The genetic consequences of dog breed formation-Accumulation of deleterious genetic variation and fixation of mutations associated with myxomatous mitral valve disease in cavalier King Charles spaniels

Selective breeding for desirable traits in strictly controlled populations has generated an extraordinary diversity in canine morphology and behaviour, but has also led to loss of genetic variation and random entrapment of disease alleles. As a consequence, specific diseases are now prevalent in certain breeds, but whether the recent breeding practice led to an overall increase in genetic load remains unclear. Here we generate whole genome sequencing (WGS) data from 20 dogs per breed from eight breeds and document a similar to 10% rise in the number of derived alleles per genome at evolutionarily conserved sites in the heavily bottlenecked cavalier King Charles spaniel breed (cKCs) relative to in most breeds studied here. Our finding represents the first clear indication of a relative increase in levels of deleterious genetic variation in a specific breed, arguing that recent breeding practices probably were associated with an accumulation of genetic load in dogs. We then use the WGS data to identify candidate risk alleles for the most common cause for veterinary care in cKCs-the heart disease myxomatous mitral valve disease (MMVD). We verify a potential link to MMVD for candidate variants near the heart specific NEBL gene in a dachshund population and show that two of the NEBL candidate variants have regulatory potential in heartderived cell lines and are associated with reduced NEBL isoform nebulette expression in papillary muscle (but not in mitral valve, nor in left ventricular wall). Alleles linked to reduced nebulette expression may hence predispose cKCs and other breeds to MMVD via loss of papillary muscle integrity

Improving the assessment of risk factors relevant to potential carcinogenicity of gene therapies: a consensus article

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

Etude des polymorphismes génétiques de différentes populations de cynomolgus (implication en toxicologie)

PARIS-BIUP (751062107) / SudocSudocFranceF

Identification et conséquences des polymorphismes chez différentes populations de cynomolgus (“Macaca fascicularis”) Implication pour les études de Toxicologie

Le Macaca fascicularis ou cynomolgus est une espèce de primates non humains très utilisée dans la recherche médicale, tout particulièrement pour le développement de médicaments issus des biotechnologies. Tout comme chez l’Homme, il existe des polymorphismes liés à l’origine géographique chez les individus chez cette espèce. Afin d’optimiser le choix des animaux utilisés pour les essais de recherche préclinique et d’augmenter la prédictibilité des effets du traitement chez l’Homme, une base de données intégrant des cynomolgus issus de plusieurs origines a été mise en place par le laboratoire Novartis. Cette base a été générée à partir d’un séquençage haut débit basé sur la capture d’exons suivie d’une reconstitution du génome par cartographie à partir de séquences de références. L’étude réalisée ici a permis, dans un premier temps, d’identifier un certain nombre de polymorphismes spécifiques de l’origine des Macaca fascicularis à partir de l’analyse de données bibliographiques. Situés sur l’ADN mitochondrial ainsi que sur le chromosome Y, ces polymorphismes vont permettre la mise en place d’un essai d’identification de l’origine paternelle et maternelle des animaux, permettant ainsi de s’assurer de la provenance des cynomolgus utilisés par les équipes de développement. Dans un second temps l’étude a permis de mettre en application les données contenues dans la base. En mettant son contenu en regard de données issues de la bibliographie ainsi qu’en l’utilisant afin d’étudier des cibles thérapeutiques, il a put être mis en évidence que son contenu nécessitait encore des étapes de vérification par un séquençage classique de Sanger. L’étude a également permis de montrer l’importance de l’étape de cartographie ainsi que l’utilité de cette base pour améliorer la prédictibilité des études précliniques

Translational Safety Genetics - Leveraging Genetic Variation for Enhanced Safety Assessment

The emerging field of translational safety genetics is providing new opportunities to enhance drug discovery and development. Genetic variation in therapeutic drug targets, off-target interactors, and relevant drug metabolism/disposition pathways can contribute to diverse drug pharmacologic and toxicologic responses between different animal species, strains and geographic origins. Recent advances in the sequencing of rodent, canine, non-human primate, and minipig genomes have dramatically improved the ability to select the most appropriate animal species for preclinical drug toxicity studies based on genotypic characterization of drug targets/pathways and drug metabolism and/or disposition, thus avoiding inconclusive or misleading animal studies, consistent with the principles of the 3Rs (Replacement, Reduction and Refinement). The genetic background of individual animals should also be taken into consideration when interpreting phenotypic outcomes from toxicity studies & susceptibilities to spontaneous safety-relevant background findings

