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8. ASO/siRNA Effects In Vitro, In Vivo and in Humans in HBV Infection
HepG2.2.15 cells harbor multiple copies of the HBV genome, thus providing a genetically invariable source of HBV RNA during viral replication. Early experiments with synthetic hairpin RNA (shRNA), siRNA and ASOs demonstrated antiviral activity in this and related in vitro systems [108,109,110] but without observable reductions in established cccDNA [111]. In other in vitro systems, the development of escape mutants with exogenously introduced HBV genomes were observed [112].
In vivo assessment of ASOs and siRNA almost entirely occurred in mouse models where strong antiviral responses were observed, including rapid multi-log reductions in HBsAg [113,114,115,116,117,118]. These multilog reductions in HBsAg are uniquely distinct from the normal pattern of protein response (saturation at 75–90% reduction from baseline) to ASO and siRNA observed with other liver mRNA targets in vivo [119,120,121,122,123]. However, siRNA analysis in woodchucks (which model the genetic plasticity present in human HBV infection) showed either no antiviral response or rapid rebound in viremia with continued treatment of siRNA triggers in HBsAg or HBx. This rebound was accompanied by the appearance of siRNA escape mutant mRNAs bearing point mutations in the siRNA recognition region [124], consistent with the genetic plasticity of HBV. Importantly, one of the first siRNA studies performed in the HDI mouse model of HBV infection observed cytokine induction consistent with TLR3 activation which occurred concomitantly with antiviral responses [125]. Poly I:C is a dsRNA widely used as a TLR3 agonist and in the HDI mouse model leads to a delayed and prolonged multilog HBsAg decline which is interferon dependent [126]. Moreover, polyI:C also has antiviral effects against HBV in ducks [127] and off target stimulation of innate immunity has been shown to contribute to the antiviral effects of siRNA in the woodchuck model [128].
The first clinical evaluation of siRNA in human HBV infection was performed using LNP-formulation of three siRNA triggers (2 in HBsAg and 1 in HBx, TKM-HBV/ARB-1467). This trigger combination was based on in vivo data showing futility with single triggers and designed to efficiently target mRNA produced from cccDNA or integrated HBV DNA within hepatocytes. In a phase II study conducted in ETV/TDF suppressed chronic HBV infection using ARB-1467 at doses above those saturating for the siRNA effect, mild initial responses in viremia (HBV RNA and HBcrAg) rebounded to baseline during dosing [129,130], consistent with the effects observed in woodchucks. HBsAg response was inconsistent with an siRNA effect: a highly variable and very small HBsAg reduction averaging ~0.25 log10 IU/mL from after 4 weeks followed by similarly small additional reductions following each siRNA dose. A second LNP-formulated siRNA (ARC-520) used two triggers, both in HBsAg. Phase I studies demonstrated transient increases in MCP-1 and IL-8 following dosing [131], consistent with TLR3 stimulation. In phase II studies, HBsAg response was again hypervariable and saturated with an average 0.1 log10 IU/mL reduction from baseline at 4 weeks at the lowest dose (1 mg/kg) still several times higher than required to saturate the siRNA effect with LNP formulation (0.15 mg/kg) [132]. Small but still hypervariable increases in HBsAg decline (average ~ 0.25 log10 IU/mL from baseline) required doses 4 times higher. Interestingly, the HBsAg response to ARC-520 was stronger in HBeAg-positive than HBeAg-negative patients, consistent with immunostimulatory effects as opposed to siRNA-mediated degradation (which should be the same regardless of HBeAg status). In HBeAg-positive patients, HBsAg declines following the first dose of ARC-520 approached 1.5 log10 IU/mL from baseline [132], but this was at dosing (4 mg/kg) substantially higher than required to saturate the siRNA effect. Multiple dosing of ARC-520 showed the same hypervariable and mild HBsAg response, averaging ~0.15 log10 IU/mL after 4 doses of 1 mg/kg and 0.4 log10 IU/mL after 4 doses of 2 mg/kg [133].
