RESUMEN
Air-liquid interface (ALI) culture of primary airway epithelial cells enables mucociliary differentiation providing an in vitro model of the human airway, but their proliferative potential is limited. To extend proliferation, these cells were previously transduced with viral oncogenes or mouse Bmi-1 + hTERT, but the resultant cell lines did not undergo mucociliary differentiation. We hypothesized that use of human BMI-1 alone would increase the proliferative potential of bronchial epithelial cells while retaining their mucociliary differentiation potential. Cystic fibrosis (CF) and non-CF bronchial epithelial cells were transduced by lentivirus with BMI-1 and then their morphology, replication kinetics, and karyotype were assessed. When differentiated at ALI, mucin production, ciliary function, and transepithelial electrophysiology were measured. Finally, shRNA knockdown of DNAH5 in BMI-1 cells was used to model primary ciliary dyskinesia (PCD). BMI-1-transduced basal cells showed normal cell morphology, karyotype, and doubling times despite extensive passaging. The cell lines underwent mucociliary differentiation when cultured at ALI with abundant ciliation and production of the gel-forming mucins MUC5AC and MUC5B evident. Cilia displayed a normal beat frequency and 9+2 ultrastructure. Electrophysiological characteristics of BMI-1-transduced cells were similar to those of untransduced cells. shRNA knockdown of DNAH5 in BMI-1 cells produced immotile cilia and absence of DNAH5 in the ciliary axoneme as seen in cells from patients with PCD. BMI-1 delayed senescence in bronchial epithelial cells, increasing their proliferative potential but maintaining mucociliary differentiation at ALI. We have shown these cells are amenable to genetic manipulation and can be used to produce novel disease models for research and dissemination.
Asunto(s)
Bronquios/citología , Diferenciación Celular , Cilios/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Moco/metabolismo , Complejo Represivo Polycomb 1/metabolismo , Animales , Dineínas Axonemales/metabolismo , Proliferación Celular , Forma de la Célula , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Dineínas/metabolismo , Impedancia Eléctrica , Fenómenos Electrofisiológicos , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Síndrome de Kartagener/metabolismo , Síndrome de Kartagener/patología , Síndrome de Kartagener/fisiopatología , Cariotipificación , Ratones , Microtúbulos/metabolismo , Modelos Biológicos , Fenotipo , Transducción GenéticaRESUMEN
In vivo bioluminescent imaging allows the detection of reporter gene expression in rodents in real time. Here we describe a novel technology whereby we can generate somatotransgenic rodents with the use of a viral vector carrying a luciferase transgene. We are able to achieve long term luciferase expression by a single injection of lentiviral or adeno-associated virus vectors to newborn mice. Further, we describe whole body bioluminescence imaging of conscious mice in a noninvasive manner, thus enforcing the 3R's (replacement, reduction, and refinement) of biomedical animal research.
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Expresión Génica , Genes Reporteros , Mediciones Luminiscentes/métodos , Animales , Técnicas Biosensibles , Orden Génico , Vectores Genéticos/genética , Luciferasas de Luciérnaga/genética , Ratones , Plásmidos/genética , Transfección , TransgenesRESUMEN
Explosion of gene therapy approaches for treating rare monogenic and common liver disorders created an urgent need for disease models able to replicate human liver cellular environment. Available models lack 3D liver structure or are unable to survive in long-term culture. We aimed to generate and test a 3D culture system that allows long-term maintenance of human liver cell characteristics. The in vitro whole-organ "Bioreactor grown Artificial Liver Model" (BALM) employs a custom-designed bioreactor for long-term 3D culture of human induced pluripotent stem cells-derived hepatocyte-like cells (hiHEPs) in a mouse decellularized liver scaffold. Adeno-associated viral (AAV) and lentiviral (LV) vectors were introduced by intravascular injection. Substantial AAV and LV transgene expression in the BALM-grown hiHEPs was detected. Measurement of secreted proteins in the media allowed non-invasive monitoring of the system. We demonstrated that humanized whole-organ BALM is a valuable tool to generate pre-clinical data for investigational medicinal products.
