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CRISPR-Cas genome-editing nucleases hold substantial promise for developing human therapeutic applications1-6 but identifying unwanted off-target mutations is important for clinical translation7. A well-validated method that can reliably identify off-targets in vivo has not been described to date, which means it is currently unclear whether and how frequently these mutations occur. Here we describe 'verification of in vivo off-targets' (VIVO), a highly sensitive strategy that can robustly identify the genome-wide off-target effects of CRISPR-Cas nucleases in vivo. We use VIVO and a guide RNA deliberately designed to be promiscuous to show that CRISPR-Cas nucleases can induce substantial off-target mutations in mouse livers in vivo. More importantly, we also use VIVO to show that appropriately designed guide RNAs can direct efficient in vivo editing in mouse livers with no detectable off-target mutations. VIVO provides a general strategy for defining and quantifying the off-target effects of gene-editing nucleases in whole organisms, thereby providing a blueprint to foster the development of therapeutic strategies that use in vivo gene editing.
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Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Edición Génica/normas , Genoma/genética , Mutación , Especificidad por Sustrato/genética , Animales , Proteínas Asociadas a CRISPR/genética , Femenino , Humanos , Mutación INDEL , Masculino , Ratones , Ratones Endogámicos C57BL , Proproteína Convertasa 9/genética , Transgenes/genéticaRESUMEN
A cross-industry survey was conducted by EFPIA/IQ DruSafe in 2018 to provide information on photosafety evaluation of pharmaceuticals after implementation of ICH S10. This survey focused on the strategy utilized for photosafety risk assessment, the design of nonclinical (in vitro and in vivo) and clinical evaluations, the use of exposure margins in risk assessment, and regulatory interactions. The survey results indicated that a staged approach for phototoxicity assessment has been widely accepted by regulatory authorities globally. The OECD-based 3T3 NRU Phototoxicity Test is the most frequently used in vitro approach. Modifications to this assay suggested by ICH S10 are commonly applied. For in-vitro-positives, substantial margins from in vitro IC50 values under irradiation to Cmax (clinical) have enabled further development without the need for additional photosafety data. In vivo phototoxicity studies typically involve dosing rodents and exposing skin and eyes to simulated sunlight, and subsequently evaluating at least the skin for erythema and edema. However, no formal guidelines exist and protocols are less standardized across companies. A margin-of-safety approach (based on Cmax at NOAEL) has been successfully applied to support clinical development. Experience with dedicated clinical phototoxicity studies was limited, perhaps due to effective de-risking approaches employed based on ICH S10.
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Dermatitis Fototóxica/patología , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/patología , Organización para la Cooperación y el Desarrollo Económico/normas , Preparaciones Farmacéuticas/normas , Luz Solar/efectos adversosRESUMEN
In vitro genotoxicity assessment routinely employs an exogenous metabolic activation mixture to simulate mammalian metabolism. Activation mixtures commonly contain post-mitochondrial liver supernatant (i.e. S9) from chemically induced Sprague Dawley rats. Although Organization for Economic Cooperation and Development (OECD) test guidelines permit the use of other S9 preparations, assessments rarely employ human-derived S9. The objective of this study is to review and evaluate the use of human-derived S9 for in vitro genetic toxicity assessment. All available published genotoxicity assessments employing human S9 were compiled for analysis. To facilitate comparative analyses, additional matched Ames data using induced rat liver S9 were obtained for certain highly cited chemicals. Historical human and induced rat S9 quality control reports from Moltox were obtained and mined for enzyme activity and mutagenic potency data. Additional in vitro micronucleus data were experimentally generated using human and induced rat S9. The metabolic activity of induced rat S9 was found to be higher than human S9, and linked to high mutagenic potency results. This study revealed that human S9 often yields significantly lower Salmonella mutagenic potency values, especially for polycyclic aromatic hydrocarbons, aflatoxin B1 and heterocyclic amines (~3- to 350-fold). Conversely, assessment with human S9 activation yields higher potency for aromatic amines (~2- to 50-fold). Outliers with extremely high mutagenic potency results were observed in the human S9 data. Similar trends were observed in experimentally generated mammalian micronucleus cell assays, however human S9 elicited potent cytotoxicity L5178Y, CHO and TK6 cell lines. Due to the potential for reduced sensitivity and the absence of a link between enzyme activity levels and mutagenic potency, human liver S9 is not recommended for use alone in in vitro genotoxicity screening assays; however, human S9 may be extremely useful in follow-up tests, especially in the case of chemicals with species-specific metabolic differences, such as aromatic amines.
