Targeting the hepatitis B cccDNA with a sequence-specific ARCUS nuclease to eliminate hepatitis B virus in vivo

Abstract

Persistence of chronic hepatitis B (CHB) is attributed to maintenance of the intrahepatic pool of the viral covalently closed circular DNA (cccDNA), which serves as the transcriptional template for all viral gene products required for replication. Current nucleos(t)ide therapies for CHB prevent virus production and spread but have no direct impact on cccDNA or expression of viral genes. We describe a potential curative approach using a highly specific engineered ARCUS nuclease (ARCUS-POL) targeting the hepatitis B virus (HBV) genome. Transient ARCUS-POL expression in HBV-infected primary human hepatocytes produced substantial reductions in both cccDNA and hepatitis B surface antigen (HBsAg). To evaluate ARCUS-POL in vivo, we developed episomal adeno-associated virus (AAV) mouse and non-human primate (NHP) models containing a portion of the HBV genome serving as a surrogate for cccDNA. Clinically relevant delivery was achieved through systemic administration of lipid nanoparticles containing ARCUS-POL mRNA. In both mouse and NHP, we observed a significant decrease in total AAV copy number and high on-target indel frequency. In the case of the mouse model, which supports HBsAg expression, circulating surface antigen was durably reduced by 96%. Together, these data support a gene-editing approach for elimination of cccDNA toward an HBV cure. Persistence of chronic hepatitis B (CHB) is attributed to maintenance of the intrahepatic pool of the viral covalently closed circular DNA (cccDNA), which serves as the transcriptional template for all viral gene products required for replication. Current nucleos(t)ide therapies for CHB prevent virus production and spread but have no direct impact on cccDNA or expression of viral genes. We describe a potential curative approach using a highly specific engineered ARCUS nuclease (ARCUS-POL) targeting the hepatitis B virus (HBV) genome. Transient ARCUS-POL expression in HBV-infected primary human hepatocytes produced substantial reductions in both cccDNA and hepatitis B surface antigen (HBsAg). To evaluate ARCUS-POL in vivo, we developed episomal adeno-associated virus (AAV) mouse and non-human primate (NHP) models containing a portion of the HBV genome serving as a surrogate for cccDNA. Clinically relevant delivery was achieved through systemic administration of lipid nanoparticles containing ARCUS-POL mRNA. In both mouse and NHP, we observed a significant decrease in total AAV copy number and high on-target indel frequency. In the case of the mouse model, which supports HBsAg expression, circulating surface antigen was durably reduced by 96%. Together, these data support a gene-editing approach for elimination of cccDNA toward an HBV cure. IntroductionFollowing hepatitis B virus (HBV) infection, 5%–10% of adults and up to 90% of young children fail to produce an immune response adequate to clear the infection and subsequently develop chronic hepatitis B (CHB).1Zuckerman A.J. Introduction. Windsor, Berkshire, United Kingdom, 25-26 July 1995.Gut. 1996; 38: S1-S70https://doi.org/10.1136/gut.38.suppl_2.s1Crossref PubMed Scopus (10) Google Scholar Worldwide, approximately 240 million people have CHB, and these patients often progress to liver cirrhosis, hepatocellular carcinoma, and liver failure.2Trépo C. Chan H.L.Y. Lok A. Hepatitis B virus infection.Lancet. 2014; 384: 2053-2063https://doi.org/10.1016/s0140-6736(14)60220-8Abstract Full Text Full Text PDF PubMed Google Scholar,3Polaris Observatory CollaboratorsGlobal prevalence, treatment, and prevention of hepatitis B virus infection in 2016: a modelling study. The Lancet.Gastroenterol. Hepatol. 