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1.
Sheng Wu Gong Cheng Xue Bao ; 37(8): 2786-2793, 2021 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-34472296

RESUMO

To obtain chicken CD40L protein, the cDNA was prepared from chicken splenic cells and used as a template to clone and amplify CD40L by PCR. The target gene was cloned into pFastBac vector to construct a pFastBac-chCD40L donor plasmid. Recombinant plasmid was transformed into DH10Bac and recombinant Bacmid-chCD40L was obtained. The Bacmid-chCD40L plasmid was transfected into sf9 insect cells to obtain His-chCD40L protein. In addition, the target gene was cloned into pQM01 vector to construct a pQM01-chCD40L plasmid, recombinant plasmid was transfected into HEK 293T cells to obtain Strep-chCD40L protein. The chCD40L protein was purified by affinity chromatography, and the concentration of purified chCD40L protein was determined to be 0.01 mg/mL. Primary cells were isolated from the bursal tissue of 3-week old SPF chickens, and the chCD40L protein was added to the culture medium to stimulate cells. The chCD40L could bind to CD40 on B cells as examined by Western blotting, indirect immunofluorescence assay and flow cytometry, suggesting that chCD40L protein is biologically active. We successfully obtained chicken CD40L protein of biological activity, which laid the foundation in the in vitro culture of primary B lymphocytes for the isolation and diagnosis of virulent IBDV.


Assuntos
Baculoviridae , Galinhas , Animais , Baculoviridae/genética , Ligante de CD40/genética , Clonagem Molecular , Vetores Genéticos/genética , Proteínas Recombinantes/genética
2.
Front Immunol ; 12: 728513, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34484238

RESUMO

VITT is a rare, life-threatening syndrome characterized by thrombotic symptoms in combination with thrombocytopenia, which may occur in individuals receiving the first administration of adenoviral non replicating vectors (AVV) anti Covid19 vaccines. Vaccine-induced immune thrombotic thrombocytopenia (VITT) is characterized by high levels of serum IgG that bind PF4/polyanion complexes, thus triggering platelet activation. Therefore, identification of the fine pathophysiological mechanism by which vaccine components trigger platelet activation is mandatory. Herein, we propose a multistep mechanism involving both the AVV and the neo-synthetized Spike protein. The former can: i) spread rapidly into blood stream, ii), promote the early production of high levels of IL-6, iii) interact with erythrocytes, platelets, mast cells and endothelia, iv) favor the presence of extracellular DNA at the site of injection, v) activate platelets and mast cells to release PF4 and heparin. Moreover, AVV infection of mast cells may trigger aberrant inflammatory and immune responses in people affected by the mast cell activation syndrome (MCAS). The pre-existence of natural antibodies binding PF4/heparin complexes may amplify platelet activation and thrombotic events. Finally, neosynthesized Covid 19 Spike protein interacting with its ACE2 receptor on endothelia, platelets and leucocyte may trigger further thrombotic events unleashing the WITT syndrome.


Assuntos
Anticorpos/efeitos adversos , Vacinas contra COVID-19/efeitos adversos , COVID-19/prevenção & controle , Púrpura Trombocitopênica Idiopática/induzido quimicamente , Púrpura Trombocitopênica Idiopática/fisiopatologia , Adenoviridae/genética , Animais , Plaquetas/imunologia , Plaquetas/patologia , Vacinas contra COVID-19/imunologia , Modelos Animais de Doenças , Vetores Genéticos , Humanos , Camundongos , Ativação Plaquetária/imunologia , Fator Plaquetário 4 , Coelhos
3.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 37(9): 788-793, 2021 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-34533125

RESUMO

Objective To construct a lentiviral expression vector of chimeric antigen receptor (CAR) recognizing hepatitis B surface antigen (HBsAg) and make it expressed in NK92MI cells. Methods The CAR was designed and synthesized with the sequence of single chain variable fragment (scFv). Next it was cloned into a lentivirus expression vector, followed by packaging, concentration and titer determination. Then HEK293T cells were transduced with the concentrated lentivirus. The expression of CD3 zeta chain and the infection efficiency in the NK92MI cells were detected by Western blotting. Results The lentiviral expression vector of CAR for HBsAg recognition was successfully constructed, and the titer was ≥107 transducing units (TU)/mL. The expression of protein CD3zeta was verified in the NK92MI cells after infected with the lentiviral vector. Conclusion CAR-NK92MI cells recognizing HBsAg has been established successfully.


