RESUMEN
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.
Asunto(s)
Virus de la Hepatitis B , Hepatitis B Crónica , Ratones , Ratas , Animales , Perros , Virus de la Hepatitis B/genética , Antígenos de Superficie de la Hepatitis B , Antivirales/uso terapéutico , Hepatitis B Crónica/tratamiento farmacológico , ARN Viral/genética , Relación Estructura-Actividad , Naftiridinas/farmacología , Naftiridinas/uso terapéutico , ADN Viral/genética , Replicación ViralRESUMEN
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.
Asunto(s)
Antivirales , Proteínas de la Cápside , Cápside , Animales , Ratones , Antivirales/farmacología , Modelos Animales de Enfermedad , Relación Estructura-Actividad , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/metabolismoRESUMEN
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.
Asunto(s)
Antivirales , Hepatitis B , Humanos , Antivirales/efectos adversos , Cápside , Proteínas de la Cápside , ADN Viral , Estudios de Seguimiento , Voluntarios Sanos , Hepatitis B/tratamiento farmacológico , Antígenos e de la Hepatitis B , Virus de la Hepatitis B/genéticaRESUMEN
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: â¼3.0 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.
Asunto(s)
Antivirales/farmacología , Virus de la Hepatitis B/efectos de los fármacos , Proteínas del Núcleo Viral/antagonistas & inhibidores , Replicación Viral/efectos de los fármacos , Animales , Antivirales/farmacocinética , Células Cultivadas , Evaluación Preclínica de Medicamentos , Femenino , Células Hep G2 , Hepatocitos/efectos de los fármacos , Hepatocitos/virología , Humanos , Ratones , Ratas , Ensamble de Virus/efectos de los fármacosRESUMEN
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 log10. 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.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Virus de la Hepatitis B/genética , Hepatitis B/virología , ARN Nucleotidiltransferasas/metabolismo , Estabilidad del ARN , ARN Viral/química , Replicación Viral , Animales , Antivirales/farmacología , Proteínas Cromosómicas no Histona/antagonistas & inhibidores , Proteínas Cromosómicas no Histona/genética , ADN Polimerasa Dirigida por ADN/genética , Inhibidores Enzimáticos/farmacología , Células Hep G2 , Hepatitis B/genética , Hepatitis B/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Nucleotidiltransferasas/antagonistas & inhibidores , ARN Nucleotidiltransferasas/genética , ARN Viral/genéticaRESUMEN
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.
Asunto(s)
Acetilgalactosamina/química , Receptor de Asialoglicoproteína/metabolismo , ARN Interferente Pequeño/administración & dosificación , Animales , Femenino , Células Hep G2 , Humanos , Inyecciones Subcutáneas , Ligandos , Liposomas , Masculino , Ratones , Nanopartículas , Primates , ARN Interferente Pequeño/química , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Antígeno B7-H1/metabolismo , Endocitosis , Inhibidores de Puntos de Control Inmunológico/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Antineoplásicos/farmacología , Antivirales/farmacología , Células CHO , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/patología , Cricetulus , Modelos Animales de Enfermedad , Femenino , Virus de la Hepatitis B/efectos de los fármacos , Humanos , Ratones Endogámicos C57BL , Receptor de Muerte Celular Programada 1/metabolismo , Multimerización de Proteína/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
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.
Asunto(s)
Antivirales/uso terapéutico , Virus de la Hepatitis B/efectos de los fármacos , Hepatitis B Crónica/tratamiento farmacológico , Interferencia de ARN , ARN Interferente Pequeño/uso terapéutico , Animales , Genoma Viral , Humanos , Inmunidad Innata , Masculino , Ratones , Ratones Endogámicos C57BL , Nanopartículas/administración & dosificación , Nanopartículas/química , ARN Interferente Pequeño/química , Replicación Viral/efectos de los fármacosRESUMEN
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.
