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1.
Nature ; 572(7768): 199-204, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31292543

RESUMO

The human liver is an essential multifunctional organ. The incidence of liver diseases is rising and there are limited treatment options. However, the cellular composition of the liver remains poorly understood. Here we performed single-cell RNA sequencing of about 10,000 cells from normal liver tissue from nine human donors to construct a human liver cell atlas. Our analysis identified previously unknown subtypes of endothelial cells, Kupffer cells, and hepatocytes, with transcriptome-wide zonation of some of these populations. We show that the EPCAM+ population is heterogeneous, comprising hepatocyte-biased and cholangiocyte populations as well as a TROP2int progenitor population with strong potential to form bipotent liver organoids. As a proof-of-principle, we used our atlas to unravel the phenotypic changes that occur in hepatocellular carcinoma cells and in human hepatocytes and liver endothelial cells engrafted into a mouse liver. Our human liver cell atlas provides a powerful resource to enable the discovery of previously unknown cell types in normal and diseased livers.


Assuntos
Células Epiteliais/citologia , Hepatócitos/citologia , Fígado/citologia , Células-Tronco/citologia , Adulto , Animais , Antígenos de Neoplasias/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Moléculas de Adesão Celular/metabolismo , Quimera/imunologia , Quimera/metabolismo , Células Endoteliais/citologia , Células Endoteliais/imunologia , Células Epiteliais/imunologia , Feminino , Regulação da Expressão Gênica , Hepatócitos/imunologia , Hepatócitos/metabolismo , Humanos , Fígado/imunologia , Masculino , Camundongos , Organoides/metabolismo , RNA Citoplasmático Pequeno/genética , RNA-Seq , Reprodutibilidade dos Testes , Células-Tronco/imunologia
2.
J Hepatol ; 78(2): 343-355, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36309131

RESUMO

BACKGROUND & AIMS: Despite recent approvals, the response to treatment and prognosis of patients with advanced hepatocellular carcinoma (HCC) remain poor. Claudin-1 (CLDN1) is a membrane protein that is expressed at tight junctions, but it can also be exposed non-junctionally, such as on the basolateral membrane of the human hepatocyte. While CLDN1 within tight junctions is well characterized, the role of non-junctional CLDN1 and its role as a therapeutic target in HCC remains unexplored. METHODS: Using humanized monoclonal antibodies (mAbs) specifically targeting the extracellular loop of human non-junctional CLDN1 and a large series of patient-derived cell-based and animal model systems we aimed to investigate the role of CLDN1 as a therapeutic target for HCC. RESULTS: Targeting non-junctional CLDN1 markedly suppressed tumor growth and invasion in cell line-based models of HCC and patient-derived 3D ex vivo models. Moreover, the robust effect on tumor growth was confirmed in vivo in a large series of cell line-derived xenograft and patient-derived xenograft mouse models. Mechanistic studies, including single-cell RNA sequencing of multicellular patient HCC tumorspheres, suggested that CLDN1 regulates tumor stemness, metabolism, oncogenic signaling and perturbs the tumor immune microenvironment. CONCLUSIONS: Our results provide the rationale for targeting CLDN1 in HCC and pave the way for the clinical development of CLDN1-specific mAbs for the treatment of advanced HCC. IMPACT AND IMPLICATIONS: Hepatocellular carcinoma (HCC) is associated with high mortality and unsatisfactory treatment options. Herein, we identified the cell surface protein Claudin-1 as a treatment target for advanced HCC. Monoclonal antibodies targeting Claudin-1 inhibit tumor growth in patient-derived ex vivo and in vivo models by modulating signaling, cell stemness and the tumor immune microenvironment. Given the differentiated mechanism of action, the identification of Claudin-1 as a novel therapeutic target for HCC provides an opportunity to break the plateau of limited treatment response. The results of this preclinical study pave the way for the clinical development of Claudin-1-specific antibodies for the treatment of advanced HCC. It is therefore of key impact for physicians, scientists and drug developers in the field of liver cancer and gastrointestinal oncology.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Animais , Camundongos , Carcinoma Hepatocelular/genética , Claudina-1/genética , Neoplasias Hepáticas/genética , Carcinógenos , Microambiente Tumoral , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Linhagem Celular Tumoral
3.
Gut ; 2022 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-36591611

