Les furocoumarines sont des composés phénoliques impliqués dans la défense contre les herbivores. Ces molécules sont majoritairement décrites dans quatre familles botaniques, notamment les Rutaceae, dont font partie les agrumes. Ces molécules sont phototoxiques ce qui peut poser des problèmes pour leur utilisation comme par exemple en cosmétique ou en phytothérapie. D’autre part, en cas d’ingestion par exemple via la consommation de jus de certains agrumes, elles ont responsables de l’inhibition d’enzymes de détoxication comme le CYP3A4 humain. Cela peut conduire à des surdosages médicamenteux connus sous le nom d’Effet Pomelo. Ce travail de thèse a consisté à réfléchir et à développer, des outils qui permettront de générer de manière ciblée des variétés de pomelo qui ne produisent plus de furocoumarines. Nous avons abordé l’ensemble des étapes essentielles pour la mise en place d’une stratégie global : i) des méthodes reproductibles ont été développées pour la production de protoplastes et de cultures cellulaires de pomelo Star Ruby ; ii) des conditions de transformation de protoplastes par électroporation ont également été mises au point ; iii) finalement, pour inhiber de manière spécifique la voie de biosynthèse des furocoumarines, nous avons choisi de mettre en œuvre une approche d’édition de génome en utilisant une méthodologie CRISPR/Cas9. La mise au point de la méthode a été réalisée avec un gène codant pour une umbelliferone 6-dimethylallyl transférase. Les résultats obtenus indiquent que la stratégie est envisageable. Pour renforcer la stratégie CRISPR/Cas9, nous avons mis en œuvre une démarche d’identification de gènes cibles additionnels. En utilisant une approche de data mining de bases de données génomiques et transcriptomiques nous avons identifié 18 séquences candidates, potentiellement impliquées dans la voie de biosynthèse des furocoumarines. L’expression hétérologue des protéines correspondantes et leur caractérisation fonctionnelle a permis de montrer que CYP706J12 est en mesure de métaboliser l’hérniarine, une coumarine. Ce résultat apporte des éléments pour émettre des hypothèses sur l’évolution convergente de la synthèse des coumarines et des furocoumarines chez les végétaux supérieurs. / Furanocoumarins are phenolic compounds involved in defense against herbivores. These molecules are mainly described in four botanical families. Rutaceae, one of those families, includes Citrus species. Furanocoumarins are phototoxic compounds, which can be problematic for their use in cosmetics or in phytotherapy. Furanocoumarin ingestion via citrus juice consumption, may inhibit human enzymes of detoxification, such as human CYP3A4. This can lead to drug overdoses known as the “Grapefruit Juice Effect”. This work consisted in the development of tools that will allow to generate new varieties of pomelo that no longer produce furanocoumarins by targeted genome edition. We have covered the essential steps for the implementation of a global strategy: i) reproducible methods have been developed for the production of protoplasts and cell cultures of Star Ruby grapefruit; ii) conditions for protoplast transformation by electroporation have also been developed; iii) finally, to specifically inhibit the furanocoumarin biosynthetic pathway, we chose to implement a genome editing approach using a CRISPR / Cas9 methodology. The development of the method was carried out with a gene encoding umbelliferon 6-dimethylallyltransferase. The results obtained indicate that the strategy is feasible. To strengthen the CRISPR / Cas9 strategy, we implemented a method to identify additional target genes. Using a data mining approach of available genomic and transcriptomic databases we identified 18 candidate sequences potentially involved in the furanocoumarin biosynthetic pathway. Heterologous expression of the corresponding proteins and their functional characterization made it possible to show that CYP706J12 is able to metabolize herniarin (a coumarin). This result provides elements to hypothesize about the convergent evolution of coumarin and furanocoumarin synthesis in higher plants.

