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

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. Bassit L. Asselah T. Towards HBV curative therapies.Liver Int. 2018; 38: 102-114https://doi.org/10.1111/liv.13656Crossref PubMed Scopus (56) Google ScholarHepatitis B surface antigen (HBsAg) is the major viral component of the envelope for infectious HBV particles. It is secreted in excess into patients’ serum and thought to contribute to chronic immune dysfunction in patients.9Burton A.R. Pallett L.J. McCoy L.E. Suveizdyte K. Amin O.E. Swadling L. Alberts E. Davidson B.R. Kennedy P.T. Gill U.S. et al.Circulating and intrahepatic antiviral B cells are defective in hepatitis B.J. Clin. Invest. 2018; 128: 4588-4603https://doi.org/10.1172/jci121960Crossref PubMed Scopus (0) Google Scholar, 10le Bert N. Gill U.S. Hong M. Kunasegaran K. Tan D.Z.M. Ahmad R. Cheng Y. Dutertre C.-A. Heinecke A. Rivino L. et al.Effects of hepatitis B surface antigen on virus-specific and global T cells in patients with chronic hepatitis B virus infection.Gastroenterology. 2020; 159: 652-664https://doi.org/10.1053/j.gastro.2020.04.019Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 11Salimzadeh L. le Bert N. Dutertre C.-A. Gill U.S. Newell E.W. Frey C. Hung M. Novikov N. Fletcher S. Kennedy P.T. et al.PD-1 blockade partially recovers dysfunctional virus-specific B cells in chronic hepatitis B infection.J. Clin. Invest. 2018; 128: 4573-4587https://doi.org/10.1172/jci121957Crossref PubMed Scopus (0) Google Scholar In addition to cccDNA-derived HBsAg, HBsAg can also originate from integrated forms of HBV DNA.12Tu T. Budzinska M.A. Shackel N.A. Urban S. HBV DNA integration: molecular mechanisms and clinical implications.Viruses. 2017; 9: 75https://doi.org/10.3390/v9040075Crossref PubMed Scopus (194) Google Scholar, 13Podlaha O. Wu G. Downie B. Ramamurthy R. Gaggar A. Subramanian M. Ye Z. Jiang Z. Genomic modeling of hepatitis B virus integration frequency in the human genome.PLoS One. 2019; 14: e0220376https://doi.org/10.1371/journal.pone.0220376Crossref PubMed Scopus (28) Google Scholar, 14Wooddell C.I. Yuen M.-F. Chan H.L.-Y. Gish R.G. Locarnini S.A. Chavez D. Ferrari C. Given B.D. Hamilton J. Kanner S.B. et al.RNAi-based treatment of chronically infected patients and chimpanzees reveals that integrated hepatitis B virus DNA is a source of HBsAg.Sci. Transl. Med. 2017; 9: eaan0241https://doi.org/10.1126/scitranslmed.aan0241Crossref PubMed Scopus (259) Google Scholar The loss of HBsAg has been associated with improved patient outcomes, including reversal of cirrhosis, decreased risk of HCC, and prolonged survival. Sustained loss of HBsAg is considered an important clinical endpoint for HBV therapies and therefore a key parameter associated with functional cure.15Benias P.C. Min A.D. Goals of antiviral therapy for hepatitis B: HBeAg seroconversion, HBsAg seroconversion, histologic improvement, and possible impact on risk of hepatocellular carcinoma.Curr. Hepat. Rep. 2011; 10: 292-296https://doi.org/10.1007/s11901-011-0112-4Crossref Scopus (3) Google Scholar, 16European Association For The Study Of The LiverEASL clinical practice guidelines: management of chronic hepatitis B virus infection.J. Hepatol. 2012; 57: 167-185https://doi.org/10.1016/j.jhep.2012.02.010Abstract Full Text Full Text PDF PubMed Scopus (2658) Google Scholar, 17Kim G.-A. Lim Y.-S. An J. Lee D. Shim J.H. Kim K.M. Lee H.C. Chung Y.-H. Lee Y.S. Suh D.J. HBsAg seroclearance after nucleoside analogue therapy in patients with chronic hepatitis B: clinical outcomes and durability.Gut. 2014; 63: 1325-1332https://doi.org/10.1136/gutjnl-2013-305517Crossref PubMed Scopus (224) Google Scholar, 18Zoulim F. Durantel D. Antiviral therapies and prospects for a cure of chronic hepatitis B.Cold Spring Harb. Perspect. Med. 2015; 5: a021501https://doi.org/10.1101/cshperspect.a021501Crossref PubMed Scopus (119) Google ScholarGenome editing has recently emerged as an attractive therapeutic approach, potentially offering a single-administration treatment with a lasting effect.19Yang Y.