RESUMO

OBJECTIVES: Chronic hepatitis B virus (HBV) infection is a leading cause of liver disease and hepatocellular carcinoma. A key feature of HBV replication is the synthesis of the covalently close circular (ccc)DNA, not targeted by current treatments and whose elimination would be crucial for viral cure. To date, little is known about cccDNA formation. One major challenge to address this urgent question is the absence of robust models for the study of cccDNA biology. DESIGN: We established a cell-based HBV cccDNA reporter assay and performed a loss-of-function screen targeting 239 genes encoding the human DNA damage response machinery. RESULTS: Overcoming the limitations of current models, the reporter assay enables to quantity cccDNA levels using a robust ELISA as a readout. A loss-of-function screen identified 27 candidate cccDNA host factors, including Y box binding protein 1 (YBX1), a DNA binding protein regulating transcription and translation. Validation studies in authentic infection models revealed a robust decrease in HBV cccDNA levels following silencing, providing proof-of-concept for the importance of YBX1 in the early steps of the HBV life cycle. In patients, YBX1 expression robustly correlates with both HBV load and liver disease progression. CONCLUSION: Our cell-based reporter assay enables the discovery of HBV cccDNA host factors including YBX1 and is suitable for the characterisation of cccDNA-related host factors, antiviral targets and compounds.

4.
J Hepatol ; 75(1): 64-73, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33516779

RESUMO

BACKGROUND & AIMS: Hypoxia inducible factors (HIFs) are a hallmark of inflammation and are key regulators of hepatic immunity and metabolism, yet their role in HBV replication is poorly defined. HBV replicates in hepatocytes within the liver, a naturally hypoxic organ, however most studies of viral replication are performed under conditions of atmospheric oxygen, where HIFs are inactive. We therefore investigated the role of HIFs in regulating HBV replication. METHODS: Using cell culture, animal models, human tissue and pharmacological agents inhibiting the HIF-prolyl hydroxylases, we investigated the impact of hypoxia on the HBV life cycle. RESULTS: Culturing liver cell-based model systems under low oxygen uncovered a new role for HIFs in binding HBV DNA and activating the basal core promoter, leading to increased pre-genomic RNA and de novo HBV particle secretion. The presence of hypoxia responsive elements among all primate members of the hepadnaviridae highlights an evolutionary conserved role for HIFs in regulating this virus family. CONCLUSIONS: Identifying a role for this conserved oxygen sensor in regulating HBV transcription suggests that this virus has evolved to exploit the HIF signaling pathway to persist in the low oxygen environment of the liver. Our studies show the importance of considering oxygen availability when studying HBV-host interactions and provide innovative routes to better understand and target chronic HBV infection. LAY SUMMARY: Viral replication in host cells is defined by the cellular microenvironment and one key factor is local oxygen tension. Hepatitis B virus (HBV) replicates in the liver, a naturally hypoxic organ. Hypoxia inducible factors (HIFs) are the major sensors of low oxygen; herein, we identify a new role for these factors in regulating HBV replication, revealing new therapeutic targets.


Assuntos
Vírus da Hepatite B , Fator 1 Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia , Fator 6 Semelhante a Kruppel/metabolismo , Oxigênio/metabolismo , Replicação Viral/fisiologia , Animais , Microambiente Celular , Hepadnaviridae/fisiologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Hepatite B Crônica/metabolismo , Hepatite B Crônica/virologia , Interações entre Hospedeiro e Microrganismos , Humanos , Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/antagonistas & inibidores , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Fígado/metabolismo , Transdução de Sinais , Ativação Transcricional
5.
PLoS Pathog ; 14(3): e1006908, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29505618