Assuntos
Antígenos de Superfície da Hepatite B , Receptores de Antígenos Quiméricos , Linhagem Celular Tumoral , Vetores Genéticos/genética , Células HEK293 , Antígenos de Superfície da Hepatite B/genética , Humanos , Lentivirus/genética
4.
Clin Appl Thromb Hemost ; 27: 10760296211040110, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34541935

RESUMO

Since the outbreak of Covid-19 in December, 2019, scientists worldwide have been committed to developing COVID-19 vaccines. Only when most people have immunity to SARS-CoV-2, COVID-19 can reduce even wholly overcome. So far, nine kinds of COVID-19 vaccines have passed the phase III clinical trials and have approved for use. At the same time, adverse reactions after COVID-19 vaccination have also reported. This paper focuses on the adverse effects of thrombosis and thrombocytopenia caused by the COVID-19 vaccine, especially the adenovirus-vector vaccine from AstraZeneca and Pfizer, and discusses its mechanism and possible countermeasures.


Assuntos
Adenoviridae/genética , Vacinas contra COVID-19/efeitos adversos , Vetores Genéticos , Trombocitopenia/induzido quimicamente , Trombose/induzido quimicamente , Vacinação/efeitos adversos , Anticorpos/sangue , Vacinas contra COVID-19/genética , Vacinas contra COVID-19/imunologia , Humanos , Fator Plaquetário 4/imunologia , Medição de Risco , Fatores de Risco , Trombocitopenia/sangue , Trombocitopenia/imunologia , Trombose/sangue , Trombose/imunologia
5.
Enzyme Microb Technol ; 150: 109894, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34489047

RESUMO

Aspergillus oryzae is a safe microorganism that is commonly used in food production. We constructed a self-cloning vector capable of high expression in A. oryzae. Using the vector, three putative pectin methylesterase (PME) genes belonging to Carbohydrate Esterase family 8 derived from A. oryzae were expressed, and several characteristics of the gene products were examined. The effects of temperature and pH on the three enzymes (AoPME1, 2, and 3) were similar, with optimal reaction temperatures of 50 - 60 °C and optimal reaction pH range of 5 - 6. The specific activities of AoPME1, 2, and 3 for apple pectin were significantly different (34, 7,601, and 2 U/mg, respectively). When the substrate specificity was examined, AoPME1 showed high activity towards pectin derived from soybean and pea. Although AoPME2 showed little activity towards these pectins, it showed very high activity towards apple- and citrus-derived pectins. AoPME3 showed low specific activity towards all substrates tested. Sugar composition analysis revealed that apple- and citrus-derived pectins were rich in homogalacturonan, while soybean- and pea-derived pectins were rich in xylogalacturonan. When pea pectin was treated with endo-polygalacturonase or endo-xylogalacturonase in the presence of each PME, specific synergistic actions were observed (endo-polygalacturonase with AoPME1 or AoPME2 and endo-xylogalacturonase with AoPME1 or AoPME3). Thus, AoPME1 and AoPME3 hydrolyzed the methoxy group in xylogalacturonan. This is the first report of this activity in microbial enzymes. Our findings on the substrate specificity of PMEs should lead to the determination of the distribution of methoxy groups in pectin and the development of new applications in the field of food manufacturing.


Assuntos
Aspergillus oryzae , Aspergillus oryzae/genética , Hidrolases de Éster Carboxílico/genética , Vetores Genéticos , Ácidos Hexurônicos , Pectinas
6.
Indian J Ophthalmol ; 69(9): 2257-2265, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34427196

RESUMO

Over 2 million people worldwide are suffering from gene-related retinal diseases, inherited or acquired, and over 270 genes have been identified which are found to be responsible for these conditions. This review article touches upon the mechanisms of gene therapy, various enzymes of the visual cycle responsible for different genetic diseases, Luxturna-the first US Food and Drug Administration (FDA)-approved therapeutic gene product, and several ongoing trials of gene therapy for age-related macular degeneration. Gene therapy has tremendous potential for retinal conditions due to its ease of accessibility, immune-privileged status, and tight blood-retinal barriers, limiting systemic side effects of the drug. In recent years, advances in gene therapy in retinal conditions have increasing significantly, with progress in cell-specific targeting and transduction efficiency of gene products through the use of adeno-associated viral vectors (AAVs), suggesting that even greater success in future clinical trials is possible.


