RESUMO
Chronic hepatitis B virus (HBV) infection persists due to the lack of therapies that effectively target the HBV covalently closed circular DNA (cccDNA). We used HBV-specific guide RNAs (gRNAs) and CRISPR-Cas9 and determined the fate of cccDNA after gene editing. We set up a ribonucleoprotein (RNP) delivery system in HBV-infected HepG2-NTCP cells. HBV parameters after Cas9 editing were analyzed. Southern blot (SB) analysis and DNA/RNA sequencing (DNA/RNA-seq) were performed to determine the consequences of cccDNA editing and transcriptional activity of mutated cccDNA. Treatment of infected cells with HBV-specific gRNAs showed that CRISPR-Cas9 can efficiently affect HBV replication. The appearance of episomal HBV DNA variants after dual gRNA treatment was observed by PCR, SB analysis, and DNA/RNA-seq. These transcriptionally active variants are the products of simultaneous Cas9-induced double-strand breaks in two target sites, followed by repair and religation of both short and long fragments. Following suppression of HBV DNA replicative intermediates by nucleoside analogs, mutations and formation of smaller transcriptionally active HBV variants were still observed, suggesting that established cccDNA is accessible to CRISPR-Cas9 editing. Targeting HBV DNA with CRISPR-Cas9 leads to cleavage followed by appearance of episomal HBV DNA variants. Effects induced by Cas9 were sustainable after RNP degradation/loss of detection, suggesting permanent changes in the HBV genome instead of transient effects due to transcriptional interference. IMPORTANCE Hepatitis B virus infection can develop into chronic infection, cirrhosis, and hepatocellular carcinoma. Treatment of chronic hepatitis B requires novel approaches to directly target the viral minichromosome, which is responsible for the persistence of the disease. Designer nuclease approaches represent a promising strategy to treat chronic infectious diseases; however, comprehensive knowledge about the fate of the HBV minichromosome is needed before this potent tool can be used as a potential therapeutic approach. This study provides an in-depth analysis of CRISPR-Cas9 targeting of HBV minichromosome.
Assuntos
Vírus da Hepatite B , Hepatite B Crônica , Sistemas CRISPR-Cas , DNA Circular/genética , DNA Viral/genética , Vírus da Hepatite B/genética , Hepatite B Crônica/tratamento farmacológico , Humanos , RNA Guia de Cinetoplastídeos/genéticaRESUMO
Optogenetics enables genome manipulations with high spatiotemporal resolution, opening exciting possibilities for fundamental and applied biological research. Here, we report the development of LiCre, a novel light-inducible Cre recombinase. LiCre is made of a single flavin-containing protein comprising the AsLOV2 photoreceptor domain of Avena sativa fused to a Cre variant carrying destabilizing mutations in its N-terminal and C-terminal domains. LiCre can be activated within minutes of illumination with blue light without the need of additional chemicals. When compared to existing photoactivatable Cre recombinases based on two split units, LiCre displayed faster and stronger activation by light as well as a lower residual activity in the dark. LiCre was efficient both in yeast, where it allowed us to control the production of ß-carotene with light, and human cells. Given its simplicity and performances, LiCre is particularly suited for fundamental and biomedical research, as well as for controlling industrial bioprocesses.
In a biologist's toolkit, the Cre protein holds a special place. Naturally found in certain viruses, this enzyme recognises and modifies specific genetic sequences, creating changes that switch on or off whatever gene is close by. Genetically engineering cells or organisms so that they carry Cre and its target sequences allows scientists to control the activation of a given gene, often in a single tissue or organ. However, this relies on the ability to activate the Cre protein 'on demand' once it is in the cells of interest. One way to do so is to split the enzyme into two pieces, which can then reassemble when exposed to blue light. Yet, this involves the challenging step of introducing both parts separately into a tissue. Instead, Duplus-Bottin et al. engineered LiCre, a new system where a large section of the Cre protein is fused to a light sensor used by oats to detect their environment. LiCre is off in the dark, but it starts to recognize and modify Cre target sequences when exposed to blue light. Duplus-Bottin et al. then assessed how LiCre compares to the two-part Cre system in baker's yeast and human kidney cells. This showed that the new protein is less 'incorrectly' active in the dark, and can switch on faster under blue light. The improved approach could give scientists a better tool to study the role of certain genes at precise locations and time points, but also help them to harness genetic sequences for industry or during gene therapy.