RESUMEN
We have previously designed a library of lentiviral vectors to generate somatic-transgenic rodents to monitor signalling pathways in diseased organs using whole-body bioluminescence imaging, in conscious, freely moving rodents. We have now expanded this technology to adeno-associated viral vectors. We first explored bio-distribution by assessing GFP expression after neonatal intravenous delivery of AAV8. We observed widespread gene expression in, central and peripheral nervous system, liver, kidney and skeletal muscle. Next, we selected a constitutive SFFV promoter and NFκB binding sequence for bioluminescence and biosensor evaluation. An intravenous injection of AAV8 containing firefly luciferase and eGFP under transcriptional control of either element resulted in strong and persistent widespread luciferase expression. A single dose of LPS-induced a 10-fold increase in luciferase expression in AAV8-NFκB mice and immunohistochemistry revealed GFP expression in cells of astrocytic and neuronal morphology. Importantly, whole-body bioluminescence persisted up to 240 days. We have validated a novel biosensor technology in an AAV system by using an NFκB response element and revealed its potential to monitor signalling pathway in a non-invasive manner in a model of LPS-induced inflammation. This technology complements existing germline-transgenic models and may be applicable to other rodent disease models.
Asunto(s)
Dependovirus/genética , Vectores Genéticos/genética , Ratones Transgénicos/genética , Animales , Técnicas Biosensibles/métodos , Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Inflamación/genética , Luciferasas de Luciérnaga/genética , Ratones , FN-kappa B/genética , Regiones Promotoras Genéticas/genética , Transducción de Señal/genética , Virus Formadores de Foco en el Bazo/genética , Transcripción Genética/genéticaRESUMEN
Human neural development begins at embryonic day 19 and marks the beginning of organogenesis. Neural stem cells in the neural tube undergo profound functional, morphological, and metabolic changes during neural specification, coordinated by a combination of exogenous and endogenous cues. The temporal cell signaling activities that mediate this process, during development and in the postnatal brain, are incompletely understood. We have applied gene expression studies and transcription factor-activated reporter lentiviruses during in vitro neural specification of human pluripotent stem cells. We show that nuclear factor κB orchestrates a multi-faceted metabolic program necessary for the maturation of neural progenitor cells during neurogenesis.
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Diferenciación Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Metabolismo Energético , FN-kappa B/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Autofagia , Biomarcadores , Ciclo Celular , Diferenciación Celular/genética , Células Cultivadas , Biología Computacional/métodos , Perfilación de la Expresión Génica , Ontología de Genes , Humanos , Inmunohistoquímica , Modelos Biológicos , Neurogénesis/genética , Fenotipo , Transducción de SeñalRESUMEN
PURPOSE OF REVIEW: Alternative approaches to conventional drug-based cancer treatments have seen T cell therapies deployed more widely over the last decade. This is largely due to their ability to target and kill specific cell types based on receptor recognition. Introduction of recombinant T cell receptors (TCRs) using viral vectors and HLA-independent T cell therapies using chimeric antigen receptors (CARs) are discussed. This article reviews the tools used for genome editing, with particular emphasis on the applications of site-specific DNA nuclease mediated editing for T cell therapies. RECENT FINDINGS: Genetic engineering of T cells using TCRs and CARs with redirected antigen-targeting specificity has resulted in clinical success of several immunotherapies. In conjunction, the application of genome editing technologies has resulted in the generation of HLA-independent universal T cells for allogeneic transplantation, improved T cell sustainability through knockout of the checkpoint inhibitor, programmed cell death protein-1 (PD-1), and has shown efficacy as an antiviral therapy through direct targeting of viral genomic sequences and entry receptors. SUMMARY: The combined use of engineered antigen-targeting moieties and innovative genome editing technologies have recently shown success in a small number of clinical trials targeting HIV and hematological malignancies and are now being incorporated into existing strategies for other immunotherapies.
RESUMEN
This article focuses on clinical applications of T cells transduced to express recombinant T cell receptor and chimeric antigen receptor constructs directed toward hematological malignancies, and considers newer strategies incorporating gene-editing technologies to address GvHD and host-mediated rejection. Recent data from clinical trials are reviewed, and an overview is provided of current and emerging manufacturing processes; consideration is also given to new developments in the pipeline.
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Tratamiento Basado en Trasplante de Células y Tejidos , Terapia Genética , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/terapia , Linfocitos T/metabolismo , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Biotecnología , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Edición Génica , Ingeniería Genética , Terapia Genética/métodos , Vectores Genéticos/genética , Neoplasias Hematológicas/inmunología , Humanos , Inmunoterapia Adoptiva , Receptores de Antígenos de Linfocitos T/genética , Proteínas Recombinantes de Fusión/genética , Linfocitos T/inmunologíaRESUMEN
The application of luciferase reporter genes to provide quantitative outputs for the activation of promoters is a well-established technique in molecular biology. Luciferase catalyzes an enzymatic reaction, which in the presence of the substrate luciferin produces photons of light relative to its molar concentration. The luciferase transgene can be genetically inserted at the first intron of a target gene to act as a surrogate for the gene's endogenous expression in cells and transgenic mice. Alternatively, promoter sequences can be excised and/or amplified from genomic sources or constructed de novo and cloned upstream of luciferase in an expression cassette transfected into cells. More recently, the development of synthetic promoters where the essential components of an RNA polymerase binding site and transcriptional start site are fused with various upstream regulatory sequences are being applied to drive reporter gene expression. We have developed a high-throughput cloning strategy to develop lentiviral luciferase reporters driven by transcription factor activated synthetic promoters. Lentiviruses integrate their payload cassette into the host cell genome, thereby facilitating the study of gene expression not only in the transduced cells but also within all subsequent daughter cells. In this manuscript we describe the design, vector construction, lentiviral transduction, and luciferase quantitation of transcription factor activated reporters (TFARs) in vitro and in vivo.