-C. Chen Y.-H. Kao J.-H. Ching C. Liu I.-J. Wang C.-C. Tsai C.-H. Wu F.-Y. Liu C.-J. Chen P.-J. et al.Permanent inactivation of HBV genomes by CRISPR/Cas9-Mediated non-cleavage base editing.Mol. Ther. Nucleic Acids. 2020; 20: 480-490https://doi.org/10.1016/j.omtn.2020.03.005Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 20Stone D. Long K.R. Loprieno M.A. de Silva Feelixge H.S. Kenkel E.J. Liley R.M. Rapp S. Roychoudhury P. Nguyen T. Stensland L. et al.CRISPR-Cas9 gene editing of hepatitis B virus in chronically infected humanized mice.Methods Clin. Dev. 2021; 20: 258-275https://doi.org/10.1016/j.omtm.2020.11.014Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 21Yan K. Feng J. Liu X. Wang H. Li Q. Li J. Xu T. Sajid M. Ullah H. Zhou L. et al.Inhibition of hepatitis B virus by AAV8-derived CRISPR/SaCas9 expressed from liver-specific promoters.Front. Microbiol. 2021; 12: 665184https://doi.org/10.3389/fmicb.2021.665184Crossref PubMed Scopus (13) Google Scholar, 22Doudna J.A. The promise and challenge of therapeutic genome editing.Nature. 2020; 578: 229-236https://doi.org/10.1038/s41586-020-1978-5Crossref PubMed Scopus (303) Google Scholar ARCUS endonucleases have demonstrated high levels of editing at various target sites in numerous models.23Wang L. Breton C. Warzecha C.C. Bell P. Yan H. He Z. White J. Zhu Y. Li M. Buza E.L. et al.Long-term stable reduction of low-density lipoprotein in nonhuman primates following in vivo genome editing of PCSK9.Mol. Ther. J. Am. Soc. Gene Ther. 2021; 29: 2019-2029https://doi.org/10.1016/j.ymthe.2021.02.020Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 24Wang L. Smith J. Breton C. Clark P. Zhang J. Ying L. Che Y. Lape J. Bell P. Calcedo R. et al.Meganuclease targeting of PCSK9 in macaque liver leads to stable reduction in serum cholesterol.Nat. Biotechnol. 2018; 36: 717-725https://doi.org/10.1038/nbt.4182Crossref PubMed Scopus (63) Google Scholar, 25Zekonyte U. Bacman S.R. Smith J. Shoop W. Pereira C.V. Tomberlin G. Stewart J. Jantz D. Moraes C.T. Mitochondrial targeted meganuclease as a platform to eliminate mutant mtDNA in vivo.Nat. Commun. 2021; 12: 3210https://doi.org/10.1038/s41467-021-23561-7Crossref PubMed Scopus (13) Google Scholar, 26MacLeod D.T. Antony J. Martin A.J. Moser R.J. Hekele A. Wetzel K.J. Brown A.E. Triggiano M.A. Hux J.A. Pham C.D. et al.Integration of a CD19 CAR into the TCR alpha chain locus streamlines production of allogeneic gene-edited CAR T cells.Mol. Ther. J. Am. Soc. Gene Ther. 2017; 25: 949-961https://doi.org/10.1016/j.ymthe.2017.02.005Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar Additionally, ARCUS nucleases possess several attractive attributes for therapeutic application, including a single-component protein containing both a site-specific DNA recognition interface and endonuclease activity. The combination of the substrate-recognition and catalytic motifs into a single protein, encoded by ∼1,100 bp, allows for both viral and non-viral delivery modalities and iterative improvements in both activity and specificity through protein engineering. In an effort toward achieving HBV cure, we engineered and optimized gene-editing ARCUS nucleases (ARCUS-POL) capable of specifically cleaving a 22-bp sequence in the HBV polymerase open reading frame (ORF). We hypothesized that a nuclease-mediated double-stranded break (DSB) would lead to degradation of cccDNA or generate mutated, replication-incompetent cccDNA, with both outcomes potentially reducing HBsAg and persistent viremia (Figure S1). Here, we demonstrate sustained reductions of extracellular HBsAg, reduction in cccDNA, and on-target editing in HBV-infected primary human hepatocytes (PHHs) following ARCUS-POL nuclease activity. Potential risks of utilizing a gene-editing approach to cut and degrade cccDNA include the possibility of integrating