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Activación Metabólica , Sistema Libre de Células , Hepatocitos/metabolismo , Pruebas de Mutagenicidad/métodos , Animales , Carcinógenos/toxicidad , Línea Celular , Activación Enzimática , Hepatocitos/efectos de los fármacos , Hepatocitos/enzimología , Humanos , Ratones , Pruebas de Micronúcleos , Mutágenos/toxicidad , Ratas , Salmonella/efectos de los fármacos , Salmonella/genéticaRESUMEN
The ICH S6(R1) recommendations on safety evaluation of biotherapeutics have led to uncertainty in determining what would constitute a cause for concern that would require genotoxicity testing. A Health and Environmental Sciences Institute's Genetic Toxicology Technical Committee Workgroup was formed to review the current practice of genotoxicity assessment of peptide/protein-related biotherapeutics. There are a number of properties of peptide/protein-related biotherapeutics that distinguish such products from traditional 'small molecule' drugs and need to be taken into consideration when assessing whether genotoxicity testing may be warranted and if so, how to do it appropriately. Case examples were provided by participating companies and decision trees were elaborated to determine whether and when genotoxicity evaluation is needed for peptides containing natural amino acids, non-natural amino acids and other chemical entities and for unconjugated and conjugated proteins. From a scientific point of view, there is no reason for testing peptides containing exclusively natural amino acids irrespective of the manufacturing process. If non-natural amino acids, organic linkers and other non-linker chemical components have already been tested for genotoxicity, there is no need to re-evaluate them when used in different peptide/protein-related biotherapeutics. Unless the peptides have been modified to be able to enter the cells, it is generally more appropriate to evaluate the peptides containing the non-natural amino acids and other non-linker chemical moieties in vivo where the cleavage products can be formed. For linkers, it is important to determine if exposure to reactive forms are likely to occur and from which origin. When the linkers are anticipated to be potential mutagenic impurities they should be evaluated according to ICH M7. If linkers are expected to be catabolic products, it is recommended to test the entire conjugate in vivo, as this would ensure that the relevant 'free' linker forms stemming from in vivo catabolism are tested.
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Guías como Asunto , Pruebas de Mutagenicidad/métodos , Mutágenos/toxicidad , Péptidos/toxicidad , Animales , Humanos , Mutágenos/efectos adversos , Péptidos/efectos adversos , Péptidos/uso terapéuticoRESUMEN
We report the discovery of benzothiazoles, a novel anti-mycobacterial series, identified from a whole cell based screening campaign. Benzothiazoles exert their bactericidal activity against Mycobacterium tuberculosis (Mtb) through potent inhibition of decaprenylphosphoryl-ß-d-ribose 2'-oxidase (DprE1), the key enzyme involved in arabinogalactan synthesis. Specific target linkage and mode of binding were established using co-crystallization and protein mass spectrometry studies. Most importantly, the current study provides insights on the utilization of systematic medicinal chemistry approaches to mitigate safety liabilities while improving potency during progression from an initial genotoxic hit, the benzothiazole N-oxides (BTOs) to the lead-like AMES negative, crowded benzothiazoles (cBTs). These findings offer opportunities for development of safe clinical candidates against tuberculosis. The design strategy adopted could find potential application in discovery of safe drugs in other therapy areas too.
