BACKGROUND:Functional cure of chronic hepatitis B requires a sustained loss of HBsAg and hepatitis B virus (HBV) DNA, which is unlikely with current therapies. We aimed to investigate the safety, antiviral activity, and anti-HBV immune responses of the N-acetylgalactosamine-conjugated, small interfering RNA imdusiran, a therapeutic strategy designed to reduce viral antigens on the path to achieving immune control. METHODS:AB-729-001 was a phase 1a/b trial conducted at 11 centres and hospitals: three in Australia, one in New Zealand, three in Thailand, one in Hong Kong, two in South Korea, and one in Moldova. In part 1, which had a double-blinded, single ascending dose design consisting of four sequential dose groups, healthy individuals aged 18-45 years were randomly assigned (2:1), using fixed block randomisation (block number of two for 1:1 active:placebo sentinel dosing, followed by block number of four for 3:1 active:placebo for the next four participants), into three equal-sized cohorts and received subcutaneous imdusiran or placebo as a single dose of 60 mg, 180 mg, or 360 mg. Participants and investigators were masked to treatment assignment until part 1 was complete. Parts 2 and 3 were open-label and enrolled individuals aged 18-65 years with chronic hepatitis B, who were HBeAg-positive or HBeAg-negative and were taking or not taking nucleoside or nucleotide (nucleos[t]ide) analogue therapy (continued during the imdusiran treatment period), and who had HBsAg concentrations of at least 250 international units per mL, no clinically significant abnormalities on liver ultrasound, and an absence of cirrhosis. These participants received subcutaneous imdusiran as a single dose of 60 mg, 90 mg, or 180 mg (part 2) or as repeat doses of 60 mg or 90 mg every 4, 8, or 12 weeks for up to 48 weeks (part 3). The primary endpoint for all parts of the study was to evaluate the frequency and severity of treatment-emergent adverse events, discontinuations due to adverse events, and laboratory abnormalities following the administration of single doses to healthy participants (evaluated until day 29 after dosing) and of single and multiple doses of imdusiran to participants with chronic hepatitis B (evaluated until week 48 of follow-up and until month 36 of follow-up for participants who discontinued nucleos[t]ide analogue therapy after completing imdusiran treatment and meeting eligibility criteria). All individuals who received at least one dose of study drug comprised the safety population and were included in safety analyses. This study was registered with the Australia New Zealand Clinical Trials Registry-ACTRN12619000954123 (part 1), ACTRN12619001197123 (part 2), and ACTRN12620000295943 (part 3)-and is complete. FINDINGS:Between July 16 and Aug 30, 2019, 18 individuals (18 [100%] male) were enrolled in part 1 and randomly assigned (12 [67%] to imdusiran and six [33%] to placebo). Between Sept 3, 2019, and June 11, 2020, of 43 individuals assessed for eligibility, 22 (51%) were enrolled in part 2, of whom 15 (68%) were male and seven (32%) were female. 78 individuals were assessed for eligibility in part 3 between April 30, 2020, and June 26, 2021, of whom 43 (55%) were enrolled (26 [60%] male and 17 [40%] female). All 83 enrolled participants received at least one dose of imdusiran or placebo and were included in the safety analyses. Across all parts, no participants discontinued imdusiran owing to an adverse event and no dose-related trends in reported treatment-emergent adverse events were observed across groups. There were no deaths. The most commonly reported treatment-emergent adverse events occurring in two or more participants in part 1 were an increase in alanine aminotransferase concentration (n=2; both reported as treatment-related), dizziness (n=2), medical device site reaction (n=3), headache (n=2), and oropharyngeal pain (n=2). The most commonly reported treatment-emergent adverse events in part 2 were transient injection-site pain (n=5; all reported as treatment-related), headache (n=4; two treatment-related), an increase in alanine aminotransferase (n=3; two treatment-related) and aspartate aminotransferase (n=2; one treatment-related) concentrations, and dizziness (n=2). The most commonly reported treatment-emergent adverse events in part 3 were coronavirus infection (n=18), injection-site pain (n=8; all of which were reported to be treatment-related), injection-site erythema (n=4; all treatment-related), headache (n=4), upper respiratory tract infection (n=4), pyrexia (n=4), fatigue (n=3; one treatment-related), and injection-site bruising (n=3; all treatment-related). All were reported as grade 1. No clinically relevant changes in clinical laboratory, vital signs, and electrocardiogram values over time were noted in any part of the study. INTERPRETATION:Single and multiple doses of imdusiran were safe and well tolerated in healthy individuals and in individuals with chronic hepatitis B, supporting drug development of imdusiran as a future treatment targeting functional cure for chronic hepatitis B. FUNDING:Arbutus Biopharma.