An afucosylated anti-CD32b monoclonal antibody induced platelet-mediated adverse events in human Fcg receptor transgenic mouse model and its potential human translatability

A monoclonal, afucosylated, anti-CD32b (FCGR2B) antibody, NVS32b, was developed internally as a therapeutic candidate for treatment of B-cell malignancies. To assess its safety and tolerability, a humanized transgenic (Tg) mouse model that reportedly expresses all human Fc gamma receptors (FCGRs) while lacking all mouse FCGRs was used. Prior to its use, the model was extensively characterized and found to express all human FCGRs in a pattern similar to humans, with some deviations, such as low CD32 expression on T cells, substantial individual variation in the transgene copy number, integration of additional human genes, and overall higher expression of all FCGRs on myeloid cells compared to human. Unexpectedly, NVS32b induced severe thrombosis in huFCGR mice. The mechanism and relevance for human was further investigated. Species differences, in the NVS32b-driven in vitro platelet binding, activation and aggregation were observed (CD32a-binding by Fc and CDR of NVS32b causing platelet activation in huFCGR mice opposing to CD32a-binding by Fc and off-target-binding by CDR of NVS32b only after platelet activation in human). Therefore huFCGR mice may not be fully predictive of the risk of NVS32b-induced thromboembolic events in the clinic. Nevertheless the program was terminated as a result of this potential safety liability. This model could be considered beneficial in the pre-clinical research of immunotherapies targeting or involving FCGRs. While potential biological implications resulting from the differences in theFCGR expression pattern in humans cannot be predicted, these deviations should be considered and further evaluated when using this huFCGR mouse model

In vitro transformation assays for non-clinical safety assessment of CRISPR/Cas9 genome-edited cells

Off-target editing is one of the main safety concerns for the use of CRISPR/Cas9 genome editing in gene therapy. Although theoretically rare, these unwanted modifications could lead to malignant transformation, which renders tumorigenicity assessment of the cell therapy product indispensable. Here, we establish two in vitro assays, the soft agar colony forming assay (SACF) and growth in low attachment plates (GILA), as valid, quick and cost-efficient methods for tumorigenicity assessment of genome-edited cells. Using a CRISPR/Cas9 based approach to transform immortalized MCF10A cells, we identified PTPN12, a known tumor suppressor, as first true positive control in GILA and SACF. Next, we assessed the limit of detection for both assays and found that SACF is more sensitive than GILA (0.8% vs. 3.2% transformed cells). We further validated SACF and GILA by identifying a set of positive and negative controls. In contrast to SACF and GILA, an in vivo tumorigenicity study failed to detect the known tumorigenic potential of PTPN12-/- demonstrating the importance of including GILA and SACF in tumorigenicity testing. In conclusion, SACF and GILA are both attractive and valuable additions to non-clinical safety assessment of genome-edited cells

Retinoic-acid-orphan-receptor-C inhibition suppresses Th17 cells and induces thymic aberrations

Retinoic-acid-orphan-receptor-C (RORC) is a master regulator of Th17 cells, which are pathogenic in several autoimmune diseases. Genetic Rorc deficiency in mice, while preventing autoimmunity, causes early lethality due to metastatic thymic T cell lymphomas. We sought to determine whether pharmacological RORC inhibition could be an effective and safe therapy for autoimmune diseases by evaluating its effects on Th17 cell functions and intrathymic T cell development. RORC inhibitors effectively inhibited Th17 differentiation and IL-17A production, and delayed-type hypersensitivity reactions. In vitro, RORC inhibitors induced apoptosis, as well as Bcl2l1 and BCL2L1 mRNA downregulation, in mouse and nonhuman primate thymocytes, respectively. Chronic, 13-week RORC inhibitor treatment in rats caused progressive thymic alterations in all analyzed rats similar to those in Rorc-deficient mice prior to T cell lymphoma development. One rat developed thymic cortical hyperplasia with preneoplastic features, including increased mitosis and reduced IKAROS expression, albeit without skewed T cell clonality. In summary, pharmacological inhibition of RORC not only blocks Th17 cell development and related cytokine production, but also recapitulates thymic aberrations seen in Rorc-deficient mice. While RORC inhibition may offer an effective therapeutic principle for Th17-mediated diseases, T cell lymphoma with chronic therapy remains an apparent risk