2018; 3: 383-403Google Scholar Long-term treatment with nucleos(t)ide analogues (NAs) provides durable on-treatment suppression of viral replication but is unable to directly target the covalently closed circular DNA (cccDNA) leading to life-long therapy. Furthermore, NAs are unable to completely suppress viral replication; therefore, low-level infectious virus persists for the majority of patients.4Burdette D.L. Lazerwith S. Yang J. Chan H.L.Y. Delaney Iv W.E. Fletcher S.P. Cihlar T. Feierbach B. Ongoing viral replication and production of infectious virus in patients with chronic hepatitis B virus suppressed below the limit of quantitation on long-term nucleos(t)ide therapy.PLoS One. 2022; 17: e0262516https://doi.org/10.1371/journal.pone.0262516Crossref PubMed Scopus (2) Google ScholarHBV is a hepatotropic, partially double-stranded 3.2-kb DNA virus. On infection of human hepatocytes, the HBV genome enters the nucleus, undergoes a repair process, and is converted to cccDNA. Five overlapping mRNAs are produced from the HBV cccDNA, which results in the expression of HBV proteins necessary to complete the remainder of the viral life cycle.5Urban S. Schulze A. Dandri M. Petersen J. The replication cycle of hepatitis B virus.J. Hepatol. 2010; 52: 282-284https://doi.org/10.1016/j.jhep.2009.10.031Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 6Nassal M. HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B.Gut. 2015; 64: 1972-1984https://doi.org/10.1136/gutjnl-2015-309809Crossref PubMed Scopus (518) Google Scholar, 7Tong S. Revill P. Overview of hepatitis B viral replication and genetic variability.J. Hepatol. 2016; 64: S4-S16https://doi.org/10.1016/j.jhep.2016.01.027Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 8Schinazi R.F. Ehteshami M. T. HBV curative 2018; 38: PubMed Scopus Google B surface antigen is the viral of the for infectious HBV is in and to to chronic immune in and intrahepatic B are in hepatitis 2018; PubMed Scopus Google Scholar, M. A. of hepatitis B surface antigen on and in patients with chronic hepatitis B virus Full Text Full Text PDF PubMed Scopus Google Scholar, C. M. Fletcher S. partially B in chronic hepatitis B 2018; PubMed Scopus Google Scholar In to HBsAg from of HBV T. S. HBV DNA and PubMed Scopus Google Scholar, B. A. M. of hepatitis B virus in the human One. PubMed Scopus Google Scholar, Chan C. J. treatment of patients and hepatitis B virus DNA is a of PubMed Scopus Google Scholar The of HBsAg with of cirrhosis, of and of HBsAg is an for HBV therapies and a key with of for hepatitis HBsAg and impact on of hepatocellular Scopus Google Scholar, The The of chronic hepatitis B virus Hepatol. Full Text Full Text PDF PubMed Scopus Google Scholar, J. HBsAg in patients with chronic hepatitis and 2014; PubMed Scopus Google Scholar, therapies and for a cure of chronic hepatitis 2015; PubMed Scopus Google as an a treatment with a C. of HBV by Full Text Full Text PDF PubMed Scopus Google Scholar, S. P. T. gene of hepatitis B virus in Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. T. M. of hepatitis B virus by from PubMed Scopus Google Scholar, The and of genome PubMed Scopus Google Scholar ARCUS have high of target in C. P. J. M. of in in genome of J. Full Text Full Text PDF PubMed Scopus Google Scholar, J. C. P. J. J. P. targeting of in liver to in 2018; PubMed Scopus Google Scholar, J. J. as a to in PubMed Scopus Google Scholar, J. A.J. A. of a the production of J. Full Text Full Text PDF PubMed Scopus Google Scholar ARCUS for a containing both a DNA and The of the and a by for both viral and delivery and in both and through In an toward HBV we engineered and gene-editing ARCUS (ARCUS-POL) of a in the HBV We a double-stranded to of cccDNA or cccDNA, with both HBsAg and we reductions of in cccDNA, and on-target in HBV-infected primary human hepatocytes ARCUS-POL nuclease of a gene-editing approach to and cccDNA the of the viral DNA the genome and the of We nuclease in viral DNA and elimination of models cccDNA are of of to HBV we have developed an episomal adeno-associated virus (AAV) mouse and a non-human primate (NHP) to the of the ARCUS-POL nuclease a cccDNA surrogate in of and of the episomal AAV lipid delivery of the ARCUS-POL nuclease observed in both these data the of a gene-editing approach using the ARCUS-POL nuclease to decrease cccDNA and HBsAg with the of HBV of targeting the HBV J. A.J. A. of a the production of J. Full Text Full Text PDF PubMed Scopus Google Scholar ARCUS are engineered of the from C. are to and a of To a gene-editing for a target the HBV with high HBV and in the human