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Genes Reporteros , Luciferasas de Luciérnaga/análisis , Sustancias Luminiscentes/análisis , Mediciones Luminiscentes/métodos , Regiones Promotoras Genéticas , Activación Transcripcional , Animales , Clonación Molecular , Luciérnagas/enzimología , Luciérnagas/genética , Células HEK293 , Humanos , Lentivirus/genética , Luciferasas de Luciérnaga/genética , Sustancias Luminiscentes/metabolismo , Ratones , Factores de Transcripción/metabolismo , Transducción Genética/métodos , TransgenesRESUMEN
Molecular mechanisms regulating liver repair following cholestatic injury remain largely unknown. We have combined a mouse model of acute cholestatic liver injury, partial bile duct ligation (pBDL), with a novel longitudinal bioimaging methodology to quantify transcription factor activity during hepatic injury and repair. We administered lentiviral transcription factor activated luciferase/eGFP reporter (TFAR) cassettes to neonatal mice enabling longitudinal TFAR profiling by continued bioimaging throughout the lives of the animals and following pBDL in adulthood. Neonatal intravascular injection of VSV-G pseudotyped lentivirus resulted in almost exclusive transduction of hepatocytes allowing analysis of hepatocyte-specific transcription factor activity. We recorded acute but transient responses with NF-κB and Smad2/3 TFAR whilst our Notch reporter was repressed over the 40 days of evaluation post-pBDL. The bipotent hepatic progenitor cell line, HepaRG, can be directed to differentiate into hepatocytes and biliary epithelia. We found that forced expression of the Notch inhibitor NUMB in HepaRG resulted in enhanced hepatocyte differentiation and proliferation whereas over-expressing the Notch agonist JAG1 resulted in biliary epithelial differentiation. In conclusion, our data demonstrates that hepatocytes rapidly upregulate NF-κB and Smad2/3 activity, whilst repressing Notch signalling. This transcriptional response to cholestatic liver injury likely promotes partial de-differentiation to allow pro-regenerative proliferation of hepatocytes.
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Colestasis/metabolismo , Hepatocitos/metabolismo , Hepatopatías/metabolismo , Transducción de Señal , Células 3T3 , Animales , Diferenciación Celular , Proliferación Celular , Colestasis/complicaciones , Colestasis/diagnóstico por imagen , Células Epiteliales/citología , Células Epiteliales/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Células HeLa , Hepatocitos/citología , Hepatocitos/fisiología , Humanos , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Lentivirus/genética , Hepatopatías/diagnóstico por imagen , Hepatopatías/etiología , Luciferasas/genética , Luciferasas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , FN-kappa B/genética , FN-kappa B/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína smad3/genética , Proteína smad3/metabolismoRESUMEN
Luciferase bioimaging in living animals is increasingly being applied in many fields of biomedical research. Rodent imaging usually involves anaesthetising the animal during data capture, however, the biological consequences of anaesthesia have been largely overlooked. We have evaluated luciferase bioimaging in conscious, unrestrained mice after neonatal intracranial or intravascular administration of lentiviral, luciferase reporter cassettes (biosensors); we present real-time analyses from the first day of life to adulthood. Anaesthetics have been shown to exert both neurotoxic and neuroprotective effects during development and in models of brain injury. Mice subjected to bioimaging after neonatal intracranial or intravascular administration of biosensors, targeting the brain and liver retrospectively showed no significant difference in luciferase expression when conscious or unconscious throughout development. We applied conscious bioimaging to the assessment of NFκB and STAT3 transcription factor activated reporters during the earliest stages of development in living, unrestrained pups. Our data showed unique longitudinal activities for NFκB and STAT3 in the brain of conscious mice. Conscious bioimaging was applied to a neonatal mouse model of cerebral palsy (Hypoxic-Ischaemic Encephalopathy). Imaging of NFκB reporter before and after surgery showed a significant increase in luciferase expression, coinciding with secondary energy failure, in lesioned mice compared to controls.