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Oxidorreductasas de Alcohol/metabolismo , Antituberculosos/química , Antituberculosos/farmacología , Proteínas Bacterianas/metabolismo , Benzotiazoles/química , Benzotiazoles/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Oxidorreductasas de Alcohol/antagonistas & inhibidores , Proteínas Bacterianas/antagonistas & inhibidores , Diseño de Fármacos , Humanos , Simulación del Acoplamiento Molecular , Relación Estructura-Actividad , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiologíaRESUMEN
Microphysiological systems (MPS) are gaining broader application in the pharmaceutical industry but have primarily been leveraged in early discovery toxicology and pharmacology studies with small molecules. The adoption of MPS offers a promising avenue to reduce animal use, improve in-vitro-to-in-vivo translation of pharmacokinetics/pharmacodynamics and toxicity correlation, and provide mechanistic understanding of model species suitability. While MPS have demonstrated utility in these areas with small molecules and biologics, MPS models in cell therapy development have not been fully explored, let alone validated. Distinguishing features of MPS, including long-term viability and physiologically relevant expression of functional enzymes, receptors, and pharmacological targets make them attractive tools for nonclinical characterization. However, there is currently limited published evidence of MPS being utilized to study the disposition, metabolism, pharmacology, and toxicity profiles of cell therapies. This review provides an industry perspective on the nonclinical application of MPS on cell therapies, first with a focus on oncology applications followed by examples in regenerative medicine.
Microphysiological systems (MPS) are advanced cell models, applied in the pharmaceutical industry to characterize novel therapies. While their application in studies of small molecule therapies has been very successful, the use of these models to study cell therapies has been limited. Cell therapies consist of cells and are living drugs, often with complex biological mechanisms of action, which can be very challenging to study. However, MPS have several features that make them attractive for studying cell therapies, including possibilities for longer-term studies and the ability to mimic physiologically relevant biological functions. MPS can mimic complex biological systems and processes, as such, the adoption of MPS offers a promising avenue to reduce the use of animals in the characterization of novel therapies. This review provides an industry perspective on current challenges and highlights opportunities for using MPS in the development of cell therapies.
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Alternativas a las Pruebas en Animales , Tratamiento Basado en Trasplante de Células y Tejidos , Animales , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Humanos , Medicina Regenerativa/métodos , Sistemas MicrofisiológicosRESUMEN
Regulators and industry are actively seeking improvements and alternatives to current models and approaches to evaluate potential carcinogenicity of gene therapies (GTs). A meeting of invited experts was organized by NC3Rs/UKEMS (London, March 2023) to discuss this topic. This article describes the consensus reached among delegates on the definition of vector genotoxicity, sources of uncertainty, suitable toxicological endpoints for genotoxic assessment of GTs, and future research needs. The collected recommendations should inform the further development of regulatory guidelines for the nonclinical toxicological assessment of GT products.
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Terapia Genética , Terapia Genética/efectos adversos , Terapia Genética/métodos , Humanos , Factores de Riesgo , Animales , Vectores Genéticos/efectos adversos , Consenso , Neoplasias/terapia , Neoplasias/genética , Medición de RiesgoRESUMEN
Aim & methods: The Health and Environmental Sciences Institute Cell Therapy-TRAcking, Circulation & Safety Technical Committee launched an international, multisite study to evaluate the sensitivity and reproducibility of the highly efficient culture (HEC) assay, an in vitro assay to detect residual undifferentiated human pluripotent stem cells (hPSCs) in cell therapy products. Results: All facilities detected colonies of human induced pluripotent stem cells (hiPSCs) when five hiPSCs were spiked into 1 million hiPSC-derived cardiomyocytes. Spiking with a trace amount of hiPSCs revealed that repeatability accounts for the majority of reproducibility while the true positive rate was high. Conclusion: The results indicate that the HEC assay is highly sensitive and robust and can be generally applicable for tumorigenicity evaluation of hPSC-derived cell therapy products.