genome for on-target of the in the human containing of HBV was using a To potential was in a to a of a HBV genome of ARCUS-POL and for and through an nuclease data from are in of from in on-target or on-target of a nuclease with high of both on-target and of ARCUS-POL of and ARCUS-POL in HBV-infected we with the in the and using all the nuclease reduced with the nuclease with indel for and and below the limit of for in cccDNA and for the and ARCUS-POL these data the ARCUS-POL nuclease is both and specific for the target of and ARCUS-POL in cccDNA and in the by on-target in cccDNA, total DNA was with to for cccDNA to HBV and DNA by of treatment or treatment with or ARCUS by as potential are as with genes. with a by the of a gene-editing approach for and cccDNA is the potential of the viral DNA to in the genome. To we by to viral DNA with to the in HBV-infected or with or ARCUS-POL and C. M. and transcriptional from hepatitis B and in hepatocellular PubMed Google Scholar observed with the with containing the genome and the HBV genome the ARCUS target for the ARCUS-POL we and for with we observed of the HBV DNA and no in the DNA with the ARCUS-POL Together, these data the high of the ARCUS-POL nuclease results in of the HBV DNA the DNA and of ARCUS-POL in HBV-infected to the of the ARCUS-POL nuclease to and cccDNA and HBsAg with ARCUS-POL nuclease or nuclease on and and DNA the in and for cccDNA and HBV and HBsAg to in both circular HBV DNA and cccDNA ARCUS-POL nuclease with nuclease ARCUS-POL nuclease treatment in an in cccDNA with a nuclease of ARCUS-POL nuclease in HBV-infected of HBV-infected of ARCUS-POL or a was to cccDNA in with ARCUS-POL or a nuclease from the cccDNA to and are as a of the cccDNA of the nuclease in cccDNA by to total DNA was with to for cccDNA. HBV DNA was by a from HBsAg was by from was by from and to the of cccDNA, is for to the nuclease target we the cccDNA in with ARCUS-POL nuclease by the target cccDNA and in an in HBV DNA and a in HBsAg and significant was observed in with the ARCUS-POL nuclease or the the in HBsAg is specific to the nuclease data by ARCUS-POL results in the of cccDNA a the of indel the nuclease target to the observed in viral DNA and of ARCUS-POL nuclease HBV viral DNA the genome serves as a significant of Chan C. J. treatment of patients and hepatitis B virus DNA is a of PubMed Scopus Google Scholar ARCUS-POL nuclease HBV was using an engineered containing a of a HBV with ARCUS-POL nuclease or and indel and HBsAg The by and in a and of In both achieved HBsAg on with the and and of data the ARCUS-POL nuclease is of HBsAg from HBV viral DNA with ARCUS-POL with ARCUS-POL nuclease or of and or On and for on-target indel and was for HBsAg and of nuclease in an episomal AAV mouse is to human hepatocytes, a for in To evaluate the ARCUS-POL nuclease in we developed an episomal containing a HBV by a to as a surrogate for cccDNA. On of the episomal the AAV with an or to to cccDNA and with an containing the ARCUS-POL nuclease or HBsAg was and liver AAV and AAV copy number was for both and to nuclease ARCUS-POL nuclease reduced AAV copy number with the the in was in the the and indel by the ARCUS-POL nuclease to a of HBsAg with administration and with the of HBsAg HBsAg on liver a in HBsAg in with in and Together, these data the of the ARCUS-POL nuclease to episomal DNA leading to both and indel the target the ARCUS-POL is of the HBsAg the indel and AAV to HBsAg in ARCUS-POL nuclease in an episomal AAV mouse with an containing ARCUS-POL nuclease and and DNA was and for AAV copy number and indel the target on the was from and for HBsAg for HBsAg was on liver from and was using nuclease in a episomal HBV significant in HBV is the of models to HBV B. Yang models for the of hepatitis B virus 2018; PubMed Scopus Google Scholar To the ARCUS-POL nuclease in we developed an to the mouse liver in we the to A. 