Assuntos
Integrases/genética , Optogenética/métodos , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Humanos , Integrases/metabolismo , Luz , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
BACKGROUND: Duchenne muscular dystrophy (DMD) is a devastating X-linked recessive genetic myopathy. DMD physiopathology is still not fully understood and a prenatal onset is suspected but difficult to address. METHODS: The bone morphogenetic protein 4 (BMP4) is a critical signaling molecule involved in mesoderm commitment. Human induced pluripotent stem cells (hiPSCs) from DMD and healthy individuals and human embryonic stem cells (hESCs) treated with BMP4 allowed us to model the early steps of myogenesis in normal and DMD contexts. RESULTS: Unexpectedly, 72h following BMP4 treatment, a new long DMD transcript was detected in all tested hiPSCs and hESCs, at levels similar to that found in adult skeletal muscle. This novel transcript named "Dp412e" has a specific untranslated first exon which is conserved only in a sub-group of anthropoids including human. The corresponding novel dystrophin protein of 412-kiloDalton (kDa), characterized by an N-terminal-truncated actin-binding domain, was detected in normal BMP4-treated hiPSCs/hESCs and in embryoid bodies. Finally, using a phosphorodiamidate morpholino oligomer (PMO) targeting the DMD exon 53, we demonstrated the feasibility of exon skipping validation with this BMP4-inducible hiPSCs model. CONCLUSIONS: In this study, the use of hiPSCs to analyze early phases of human development in normal and DMD contexts has led to the discovery of an embryonic 412 kDa dystrophin isoform. Deciphering the regulation process(es) and the function(s) associated to this new isoform can contribute to a better understanding of the DMD physiopathology and potential developmental defects. Moreover, the simple and robust BMP4-inducible model highlighted here, providing large amount of a long DMD transcript and the corresponding protein in only 3 days, is already well-adapted to high-throughput and high-content screening approaches. Therefore, availability of this powerful cell platform can accelerate the development, validation and improvement of DMD genetic therapies.
Assuntos
Terapia Genética , Vetores Genéticos , Imunoterapia Ativa , Lentivirus , Glicoproteínas de Membrana/genética , Receptores de LDL/metabolismo , Transdução Genética , Proteínas do Envelope Viral/genética , Linfócitos B/imunologia , Linfócitos B/transplante , Células Cultivadas , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Humanos , Ativação Linfocitária , Linfócitos T/imunologia , Linfócitos T/transplante , Falha de Tratamento , Internalização do VírusRESUMO
Influenza A NS1 and NS2 proteins are encoded by the RNA segment 8 of the viral genome. NS1 is a multifunctional protein and a virulence factor while NS2 is involved in nuclear export of viral ribonucleoprotein complexes. A yeast two-hybrid screening strategy was used to identify host factors supporting NS1 and NS2 functions. More than 560 interactions between 79 cellular proteins and NS1 and NS2 proteins from 9 different influenza virus strains have been identified. These interacting proteins are potentially involved in each step of the infectious process and their contribution to viral replication was tested by RNA interference. Validation of the relevance of these host cell proteins for the viral replication cycle revealed that 7 of the 79 NS1 and/or NS2-interacting proteins positively or negatively controlled virus replication. One of the main factors targeted by NS1 of all virus strains was double-stranded RNA binding domain protein family. In particular, adenosine deaminase acting on RNA 1 (ADAR1) appeared as a pro-viral host factor whose expression is necessary for optimal viral protein synthesis and replication. Surprisingly, ADAR1 also appeared as a pro-viral host factor for dengue virus replication and directly interacted with the viral NS3 protein. ADAR1 editing activity was enhanced by both viruses through dengue virus NS3 and influenza virus NS1 proteins, suggesting a similar virus-host co-evolution.
