1,721,001 research outputs found
Towards the correction of genetic defect in corneal keratinocytes from patient with macular corneal Dystrophy type II
No full textTowards the correction of genetic defect in corneal keratinocytes from patient with macular corneal Dystrophy type I
Concise Review: Epigenetic Regulation of Hematopoiesis: Biological Insights and Therapeutic Applications
Get PDFHematopoiesis is the process of blood cell formation starting from hematopoietic stem/progenitor cells (HSPCs). The understanding of regulatory networks involved in hematopoiesis and their impact on gene expression is crucial to decipher the molecular mechanisms that control hematopoietic development in physiological and pathological conditions, and to develop novel therapeutic strategies. An increasing number of epigenetic studies aim at defining, on a genome-wide scale, the cis-regulatory sequences (e.g., promoters and enhancers) used by human HSPCs and their lineage-restricted progeny at different stages of development. In parallel, human genetic studies allowed the discovery of genetic variants mapping to cis-regulatory elements and associated with hematological phenotypes and diseases. Here, we summarize recent epigenetic and genetic studies in hematopoietic cells that give insights into human hematopoiesis and provide a knowledge basis for the development of novel therapeutic approaches. As an example, we discuss the therapeutic approaches targeting cis-regulatory regions to reactivate fetal hemoglobin for the treatment of Î2-hemoglobinopathies. Epigenetic studies allowed the definition of cis-regulatory sequences used by human hematopoietic cells. Promoters and enhancers are targeted by transcription factors and are characterized by specific histone modifications. Genetic variants mapping to cis-regulatory elements are often associated with hematological phenotypes and diseases. In some cases, these variants can alter the binding of transcription factors, thus changing the expression of the target genes. Targeting cis-regulatory sequences represents a promising therapeutic approach for many hematological diseases. Stem Cells Translational Medicine 2017;6:2106â2114
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
RECOMBINANT LENTIVIRAL VECTOR FOR STEM CELL-BASED GENE THERAPY OF SICKLE CELL DISORDER
No full textThis invention relates to recombinant lentiviral vectors, compositions thereof, the use of the vectors or the compositions thereof, kits of parts comprising said vectors or compositions thereof and a catalytically active Cas9 or Cpf1 protein, methods for modifying the genome of a hematopoietic stem/progenitor cell (HSPC), and the HSPC obtainable by such methods
VIRAL VECTOR COMBINING GENE THERAPY AND GENOME EDITING APPROACHES FOR GENE THERAPY OF GENETIC DISORDERS
No full textThis invention relates to recombinant viral vectors, preferably retroviral (RV), lentiviral (LV) or adeno-associated viral (AAV) vectors, compositions thereof, the use of the recombinant viral vectors or the compositions thereof, kits of parts comprising said recombinant viral vectors or compositions thereof and a catalytically active Cas9 or Cpfl protein, methods for modifying the genome of a cell, and the cells obtainable by such methods
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Approches d'édition du génome et de l'épigénome pour traiter les β-hémoglobinopathies
No full textDrépanocytose et bêta-thalassémie sont dues à des mutations affectant la production de la chaîne de globine β. La sévérité clinique est atténuée par des mutations augmentant la quantité d'hémoglobine fœtale (HbF), une condition appelée persistance héréditaire d'HbF à l'âge adulte. La transplantation autologue de cellules souches/progénitrices hématopoïétiques (CSPH) génétiquement corrigées est prometteuse. Une approche CRISPR/Cas9 pour réprimer BCL11A, un répresseur de la globine γ, a été approuvée mais comporte des risques de génotoxicité dues aux cassures double brin (CDB). Nous avons ciblé les sites de liaison (SL) des activateurs transcriptionnels GATA1 et ATF4, présents dans les régions +58-kb et +55-kb, respectivement, des enhancers érythroïdes de BCL11A, avec des éditeurs de base (BEs) pour introduire des mutations ponctuelles précises sans créer de CDBs. Des ARN guides (ARNg) ont été testés dans des cellules souches hématopoïétiques (CSH) de patients drépanocytaires en combinaison avec des BEs, générant différents nucléotides dans les motifs de liaison avec une efficacité d'édition atteignant 90 %, avec peu ou pas d'indels induits par les CDB. La réactivation d'HbF a été observée dans tous les échantillons édités, mesurée par HPLC, mais pas suffisante pour un sauvetage complet du phénotype dans les cellules érythroïdes issues des CSPHs drépanocytaires éditées. Nous avons donc ciblé simultanément les SL de GATA1 et ATF4 pour augmenter les niveaux d'HbF. Les