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1.
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
2.
Comp Immunol Microbiol Infect Dis ; 78: 101693, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34399377

RESUMO

The objective of this study was to verify the presence of small ruminant lentivirus in the amniotic fluid of goats using molecular tests and viral isolation by cocultivation in the amniotic fluid of naturally infected goats. The study analyzed eight goats: seven were small ruminant lentivirus-positive and one was negative. The amniotic fluid was collected from each of the eight animals during cesarean section at 147 days of pregnancy. Cocultivation was undertaken using secondary goat nictitating membrane cell cultures obtained by explant from a small ruminant lentivirus-negative calf followed by trypsinization and sub-cultivation of the cells for 63 days. During this period, five supernatant collections were performed for DNA extraction and subsequent nested polymerase chain reaction. DNA was extracted from the amniotic fluid after 3 h of cellular sedimentation, from which a sample of 600 µL was taken from the sediment and another 600 µL sample from the supernatant. After DNA extraction, nested polymerase chain reaction was performed. Of the eight goats, 62.5 % (05/08) were small ruminant lentivirus-positive, with 43.75 % (07/16) of the total samples positive when considering the two repetitions (supernatant and cell sediment). Moreover, positivity was confirmed by small ruminant lentivirus pro-viral DNA amplification in the cell supernatant throughout the cocultivation period. Small ruminant lentivirus were present in the amniotic fluid samples from the naturally infected goats indicating an intrauterine transmission route. Moreover, this biological fluid can be adopted for the diagnosis of these lentiviruse because it is an important risk factor related to intrauterine transmission.


Assuntos
Doenças das Cabras , Infecções por Lentivirus , Doenças dos Ovinos , Líquido Amniótico , Animais , Cesárea/veterinária , Feminino , Doenças das Cabras/diagnóstico , Cabras , Lentivirus/genética , Infecções por Lentivirus/veterinária , Gravidez , Ruminantes , Ovinos
3.
Viruses ; 13(8)2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34452443

RESUMO

The novel coronavirus SARS-CoV-2 is the seventh identified human coronavirus. Understanding the extent of pre-existing immunity induced by seropositivity to endemic seasonal coronaviruses and the impact of cross-reactivity on COVID-19 disease progression remains a key research question in immunity to SARS-CoV-2 and the immunopathology of COVID-2019 disease. This paper describes a panel of lentiviral pseudotypes bearing the spike (S) proteins for each of the seven human coronaviruses (HCoVs), generated under similar conditions optimized for high titre production allowing a high-throughput investigation of antibody neutralization breadth. Optimal production conditions and most readily available permissive target cell lines were determined for spike-mediated entry by each HCoV pseudotype: SARS-CoV-1, SARS-CoV-2 and HCoV-NL63 best transduced HEK293T/17 cells transfected with ACE2 and TMPRSS2, HCoV-229E and MERS-CoV preferentially entered HUH7 cells, and CHO cells were most permissive for the seasonal betacoronavirus HCoV-HKU1. Entry of ACE2 using pseudotypes was enhanced by ACE2 and TMPRSS2 expression in target cells, whilst TMPRSS2 transfection rendered HEK293T/17 cells permissive for HCoV-HKU1 and HCoV-OC43 entry. Additionally, pseudotype viruses were produced bearing additional coronavirus surface proteins, including the SARS-CoV-2 Envelope (E) and Membrane (M) proteins and HCoV-OC43/HCoV-HKU1 Haemagglutinin-Esterase (HE) proteins. This panel of lentiviral pseudotypes provides a safe, rapidly quantifiable and high-throughput tool for serological comparison of pan-coronavirus neutralizing responses; this can be used to elucidate antibody dynamics against individual coronaviruses and the effects of antibody cross-reactivity on clinical outcome following natural infection or vaccination.