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Encéfalo/metabolismo , Parálisis Cerebral/metabolismo , Hígado/metabolismo , Luciferasas/metabolismo , Imagen Molecular/métodos , Animales , Animales Recién Nacidos , Técnicas Biosensibles/métodos , Parálisis Cerebral/cirugía , Estado de Conciencia , Modelos Animales de Enfermedad , Vectores Genéticos/administración & dosificación , Inyecciones Intraarteriales , Lentivirus/genética , Luciferasas/genética , Ratones , Ratones Transgénicos , FN-kappa B/genética , FN-kappa B/metabolismo , Proteínas Recombinantes/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismoRESUMEN
The potential of induced pluripotent stem cells (iPSCs) in disease modeling and regenerative medicine is vast, but current methodologies remain inefficient. Understanding the cellular mechanisms underlying iPSC reprogramming, such as the metabolic shift from oxidative to glycolytic energy production, is key to improving its efficiency. We have developed a lentiviral reporter system to assay longitudinal changes in cell signaling and transcription factor activity in living cells throughout iPSC reprogramming of human dermal fibroblasts. We reveal early NF-κB, AP-1, and NRF2 transcription factor activation prior to a temporal peak in hypoxia inducible factor α (HIFα) activity. Mechanistically, we show that an early burst in oxidative phosphorylation and elevated reactive oxygen species generation mediates increased NRF2 activity, which in turn initiates the HIFα-mediated glycolytic shift and may modulate glucose redistribution to the pentose phosphate pathway. Critically, inhibition of NRF2 by KEAP1 overexpression compromises metabolic reprogramming and results in reduced efficiency of iPSC colony formation.
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Reprogramación Celular , Fibroblastos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/genética , Factor 2 Relacionado con NF-E2/genética , Dermis/citología , Dermis/metabolismo , Fibroblastos/citología , Regulación de la Expresión Génica , Genes Reporteros , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Glucólisis/genética , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Células Madre Pluripotentes Inducidas/citología , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Lentivirus/genética , Lentivirus/metabolismo , Luciferasas/genética , Luciferasas/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Fosforilación Oxidativa , Vía de Pentosa Fosfato/genética , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , Transducción GenéticaRESUMEN
The application of transcription factor activated luciferase reporter cassettes in vitro is widespread but potential for in vivo application has not yet been realized. Bioluminescence imaging enables non-invasive tracking of gene expression in transfected tissues of living rodents. However the mature immune response limits luciferase expression when delivered in adulthood. We present a novel approach of tissue-targeted delivery of transcription factor activated luciferase reporter lentiviruses to neonatal rodents as an alternative to the existing technology of generating germline transgenic light producing rodents. At this age, neonates acquire immune tolerance to the conditionally responsive luciferase reporter. This simple and transferrable procedure permits surrogate quantitation of transcription factor activity over the lifetime of the animal. We show principal efficacy by temporally quantifying NFκB activity in the brain, liver and lungs of somatotransgenic reporter mice subjected to lipopolysaccharide (LPS)-induced inflammation. This response is ablated in Tlr4(-/-) mice or when co-administered with the anti-inflammatory glucocorticoid analogue dexamethasone. Furthermore, we show the malleability of this technology by quantifying NFκB-mediated luciferase expression in outbred rats. Finally, we use somatotransgenic bioimaging to longitudinally quantify LPS- and ActivinA-induced upregulation of liver specific glucocorticoid receptor and Smad2/3 reporter constructs in somatotransgenic mice, respectively.
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Luciferasas de Luciérnaga/genética , Factores de Transcripción/fisiología , Activación Transcripcional/inmunología , Animales , Genes Reporteros , Vectores Genéticos , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Células HeLa , Humanos , Lentivirus/genética , Lipopolisacáridos/farmacología , Luciferasas de Luciérnaga/biosíntesis , Ratones , Células 3T3 NIH , Especificidad de Órganos , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Surrogate genetically encoded markers have been utilized in order to analyze gene transfer efficacy, location, and persistence. These marker genes have greatly accelerated the development of gene transfer vectors for the ultimate application of gene therapy using therapeutic genes. They have also been used in many other applications, such as gene marking in order to study developmental cell lineages, to track cell migration, and to study tumor growth and metastasis. This chapter aims to describe the analysis of several commonly used marker genes: green fluorescent protein (GFP), ß-galactosidase, firefly luciferase, human factor IX, and alkaline phosphatase. The merits and disadvantages of each are briefly discussed. In addition a few short examples are provided for continual and endpoint analysis in different disease models including hemophilia, cystic fibrosis, ornithine transcarbamylase deficiency and Gaucher disease.