cellules éditées au niveau des enhancers +58 et +55 ont montré une augmentation de l'expression d'HbF par rapport aux cellules recevant des ARNg individuels, dépassant les niveaux atteints avec la stratégie CRISPR/Cas9. Enfin, la réactivation de l'HbF était suffisante pour permettre un sauvetage substantiel du phénotype malade, diminuant le nombre de cellules drépanocytaires à 16,4%. Pour évaluer les effets hors cibles, nous avons utilisé le GUIDE-seq couplé au séquençage ciblé, le séquençage de l'exome entier et le RNA-seq, tandis que les effets indésirables présent dans les sites cibles ont été évalués par séquençage Long read. L'efficacité du BE pour repeupler les CSH a été démontrée en transplantant des CSH éditées dans des souris immunodéficientes, prouvant l'efficacité de l'édition simultanée des éléments transrégulateurs de la globine γ pour la réactivation de l'HbF. Cette preuve de concept permettra le développement préclinique et clinique de CSH modifiées pour le traitement des β-hémoglobinopathies. Cependant, des travaux récents montrent que les BEs peuvent également générer de larges délétions ou indels (Antoniou et al. 2022). Ainsi, une nouvelle stratégie basée sur l'édition de l'épigénome a été développée pour moduler l'expression des gènes sans modifier la séquence d'ADN sous-jacente. Nous avons analysé les marques épigénétiques dans deux régions cis-régulatrices clés, les promoteurs de HBG et les enhancers de BCL11A, dans des cellules érythroïdes dérivées de CSPH. Des marques épigénétiques répressives, telles que la méthylation de l'ADN, ont été détectées au niveau des promoteurs de HBG dans des cellules érythroïdes adultes n'exprimant pas la globine γ. En revanche, la déméthylation de l'ADN et les marques épigénétiques activatrices telles que l'acétylation de la lysine 27 de l'histone 3 (H3K27ac) et la triméthylation de la lysine 4 (H3K4me3) ont été détectées au niveau des promoteurs de HBG dans les cellules érythroïdes exprimant la globine γ. La forte expression de BCL11A dans les cellules érythroïdes adultes est associée à de faibles niveaux de méthylation de l'ADN au niveau des enhancers de BCL11A. L'inactivation des enhancers de BCL11A est associée à une augmentation de la méthylation de l'ADN. Ces données nous ont permis de concevoir des modificateurs de l'épigénome pour manipuler l'architecture épigénétique des promoteurs de HBG et des enhancers de BCL11A afin d'atteindre des niveaux thérapeutiques d'HbF.Bêta-thalassemia and sickle cell disease (SCD) result from mutations that affect the synthesis or structure of adult hemoglobin. Historically, allogeneic hematopoietic stem cell (HSC) transplantation from a compatible donor was the only curative treatment. Transplantation of autologous, genetically modified HSCs offers a promising therapeutic alternative for patients lacking a suitable donor. The clinical severity in β-hemoglobinopathies is mitigated by co-inheritance of hereditary persistence of fetal hemoglobin (HPFH), a benign condition characterized by mutations occurring in the genes encoding the fetal γ-globin chains, which lead to increased fetal hemoglobin (HbF, α2γ2) expression, which can rescue the β-thalassemic and SCD phenotypes. HbF reactivation can be achieved by down-regulating BCL11A, encoding a key repressor of HbF. A CRISPR/Cas9 strategy targeting the GATA1 binding site (BS) within the +58-kb erythroid-specific enhancer of BCL11A has recently been approved as the first gene-editing therapy for β-thalassemia and SCD. Indeed, the targeting of the BCL11A erythroid-specific enhancer led to an efficient reduction of BCL11A in the erythroid cells, without impacting the differentiation of HSPCs in the other cell lineages. However, site-specific nucleases induce double strand breaks (DSBs), posing significant risks, such apoptosis and generation of large genomic rearrangements. In addition, to obtain an adequate number of corrected cells to transplant, several collections of HSCs are necessary to compensate for the cell loss due to DSB-induced apoptosis. Finally, the clinical study showed variability in the extent of HbF reactivation, still high HbS levels and modest correction of ineffective erythropoiesis. Novel CRISPR/Cas9 derived tools are currently available and can be used to develop therapeutic strategies associated with a low risk of DSB generation and increased HbF expression. In this project, we intend to develop universal, safe and efficacious therapeutic strategies for β-hemoglobinopathies aimed at modifying HSCs using base editors (BEs) and epigenome editors to reactivate HbF expression in their erythroid progeny. BEs are a CRISPR-Cas9-based genome editing technology that allows the introduction of point mutations with little DSB generation. In this work we used this technology to inactivate the GATA1 or the ATF4 transcriptional activator BS in the +58-kb and +55-kb BCL11A