Assuntos
Anticorpos Antivirais/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , COVID-19/imunologia , Coronavirus/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Antivirais/sangue , Anticorpos Amplamente Neutralizantes/sangue , Linhagem Celular , Coronavirus Humano 229E/imunologia , Coronavirus Humano 229E/fisiologia , Coronavirus Humano NL63/imunologia , Coronavirus Humano NL63/fisiologia , Coronavirus Humano OC43/imunologia , Coronavirus Humano OC43/fisiologia , Reações Cruzadas , Humanos , Lentivirus/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , Testes de Neutralização , Plasmídeos , SARS-CoV-2/fisiologia , Transfecção , Internalização do Vírus
4.
FASEB J ; 35(9): e21801, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34365657

RESUMO

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a crucial role in mediating viral entry into host cells. However, whether it contributes to pulmonary hyperinflammation in patients with coronavirus disease 2019 is not well known. In this study, we developed a spike protein-pseudotyped (Spp) lentivirus with the proper tropism of the SARS-CoV-2 spike protein on the surface and determined the distribution of the Spp lentivirus in wild-type C57BL/6J male mice that received an intravenous injection of the virus. Lentiviruses with vesicular stomatitis virus glycoprotein (VSV-G) or with a deletion of the receptor-binding domain (RBD) in the spike protein [Spp (∆RBD)] were used as controls. Two hours postinfection (hpi), there were 27-75 times more viral burden from Spp lentivirus in the lungs than in other organs; there were also about 3-5 times more viral burden from Spp lentivirus than from VSV-G lentivirus in the lungs, liver, kidney, and spleen. Deletion of RBD diminished viral loads in the lungs but not in the heart. Acute pneumonia was observed in animals 24 hpi. Spp lentivirus was mainly found in SPC+ and LDLR+ pneumocytes and macrophages in the lungs. IL6, IL10, CD80, and PPAR-γ were quickly upregulated in response to infection in the lungs as well as in macrophage-like RAW264.7 cells. Furthermore, forced expression of the spike protein in RAW264.7 cells significantly increased the mRNA levels of the same panel of inflammatory factors. Our results demonstrated that the spike protein of SARS-CoV-2 confers the main point of viral entry into the lungs and can induce cellular pathology. Our data also indicate that an alternative ACE2-independent viral entry pathway may be recruited in the heart and aorta.


Assuntos
Macrófagos/imunologia , Pneumonia Viral/imunologia , Pneumonia Viral/patologia , Glicoproteína da Espícula de Coronavírus/imunologia , Doença Aguda , Células Epiteliais Alveolares/virologia , Animais , Antígeno B7-1 , Linhagem Celular , Mediadores da Inflamação , Interleucina-10 , Interleucina-6 , Lentivirus/genética , Lentivirus/isolamento & purificação , Lentivirus/metabolismo , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Macrófagos/virologia , Masculino , Glicoproteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , PPAR gama , Células RAW 264.7 , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Proteínas do Envelope Viral
5.
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
6.
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
7.
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
8.
Methods Mol Biol ; 2352: 117-126, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34324183

RESUMO

Somatic cell nuclear transfer and in vitro induction of pluripotency in somatic cells by defined factors provided unambiguous evidence that the epigenetic state of terminally differentiated somatic cells is not static and can be reversed to a more primitive one. Inspired by these results, stem cell biologists have identified approaches to directly convert fibroblasts into induced neuronal (iN) cells, indicating that direct lineage conversions are possible between distantly related cell types. More recently, we took advantages of pro-neurogenic capacity of iN factors and developed methods to rapidly derive functionally mature neurons directly from human pluripotent stem cells (hPSCs) through a brief induction of defined transcription factors. In this chapter, we describe the detailed methods used to attain the direct conversion from hPSCs to glutamatergic and GABAergic iN cells.


Assuntos
Diferenciação Celular , Neurônios/citologia , Neurônios/metabolismo , Células-Tronco Pluripotentes/citologia , Linhagem Celular , Separação Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Imunofluorescência , Neurônios GABAérgicos/citologia , Neurônios GABAérgicos/metabolismo , Vetores Genéticos/administração & dosagem , Vetores Genéticos/biossíntese , Vetores Genéticos/genética , Humanos , Lentivirus/genética , Neurogênese , Neuroglia/citologia , Neuroglia/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição , Transdução Genética
9.
Methods Mol Biol ; 2352: 149-170, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34324186