RUNX1 est un facteur de transcription essentiel pour l’hématopoïèse et joue un rôle important dans la fonction immunitaire. Des mutations surviennent dans ce gène chez 5 à 13% des patients atteints de leucémie myéloïde aiguë (LMA) (RUNX1mut) et définissent un sous-groupe particulier de LMA associé à un pronostic défavorable. En conséquence, il est nécessaire de procéder à une meilleure caractérisation génétique et de concevoir des stratégies thérapeutiques plus efficaces pour ce sousgroupe particulier de LMA. Bien que la plupart des mutations trouvées dans le gène RUNX1 dans la LMA soient supposément acquises, des mutations germinales dans RUNX1 sont observées chez les patients atteints du syndrome plaquettaire familial avec prédisposition aux hémopathies malignes (RUNX1-FPD, FPD/AML). En outre, 44 % des individus atteints évoluent vers le développement d’une LMA. Suite au séquençage du transcriptome (RNA-Seq) d’échantillons de la cohorte Leucégène, nous avons montré que le dosage allélique de RUNX1 influence l’association avec des mutations coopérantes, le profil d’expression génique et la sensibilité aux médicaments dans les échantillons primaires de LMA RUNX1mut. Aussi, la validation des mutations trouvées chez RUNX1 a mené à la découverte que 30% des mutations identifiées dans notre cohorte de LMA étaient d’origine germinale, révélant une proportion plus élevée qu’attendue de cas de mutations RUNX1 familiales. Un crible chimique a, quant à lui, révélé que la plupart des échantillons RUNX1mut sont sensibles aux glucocorticoïdes (GCs) et nous avons confirmé que les GCs inhibent la prolifération des cellules de LMA et ce, via l’interaction avec le récepteur des glucocorticoïdes (Glucocorticoid Receptor, GR). De plus, nous avons observé que les échantillons contenant des mutations RUNX1 censées entraîner une faible activité résiduelle étaient plus sensibles aux GCs. Nous avons aussi observé que la co-association de certaines mutations, SRSF2mut par exemple, et les niveaux de GR contribuaient à la sensibilité aux GCs. Suite à cela, la sensibilité acquise aux GCs a été obtenue en régulant négativement l’expression de RUNX1 dans des cellules LMA humaines, ce qui a été accompagné par une régulation positive de GR. L’analyse de transcriptome induit par GC a révélé que la différenciation des cellules de LMA induite par GCs pourrait être un mécanisme en jeu dans la réponse antiproliférative associée à ces médicaments. Plus important encore, un criblage génomique fonctionnel a identifié le répresseur transcriptionnel PLZF (ZBTB16) comme un modulateur spécifique de la réponse aux GCs dans les cellules LMA sensibles et résistantes. Ces observations fournissent une caractérisation supplémentaire de la LMA RUNX1mut, soulignant l’importance de procéder à des tests germinaux pour les patients porteurs de mutations RUNX1 délétères. Nos résultats ont également identifié un nouveau rôle pour RUNX1 dans le réseau de signalisation de GR et montrent l’importance d’investiguer le repositionnement des GCs pour traiter la LMA RUNX1mut dans des modèles précliniques. Enfin, nous avons fourni des indications sur le mécanisme d’action des GCs, en montrant que PLZF s’avère un facteur important favorisant la résistance aux GCs dans la LMA. / RUNX1 is an essential transcription factor for definite hematopoiesis and plays important roles in immune function. Mutations in RUNX1 occur in 5-13% of Acute Myeloid Leukemia (AML) patients (RUNX1mut ) and are associated with adverse outcome, thus highlighting the need for better genetic characterization and for the design of efficient therapeutic strategies for this particular AML subgroup. Although most RUNX1 mutations in AML are believed to be acquired, germline RUNX1 mutations are observed in the familial platelet disorder with predisposition to hematologic malignancies (RUNX1-FPD, FPD/AML) in which about 44% of affected individuals progress to AML. By performing RNA-sequencing of the Leucegene collection, we revealed that RUNX1 allele dosage influences the association with cooperating mutations, gene expression profile, and drug sensitivity in RUNX1mut primary AML specimens. Validation of RUNX1 mutations led to the discovery that 30% of RUNX1 mutations in our AML cohort are of germline origin, indicating a greater than expected proportion of cases with familial RUNX1 mutations. Chemical screening showed that most RUNX1mut specimens are sensitive to