Approved therapies for hepatitis B virus (HBV) treatment include nucleos(t)ides and interferon alpha (IFN-α) which effectively suppress viral replication, but they rarely lead to cure. Expression of viral proteins, especially surface antigen of the hepatitis B virus (HBsAg) from covalently closed circular DNA (cccDNA) and the integrated genome, is believed to contribute to the persistence of HBV. This work focuses on therapies that target the expression of HBV proteins, in particular HBsAg, which differs from current treatments. Here we describe the identification of AB-452, a dihydroquinolizinone (DHQ) analogue. AB-452 is a potent HBV RNA destabilizer by inhibiting PAPD5/7 proteins in vitro with good in vivo efficacy in a chronic HBV mouse model. AB-452 showed acceptable tolerability in 28-day rat and dog toxicity studies, and a high degree of oral exposure in multiple species. Based on its in vitro and in vivo profiles, AB-452 was identified as a clinical development candidate.

Inhibition of Hepatitis B Virus (HBV) replication by small molecules that modulate capsid assembly and the encapsidation of pgRNA and viral polymerase by HBV core protein is a clinically validated approach toward the development of new antivirals. Through definition of a minimal pharmacophore, a series of isoquinolinone-based capsid assembly modulators (CAMs) was identified. Structural biology analysis revealed that lead molecules possess a unique binding mode, exploiting electrostatic interactions with accessible phenylalanine and tyrosine residues. Key analogs demonstrated excellent primary potency, absorption, distribution, metabolism, and excretion (ADME) and pharmacokinetic properties, and efficacy in a mouse model of HBV. The optimized lead also displayed potent inhibition of capsid uncoating in HBV-infected HepG2 cells expressing the sodium-taurocholate cotransporting polypeptide (NTCP) receptor, affecting the generation of HBsAg and cccDNA establishment. Based on these results, isoquinolinone derivative AB-836 was advanced into clinical development. In Phase 1b trials, AB-836 demonstrated >3 log10 reduction in serum HBV DNA, however, further development was discontinued due to the observation of incidental alanine aminotransferase (ALT) elevations.

Chronic hepatitis B is a global health concern with a high risk of end-stage liver disease. Current standard-of-care agents have low cure rates, and new therapies are needed. Small interfering RNAs (siRNAs) that target viral RNAs fulfill a gap not addressed by standard-of-care agents and may contribute to a functional cure. Here, we describe the preclinical characterization of imdusiran (AB-729), a novel, pan-genotypic siRNA therapeutic that effectively reduces HBsAg, viral antigens, and viral replication in chronic hepatitis B patients and is currently in Phase 2 clinical studies. In hepatitis B virus (HBV) cell-based systems, imdusiran possessed pan-genotypic nanomolar potency and retained activity against HBV target site polymorphisms. Imdusiran was active against nucleos(t)ide analogue- and capsid assembly modulator-resistant HBV isolates, and combination with standard-of-care agents was additive. In an HBV adeno-associated virus mouse model, HBsAg was reduced up to 3.7 log10 after a single imdusiran dose, with sustained suppression for 10 weeks. Imdusiran did not intrinsically stimulate cytokine release in healthy donor human whole blood, supportive of its mechanism of action as a direct acting RNA interference antiviral. Taken together, these data support imdusiran in combination treatment approaches toward chronic hepatitis B functional cure.

Disruption of the HBV capsid assembly process through small-molecule interaction with HBV core protein is a validated target for the suppression of hepatitis B viral replication and the development of new antivirals. Through combination of key structural features associated with two distinct series of capsid assembly modulators, a novel aminochroman-based chemotype was identified. Optimization of anti-HBV potency through generation of SAR in addition to further core modifications provided a series of related functionalized aminoindanes. Key compounds demonstrated excellent cellular potency in addition to favorable ADME and pharmacokinetic profiles and were shown to be highly efficacious in a mouse model of HBV replication. Aminoindane derivative AB-506 was subsequently advanced into clinical development.

