloned integratedHBV DNA genomes

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Characterization of integrated hepatitis B viral DNA cloned from a human hepatoma and the hepatoma-derived cell line PLC/PRF/5

ANNE DEJEAN, CHRISTIAN BRECHOT, PIERRE TIOLLAIS, AND SIMON WAIN-HOBSON

Proc. Natl Acad. Sci. USAVol. 80, pp. 2505-2509, May 1983

HBsAg expression upontransfection of HBV DNA integratedsequences

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HBsAg expression upontransfection of HBV DNA integratedsequences

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DevBiolStandard1983Vol 54

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

Wooddell et al., Sci. Transl. Med. 9, 27 September 2017

HBsAg reduction in ETV naive patients (single 4 mg dose (cohort 7))

HBsAg reduction in Chimpanzees

Courtesy: Fabien Zoulim

RNAi-based treatment of chronically infectedpatients and chimpanzees reveals that integratedhepatitis B virus DNA is a source of HBsAg

C.I.Woodell et alScience TranslationalMedecine 2017

HBV DNA integration into cellular DNA is an early eventand is associated to clonal hepatocyte expansion

Integration occurs very early during HBV infection:Acute in vitro and in vivo infection

A very high percentage of Woodchucks hepatocytesIs infected during acute infection

Liver cells containing integrated HBV DNA sequencesundergo clonal expansion from acute to chronic stages of the viral infection

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HBV DNA Integration and Clonal Hepatocyte Expansion in Chronic Hepatitis B Patients

Considered Immune TolerantWilliam S. Mason, Upkar S. Gill, Samuel Litwin, Yan Zhou, Suraj Peri, Oltin Pop,Michelle L.W. Hong, Sandhia Naik, Alberto Quaglia, Antonio Bertoletti, Patrick T.F.Kennedy Gastroenterology 2016

Inverse PCR for detection of clonal expansion of hepatocytes

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William S. Mason, et alGastroenterology2016

Around 5x10-6HBV

integrants/liver

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Clonal expansion of hepatocytes with a selective advantageoccurs during all stages of chronic hepatitis B virus infectionT. Tu et al ; J Viral Hepatitis 2015

Cloned Hepatocyte size independent of sites of HBV DNA integrationand HBsAg expression?

Clonal expansion in histologically normal hepatocytes

Mechanisms; molecular bases?

Detection of Clonally Expanded Hepatocytes in Chimpanzees and Woodchucks with Chronic HBV and WHV infectionsW S. Mason et al ; J Virol 2009; Proc Natl Acad Sci USA 2005

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- Novel integration sites generated through ongoingreplication of HBV DNADouble stranded linear DNA (dslDNA):

Mispriming eventsSplicing?

- Rearrengements of integrated HBV DNA sequences

- Novel integration events from previously integratedsequences (translocation etc..)

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HBV DNA integration is a dynamic process duringchronic infection

Interrupted Replication of Hepatitis B Virus in Liver Tissue of HBsAgCarriers with Hepatocellular Carcinoma

GIOVANNI RAIMONDO,* ROBERT D. BURK,t+ HARVEY M. LIEBERMAN,* JOSEPH MUSCHEL,tSTEPHANOS J. HADZIYANNIS,§ HANS WILL,” MICHAEL C. KEW,lI GEOFFREY M. DUSHEIKO,lTAND DAVID A. SHAFRITZVIROLOGY 166, 103-l 12 (1988)

- Blocked virus assembly/secretion and accumulation of unencapsidated HBV DNA replicative intermediates in the liver cell.

- Accumulation of such HBV DNA molecular forms in the liver may lead to an increasedpropensity for HBV DNA to integrate into the host genome

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Direct and indirect effects of HBV chronic infection

Liver carcinogenesis

Host immune responseInflammation; oxydative stress; fibrosis

Genetic instabilityActivation or inactivation of cellular genes

Prolonged expression of viral genesHBx, COOH-terminally deleted HBx

PreS1/PreS2/S: LHBsModulation of liver cell proliferation

and viability

Integration of HBV DNA:Translocations, chromosomalrearrengements

Insertional mutagenesisof cellular genes

HBV DNA integration : which impact?