glucocorticoids (GC) and we confirmed that GCs inhibit AML cell proliferation via interaction with the Glucocorticoid Receptor (GR). We observed that specimens harboring RUNX1 mutations expected to result in low residual RUNX1 activity were most sensitive to GCs, and that co-associating mutations, such as SRSF2mut, as well as GR levels contribute to GC-sensitivity. Accordingly, acquired GC-sensitivity was achieved by negatively regulating RUNX1 expression in human AML cells, which was accompanied by upregulation of the GR. GC-induced transcriptome analysis revealed that GC-induced differentiation of AML cells might be a mechanism at play in the antiproliferative response to these drugs. Most critically, functional genomic screening identified the transcriptional repressor PLZF (ZBTB16) as a specific modulator of the GC response in sensitive and resistant AML cells. These findings provide additional characterization of RUNX1mut AML, further stressing the importance of germline testing for patients carrying deleterious RUNX1 mutations. Our results also identified a novel role for RUNX1 in the GR signaling network and support the rationale of investigating GC repurposing for RUNX1mut AML in preclinical models. Finally, we provided insights into the mechanism of action of GCs, which positions PLZF as an important factor promoting resistance to glucocorticoids in AML.

Die physische Nähe zwischen Genpromotoren und regulatorischen Elementen (Enhancer) spielt eine entscheidene Rolle in der Genexpression, um präzise räumliche und zeitliche Genexpressionmuster während der Embryogenese zu erzeugen. Abhängig von der Aktivität der Zielgene lassen sich zwei Typen von Interaktionen unterscheiden. Zum einen führen dynamische Enhancer-Promoter Interaktionen unmittelbar zur Genexpression, wohingegen in anderen Fällen stabile Interaktionen bereits vor der Genexpression existieren. In der vorliegenden Studie wurde die Rolle der stabilen Interaktion zwischen dem Shh Gen und dem Extremitätenenhancer, der ZRS, während der Embryonalentwicklung in der Maus untersucht. Der Verlust der konstitutiven Transkription, die den ZRS Enhancer abdeckt, führte zu einer Verschiebung innerhalb der Shh-ZRS Kontakte und einer moderaten Reduzierung der Shh Genexpression. Im Gegensatz dazu führte die Mutation von CTCF Bindungsstellen, die den ZRS Enhancer umgeben, zu einem Verlust der stabilen Shh-ZRS Interaktion und einem 50%igen Rückgang in der Shh Genexpression. Dieser Expressionsverlust hatte jedoch keine phänotypischen Auswirkungen in den Deletionsmutanten, was darauf hindeutet, dass die restliche Genaktivität und Enhancer-Promotor-Interaktion über einen zusätzlichen, CTCF-unabhängigen Mechanismus erfolgt. Erst die kombinierte Deletion von CTCF-Bindungsmotiven und einem hypomorphen ZRS-Allel führte zu einem fast vollständigen Expressionsverlust von Shh und damit zu einem schweren Funktionsverlust und Gliedmaßen-Agenesie. Die hier präsentierten Ergebnisse zeigen, dass die stabile Chromatinstruktur am Shh Locus von mehreren Komponenten getragen wird und die physicalische Interaktion zwischen Enhancern und Promotern für eine robuste Transkription während der Embryonalentwicklung benötigt werden. / Long-range gene regulation involves physical proximity between enhancers and promoters to generate precise patterns of gene expression in space and time. However, in some cases proximity coincides with gene activation, whereas in others preformed topologies already exist before activation. In this study, we investigate the preformed configuration underlying the regulation of the Shh gene by its unique limb enhancer, the ZRS, in vivo during mouse development. Abrogating the constitutive transcription covering the ZRS region led to a shift within the Shh-ZRS contacts and a moderate reduction in Shh transcription. Deletion of the CTCF binding sites around the ZRS resulted in a loss of the Shh-ZRS preformed interaction and a 50% decrease in Shh expression but no phenotype, suggesting an additional, CTCF-independent mechanism of promoter-enhancer communication. This residual activity, however, was diminished by combining the loss of CTCF binding with a hypomorphic ZRS allele resulting in severe Shh loss-of-function and digit agenesis. Our results indicate that the preformed chromatin structure of the Shh locus is sustained by multiple components and acts to reinforce enhancer-promoter communication for robust transcription.

