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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.
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
3.
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
4.
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
5.
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
6.
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
7.
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
8.
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
9.
Viruses ; 13(6)2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34207354

RESUMO

Gene/cell therapies are promising strategies for the many presently incurable diseases. A key step in this process is the efficient delivery of genes and gene-editing enzymes to many cell types that may be resistant to lentiviral vector transduction. Herein we describe tuning of a lentiviral gene therapy platform to focus on genetic modifications of resting CD4+ T cells. The motivation for this was to find solutions for HIV gene therapy efforts. Through selection of the optimal viral envelope and further modification to its expression, lentiviral fusogenic delivery into resting CD4+ T cells exceeded 80%, yet Sterile Alpha Motif and HD domain 1 (SAMHD1) dependent and independent intracellular restriction factors within resting T cells then dominate delivery and integration of lentiviral cargo. Overcoming SAMHD1-imposed restrictions, only observed up to 6-fold increase in transduction, with maximal gene delivery and expression of 35%. To test if the biologically limiting steps of lentiviral delivery are reverse transcription and integration, we re-engineered lentiviral vectors to simply express biologically active mRNA to direct transgene expression in the cytoplasm. In this setting, we observed gene expression in up to 65% of resting CD4+ T cells using unconcentrated MS2 lentivirus-like particles (MS2-LVLPs). Taken together, our findings support a gene therapy platform that could be readily used in resting T cell gene editing.


Assuntos
Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Lentivirus/genética , Fase de Repouso do Ciclo Celular , Transgenes , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Citometria de Fluxo , Genótipo , Humanos , Linfócitos T/metabolismo , Transdução Genética
10.
Viruses ; 13(6)2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34208264

RESUMO

Oncolytic virotherapy (OV) is an emerging class of immunotherapeutic drugs. Their mechanism of action is two-fold: direct cell lysis and unmasking of the cancer through immunogenic cell death, which allows the immune system to recognize and eradicate tumours. Breast cancer is the most common cancer in women and is challenging to treat with immunotherapy modalities because it is classically an immunogenically "cold" tumour type. This provides an attractive niche for OV, given viruses have been shown to turn "cold" tumours "hot," thereby opening a plethora of treatment opportunities. There has been a number of pre-clinical attempts to explore the use of OV in breast cancer; however, these have not led to any meaningful clinical trials. This review considers both the potential and the barriers to OV in breast cancer, namely, the limitations of monotherapy and the scope for combination therapy, improving viral delivery and challenges specific to the breast cancer population (e.g., tumour subtype, menopausal status, age).


Assuntos
Neoplasias da Mama/terapia , Terapia Genética , Terapia Viral Oncolítica , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/etiologia , Estudos Clínicos como Assunto , Terapia Combinada , Avaliação Pré-Clínica de Medicamentos , Feminino , Terapia Genética/efeitos adversos , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Terapia Viral Oncolítica/efeitos adversos , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/genética , Resultado do Tratamento
11.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204559

RESUMO

Recent data indicate that modifications to carotenoid biosynthesis pathway in plants alter the expression of genes affecting chemical composition of the cell wall. Phytoene synthase (PSY) is a rate limiting factor of carotenoid biosynthesis and it may exhibit species-specific and organ-specific roles determined by the presence of psy paralogous genes, the importance of which often remains unrevealed. Thus, the aim of this work was to elaborate the roles of two psy paralogs in a model system and to reveal biochemical changes in the cell wall of psy knockout mutants. For this purpose, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas9) proteins (CRISPR/Cas9) vectors were introduced to carotenoid-rich carrot (Daucus carota) callus cells in order to induce mutations in the psy1 and psy2 genes. Gene sequencing, expression analysis, and carotenoid content analysis revealed that the psy2 gene is critical for carotenoid biosynthesis in this model and its knockout blocks carotenogenesis. The psy2 knockout also decreased the expression of the psy1 paralog. Immunohistochemical staining of the psy2 mutant cells showed altered composition of arabinogalactan proteins, pectins, and extensins in the mutant cell walls. In particular, low-methylesterified pectins were abundantly present in the cell walls of carotenoid-rich callus in contrast to the carotenoid-free psy2 mutant. Transmission electron microscopy revealed altered plastid transition to amyloplasts instead of chromoplasts. The results demonstrate for the first time that the inhibited biosynthesis of carotenoids triggers the cell wall remodelling.