Assuntos
Degeneração Macular , Preparações Farmacêuticas , Doenças Retinianas , Terapia Genética , Vetores Genéticos , Humanos , Degeneração Macular/terapia , Doenças Retinianas/genética , Doenças Retinianas/terapia , Estados Unidos
7.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34361005

RESUMO

Although the development of gene delivery systems based on non-viral vectors is advancing, it remains a challenge to deliver plasmid DNA into human blood cells. The current "gold standard", namely linear polyethyleneimine (l-PEI 25 kDa), in particular, is unable to produce transgene expression levels >5% in primary human B lymphocytes. Here, it is demonstrated that a well-defined 24-armed poly(2-dimethylamino) ethyl methacrylate (PDMAEMA, 755 kDa) nano-star is able to reproducibly elicit high transgene expression (40%) at sufficient residual viability (69%) in primary human B cells derived from tonsillar tissue. Moreover, our results indicate that the length of the mitogenic stimulation prior to transfection is an important parameter that must be established during the development of the transfection protocol. In our hands, four days of stimulation with rhCD40L post-thawing led to the best transfection results in terms of TE and cell survival. Most importantly, our data argue for an impact of the B cell subsets on the transfection outcomes, underlining that the complexity and heterogeneity of a given B cell population pre- and post-transfection is a critical parameter to consider in the multiparametric approach required for the implementation of the transfection protocol.


Assuntos
Linfócitos B/metabolismo , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Linfócitos B/citologia , Células Cultivadas , Humanos , Metacrilatos/química , Nylons/química , Transgenes
8.
Science ; 373(6557): 882-889, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34413232

RESUMO

Eukaryotic genomes contain domesticated genes from integrating viruses and mobile genetic elements. Among these are homologs of the capsid protein (known as Gag) of long terminal repeat (LTR) retrotransposons and retroviruses. We identified several mammalian Gag homologs that form virus-like particles and one LTR retrotransposon homolog, PEG10, that preferentially binds and facilitates vesicular secretion of its own messenger RNA (mRNA). We showed that the mRNA cargo of PEG10 can be reprogrammed by flanking genes of interest with Peg10's untranslated regions. Taking advantage of this reprogrammability, we developed selective endogenous encapsidation for cellular delivery (SEND) by engineering both mouse and human PEG10 to package, secrete, and deliver specific RNAs. Together, these results demonstrate that SEND is a modular platform suited for development as an efficient therapeutic delivery modality.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação a DNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/genética , Capsídeo/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Vesículas Extracelulares/metabolismo , Edição de Genes , Vetores Genéticos , Humanos , Camundongos , Neurônios/metabolismo , Domínios Proteicos , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Retroelementos , Transfecção , Regiões não Traduzidas , Regulação para Cima
9.
Nat Commun ; 12(1): 4734, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354077

RESUMO

The tumor microenvironment (TME) is a complex amalgam of tumor cells, immune cells, endothelial cells and fibroblastic stromal cells (FSC). Cancer-associated fibroblasts are generally seen as tumor-promoting entity. However, it is conceivable that particular FSC populations within the TME contribute to immune-mediated tumor control. Here, we show that intratumoral treatment of mice with a recombinant lymphocytic choriomeningitis virus-based vaccine vector expressing a melanocyte differentiation antigen resulted in T cell-dependent long-term control of melanomas. Using single-cell RNA-seq analysis, we demonstrate that viral vector-mediated transduction reprogrammed and activated a Cxcl13-expressing FSC subset that show a pronounced immunostimulatory signature and increased expression of the inflammatory cytokine IL-33. Ablation of Il33 gene expression in Cxcl13-Cre-positive FSCs reduces the functionality of intratumoral T cells and unleashes tumor growth. Thus, reprogramming of FSCs by a self-antigen-expressing viral vector in the TME is critical for curative melanoma treatment by locally sustaining the activity of tumor-specific T cells.