Mechanisms of HBV DNA integration:. Random integration followed by selection of clonally

expanding cells ?

. Family of preferential sites for insertion?Fragiles sitesTranscriptionnally active genes (chromatin remodelling)

Insertion into cellular genes: cause or consequence of liver cellproliferation?Insertion in key driver genes for carcinogenesisInsertional mutagenesis of the target gene

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IP3

Ca2+

Ca2+

Ca2+

GrowthFactors

Cytoplasm

Ca2+Ca2+

EndoplasmicReticulum

hTERT

IP3R

Ca2+

Ca2+

S1

Ca2+Ca2+

Ca2+

HBV

Ca2+

MCM8

HBV

RARβ

HBV

CycA

NRTK 2

Ca2+

Ca2+

RB

TRUPHBVFR7

RASHBV

MEVAL. KINASE

E2FEMX1/2-like

TRANSCRIPTION

TR

TRAP150

NMP

MAPK1

IRAK2

Unique targetgenes:

RAR betaCyclin AMeval. kinaseMCMSERCA1TRAP150FR7EMX2-likeMAPK1IRAK2TRUPNRTK2Ras-REBP-1Calmodulin 1Recurrent

targetgenes:

hTERTIP3RMLL2

Ras-REBP-1

Calmodulin

p21

Paterlini-Brechot oncogene 2003, Murakami Gut 2005,

Insertional mutagenesisHBV insertion targets a variety of genesthat regulate key cellular pathways

Brechot Seminars in Cancer Biology 2001; Paterlini-Brechot Oncogene 2003, Saigo HumanMutation 2008; Murakami Gut 2005; Tamori Clin Cancer Res 2005

Alu

Cellular DNA

HBV

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Genome-wide survey of recurrent HBV integration in hepatocellular carcinoma

Massively parallel sequencing of 81 HBV-positive and 7 HBV-negative hepatocellular carcinomas (HCCs) and adjacent normal tissues.

HBV integration observed more frequently in the tumors (86.4%) than in adjacent liver tissues (30.7%).

Recurrent HBV integration events validated by RNA and Sanger sequencing at the known and putative cancer-related TERT, MLL4 and CCNE1 genes, which showed upregulated gene expression in tumor versus normal tissue.

Sung et al Nature 2013

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In total: 1377 Viral Integration Sites (VIS) within close proximity to protein coding genes and 767 with noncoding genes, respectively.

23.1% of protein-coding genes and 24.7% of long noncoding RNAs (lncRNA) targeted more than two times.

Only 4.8% of Viral Integration Sites common between HCC and non-tumor tissues. Total number of VIS in tumorous tissues: 2-fold higher than non-tumorous tissue.

The number of integration sites on each chromosome correlated with the number offragile sites in non-tumorous tissue but not in HCC tissue.Enrichment of cancer- related gene at or in close proximity to HBV integration sites in HCC tissue

Distinct hepatitis B virus integration patterns in hepatocellular carcinoma andadjacent normal liver tissueX Yang et al Int J Cancer 2017

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HBV DNA Integration into cellular DNA is a dynamic process

- Novel integration sites generated through ongoingreplication of HBV DNA

- Rearrengements of integrated HBV DNA sequences

- Novel integration events from previously integratedsequences (translocation etc..)

- Novel integrations generated through spliced HBV RNAs?