Assuntos
Vias Biossintéticas/genética , Sistemas CRISPR-Cas , Carotenoides/metabolismo , Parede Celular/metabolismo , Daucus carota/fisiologia , Edição de Genes , Sequência de Bases , Parede Celular/ultraestrutura , Daucus carota/ultraestrutura , Marcação de Genes , Genes de Plantas , Vetores Genéticos/genética , Mutação , Fenótipo , Plastídeos/genética , Plastídeos/ultraestrutura
12.
Anticancer Res ; 41(8): 3731-3740, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34281831

RESUMO

BACKGROUND: The clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) is thought to have promising clinical potential. However, the off-target effects of Cas9 are a major concern for its application. Therefore, we hypothesized that the adverse effects of off-target gene editing might be minimized if the human codon-optimized Streptococcus pyogenes Cas9 (hCas9) could be specifically expressed in cancer cells. MATERIALS AND METHODS: We constructed a chimeric adenoviral vector, Ad5F35-MKp-hCas9, and infected human bladder cancer cell lines with this vector. The confirmation of hCas9 gene expression was performed in 3-4 days after from infection. RESULTS: hCas9 gene expression was observed in Ad5F35-MKp-hCas9 infected bladder cancer cells but not in non-malignant cells. CONCLUSION: Our study showed that the Ad5F35-MKp-hCas9 vector is capable of expressing the hCas9 gene with high specificity in bladder cancer cells. These findings may help in minimizing the risk of off-target effects of gene editing.


Assuntos
Adenoviridae/genética , Proteína 9 Associada à CRISPR/genética , Vetores Genéticos/genética , Transfecção/métodos , Neoplasias da Bexiga Urinária/genética , Proteína 9 Associada à CRISPR/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Edição de Genes/métodos , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Regiões Promotoras Genéticas , Neoplasias da Bexiga Urinária/patologia
13.
Int J Mol Sci ; 22(14)2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-34299360

RESUMO

Nonviral vectors for gene therapy such as lipoplexes are characterized by low toxicity, high biocompatibility, and good transfection efficiency. Specifically, lipoplexes based on polymeric surfactants and phospholipids have great potential as gene carriers due to the increased ability to bind genetic material (multiplied positive electric charge) while lowering undesirable effects (the presence of lipids makes the system more like natural membranes). This study aimed to test the ability to bind and release genetic material by lipoplexes based on trimeric surfactants and lipid formulations of different compositions and to characterize formed complexes by circular dichroism (CD) spectroscopy and atomic force microscopy (AFM). The cytotoxicity of studied lipoplexes was tested on HeLa cells by the MTT cell viability assay and the dye exclusion test (trypan blue). The presence of lipids in the system lowered the surfactant concentration required for complexation (higher efficiency) and reduced the cytotoxicity of lipoplexes. Surfactant/lipids/DNA complexes were more stable than surfactant/DNA complexes. Surfactant molecules induced the genetic material condensation, but the presence of lipids significantly intensified this process. Systems based on trimeric surfactants and lipid formulations, particularly TRI_N and TRI_IMI systems, could be used as delivery carrier, and have proven to be highly effective, nontoxic, and universal for DNA of various lengths.


Assuntos
Vetores Genéticos/genética , Fosfolipídeos/química , Tensoativos/química , Linhagem Celular Tumoral , Dicroísmo Circular/métodos , DNA/química , Técnicas de Transferência de Genes , Células HeLa , Humanos , Lipídeos/química , Microscopia de Força Atômica/métodos
14.
Viruses ; 13(6)2021 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-34198859

RESUMO

Oncolytic viruses have emerged as a promising strategy for cancer therapy due to their dual ability to selectively infect and lyse tumor cells and to induce systemic anti-tumor immunity. Among various candidate viruses, coxsackievirus group B (CVBs) have attracted increasing attention in recent years. CVBs are a group of small, non-enveloped, single-stranded, positive-sense RNA viruses, belonging to species human Enterovirus B in the genus Enterovirus of the family Picornaviridae. Preclinical studies have demonstrated potent anti-tumor activities for CVBs, particularly type 3, against multiple cancer types, including lung, breast, and colorectal cancer. Various approaches have been proposed or applied to enhance the safety and specificity of CVBs towards tumor cells and to further increase their anti-tumor efficacy. This review summarizes current knowledge and strategies for developing CVBs as oncolytic viruses for cancer virotherapy. The challenges arising from these studies and future prospects are also discussed in this review.