RESUMO

Oligodendrocytes are the main glial cell type in the central nervous system supporting the axonal part of neurons via myelin and lactate delivery. Both the conductive myelin formation and the energy support via lactate can be affected in diseases, such as multiple sclerosis and amyotrophic lateral sclerosis, respectively. Therefore, human disease modeling is needed to gain more mechanistic insights to drive drug discovery research. Here, patient-derived induced pluripotent stem cells (iPSCs) serve as a necessary tool providing an infinite cell source for patient-specific disease modeling, which allows investigation of oligodendrocyte involvement in human disease.Small molecule-based differentiation protocols to generate oligodendrocytes from pluripotent stem cells can last more than 90 days. Here, we provide a transcription factor-based, fast and efficient protocol for generating O4+ oligodendrocytes in just 20-24 days. After a neural induction phase of 8-12 days, SOX10 is overexpressed either with the use of lentiviral vectors or via engineered iPSCs, which inducibly overexpress SOX10 after doxycycline addition. Using this last method, a pure O4+ cell population is achieved after keeping the SOX10-overexpressing neural stem cells in culture for an additional 10 days. Furthermore, these O4+ cells can be co-cultured with iPSC-derived cortical neurons in 384-well format, allowing pro-myelinating drug screens. In conclusion, we provide a fast and efficient oligodendrocyte differentiation protocol allowing both in vitro human disease modeling and a high-throughput co-culture system for drug discovery.


Assuntos
Diferenciação Celular/genética , Expressão Gênica , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Fatores de Transcrição SOXE/genética , Fatores de Transcrição/genética , Técnicas de Cultura de Células , Células Cultivadas , Clonagem Molecular , Ordem dos Genes , Vetores Genéticos/administração & dosagem , Vetores Genéticos/biossíntese , Vetores Genéticos/genética , Humanos , Separação Imunomagnética , Lentivirus/genética , Neurogênese
10.
Methods Mol Biol ; 2352: 183-199, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34324188

RESUMO

Direct reprogramming is an emerging research field where you can generate neurons from a somatic cell, such as a skin or glial cell by overexpressing neurogenic transcription factors. This technique allows fast generation of subtype-specific and functional neurons from both human and mouse cells. Despite the fact that neurons have been successfully generated both in vitro and in vivo, a more extensive analysis of the induced neurons including phenotypic functional identity or gradual maturity is still lacking. This is an important step for a further development of induced neurons towards cell therapy or disease modeling of neurological diseases. In this protocol, we describe a method for functional assessment of direct reprogrammed neuronal cells both in vitro and in vivo. Using a synapsin-driven reporter, our protocol allows for a direct identification of the reprogrammed neurons that permits functional assessment using patch-clamp electrophysiology. For in vitro reprogramming we further provide an optimized coating condition that allows a long-term maturation of human induced neurons in vitro.


Assuntos
Diferenciação Celular/genética , Reprogramação Celular , Neurogênese , Neurônios/citologia , Neurônios/metabolismo , Animais , Técnicas de Cultura de Células , Células Cultivadas , Reprogramação Celular/genética , Técnicas de Reprogramação Celular , Dependovirus/genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/biossíntese , Vetores Genéticos/genética , Vetores Genéticos/isolamento & purificação , Humanos , Lentivirus/genética , Camundongos , Técnicas de Patch-Clamp , Fatores de Transcrição/genética , Transdução Genética
11.
Sheng Wu Gong Cheng Xue Bao ; 37(7): 2283-2292, 2021 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-34327895

RESUMO

Immunotherapy is becoming an effective and less invasive strategy that can be applied to the treatment of various malignancies. Lentiviral vectors (LVs) have shown great potential in immunotherapy as they can stably integrate relatively large foreign DNA, and effectively transduce dividing and non-dividing cells. Clinical application needs high quality LVs, and therefore strict quality control of the final products is necessary to ensure their purity, efficacy and safety. The quantitative detection of LVs is among the key parts of product development and quality control. In this paper, the existing methods for quantitative detection of LVs are summarized, including fluorescence activated cell sorter (FACS), P24 enzyme-linked immuno sorbent assay (P24 ELISA), real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), nanoparticle tracking analysis (NTA), tunable resistive pulse sensing(TRPS) and virus counter(VC).Their advantages and disadvantages are listed, and future development and challenges are discussed.