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Células Madre Pluripotentes Inducidas , Células Madre Pluripotentes , Humanos , Reproducibilidad de los Resultados , Academias e Institutos , BioensayoRESUMEN
This is the report from the "ECVAM-EFPIA workshop on 3T3 NRU Phototoxicity Test: Practical Experience and Implications for Phototoxicity Testing", jointly organized by ECVAM and EFPIA and held on the 25-27 October 2010 in Somma Lombardo, Italy. The European Centre for the Validation of Alternative Methods (ECVAM) was established in 1991 within the European Commission Joint Research, based on a Communication from the European Commission (1991). The main objective of ECVAM is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences and which reduce, refine and replace the use of laboratory animals. The European Federation of Pharmaceuticals Industries and Association (EFPIA) represent the pharmaceutical industry operating in Europe. Through its direct membership of 31 national associations and 40 leading pharmaceutical companies, EFPIA is the voice on the EU scene of 2200 companies committed to researching, developing and bringing to patients new medicines that improve health and the quality of life around the world. The workshop, co-chaired by Joachim Kreysa (ECVAM) and Phil Wilcox (GSK, EFPIA) involved thirty-five experts from academia, regulatory authorities and industry, invited to contribute with their experiences in the field of phototoxicology. The main objectives of the workshop were: -to present 'in use' experience of the pharmaceutical industry with the 3T3 Neutral Red Uptake Phototoxicity Test (3T3 NRU-PT), -to discuss why it differs from the results in the original validation exercise, -to discuss technical issues and consider ways to improve the usability of the 3T3 NRU-PT for (non-topical) pharmaceuticals, e.g., by modifying the threshold of chemical light absorption to trigger photo-toxicological testing, and by modifying technical aspects of the assay, or adjusting the criteria used to classify a positive response. During the workshop, the assay methodology was reviewed by comparing the OECD Test Guideline (TG 432) with the protocols used in testing laboratories, data from EFPIA and JPMA 'surveys' were presented and possible reasons for the outcomes were discussed. Experts from cosmetics and pharmaceutical industries reported on their experience with the 3T3 NRU-PT and evidence was presented for phototoxic clinical symptoms that could be linked to certain relevant molecules. Brainstorming sessions discussed if the 3T3 NRU-PT needed to be improved and whether alternatives to the 3T3 NRU-PT exist. Finally, the viewpoint from EU and US regulators was presented. In the final session, the conclusions of the meeting were summarized, with action points. It was concluded that the 3T3 NRU-PT identifies phototoxicological hazards with a 100% sensitivity, and thus is accepted as the tier one test that correctly identifies the absence of phototoxic potential. Consequently, positive results in the 3T3 NRU-PT often do not translate into a clinical phototoxicity risk. Possible ways to improve the practical use of this assay include: (i) adaptation of changed UV/vis-absorption criteria as a means to reduce the number of materials tested, (ii) reduction of the highest concentration to be tested, and (iii) consideration of modifying the threshold criteria for the prediction of a positive call in the test.
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Alternativas a las Pruebas en Animales/métodos , Dermatitis Fototóxica , Rojo Neutro/metabolismo , Fármacos Fotosensibilizantes/toxicidad , Pruebas de Toxicidad/métodos , Células 3T3 , Animales , Bioensayo/métodos , Seguridad de Productos para el Consumidor , Cosméticos/toxicidad , Dermatitis Fototóxica/etiología , Industria Farmacéutica , Ratones , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Understanding how breaks form and are repaired in the genome depends on the accurate measurement of the frequency and position of DNA double strand breaks (DSBs). This is crucial for identification of a chemical's DNA damage potential and for safe development of therapies, including genome editing technologies. Current DSB sequencing methods suffer from high background levels, the inability to accurately measure low frequency endogenous breaks and high sequencing costs. Here we describe INDUCE-seq, which overcomes these problems, detecting simultaneously the presence of low-level endogenous DSBs caused by physiological processes, and higher-level recurrent breaks induced by restriction enzymes or CRISPR-Cas nucleases. INDUCE-seq exploits an innovative NGS flow cell enrichment method, permitting the digital detection of breaks. It can therefore be used to determine the mechanism of DSB repair and to facilitate safe development of therapeutic genome editing. We further discuss how the method can be adapted to detect other genomic features.