Assuntos
Melanoma Experimental/terapia , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Fibroblastos Associados a Câncer/imunologia , Fibroblastos Associados a Câncer/patologia , Técnicas de Reprogramação Celular/métodos , Quimiocina CXCL13/genética , Quimiocina CXCL13/imunologia , Feminino , Vetores Genéticos , Interleucina-33/deficiência , Interleucina-33/genética , Interleucina-33/imunologia , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/imunologia , Vírus da Coriomeningite Linfocítica/genética , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células Estromais/imunologia , Células Estromais/patologia , Linfócitos T/imunologia , Linfócitos T/patologia , Microambiente Tumoral/imunologia
11.
Viruses ; 13(7)2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34372506

RESUMO

Adenovirus-based vectors are playing an important role as efficacious genetic vaccines to fight the current COVID-19 pandemic. Furthermore, they have an enormous potential as oncolytic vectors for virotherapy and as vectors for classic gene therapy. However, numerous vector-host interactions on a cellular and noncellular level, including specific components of the immune system, must be modulated in order to generate safe and efficacious vectors for virotherapy or classic gene therapy. Importantly, the current widespread use of Ad vectors as vaccines against COVID-19 will induce antivector immunity in many humans. This requires the development of strategies and techniques to enable Ad-based vectors to evade pre-existing immunity. In this review article, we discuss the current status of genetic and chemical capsid modifications as means to modulate the vector-host interactions of Ad-based vectors.


Assuntos
Adenoviridae/genética , COVID-19/prevenção & controle , Capsídeo/química , Adenoviridae/imunologia , COVID-19/imunologia , COVID-19/terapia , Vacinas contra COVID-19/administração & dosagem , Vacinas contra COVID-19/imunologia , Genes Virais , Vetores Genéticos , Humanos , Imunidade , Terapia Viral Oncolítica/métodos , Pandemias , SARS-CoV-2/imunologia , SARS-CoV-2/isolamento & purificação
12.
Viruses ; 13(7)2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34372567

RESUMO

Glioblastoma is the most malignant and most common form of brain tumor, still today associated with a poor 14-months median survival from diagnosis. Protein kinase A, particularly its regulatory subunit R2Alpha, presents a typical intracellular distribution in glioblastoma cells compared to the healthy brain parenchyma and this peculiarity might be exploited in a therapeutic setting. In the present study, a third-generation lentiviral system for delivery of shRNA targeting the regulatory subunit R2Alpha of protein kinase A was developed. Generated lentiviral vectors are able to induce an efficient and stable downregulation of R2Alpha in different cellular models, including non-stem and stem-like glioblastoma cells. In addition, our data suggest a potential correlation between silencing of the regulatory subunit of protein kinase A and reduced viability of tumor cells, apparently due to a reduction in replication rate. Thus, our findings support the role of protein kinase A as a promising target for novel anti-glioma therapies.


Assuntos
Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Glioblastoma/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Glioblastoma/genética , Glioblastoma/fisiopatologia , Glioma/genética , Glioma/metabolismo , Células HEK293 , Humanos , Lentivirus/genética , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Transdução Genética/métodos
13.
Viruses ; 13(7)2021 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-34372571