All siRNA drugs which followed ARC-520 transitioned away from LNP formulation to GalNAc conjugation (JNJ-3839, AB-729, VIR-2218 and RG6346), which is associated with accumulation of these siRNAs in KCs and LSECs. For all these GalANc-siRNA, single or multiple dose HBsAg responses were very similar (Figure 5) [134,135,136,137]. In the majority of patients, HBsAg response was either absent until 4 weeks following the first dose (similar to poly I:C response in mice) or was very mild and hypervariable with negligible HBsAg response observed at 15 days. After 4 weeks, a universal decline in HBsAg was observed in all patients saturating at 1.5–2.5 log10 IU/mL reduction from baseline. Despite delayed HBsAg declines in most patients, declines in markers of cccDNA activity (HBV DNA, HBV RNA and HBcrAg) were rapid [134], consistent with cccDNA inactivation. Extension of therapy to 48 weeks in NUC suppressed subjects with the addition a capsid assembly modulator did not significantly improve this response, and HBsAg rebound following cessation of treatment has been slow but continuous [134]. The addition of pegIFN only improves HBsAg decline seen with siRNA by an additional 0.5 log10 IU/mL [138]. To date, HBsAg loss has not occurred with any siRNA agent, except for 2 cases with ARC-520 with very low baseline HBsAg (<10 IU/mL) and with several years of exposure in the presence of ETV or TDF [139]. Importantly, HBsAg isoform response to siRNA treatment has been performed with AB-729 using the identical assay platform used for NAPs: no selective decline in the small isoform of HBsAg was observed, indicating that SVP are not targeted and that an siRNA effect is absent. Moreover, selective declines in the virus-specific large HBsAg isoform are observed, suggesting that cccDNA inactivation or other off-target effects are occurring. This is supported by additional recent observations that HBsAg response to siRNA (AB-729) is correlated with increased immunostimulatory activity [140,141].
HBsAg response to GalNAc-ASOs (RG6004/RO7062931 and GSK3389404) has also been inconsistent with an ASO effect [142,143]. No HBsAg response to RG6004 was observed with 0.5 mg/kg dosing, a dose level which yields easily observable protein reductions with Gal-NAc ASOs for other target mRNAs within 2 weeks after the first dose. Elevated dosing with RG6004 to 3 mg/kg produced a highly variable HBsAg decline between subjects which averaged only 0.4 log10 IU/mL after 8 weeks. Responses with GSK3398404 were almost identical to RG6004 in dose response, variability and average HBsAg decline. Bepirovirsen is the unconjugated variant of GSK3389404 carrying the identically modified oligonucleotide. This ASO is a 2′MOE gapmer designed to target mRNA cleavage in HBsAg but it also contains a class II CpG motif within its middle segment of DNA which is not shielded by 2′ ribose modification [79,144]. In contrast to administration of GSK3389404, where this oligonucleotide is delivered primarily to hepatocytes, administration of bepirovirsen results in the delivery of this oligonucleotide mainly to LSECs and KCs. With bepirovirsen, no HBV DNA or HBsAg response was observed in 4/6 subjects at the 150 mg dose [145], a dose which results in easily observed rapid protein reductions with unconjugated ASOs against other liver targets [146,147]. HBsAg responses were limited to the 300 mg dose, but in contrast to GSK 3389404, strong and rapid HBsAg declines (accompanied by host mediated transaminase flares) were restricted to subjects with baseline HBsAg < 1000 IU/mL [145]. In subjects with baseline HBsAg > 1000 IU/mL, HBsAg declines were indistinguishable from those observed with GSK3389404. Recently, HBsAg declines with bepirovirsen have also been correlated with its immunostimulatory activity [148]. These immunostimulatory activities have been attributed to TLR8 based on studies in mice but mice do not accurately model TLR reactivity in humans (Table 1) and this finding is at odds with the well understood biochemical properties of oligonucleotides: TLR8 reacts to unmodified ssRNA and TLR reactivity to TLR7/8 is well shielded by 2′MOE modification in bepirovirsen. Moreover, the oral TLR8 antagonist selgantolimod (GS-9688) did not elicit any HBsAg responses in human infection [149] despite clear immunostimulatory effects [149,150].
9. Conclusions and Perspective
HBsAg loss is an important milestone in the establishment of a functional cure for chronic hepatitis B infection. The molecular biology of HBV, and clinical experience with pegIFN and NAPs (the only agents currently capable of achieving HBsAg loss on therapy) indicate the antiviral effects on HBsAg required to achieve functional cure are 1) SVP particle production must be controlled and 2) HBsAg reduction > 4 log10 IU/mL from baseline must occur during therapy.
