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To (Hep)B or not to (Hep)B? Hepatitis B cure in reach for oligonucleotide therapeutics
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The Hepatitis B virus (HBV) was first identified in 1965 by Dr. Baruch Blumberg who won the 1976 Nobel Prize in Physiology or Medicine for this discovery. He also determined that HBV infection is strongly linked to the development of liver cirrhosis and hepatocellular carcinoma (HCC) as well as developing a diagnostic test and vaccine for the virus. Although a
HBV vaccine has been available since 1981, in 2015 an estimated 2 billion people had been infected with the virus, 257 million were living with chronic HBV infection and 887 000 died from HBV related liver disease.1
Hepatitis B is most commonly spread perinatally (up to 90% risk if the mother is positive for both HBsAg and HBeAg) or horizontally between children. This is of importance because the likelihood that an infection becomes chronic is inversely related to age at infection, such that 80-90% of infants infected during their first year of life, 30-50% of children infected before the age of 6 and less than 5% of otherwise healthy adults will develop a chronic infection. Up to 30% of chronically infected adults will develop cirrhosis and/or liver cancer.1,2 It is not the virus itself that causes the liver damage but rather the body’s own adaptive immune response, specifically cytotoxic T lymphocytes attacking infected hepatocytes and secreting antiviral cytokines.
The current standard of care for chronic infection with signs of liver damage is treatment with nucleos(t)ide analogues (NUC) such as entecavir (ETV) or tenofovir which lack a 3′ hydroxyl group and thus act as DNA chain terminators, sometimes combined with interferon-2α.3 However, even extended treatment over 6-12 months cannot clear the virus completely although it limits virus replication and thus liver damage. The difficulty with clearing HBV completely is due to the virus’s complex life cycle and how it interacts with the host’s immune response.3
The HBV genome contains four overlapping open reading frames which are transcribed as subgenomic RNAs (ie they share a common poly-A tail) and translated into seven different proteins. These include the immunosuppressive HBsAg (surface antigen) and HBeAg, an indicator of active viral replication. Upon initial infection, entry into hepatocytes and transport into the nucleus, viral DNA is converted from its relaxed circular form (rcDNA) into a covalently closed circular form (cccDNA).2,4 This acts as a template for viral mRNAs and pregenomic RNA which is reverse transcribed into rcDNA for packaging into nucleocapsids and subsequent secretion of viral particles. Up to 30% of the reverse transcripts form double stranded linear DNA (dslDNA) and this may integrate into the host cell genome at naturally occurring double strand breaks. Deletions and multiple rearrangements of the HBV genome are common in these integration events and resulting expression of mutated HBV proteins may play a role in HCC development.4 The integrated DNA is replication defective and as such not susceptible to NUC treatments.
High levels of HBV DNA and HBsAg combined with the presence of HBeAg are early markers of HBV infection.3 Effective host immune response is characterised by loss of HBeAg, appearance of anti-HBe antibodies, and slow clearance of HBV DNA and HBsAg. Continued viral replication and detectable HBV DNA, HBsAg and HBeAg in serum, often at high titers, mark chronic hepatitis and are generally associated with elevated and spiking serum alanine and aspartate aminotransferase (ALT/AST) levels. Around 10-20% of chronically infected patients per year seroconvert to the HBeAg-negative, anti-HBe-positive inactive carrier state with HBV DNA concentrations below 2000 IU/ml. However, 20-30% of inactive carriers experience later re-activation of the HBV infection and subsequent increased risk of liver damage. Only 0.5-1% achieve spontaneous HBsAg clearance, classed as functional cure, with undetectable serum HBV DNA despite persistence of integrated and cccDNA. Patients with HBsAg concentrations of less than 1000 IU/ml are more likely to achieve this state, probably because low levels of the immunosuppressive HBsAg allow recovery of effective host immune control. Thus, current experimental treatment strategies mainly focus on sustained HBsAg reduction.
Considering that the target organ is the liver, oligonucleotide therapeutics based on RNA interference or RNaseH antisense should be highly successful in achieving dramatic reductions in targeted mRNA. Due to the subgenomic nature of the viral mRNAs, targeting of all viral mRNAs as well as pregenomic RNA with the same therapeutic oligonucleotide(s) is possible.2
In a recent paper, Woodell and colleagues5 report on the results of a phase II trial (Heparc-2001; NCT02065336) in chronic HBV patients and a complementary study in HBV infected chimpanzees using the RNAi therapeutic ARC-520. ARC-520 consists of an equimolar mixture of cholesterol-conjugated siRNAs targeted to 118 (siHBV-74) and 71 (siHBV-77) bases upstream of the common poly-A signal.6 Patients in the study were allocated to cohorts as shown in the table below; all had HBsAg levels of 3.5±0.7 log10 IU/mL.