Assuntos
Enterovirus Humano B/genética , Engenharia Genética , Vetores Genéticos/genética , Vírus Oncolíticos/genética , Animais , Ensaios Clínicos como Assunto , Avaliação Pré-Clínica de Medicamentos , Enterovirus Humano B/fisiologia , Engenharia Genética/métodos , Terapia Genética/efeitos adversos , Terapia Genética/métodos , Humanos , Neoplasias/terapia , Terapia Viral Oncolítica/efeitos adversos , Terapia Viral Oncolítica/métodos , Resultado do Tratamento , Replicação Viral
15.
Emerg Microbes Infect ; 10(1): 1574-1588, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34289779

RESUMO

A safe and effective vaccine is urgently needed to control the unprecedented COVID-19 pandemic. Four adenovirus-vectored vaccines expressing spike (S) protein have been approved for use. Here, we generated several recombinant chimpanzee adenovirus (AdC7) vaccines expressing S, receptor-binding domain (RBD), or tandem-repeat dimeric RBD (RBD-tr2). We found vaccination via either intramuscular or intranasal route was highly immunogenic in mice to elicit both humoral and cellular immune responses. AdC7-RBD-tr2 showed higher antibody responses compared to either AdC7-S or AdC7-RBD. Intranasal administration of AdC7-RBD-tr2 additionally induced mucosal immunity with neutralizing activity in bronchoalveolar lavage fluid. Either single-dose or two-dose mucosal administration of AdC7-RBD-tr2 protected mice against SARS-CoV-2 challenge, with undetectable subgenomic RNA in lung and relieved lung injury. AdC7-RBD-tr2-elicted sera preserved the neutralizing activity against the circulating variants, especially the Delta variant. These results support AdC7-RBD-tr2 as a promising COVID-19 vaccine candidate.


Assuntos
Adenoviridae/genética , Anticorpos Antivirais/sangue , Vacinas contra COVID-19/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Administração Intranasal , Animais , Anticorpos Neutralizantes/sangue , COVID-19 , Vacinas contra COVID-19/administração & dosagem , Vacinas contra COVID-19/genética , Chlorocebus aethiops , Feminino , Vetores Genéticos/genética , Células HEK293 , Humanos , Imunogenicidade da Vacina , Injeções Intramusculares , Camundongos , Camundongos Endogâmicos BALB C , Pan troglodytes/virologia , Ligação Proteica , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/administração & dosagem , Glicoproteína da Espícula de Coronavírus/genética , Vacinação , Células Vero
16.
Methods Mol Biol ; 2352: 1-12, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34324176

RESUMO

Forced expression of specific neuronal transcription factors in mouse embryonic fibroblasts (MEFs) can lead to their direct conversion into functional neurons. Direct neuronal reprogramming has become a powerful tool to characterize individual factors and molecular mechanisms involved in forced and normal neurogenesis and to generate neuronal cell types for in vitro studies. Here we provide a detailed protocol for the isolation of MEFs devoid of neural tissue and their direct reprogramming into functional neurons by overexpression of neuronal reprogramming factors (Ascl1, Brn2, and Myt1l) using lentiviral vectors. This method enables quick and efficient generation of mouse neurons in vitro for versatile functional and mechanistic characterization.


Assuntos
Separação Celular/métodos , Técnicas de Reprogramação Celular , Reprogramação Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Animais , Técnicas de Cultura de Células , Diferenciação Celular/genética , Reprogramação Celular/genética , Criopreservação , Vetores Genéticos/biossíntese , Vetores Genéticos/genética , Humanos , Camundongos , Neurogênese/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transdução Genética
17.
Methods Mol Biol ; 2352: 31-43, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34324178

RESUMO

Astrocytes play an important role in maintaining brain homeostasis and their dysfunction is involved in a number of neurological disorders. An accessible source of astrocytes is essential to model neurological diseases and potential cell therapy approaches. Cell reprogramming techniques offer possibilities to reprogram terminally differentiated cells into other cell types. By overexpressing the three astrocytic transcription factors NFIA, NFIB, and SOX9, we showed that it is possible to directly transdifferentiate fibroblasts into functional astrocytes. These induced astrocytes (iAstrocytes) express glial fibrillary acidic protein (GFAP) and S100 calcium binding protein B (S100B), as well as other astrocytic markers. Moreover, electrophysiological properties indicate that iAstrocytes are functionally comparable to native brain astrocytes. Here we describe an optimized protocol to generate iAstrocytes starting from skin fibroblasts and this approach can be adapted for a wide range of somatic cell types.