RESUMO

Amino-acid coevolution can be referred to mutational compensatory patterns preserving the function of a protein. Viral envelope glycoproteins, which mediate entry of enveloped viruses into their host cells, are shaped by coevolution signals that confer to viruses the plasticity to evade neutralizing antibodies without altering viral entry mechanisms. The functions and structures of the two envelope glycoproteins of the Hepatitis C Virus (HCV), E1 and E2, are poorly described. Especially, how these two proteins mediate the HCV fusion process between the viral and the cell membrane remains elusive. Here, as a proof of concept, we aimed to take advantage of an original coevolution method recently developed to shed light on the HCV fusion mechanism. When first applied to the well-characterized Dengue Virus (DENV) envelope glycoproteins, coevolution analysis was able to predict important structural features and rearrangements of these viral protein complexes. When applied to HCV E1E2, computational coevolution analysis predicted that E1 and E2 refold interdependently during fusion through rearrangements of the E2 Back Layer (BL). Consistently, a soluble BL-derived polypeptide inhibited HCV infection of hepatoma cell lines, primary human hepatocytes and humanized liver mice. We showed that this polypeptide specifically inhibited HCV fusogenic rearrangements, hence supporting the critical role of this domain during HCV fusion. By combining coevolution analysis and in vitro assays, we also uncovered functionally-significant coevolving signals between E1 and E2 BL/Stem regions that govern HCV fusion, demonstrating the accuracy of our coevolution predictions. Altogether, our work shed light on important structural features of the HCV fusion mechanism and contributes to advance our functional understanding of this process. This study also provides an important proof of concept that coevolution can be employed to explore viral protein mediated-processes, and can guide the development of innovative translational strategies against challenging human-tropic viruses.


Assuntos
Evolução Molecular , Hepacivirus/fisiologia , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus , Animais , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Hepatite C/metabolismo , Hepatite C/patologia , Hepatite C/virologia , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Células Tumorais Cultivadas , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Replicação Viral
6.
Hepatology ; 68(5): 1695-1709, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29679386

RESUMO

Chronic hepatitis B virus (HBV) infection is a major cause of chronic liver disease and cancer worldwide. The mechanisms of viral genome sensing and the evasion of innate immune responses by HBV infection are still poorly understood. Recently, the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) was identified as a DNA sensor. In this study, we investigated the functional role of cGAS in sensing HBV infection and elucidate the mechanisms of viral evasion. We performed functional studies including loss-of-function and gain-of-function experiments combined with cGAS effector gene expression profiling in an infectious cell culture model, primary human hepatocytes, and HBV-infected human liver chimeric mice. Here, we show that cGAS is expressed in the human liver, primary human hepatocytes, and human liver chimeric mice. While naked relaxed-circular HBV DNA is sensed in a cGAS-dependent manner in hepatoma cell lines and primary human hepatocytes, host cell recognition of viral nucleic acids is abolished during HBV infection, suggesting escape from sensing, likely during packaging of the genome into the viral capsid. While the hepatocyte cGAS pathway is functionally active, as shown by reduction of viral covalently closed circular DNA levels in gain-of-function studies, HBV infection suppressed cGAS expression and function in cell culture models and humanized mice. Conclusion: HBV exploits multiple strategies to evade sensing and antiviral activity of cGAS and its effector pathways.


Assuntos
Vírus da Hepatite B/patogenicidade , Hepatite B/fisiopatologia , Hepatócitos/virologia , Evasão da Resposta Imune/fisiologia , Nucleotídeos Cíclicos/metabolismo , Animais , Western Blotting , Técnicas de Cultura de Células , DNA Viral/imunologia , Perfilação da Expressão Gênica/métodos , Hepatite B/imunologia , Hepatócitos/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Evasão da Resposta Imune/imunologia , Hibridização in Situ Fluorescente/métodos , Camundongos , Reação em Cadeia da Polimerase em Tempo Real
7.
Gut ; 67(4): 736-745, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-28360099