Asunto(s)
Antivirales/uso terapéutico , Hepatitis D/tratamiento farmacológico , Virus de la Hepatitis Delta/efectos de los fármacos , Interferencia de ARN , Animales , Animales Modificados Genéticamente , Modelos Animales de Enfermedad , Femenino , Virus de la Hepatitis B/efectos de los fármacos , Humanos , RatonesRESUMEN
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 [EC50] = 0.08 to 0.27 µM; EC90 = 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 EC50s. 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.
Asunto(s)
Antivirales/farmacología , Cápside/metabolismo , Virus de la Hepatitis B/efectos de los fármacos , Hepatitis B/tratamiento farmacológico , Ensamble de Virus/efectos de los fármacos , Animales , Sitios de Unión , Línea Celular Tumoral , ADN Circular/metabolismo , ADN Viral/sangre , ADN Viral/metabolismo , Femenino , Guanina/análogos & derivados , Guanina/farmacología , Virus de la Hepatitis B/crecimiento & desarrollo , Humanos , Ratones , Simulación del Acoplamiento Molecular , Unión Proteica , ARN Viral/genéticaRESUMEN
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.
Asunto(s)
Antivirales/farmacología , Antígenos de Superficie de la Hepatitis B/genética , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/genética , Procesamiento Postranscripcional del ARN/efectos de los fármacos , ARN Mensajero/genética , ARN Viral/genética , Elementos de Respuesta , Sitios de Unión , Genotipo , Humanos , Unión Proteica , Estabilidad del ARN/efectos de los fármacos , Transfección , Replicación ViralRESUMEN
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.
Asunto(s)
Fiebre Hemorrágica Ebola/terapia , Lípidos , Interferencia de ARN , ARN Interferente Pequeño/uso terapéutico , Animales , Anticuerpos Antivirales , Modelos Animales de Enfermedad , Composición de Medicamentos , Ebolavirus/aislamiento & purificación , Ebolavirus/patogenicidad , Fiebre Hemorrágica Ebola/virología , Células Hep G2 , Humanos , Macaca mulatta , Nanopartículas/administración & dosificación , Nanopartículas/química , ARN Interferente Pequeño/genética , Sudán , Carga Viral/efectos de los fármacos , Proteínas Reguladoras y Accesorias Virales/genética , Proteínas Reguladoras y Accesorias Virales/metabolismo , Viremia/terapia , Replicación ViralRESUMEN
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.
Asunto(s)
Ebolavirus/inmunología , Fiebre Hemorrágica Ebola/prevención & control , Inmunización Pasiva , Animales , Convalecencia , Femenino , Fiebre Hemorrágica Ebola/virología , Humanos , Macaca mulatta , Masculino , Suero/inmunología , ViremiaRESUMEN
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.
Asunto(s)
Ebolavirus/efectos de los fármacos , Ebolavirus/genética , Fiebre Hemorrágica Ebola/terapia , Fiebre Hemorrágica Ebola/virología , Nanopartículas/administración & dosificación , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/uso terapéutico , Animales , Secuencia de Bases , Modelos Animales de Enfermedad , Ebolavirus/clasificación , Femenino , Fiebre Hemorrágica Ebola/patología , Fiebre Hemorrágica Ebola/prevención & control , Humanos , Macaca mulatta/virología , Masculino , ARN Interferente Pequeño/farmacología , Análisis de Supervivencia , Factores de Tiempo , Resultado del Tratamiento , Carga Viral/efectos de los fármacosRESUMEN
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.