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dHBV particles and HBSP protein

2.2 Kb Alternative single splicing

SP1RNA

AAAA

AUG HBSP (Pol) Stop HBSP

AAAA

AUG Pol Stop PolpgRNA

Reverse transcription

Defective HBV particles

HBSP 12.4 kDa

Translation

2447 489

Soussan et al. JCI. 2000 ; Soussan et al. J.Hepatol. 2003 ; Kremsdorf et al. Oncogene. 2006; Mancini-Bourgine et al. J. Virol. 2007 ; Soussan et al. JID. 2008; Asrir et al. Frontiers in bioscience. 2010; Bayard et al. J.Viral.Hepat. 2012; Redelsperger et al. Virology. 2012; Pol et al. Faseb J. 2015.

dHBV particles and HBSP protein

Integration?

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HBSP contributes to limit liver immune recruitment

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*

02468

10

20

40

60

80

Duriez et alJournal of Hepatology 2017 vol. 67 j 687–699

Anti-HBSP: 40% of HBV chronic carriers

Patrick Soussan and Dina Kremsdorf

Novel pre-mRNA splicing of intronically integrated HBV generates oncogenic chimera in hepatocellular carcinoma Yung-Tuen Chiu et al; J Hepatol 2016

(HBV 282 and 458 nt.

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6/48 HCC vs 0/22 Non-HCC

The actual impact of HBV DNA persistence afterserum HBsAg negativation ?

« signature » of the HBV infection with no functionalconsequences?

Potential persistent risk?:

Reactivation of HBV multiplication?

HCC development?

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Systematic review with meta-analysis: development ofhepatocellular carcinoma in chronic hepatitis B patients withhepatitis B surface antigen seroclearanceF. Liu et al Aliment Pharmacol Ther 2016; 43: 1253–1261

Twenty-eight studies, involving 34 952 patients withHBsAg seroclearance.

The overall pooled proportion suggested that 2.29% (95% CI: 1.19–4.37%) CHB patients would develop HCC despiteHBsAg seroclearance.

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Decreased cumulative risk of HCC after HBsAg loss

Yuen et al, Gastroenterology 2008 29

The impact of occult HBV infections?A debated issue

Statements from the Taormina expert meeting on occult hepatitisB virus infections

Giovanni Raimondo, Jean-Pierre Allain, Maurizia R. Brunetto,Marie-Annick Buendia, Ding-Shinn Chen, Massimo Colombo, Antonio Craxı`,Francesco Donato, Carlo Ferrari, Giovanni B. Gaeta, Wolfram H. Gerlich, Massimo Levrero, Stephen Locarnini, Thomas Michalak, Mario U. Mondelli, Jean-Michel Pawlotsky, Teresa Pollicino, Daniele Prati, Massimo Puoti,Didier Samuel, Daniel Shouval, Antonina Smedile, Giovanni Squadrito,Christian Trepo, Erica Villa, Hans Will, Alessandro R. Zanetti, Fabien Zoulim

Journal of Hepatology 49 (2008) 652–657

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(HEPATOLOGY1993;17:20-29.)

Persistence of intrahepatic replicationin the liver of HBsAG negative patients

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IP3

Ca2+

Ca2+

Ca2+

GrowthFactors

Cytoplasm

Ca2+Ca2+

EndoplasmicReticulum

hTERT

IP3R

Ca2+

Ca2+

S1

Ca2+Ca2+

Ca2+

HBV

Ca2+

MCM8

HBV

RARβ

HBV

CycA

NRTK 2

Ca2+

Ca2+

RB

TRUPHBVFR7

RASHBV

MEVAL. KINASE

E2FEMX1/2-like

TRANSCRIPTION

TR

TRAP150

NMP

MAPK1

IRAK2

Unique targetgenes:

RAR betaCyclin AMeval. kinaseMCMSERCA1TRAP150FR7EMX2-likeMAPK1IRAK2TRUPNRTK2Ras-REBP-1Calmodulin 1Recurrent

targetgenes:

hTERTIP3RMLL2

Ras-REBP-1

Calmodulin

p21

Paterlini-Brechot oncogene 2003, Murakami Gut 2005,

Insertional mutagenesisHBV insertion targets a variety of genesthat regulate key cellular pathways

Brechot Seminars in Cancer Biology 2001; Paterlini-Brechot Oncogene 2003, Saigo HumanMutation 2008; Murakami Gut 2005; Tamori Clin Cancer Res 2005

Alu

Cellular DNA

HBV

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Hepatitis B virus (HBV) DNA integrationin patients with occult HBV infection and hepatocellular carcinoma

Carlo Saitta et alLiver International 2015; 35: 2311–2317.