erythroid-specific enhancers through the insertion of point mutations. In particular, to reach levels of HbF sufficient to rescue the sickling phenotype, we performed simultaneous targeting of the two BS, achieving similar HbF levels compared to CRISPR/Cas9 nuclease-based approach. Additionally, we showed that BEs generated fewer DSBs and genomic rearrangements compared to the CRISPR/Cas9 nuclease approach. In parallel, we developed a novel epigenome-editing strategy aimed at modulating gene expression without altering the DNA sequence (e.g. without generating DSBs). We designed two approaches to upregulate HbF expression: a first strategy targeting and activating the γ-globin promoters and a second approach downregulating BCL11A by targeting its erythroid-specific enhancers. We first identified the epigenetic marks in these trans- and cis-regulatory regions that are associated with active or inactive transcription in adult versus fetal erythroid cells. Then we used epigenome editors to deposit active histone modifications at the γ-globin promoters and remove inactive marks such as DNA methylation. In parallel, we decorated the BCL11A enhancers with inactive epigenetic marks. Preliminary results demonstrated γ-globin reactivation using both strategies, though the effects diminished over time, indicating the need for further optimization. In conclusion, we proposed two different editing approaches that allow to reduce DSB-associate issues as strategies to treat β-hemoglobinopathies
Approches d'édition de base pour le traitement des β-hémoglobinopathies
No full textLes beta-hémoglobinopathies sont dues à des mutations affectant la synthèse ou la structure de l'hémoglobine (Hb) adulte. La transplantation de cellules souches hématopoïétiques (CSH) autologues et génétiquement modifiées est une option thérapeutique intéressante pour les patients qui n'ont pas de donneur de CSH compatible. La gravité clinique des beta-hémoglobinopathies est atténuée par la présence de mutations provoquant l'expression de l'hémoglobine fœtale (HbF) dans la vie adulte - une condition appelée persistance héréditaire de l'HbF (HPFH). Pour réactiver l'expression de l'HbF, des approches d'édition du génome basées sur des nucléases spécifiques ont été explorées. Cependant, les nucléases spécifiques génèrent des cassures double brin (DSB) dans le génome et soulèvent des problèmes de sécurité pour les applications cliniques, en particulier lorsqu'elles sont utilisées dans des CSH sensibles aux DSB. L'édition de base est une technologie d'édition du génome basée sur CRISPR-Cas9 qui permet l'introduction de mutations ponctuelles (C>T par la cytidine désaminase ou CBE et A>G par l'adénine désaminase ou ABE) dans l'ADN sans générer de DSB. Les mutations HPFH dans les 2 promoteurs de la globine fœtale génèrent des sites de liaison pour les activateurs de l'HbF (par exemple KLF1, TAL1 et GATA1) ou perturbent les sites de liaison des répresseurs de l'HbF (par exemple LRF et BCL11A). De même, les SNP situés dans l'enhancer de BCL11A régulent négativement l'expression de BCL11A, un répresseur majeur de la synthèse de l'HbF. L'objectif de cette thèse de doctorat est d'utiliser l'édition de base pour cibler des séquences d'ADN spécifiques dans les promoteurs HBG et l'enhancer de BCL11A afin d'imiter l'effet des mutations HPFH.Beta-hemoglobinopathies are caused by mutations affecting the synthesis or the structure of the adult hemoglobin (Hb). Transplantation of autologous, genetically modified hematopoietic stem cells (HSCs) is an attractive therapeutic option for patients lacking a suitable HSC donor. The clinical severity of beta-hemoglobinopathies is alleviated by the co-inheritance of mutations causing fetal gamma-globin expression in adult life - a condition termed hereditary persistence of fetal Hb (HPFH). To reactivate gamma-globin expression, genome editing approaches based on site-specific nucleases have been explored. Site-specific nucleases, however, generate double-strand breaks (DSBs) in the genome and raise safety concerns for clinical applications, particularly when used in DSB-sensitive HSCs. Base editing (BE) is a CRISPR-Cas9-based genome editing technology that allows the introduction of point mutations (C>T by cytidine deaminase or CBE and A>G by adenine deaminase or ABE) in the DNA without generating DSBs. HPFH mutations in the 2 HBG gamma-globin promoters either generate binding sites (BS) for fetal Hb (HbF) activators (e.g. KLF1, TAL1 and GATA1) or disrupt the BS of HbF repressors (e.g. LRF and BCL11A). Similarly, SNPs located in the BCL11A enhancer downregulate the expression of BCL11A, a master repressor of HbF synthesis. The aim of this PhD thesis is to use BE and sgRNAs to target specific DNA sequences in the HBG promoters and the BCL11A enhancer and mimic the effect of HPFH mutations
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