Assuntos
Adenosina Desaminase/metabolismo , Interações Hospedeiro-Patógeno , Vírus da Influenza A/fisiologia , Proteínas não Estruturais Virais/metabolismo , Fatores de Virulência/metabolismo , Replicação Viral , Adenosina Desaminase/química , Adenosina Desaminase/genética , Transporte Biológico , Linhagem Celular , Vírus da Dengue/enzimologia , Humanos , Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/metabolismo , Influenza Humana/patologia , Influenza Humana/virologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/metabolismo , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Mucosa Respiratória/virologia , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Especificidade da Espécie , Técnicas do Sistema de Duplo-Híbrido , Proteínas não Estruturais Virais/genética , Fatores de Virulência/genéticaRESUMO
Identification of new techniques to express proteins into mammal cells is of particular interest for both research and medical purposes. The present study describes the use of engineered vesicles to deliver exogenous proteins into human cells. We show that overexpression of the spike glycoprotein of the vesicular stomatitis virus (VSV-G) in human cells induces the release of fusogenic vesicles named gesicles. Biochemical and functional studies revealed that gesicles incorporated proteins from producer cells and could deliver them to recipient cells. This protein-transduction method allows the direct transport of cytoplasmic, nuclear or surface proteins in target cells. This was demonstrated by showing that the TetR transactivator and the receptor for the murine leukemia virus (MLV) envelope [murine cationic amino acid transporter-1 (mCAT-1)] were efficiently delivered by gesicles in various cell types. We further shows that gesicle-mediated transfer of mCAT-1 confers to human fibroblasts a robust permissiveness to ecotropic vectors, allowing the generation of human-induced pluripotent stem cells in level 2 biosafety facilities. This highlights the great potential of mCAT-1 gesicles to increase the safety of experiments using retro/lentivectors. Besides this, gesicles is a versatile tool highly valuable for the nongenetic delivery of functions such as transcription factors or genome engineering agents.
Assuntos
Técnicas de Transferência de Genes , Vetores Genéticos , Glicoproteínas de Membrana/metabolismo , Proteínas do Envelope Viral/metabolismo , Animais , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Biologia Computacional , Citometria de Fluxo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Imuno-Histoquímica , Vírus da Leucemia Murina/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Microscopia Eletrônica de Transmissão , Células-Tronco Pluripotentes/metabolismo , Proteômica/métodos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Fatores de Transcrição/genética , Transdução Genética , Vesiculovirus/genéticaRESUMO
Hepatitis C virus (HCV) exploits serum-dependent mechanisms that inhibit neutralizing antibodies. Here we demonstrate that high density lipoprotein (HDL) is a key serum factor that attenuates neutralization by monoclonal and HCV patient-derived polyclonal antibodies of infectious pseudo-particles (HCVpp) harboring authentic E1E2 glycoproteins and cell culture-grown genuine HCV (HCVcc). Over 10-fold higher antibody concentrations are required to neutralize either HCV-enveloped particles in the presence of HDL or human serum, and less than 3-5-fold reduction of infectious titers are obtained at saturating antibody concentrations, in contrast to complete inhibition in serum-free conditions. We show that HDL interaction with the scavenger receptor BI (SR-BI), a proposed cell entry co-factor of HCV and a receptor mediating lipid transfer with HDL, strongly reduces neutralization of HCVpp and HCVcc. We found that HDL activation of target cells strongly stimulates cell entry of viral particles by accelerating their endocytosis, thereby suppressing a 1-h time lag during which cell-bound virions are not internalized and can be targeted by antibodies. Compounds that inhibit lipid transfer functions of SR-BI fully restore neutralization by antibodies in human serum. We demonstrate that this functional HDL/SR-BI interaction only interferes with antibodies blocking HCV-E2 binding to CD81, a major HCV receptor, reflecting its prominent role during the cell entry process. Moreover, we identify monoclonal antibodies targeted to epitopes in the E1E2 complex that are not inhibited by HDL. Consistently, we show that antibodies targeted to HCV-E1 efficiently neutralize HCVpp and HCVcc in the presence of human serum.