the cut viral DNA into the host genome and the introduction of chromosomal rearrangements. We show that nuclease specificity improvements result in decreased off-target editing, fewer nuclease-mediated viral DNA integrations, and elimination of chromosomal rearrangements.HBV animal models supporting cccDNA formation are limited because of lack of susceptibility to HBV infection. Therefore, we have developed an episomal adeno-associated virus (AAV) mouse model and a novel non-human primate (NHP) model to assess the activity of the ARCUS-POL nuclease against a cccDNA surrogate in vivo. High levels of editing and degradation of the episomal AAV vector after lipid nanoparticle (LNP) delivery of the ARCUS-POL nuclease mRNA were observed in both models. Together these data demonstrate the viability of a gene-editing approach using the ARCUS-POL nuclease to decrease cccDNA and secreted HBsAg with the goal of achieving HBV cure.ResultsOptimization of nucleases targeting the HBV polymerase geneAs previously reported,26MacLeod D.T. Antony J. Martin A.J. Moser R.J. Hekele A. Wetzel K.J. Brown A.E. Triggiano M.A. Hux J.A. Pham C.D. et al.Integration of a CD19 CAR into the TCR alpha chain locus streamlines production of allogeneic gene-edited CAR T cells.Mol. Ther. J. Am. Soc. Gene Ther. 2017; 25: 949-961https://doi.org/10.1016/j.ymthe.2017.02.005Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar ARCUS nucleases are engineered variants of the I-CreI homing endonuclease from C. reinhardtii that are designed to specifically recognize and cut a 22-bp sequence of choice. To assess a gene-editing strategy for HBV, nucleases were designed against a target site within the HBV polymerase ORF (nt 1,259–1,280) with high conservation across HBV genotypes and low predicted off-target editing activity in the human genome (Figure S2). Nucleases were initially characterized for on-target editing activity following electroporation of the mRNA in the human HCC-derived Hep3B cell line containing multiple integrated copies of HBV sequences. Specificity analysis was assessed using a semi-quantitative, unbiased oligo-capture assay. To simplify potential off-targeting readouts, this assay was performed in a HepG2 cell line generated to contain a single integration of a partial HBV genome (HepG2-HBV).Five generations of ARCUS-POL nucleases (Gens 1–5) were created and optimized for activity and specificity through an iterative approach; representative nuclease data from each generation are shown in Figure 1. Initial rounds of optimization from Gen 1 nucleases yielded improvements in either on-target activity or on-target specificity. A final round of optimization aimed at specificity improvements yielded a Gen 5 nuclease with high levels of both on-target activity and specificity.Specificity characterization of ARCUS-POL nucleasesTo quantitate off-target editing frequencies of Gen 4 and Gen 5 ARCUS-POL nucleases in HBV-infected PHH cells, we designed PCR amplicons against sites, with the top three highest read counts recovered in the oligo-capture analysis, and indels were quantitated using next generation sequencing (NGS) (Table S1). At all off-target sites tested, the Gen 5 nuclease showed reduced off-target editing compared with the Gen 4 nuclease with less than 1% indel formation for sites 1 and 2 and below the limit of detection (0.1% indels) for site 3 (Figure 2A ). On-target indels in cccDNA were detected at 21% and 29% for the Gen 4 and Gen 5 ARCUS-POL nucleases, respectively (Figure 2A). Overall, these data indicate that the Gen 5 ARCUS-POL nuclease is both robust and specific for the intended target sequence.Figure 2Specificity of Gen 4 and Gen 5 ARCUS-POL nucleasesShow full caption(A) On-target indels in cccDNA and indels at three putative off-target sites identified in the oligo-capture assay were quantified by NGS. For on-target indels in cccDNA, total cellular DNA was treated with T5 exonuclease to enrich for cccDNA