The genetic plasticity of chronic HBV infection is at odds with the notion that sequence-dependent mRNA cleavage of HBV mRNA by ASO or siRNA approaches are by themselves capable of achieving HBsAg responses below the threshold required for achieving functional cure. The failure of siRNA in preclinical development in models of HBV infection that model this genetic plasticity and the seminal siRNA assessment with ARB-1467, where rebound of viremia in the presence of three siRNA triggers occurred are consistent with these realities.
GalNAc-ASOs and GalNAc-siRNA have an identical pharmacodynamic protein response signature, susceptibility to single point mutation and share similar, very well-established threshold dosing for effects in humans. As such, clinical evaluations with ASOs in chronic HBV infection inform on the potential for siRNA effects and vice versa in human studies. GalNAc-ASOs are accompanied by little to no HBsAg response at doses saturatingthe ASO effect for other mRNA targets in the liver and at higher doses experience weak and hypervariable HBsAg responses still inconsistent with the ASO effect. Strong HBsAg responses are only observed when an ASO containing a TLR9 stimulatory motif is targeted to immunoreactive cells in the liver (bepirovirsen) and then only in patients with low baseline HBsAg (where immune functioning against HBV is more permissive). Consistent with these ASO findings in chronic HBV infection, HBsAg responses to all GalANc-siRNA are also inconsistent with the cleavage of mRNA. Rebound of infection occurs with LNP-RNA or mild HBsAg response is restricted to immunosensitive (HBeAg+) patient populations at siRNA doses far above the threshold required to saturate the siRNA effect. All GalNAc siRNAs have a delayed HBsAg response in the majority of patients which excludes an siRNA effect but is consistent with the exposure of immunoreactive siRNA in LSECs and KCs with GalNAc conjugation and with the delayed HBsAg responses observed with TLR 3 stimulation in mouse HBV models with dsRNA (poly I:C). These off target effects have been previously observed during the development of siRNA for influenza infection [151]. Additionally, siRNA has been shown to not target the production of SVP both directly by HBsAg isoform analysis and indirectly by the ubiquitously saturated HBsAg decline of 1.5–2.5 log10 IU/mL from baseline with all siRNA. SVP constitute > 99.99% of circulating HBsAg so the relatively mild HBsAg reduction threshold with siRNA indicates SVP are not being targeted.
The residence time of NAPs differs from GalNAc-ASO and GalNAc-siRNA. NAPs are rapidly cleared from the liver [52], whereas the GalNAc conjugation acts as a depot for ASO and siRNA in the liver [75]. Caution must be taken when interpreting the off-therapy antiviral responses to GalNAc-ASO and GalNAC-siRNA as the pharmacological effects of these agents can linger for months. As such, the delayed or slow rebound of HBsAg observed following cessation of GalNAc-ASO or GalNAc siRNA may reflect persistent pharmacological effects of these agents instead of real host antiviral responses.
Based on the substantial amount of available clinical data, the persisting or rapid development of escape mutants to ASO or siRNA exposure appear to restrict the effects of siRNA and ASO in human HBV infection to inactivation of cccDNA and or autophagy of HBsAg destined to form virus or subviral filaments: both activities are known to be stimulated by TLR activation. Neither ASO or siRNA appear to directly target the degradation of mRNA and consequently, the production of SVP from integrated HBV DNA. This is an important distinction as integrated HBV DNA is the source of the bulk of HBsAg in HBeAg negative chronic HBV infection [79]. On the other hand, NAPs do not directly affect viral replication but very effectively target SVP production from cccDNA and integrated HBV DNA, leading to much stronger HBsAg declines to below the limit of detection of the most sensitive experimental HBsAg assay available.
It is important to point out that the apparent TLR-stimulatory effects of ASO and siRNA are the first TLR-agonist effects to yield significant antiviral responses in chronic HBV infection. This is likely a function of their ability to activate TLR3 or TLR9 and be targeted directly to the liver and to immunoreactive non-parenchymal cells. These activities will be important to consider in future combination therapies in the pursuit of HBV functional cure. In the design and reporting of future clinical data from ASO/siRNA based drugs, individual baseline HBsAg and HBsAg responses will also be important to disclose to solidify our understanding of how ASO/siRNA technologies may participate in functional cure of HBV. |
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发表于 2022-11-17 02:13