Assuntos
Astrócitos/citologia , Astrócitos/metabolismo , Transdiferenciação Celular/genética , Reprogramação Celular/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Fatores de Transcrição/genética , Animais , Cálcio , Linhagem Celular , Células Cultivadas , Expressão Gênica , Vetores Genéticos/biossíntese , Vetores Genéticos/genética , Humanos , Lentivirus/genética , Camundongos , Imagem Molecular , Fatores de Transcrição NFI/genética , Fatores de Transcrição SOX9/genética , Fatores de Transcrição/metabolismo
18.
Methods Mol Biol ; 2352: 57-71, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34324180

RESUMO

Direct neuronal reprogramming is a promising strategy to generate various types of neurons that are, otherwise, inaccessible for researchers. However, the efficiency of neuronal conversion is highly dependent on the transcription factor used, the identity of the initial cells to convert, their species' background, and the neuronal subtype to which cells will convert. Regardless of these conditioning factors, the apoptotic regulator Bcl-2 acts as a pan-neuronal reprogramming enhancer. Bcl-2 mediates its effect in reprogramming by preventing an overshot of oxidative stress during the acquisition of a neuronal oxidative metabolism, thus reducing cell death by ferroptosis and facilitating the phenotypic conversion. In this chapter, we outline two methods to obtain either mouse or human neurons derived from postnatal astrocytes and skin fibroblasts, respectively. The overall reprogramming strategy is based on the co-expression of Bcl-2 and the transcription factor Neurog2 that produces mostly excitatory neurons. However, the method can be easily adapted to achieve alternative neuronal subtypes by using additional transcription factors, such as Isl1 for motor neurons. Therefore, our approaches provide solid but flexible platforms to obtain human and mouse induced neurons in vitro that can be applied to basic or translational research.


Assuntos
Astrócitos/citologia , Astrócitos/metabolismo , Técnicas de Reprogramação Celular , Reprogramação Celular/genética , Fibroblastos/citologia , Neurônios/citologia , Neurônios/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Animais , Astrócitos/efeitos dos fármacos , Técnicas de Cultura de Células , Linhagem Celular , Células Cultivadas , Reprogramação Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Expressão Gênica , Vetores Genéticos/genética , Humanos , Camundongos , Neurônios/efeitos dos fármacos , Retroviridae/genética , Transdução Genética , Transfecção
19.
Methods Mol Biol ; 2352: 73-96, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34324181

RESUMO

Progressive aging is a physiological process that represents a central risk factor for the development of several human age-associated chronic diseases, including neurodegenerative diseases. A major focus in biomedical research is the pursuit for appropriate model systems to better model the biology of human aging and the interface between aging and disease mechanisms. Direct conversion of human fibroblasts into induced neurons (iNs) has emerged as a novel technology for the in vitro modeling of age-dependent neurological diseases. Similar to other cellular reprogramming techniques, e.g., iPSC-based cellular reprograming, direct conversion relies on the ectopic overexpression of transcription factors, typically including well-known pioneer factors. However, in contrast to alternative technologies to generate neurons, the entire process of direct conversion bypasses any proliferative or stem cell-like stage, which in fact renders it the unique aptitude of preserving age-associated hallmarks from the initial fibroblast source. In this chapter, we introduce direct conversion as a practical and easy-to-approach disease model for aging and neurodegenerative disease research. A focus here is to provide a stepwise protocol for the efficient and highly reproducible generation of iNs from adult dermal fibroblasts from human donors.


Assuntos
Técnicas de Reprogramação Celular , Reprogramação Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Biomarcadores , Reprogramação Celular/genética , Derme/citologia , Citometria de Fluxo , Vetores Genéticos/administração & dosagem , Vetores Genéticos/biossíntese , Vetores Genéticos/genética , Humanos , Imunofenotipagem , Lentivirus/genética , Transdução Genética
20.
Methods Mol Biol ; 2352: 97-115, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34324182

RESUMO

Since the first demonstration of direct dopaminergic neuronal reprogramming, over a dozen methods have been developed to generate induced dopaminergic neurons from various sources of cells. Here, we first present an overview of the different methods to generate induced neurons of a generic type and of different subtypes, with a particular focus on induced dopaminergic neurons generated from human fibroblasts. We then describe a protocol to generate induced dopaminergic neurons from commercially available human fetal lung fibroblasts. These cells could serve for various biomedical application, including regenerative medicine for conditions such as Parkinson's disease.


Assuntos
Transdiferenciação Celular , Técnicas de Reprogramação Celular , Reprogramação Celular , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Células Cultivadas , Expressão Gênica , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Lentivirus/genética , Fatores de Transcrição/genética
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