Assuntos
Lentivirus , Neoplasias , Vetores Genéticos/genética , Humanos , Imunoterapia , Lentivirus/genética , Transdução Genética
12.
Blood Adv ; 5(13): 2701-2706, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34196676

RESUMO

Betibeglogene autotemcel (beti-cel) gene therapy (GT) for patients with transfusion-dependent ß-thalassemia uses autologous CD34+ cells transduced with BB305 lentiviral vector (LVV), which encodes a modified ß-globin gene. BB305 LVV also contains select HIV sequences for viral packaging, reverse transcription, and integration. This case report describes a patient successfully treated with beti-cel in a phase 1/2 study (HGB-204; #NCT01745120) and subsequently diagnosed with wild-type (WT) HIV infection. From 3.5 to 21 months postinfusion, the patient stopped chronic red blood cell transfusions; total hemoglobin (Hb) and GT-derived HbAT87Q levels were 6.6 to 9.5 and 2.8 to 3.8 g/dL, respectively. At 21 months postinfusion, the patient resumed transfusions for anemia that coincided with an HIV-1 infection diagnosis. Quantitative polymerase chain reaction assays detected no replication-competent lentivirus. Next-generation sequencing confirmed WT HIV sequences. Six months after starting antiretroviral therapy, total Hb and HbAT87Q levels recovered to 8.6 and 3.6 g/dL, respectively, and 3.5 years postinfusion, 13.4 months had elapsed since the patient's last transfusion. To our knowledge, this is the first report of WT HIV infection in an LVV-based GT recipient and demonstrates persistent long-term hematopoiesis after treatment with beti-cel and the ability to differentiate between WT HIV and BB305-derived sequences.


Assuntos
Infecções por HIV , Talassemia beta , Terapia Genética , Vetores Genéticos/genética , Infecções por HIV/complicações , Infecções por HIV/terapia , Humanos , Lentivirus/genética , Talassemia beta/genética , Talassemia beta/terapia
13.
Methods Mol Biol ; 2312: 321-328, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34228300

RESUMO

Single-cell engineering via virus based genetic manipulation allows the possibility of understanding of complex tissues. However, current delivery methods for the genetic engineering of single cells via viral transduction suffer from limitations that restrict their application. Here I present a protocol describing a precise technique which can be used for the targeted virus infection of single cells in a monolayer of cells that is optically accessible. The protocol, demonstrated here by stamping cultured Hela cells with lentiviruses (LVs), completes in a few minutes and allows stable transgene expression within a few days, at success rates approaching 80%.


Assuntos
Engenharia Celular , Vetores Genéticos , Lentivirus/genética , Magnetismo , Nanopartículas de Magnetita , Análise de Célula Única , Transdução Genética , Técnicas de Cultura de Células , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Células HeLa , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência
14.
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
15.
Methods Mol Biol ; 2320: 261-281, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302664

RESUMO

Identifying causative genes in a given phenotype or disease model is important for biological discovery and drug development. The recent development of the CRISPR/Cas9 system has enabled unbiased and large-scale genetic perturbation screens to identify causative genes by knocking out many genes in parallel and selecting cells with desired phenotype of interest. However, compared to cancer cell lines, human somatic cells including cardiomyocytes (CMs), neuron cells, and endothelial cells are not easy targets of CRISPR screens because CRISPR screens require a large number of isogenic cells to be cultured and thus primary cells from patients are not ideal. The combination of CRISPR screens with induced pluripotent stem cell (iPSC) technology would be a powerful tool to identify causative genes and pathways because iPSCs can be expanded easily and differentiated to any cell type in principle. Here we describe a robust protocol for CRISPR screening using human iPSCs. Because each screening is different and needs to be customized depending on the cell types and phenotypes of interest, we show an example of CRISPR knockdown screening using CRISPRi system to identify essential genes to differentiate iPSCs to CMs.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Sequência de Bases , Causalidade , Células Cultivadas , Cromatografia Líquida/métodos , DNA/isolamento & purificação , Doxiciclina/farmacologia , Citometria de Fluxo , Estudos de Associação Genética , Vetores Genéticos/genética , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Lentivirus/genética , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , RNA Guia/genética , Transfecção
16.
Nat Commun ; 12(1): 4559, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315896

