ABSTRACT
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.
Subject(s)
Antiviral Agents , Capsid Proteins , Capsid , Animals , Mice , Antiviral Agents/pharmacology , Disease Models, Animal , Structure-Activity Relationship , Hepatitis B virus/drug effects , Hepatitis B virus/metabolismABSTRACT
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.
Subject(s)
Acetylgalactosamine/chemistry , Asialoglycoprotein Receptor/metabolism , RNA, Small Interfering/administration & dosage , Animals , Female , Hep G2 Cells , Humans , Injections, Subcutaneous , Ligands , Liposomes , Male , Mice , Nanoparticles , Primates , RNA, Small Interfering/chemistry , Structure-Activity RelationshipABSTRACT
The current outbreak of Ebola virus in West Africa is unprecedented, causing more cases and fatalities than all previous outbreaks combined, and has yet to be controlled. Several post-exposure interventions have been employed under compassionate use to treat patients repatriated to Europe and the United States. However, the in vivo efficacy of these interventions against the new outbreak strain of Ebola virus is unknown. Here we show that lipid-nanoparticle-encapsulated short interfering RNAs (siRNAs) rapidly adapted to target the Makona outbreak strain of Ebola virus are able to protect 100% of rhesus monkeys against lethal challenge when treatment was initiated at 3 days after exposure while animals were viraemic and clinically ill. Although all infected animals showed evidence of advanced disease including abnormal haematology, blood chemistry and coagulopathy, siRNA-treated animals had milder clinical features and fully recovered, while the untreated control animals succumbed to the disease. These results represent the first, to our knowledge, successful demonstration of therapeutic anti-Ebola virus efficacy against the new outbreak strain in nonhuman primates and highlight the rapid development of lipid-nanoparticle-delivered siRNA as a countermeasure against this highly lethal human disease.
Subject(s)
Ebolavirus/drug effects , Ebolavirus/genetics , Hemorrhagic Fever, Ebola/therapy , Hemorrhagic Fever, Ebola/virology , Nanoparticles/administration & dosage , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/therapeutic use , Animals , Base Sequence , Disease Models, Animal , Ebolavirus/classification , Female , Hemorrhagic Fever, Ebola/pathology , Hemorrhagic Fever, Ebola/prevention & control , Humans , Macaca mulatta/virology , Male , RNA, Small Interfering/pharmacology , Survival Analysis , Time Factors , Treatment Outcome , Viral Load/drug effectsABSTRACT
BACKGROUND: Convalescent serum and blood were used to treat patients during outbreaks of Zaire ebolavirus (ZEBOV) infection in 1976 and 1995, with inconclusive results. During the recent 2013-2016 West African epidemic, serum/plasma from survivors of ZEBOV infection was used to treat patients in the affected countries and several repatriated patients. The effectiveness of this strategy remains unknown. METHODS: Nine rhesus monkeys were experimentally infected with ZEBOV-Makona. Beginning on day 3 after exposure (at the onset of viremia), 4 animals were treated with homologous ZEBOV-Makona convalescent macaque sera, 3 animals were treated in parallel with heterologous Sudan ebolavirus (SEBOV) convalescent macaque sera, and 2 animals served as positive controls and were not treated. Surviving animals received additional treatments on days 6 and 9. RESULTS: Both untreated control animals died on postinfection day 9. All 4 ZEBOV-Makona-infected macaques treated with homologous ZEBOV-Makona convalescent sera died on days 8-9. One macaque treated with heterologous SEBOV convalescent sera survived, while the other animals treated with the heterologous SEBOV sera died on days 7 and 9. CONCLUSIONS: The findings suggest that convalescent sera alone is not sufficient for providing 100% protection against lethal ZEBOV infection when administered at the onset of viremia.