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Roturas del ADN de Doble Cadena , Edición Génica , Sistemas CRISPR-Cas/genética , ADN/genética , Reparación del ADN/genética , Endonucleasas/genética , Edición Génica/métodos , GenómicaRESUMEN
Heart regeneration is an unmet clinical need, hampered by limited renewal of adult cardiomyocytes and fibrotic scarring. Pluripotent stem cell-based strategies are emerging, but unravelling cellular dynamics of host-graft crosstalk remains elusive. Here, by combining lineage tracing and single-cell transcriptomics in injured non-human primate heart biomimics, we uncover the coordinated action modes of human progenitor-mediated muscle repair. Chemoattraction via CXCL12/CXCR4 directs cellular migration to injury sites. Activated fibroblast repulsion targets fibrosis by SLIT2/ROBO1 guidance in organizing cytoskeletal dynamics. Ultimately, differentiation and electromechanical integration lead to functional restoration of damaged heart muscle. In vivo transplantation into acutely and chronically injured porcine hearts illustrated CXCR4-dependent homing, de novo formation of heart muscle, scar-volume reduction and prevention of heart failure progression. Concurrent endothelial differentiation contributed to graft neovascularization. Our study demonstrates that inherent developmental programmes within cardiac progenitors are sequentially activated in disease, enabling the cells to sense and counteract acute and chronic injury.
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Proteínas del Tejido Nervioso , Células Madre Pluripotentes , Animales , Diferenciación Celular , Cicatriz/patología , Cicatriz/prevención & control , Fibrosis , Humanos , Miocardio/patología , Miocitos Cardíacos/patología , Células Madre Pluripotentes/patología , Receptores Inmunológicos , PorcinosRESUMEN
A semi-automated scoring system has been developed to provide rapid, accurate assessment of micronuclei in preparations of mononuclear mouse lymphoma L5178Y cells. Following exposure to a range of test agents, flat, single-cell preparations were produced from exponentially growing cultures by cytocentrifugation. Following staining with 4'-6-diamidino-2-phenylindole (DAPI), cells were scanned by use of the MicroNuc module of Metafer 4 v 3.4.102, after modifying the classifier developed for selecting micronuclei in binucleate cells to increase its sensitivity. The image gallery of all cells was then sorted to bring aberrant cells to the top of the gallery to assess visually the numbers of cells with micronuclei, as distinct from other debris. Slide quality was shown to be paramount in obtaining accurate results from an automated scan and the data obtained compared very well with the incidence of micronuclei scored conventionally by microscopy. Compared with manual scoring the time saving is considerable, as more than 2000 images are captured in approximately 2min, with subsequent visual assessment of aberrant cells in the image gallery taking about 1-2min/slide. By scanning all aberrant cells, the system also captures additional information on necrotic, apoptotic and fragmented cells. Although optimised for mouse lymphoma cells, it should be simple to adapt the method for any cell type growing in suspension.
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Procesamiento de Imagen Asistido por Computador/métodos , Pruebas de Micronúcleos/métodos , Animales , Leucemia L5178 , Ratones , Micronúcleos con Defecto Cromosómico , Mutágenos/toxicidad , Sensibilidad y EspecificidadRESUMEN
The selection of maximum concentrations for in vitro mammalian cell genotoxicity assays was reviewed at the 5th International Workshop on Genotoxicity Testing (IWGT), 2009. Currently, the top concentration recommended when toxicity is not limiting is 10mM or 5mg/ml, whichever is lower. The discussion was whether to reduce the limit, and if so whether the 1mM limit proposed for human pharmaceuticals was appropriate for testing other chemicals. The consensus was that there was reason to consider reducing the 10mM limit, and many, but not all, attendees favored a reduction to 1mM. Several proposals are described here for the concentration limit. The in vitro cytogenetics expert working group also discussed appropriate measures and level of cytotoxicity. Data were reviewed from a multi-laboratory trial of the in vitro micronucleus (MN) assay with multiple cell types and several types of toxicity measurements. The group agreed on a preference for toxicity measures that take cell proliferation after the beginning of treatment into account (relative increase in cell counts, relative population doubling, cytokinesis block proliferation index or replicative index), and that this applies both to in vitro MN assays and to in vitro chromosome aberration assays. Since relative cell counts (RCC) underestimate toxicity, many group members favored making a recommendation against the use of RCC as a toxicity measure for concentration selection. All 14 chemicals assayed for MN induction in the multi-laboratory trial were detected without exceeding 50% toxicity by any measure, but some were positive only at concentrations with toxicity quite close to 50%. The expert working group agreed to accept the cytotoxicity range recommended by OECD guideline 487 (55±5% toxicity at the top concentration scored). This also reinforces the original intent of the guidance for the in vitro chromosome aberration assay, where ">50%" was intended to target the range close to 50% toxicity.