RESUMO

OBJECTIVE: HCV infection is a leading cause of chronic liver disease and a major indication for liver transplantation. Although direct-acting antivirals (DAAs) have much improved the treatment of chronic HCV infection, alternative strategies are needed for patients with treatment failure. As an essential HCV entry factor, the tight junction protein claudin-1 (CLDN1) is a promising antiviral target. However, genotype-dependent escape via CLDN6 and CLDN9 has been described in some cell lines as a possible limitation facing CLDN1-targeted therapies. Here, we evaluated the clinical potential of therapeutic strategies targeting CLDN1. DESIGN: We generated a humanised anti-CLDN1 monoclonal antibody (mAb) (H3L3) suitable for clinical development and characterised its anti-HCV activity using cell culture models, a large panel of primary human hepatocytes (PHH) from 12 different donors, and human liver chimeric mice. RESULTS: H3L3 pan-genotypically inhibited HCV pseudoparticle entry into PHH, irrespective of donor. Escape was likely precluded by low surface expression of CLDN6 and CLDN9 on PHH. Co-treatment of a panel of PHH with a CLDN6-specific mAb did not enhance the antiviral effect of H3L3, confirming that CLDN6 does not function as an entry factor in PHH from multiple donors. H3L3 also inhibited DAA-resistant strains of HCV and synergised with current DAAs. Finally, H3L3 cured persistent HCV infection in human-liver chimeric uPA-SCID mice in monotherapy. CONCLUSIONS: Overall, these findings underscore the clinical potential of CLDN1-targeted therapies and describe the functional characterisation of a humanised anti-CLDN1 antibody suitable for further clinical development to complement existing therapeutic strategies for HCV.


Assuntos
Anticorpos Monoclonais/farmacologia , Antivirais/farmacologia , Claudina-1/antagonistas & inibidores , Hepacivirus/efeitos dos fármacos , Hepatite C/prevenção & controle , Hepatócitos/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Animais , Claudina-1/imunologia , Hepatite C/imunologia , Hepatócitos/imunologia , Hepatócitos/virologia , Humanos , Camundongos , Camundongos SCID , Resultado do Tratamento
8.
J Hepatol ; 66(5): 919-929, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28126468

RESUMO

BACKGROUND & AIMS: Chronic hepatitis C is a global health problem with an estimated 170 million hepatitis C virus (HCV) infected individuals at risk of progressive liver disease and hepatocellular carcinoma (HCC). Autotaxin (ATX, gene name: ENPP2) is a phospholipase with diverse roles in the physiological and pathological processes including inflammation and oncogenesis. Clinical studies have reported increased ATX expression in chronic hepatitis C, however, the pathways regulating ATX and its role in the viral life cycle are not well understood. METHODS: In vitro hepatocyte and ex vivo liver culture systems along with chimeric humanized liver mice and HCC tissue enabled us to assess the interplay between ATX and the HCV life cycle. RESULTS: HCV infection increased hepatocellular ATX RNA and protein expression. HCV infection stabilizes hypoxia inducible factors (HIFs) and we investigated a role for these transcription factors to regulate ATX. In vitro studies show that low oxygen increases hepatocellular ATX expression and transcriptome analysis showed a positive correlation between ATX mRNA levels and hypoxia gene score in HCC tumour tissue associated with HCV and other aetiologies. Importantly, inhibiting ATX-lysophosphatidic acid (LPA) signalling reduced HCV replication, demonstrating a positive role for this phospholipase in the viral life cycle. LPA activates phosphoinositide-3-kinase that stabilizes HIF-1α and inhibiting the HIF signalling pathway abrogates the pro-viral activity of LPA. CONCLUSIONS: Our data support a model where HCV infection increases ATX expression which supports viral replication and HCC progression. LAY SUMMARY: Chronic hepatitis C is a global health problem with infected individuals at risk of developing liver disease that can progress to hepatocellular carcinoma. Autotaxin generates the biologically active lipid lysophosphatidic acid that has been reported to play a tumorigenic role in a wide number of cancers. In this study we show that hepatitis C virus infection increases autotaxin expression via hypoxia inducible transcription factor and provides an environment in the liver that promotes fibrosis and liver injury. Importantly, we show a new role for lysophosphatidic acid in positively regulating hepatitis C virus replication.


