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Chronic hepatitis B virus (HBV) infection is a common cause of liver disease globally, with a disproportionately high burden in South-East Asia. Vaccines and nucleoside or nucleotide drugs are available and reduce both new infection rates and the development of liver disease in HBV-positive persons who adhere to long-term suppressive treatment. Although there is still considerable value in optimizing access to virus-suppressing regimens, the scientific and medical communities have embarked on a concerted journey to identify new antiviral drugs and immune interventions aimed at curing infection. The mechanisms and drug targets being explored are diverse; however, the field universally recognizes the importance of addressing the persistence of episomal covalently closed circular DNA, the existence of integrated HBV DNA in the host genome and the large antigen load, particularly of hepatitis B surface antigen. Another major challenge is to reinvigorate the exhausted immune response within the liver microenvironment. Ultimately, combinations of new drugs will be required to cure infection. Here we critically review the recent literature that describes the rationale for curative therapies and the resulting compounds that are being tested in clinical trials for hepatitis B.
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Antivirais/uso terapêutico , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B/tratamento farmacológico , Animais , Quimioterapia Combinada , Hepatite B/virologia , HumanosRESUMO
Hepatitis C virus (HCV) infection is a major global health burden and is associated with an increased risk of liver cirrhosis and hepatocellular carcinoma. There remains an unmet medical need for efficacious and safe direct antivirals with complementary modes of action for combination in treatment regimens to deliver a high cure rate with a short duration of treatment for HCV patients. Here we report the in vitro inhibitory activity, mode of action, binding kinetics, and resistance profile of TMC647055, a novel and potent nonnucleoside inhibitor of the HCV NS5B RNA-dependent RNA polymerase. In vitro combination studies with an HCV NS3/4A protease inhibitor demonstrated potent suppression of HCV RNA replication, confirming the potential for combination of these two classes in the treatment of chronic HCV infection. TMC647055 is a potent nonnucleoside NS5B polymerase inhibitor of HCV replication with a promising in vitro biochemical, kinetic, and virological profile that is currently undergoing clinical evaluation.
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Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Hepacivirus/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Sulfonamidas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Linhagem Celular , Clonagem Molecular , Combinação de Medicamentos , Sinergismo Farmacológico , Escherichia coli/genética , Genes Reporter , Hepacivirus/enzimologia , Hepacivirus/genética , Hepacivirus/crescimento & desenvolvimento , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/virologia , Humanos , Plasmídeos , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Transfecção , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: Expressed short hairpin RNAs (shRNA) used in mammalian RNA interference (RNAi) are often designed around a specific short interfering RNA (siRNA) core. Whilst there are algorithms to aid siRNA design, hairpin-specific characteristics such as stem-length and siRNA core placement within the stem are not well defined. RESULTS: Using more than 91 hairpins designed against HIV-1 Tat and Vpu, we investigated the influence of both of these factors on suppressive activity, and found that stem length does not correspond with predictable changes in suppressive activity. We also detected multiple processed products for all stem lengths tested. However, the entire length of the hairpin stem was not equally processed into active products. As such, the placement of the siRNA core at the base terminus was critical for activity. CONCLUSION: We conclude that there is no fixed correlation between stem length and suppressive activity. Instead, core selection and placement likely have a greater influence on the effectiveness of shRNA-based silencing.
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RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Sequência de Bases , Vetores Genéticos , Células HEK293 , Humanos , Dados de Sequência Molecular , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
Chronic infection with hepatitis C virus (HCV) is a major global health burden and is associated with an increased risk of liver cirrhosis and hepatocellular carcinoma. Current therapy for HCV infection has limited efficacy, particularly against genotype 1 virus, and is hampered by a range of adverse effects. Therefore, there is a clear unmet medical need for efficacious and safe direct antiviral drugs for use in combination with current treatments to increase cure rates and shorten treatment times. The broad genotypic coverage achievable with nucleosides or nucleotides and the high genetic barrier to resistance of these compounds observed in vitro and in vivo suggest that this class of inhibitors could be a valuable component of future therapeutic regimens. Here, we report the in vitro inhibitory activity and mode of action of 2'-deoxy-2'-spirocyclopropylcytidine (TMC647078), a novel and potent nucleoside inhibitor of the HCV NS5B RNA-dependent RNA polymerase that causes chain termination of the nascent HCV RNA chain. In vitro combination studies with a protease inhibitor resulted in additive efficacy in the suppression of HCV RNA replication, highlighting the potential for the combination of these two classes in the treatment of chronic HCV infection. No cytotoxic effects were observed in various cell lines. Biochemical studies indicated that TMC647078 is phosphorylated mainly by deoxycytidine kinase (dCK) without inhibiting the phosphorylation of the natural substrate, and high levels of triphosphate were observed in Huh7 cells and in primary hepatocytes in vitro. TMC647078 is a potent novel nucleoside inhibitor of HCV replication with a promising in vitro virology and biology profile.