specifically prior to NGS. (B) Junctions between HBV and host DNA identified by targeted sequencing of PHHs mock treatment or treatment with Gen 4 or Gen 5 ARCUS nucleases. Junction points identified by oligo-capture analysis as potential off-target loci are shown as rhombi. Shape size reflects junction-site frequency after normalization with control host genes. Shape color indicates host chromosomes. Gray lines link junctions with at least 20 reads supporting a chromosomal translocation bridged by the integrated HBV. See also Figure S3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)A risk of utilizing a gene-editing approach for cutting and degrading cccDNA is the potential of the resulting linear viral DNA to integrate within DSBs in the host genome. To assess this risk, we utilized hybrid capture followed by long-read sequencing to identify viral DNA sequences with junctions to the host genomic sequence in HBV-infected PHH cells following mock transfection or transfection with Gen 4 or Gen 5 ARCUS-POL nucleases (Figures S3 and 2B).27Ramirez R. van Buuren N. Gamelin L. Soulette C. May L. Han D. Yu M. Choy R. Cheng G. Bhardwaj N. et al.Targeted long-read sequencing reveals comprehensive architecture, burden, and transcriptional signatures from hepatitis B virus-associated integrations and translocations in hepatocellular carcinoma cell lines.J. Virol. 2021; 95: e0029921https://doi.org/10.1128/jvi.00299-21Crossref PubMed Google Scholar Integrations were observed at higher rates with the Gen 4 nuclease, with most insertions containing junctions between the host genome and the HBV genome at the ARCUS target site. Additionally, for the Gen 4 ARCUS-POL nuclease, we found that most integrations and translocations occurred at known off-target sites for this nuclease. Consistent with our specificity analysis, we observed fewer integrations of the HBV DNA and no detectable translocations in the host genomic DNA with the Gen 5 ARCUS-POL nuclease. Together, these data suggest that the high specificity of the Gen 5 ARCUS-POL nuclease results in fewer integrations of the HBV DNA into the host DNA and eliminated chromosomal rearrangements.Antiviral activity of ARCUS-POL in HBV-infected PHHsHBV-infected PHHs were used to assess the ability of the Gen 5 ARCUS-POL nuclease to specifically cut and degrade cccDNA and diminish HBsAg levels. Cells were transfected with Gen 5 ARCUS-POL nuclease mRNA or non-HBV-targeting nuclease mRNA on days 3 and 6 post-HBV infection. Supernatant and cellular DNA were collected at the time points indicated in Figure 3 and evaluated for cccDNA levels and editing, extracellular HBV DNA, and HBsAg levels. Human albumin levels were monitored to assess cell viability. Southern blot analysis revealed progressive declines in both relaxed circular HBV DNA (rcDNA) and cccDNA following Gen 5 ARCUS-POL nuclease mRNA transfection compared with non-targeting nuclease mRNA (Figure 3A). By day 17, Gen 5 ARCUS-POL nuclease treatment resulted in an ∼85% reduction in cccDNA compared with cells receiving a non-targeting nuclease mRNA (Figure 3B).Figure 3Antiviral effect of Gen 5 ARCUS-POL nuclease in HBV-infected PHHsShow full caption(A) Southern blot time-course analysis of HBV-infected PHHs following transfection of either Gen 5 ARCUS-POL or a non-HBV-targeting nuclease. (B) Densitometry was used to quantify cccDNA levels in PHH cells treated with Gen 5 ARCUS-POL or a non-targeting nuclease from the Southern blot. cccDNA levels were normalized to mtDNA and are shown as a percent of the normalized cccDNA levels of the non-targeting nuclease at day 3 post-HBV infection. (C) On-target indels in cccDNA were quantified by NGS. Prior to NGS, total cellular DNA was treated with T5 exonuclease to enrich for cccDNA. (D) Extracellular HBV DNA was quantified by a qPCR assay from PHH supernatant. (E) Extracellular HBsAg was quantified by CLIA from PHH supernatant. (F) Extracellular albumin was quantified by ELISA from PHH supernatant. Lines and error bars represent mean ± SD.