Abstract AB‐506 is a potent, pan‐genotypic small molecule capsid inhibitor that inhibits hepatitis B virus (HBV) pregenomic RNA encapsidation. We assessed the safety, pharmacokinetics, and antiviral activity of AB‐506 in two randomized, double‐blinded Phase 1 studies in healthy subjects (HS) and subjects with chronic HBV infection (CHB). Single ascending and multiple doses of AB‐506 or placebo (30–1000 mg or 400 mg daily for 10 days) were assessed in HS. AB‐506 or placebo was assessed at either 160 mg or 400 mg daily for 28 days in subjects with CHB. A second follow‐up study examined AB‐506 or placebo at 400 mg daily for 28 days in 14 Caucasian and 14 East‐Asian HS. Twenty‐eight days of AB‐506 at 160 mg and 400 mg produced mean HBV‐DNA declines from baseline of 2.1 log10 IU/ml and 2.8 log10 IU/ml, respectively. Four subjects with CHB (all Asian) had Grade 4 alanine aminotransferase (ALT) elevations (2 at each dose) as HBV DNA was declining; three events led to treatment discontinuation. In the second follow‐up study, 2 Asian HS had serious transaminitis events leading to treatment and study termination. No subjects had bilirubin elevations or signs of hepatic decompensation. Conclusion: AB‐506 demonstrated mean HBV‐DNA declines of >2 log10; however, transient but severe ALT flares were observed in 4 Asian subjects with CHB. In the follow‐up study in HS, 2 additional Asian HS had Grade 4 flares, suggesting that AB‐506 hepatotoxicity contributed to the ALT elevations. The AB‐506 development program was terminated because of these findings.

AB-506, a small-molecule inhibitor targeting the HBV core protein, inhibits viral replication in vitro (HepAD38 cells: EC50 of 0.077 μM, CC50 > 25 μM) and in vivo (HBV mouse model: ∼3.0 log10 reductions in serum HBV DNA compared to the vehicle control). Binding of AB-506 to HBV core protein accelerates capsid assembly and inhibits HBV pgRNA encapsidation. Furthermore, AB-506 blocks cccDNA establishment in HBV-infected HepG2-hNTCP-C4 cells and primary human hepatocytes, leading to inhibition of viral RNA, HBsAg, and HBeAg production (EC50 from 0.64 μM to 1.92 μM). AB-506 demonstrated activity across HBV genotypes A-H and maintains antiviral activity against nucleos(t)ide analog-resistant variants in vitro. Evaluation of AB-506 against a panel of core variants showed that T33N/Q substitutions results in >200-fold increase in EC50 values, while L30F, L37Q, and I105T substitutions showed an 8 to 20-fold increase in EC50 values in comparison to the wild-type. In vitro combinations of AB-506 with NAs or an RNAi agent were additive to moderately synergistic. AB-506 exhibits good oral bioavailability, systemic exposure, and higher liver to plasma ratios in rodents, a pharmacokinetic profile supporting clinical development for chronic hepatitis B.