RESUMO

Activating mutations in the BRAF-MAPK pathway have been reported in histiocytoses, hematological inflammatory neoplasms characterized by multi-organ dissemination of pro-inflammatory myeloid cells. Here, we generate a humanized mouse model of transplantation of human hematopoietic stem and progenitor cells (HSPCs) expressing the activated form of BRAF (BRAFV600E). All mice transplanted with BRAFV600E-expressing HSPCs succumb to bone marrow failure, displaying myeloid-restricted hematopoiesis and multi-organ dissemination of aberrant mononuclear phagocytes. At the basis of this aggressive phenotype, we uncover the engagement of a senescence program, characterized by DNA damage response activation and a senescence-associated secretory phenotype, which affects also non-mutated bystander cells. Mechanistically, we identify TNFα as a key determinant of paracrine senescence and myeloid-restricted hematopoiesis and show that its inhibition dampens inflammation, delays disease onset and rescues hematopoietic defects in bystander cells. Our work establishes that senescence in the human hematopoietic system links oncogene-activation to the systemic inflammation observed in histiocytic neoplasms.


Assuntos
Senescência Celular , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Histiocitose/patologia , Inflamação/patologia , Células Mieloides/patologia , Oncogenes , Animais , Medula Óssea/patologia , Pontos de Checagem do Ciclo Celular/genética , Senescência Celular/genética , Doença Crônica , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Histiocitose/complicações , Humanos , Inflamação/complicações , Lentivirus/genética , Camundongos , Mutação/genética , Comunicação Parácrina , Análise de Componente Principal , Proteínas Proto-Oncogênicas B-raf/genética , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismo
17.
J Virol Methods ; 295: 114221, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34182038

RESUMO

SARS-CoV-2 is the culprit causing Coronavirus Disease 2019 (COVID-19). For the study of SARS-CoV-2 infection in a BSL-2 laboratory, a SARS-CoV-2 pseudovirus particle (SARS2pp) production and infection system was constructed by using a lentiviral vector bearing dual-reporter genes eGFP and firefly luciferase (Luc2) for easy observation and analysis. Comparison of SARS2pp different production conditions revealed that the pseudovirus titer could be greatly improved by: 1) removing the last 19 amino acids of the spike protein and replacing the signal peptide with the mouse Igk signal sequence; 2) expressing the spike protein using CMV promoter other than CAG (a hybrid promoter consisting of a CMV enhancer, beta-actin promoter, splice donor, and a beta-globin splice acceptor); 3) screening better optimized spike protein sequences for SARS2pp production; and 4) adding 1 % BSA in the SARS2pp production medium. For infection, this SARS2pp system showed a good linear relationship between MOI 2-0.0002 and then was successfully used to evaluate SARS-CoV-2 infection inhibitors including recombinant human ACE2 proteins and SARS-CoV-2 neutralizing antibodies. The kidney, liver and small intestine-derived cell lines were also found to show different susceptibility to SARSpp and SARS2pp. Given its robustness and good performance, it is believed that this pseudovirus particle production and infection system will greatly promote future research for SARS-CoV-2 entry mechanisms and inhibitors and can be easily applied to study new emerging SARS-CoV-2 variants.


Assuntos
Testes de Neutralização/métodos , SARS-CoV-2/fisiologia , Internalização do Vírus , Enzima de Conversão de Angiotensina 2/farmacologia , Animais , Anticorpos Neutralizantes/farmacologia , Antivirais/farmacologia , Linhagem Celular , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Lentivirus/genética , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Proteínas Recombinantes/farmacologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Vírion , Internalização do Vírus/efeitos dos fármacos
18.
Commun Biol ; 4(1): 713, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34112936

RESUMO

We report a lentiviral vector harboring the human ß2-microglobulin promoter, with predominant expression in immune cells and minimal proximal enhancers to improve vector safety. This lentiviral vector efficiently transduces major dendritic cell subsets in vivo. With a mycobacterial immunogen, we observed distinct functional signatures and memory phenotype in lentiviral vector- or Adenovirus type 5 (Ad5)-immunized mice, despite comparable antigen-specific CD8+ T cell magnitudes. Compared to Ad5, lentiviral vector immunization resulted in higher multifunctional and IL-2-producing CD8+ T cells. Furthermore, lentiviral vector immunization primed CD8+ T cells towards central memory phenotype, while Ad5 immunization favored effector memory phenotype. Studies using HIV antigens in outbred rats demonstrated additional clear-cut evidence for an immunogenic advantage of lentiviral vector over Ad5. Additionally, lentiviral vector provided enhance therapeutic anti-tumor protection than Ad5. In conclusion, coupling lentiviral vector with ß2-microglobulin promoter represents a promising approach to produce long-lasting, high-quality cellular immunity for vaccinal purposes.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Vetores Genéticos/genética , Lentivirus/genética , Transdução Genética , Microglobulina beta-2/genética , Animais , Linfócitos T CD8-Positivos/metabolismo , Células Dendríticas/metabolismo , Feminino , Engenharia Genética , Humanos , Imunidade Celular , Memória Imunológica , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Transgenes
19.
Int J Mol Sci ; 22(11)2021 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-34070997