Subject(s)
Ebolavirus/immunology , Hemorrhagic Fever, Ebola/prevention & control , Immunization, Passive , Animals , Convalescence , Female , Hemorrhagic Fever, Ebola/virology , Humans , Macaca mulatta , Male , Serum/immunology , ViremiaABSTRACT
Using a genetic screen in yeast we found that Mycobacterium tuberculosis PE-PGRS62 was capable of disrupting yeast vacuolar protein sorting, suggesting effects on endosomal trafficking. To study the impact of PE-PGRS62 on macrophage function, we infected murine macrophages with Mycobacterium smegmatis expressing PE-PGRS62. Infected cells displayed phagosome maturation arrest. Phagosomes acquired Rab5, but displayed a significant defect in Rab7 and LAMP-1 acquisition. Macrophages infected with M. smegmatis expressing PE-PGRS62 also expressed two- to threefold less iNOS protein when compared with cells infected with wild-type bacteria. Consistent with this, cells infected with a Mycobacterium marinum transposon mutant for the PE-PGRS62 orthologue showed greater iNOS protein expression when compared to cells infected with wild-type organisms. Complementation restored the ability of the mutant to inhibit iNOS expression. No differences in iNOS transcript levels were observed, suggesting that PE-PGRS62 effects on iNOS expression occurred post-transcriptionally. Marked differences in colony morphology were also seen in M. smegmatis expressing PE-PGRS62 and in the M. marinum transposon mutant, suggesting that PE-PGRS62 may affect cell wall composition. These findings suggest that PE-PGRS62 supports virulence via inhibition of phagosome maturation and iNOS expression, and these phenotypes may be linked to effects on bacterial cell wall composition.
Subject(s)
Bacterial Proteins/isolation & purification , Macrophages/metabolism , Mycobacterium tuberculosis/genetics , Nitric Oxide Synthase Type II/metabolism , Animals , Bacterial Proteins/genetics , Gene Expression Regulation, Bacterial , Humans , Macrophages/microbiology , Macrophages/pathology , Mice , Mycobacterium smegmatis/metabolism , Mycobacterium smegmatis/pathogenicity , Mycobacterium tuberculosis/pathogenicity , Nitric Oxide Synthase Type II/antagonists & inhibitors , Phagosomes/metabolism , Phagosomes/microbiologyABSTRACT
HBV RNA destabilizers are a class of small-molecule compounds that target the noncanonical poly(A) RNA polymerases PAPD5 and PAPD7, resulting in HBV RNA degradation and the suppression of viral proteins including the hepatitis B surface antigen (HBsAg). AB-161 is a next-generation HBV RNA destabilizer with potent antiviral activity, inhibiting HBsAg expressed from cccDNA and integrated HBV DNA in HBV cell-based models. AB-161 exhibits broad HBV genotype coverage, maintains activity against variants resistant to nucleoside analogs, and shows additive effects on HBV replication when combined with other classes of HBV inhibitors. In AAV-HBV-transduced mice, the dose-dependent reduction of HBsAg correlated with concentrations of AB-161 in the liver reaching above its effective concentration mediating 90% inhibition (EC90), compared to concentrations in plasma which were substantially below its EC90, indicating that high liver exposure drives antiviral activities. In preclinical 13-week safety studies, minor non-adverse delays in sensory nerve conductance velocity were noted in the high-dose groups in rats and dogs. However, all nerve conduction metrics remained within physiologically normal ranges, with no neurobehavioral or histopathological findings. Despite the improved neurotoxicity profile, microscopic findings associated with male reproductive toxicity were detected in dogs, which subsequently led to the discontinuation of AB-161's clinical development.
Subject(s)
Coordination Complexes , Hepatitis B virus , Hepatitis B, Chronic , Naphthalenesulfonates , Male , Mice , Rats , Animals , Dogs , Hepatitis B virus/physiology , Hepatitis B Surface Antigens/genetics , RNA, Viral , RNA, Messenger , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , DNA, Viral/genetics , Hepatitis B, Chronic/drug therapy , DNA, CircularABSTRACT
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.