RESUMO

Anti-cancer activity can be improved by engineering immune cells to express chimeric antigen receptors (CARs) that recognize tumor-associated antigens. Retroviral vector gene transfer strategies allow stable and durable transgene expression. Here, we used alpharetroviral vectors to modify NK-92 cells, a natural killer cell line, with a third-generation CAR designed to target the IL-3 receptor subunit alpha (CD123), which is strongly expressed on the surface of acute myeloid leukemia (AML) cells. Alpharetroviral vectors also contained a transgene cassette to allow constitutive expression of human IL-15 for increased NK cell persistence in vivo. The anti-AML activity of CAR-NK-92 cells was tested via in vitro cytotoxicity assays with the CD123+ AML cell line KG-1a and in vivo in a patient-derived xenotransplantation CD123+ AML model. Unmodified NK-92 cells or NK-92 cells modified with a truncated version of the CAR that lacked the signaling domain served as controls. Alpharetroviral vector-modified NK-92 cells stably expressed the transgenes and secreted IL-15. Anti-CD123-CAR-NK-92 cells exhibited enhanced anti-AML activity in vitro and in vivo as compared to control NK-92 cells. Our data (1) shows the importance of IL-15 expression for in vivo persistence of NK-92 cells, (2) supports continued investigation of anti-CD123-CAR-NK cells to target AML, and (3) points towards potential strategies to further improve CAR-NK anti-AML activity.


Assuntos
Imunoterapia Adotiva/métodos , Subunidade alfa de Receptor de Interleucina-3/imunologia , Leucemia Mieloide Aguda/tratamento farmacológico , Idoso , Alpharetrovirus/genética , Animais , Linhagem Celular Tumoral , Feminino , Terapia Genética , Vetores Genéticos/genética , Humanos , Subunidade alfa de Receptor de Interleucina-3/genética , Subunidade alfa de Receptor de Interleucina-3/metabolismo , Células Matadoras Naturais/imunologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/imunologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Cultura Primária de Células , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologia , Transdução Genética , Transgenes , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Methods Mol Biol ; 2351: 321-334, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382198

RESUMO

Loss-of-function experiments are essential for the functional investigation of cis-regulatory elements (CREs), such as transcriptional enhancers. This can be achieved with CRISPR-Cas9 using pairs of single guide RNAs (sgRNAs) to target the flanking regions of a CRE. Here, I describe a single-step protocol to rapidly and inexpensively generate vectors co-expressing two sgRNAs, which allows re-usage of gRNAs oligonucleotides from one experimental design to another. This protocol is applicable to cloning sgRNAs into virtually any CRISPR-Cas9 backbone that allows cloning using Golden Gate, by adapting the primer design.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Marcação de Genes , Vetores Genéticos/genética , Sequências Reguladoras de Ácido Nucleico , Deleção de Sequência , Clonagem Molecular , Ordem dos Genes , Marcação de Genes/métodos , Humanos , Células Secretoras de Insulina/metabolismo , RNA Guia/química , RNA Guia/genética
15.
Nat Commun ; 12(1): 4934, 2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34400638

RESUMO

Rhodopsin (RHO) gene mutations are a common cause of autosomal dominant retinitis pigmentosa (ADRP). The need to suppress toxic protein expression together with mutational heterogeneity pose challenges for treatment development. Mirtrons are atypical RNA interference effectors that are spliced from transcripts as short introns. Here, we develop a novel mirtron-based knockdown/replacement gene therapy for the mutation-independent treatment of RHO-related ADRP, and demonstrate efficacy in a relevant mammalian model. Splicing and potency of rhodopsin-targeting candidate mirtrons are initially determined, and a mirtron-resistant codon-modified version of the rhodopsin coding sequence is validated in vitro. These elements are then combined within a single adeno-associated virus (AAV) and delivered subretinally in a RhoP23H knock-in mouse model of ADRP. This results in significant mouse-to-human rhodopsin RNA replacement and is associated with a slowing of retinal degeneration. This provides proof of principle that synthetic mirtrons delivered by AAV are capable of reducing disease severity in vivo.


Assuntos
Terapia Genética , RNA/genética , Retinite Pigmentosa/genética , Retinite Pigmentosa/terapia , Animais , Dependovirus/genética , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Vetores Genéticos , Células HEK293 , Humanos , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , RNA/metabolismo , Interferência de RNA , Splicing de RNA , Retina , Degeneração Retiniana , Rodopsina/genética , Rodopsina/metabolismo
16.
Gene ; 803: 145889, 2021 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-34371094