Assuntos
Hepacivirus/fisiologia , Diester Fosfórico Hidrolases/fisiologia , Receptores de Ácidos Lisofosfatídicos/fisiologia , Replicação Viral , Animais , Linhagem Celular , Hepatite C Crônica/complicações , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Neoplasias Hepáticas/etiologia , Camundongos , Diester Fosfórico Hidrolases/genética , Regiões Promotoras Genéticas , RNA Mensageiro/análise , Transdução de Sinais
9.
Gut ; 64(3): 483-94, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24848265

RESUMO

OBJECTIVE: Although direct-acting antiviral agents (DAAs) have markedly improved the outcome of treatment in chronic HCV infection, there continues to be an unmet medical need for improved therapies in difficult-to-treat patients as well as liver graft infection. Viral entry is a promising target for antiviral therapy. DESIGN: Aiming to explore the role of entry inhibitors for future clinical development, we investigated the antiviral efficacy and toxicity of entry inhibitors in combination with DAAs or other host-targeting agents (HTAs). Screening a large series of combinations of entry inhibitors with DAAs or other HTAs, we uncovered novel combinations of antivirals for prevention and treatment of HCV infection. RESULTS: Combinations of DAAs or HTAs and entry inhibitors including CD81-, scavenger receptor class B type I (SR-BI)- or claudin-1 (CLDN1)-specific antibodies or small-molecule inhibitors erlotinib and dasatinib were characterised by a marked and synergistic inhibition of HCV infection over a broad range of concentrations with undetectable toxicity in experimental designs for prevention and treatment both in cell culture models and in human liver-chimeric uPA/SCID mice. CONCLUSIONS: Our results provide a rationale for the development of antiviral strategies combining entry inhibitors with DAAs or HTAs by taking advantage of synergy. The uncovered combinations provide perspectives for efficient strategies to prevent liver graft infection and novel interferon-free regimens.


Assuntos
Antivirais/uso terapêutico , Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Internalização do Vírus/efeitos dos fármacos , Animais , Antivirais/administração & dosagem , Linhagem Celular , Quimera , Sinergismo Farmacológico , Quimioterapia Combinada , Hepatite C/prevenção & controle , Hepatócitos/efeitos dos fármacos , Hepatócitos/virologia , Humanos , Camundongos , Camundongos SCID
10.
Biochem Biophys Res Commun ; 465(4): 658-64, 2015 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-26241675

RESUMO

High-risk human papillomavirus (HPV) types 16 and 18 are associated with more than 70% of cervical cancer cases. The oncoprotein E6 is multifunctional and has numerous cellular partners. The best-known activity of E6 is the polyubiquination of the pro-apoptotic tumor suppressor p53, targeting it for degradation by the 26S proteasome. Loss of p53 triggers genomic instability and favors cancer development. Here, we generated recombinant adenovirus (Ad) vectors expressing artificial microRNAs directed against HPV16 E6 (Ad16_1) or HPV18 E6 (Ad18_2). E6-knockdown was observed in HeLa after treatment with Ad18_2 and in SiHa with Ad16_1. Western-blot experiments found an increase in p53 levels after treatment in both cell lines. Cell death was observed in both cell lines after knockdown of E6. Further analysis such as cleavage of caspases (3 and 7) as well as of PARP1 indicated that treated HeLa and SiHa cells underwent apoptosis. The growth of HeLa-derived tumors developed in nude mice was significantly reduced after intra-tumoral injection of Ad18_2. Therefore, vectorisation of artificial miRNA against E6 oncoprotein by means of recombinant adenoviruses might represent a valuable therapeutic approach for treating HPV-positive cancers.


Assuntos
Apoptose/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , MicroRNAs/genética , Proteínas Oncogênicas Virais/antagonistas & inibidores , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/terapia , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Neoplasias do Colo do Útero/terapia , Adenoviridae/genética , Animais , Linhagem Celular , Feminino , Técnicas de Silenciamento de Genes , Terapia Genética , Vetores Genéticos , Células HeLa , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/patogenicidade , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/patogenicidade , Humanos , Camundongos , Camundongos Nus , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Proteína Supressora de Tumor p53/metabolismo , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologia , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Mol Ther ; 22(3): 634-644, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24445938