ABSTRACT
The recent COVID-19 pandemic underscored the limitations of currently available direct-acting antiviral treatments against acute respiratory RNA-viral infections and stimulated major research initiatives targeting anticoronavirus agents. Two novel nsp5 protease (MPro) inhibitors have been approved, nirmatrelvir and ensitrelvir, along with two existing nucleos(t)ide analogues repurposed as nsp12 polymerase inhibitors, remdesivir and molnupiravir, but a need still exists for therapies with improved potency and systemic exposure with oral dosing, better metabolic stability, and reduced resistance and toxicity risks. Herein, we summarize our research toward identifying nsp12 inhibitors that led to nucleoside analogues 10e and 10n, which showed favorable pan-coronavirus activity in cell-infection screens, were metabolized to active triphosphate nucleotides in cell-incubation studies, and demonstrated target (nsp12) engagement in biochemical assays.
Subject(s)
Antiviral Agents , COVID-19 Drug Treatment , Nucleosides , SARS-CoV-2 , Antiviral Agents/pharmacology , Antiviral Agents/chemistry , SARS-CoV-2/drug effects , Humans , Nucleosides/pharmacology , Nucleosides/chemistry , Animals , Drug Discovery , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/metabolism , Chlorocebus aethiops , Vero Cells , COVID-19/virology , Coronavirus RNA-Dependent RNA PolymeraseABSTRACT
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.
Subject(s)
Hepatitis B virus , Hepatitis B , Humans , Hepatitis B virus/genetics , Reproducibility of Results , RNA, Viral/geneticsABSTRACT
The incorporation of nanoparticles (NPs) in industrial and biomedical applications has increased significantly in recent years, yet their hazardous and toxic effects have not been studied extensively. Here, we studied the effects of 24 nm silver NPs (AgNPs) on a panel of bacteria isolated from medical devices used in a hospital intensive care unit. The cytotoxic effects were evaluated in macrophages and the expression of the inflammatory cytokines IL-6, IL-10 and TNF-α were quantified. The effects of NPs on coagulation were tested in vitro in plasma-based assays. We demonstrated that 24 nm AgNPs were effective in suppressing the growth of clinically relevant bacteria with moderate to high levels of antibiotic resistance. The NPs had a moderate inhibitory effect when coagulation was initiated through the intrinsic pathway. However, these NPs are cytotoxic to macrophages and are able to elicit an inflammatory response. Thus, beneficial and potential harmful effects of 24 nm AgNPs on biomedical devices must be weighed in further studies in vivo. From the Clinical Editor: The authors of this study demonstrate that gallic acid reduced 24 nm Ag NPs are effective in suppressing growth of clinically relevant antibiotic resistant bacteria. However, these NPs also exhibit cytotoxic properties to macrophages and may trigger an inflammatory response. Thus, the balance of beneficial and potential harmful effects must be weighed carefully in further studies.
Subject(s)
Anti-Bacterial Agents/pharmacology , Blood Coagulation/drug effects , Inflammation/pathology , Metal Nanoparticles/toxicity , Silver/pharmacology , Silver/toxicity , Bacteria/drug effects , Cell Death/drug effects , Cell Line , Cytokines/metabolism , Humans , Inflammation Mediators/metabolism , Light , Macrophages/cytology , Macrophages/drug effects , Metal Nanoparticles/ultrastructure , Microbial Sensitivity Tests , Particle Size , Scattering, RadiationABSTRACT
Disruption of the HBV viral life cycle with small molecules that prevent the encapsidation of pregenomic RNA and viral polymerase through binding to HBV core protein is a clinically validated approach to inhibiting HBV viral replication. Herein we report the further optimisation of clinical candidate AB-506 through core modification with a focus on increasing oral exposure and oral half-life. Maintenance of high levels of anti-HBV cellular potency in conjunction with improvements in pharmacokinetic properties led to multi-log10 reductions in serum HBV DNA following low, once-daily oral dosing for key analogues in a preclinical animal model of HBV replication.