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Antivirais/farmacologia , Citidina/análogos & derivados , Hepacivirus/efeitos dos fármacos , Compostos de Espiro/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/metabolismo , Linhagem Celular , Citidina/metabolismo , Citidina/farmacologia , Desoxicitidina Quinase/metabolismo , Humanos , Mitocôndrias/efeitos dos fármacos , Fenótipo , Fosforilação , Inibidores de Proteases/metabolismo , Inibidores de Proteases/farmacologia , RNA Viral/genética , RNA Viral/metabolismo , Compostos de Espiro/metabolismo , Proteínas não Estruturais Virais/genéticaRESUMO
RNAi gene therapies for HIV-1 will likely need to employ multiple shRNAs to counter resistant strains. We evaluated 3 shRNA co-expression methods to determine their suitability for present use; multiple expression vectors, multiple expression cassettes and single transcripts comprised of several dsRNA units (aka domains) with each being designed to a different target. Though the multiple vector strategy was effective with 2 shRNAs, the increasing number of vectors required is a major shortcoming. With single transcript configurations we only saw adequate activity from 1 of 10 variants tested, the variants being comprised of 2 - 3 different target domains. Whilst single transcript configurations have the most advantages on paper, these configurations can not yet be rapidly and reliably re-configured for new targets. However, our multiple cassette combinations of 2, 3 and 4 (29 bp) shRNAs were all successful, with suitable activity maintained in all positions and net activities comparable to that of the corresponding single shRNAs. We conclude that the multiple cassette strategy is the most suitably developed for present use as it is easy to design, assemble, is directly compatible with pre-existing shRNA and can be easily expanded.
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TMC435 is a small-molecule inhibitor of the NS3/4A serine protease of hepatitis C virus (HCV) currently in phase 2 development. The in vitro resistance profile of TMC435 was characterized by selection experiments with HCV genotype 1 replicon cells and the genotype 2a JFH-1 system. In 80% (86/109) of the sequences from genotype 1 replicon cells analyzed, a mutation at NS3 residue D168 was observed, with changes to V or A being the most frequent. Mutations at NS3 positions 43, 80, 155, and 156, alone or in combination, were also identified. A transient replicon assay confirmed the relevance of these positions for TMC435 inhibitory activity. The change in the 50% effective concentrations (EC(50)s) observed for replicons with mutations at position 168 ranged from <10-fold for those with the D168G or D168N mutation to approximately 2,000-fold for those with the D168V or D168I mutation, compared to the EC(50) for the wild type. Of the positions identified, mutations at residue Q80 had the least impact on the activity of TMC435 (<10-fold change in EC(50)s), while greater effects were observed for some replicons with mutations at positions 43, 155, and 156. TMC435 remained active against replicons with the specific mutations observed after in vitro or in vivo exposure to telaprevir or boceprevir, including most replicons with changes at positions 36, 54, and 170 (<3-fold change in EC(50)s). Replicons carrying mutations affecting the activity of TMC435 remained fully susceptible to alpha interferon and NS5A and NS5B inhibitors. Finally, combinations of TMC435 with alpha interferon and NS5B polymerase inhibitors prevented the formation of drug-resistant replicon colonies.