RESUMO

Cell therapy based on alloreactivity has completed clinical proof of concept against hematological malignancies. However, the efficacy of alloreactivity as a therapeutic approach to treat solid tumors is unknown. Using cell culture and animal models, we aimed to investigate the efficacy and safety of allogeneic suicide gene-modified killer cells as a cell-based therapy for hepatocellular carcinoma (HCC), for which treatment options are limited. Allogeneic killer cells from healthy donors were isolated, expanded, and phenotypically characterized. Antitumor cytotoxic activity and safety were studied using a panel of human or murine HCC cell lines engrafted in immunodeficient or immunocompetent mouse models. Human allogeneic suicide gene-modified killer cells (aSGMKCs) exhibit a high, rapid, interleukin-2-dependent, and non-major histocompatibility complex class I-restricted in vitro cytotoxicity toward human hepatoma cells, mainly mediated by natural killer (NK) and NK-like T cells. In vivo evaluation of this cell therapy product demonstrates a marked, rapid, and sustained regression of HCC. Preferential liver homing of effector cells contributed to its marked efficacy. Calcineurin inhibitors allowed preventing rejection of allogeneic lymphocytes by the host immune system without impairing their antitumor activity. Our results demonstrate proof of concept for aSGMKCs as immunotherapy for HCC and open perspectives for the clinical development of this approach.


Assuntos
Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/terapia , Citotoxicidade Imunológica , Neoplasias Hepáticas/imunologia , Transplante de Neoplasias/imunologia , Linfócitos T/imunologia , Transplante Homólogo/métodos , Animais , Linhagem Celular Tumoral , Células HeLa , Humanos , Imunoterapia Adotiva , Neoplasias Hepáticas/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Linfócitos T/transplante
12.
J Virol ; 86(19): 10418-31, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22787235

RESUMO

In this study, our goal was to generate a chimeric adenovirus-parvovirus (Ad-PV) vector that combines the high-titer and efficient gene transfer of adenovirus with the anticancer potential of rodent parvovirus. To this end, the entire oncolytic PV genome was inserted into a replication-defective E1- and E3-deleted Ad5 vector genome. As we found that parvoviral NS expression inhibited Ad-PV chimera production, we engineered the parvoviral P4 early promoter, which governs NS expression, by inserting into its sequence tetracycline operator elements. As a result of these modifications, P4-driven expression was blocked in the packaging T-REx-293 cells, which constitutively express the tetracycline repressor, allowing high-yield chimera production. The chimera effectively delivered the PV genome into cancer cells, from which fully infectious replication-competent parvovirus particles were generated. Remarkably, the Ad-PV chimera exerted stronger cytotoxic activities against various cancer cell lines, compared with the PV and Ad parental viruses, while being still innocuous to a panel of tested healthy primary human cells. This Ad-PV chimera represents a novel versatile anticancer agent which can be subjected to further genetic manipulations in order to reinforce its enhanced oncolytic capacity through arming with transgenes or retargeting into tumor cells.


Assuntos
Adenoviridae/metabolismo , Vírus Oncolíticos/metabolismo , Parvovirus/metabolismo , Animais , Sequência de Bases , Proliferação de Células , Sobrevivência Celular , Clonagem Molecular , Fibroblastos/citologia , Deleção de Genes , Células HEK293 , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Sais de Tetrazólio/farmacologia , Tiazóis/farmacologia , Virologia/métodos
14.
Sci Transl Med ; 14(676): eabj4221, 2022 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-36542691

RESUMO

Tissue fibrosis is a key driver of end-stage organ failure and cancer, overall accounting for up to 45% of deaths in developed countries. There is a large unmet medical need for antifibrotic therapies. Claudin-1 (CLDN1) is a member of the tight junction protein family. Although the role of CLDN1 incorporated in tight junctions is well established, the function of nonjunctional CLDN1 (njCLDN1) is largely unknown. Using highly specific monoclonal antibodies targeting a conformation-dependent epitope of exposed njCLDN1, we show in patient-derived liver three-dimensional fibrosis and human liver chimeric mouse models that CLDN1 is a mediator and target for liver fibrosis. Targeting CLDN1 reverted inflammation-induced hepatocyte profibrogenic signaling and cell fate and suppressed the myofibroblast differentiation of hepatic stellate cells. Safety studies of a fully humanized antibody in nonhuman primates did not reveal any serious adverse events even at high steady-state concentrations. Our results provide preclinical proof of concept for CLDN1-specific monoclonal antibodies for the treatment of advanced liver fibrosis and cancer prevention. Antifibrotic effects in lung and kidney fibrosis models further indicate a role of CLDN1 as a therapeutic target for tissue fibrosis across organs. In conclusion, our data pave the way for further therapeutic exploration of CLDN1-targeting therapies for fibrotic diseases in patients.