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.
Subject(s)
Antiviral Agents , Hepatitis B , Humans , Antiviral Agents/adverse effects , Capsid , Capsid Proteins , DNA, Viral , Follow-Up Studies , Healthy Volunteers , Hepatitis B/drug therapy , Hepatitis B e Antigens , Hepatitis B virus/geneticsABSTRACT
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.
Subject(s)
B7-H1 Antigen/metabolism , Endocytosis , Immune Checkpoint Inhibitors/pharmacology , Small Molecule Libraries/pharmacology , Animals , Antineoplastic Agents/pharmacology , Antiviral Agents/pharmacology , CHO Cells , Cell Proliferation/drug effects , Colorectal Neoplasms/pathology , Cricetulus , Disease Models, Animal , Female , Hepatitis B virus/drug effects , Humans , Mice, Inbred C57BL , Programmed Cell Death 1 Receptor/metabolism , Protein Multimerization/drug effects , Small Molecule Libraries/chemistrySubject(s)
Adaptation, Physiological/immunology , Blood-Borne Pathogens , Host-Pathogen Interactions , Immune Evasion/immunology , Macrophage Activation , Macrophages/immunology , Bacteria/pathogenicity , Fungi/pathogenicity , Macrophages/metabolism , Macrophages/microbiology , Phagosomes/immunology , Phagosomes/metabolism , Reactive Nitrogen Species/metabolism , Respiratory Burst/immunology , Viruses/pathogenicityABSTRACT
Hepatitis delta virus (HDV) infects 10-20 million individuals worldwide and causes severe fulminant hepatitis with high likelihood of cirrhosis and hepatocellular carcinoma. HDV infection cannot occur in the absence of the surface antigen (HBsAg) of the hepatitis B virus. RNA interference is an effective mechanism by which to inhibit viral transcripts, and siRNA therapeutics sharing this mechanism have begun to demonstrate clinical efficacy. Here we assessed the outcome of HBV-targeting siRNA intervention against HDV and compared it to a direct anti-HDV siRNA approach in dually infected humanized mice. Treatment with ARB-1740, a clinical stage HBV-targeting siRNA agent delivered using lipid nanoparticle (LNP) technology, effectively reduced HBV viremia by 2.3 log10 and serum HBsAg by 2.6 log10, leading to 1.6 log10 reduction of HDV viremia. In contrast, HDV-targeting siRNA inhibited HDV in both blood and liver compartments without affecting HBV and PEGylated interferon-alpha reduced HBV viremia by 2.0 log10 but had no effect on HDV viremia under these study conditions. These results illustrate the inhibitory effects of siRNAs against these two viral infections and suggest that ARB-1740 may be of therapeutic benefit for hepatitis delta patients, a subpopulation with high unmet medical need.
Subject(s)
Antiviral Agents/therapeutic use , Hepatitis D/drug therapy , Hepatitis Delta Virus/drug effects , RNA Interference , Animals , Animals, Genetically Modified , Disease Models, Animal , Female , Hepatitis B virus/drug effects , Humans , MiceABSTRACT
Current approved nucleoside analogue treatments for chronic hepatitis B virus (HBV) infection are effective at controlling viral titer but are not curative and have minimal impact on the production of viral proteins such as surface antigen (HBsAg), the HBV envelope protein believed to play a role in maintaining the immune tolerant state required for viral persistence. Novel agents are needed to effect HBV cure, and reduction of HBV antigenemia may potentiate activation of effective and long-lasting host immune control. ARB-1740 is a clinical stage RNA interference agent composed of three siRNAs delivered using lipid nanoparticle technology. In a number of cell and animal models of HBV, ARB-1740 caused HBV RNA reduction, leading to inhibition of multiple elements of the viral life cycle including HBsAg, HBeAg, and HBcAg viral proteins as well as replication marker HBV DNA. ARB-1740 demonstrated pan-genotypic activity in vitro and in vivo, targeting three distinct highly conserved regions of the HBV genome, and effectively inhibited replication of nucleoside analogue-resistant HBV variants. Combination of ARB-1740 with a capsid inhibitor and pegylated interferon-alpha led to greater liver HBsAg reduction which correlated with more robust induction of innate immune responses in a human chimeric mouse model of HBV. The preclinical profile of ARB-1740 demonstrates the promise of RNA interference and HBV antigen reduction in treatment strategies driving toward a cure for HBV.