Knowledge on the human genome and specific sequences associated with human diseases is continuously growing. The ability to connect human genetics to cellular mechanisms and physiology raises the need for medicine to get to gene specific therapeutics. In order to achieve gene-specific modification, tools are required to enable sequence-specific DNA cleavage. Not long ago, the RNA-guided endonuclease Cas9 was shown to effectively facilitate gene editing in humans. Cas9 endonuclease, which is naturally part of an adaptable bacterial immune system, can be easily adjusted to recognize and cleave specific DNA sequences in a 20 nt RNA-DNA complementary manner. The easy adjustability and high efficiency of Cas9 gave rise to hopes that this genome engineering tool could pave the way to ‘gene surgery’ in humans. However, to achieve DNA cleavage, the endonuclease and its guiding RNA need to be sufficiently accessible in the nucleus of target cells. Viruses, which evolution has made well adapted to transfer their own genetic information into cells can be exploited for transfer of foreign genetic material. Replication deficient retroviruses therefore represent interesting vehicles for gene delivery. Retroviruses preferentially incorporate their own genetic information in the form of RNA into viral particles. Typically, viral RNA of retroviruses is reverse transcribed into DNA during viral infection and integrated into host cell chromosomes. In this respect, integration-competent or integration-deficient lentiviral (HIV-derived) vectors (ICLV/IDLV) were reported to be efficient ‘gene shuttles’ for Cas9 delivery. In contrast, up to now Foamy viruses (FV), which represent a distinct subfamily in the family of retroviruses have not previously been tested for their efficiency to transduce CRISPR/Cas9 components. FV show several unique characteristics some of which make them interesting candidates for gene therapy, such as high transduction efficiency on a wide variety of human cell lines or a special capability to efficiently transfer and provide non-viral RNA in target cells. In this thesis the unique characteristic of FVs, which allow for the efficient transduction of non-viral RNAs, was exploited for transient FV mediated (TraFo) Cas9 expression. It is shown in this thesis that gene knock-out (KO) achieved with TraFo Cas9 particles appears to have several advantages over ICLV or IDLV mediated Cas9 transduction. In this work, it could be demonstrated that a single application of TraFo Cas9 supernatant results in high efficiency of GFP KO in osteosarcoma cells (U2OS). The efficiency of gene KO with TraFo Cas9 particles exceeded gene KO frequencies achieved with similar volumes of ICLV or IDLV supernatant for Cas9 transduction. In addition, transient Cas9 delivery achieved with TraFo particle supernatant resulted in remarkably reduced Cas9 off-target cleavage compared to corresponding infections with ICLV or IDLV particles. The results show, that TraFo Cas9 represents an interesting addition to the currently utilized methods for transient Cas9 delivery. One particular feature of TraFo particle transduction is especially noteworthy – TraFo mediated transduction does not depend on any particular adjustment on the encapsidated non-viral RNA sequence (such RNA only needs to be present in sufficient amounts during virus assembly) nor does it depend on any modification of viral proteins. The easy adaptability of TraFo mediated non-viral RNA transfer is an especially remarkable feature, since science continues to both developing new variants of Cas9 and continues to find new and interesting members of the pool of CRISPR enzymes. In this regard TraFo particles represent interesting vehicles to transiently provide mRNA transcripts of such new protein candidates in cells. The ability of TraFo particles to provide the RNA sequence needed to guide Cas9 (termed sgRNA) to its target DNA sequence in cells was