Noncanonical poly(A) polymerases PAPD5 and PAPD7 (PAPD5/7) stabilize hepatitis B virus (HBV) RNA via the interaction with the viral posttranscriptional regulatory element (PRE), representing new antiviral targets to control HBV RNA metabolism, hepatitis B surface antigen (HBsAg) production, and viral replication. Inhibitors targeting these proteins are being developed as antiviral therapies; therefore, it is important to understand how PAPD5/7 coordinate to stabilize HBV RNA. Here, we utilized a potent small molecule AB-452 as a chemical probe, along with genetic analyses to dissect the individual roles of PAPD5/7 in HBV RNA stability. AB-452 inhibits PAPD5/7 enzymatic activities and reduces HBsAg both in vitro (50% effective concentration [EC50] ranged from 1.4 to 6.8 nM) and in vivo by 0.94 log(10). Our genetic studies demonstrate that the stem-loop alpha sequence within PRE is essential for both maintaining HBV poly(A) tail integrity and determining sensitivity toward the inhibitory effect of AB-452. Although neither single knockout (KO) of PAPD5 nor PAPD7 reduces HBsAg RNA and protein production, PAPD5 KO does impair poly(A) tail integrity and confers partial resistance to AB-452. In contrast, PAPD7 KO did not result in any measurable changes within the HBV poly(A) tails, but cells with both PAPD5 and PAPD7 KO show reduced HBsAg production and conferred complete resistance to AB-452 treatment. Our results indicate that PAPD5 plays a dominant role in stabilizing viral RNA by protecting the integrity of its poly(A) tail, while PAPD7 serves as a second line of protection. These findings inform PAPD5-targeted therapeutic strategies and open avenues for further investigating PAPD5/7 in HBV replication. IMPORTANCE Chronic hepatitis B affects more than 250 million patients and is a major public health concern worldwide. HBsAg plays a central role in maintaining HBV persistence, and as such, therapies that aim at reducing HBsAg through destabilizing or degrading HBV RNA have been extensively investigated. Besides directly degrading HBV transcripts through antisense oligonucleotides or RNA silencing technologies, small-molecule compounds targeting host factors such as the noncanonical poly(A) polymerase PAPD5 and PAPD7 have been reported to interfere with HBV RNA metabolism. Herein, our antiviral and genetic studies using relevant HBV infection and replication models further characterize the interplays between the cis element within the viral sequence and the trans elements from the host factors. PAPD5/7-targeting inhibitors, with oral bioavailability, thus represent an opportunity to reduce HBsAg through destabilizing HBV RNA.

N-Acetylgalactosamine (GalNAc) conjugated short interfering RNAs (siRNAs) are a leading RNA interference (RNAi) platform allowing targeted inhibition of disease-causing genes in hepatocytes. More than a decade of development has recently resulted in the first approvals for this class of drugs. While substantial effort has been made to improve nucleic acid modification patterns for better payload stability and efficacy, relatively little attention has been given to the GalNAc targeting ligand. In addition, the lack of an intrinsic endosomal release mechanism has limited potency. Here, we report a stepwise analysis of the structure activity relationships (SAR) of the components comprising these targeting ligands. We show that there is relatively little difference in biological performance between bi-, tri-, and tetravalent ligand structures while identifying other features that affect their biological activity more significantly. Further, we demonstrate that subcutaneous co-administration of a GalNAc-functionalized, pH responsive endosomal release agent markedly improved the activity and duration of effect for siRNA conjugates, without compromising tolerability, in non-human primates. These findings could address a significant bottleneck for future siRNA ligand conjugate development.

Programmed death-ligand 1 is a glycoprotein expressed on antigen presenting cells, hepatocytes, and tumors which upon interaction with programmed death-1, results in inhibition of antigen-specific T cell responses. Here, we report a mechanism of inhibiting programmed death-ligand 1 through small molecule-induced dimerization and internalization. This represents a mechanism of checkpoint inhibition, which differentiates from anti-programmed death-ligand 1 antibodies which function through molecular disruption of the programmed death 1 interaction. Testing of programmed death ligand 1 small molecule inhibition in a humanized mouse model of colorectal cancer results in a significant reduction in tumor size and promotes T cell proliferation. In addition, antigen-specific T and B cell responses from patients with chronic hepatitis B infection are significantly elevated upon programmed death ligand 1 small molecule inhibitor treatment. Taken together, these data identify a mechanism of small molecule-induced programmed death ligand 1 internalization with potential therapeutic implications in oncology and chronic viral infections.