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Lazerwith S. Yang J. Chan H.L.Y. Delaney Iv W.E. Fletcher S.P. Cihlar T. Feierbach B. Ongoing viral replication and production of infectious virus in patients with chronic hepatitis B virus suppressed below the limit of quantitation on long-term nucleos(t)ide therapy.PLoS One. 2022; 17: e0262516https://doi.org/10.1371/journal.pone.0262516Crossref PubMed Scopus (2) Google Scholar data have of and viral the genome is for HBV DNA of viral replication hepatitis B virus with on gene 2022; Full Text Full Text PDF PubMed Scopus Google Scholar a the elimination of cccDNA is often as to HBV Dandri M. A.J. J. A. P. to cure hepatitis Hepatol. 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S. 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DNA for HBV by with pool for with for and using the and DNA using the DNA on the the the of converted to circular for with to the HBV DNA using HBV DNA was by using with the in DNA was using the on and HBV infection, and HBV DNA was using to the total of DNA was by by and and using the on the from was using a human to the In was and in a to and for The was an of on a and using a from and containing a HBV and by a was with for The was in the of was by on the and to which for expression by HBV copy and the to produce The HBV and The was using the but with a HBV mouse for by the and in with from The on a to the On all of the On or an containing ARCUS-POL nuclease of was from all by the study. On was and the with and of the liver was on for and of the liver was and to for by and The primary was to on the for and the was for was on using and on the and AAV DNA was from mouse or liver using the and from using to the The DNA was by the indel and copy number was using in in a containing for of of of and using a and was on a as cycle of for of for for for cycle of for using a and was to and by the number of for the by the number of for the AAV by the number of for the AAV copy number by the number of of the and by to for in total DNA was using in and was to To on-target in we total DNA and to the in to the nuclease target and was to quantitation of using both and for using the for using a on the from and using and to a using developed to and the target and to of by the number of containing by the total number of in and in HBsAg in mouse and with an to the using a and using a of in a study. The was a is of for and of and in in with the for the and of and of in the below the limit of for was on from to AAV The in the the gene by a and was by the the of The are as the of the in by with a mouse The limit of for the is The of the is a with on through of the The was on and ARCUS-POL on and was on and on and was on and on and The AAV was in and a a of a of The was in a and a a of a the to On the of with to and and was direct of the on to on and and and on and of the to or and approximately and or was direct of the on and and of and HBsAg or and and and or and HBsAg was direct of the on and on and of the to or and approximately and or from the of the liver from all on and of the with an of and to was with an of with a of the The was the and was using a all with and The liver and was from all the approximately from of the for for to with and by a liver using and by in with to the IntroductionFollowing hepatitis B virus (HBV) infection, 5%–10% of adults and up to 90% of young children fail to produce an immune response adequate to clear the infection and subsequently develop chronic hepatitis B (CHB).1Zuckerman A.J. Introduction. Windsor, Berkshire, United Kingdom, 25-26 July 1995.Gut. 1996; 38: S1-S70https://doi.org/10.1136/gut.38.suppl_2.s1Crossref PubMed Scopus (10) Google Scholar Worldwide, approximately 240 million people have CHB, and these patients often progress to liver cirrhosis, hepatocellular carcinoma, and liver failure.2Trépo C. Chan H.L.Y. Lok A. Hepatitis B virus infection.Lancet. 2014; 384: 2053-2063https://doi.org/10.1016/s0140-6736(14)60220-8Abstract Full Text Full Text PDF PubMed Google Scholar,3Polaris Observatory CollaboratorsGlobal prevalence, treatment, and prevention of hepatitis B virus infection in 2016: a modelling study. The Lancet.Gastroenterol. Hepatol. 