Assuntos
Anticorpos Monoclonais , Plasticidade Celular , Animais , Camundongos , Humanos , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Claudina-1 , Cirrose Hepática/tratamento farmacológico
15.
Nat Commun ; 12(1): 1658, 2021 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-33712578

RESUMO

Chronic hepatitis B virus (HBV) infection is a major cause of liver disease and cancer worldwide for which there are no curative therapies. The major challenge in curing infection is eradicating or silencing the covalent closed circular DNA (cccDNA) form of the viral genome. The circadian factors BMAL1/CLOCK and REV-ERB are master regulators of the liver transcriptome and yet their role in HBV replication is unknown. We establish a circadian cycling liver cell-model and demonstrate that REV-ERB directly regulates NTCP-dependent hepatitis B and delta virus particle entry. Importantly, we show that pharmacological activation of REV-ERB inhibits HBV infection in vitro and in human liver chimeric mice. We uncover a role for BMAL1 to bind HBV genomes and increase viral promoter activity. Pharmacological inhibition of BMAL1 through REV-ERB ligands reduces pre-genomic RNA and de novo particle secretion. The presence of conserved E-box motifs among members of the Hepadnaviridae family highlight an evolutionarily conserved role for BMAL1 in regulating this family of small DNA viruses.


Assuntos
Relógios Biológicos/fisiologia , Ritmo Circadiano/fisiologia , Vírus da Hepatite B/fisiologia , Replicação Viral/fisiologia , Animais , Relógios Biológicos/efeitos dos fármacos , Relógios Biológicos/genética , Ritmo Circadiano/genética , DNA Circular , DNA Viral/metabolismo , Regulação da Expressão Gênica , Genoma Viral , Células Hep G2 , Hepatite B/virologia , Vírus da Hepatite B/genética , Hepatite B Crônica/genética , Hepatócitos/metabolismo , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Fígado/metabolismo , Camundongos , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Regiões Promotoras Genéticas , Simportadores/metabolismo , Transcriptoma , Vírion/metabolismo , Internalização do Vírus
16.
bioRxiv ; 2021 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-33758862

RESUMO

The COVID-19 pandemic, caused by SARS-CoV-2 coronavirus, is a global health issue with unprecedented challenges for public health. SARS-CoV-2 primarily infects cells of the respiratory tract, via Spike glycoprotein binding angiotensin-converting enzyme (ACE2). Circadian rhythms coordinate an organism’s response to its environment and can regulate host susceptibility to virus infection. We demonstrate a circadian regulation of ACE2 in lung epithelial cells and show that silencing BMAL1 or treatment with a synthetic REV-ERB agonist SR9009 reduces ACE2 expression and inhibits SARS-CoV-2 entry. Treating infected cells with SR9009 limits viral replication and secretion of infectious particles, showing that post-entry steps in the viral life cycle are influenced by the circadian system. Transcriptome analysis revealed that Bmal1 silencing induced a wide spectrum of interferon stimulated genes in Calu-3 lung epithelial cells, providing a mechanism for the circadian pathway to dampen SARS-CoV-2 infection. Our study suggests new approaches to understand and improve therapeutic targeting of SARS-CoV-2.

17.
iScience ; 24(10): 103144, 2021 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-34545347

RESUMO

The coronavirus disease 2019 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus, is a global health issue with unprecedented challenges for public health. SARS-CoV-2 primarily infects cells of the respiratory tract via spike glycoprotein binding to angiotensin-converting enzyme (ACE2). Circadian rhythms coordinate an organism's response to its environment and can regulate host susceptibility to virus infection. We demonstrate that silencing the circadian regulator Bmal1 or treating lung epithelial cells with the REV-ERB agonist SR9009 reduces ACE2 expression and inhibits SARS-CoV-2 entry and replication. Importantly, treating infected cells with SR9009 limits SARS-CoV-2 replication and secretion of infectious particles, showing that post-entry steps in the viral life cycle are influenced by the circadian system. Transcriptome analysis revealed that Bmal1 silencing induced interferon-stimulated gene transcripts in Calu-3 lung epithelial cells, providing a mechanism for the circadian pathway to limit SARS-CoV-2 infection. Our study highlights alternative approaches to understand and improve therapeutic targeting of SARS-CoV-2.