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In addition to the loss of cccDNA, it is possible for indels to occur at the nuclease target site (Figure S1). Using NGS, we found that the remaining cccDNA in cells treated with Gen 5 ARCUS-POL nuclease mRNA contained 29% indels by day 17 at the intended target site (Figure 3C). cccDNA editing and depletion resulted in an 80% reduction in extracellular HBV DNA and a 77% reduction in secreted HBsAg (Figures 3D and 3E). No significant difference was observed in secreted albumin with either the ARCUS-POL nuclease or the non-HBV-targeting nuclease, suggesting the reduction in HBsAg is specific to the HBV-targeting nuclease activity (Figure 3F). These data suggest that cutting by ARCUS-POL results predominantly in the degradation of cccDNA or, at a lower frequency, the introduction of indel mutations at the nuclease target site. Both outcomes likely contribute to the observed decreases in secreted viral DNA and HBsAg.Activity of ARCUS-POL nuclease against integrated HBV DNARecent findings show integrated viral DNA within the host genome serves as a significant source of secreted HBsAg.14Wooddell C.I. Yuen M.-F. Chan H.L.-Y. Gish R.G. Locarnini S.A. Chavez D. Ferrari C. Given B.D. Hamilton J. Kanner S.B. et al.RNAi-based treatment of chronically infected patients and chimpanzees reveals that integrated hepatitis B virus DNA is a source of HBsAg.Sci. Transl. Med. 2017; 9: eaan0241https://doi.org/10.1126/scitranslmed.aan0241Crossref PubMed Scopus (259) Google Scholar ARCUS-POL nuclease activity against integrated HBV was evaluated using an engineered HepG2 cell line (HepG2-sAg) containing a single integration of a partial HBV sequence that produces HBsAg. HepG2-sAg cells were electroporated with Gen 5 ARCUS-POL nuclease or mCherry mRNA, and indel formation and secreted HBsAg were evaluated. The average indels by day 9 post-transfection were 53% and 87% in cells receiving a 10 and 100 ng dose of mRNA, respectively (Figure 4A ). In both doses, cells achieved maximal HBsAg reduction on day 6 post-transfection, with the 10 and 100 ng doses achieving 72% and 83% reduction of HBsAg, respectively (Figure 4B). These data demonstrate that the ARCUS-POL nuclease is also capable of reducing extracellular HBsAg from integrated HBV DNA.Figure 4Targeting integrated viral DNA with Gen 5 ARCUS-POL nucleaseShow full captionHepG2-sAg cells were electroporated with Gen 5 ARCUS-POL nuclease mRNA or control mCherry mRNA at concentrations of 10 and 100 ng or were mock transfected. On days 3, 6, and 9, cells were harvested for (A) on-target indel frequency analysis via ddPCR, and (B) supernatant was collected for extracellular HBsAg analysis via CLIA. Lines and error bars represent mean ± SD of biological replicates.View Large Image Figure ViewerDownload Hi-res image Download (PPT)ARCUS-POL nuclease activity in an episomal AAV mouse modelHBV tropism is limited to human hepatocytes, posing a challenge for testing therapeutic approaches in vivo. To evaluate the ARCUS-POL nuclease in mice, we developed an episomal AAV9 (AAV subtype 9) vector (AAV9-HBsAg) containing a partial HBV sequence driven by a liver-specific promoter to serve as a surrogate for cccDNA. On cutting of the episomal AAV, the AAV can either be degraded, repaired with an indel, or repaired back to wild-type, similar to cccDNA (Figure S1). NSG mice were administered AAV9-HBsAg vector intravenously (i.v.) and were then i.v. dosed with either an LNP containing the Gen 5 ARCUS-POL nuclease mRNA or phosphate-buffered saline (PBS) 3 weeks later. Serum HBsAg was monitored weekly, and liver AAV copies and indels assessed 4 weeks post-LNP administration.Absolute AAV copy number was determined for both PBS- and nuclease-treated groups to assess degradation after nuclease cutting. Gen 5 ARCUS-POL nuclease significantly reduced AAV copy number compared