Background A link between HBV and PLK1 was clearly evidenced in HBV-driven carcinogenesis, and we have also recently shown that PLK1 is a proviral factor in the early phases of HBV infection. Moreover, we have shown that BI-2536, a small molecule PLK1 inhibitor, was very efficient at inhibiting HBV DNA neosynthesis, notably by affecting nucleocapsid assembly as a result of the modulation of HBc phosphorylation. Yet, as small molecule kinase inhibitors often feature poor selectivity, a more specific and safer strategy to target PLK1 would be needed for a potential development against chronic HBV infections. Methods Here, we analysed using both freshly isolated primary human hepatocytes and differentiated HepaRG, the anti-HBV properties of an LNP-encapsulated PLK1-targeting siRNA. Standard assays were used to monitor the effect of LNP siPLK1, or controls (LNP siHBV and LNP siNon-targeting), on HBV replication and cell viability. Results A dose as low as 100 ng/ml of LNP-siPLK1 resulted in a >75% decrease in secreted HBV DNA (viral particles), which was comparable to that obtained with LNP siHBV or 10 μM of tenofovir (TFV), without affecting cell viability. Interestingly, and in contrast to that obtained with TFV, a strong inhibition of viral RNA and HBe/HBsAg secretions was also observed under LNP siPLK1 treatment. This correlated with a significant intracellular decrease of vRNA accumulation, which was independent of any change in cccDNA levels, thus suggesting a transcriptional or post-transcriptional modulation. Such an effect was not obtained with a biochemical approach of PLK1 inhibition, suggesting an enzymatic-independent role of PLK1. Conclusions This study emphasizes that a specific PLK1 inhibition could help in achieving an improved HBsAg loss in CHB patients, likely in combination with other HBsAg-targeting strategies.

In pursuit of novel therapeutics targeting the hepatitis B virus (HBV) infection, we evaluated a dihydroquinolizinone compound (DHQ-1) that in the nanomolar range reduced the production of virion and surface protein (HBsAg) in tissue culture. This compound also showed broad HBV genotype coverage, but was inactive against a panel of DNA and RNA viruses of other species. Oral administration of DHQ-1 in the AAV-HBV mouse model resulted in a significant reduction of serum HBsAg as soon as 4 days following the commencement of treatment. Reduction of HBV markers in both in vitro and in vivo experiments was related to the reduced amount of viral RNA including pre-genomic RNA (pgRNA) and 2.4/2.1 kb HBsAg mRNA. Nuclear run-on and subcellular fractionation experiments indicated that DHQ-1 mediated HBV RNA reduction was the result of accelerated viral RNA degradation in the nucleus, rather than the consequence of inhibition of transcription initiation. Through mutagenesis of HBsAg gene sequences, we found induction of HBsAg mRNA decay by DHQ-1 required the presence of the HBV posttranscriptional regulatory element (HPRE), with a 109 nucleotides sequence within the central region of the HPRE alpha sub-element being the most critical. Taken together, the current study shows that a small molecule can reduce the overall levels of HBV RNA, especially the HBsAg mRNA, and viral surface proteins. This may shed light on the development of a new class of HBV therapeutics.

ABSTRACT AB-423 is a member of the sulfamoylbenzamide (SBA) class of hepatitis B virus (HBV) capsid inhibitors in phase 1 clinical trials. In cell culture models, AB-423 showed potent inhibition of HBV replication (50% effective concentration [EC 50 ] = 0.08 to 0.27 μM; EC 90 = 0.33 to 1.32 μM) with no significant cytotoxicity (50% cytotoxic concentration > 10 μM). Addition of 40% human serum resulted in a 5-fold increase in the EC 50 s. AB-423 inhibited HBV genotypes A through D and nucleos(t)ide-resistant variants in vitro . Treatment of HepDES19 cells with AB-423 resulted in capsid particles devoid of encapsidated pregenomic RNA and relaxed circular DNA (rcDNA), indicating that it is a class II capsid inhibitor. In a de novo infection model, AB-423 prevented the conversion of encapsidated rcDNA to covalently closed circular DNA, presumably by interfering with the capsid uncoating process. Molecular docking of AB-423 into crystal structures of heteroaryldihydropyrimidines and an SBA and biochemical studies suggest that AB-423 likely also binds to the dimer-dimer interface of core protein. In vitro dual combination studies with AB-423 and anti-HBV agents, such as nucleos(t)ide analogs, RNA interference agents, or interferon alpha, resulted in additive to synergistic antiviral activity. Pharmacokinetic studies with AB-423 in CD-1 mice showed significant systemic exposures and higher levels of accumulation in the liver. A 7-day twice-daily administration of AB-423 in a hydrodynamic injection mouse model of HBV infection resulted in a dose-dependent reduction in serum HBV DNA levels, and combination with entecavir or ARB-1467 resulted in a trend toward antiviral activity greater than that of either agent alone, consistent with the results of the in vitro combination studies. The overall preclinical profile of AB-423 supports its further evaluation for safety, pharmacokinetics, and antiviral activity in patients with chronic hepatitis B.