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Pruebas de Micronúcleos/normas , Pruebas de Mutagenicidad/normas , Animales , Aberraciones Cromosómicas , Guías como Asunto , Humanos , Mamíferos , Mutágenos/administración & dosificaciónRESUMEN
Improving current in vitro genotoxicity tests is an ongoing task for genetic toxicologists. Further, the question on how to deal with positive in vitro results that are demonstrated to not predict genotoxicity or carcinogenicity potential in rodents or humans is a challenge. These two aspects were addressed at the 5th International Workshop on Genotoxicity Testing (IWGT) held in Basel, Switzerland, on August 17-19, 2009. The objectives of the working group (WG) were to make recommendations on the use of cell types or lines, if possible, and to provide evaluations of promising new approaches. Results obtained in rodent cell lines with impaired p53 function (L5178Y, V79, CHL and CHO cells) and human p53-competent cells (peripheral blood lymphocytes, TK6 and HepG2 cells) suggest that a reduction in the percentage of non-relevant positive results for carcinogenicity prediction can be achieved by careful selection of cells used without decreasing the sensitivity of the assays. Therefore, the WG suggested using p53- competent - preferably human - cells in in vitro micronucleus or chromosomal aberration tests. The use of the hepatoma cell line HepaRG for genotoxicity testing was considered promising since these cells possess better phase I and II metabolizing potential compared to cell lines commonly used in this area and may overcome the need for the addition of S9. For dermally applied compounds, the WG agreed that in vitro reconstructed skin models, once validated, will be useful to follow up on positive results from standard in vitro assays as they resemble the properties of human skin (barrier function, metabolism). While the reconstructed skin micronucleus assay has been shown to be further advanced, there was also consensus that the Comet assay should be further evaluated due to its independence from cell proliferation and coverage of a wider spectrum of DNA damage.
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Pruebas de Mutagenicidad/métodos , Pruebas de Mutagenicidad/tendencias , Animales , Línea Celular , Aberraciones Cromosómicas , Guías como Asunto , Humanos , Pruebas de Micronúcleos/métodos , Valor Predictivo de las PruebasRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the cause of a present pandemic, infects human lung alveolar type 2 (hAT2) cells. Characterizing pathogenesis is crucial for developing vaccines and therapeutics. However, the lack of models mirroring the cellular physiology and pathology of hAT2 cells limits the study. Here, we develop a feeder-free, long-term, three-dimensional (3D) culture technique for hAT2 cells derived from primary human lung tissue and investigate infection response to SARS-CoV-2. By imaging-based analysis and single-cell transcriptome profiling, we reveal rapid viral replication and the increased expression of interferon-associated genes and proinflammatory genes in infected hAT2 cells, indicating a robust endogenous innate immune response. Further tracing of viral mutations acquired during transmission identifies full infection of individual cells effectively from a single viral entry. Our study provides deep insights into the pathogenesis of SARS-CoV-2 and the application of defined 3D hAT2 cultures as models for respiratory diseases.