RESUMO

Although seen as a revolution in modern science, gene therapy has been plagued by failed clinical trials and controversial ethics in the last thirty years. Moreover, there is no comprehensive, in-depth, high-quality analysis of global gene therapy patents. This paper proposes a method to correctly retrieve patents to address the issue and use it for the patent landscape. The results show the global patent landscape of gene therapy, with the United States dominating the field, while China has emerged as a leader in recent years. For various reasons, the EU, Korea, and Japan lag in the development of patented technologies. China has edged closer to the US in both live and indefinite patents, with the Chinese Academy of Military Medical Sciences and the Chinese Academy of Sciences leading the way, surpassing primary applicants such as the US Department of Health and Human Services, the University of California, and the University of Pennsylvania. The study also reveals four broad categories of technologies that have been extensively studied in gene therapy: basic biology of the gene and diseases, diseases being treated, gene delivery methods, and potential adverse events. What is more, Adeno-Associated Virus, Retrovirus, and Lentivirus are the most prevalent gene therapy delivery vectors after 2014. The industrial development trend revealed in this paper can provide an evidence-based basis for scientific research management and decision-making.


Assuntos
Terapia Genética , Vetores Genéticos/classificação , Patentes como Assunto , China , Dependovirus/genética , União Europeia , Humanos , Japão , Lentivirus/genética , República da Coreia , Retroviridae/genética , Estados Unidos
17.
Molecules ; 26(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34443540

RESUMO

Helicobacter pylori (H. pylori) is a pathogen that can remain in the stomach of an infected person for their entire life. As a result, this leads to the development of severe gastric diseases such as gastric cancer. In addition, current therapies have several problems including antibiotics resistance. Therefore, new practical options to eliminate this bacterium, and its induced affections, are required to avoid morbidity and mortality worldwide. One strategy in the search for new drugs is to detect compounds that inhibit a limiting step in a central metabolic pathway of the pathogen of interest. In this work, we tested 55 compounds to gain insights into their possible use as new inhibitory drugs of H. pylori glucose-6-phosphate dehydrogenase (HpG6PD) activity. The compounds YGC-1; MGD-1, MGD-2; TDA-1; and JMM-3 with their respective scaffold 1,3-thiazolidine-2,4-dione; 1H-benzimidazole; 1,3-benzoxazole, morpholine, and biphenylcarbonitrile showed the best inhibitory activity (IC50 = 310, 465, 340, 204 and 304 µM, respectively). We then modeled the HpG6PD protein by homology modeling to conduct an in silico study of the chemical compounds and discovers its possible interactions with the HpG6PD enzyme. We found that compounds can be internalized at the NADP+ catalytic binding site. Hence, they probably exert a competitive inhibitory effect with NADP+ and a non-competitive or uncompetitive effect with G6P, that of the compounds binding far from the enzyme's active site. Based on these findings, the tested compounds inhibiting HpG6PD represent promising novel drug candidates against H. pylori.


Assuntos
Simulação por Computador , Inibidores Enzimáticos/farmacologia , Glucosefosfato Desidrogenase/antagonistas & inibidores , Helicobacter pylori/enzimologia , Vetores Genéticos/metabolismo , Glucosefosfato Desidrogenase/química , Glucosefosfato Desidrogenase/metabolismo , Helicobacter pylori/efeitos dos fármacos , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas Recombinantes/isolamento & purificação , Homologia Estrutural de Proteína
18.
Biomaterials ; 276: 121062, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34418816

RESUMO

Adenovirus (Ad) has been extensively developed as a gene delivery vector, but the potential side effect caused by systematic immunization remains one major obstacle for its clinical application. Needle-free mucosal immunization with Ad-based vaccine shows advantages but still faces poor mucosal responses. We herein report that the chemical engineering of single live viral-based vaccine effectively modulated the location and pattern of the subsequently elicited immunity. Through precisely assembly of functional materials onto single live Ad particle, the modified virus entered host cell in a phagocytosis-dependent manner, which is completely distinct from the receptor-mediated entry of native Ad. RNA-Seq data further demonstrated that the modified Ad-induced innate immunity was sharply reshaped via phagocytosis-related pathway, therefore promoting the activation and mature of antigen presentation cells (APC). Moreover, the functional shell enabled the modified Ad-based vector with enhanced muco-adhesion to nasal tissues in mice, and then prolonged resident time onto mucosal surface, leading to the robust mucosal IgA production and T cell immunity at local and even remote mucosal-associated lymphoid tissues. This study demonstrated that vaccine-induced immunity can be well modulated by chemistry engineering, and this method provides the rational design for needle-free mucosa-targeting vaccine against a variety of emerging infectious diseases.