Subject(s)
Antiviral Agents/therapeutic use , Hepatitis B virus/drug effects , Hepatitis B, Chronic/drug therapy , RNA Interference , RNA, Small Interfering/therapeutic use , Animals , Genome, Viral , Humans , Immunity, Innate , Male , Mice , Mice, Inbred C57BL , Nanoparticles/administration & dosage , Nanoparticles/chemistry , RNA, Small Interfering/chemistry , Virus Replication/drug effectsABSTRACT
Caspases play an essential role during programmed cell death in all metazoans. These enzymes are cysteine proteases and comprise a multi-gene family with more than a dozen mammalian family members. Although caspases have been characterized in many animals, including Drosophila melanogaster, little is known about the caspases that exist in mosquitoes. Here we describe the identification and characterization of Aedes Dredd (AeDredd), a novel caspase in the yellow fever mosquito, Aedes aegypti. AeDredd contains two N-terminal death effector domains and the well conserved caspase catalytic domain. Multiple sequence alignments and functional substrate assays of recombinant protein suggest that AeDredd is an orthologue of Drosophila Dredd and human caspase-8, both central effectors of the death receptor-mediated apoptotic pathway. AeDredd exhibits substrate specificity most similar to human caspase-8. AeDredd transcripts were found in all developmental stages with highest expression in early pupae. Within adults, AeDredd was found in all the tissues examined, with the highest transcript levels detected in fat body tissues. This is the first functional characterization of a death domain-containing caspase in an insect vector of human disease, and will initiate studies on the role of apoptosis in the innate immune response of vectors towards intracellular parasites such as viruses.
Subject(s)
Aedes/enzymology , Caspases/genetics , Caspases/metabolism , Insect Proteins/genetics , Insect Proteins/metabolism , Aedes/metabolism , Amino Acid Sequence , Animals , Apoptosis , Caspases/chemistry , Ecdysone/metabolism , Gene Expression/radiation effects , Gene Expression Regulation, Developmental , Insect Proteins/chemistry , Models, Molecular , Molecular Sequence Data , Organ Specificity , Protein Conformation , Sequence Alignment , Substrate Specificity , Ultraviolet RaysABSTRACT
Ebolaviruses and marburgviruses belong to the family Filoviridae and cause high lethality in infected patients. There are currently no licensed filovirus vaccines or antiviral therapies. The development of broad-spectrum therapies against members of the Marburgvirus genus, including Marburg virus (MARV) and Ravn virus (RAVV), is difficult because of substantial sequence variability. RNAi therapeutics offer a potential solution, as identification of conserved target nucleotide sequences may confer activity across marburgvirus variants. Here, we assessed the therapeutic efficacy of lipid nanoparticle (LNP) delivery of a single nucleoprotein-targeting (NP-targeting) siRNA in nonhuman primates at advanced stages of MARV or RAVV disease to mimic cases in which patients begin treatment for fulminant disease. Sixteen rhesus monkeys were lethally infected with MARV or RAVV and treated with NP siRNA-LNP, with MARV-infected animals beginning treatment four or five days after infection and RAVV-infected animals starting treatment three or six days after infection. While all untreated animals succumbed to disease, NP siRNA-LNP treatment conferred 100% survival of RAVV-infected macaques, even when treatment began just 1 day prior to the death of the control animals. In MARV-infected animals, day-4 treatment initiation resulted in 100% survival, and day-5 treatment resulted in 50% survival. These results identify a single siRNA therapeutic that provides broad-spectrum protection against both MARV and RAVV.