additionally investigated. It was assumed that typically engaged RNA polymerase (RNAP) III transcription of sgRNAs hampers transduction with TraFo particles, since RNAP III-derived transcripts are not actively exported into the cytoplasm and show low stability. An additional CRISPR enzyme Csy4 was used, which is able to specifically cleave RNA. This enabled TraFo mediated transfer of RNAP II transcripts (with active nuclear export and higher stability than RNAP III transcripts) with embedded sgRNA sequences. It was demonstrated that a simultaneous infection of cells with TraFo particles providing bicistronic transcripts of Cas9 and Csy4 on the one side and RNAP II-derived transcripts with embedded sgRNA sequences on the other, enabled reasonable GFP gene inactivation in U2OS cells. Gene KO with RNAP II transcripts as a result significantly exceeds TraFo transduction of RNAP III-derived sgRNA. Interestingly, with regard to gene KO, it was found that de novo transcription of sgRNAs from viral DNA (by integration-competent or integration-deficient retroviral vector [ICRV/IDRV] transduction) when combined with TraFo Cas9 transduction was superior to a TraFo transduction of sgRNA transcripts. IDRV mediated transduction was optimized in order to minimize the risk of unfavorable genome modification of cells by viral DNA integration. By adding the coding sequence of a fluorescent marker to the viral vector, it was demonstrated that a smaller number of viral particles helps to significantly lower the frequency of viral DNA integration. In addition, the expression of a fluorescent marker opened up the opportunity to further reduce the cell fraction with continuous marker gene expression by flow cytometric cell sorting. The IDRV/ICRV sgRNA and TraFo Cas9 delivery system was then challenged for use on immortalized and primary T cells. Primary T cells represent interesting targets for genetic engineering since modified T cells can be utilized as ‘living drugs’ (by expression of chimeric antigen receptors – CARs) against cancer cells. Efficient gene inactivation was observed on the immortalized T cell line – Jurkat. Transduction of primary T cells pointed to certain restrictions of the split two-virus delivery system for sgRNA and Cas9 transduction. However, despite certain limitations, it was possible to demonstrate that this FV-derived Cas9 delivery system is also feasible on primary tissue, and further optimization could make it an interesting alternative delivery method for CAR therapy. The ability of IDRV vector genomes to provide repair template donor DNA to induce homologous recombination (HR) was additionally investigated. DNA double-strand breaks in eukaryotic cells are typically repaired by the error prone non-homologous end joining pathway (often leading to frame-shift mutations by small insertions or deletions) or HR. Delivery of a homologous DNA sequence during DNA cleavage enables site-specific integration of exogenous DNA sequences. The work of this thesis showed that IDRV vector genomes providing repair template donor DNA allow for HR in a homology length dependent manner. Besides the length of homology, it was also observed, that the length of sequence which should be integrated (KI) remarkably influences the frequency of HR. HR is therefore engaged significantly more frequently if single nucleotides, rather than a whole gene, are provided as sequences within a repair template. In addition, viral vectors were augmented with additional fluorescent marker sequences. It could subsequently be demonstrated that the majority of cells showed accurate sequence-specific DNA integration. Furthermore, several indications were found, which lead to the assumption that the ratio of KI to homologous sequence markedly influences the accuracy of HR. Using the previously obtained knowledge it was further possible to tag an essential human protein by FV vector mediated transient Cas9 and repair