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COVID-19 , Alveolos Pulmonares/virología , SARS-CoV-2/fisiología , Células Madre/virología , COVID-19/virología , Técnicas de Cultivo de Célula , Medios de Cultivo , Humanos , Interferones/metabolismo , Modelos Biológicos , Alveolos Pulmonares/metabolismo , Alveolos Pulmonares/ultraestructura , SARS-CoV-2/ultraestructura , Transcriptoma , Internalización del Virus , Replicación ViralRESUMEN
Extracellular vesicles (EVs) mediate cellular communication through the transfer of active biomolecules, raising interest in using them as biological delivery vehicles for therapeutic drugs. For drug delivery applications, it is important to understand the intrinsic safety and toxicity liabilities of EVs. Nanoparticles, including EVs, typically demonstrate significant accumulation in the liver after systemic administration in vivo. We confirmed uptake of EVs derived from Expi293F cells into HepG2 cells and did not detect any signs of hepatotoxicity measured by cell viability, functional secretion of albumin, plasma membrane integrity, and mitochondrial and lysosomal activity even at high exposures of up to 5 × 1010 EVs per mL. Whole genome transcriptome analysis was used to measure potential effects on the gene expression in the recipient HepG2 cells at 24 h following exposure to EVs. Only 0.6% of all genes were found to be differentially expressed displaying less than 2-fold expression change, with genes related to inflammation or toxicity being unaffected. EVs did not trigger any proinflammatory cytokine response in HepG2 cells. However, minor changes were noted in human blood for interleukin (IL)-8, IL-6, and monocyte chemotactic protein 1 (MCP-1). Administration of 5 × 1010 Expi293F-derived EVs to BALB/c mice did not result in any histopathological changes or increases of liver transaminases or cytokine levels, apart from a modest increase in keratinocyte chemoattractant (KC). The absence of any significant toxicity associated with EVs in vitro and in vivo supports the prospective use of EVs for therapeutic applications and for drug delivery.
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Vesículas Extracelulares/fisiología , Hígado/patología , Animales , Citocinas/metabolismo , Vesículas Extracelulares/trasplante , Células HEK293 , Células Hep G2 , Humanos , Mediadores de Inflamación/metabolismo , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Nanopartículas/química , Albúmina Sérica/metabolismo , Transaminasas/metabolismo , TranscriptomaRESUMEN
Nucleoside analogs with 2'-modified sugar moieties are often used to improve the RNA target affinity and nuclease resistance of therapeutic oligonucleotides in preclinical and clinical development. Despite their enhanced nuclease resistance, oligonucleotides could slowly degrade releasing nucleoside analogs that have the potential to become phosphorylated and incorporated into cellular DNA and RNA. For the first time, the phosphorylation and DNA/RNA incorporation of 2'-O-(2-methoxyethyl) (2'-O-MOE) nucleoside analogs have been investigated. Using liquid chromatography/tandem mass spectrometry, we showed that enzymes in the nucleotide salvage pathway including deoxycytidine kinase (dCK) and thymidine kinase (TK1) displayed poor reactivity toward 2'-O-MOE nucleoside analogs. On the other hand, 2'-fluoro (F) nucleosides, regardless of the nucleobase, were efficiently phosphorylated to their monophosphate forms by dCK and TK1. Consistent with their efficient phosphorylation by dCK and TK1, 2'-F nucleoside analogs were incorporated into cellular DNA and RNA while no incorporation was detected with 2'-O-MOE nucleoside analogs. In conclusion, these data suggest that the inability of dCK and TK1 to create the monophosphates of 2'-O-MOE nucleoside analogs reduces the risk of their incorporation into cellular DNA and RNA.
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Núcleo Celular/efectos de los fármacos , ADN/metabolismo , Genoma Humano , Nucleósidos/farmacología , ARN/metabolismo , Línea Celular Tumoral , Núcleo Celular/metabolismo , Desoxicitidina Quinasa/metabolismo , Humanos , Nucleósidos/química , Fosforilación , Especificidad por Sustrato , Timidina Quinasa/metabolismoRESUMEN
The recent revisions of the Organisation for Economic Co-operation and Development (OECD) genetic toxicology test guidelines emphasize the importance of historical negative controls both for data quality and interpretation. The goal of a HESI Genetic Toxicology Technical Committee (GTTC) workgroup was to collect data from participating laboratories and to conduct a statistical analysis to understand and publish the range of values that are normally seen in experienced laboratories using TK6 cells to conduct the in vitro micronucleus assay. Data from negative control samples from in vitro micronucleus assays using TK6 cells from 13 laboratories were collected using a standard collection form. Although in some cases statistically significant differences can be seen within laboratories for different test conditions, they were very small. The mean incidence of micronucleated cells/1000 cells ranged from 3.2/1000 to 13.8/1000. These almost four-fold differences in micronucleus levels cannot be explained by differences in scoring method, presence or absence of exogenous metabolic activation (S9), length of treatment, presence or absence of cytochalasin B or different solvents used as vehicles. The range of means from the four laboratories using flow cytometry methods (3.7-fold: 3.5-12.9 micronucleated cells/1000 cells) was similar to that from the nine laboratories using other scoring methods (4.3-fold: 3.2-13.8 micronucleated cells/1000 cells). No laboratory could be identified as an outlier or as showing unacceptably high variability. Quality Control (QC) methods applied to analyse the intra-laboratory variability showed that there was evidence of inter-experimental variability greater than would be expected by chance (i.e. over-dispersion). However, in general, this was low. This study demonstrates the value of QC methods in helping to analyse the reproducibility of results, building up a 'normal' range of values, and as an aid to identify variability within a laboratory in order to implement processes to maintain and improve uniformity.