RESUMO

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder based on a mutation in the IDS gene that encodes iduronate 2-sulphatase. As a result, there is an accumulation of glycosaminoglycans-heparan sulphate and dermatan sulphate-in almost all body tissues, which leads to their dysfunction. Currently, the primary treatment is enzyme replacement therapy, which improves the course of the disease by reducing somatic symptoms, including hepatomegaly and splenomegaly. The enzyme, however, does not cross the blood-brain barrier, and no improvement in the function of the central nervous system has been observed in patients with the severe form of the disease. An alternative method of treatment that solves typical problems of enzyme replacement therapy is gene therapy, i.e., delivery of the correct gene to target cells through an appropriate vector. Much progress has been made in applying gene therapy for MPS II, from cellular models to human clinical trials. In this article, we briefly present the history and basics of gene therapy and discuss the current state of knowledge about the methods of this therapy in mucopolysaccharidosis type II.


Assuntos
Glicoproteínas/genética , Mucopolissacaridose II/terapia , Adolescente , Animais , Barreira Hematoencefálica , Sistemas CRISPR-Cas , Criança , Pré-Escolar , Ensaios Clínicos como Assunto , Dependovirus/genética , Modelos Animais de Doenças , Portadores de Fármacos , Eletroporação , Terapia de Reposição de Enzimas/métodos , Edição de Genes , Terapia Genética , Vetores Genéticos/efeitos adversos , Vetores Genéticos/uso terapêutico , Glicoproteínas/farmacocinética , Glicoproteínas/uso terapêutico , Transplante de Células-Tronco Hematopoéticas , Humanos , Imunoconjugados/administração & dosagem , Imunoconjugados/uso terapêutico , Lactente , Injeções Intraventriculares , Injeções Espinhais , Lentivirus/genética , Camundongos , Mucopolissacaridose II/genética , Estudos Multicêntricos como Assunto , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/uso terapêutico , Retroviridae/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição
20.
Methods Mol Biol ; 2308: 281-300, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34057730

RESUMO

Cellular barcoding is a powerful technique that allows for high-throughput mapping of the fate of single cells, notably hematopoietic stem and progenitor cells (HSPCs) after transplantation. Unique artificial DNA fragments, termed barcodes, are stably inserted into HSPCs using lentiviral transduction, making sure that each individual cell receives a single unique barcode. Barcoded HSPCs are transplanted into sublethally irradiated mice where they reconstitute the hematopoietic system through proliferation and differentiation. During this process, the barcode of each HSPC is inherited by all of its daughter cells and their subsequent mature hematopoietic cell progeny. After sorting mature hematopoietic cell subsets, their barcodes can be retrieved from genomic DNA through nested PCR and sequencing. Analysis of barcode sequencing results allows for determination of clonal relationships between the mature cells, that is, which cell types were produced by a single barcoded HSPC, as well as the heterogeneity of the initial HSPC population. Here, we give a detailed protocol of a complete HSPC cellular barcoding experiment, starting with barcode lentivirus production, isolation, transduction, and transplantation of HSPCs, isolation of target cells followed by PCR amplification and sequencing of DNA barcodes. Finally, we describe the basic filtering and analysis steps of barcode sequencing data to ensure high-quality results.


Assuntos
Linhagem da Célula , Rastreamento de Células , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/fisiologia , Sequenciamento de Nucleotídeos em Larga Escala , Transdução Genética , Animais , Proliferação de Células , Separação Celular , Vetores Genéticos , Células HEK293 , Humanos , Lentivirus/genética , Camundongos , Camundongos Transgênicos , Fenótipo , Reação em Cadeia da Polimerase
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