template transduction. It was found that the large packaging capacity of FV vectors can be exploited to enable selection and flow cytometric sorting of cells with correct site-specific DNA integration. In summary, the results of this thesis demonstrate for the first time that FV mediated non-viral mRNA Cas9 transduction in combination with retroviral delivery of sgRNA (and repair template sequence) are a promising basis for several different interesting applications with relevance for not only basic research, but also for gene therapy.:1. Introduction 1 1.1 Gene therapy 1 1.2 Viral vectors for gene therapy 2 1.3 History of retroviral research 2 1.4 Taxonomy of Retroviruses 3 1.5 Foamy Viruses 4 1.5.1 Morphology of Foamy Virus 6 1.5.2 Foamy Virus replication 7 1.5.3 Foamy virus proteins, as part of a viral vector system 10 1.6 Genetic engineering 14 1.6.1 ‘DNA scissors’ – Zinc-finger and Transcription-activator like effector nucleases 15 1.6.2 History of CRISPR/Cas9 as a tool for genetic engineering 16 1.7 CRISPR/Cas immunity in prokaryotes 18 1.8 CRISPR/Cas9 functioning 21 1.9 Double-strand break repair in eukaryotic cells 21 1.9.1 Classical NHEJ 23 1.9.2 Homologous recombination 24 1.9.3 DSB repair in vertebrates 26 1.10 DSBs in context of CRISPR/Cas9 cleavage 27 1.11 Thesis Aim: CRISPR/Cas9 transduction with FV particles 28 2. Materials and Methods 30 2.1 Materials 30 2.1.1 Chemicals 30 2.1.2 Buffers and Solutions 30 2.1.3 Bacterial Growth Media 33 2.1.4 Cell Culture Media 34 2.1.5 Antibodies 34 2.1.6 Enzymes 35 2.1.7 Commercial Kits and additional reagents 36 2.1.8 Size Markers 36 2.1.9 Antibiotics 36 2.1.10 Bacterial strains 37 2.1.11 Cell lines 37 2.1.12 Devices and Software 37 2.1.13 Oligonucleotides 38 2.1.14 Plasmids 46 2.1.15 sgRNA sequences 56 2.1.16 Consumable material 57 2.2 Molecular Biology Methods 58 2.2.1 Restriction of DNA 58 2.2.2 Polymerase chain reaction 59 2.2.3 Gibson assembly 60 2.2.4 Agarose gel electrophoresis 60 2.2.5 Ligation 61 2.2.6 Cultivation of bacteria 62 2.2.7 Transformation 62 2.2.8 Plasmid Preparation 63 2.2.9 Sequencing 65 2.3 Cell culture methods 66 2.3.1 Passaging of cells 66 2.3.2 Cell counting 66 2.3.3 Freezing and thawing of cells 66 2.3.4 Seeding and fixation of cells for microscopy 67 2.4 Virological Methods 67 2.4.1 Polyethyleneimine transfection 67 2.4.2 Integration-competent, integration-deficient and ‘transient’ retroviral vectors 68 2.4.3 Infection of adherent cells 70 2.4.4 Infection of suspension cells 71 2.4.5 Flow cytometry 72 2.4.6 Multiplicity of infection (MOI) 72 2.4.7 Particle preparation 73 2.5 Nucleic acid composition in viral particles and culture cells 73 2.5.1 Isolation of total RNA from viral particles 73 2.5.2 RNA isolation from culture cells 73 2.5.3 Reverse transcription of viral or cellular RNA 73 2.5.4 DNA isolation from culture cells 74 2.5.5 Quantitative PCR (qPCR) analysis 74 2.5.6 T7 endonuclease assay 75 2.6 Protein biochemistry methods 76 2.6.1 Cell lysates 76 2.6.2 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 76 2.6.3 Semi-dry Western Blot 77 2.6.4 Immunodetection 78 2.6.5 Stripping of Western blot membranes 78 2.6.6 Immunostaining of cells for FACS analysis 78 2.7 Microscopy methods 79 2.7.1 Fluorescence microscopy 79 2.7.2 Confocal Laser scanning Microscopy (CLSM) 79 2.7.3 Live-cell imaging 79 3. Results 80 3.1 Transient foamy virus transduction of non-viral mRNA transcripts 80 3.2 Transient foamy virus transduction of Cas9-encoding mRNA transcripts 81 3.3 Cas9-encoding nucleic acids and their ‘effects’ in cells after retroviral transduction 84 3.4 Off-target analysis after TraFo Cas9 delivery 87 3.5 Transient fomy virus transduction of Cas9 and sgRNAs 89 3.6 Retroviral vectors providing sgRNAs and a fluorescent protein 92 3.6.1 Gene knock-out with retroviral vectors under saturated conditions 92 3.6.2 MOI adjusted ID sgRNA vector supernatants for comparison of residual vector integration 94 3.6.3 