RÉSUMÉ
Bringing a new drug to the market costs an average of US$2.6 billion and takes more than 10 years from discovery to regulatory approval. Despite the need to reduce cost and time to increase productivity, pharma companies tend to crowd their efforts in the same indications and drug targets. This results in the commercialization of drugs that share the same mechanism of action (MoA) and, in many cases, equivalent efficacies among them-an outcome that helps neither patients nor the balance sheet of the companies trying to bring therapeutics to the same patient population. Indeed, the discovery of new therapeutic targets, based on a deeper understanding of the disease biology, would likely provide more innovative MoAs and potentially greater drug efficacies. It would also bring better chances for identifying appropriate treatments according to the patient's genetic stratification. Nowadays, we count with an enormous amount of unprocessed information on potential disease targets that could be extracted from omics data obtained from patient samples. In addition, hundreds of pharmacological and genetic screenings have been performed to identify innovative drug targets. Traditionally, rodents have been the animal models of choice to perform functional genomic studies. The high experimental cost, combined with the low throughput provided by those models, however, is a bottleneck for discovering and validating novel genetic disease associations. To overcome these limitations, we propose that zebrafish, in conjunction with the use of CRISPR/Cas9 genome-editing tools, could streamline functional genomic processes to bring biologically relevant knowledge on innovative disease targets in a shorter time frame.
Sujet(s)
Systèmes CRISPR-Cas/génétique , Découverte de médicament/tendances , Édition de gène/tendances , Tests de criblage à haut débit/tendances , Animaux , Humains , Danio zébré/génétiqueRÉSUMÉ
The xenograft of human cancer cells in model animals is a powerful tool for understanding tumor progression and metastatic potential. Mice represent a validated host, but their use is limited by the elevated experimental costs and low throughput. To overcome these restrictions, zebrafish larvae might represent a valuable alternative. Their small size and transparency allow the tracking of transplanted cells. Therefore, tumor growth and early steps of metastasis, which are difficult to evaluate in mice, can be addressed. In spite of its advantages, the use of this model has been hindered by lack of experimental homogeneity and validation. Considering these facts, the aim of our work was to standardize, automate, and validate a zebrafish larvae xenograft assay with increased translatability and higher drug screening throughput. The ZeOncoTest reliability is based on the optimization of different experimental parameters, such as cell labeling, injection site, automated individual sample image acquisition, and analysis. This workflow implementation finally allows a higher precision and experimental throughput increase, when compared to previous reports. The approach was validated with the breast cancer cell line MDA-MB-231, the colorectal cancer cells HCT116, and the prostate cancer cells PC3; and known drugs, respectively RKI-1447, Docetaxel, and Mitoxantrone. The results recapitulate growth and invasion for all tested tumor cells, along with expected efficacy of the compounds. Finally, the methodology has proven useful for understanding specific drugs mode of action. The insights gained bring a step further for zebrafish larvae xenografts to enter the regulated preclinical drug discovery path.
RÉSUMÉ
Cardiovascular drug toxicity is responsible for 17% of drug withdrawals in clinical phases, half of post-marketed drug withdrawals and remains an important adverse effect of several marketed drugs. Early assessment of drug-induced cardiovascular toxicity is mandatory and typically done in cellular systems and mammals. Current in vitro screening methods allow high-throughput but are biologically reductionist. The use of mammal models, which allow a better translatability for predicting clinical outputs, is low-throughput, highly expensive, and ethically controversial. Given the analogies between the human and the zebrafish cardiovascular systems, we propose the use of zebrafish larvae during early drug discovery phases as a balanced model between biological translatability and screening throughput for addressing potential liabilities. To this end, we have developed a high-throughput screening platform that enables fully automatized in vivo image acquisition and analysis to extract a plethora of relevant cardiovascular parameters: heart rate, arrhythmia, AV blockage, ejection fraction, and blood flow, among others. We have used this platform to address the predictive power of zebrafish larvae for detecting potential cardiovascular liabilities in humans. We tested a chemical library of 92 compounds with known clinical cardiotoxicity profiles. The cross-comparison with clinical data and data acquired from human induced pluripotent stem cell cardiomyocytes calcium imaging showed that zebrafish larvae allow a more reliable prediction of cardiotoxicity than cellular systems. Interestingly, our analysis with zebrafish yields similar predictive performance as previous validation meta-studies performed with dogs, the standard regulatory preclinical model for predicting cardiotoxic liabilities prior to clinical phases.