Asunto(s)
Lípidos/uso terapéutico , Macaca mulatta/virología , Enfermedad del Virus de Marburg/virología , Marburgvirus/fisiología , Nanopartículas/uso terapéutico , ARN Interferente Pequeño/uso terapéutico , Animales , Antígenos Virales/inmunología , Humanos , Macaca mulatta/inmunología , Enfermedad del Virus de Marburg/patología , Enfermedad del Virus de Marburg/terapia , Marburgvirus/inmunología , Nanopartículas/química , ARN Viral/metabolismo , Análisis de Supervivencia , Resultado del Tratamiento , Viremia/patologíaRESUMEN
BACKGROUND: We previously showed that small interfering RNAs (siRNAs) targeting the Zaire Ebola virus (ZEBOV) RNA polymerase L protein formulated in stable nucleic acid-lipid particles (SNALPs) completely protected guineapigs when administered shortly after a lethal ZEBOV challenge. Although rodent models of ZEBOV infection are useful for screening prospective countermeasures, they are frequently not useful for prediction of efficacy in the more stringent non-human primate models. We therefore assessed the efficacy of modified non-immunostimulatory siRNAs in a uniformly lethal non-human primate model of ZEBOV haemorrhagic fever. METHODS: A combination of modified siRNAs targeting the ZEBOV L polymerase (EK-1 mod), viral protein (VP) 24 (VP24-1160 mod), and VP35 (VP35-855 mod) were formulated in SNALPs. A group of macaques (n=3) was given these pooled anti-ZEBOV siRNAs (2 mg/kg per dose, bolus intravenous infusion) after 30 min, and on days 1, 3, and 5 after challenge with ZEBOV. A second group of macaques (n=4) was given the pooled anti-ZEBOV siRNAs after 30 min, and on days 1, 2, 3, 4, 5, and 6 after challenge with ZEBOV. FINDINGS: Two (66%) of three rhesus monkeys given four postexposure treatments of the pooled anti-ZEBOV siRNAs were protected from lethal ZEBOV infection, whereas all macaques given seven postexposure treatments were protected. The treatment regimen in the second study was well tolerated with minor changes in liver enzymes that might have been related to viral infection. INTERPRETATION: This complete postexposure protection against ZEBOV in non-human primates provides a model for the treatment of ZEBOV-induced haemorrhagic fever. These data show the potential of RNA interference as an effective postexposure treatment strategy for people infected with Ebola virus, and suggest that this strategy might also be useful for treatment of other emerging viral infections. FUNDING: Defense Threat Reduction Agency.
Asunto(s)
Ebolavirus/genética , Fiebre Hemorrágica Ebola/prevención & control , Interferencia de ARN , ARN Interferente Pequeño/uso terapéutico , Animales , Chlorocebus aethiops , Ebolavirus/aislamiento & purificación , Ebolavirus/fisiología , Femenino , Fiebre Hemorrágica Ebola/virología , Infusiones Intravenosas , Interferón-alfa/biosíntesis , Interleucina-6/biosíntesis , Macaca mulatta , Masculino , Ratones , Ratones Endogámicos ICR , ARN Interferente Pequeño/efectos adversos , ARN Interferente Pequeño/farmacología , Células Vero/virología , Proteínas Virales/genética , Viremia , Replicación ViralRESUMEN
BACKGROUND: Ebola virus (EBOV) infection causes a frequently fatal hemorrhagic fever (HF) that is refractory to treatment with currently available antiviral therapeutics. RNA interference represents a powerful, naturally occurring biological strategy for the inhibition of gene expression and has demonstrated utility in the inhibition of viral replication. Here, we describe the development of a potential therapy for EBOV infection that is based on small interfering RNAs (siRNAs). METHODS: Four siRNAs targeting the polymerase (L) gene of the Zaire species of EBOV (ZEBOV) were either complexed with polyethylenimine (PEI) or formulated in stable nucleic acid-lipid particles (SNALPs). Guinea pigs were treated with these siRNAs either before or after lethal ZEBOV challenge. RESULTS: Treatment of guinea pigs with a pool of the L gene-specific siRNAs delivered by PEI polyplexes reduced plasma viremia levels and partially protected the animals from death when administered shortly before the ZEBOV challenge. Evaluation of the same pool of siRNAs delivered using SNALPs proved that this system was more efficacious, as it completely protected guinea pigs against viremia and death when administered shortly after the ZEBOV challenge. Additional experiments showed that 1 of the 4 siRNAs alone could completely protect guinea pigs from a lethal ZEBOV challenge. CONCLUSIONS: Further development of this technology has the potential to yield effective treatments for EBOV HF as well as for diseases caused by other agents that are considered to be biological threats.