18.
Biochim Biophys Acta Biomembr ; 1862(7): 183296, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32268133

RESUMO

The hepatitis C virus (HCV) is a major cause of liver diseases ranging from liver inflammation to advanced liver diseases like cirrhosis and hepatocellular carcinoma (HCC). HCV infection is restricted to the liver, and more specifically to hepatocytes, which represent around 80% of liver cells. The mechanism of HCV entry in human hepatocytes has been extensively investigated since the discovery of the virus 30 years ago. The entry mechanism is a multi-step process relying on several host factors including heparan sulfate proteoglycan (HSPG), low density lipoprotein receptor (LDLR), tetraspanin CD81, Scavenger Receptor class B type I (SR-BI), Epidermal Growth Factor Receptor (EGFR) and Niemann-Pick C1-like 1 (NPC1L1). Moreover, in order to establish a persistent infection, HCV entry is dependent on the presence of tight junction (TJ) proteins Claudin-1 (CLDN1) and Occludin (OCLN). In the liver, tight junction proteins play a role in architecture and homeostasis including sealing the apical pole of adjacent cells to form bile canaliculi and separating the basolateral domain drained by sinusoidal blood flow. In this review, we will highlight the role of liver tight junction proteins in HCV infection, and we will discuss the potential targeted therapeutic approaches to improve virus eradication.


Assuntos
Hepatite C/genética , Interações Hospedeiro-Patógeno/genética , Junções Íntimas/genética , Internalização do Vírus , Hepacivirus/genética , Hepacivirus/patogenicidade , Hepatite C/virologia , Hepatócitos/virologia , Humanos , Fígado/patologia , Fígado/virologia , Proteínas de Membrana Transportadoras/genética , Ocludina/genética , Receptores de LDL/genética , Tetraspanina 28/genética , Junções Íntimas/virologia
20.
Antiviral Res ; 162: 136-141, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30599173

RESUMO

Despite the development of direct-acting antivirals (DAAs), hepatitis C virus (HCV) infection remains a major cause for liver disease and cancer worldwide. Entry inhibitors block virus host cell entry and, therefore, prevent establishment of chronic infection and liver disease. Due to their unique mechanism of action, entry inhibitors provide an attractive antiviral strategy in organ transplantation. In this study, we developed an innovative approach in preventing HCV infection using a synergistic combination of a broadly neutralizing human monoclonal antibody (HMAb) targeting the HCV E2 protein and a host-targeting anti-claudin 1 (CLDN1) humanized monoclonal antibody. An in vivo proof-of-concept study in human liver-chimeric FRG-NOD mice proved the efficacy of the combination therapy at preventing infection by an HCV genotype 1b infectious serum. While administration of individual antibodies at lower doses only showed a delay in HCV infection, the combination therapy was highly protective. Furthermore, the combination proved to be effective in preventing infection of primary human hepatocytes by neutralization-resistant HCV escape variants selected during liver transplantation, suggesting that a combination therapy is suited for the neutralization of difficult-to-treat variants. In conclusion, our findings suggest that the combination of two HMAbs targeting different steps of virus entry improves treatment efficacy while simultaneously reducing treatment duration and costs. Our approach not only provides a clinical perspective to employ HMAb combination therapies to prevent graft re-infection and its associated liver disease but may also help to alleviate the urgent demand for organ transplants by allowing the transplantation of organs from HCV-positive donors.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Claudina-1/imunologia , Anticorpos Anti-Hepatite C/administração & dosagem , Hepatite C/prevenção & controle , Proteínas do Envelope Viral/imunologia , Internalização do Vírus/efeitos dos fármacos , Animais , Animais Geneticamente Modificados , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/imunologia , Antivirais/administração & dosagem , Antivirais/farmacologia , Combinação de Medicamentos , Sinergismo Farmacológico , Hepacivirus/efeitos dos fármacos , Hepatite C/imunologia , Anticorpos Anti-Hepatite C/imunologia , Humanos , Camundongos Endogâmicos NOD , Estudo de Prova de Conceito
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