RÉSUMÉ
Sphingolipid imbalance is the culprit in a variety of neurological diseases, some affecting the myelin sheath. We have used whole-exome sequencing in patients with undetermined leukoencephalopathies to uncover the endoplasmic reticulum lipid desaturase DEGS1 as the causative gene in 19 patients from 13 unrelated families. Shared features among the cases include severe motor arrest, early nystagmus, dystonia, spasticity, and profound failure to thrive. MRI showed hypomyelination, thinning of the corpus callosum, and progressive thalamic and cerebellar atrophy, suggesting a critical role of DEGS1 in myelin development and maintenance. This enzyme converts dihydroceramide (DhCer) into ceramide (Cer) in the final step of the de novo biosynthesis pathway. We detected a marked increase of the substrate DhCer and DhCer/Cer ratios in patients' fibroblasts and muscle. Further, we used a knockdown approach for disease modeling in Danio rerio, followed by a preclinical test with the first-line treatment for multiple sclerosis, fingolimod (FTY720, Gilenya). The enzymatic inhibition of Cer synthase by fingolimod, 1 step prior to DEGS1 in the pathway, reduced the critical DhCer/Cer imbalance and the severe locomotor disability, increasing the number of myelinating oligodendrocytes in a zebrafish model. These proof-of-concept results pave the way to clinical translation.
Sujet(s)
Animal génétiquement modifié , Encéphale , Chlorhydrate de fingolimod/pharmacologie , Maladies démyélinisantes héréditaires du système nerveux central , Protéines de poisson-zèbre , Danio zébré , Animaux , Animal génétiquement modifié/génétique , Animal génétiquement modifié/métabolisme , Encéphale/enzymologie , Encéphale/anatomopathologie , Modèles animaux de maladie humaine , Fatty acid desaturases/génétique , Fatty acid desaturases/métabolisme , Maladies démyélinisantes héréditaires du système nerveux central/traitement médicamenteux , Maladies démyélinisantes héréditaires du système nerveux central/enzymologie , Maladies démyélinisantes héréditaires du système nerveux central/génétique , Maladies démyélinisantes héréditaires du système nerveux central/anatomopathologie , Humains , Locomotion/effets des médicaments et des substances chimiques , Oligodendroglie/enzymologie , Oligodendroglie/anatomopathologie , Danio zébré/génétique , Danio zébré/métabolisme , Protéines de poisson-zèbre/génétiqueRÉSUMÉ
The use of zebrafish larvae in basic and applied research has grown exponentially during the last 20 years. The reasons for this success lay in its specific experimental advantages: on the one hand, the small size, the large number of progeny and the fast life cycle greatly facilitate large-scale approaches while maintaining 3Rs amenability; on the other hand, high genetic and physiological homology with humans and ease of genetic manipulation make zebrafish larvae a highly robust model for understanding human disease. Together, these advantages allow using zebrafish larvae for performing high-throughput research, both in terms of chemical and genetic phenotypic screenings. Therefore, the zebrafish larva as an animal model is placed between more reductionist in vitro high-throughput screenings and informative but low-throughput preclinical assays using mammals. However, despite its biological advantages and growing translational validation, zebrafish remains scarcely used in current drug discovery pipelines. In a context in which the pharmaceutical industry is facing a productivity crisis in bringing new drugs to the market, the combined advantages of zebrafish and the CRISPR/Cas9 system, the most powerful technology for genomic editing to date, has the potential to become a valuable tool for streamlining the generation of models mimicking human disease, the validation of novel drug targets and the discovery of new therapeutics. This review will focus on the most recent advances on CRISPR/Cas9 implementation in zebrafish and all their potential uses in biomedical research and drug discovery.
RÉSUMÉ
Toxicity is one of the major attrition causes during the drug development process. In that line, cardio-, neuro-, and hepatotoxicities are among the main reasons behind the retirement of drugs in clinical phases and post market withdrawal. Zebrafish exploitation in high-throughput drug screening is becoming an important tool to assess the toxicity and efficacy of novel drugs. This animal model has, from early developmental stages, fully functional organs from a physiological point of view. Thus, drug-induced organ-toxicity can be detected in larval stages, allowing a high predictive power on possible human drug-induced liabilities. Hence, zebrafish can bridge the gap between preclinical in vitro safety assays and rodent models in a fast and cost-effective manner. ZeGlobalTox is an innovative assay that sequentially integrates in vivo cardio-, neuro-, and hepatotoxicity assessment in the same animal, thus impacting strongly in the 3Rs principles. It Reduces, by up to a third, the number of animals required to assess toxicity in those organs. It Refines the drug toxicity evaluation through novel physiological parameters. Finally, it might allow the Replacement of classical species, such as rodents and larger mammals, thanks to its high predictivity (Specificity: 89%, Sensitivity: 68% and Accuracy: 78%).