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Integrated HBV DNA persistence

Low grade inflammation Direct effects

HCV

Alcohol

MetabolicDisorders(Diabetes,Obesity)

Iron overload

ChemicalsHCC

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Occult and previous hepatitis B virus infection are not associatedwith hepatocellular carcinoma in United States patients withchronic hepatitis C

Lok AS, Everhart JE, Di Bisceglie AM, Kim HY, Hussain M, Morgan TR;HALT-C Trial Group.

Hepatology. 2011 Aug;54(2)

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HBV DNA +vsHBV DNA –8.25 fold increasedrisk of HCC

Ikeda J Viral Hepatitis2009; 16: 437

Risk of HCC in HBsAg negative, HBV DNA positivepatients Prospective studies

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Intrahepatic hepatitis B virus replication and liver histology in subjects with occult hepatitis B infection

D. K.-H. Wong et al Clin Microbiol Infect 2015;

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pgRNA 5/39

TRANSMISSION OF HEPATITIS B FROM HEPATITIS-B-SERONEGATIVE SUBJECTSV. THIERs et al Lancet

Impact on infectiosity?

Persistence of HBV DNA in extra-hepatic sites?Hepadnavirus infection of peripheral blood lymphocytes in vivo: woodchuck and chimpanzee models of viral hepatitis. Korba et al J Virol 1986

Hepatitis B virus infection of peripheral blood mononuclear cells is common in acute and chronichepatitis. Pasquinelli et al J Med Virol 1990

Detection of mononuclear cells expressing hepatitis B virus in peripheral blood from HBsAgpositive and negative patients by in situ hybridization. Hadchouel et al J Med Virol 1988

Gene copy number variations in the leukocyte genome of hepatocellular carcinoma patients with integrated hepatitis B virus DNA; Y Pang et al Oncotarget 2016

Whole-genome comparative genomic hybridization (CGH) chip array analyses to screen genecopy number variations (CNV) in the leukocyte genome,

The results were confirmed by quantitative polymerase chain reaction (qPCR).

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Hepatitis B Virus DNA in Peripheral-blood Mononuclear Cells in Chronic Hepatitis B after HBsAg Clearance A Mason et al Hepatology 1992

Can we eliminate integrated HBV DNA sequences?

Progressive elimination of hepatocytes containingintegrated HBV in the absence of ongoing HBV DNAreplication?: Hepatocyte turnover?:

But: . clonal proliferation of hepatocytes containing

integrated HBV DNA

. Frequent persistence of low rate intra-hepaticHBV DNA replication despite undetectable serum viremia.

. Reinfection events from one hepatocyte to the other

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How to get rid of integrated HBV DNA sequences?Combining different approaches

Blocking HBV genome replicationcccDNA : inhibited expression? or elimination?

Eliminating infected hepatocytes:Immunotherapy-based approaches

Eliminating integrated HBV DNA sequences?