Asunto(s)
Ebolavirus/genética , Fiebre Hemorrágica Ebola/tratamiento farmacológico , Interferencia de ARN , ARN Interferente Pequeño/administración & dosificación , Animales , Antivirales/administración & dosificación , Antivirales/farmacocinética , Antivirales/uso terapéutico , Modelos Animales de Enfermedad , Ebolavirus/efectos de los fármacos , Cobayas , Interferón-alfa/sangre , Interferón beta/sangre , Liposomas , Polietileneimina , ARN Interferente Pequeño/farmacocinética , ARN Interferente Pequeño/uso terapéutico , ARN Polimerasa Dependiente del ARN/genética , Análisis de Supervivencia , Ensayo de Placa Viral , ViremiaRESUMEN
The opportunity to harness the RNA interference (RNAi) pathway to silence disease-causing genes holds great promise for the development of therapeutics directed against targets that are otherwise not addressable with current medicines. Although there are numerous examples of in vivo silencing of target genes after local delivery of small interfering RNAs (siRNAs), there remain only a few reports of RNAi-mediated silencing in response to systemic delivery of siRNA, and there are no reports of systemic efficacy in non-rodent species. Here we show that siRNAs, when delivered systemically in a liposomal formulation, can silence the disease target apolipoprotein B (ApoB) in non-human primates. APOB-specific siRNAs were encapsulated in stable nucleic acid lipid particles (SNALP) and administered by intravenous injection to cynomolgus monkeys at doses of 1 or 2.5 mg kg(-1). A single siRNA injection resulted in dose-dependent silencing of APOB messenger RNA expression in the liver 48 h after administration, with maximal silencing of >90%. This silencing effect occurred as a result of APOB mRNA cleavage at precisely the site predicted for the RNAi mechanism. Significant reductions in ApoB protein, serum cholesterol and low-density lipoprotein levels were observed as early as 24 h after treatment and lasted for 11 days at the highest siRNA dose, thus demonstrating an immediate, potent and lasting biological effect of siRNA treatment. Our findings show clinically relevant RNAi-mediated gene silencing in non-human primates, supporting RNAi therapeutics as a potential new class of drugs.
Asunto(s)
Primates/genética , Interferencia de ARN/efectos de los fármacos , ARN Interferente Pequeño/farmacología , Animales , Apolipoproteínas B/deficiencia , Apolipoproteínas B/genética , Apolipoproteínas B/metabolismo , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismoRESUMEN
Targeted silencing of disease-associated genes by synthetic short interfering RNA (siRNA) holds considerable promise as a novel therapeutic strategy. However, unmodified siRNA can be potent triggers of the innate immune response, particularly when associated with delivery vehicles that facilitate intracellular uptake. This represents a significant barrier to the therapeutic development of siRNA due to toxicity and off-target gene effects associated with this inflammatory response. Here we show that immune stimulation by synthetic siRNA can be completely abrogated by selective incorporation of 2'-O-methyl (2'OMe) uridine or guanosine nucleosides into one strand of the siRNA duplex. These noninflammatory siRNA, containing less than 20% modified nucleotides, can be readily generated without disrupting their gene-silencing activity. We show that, coupled with an effective systemic delivery vehicle, 2'OMe-modified siRNA targeting apolipoprotein B (apoB) can mediate potent silencing of its target mRNA, causing significant decreases in serum apoB and cholesterol. This is achieved at therapeutically viable siRNA doses without cytokine induction, toxicity, or off-target effects associated with the use of unmodified siRNA. This approach to siRNA design and delivery should prove widely applicable and represents an important step in advancing synthetic siRNA into a broad range of therapeutic areas.