with the PBS-treated group (Figure 5A). Additionally, the average indels detected in remaining AAVs was 86% in the nuclease-treated group (Figure 5B). Combined, the degradation and indel formation by the Gen 5 ARCUS-POL nuclease led to a 96% reduction of serum HBsAg level compared with PBS-treated animals starting 1 week after LNP administration and persisting until necropsy at week 7 (Figure 5C). Consistent with the reduction of serum HBsAg level, HBsAg immunohistochemical (IHC) staining on liver sections collected at necropsy also demonstrated a drastic reduction in HBsAg protein in Gen 5 ARCUS-POL-treated mice compared with PBS-treated mice (representative images in Figures 5D and S4). Together, these data demonstrate the ability of the Gen 5 ARCUS-POL nuclease to effectively cut episomal DNA leading to both degradation and indel formation at the target site. Although the ARCUS-POL site is located outside of the HBsAg ORF, the resulting indel formation and AAV degradation were able to dramatically reduce HBsAg in vivo.Figure 5Gen 5 ARCUS-POL nuclease evaluation in an episomal AAV mouse modelShow full captionThree weeks after AAV9-HBsAg administration, NSG mice were i.v. dosed with an LNP containing Gen 5 ARCUS-POL nuclease mRNA at 2 mg/kg. Blood draws were performed weekly, and animals were sacrificed 4 weeks post-LNP administration. At sacrifice, livers were harvested, and genomic DNA was extracted and assessed for (A) AAV copy number per diploid cell and (B) indel analysis at the target site present on the AAV9-HBsAg vector. (C) Serum was isolated from blood samples and analyzed for HBsAg via CLIA. (D) Immunohistochemistry for HBsAg detection was performed on liver tissue from necropsy (scale bars: 400 μm). Lines and error bars represent mean ± SD. Statistical analysis was performed using Mann-Whitney’s two-tailed test. ∗p ≤ 0.05. See also Figure S4.View Large Image Figure ViewerDownload Hi-res image Download (PPT)ARCUS-POL nuclease activity in a novel episomal HBV NHP modelA significant challenge in HBV research is the lack of available NHP models susceptible to HBV infection.28Guo W.-N. Zhu B. Ai L. Yang D.-L. Wang B.-J. Animal models for the study of hepatitis B virus infection.Zool. Res. 2018; 39: 25-31https://doi.org/10.24272/j.issn.2095-8137.2018.013Crossref PubMed Scopus (27) Google Scholar To test the ARCUS-POL nuclease efficacy in cynomolgus macaques, we developed an AAV-HBsAg model similar to the mouse model described above. Because AAV8 has preferential liver tropism in NHPs, we changed the vector to AAV8-HBsAg.29Srivastava A. In vivo tissue-tropism of adeno-associated viral vectors.Curr. Opin. Virol. 2016; 21: 75-80https://doi.org/10.1016/j.coviro.2016.08.003Crossref PubMed Scopus (161) Google Scholar The AAV8-HBsAg vector contains a partial HBV sequence that includes the ARCUS-POL target site and HBsAg ORF driven by a liver-specific promoter. For this study, an alternate Gen 5 ARCUS-POL nuclease (ARCUS-POL∗) was selected because of increased on-target activity compared with the Gen 5 ARCUS-POL nuclease utilized in the other studies described (Figure S5).The groups included in the study and a study timeline are shown in Table 1 and Figure 6, respectively. Study groups included an LNP control group that received only ARCUS-POL∗ LNP, an AAV control group that received only the AAV8-HBsAg vector, and an experimental group that received both the AAV8-HBsAg vector and ARCUS-POL∗ LNP. The AAV8-HBsAg was i.v. administered on study day 8 to the AAV control and experimental groups, while the LNP control group was treated with PBS. On days 22 and 63, the LNP control and experimental groups were administered the ARCUS-POL∗ LNP, while the AAV control group was treated with PBS. Liver biopsies were performed for all groups on study days 36 and 77, and serum was collected throughout the study for HBsAg analysis.Table 1Episomal HBV-AAV NHP study groupsGroupNo. of animalsDose level AAV (vector gen