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Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Compostos Heterocíclicos com 3 Anéis/farmacologia , Inibidores de Proteases/farmacologia , Sulfonamidas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/farmacologia , Linhagem Celular , Farmacorresistência Viral/efeitos dos fármacos , Farmacorresistência Viral/genética , Sinergismo Farmacológico , Genótipo , Hepacivirus/enzimologia , Hepacivirus/genética , Hepatite C/virologia , Humanos , Técnicas In Vitro , Interferon-alfa/farmacologia , Mutagênese , Simeprevir , Proteínas não Estruturais Virais/genética , Replicação Viral/efeitos dos fármacosRESUMO
The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is an unusually attractive target for drug discovery since it contains five distinct drugable sites. The success of novel antiviral therapies will require nonnucleoside inhibitors to be active in at least patients infected with HCV of subtypes 1a and 1b. Therefore, the genotypic assessment of these agents against clinical isolates derived from genotype 1-infected patients is an important prerequisite for the selection of suitable candidates for clinical development. Here we report the 1a/1b subtype profiling of polymerase inhibitors that bind at each of the four known nonnucleoside binding sites. We show that inhibition of all of the clinical isolates tested is maintained, except for inhibitors that bind at the palm-1 binding site. Subtype coverage varies across chemotypes within this class of inhibitors, and inhibition of genotype 1a improves when hydrophobic contact with the polymerase is increased. We investigated if the polymorphism of the palm-1 binding site is the sole cause of the reduced susceptibility of subtype 1a to inhibition by 1,5-benzodiazepines by using reverse genetics, X-ray crystallography, and surface plasmon resonance studies. We showed Y415F to be a key determinant in conferring resistance on subtype 1a, with this effect being mediated through an inhibitor- and enzyme-bound water molecule. Binding studies revealed that the mechanism of subtype 1a resistance is faster dissociation of the inhibitor from the enzyme.
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Antivirais/uso terapêutico , Hepacivirus/enzimologia , Hepatite C/tratamento farmacológico , Isoenzimas/antagonistas & inibidores , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/química , Benzodiazepinas/química , Benzodiazepinas/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Descoberta de Drogas , Hepacivirus/genética , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Conformação Proteica , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Replicon/fisiologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
BACKGROUND & AIMS: The search for targeted anti-hepatitis C virus (HCV) drugs is driven by the adverse effect profile and limited efficacy of the current standard of care (pegylated interferon-alpha/ribavirin). In a first-in-human trial, we tested the safety, tolerability, and pharmacokinetics of the macrocyclic HCV NS3/4A protease inhibitor TMC435 in healthy volunteers, followed by HCV genotype 1-infected patients to assess antiviral activity. METHODS: The TMC435350-C101 study was a phase I, randomized, double-blind, placebo-controlled trial in 49 healthy volunteers, followed by an open-label, nonplacebo-controlled panel in 6 genotype 1 hepatitis C patients. Healthy volunteers received oral, single, ascending doses (up to 600 mg) or 5-day multiple ascending doses (200 mg twice daily or 100, 200, or 400 mg once daily). Patients received 200 mg once daily for 5 days. Pharmacokinetics and safety were evaluated for all panels, and plasma HCV-RNA levels were determined in patients. RESULTS: There were no serious adverse events, no grade 3 reactions, and no treatment-related discontinuations; pharmacokinetics supported a once daily dosing regimen. Plasma HCV-RNA levels dropped rapidly in all patients, with a median maximal reduction of 3.9-log(10) IU/mL and a median of 6 days to maximal reduction. The initial steep reduction of HCV-RNA (median 3.5-log(10) IU/mL at day 3) was followed by a more gradual decline that was maintained over the dosing period. No viral breakthroughs (>1-log(10) IU/mL HCV-RNA increase from nadir) were observed during treatment nor in the 3 days posttreatment; HCV-RNA returned to pretreatment levels by week 4. CONCLUSIONS: Once daily TMC435 given orally was generally safe and well tolerated and demonstrated potent antiviral activity.
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Antivirais/administração & dosagem , Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Compostos Heterocíclicos com 3 Anéis/administração & dosagem , Inibidores de Proteases/administração & dosagem , RNA Viral/sangue , Sulfonamidas/administração & dosagem , Carga Viral/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidores , Administração Oral , Adulto , Antivirais/efeitos adversos , Antivirais/farmacocinética , DNA Viral/análise , Método Duplo-Cego , Esquema de Medicação , Feminino , Genótipo , Hepacivirus/enzimologia , Hepacivirus/genética , Hepatite C/diagnóstico , Compostos Heterocíclicos com 3 Anéis/efeitos adversos , Compostos Heterocíclicos com 3 Anéis/farmacocinética , Humanos , Masculino , Pessoa de Meia-Idade , Inibidores de Proteases/efeitos adversos , Inibidores de Proteases/farmacocinética , Simeprevir , Sulfonamidas/efeitos adversos , Sulfonamidas/farmacocinética , Fatores de Tempo , Resultado do Tratamento , Proteínas não Estruturais Virais/genética , Adulto JovemRESUMO
BACKGROUND: Short hairpin RNA (shRNA) encoded within an expression vector has proven an effective means of harnessing the RNA interference (RNAi) pathway in mammalian cells. A survey of the literature revealed that shRNA vector construction can be hindered by high mutation rates and the ensuing sequencing is often problematic. Current options for constructing shRNA vectors include the use of annealed complementary oligonucleotides (74 % of surveyed studies), a PCR approach using hairpin containing primers (22 %) and primer extension of hairpin templates (4 %). RESULTS: We considered primer extension the most attractive method in terms of cost. However, in initial experiments we encountered a mutation frequency of 50 % compared to a reported 20-40 % for other strategies. By modifying the technique to be an isothermal reaction using the DNA polymerase Phi29, we reduced the error rate to 10 %, making primer extension the most efficient and cost-effective approach tested. We also found that inclusion of a restriction site in the loop could be exploited for confirming construct integrity by automated sequencing, while maintaining intended gene suppression. CONCLUSION: In this study we detail simple improvements for constructing and sequencing shRNA that overcome current limitations. We also compare the advantages of our solutions against proposed alternatives. Our technical modifications will be of tangible benefit to researchers looking for a more efficient and reliable shRNA construction process.