Retinoic-acid-orphan-receptor C inhibition suppresses Th17 cells and induces thymic aberrations

Retinoic-acid-orphan-receptor-C (RORC) is a master regulator of Th17 cells which are pathogenic in several autoimmune diseases. Genetic Rorc deficiency in mice, while preventing autoimmunity, causes early lethality due to metastatic thymic T cell lymphomas. We sought to determine whether pharmacological RORC inhibition could be an effective and safe therapy for autoimmune diseases by evaluating its effects on Th17 cell functions and intrathymic T cell development. RORC inhibitors effectively inhibited Th17 differentiation and IL-17A production, and delayed-type hypersensitivity reactions. In vitro, RORC inhibitors induced apoptosis, as well as Bcl2l1 and BCL2L1 mRNA downregulation, in mouse and nonhuman primate thymocytes, respectively. Chronic, 13-week RORC inhibitor treatment in rats caused progressive thymic alterations in all analyzed rats similar to those in Rorc-deficient mice prior to T cell lymphoma development. One rat developed thymic cortical hyperplasia with preneoplastic features, including increased mitosis and reduced IKAROS expression, albeit without skewed T cell clonality. In summary, pharmacological inhibition of RORC not only blocks Th17 cell development and related cytokine production, but also recapitulates thymic aberrations seen in Rorc-deficient mice. While RORC inhibition may offer an effective therapeutic principle for Th17-mediated diseases, T cell lymphoma with chronic therapy remains an apparent risk

Association of a genetic variant in the ALOX5AP with higher risk of ischemic stroke : a case-control, meta-analysis and functional study

Copyright © 2010 S. Karger AGBackground: Variants in the 5-lipoxygenase-activating protein (ALOX5AP) and phosphodiesterase 4D (PDE4D) genes have first been associated with ischemic stroke (IS) through whole-genome linkage screens. However, association studies obtained conflicting results. We aimed to investigate the contribution of selected single nucleotide polymorphisms (SNPs) in these genes for the first time in a large Iberian population. Methods: A case-control design was used to analyze one SNP in ALOX5AP and five SNPs in PDE4D in a total of 1,092 IS patients and 781 healthy controls of two different subsets from Spain and Portugal. The analysis was adjusted for confounding variables and the results were integrated in a meta-analysis of all case-control studies. In addition, ALOX5AP gene expression levels were determined in controls and IS cases. Results: A first meta-analysis of both subsets showed that the T allele of the SG13S114 SNP in ALOX5AP was a risk factor for IS after Bonferroni correction [OR = 1.22 (1.06–1.40); p = 0.006]. A second meta-analysis of white populations confirmed these results [OR = 1.18 (1.07–1.31); p = 0.001]. ALOX5AP gene expression analysis in a subset of controls and cases revealed that the SG13S114 genotypes modulate mRNA levels of ALOX5AP (p = 0.001) and mRNA levels were higher in IS cases (2.8 ± 2.4%) than in controls (1.4 ± 1.3%; p = 0.003). No association of the variants in PDE4D with IS was observed in our study. Conclusions: The ALOX5AP SG13S114 variant is an independent risk factor for IS in the Iberian population and is associated with ALOX5AP expression levels. The role of this gene in stroke merits further investigation.This study was funded by a grant from the Spanish government (Geno-tPA project FIS PJ060586), the stroke research network (RENEVAS) and the Ramón Areces Foundation. S.D.-M. is the recipient of a grant from the Ramón Areces Foundation, I.F.-C. a postdoctoral grant from the Vall d’Hebron Research Institute (IRVH), A.d.R.-E. received a grant from the Vall d’Hebron Research Institute (IRVH), M.M. received a grant for Formation in Investigation (FI05/00081), and J.F.-M. was a recipient of a grant from La Marató (Chromig Exp. 072310). This work was also supported in part by the Portuguese Fundação para a Ciência e a Tecnologia (FCT) grant PTDC/SAU-GMG/ 64426/2006, FCT fellowships (TK, HM), and the Fundação AstraZeneca/Faculdade de Medicina de Lisboa research fellowship (LG). The Neurovascular Research Laboratory takes part in the International Stroke Genetics Consortium ISGC and in the RENEVAS network