Assuntos
Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/imunologia , Lipoproteínas HDL/imunologia , Receptores Depuradores Classe B/imunologia , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Antígenos CD/imunologia , Antígenos CD/metabolismo , Epitopos , Hepatite C/sangue , Hepatite C/virologia , Anticorpos Anti-Hepatite C/metabolismo , Hepatite C Crônica/sangue , Hepatite C Crônica/virologia , Humanos , Imunidade Inata , Lipoproteínas HDL/metabolismo , Lipoproteínas HDL/farmacologia , Ligação Proteica/imunologia , Receptores Depuradores Classe B/metabolismo , Tetraspanina 28 , Replicação Viral/imunologiaRESUMO
Although the expression of Pitx2, a bicoid family homeodomain transcription factor, is highly regulated during hematopoiesis, its function during this process was not documented; we thus studied hematopoiesis in Pitx2-null mice. We found that Pitx2(-/-) embryos display hypoplastic livers with reduced numbers of hematopoietic cells, but these cells had normal hematopoietic potential, as evidenced by colony-forming assays, immature progenitor cell assays, and long-term repopulation assays. Because the microenvironment is also crucial to the development of normal hematopoiesis, we established Pitx2(-/-) and Pitx2(+/+) stromas from fetal liver and studied their hematopoietic supportive capacity. We showed that the frequency of cobblestone area-forming cells was 4-fold decreased when using Pitx2(-/-) stromal cells compared with Pitx2(+/+) stromal cells, whatever the Pitx2 genotype of hematopoietic cells tested in this assay. This defect was rescued by expression of Pitx2 into Pitx2(-/-) fetal liver stromal cells, demonstrating a major and direct role of Pitx2 in the hematopoietic supportive capacity of fetal liver stroma. Finally, we showed a reduced capacity of MS5 stromal cells expressing Pitx2 RNAi to support human hematopoiesis. Altogether these data showed that Pitx2 has major functions in the hematopoietic supportive capacity of fetal liver and adult bone marrow stromal cells.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Hematopoese/fisiologia , Proteínas de Homeodomínio/fisiologia , Fígado/citologia , Proteínas Nucleares/fisiologia , Células Estromais/metabolismo , Animais , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Feto , Proteínas de Homeodomínio/genética , Homozigoto , Lentivirus/genética , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/genética , RNA Interferente Pequeno/farmacologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Células Estromais/citologia , Fatores de Transcrição , Transfecção , Proteína Homeobox PITX2RESUMO
Inducible gene expression systems have contributed significantly to the understanding of molecular regulatory networks. Here we describe a simple and powerful RNA interference-based method that can silence the expression of any transgene. We first used an IRES bicistronic lentiviral vector and showed that targeting the second cistron with a specific siRNA resulted in silencing of both transgenes. We then inserted a siRNA minimal target sequence in the 3'-untranslated region (3'-UTR) of a transgene and showed that the cognate siRNA delivered by a lentiviral vector led to the partial silencing of the transgene. The multimerization of this siRNA target sequence led to the highly efficient silencing of four different transgenes. This new method to silence transgene expression is more versatile than existing methods of conditional inactivation of gene expression, such as transcriptional switches or site-specific recombination. It is applicable to a wide variety of models including primary cells, terminally differentiated cells and transgenic animals.
Assuntos
Técnicas Genéticas , Interferência de RNA , Transgenes , Animais , Células Cultivadas , Vetores Genéticos , Humanos , Lentivirus/genética , Dados de Sequência MolecularRESUMO
As major antigen-presenting cells and effectors in the maintenance of tolerance, dendritic cells (DCs) are key cells of the immune system and can thus be envisioned to have roles in immunotherapy strategies. We, and others, previously showed that simian immunodeficiency virus (SIV)-derived lentiviral vectors were able to deliver a gene into human differentiated DCs. We describe here the upgrading of the SIV vector system and the improvements of the transduction protocol, which allowed us to transduce more than 90% of human monocyte-derived DCs. We developed new SIV lentiviral vectors carrying SIV splice regulatory elements and either the woodchuck hepatitis virus regulatory element (WPRE) or the murine phosphoglycerate-kinase 1 (PGK) promoter. We show that insertion of the WPRE in the SIV vector is detrimental to gene transfer in DCs, while this sequence increases transgene expression in 293T cells. Using an optimized SIV vector, high levels of transgene expression were obtained in more than 30% of human DCs at a multiplicity of infection (MOI) of 1, and close to 100% using a MOI of 20. VSV-G pseudotyped vectors generated with only gag, pol, tat, and rev helper functions failed to transduce DCs. This defect was completely rescued when the SIV accessory gene vpx was provided in trans in vector-producing cells. Genetically modified DCs were shown to behave as bona fide DCs in both allogenic and autologous mixed leukocyte reactions. These findings allow us to propose an optimal system for efficient and safe DC transduction with improved SIV vectors.