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Núcleo Celular/genética , Proyectos de Investigación/normas , Línea Celular , Humanos , Micronúcleos con Defecto Cromosómico , Pruebas de Micronúcleos , Control de CalidadRESUMEN
Triplex forming oligonucleotides (TFOs) bind in the major groove of DNA duplex in a sequence-specific manner imparted by Hoogsteen hydrogen bonds. There have been several reports demonstrating the ability of guanine-rich TFOs to induce targeted mutagenesis on an exogenous plasmid or an endogenous chromosomal locus. In particular, a 30mer guanine-rich triplex forming oligonucleotide, AG30, optimally designed to target the supFG1 reporter gene was reported to be mutagenic in the absence of DNA reactive agents in cultured cells and in vivo Here, we investigated the mutagenic potential of AG30 using the supFG1 shuttle vector forward mutation assay under physiological conditions. We also assessed the triplex binding potential of AG30 alongside cytotoxic and mutagenic assessment. In a cell free condition, AG30 was able to bind its polypurine target site in the supFG1 gene in the absence of potassium chloride and also aligned with a 5-fold increase in the mutant frequency when AG30 was pre-incubated with the supFG1 plasmid in the absence of potassium prior to transfection into COS-7 cells. However, when we analyzed triplex formation of AG30 and the supFG1 target duplex at physiological potassium levels, triplex formation was inhibited due to the formation of competing secondary structures. Subsequent assessment of mutant frequency under physiological conditions, by pre-transfecting COS-7 cells with the supFG1 plasmid prior to AG30 treatment led to a very small increase (1.4-fold) in the mutant frequency. Transfection of cells with even higher concentrations of AG30 did result in an elevated mutagenic response but this was also seen with a scrambled sequence, and was therefore considered unlikely to be biologically relevant as an associated increase in cytotoxicity was also apparent. Our findings also provide further assurance on the low potential of triplex-mediated mutation as a consequence of unintentional genomic DNA binding by therapeutic antisense oligonucleotides.
Asunto(s)
Guanina/metabolismo , Mutágenos/farmacología , Oligonucleótidos/farmacología , Animales , Células COS , Chlorocebus aethiops , Vectores Genéticos , Mutación , Unión ProteicaRESUMEN
Nitroarenes are less preferred in drug discovery due to their potential to be mutagenic. However, several nitroarenes were shown to be promising antitubercular agents with specific modes of action, namely, nitroimidazoles and benzothiazinones. The nitro group in these compounds is activated through different mechanisms, both enzymatic and non-enzymatic, in mycobacteria prior to binding to the target of interest. From a whole-cell screening program, we identified a novel lead nitrobenzothiazole (BT) series that acts by inhibition of decaprenylphosphoryl-ß-d-ribose 2'-epimerase (DprE1) of Mycobacterium tuberculosis (Mtb). The lead was found to be mutagenic to start with. Our efforts to mitigate mutagenicity resulted in the identification of 6-methyl-7-nitro-5-(trifluoromethyl)-1,3-benzothiazoles (cBTs), a novel class of antitubercular agents that are non-mutagenic and exhibit an improved safety profile. The methyl group ortho to the nitro group decreases the electron affinity of the series, and is hence responsible for the non-mutagenic nature of these compounds. Additionally, the co-crystal structure of cBT in complex with Mtb DprE1 established the mode of binding. This investigation led to a new non-mutagenic antitubercular agent and demonstrates that the mutagenic nature of nitroarenes can be solved by modulation of stereoelectronic properties.