Assuntos
Vacinas Virais , Adenoviridae/genética , Animais , Vetores Genéticos , Imunidade nas Mucosas , Camundongos , Fagocitose
19.
Int J Mol Sci ; 22(15)2021 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-34361120

RESUMO

A major limiting factor for systemically delivered gene therapies is the lack of novel tissue specific AAV (Adeno-associated virus) derived vectors. Bispecific antibodies can be used to redirect AAVs to specific target receptors. Here, we demonstrate that the insertion of a short linear epitope "2E3" derived from human proprotein-convertase subtilisin/kexin type 9 (PCSK9) into different surface loops of the VP capsid proteins can be used for AAV de-targeting from its natural receptor(s), combined with a bispecific antibody-mediated retargeting. We chose to target a set of distinct disease relevant membrane proteins-fibroblast activation protein (FAP), which is upregulated on activated fibroblasts within the tumor stroma and in fibrotic tissues, as well as programmed death-ligand 1 (PD-L1), which is strongly upregulated in many cancers. Upon incubation with a bispecific antibody recognizing the 2E3 epitope and FAP or PD-L1, the bispecific antibody/rAAV complex was able to selectively transduce receptor positive cells. In summary, we developed a novel, rationally designed vector retargeting platform that can target AAVs to a new set of cellular receptors in a modular fashion. This versatile platform may serve as a valuable tool to investigate the role of disease relevant cell types and basis for novel gene therapy approaches.


Assuntos
Anticorpos Biespecíficos/imunologia , Proteínas do Capsídeo/imunologia , Capsídeo/imunologia , Dependovirus/genética , Endopeptidases/imunologia , Epitopos/imunologia , Vetores Genéticos/administração & dosagem , Proteínas de Membrana/imunologia , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos/genética , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/imunologia , Pró-Proteína Convertase 9/metabolismo , Transdução Genética
20.
Int J Mol Sci ; 22(15)2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34360656

RESUMO

Chikungunya virus (CHIKV) is a mosquito-transmitted infectious agent that causes an endemic or epidemic outbreak(s) of Chikungunya fever that is reported in almost all countries. This virus is an intense global threat, due to its high rate of contagion and the lack of effective remedies. In this study, we developed two baculovirus expression vector system (BEVS)-based approaches for the screening of anti-CHIKV drugs in Spodoptera frugiperda insect (Sf21) cells and U-2OS cells. First, structural protein of CHIKV was co-expressed through BEVS and thereby induced cell fusion in Sf21 cells. We used an internal ribosome entry site (IRES) to co-express the green fluorescent protein (EGFP) for identifying these fusion events. The EGFP-positive Sf21 cells fused with each other and with uninfected cells to form syncytia. We identified that ursolic acid has potential anti-CHIKV activity in vitro, by using this approach. Second, BacMam virus-based gene delivery has been successfully applied for the transient expression of non-structural proteins with a subgenomic promoter-EGFP (SP-EGFP) cassette in U-2OS cells to act as an in vitro CHIKV replicon system. Our BacMam-based screening system has identified that the potential effects of baicalin and baicalein phytocompounds can inhibit the replicon activity of CHIKV in U-2OS cells. In conclusion, our results suggested that BEVS can be a potential tool for screening drugs against CHIKV.


Assuntos
Antivirais/farmacologia , Baculoviridae/genética , Fusão Celular , Febre de Chikungunya/tratamento farmacológico , Vírus Chikungunya/efeitos dos fármacos , Proteínas do Envelope Viral/metabolismo , Replicação Viral , Animais , Febre de Chikungunya/virologia , Vetores Genéticos/genética , Proteínas de Fluorescência Verde , Ensaios de Triagem em Larga Escala , Mosquitos Vetores , Células Sf9 , Proteínas do Envelope Viral/genética
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