Imdusiran

HBV pregenomic RNA (pgRNA) is a circulating biomarker for covalently closed circular DNA activity in HBV-infected individuals and has been studied for treatment efficacy, disease staging, and off-therapy outcomes; however, data on the stability are scarce. Increasing HBV pgRNA assay sensitivity may improve its predictive value and provide additional insights at low viral levels.

Modifications to a fully automated first (v1) generation HBV pgRNA assay improved sensitivity up to 15-fold over the previous assay. Flexible sample input volumes yielded lower limits of quantitation of 10 and 22 copies/mL for 0.6 and 0.2 mL assays, respectively. Results are standardized to secondary standards that are traceable to the WHO HBV DNA standard, and internal and external controls are included.

Comparison between v1 and modified v2 assays showed increased sensitivity from 152 copies/mL with v1 to 10 (0.6 mL) and 22 (0.2 mL) copies/mL with v2, respectively. Quantitated v2 results were indistinguishable from v1, indicating that comparisons can be made to previous studies. Single timepoint treatment-naive blood donors or longitudinal draws from patients with chronic hepatitis B on AB-729, an investigational siRNA therapy, showed improved detection and quantifiable pgRNA with v2 compared with v1. Stability testing demonstrated excellent HBV pgRNA plasma stability after 3 freeze-thaw cycles, for at least 7 days at 25–37 °C and at least 30 days at 4°C, with ≤0.25 Log U/mL decrease.

HBV pgRNA v2 assays with increased sensitivity and flexible input volumes demonstrated increased detection and quantitation of low viral titer samples. Highly sensitive HBV pgRNA assays may be useful in refining predictive treatment outcomes based on this marker. HBV pgRNA was stable under multiple conditions, which increases the reliability of this marker.

The major pandemics caused by chronic viral infections is produced by HIV, hepatitis C virus (HCV), and hepatitis B virus (HBV), with estimates of 38, 70, and 250 million people worldwide, respectively (Fig. 1). During the last couple of years, the advent of direct oral antivirals has allowed pursuing global HCV eradication. In an unprecedented manner, these drugs cure more than 95% of hepatitis C patients when given for only 2-3 months. The enthusiasm on HCV has renewed the interest for curative strategies for both HIV and HBV. However, important biological differences between all three viruses may preclude envisioning a similar rapid success for either HIV or HBV than for HCV infection. As shown in figure 1, once infection of targeted cells has occurred, the viral genetic material only replicates in the cytosol for HCV whereas it enters the nucleus and integrates into the chromosomes as provirus for HIV or is converted in a circular covalently closed form (cccDNA) for HBV (Fig. 1). Blocking viral nucleic acid replication for a minimum lag of time allows definitive clearance of HCV infection, with degradation of residual cytoplasmic HCV-RNA strands. In contrast, blocking viral replication has only a transient effect on HIV or HBV, as mRNA expression resumes following treatment discontinuation, given the stability of the HIV provirus or the HBV cccDNA, respectively. The European Liver meeting took held in Paris on April 2018. A relatively large number of presentations addressed distinct new hepatitis B therapeutic strategies. Table 1 summarizes some of the molecules that have been investigated so far with more promising results, grouping them into distinct drug classes (Soriano et al. Exp Op Inv Drugs 2017;26:843-51), based on their distinct mechanism of action and targeted steps in the HBV life cycle (Fig. 2). Considering the pros and cons of novel HBV therapeutic candidates, it has become apparent new HBV gene therapies among the most attractive. Several advances have contributed to position gene therapy in front within the experimental HBV armamentarium. First, progresses in delivery systems, including the use of polymers and nanoformulations have allowed developing easier forms of administration that now are becoming subcutaneous and monthly. Second, the synthetic production of oligonucleotide formulations has reduced costs. Third, the specificity against HBV is higher than for other experimental agents, as immune modulators that enhance innate immunity, such as TLR agonists (i.e., GS-9620) or checkpoint inhibitors (i.e., nivolumab). Fourth, significant declines in serum hepatitis B surface antigen (HBsAg) are demonstrated during gene therapy, which have never been seen using the most potent polymerase inhibitors (i.e., tenofovir or entecavir). Finally, unanticipated significant reductions in cccDNA are seen with HBV gene therapy, most likely as prove of an indirect benefit of waning the immunosuppressive effect of large over amounts of HBsAg released by infected hepatocytes that contributes to T-cell exhaustion. In a pioneering study, Roche was the first to publish the potent effect of an oral small molecule that blocked HBV gene expression (Mueller et al. J Hepatol 2018;68:412-20). The drug belonged to the dihydroquinolizinone class, and directly or indirectly modified viral RNAs, promoting their degradation. This posttranscriptional silencing was accompanied by rapid drops in HBV-DNA and more importantly in serum HBsAg in the humanized mice. However, Roche decided to discontinue any further clinical development of the drug. Nowadays, two major groups of agents are being developed as HBV gene therapies. At this time, interference RNA (iRNA) molecules and nucleic acid polymers (NAPs) are the most promising. Overall, iRNA is double-stranded RNA molecules, 20 nucleotides long. One strand matches a segment of specific HBV mRNA and induces its degradation. Several iRNA molecules have entered into Phase II clinical trials (Flisiak et al. Exp Op Biol Ther, in press), including ARB-1467 and AB-729 (Arbutus), ARO-HBV (Arrowhead), ALN-HBV (Alnylam), and IONIS-HBVRx (Ionis). In most cases, they are tested as part of combination therapy with nucleos(t)ide analogs and/or peginterferon. NAPs are phosphorothioate 40 length oligonucleotides that no map any HBV sequence. However, they interact with a liver host target protein (apolipoprotein-like) and result in specific inhibition of HBV mRNAs. This is followed by rapid suppression of HBsAg release (Roehl et al. Mol Ther Nuc Acids 2017;8:1-12). In a pilot study with intravenous REP-2139, investigators from Replicor demonstrated strong reductions in HBV-DNA along with significant drops in HBsAg and seroconversion in some patients. More interestingly was the recognition of significant reductions in hepatic cccDNA, most likely a result of an indirect effect following the removal of large amounts of HBsAg from the bloodstream that contributes to impaired T-cell responses in chronic hepatitis B patients (Bazinet et al. EASL, Paris 2018; abstract FRI-343). An improved NAP, named REP-2165 and subcutaneous administration are currently being tested.