2018; 3: 383-403Google Scholar Long-term treatment with nucleos(t)ide analogues (NAs) provides durable on-treatment suppression of viral replication but is unable to directly target the covalently closed circular DNA (cccDNA) leading to life-long therapy. Furthermore, NAs are unable to completely suppress viral replication; therefore, low-level infectious virus persists for the majority of patients.4Burdette D.L. Lazerwith S. Yang J. Chan H.L.Y. Delaney Iv W.E. Fletcher S.P. Cihlar T. Feierbach B. Ongoing viral replication and production of infectious virus in patients with chronic hepatitis B virus suppressed below the limit of quantitation on long-term nucleos(t)ide therapy.PLoS One. 2022; 17: e0262516https://doi.org/10.1371/journal.pone.0262516Crossref PubMed Scopus (2) Google ScholarHBV is a hepatotropic, partially double-stranded 3.2-kb DNA virus. On infection of human hepatocytes, the HBV genome enters the nucleus, undergoes a repair process, and is converted to cccDNA. Five overlapping mRNAs are produced from the HBV cccDNA, which results in the expression of HBV proteins necessary to complete the remainder of the viral life cycle.5Urban S. Schulze A. Dandri M. Petersen J. The replication cycle of hepatitis B virus.J. Hepatol. 2010; 52: 282-284https://doi.org/10.1016/j.jhep.2009.10.031Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 6Nassal M. HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B.Gut. 2015; 64: 1972-1984https://doi.org/10.1136/gutjnl-2015-309809Crossref PubMed Scopus (518) Google Scholar, 7Tong S. Revill P. Overview of hepatitis B viral replication and genetic variability.J. Hepatol. 2016; 64: S4-S16https://doi.org/10.1016/j.jhep.2016.01.027Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 8Schinazi R.F. Ehteshami M. T. HBV curative 2018; 38: PubMed Scopus Google B surface antigen is the viral of the for infectious HBV is in and to to chronic immune in and intrahepatic B are in hepatitis 2018; PubMed Scopus Google Scholar, M. A. of hepatitis B surface antigen on and in patients with chronic hepatitis B virus Full Text Full Text PDF PubMed Scopus Google Scholar, C. M. Fletcher S. partially B in chronic hepatitis B 2018; PubMed Scopus Google Scholar In to HBsAg from of HBV T. S. HBV DNA and PubMed Scopus Google Scholar, B. A. M. of hepatitis B virus in the human One. PubMed Scopus Google Scholar, Chan C. J. treatment of patients and hepatitis B virus DNA is a of PubMed Scopus Google Scholar The of HBsAg with of cirrhosis, of and of HBsAg is an for HBV therapies and a key with of for hepatitis HBsAg and impact on of hepatocellular Scopus Google Scholar, The The of chronic hepatitis B virus Hepatol. Full Text Full Text PDF PubMed Scopus Google Scholar, J. HBsAg in patients with chronic hepatitis and 2014; PubMed Scopus Google Scholar, therapies and for a cure of chronic hepatitis 2015; PubMed Scopus Google as an a treatment with a C. of HBV by Full Text Full Text PDF PubMed Scopus Google Scholar, S. P. T. gene of hepatitis B virus in Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. T. M. of hepatitis B virus by from PubMed Scopus Google Scholar, The and of genome PubMed Scopus Google Scholar ARCUS have high of target in C. P. J. M. of in in genome of J. Full Text Full Text PDF PubMed Scopus Google Scholar, J. C. P. J. J. P. targeting of in liver to in 2018; PubMed Scopus Google Scholar, J. J. as a to in PubMed Scopus Google Scholar, J. A.J. A. of a the production of J. Full Text Full Text PDF PubMed Scopus Google Scholar ARCUS for a containing both a DNA and The of the and a by for both viral and delivery and in both and through In an toward HBV we engineered and gene-editing ARCUS (ARCUS-POL) of a in the HBV We a double-stranded to of cccDNA or cccDNA, with both HBsAg and we reductions of in cccDNA, and on-target in HBV-infected primary human hepatocytes ARCUS-POL nuclease of a gene-editing approach to and cccDNA the of the viral DNA the genome and the of We nuclease in viral DNA and elimination of models cccDNA are of of to HBV we have developed an episomal adeno-associated virus (AAV) mouse and a non-human primate (NHP) to the of the ARCUS-POL nuclease a cccDNA surrogate in of and of the episomal AAV lipid delivery of the ARCUS-POL nuclease observed in both these data the of a gene-editing approach using the ARCUS-POL nuclease to decrease cccDNA and HBsAg with the of HBV cure.

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