Subject(s)
Drug Delivery Systems/methods , Marburg Virus Disease/drug therapy , Marburgvirus , Nanoparticles/therapeutic use , RNA, Small Interfering/pharmacology , Animals , Macaca mulatta , Marburg Virus Disease/metabolism , Marburg Virus Disease/pathology , Nanoparticles/chemistry , RNA, Small Interfering/chemistryABSTRACT
Although significant progress has been made in developing therapeutics against Zaire ebolavirus, these therapies do not protect against other Ebola species such as Sudan ebolavirus (SUDV). Here, we describe an RNA interference therapeutic comprising siRNA targeting the SUDV VP35 gene encapsulated in lipid nanoparticle (LNP) technology with increased potency beyond formulations used in TKM-Ebola clinical trials. Twenty-five rhesus monkeys were challenged with a lethal dose of SUDV. Twenty animals received siRNA-LNP beginning at 1, 2, 3, 4 or 5â days post-challenge. VP35-targeting siRNA-LNP treatment resulted in up to 100% survival, even when initiated when fever, viraemia and disease signs were evident. Treatment effectively controlled viral replication, mediating up to 4 log10 reductions after dosing. Mirroring clinical findings, a correlation between high viral loads and fatal outcome was observed, emphasizing the importance of stratifying efficacy according to viral load. In summary, strong survival benefit and rapid control of SUDV replication by VP35-targeting LNP confirm its therapeutic potential in combatting this lethal disease.
Subject(s)
Hemorrhagic Fever, Ebola/therapy , Lipids , RNA Interference , RNA, Small Interfering/therapeutic use , Animals , Antibodies, Viral , Disease Models, Animal , Drug Compounding , Ebolavirus/isolation & purification , Ebolavirus/pathogenicity , Hemorrhagic Fever, Ebola/virology , Hep G2 Cells , Humans , Macaca mulatta , Nanoparticles/administration & dosage , Nanoparticles/chemistry , RNA, Small Interfering/genetics , Sudan , Viral Load/drug effects , Viral Regulatory and Accessory Proteins/genetics , Viral Regulatory and Accessory Proteins/metabolism , Viremia/therapy , Virus ReplicationABSTRACT
Marburg virus (MARV) and the closely related filovirus Ebola virus cause severe and often fatal hemorrhagic fever (HF) in humans and nonhuman primates with mortality rates up to 90%. There are no vaccines or drugs approved for human use, and no postexposure treatment has completely protected nonhuman primates against MARV-Angola, the strain associated with the highest rate of mortality in naturally occurring human outbreaks. Studies performed with other MARV strains assessed candidate treatments at times shortly after virus exposure, before signs of disease are detectable. We assessed the efficacy of lipid nanoparticle (LNP) delivery of anti-MARV nucleoprotein (NP)-targeting small interfering RNA (siRNA) at several time points after virus exposure, including after the onset of detectable disease in a uniformly lethal nonhuman primate model of MARV-Angola HF. Twenty-one rhesus monkeys were challenged with a lethal dose of MARV-Angola. Sixteen of these animals were treated with LNP containing anti-MARV NP siRNA beginning at 30 to 45 min, 1 day, 2 days, or 3 days after virus challenge. All 16 macaques that received LNP-encapsulated anti-MARV NP siRNA survived infection, whereas the untreated or mock-treated control subjects succumbed to disease between days 7 and 9 after infection. These results represent the successful demonstration of therapeutic anti-MARV-Angola efficacy in nonhuman primates and highlight the substantial impact of an LNP-delivered siRNA therapeutic as a countermeasure against this highly lethal human disease.