| cohort | Status | n | Dose mg/kg | HBV DNA
Log10 IU/mL | NUC | | PBO | mixed | 10 | 0 | BLOQ | + | | 1 | HBe-neg | 6 | 1 | BLOQ | + | | 2 | HBe-neg | 6 | 2 | BLOQ | + | | 3 | HBe-neg | 6 | 3 | BLOQ | + | | 4 | HBe-neg | 6 | 4 | BLOQ | + | | 5 | HBe-pos | 6 | 4 | BLOQ | + | | 6 | HBe-pos | 6 | 2×2 (14 d) | BLOQ | + | | 7a | HBe-pos | 6 | 4 | 7.8 | naive | | 7b | HBe-neg | 6 | 4 | 4.1 | naive |
*BLOQ (<1.46 log IU/mL)Treatment with the indicated doses of ARC-520 resulted in only modest HBsAg reductions (≤0.3 log10) in cohorts 1-6. This was not due to saturation of hepatocyte uptake (see cohort 6) or lack of RNAi efficiency as HBcrAg in cohorts 1-4 and HBeAg in cohort 5 was downregulated as much as 0.9 and 1.2 log10, respectively. However, treatment of NUC-naïve HBeAg-positive patients with high baseline HBV DNA levels (cohort 7a) dramatically decreased these levels by 4 log10 within three weeks of ARC-520 and start of daily NUC treatment. There were concomitant significant reductions in HBsAg (1.4 log10) and HBeAg (1.5 log10). In the HBeAg-negative NUC naïve group (cohort 7b), HBV DNA became undetectable and HBsAg decreases were similar to those seen in the NUC-experienced cohorts 1-6. Clearly, ARC-250 treatment was less effective in reducing HBsAg levels in HBeAg-negative patients that had undergone long-term NUC therapy.
Experiments in chronically HepB infected chimpanzees confirmed these results. To investigate the reason for the differing responses in HBeAg+ and HBeAg- chimpanzees and, by extension humans, Wooddell et al.5 measured the total HBV DNA levels in liver biopsies taken pre-study, after NUC lead-in and at various points during treatments. They found that HBeAg+ animals had 5.9-7.8 log10 copies of HBV DNA per µg of host DNA which were reduced by 0.45 log10 copies/µg per month on the lead-in NUC treatment. HBeAg- chimps only had 3.6-4.4 log10 copies/µg which were not reduced any further by NUC treatment. Since entecavir, the NUC used in this study, is a nucleoside reverse transcriptase inhibitor, these results suggest that the majority of HBV DNA in the liver of HBeAg- chimpanzees is not replicated by the viral polymerase and consequently, that it consists mainly of the integrated form of HBV DNA.
To investigate this idea, Wooddell and colleagues5 performed paired-end sequencing of fragmented liver DNA. As expected, all chimpanzees had integrated HBV DNA, with 5′ and 3′ends consistent with integration of dslDNA into the host genome. This “splitting” of the HBV genome should affect the expression of all viral proteins except HBsAg4 and thus allow the distinction of transcripts derived from cccDNA and integrated HBV DNA. Indeed, analysis of HBV transcripts by RT-qPCR, paired-end next-generation mRNA sequencing (mRNA-seq), and single-molecule real-time sequencing (Iso-seq) confirmed that the majority of transcripts in HBeAg-, but not HBeAg+, chimpanzees were templated on integrated HBV DNA. Due to the mechanism of dslDNA integration4, a large portion of these transcripts lacked the ARC-520 siRNA target sites.
When two HBeAg- animals previously treated with ARC-520 were dosed with siHBV-75, which targets a site that should be included even in integrated HBV DNA, HBsAg was reduced by up to 3 log10 after three monthly doses of 4 mg/kg. This demonstrates that as long as the target sequences are carefully designed, siRNA can significantly reduce protein expression from integrated HBV.
In an open label extension study of Heparc-2001, three patients from cohort 7a (HBeAg+) and five from cohort 7b (HBeAg-) received up to 9 additional monthly doses of 4 mg/kg ARC-520 in combination with daily NUC. As of March 2018, two of the HBeAg+ patients showed sustained HBeAg seroclearance and loss of serum HBV RNA up to a year after the end of ARC-520 treatment, while one HBeAg- patient achieved HBsAg seroclearance and one had a sustained host response.7 Considering that these results were achieved with a suboptimal siRNA therapeutic that may not efficiently target HBsAg expression from integrated HBV DNA in HBeAg- patients, they are incredibly promising.
In the meantime, Arrowhead Pharmaceuticals have quickly progressed ARO-HBV, which contains a mixture of siRNAs targeting sequences in the S and X gene, into the clinic (NCT03365947).8 Clinical trials with several competing oligonucleotide therapeutics are also ongoing. The lipid nanoparticle encapsulated ARB-1467 (previously TKM-HBV) from Arbutus Biopharma comprises three RNAi triggers (NCT02631096) and did show promising results which were clearly limited by the low dosage of ≤0.4 mg/kg.9,10 A phase II trial with GSK3389404 (previously IONIS-HBV-LRx), a GalNAc-conjugated antisense oligonucleotide (ASO), has not reported any data yet (NCT03020745). Hoffmann-La Roche have the LNA-ASO RO7062931 (also known as Roche RG6004) in phase II trials (NCT03038113).
However, it will be quite a challenge to beat the competition from small molecule capsid assembly inhibitors11 and the excellent data on the table from the nucleic acid polymer REP-2139 (NCT02565719).12,13 Yet, even if siRNA and antisense therapeutics are unable to achieve similar impressive results, they may still be valuable. According to Geoff Dusheiko, Emeritus Professor of Medicine at University College London School of Medicine and Kings College Hospital, most likely a combination of therapeutics that target distinct aspects of the viral life cycle will be required to achieve functional cures in all treated patients. Exciting times are ahead!
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发表于 2018-6-18 08:44