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Clonagem Molecular/métodos , Primers do DNA/genética , Marcação de Genes/métodos , Engenharia Genética/métodos , Vetores Genéticos/genética , Sondas RNA/genética , RNA/genética , Alinhamento de Sequência/métodos , Análise de Sequência de RNA/métodos , Sequência de Bases , Inativação Gênica , Dados de Sequência MolecularRESUMO
BACKGROUND: An anti-HIV-1 tat ribozyme, termed Rz2, has been shown to inhibit HIV-1 infection/replication and to decrease HIV-1-induced pathogenicity in T-lymphocyte cell lines and normal peripheral blood T-lymphocytes. We report here the results of a phase I gene transfer clinical trial using Rz2. METHODS: Apheresis was used to obtain a peripheral blood cell population from each of four HIV-negative donors. After enrichment for CD4+ T-lymphocytes, ex vivo expansion and genetic manipulation (approximately equal aliquots of the cells were transduced with the ribozyme-containing (RRz2) and the control (LNL6) retroviral vector), these cells were infused into the corresponding HIV-1-positive twin recipient. Marking was assessed over an initial 24-week period and in total over an approximate 4-year period. RESULTS: The gene transfer procedure was shown to be safe, and technically feasible. Both RRz2- and LNL6-gene-containing peripheral blood mononuclear cells (PBMC) were detected at all time points examined to 4 years. There was concomitant gene construct expression in the absence of the need for ex vivo peripheral blood cell stimulation and there was no evidence of immune elimination of the neoR T-lymphocytes nor of silencing of the Moloney murine leukemia virus long terminal repeat. CONCLUSIONS: The proof of principle results reported here demonstrate safety and feasibility of this type of gene transfer approach. While not specifically tested, T-lymphocytes containing an anti-HIV gene construct may impact on HIV-1 viral load and CD4+ T-lymphocyte count, potentially representing a new therapeutic modality for HIV-1 infection.
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Linfócitos T CD4-Positivos/imunologia , Doenças em Gêmeos/terapia , Terapia Genética , Infecções por HIV/terapia , HIV-1 , RNA Catalítico/farmacologia , Transdução Genética , Adulto , Contagem de Linfócito CD4 , Doenças em Gêmeos/imunologia , Expressão Gênica , Genes tat/fisiologia , Vetores Genéticos , Infecções por HIV/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , RNA Catalítico/genética , Retroviridae/genética , Taxa de Sobrevida , Fatores de Tempo , Gêmeos MonozigóticosRESUMO
A phase I gene transfer clinical study was undertaken to examine the ability to introduce a potential anti-human immunodeficiency virus (HIV) gene therapeutic into hematopoietic progenitor cells (HPC), thereby contributing to multilineage engraftment. The potential therapeutic effect of genetically modifying HPC with protective genes in HIV-infected adults depends in part on the presence of adult thymic activity and myeloid capacity in the setting of HIV replication. Herein we report the presence and expression of a retroviral vector encoding an anti-HIV-1 ribozyme in mature hematopoietic cells of different lineages, and de novo T-lymphocyte development ensuing from genetically engineered CD34(+) HPC. Sustained output of vector-containing mature myeloid and T-lymphoid cells was detected even in patients with multidrug-resistant infection. In addition, the study showed that the degree of persistence of gene-containing cells was dependent on transduced HPC dose. These novel findings support the concept of gene therapy as a modality to effect immune reconstitution with cells engineered to inhibit HIV replication and this report represents the first demonstration of long-term maintenance of a potential therapeutic transgene in HIV disease.