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Blocking expression of integrated HBV DNA:Ex: HBV RNA specific siRNA

RNAi-based treatment of chronically infected patients and chimpanzees reveals that integrated hepatitis B virus DNA is a source of HBsAgC.I.Woodell et al Science Translational Medecine 2017

The CRISPR/ Cas9 system facilitates clearance of the intrahepatic HBV templates In Vivo. Lin, S.R ,Yang,H.C,Kuo,Y.T.,Liu,C.J.,Yang,T.Y.,Sung,K.C.,Lin,Y.Y.,Wang,H.Y., Wang,C.C.,Shen,Y.C.,Wu,F.Y.,Kao,J.H.,Chen,D.S., Chen,P.JMol. Ther. Nucleic Acids 2014, 3: 186

Suppression of hepatitis B virus DNA accumulation in chronically infected cells using a bacterial CRISPR/CasRNA-guided DNA endonucleaseEdward M.Kennedy, Leda C.Bassit, Henrik Mueller, Anand V.R. Kornepati, Hal P. Bogerd , Ting Nie , Payel Chatterjee , Hassan Javanbakht c, Raymond F. Schinazi, Bryan R. CullenVirology 2015; 476: 196–205

Complete Spectrum of CRISPR/Cas9-induced Mutations on HBV cccDNAChristoph Seeger and Ji A SohnMolecular Therapy vol. 24 no. 7, 1258–1266 jul. 2016

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Removal of Integrated Hepatitis B Virus DNAusing CRISPR-Cas9

Hao Li, et alFrontiers in Cellular and Infection Microbiology March 2017; Volume 7

Hazards?« Off targets » ?:Novel rearrengements of integrated HBV DNA?Mutations in cellular genome?

Challenges:Which sequences to be used for targeting Cas9?« personalized »??

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CRISPR-Cas9 Can Inhibit HIV-1 Replication but Non Homologous end-joining (HEJ) Repair Facilitates Virus EscapeWang et al Molecular Therapy 2016

CRISPR/Cas9-Derived Mutations Both Inhibit HIV-1 Replication and Accelerate Viral EscapeZhen Wang et al Cell Reports 2016

CRISPR-Cas9: lessons from HIV

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Impact of Integrated HBV DNA?

Is it feasible to eliminate integrated HBV DNA: « real cure »?: ??

Should negative serum HBsAg be an endpoint marker of « functional cure » ? Not the only one

Hepatitis B cure: from discovery to regulatory approval:Anna Lock, Fabien Zoulim, Geoffrey Dusheiko and Marc G Ghany. Hepatology 2017

HBV cure: why, how, when? Massimo Levrero, Barbara Testoni and Fabien ZoulimCurrent Opinion in Virology 2016

Host-virus interactions in hepatitis B virus infection.Guidotti L, Isogawa M, Chisari FVCurrent Opinion Immunol; 2015 Oct;36:61-6

Antiviral strategies to eliminate hepatitis B virus covalently closed circular DNA (cccDNA).Revill, Locarnini; Current Opin Pharmacol2016 Oct;30:144

A Research Agenda for Curing Chronic Hepatitis B Virus Infection. Alter et al Hepatology 2017

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Do we need to eliminate integrated sequences?Yes

Hepatitis B CureProgress in science depends on new techniques, new

discoveries and new ideas, probably in that order.

Sydney Brenner

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Dianummer 1Dianummer 2Dianummer 3Dianummer 4Dianummer 5Dianummer 6Dianummer 7Dianummer 8RNAi-based treatment of chronically infected patients and chimpanzees reveals that integrated hepatitis B virus DNA is a source of HBsAg���Dianummer 10Dianummer 11Dianummer 12Dianummer 13Dianummer 14Dianummer 15Dianummer 16Dianummer 17Direct and indirect effects of HBV chronic infectionDianummer 19Dianummer 20Dianummer 21Dianummer 22Dianummer 23Dianummer 24Dianummer 25Dianummer 26Dianummer 27Dianummer 28Decreased cumulative risk of HCC �after HBsAg lossDianummer 30Dianummer 31Dianummer 32Dianummer 33Dianummer 34Dianummer 35Dianummer 36Dianummer 37Dianummer 38Dianummer 39Dianummer 40Dianummer 41Dianummer 42Dianummer 43Dianummer 44Dianummer 45Dianummer 46Dianummer 47