Gene knock-out in murine embryonic fibroblasts 95 3.7 Influence of Cas9 expression on IDRV vector genome integration 96 3.8 TraFo Cas9 mediated T cell receptor knock-out in immortalized and primary human T cells 97 3.9 Homology-directed repair after FV CRISPR/Cas9 mediated double-strand breaks 99 3.9.1 Length of homologous donor DNA and its influence on HDR 100 3.9.2 Effect of freezing viral supernatants on the frequency of HDR 102 3.9.3 Effect of donor DNA mismatches on the frequency of HDR 104 3.10 Investigation on donor DNA integration with additional fluorescent markers 105 3.11 Lentiviral and foamyviral transduction of HDR donor DNA 107 3.12 HDR mediated single nucleotide substitutions after TraFo CRISPR/Cas9 delivery 109 3.13 Tagging of an endogenous protein after TraFo CRISPR/Cas9 delivery 111 3.13.1 Specific CRISPR/Cas9 mediated cleavage of endogenous hPLK1 gene 111 3.13.2 Homology-directed repair of the hPLK1 gene for endogenous gene tagging 113 3.13.3 Confocal fluorescence microscopy analysis of GFP-Plk1 HeLa cell populations 118 4. Discussion 120 4.1 Genetic engineering – potential and risks 120 Chapter I Transient FV vectors – mRNA delivery vehicles for CRISPR/Cas9 mediated gene editing 122 4.2 Non-viral Cas9-encoding mRNA transfer in foamy virus particles 122 4.2.1 Fate of Cas9-encoding nucleic acids in cells after TraFo Cas9 transduction 124 4.2.2 Potential adjustments to further improve TraFo Cas9 transduction 125 4.2.3 Lentiviral in contrast to TraFo transduction of Cas9-encoding nucleic acids 126 4.3 Efficiency of Cas9-mediated gene knock-out with different retroviral vectors 127 4.4 Type of retroviral Cas transduction and its influence on the specificity of Cas9 cleavage 127 4.5 Alternative approaches to deliver Cas9-encoding mRNA in human cells 129 4.6 Transient sgRNA transduction with TraFo particles 131 Chapter II Delivery of foreign DNA with FV-derived vectors – enabling gene knock-out and homology-directed repair 133 4.7 Gene inactivation by TraFo Cas9 transduction and sgRNA expression from retroviral vector genomes 133 4.7.1 Gene editing in immortalized and primary T cells 135 4.8 Homology-directed repair with IDRV genomes 137 4.8.1 The influence of the length of sequence homology on HR 138 4.8.2 The influence of freezing viral supernatants on HR 139 4.8.3 Widening the applicability of TraFo vector particles for improved HR 140 4.8.4 The influence of mismatching nucleotides on HR 140 4.8.5 Visualization of inaccurate HR or additional dsDNA integration 141 4.8.6 The influence of the ratio of knock-in and homologous sequence on the accuracy of HR 142 4.8.7 Alternatives to double-stranded donor DNA 143 4.9 Endogenous gene tagging with IDPV donor DNA transduction 145 4.9.1 Alternative approaches for endogenous protein tagging 146 5. Conclusion 148 6. Summary 150 6.1 Summary 150 6.2 Zusammenfassung 153 7. Supplementary 157 8. References 159 9. Appendices 182 9.1 Indices 182 9.1.1 Abbreviations 182 9.1.2 Index of Figures 185 9.1.3 Index of Tables 187 9.2 Curriculum Vitae 188 9.3 Publication Record 189 9.4 Congress Contributions 189 9.5 Patent Applications 189 10. Statement of Authorship 190

L’anémie falciforme (AF) est une maladie monogénique dans laquelle la polymérisation d’une hémoglobine anormale (hémoglobine S, Hb S) entraine la déformation et la destruction du globule rouge. L’AF est associée à de nombreuses complications cliniques avec une très grande hétérogénéité dans son expression. En dehors de l’impact important de l’hémoglobine fœtale (Hb F), cette variabilité est mal comprise et peu d’outils sont disponibles afin d’identifier les patients à risque de complications. Nous avons utilisé différentes approches afin d’étudier les déterminants génétiques constitutionnels et somatiques de l’expression de l’AF ainsi que leur utilité dans la prise en charge des patients. Dans un premier temps, nous avons montré que les scores polygéniques pour les traits sanguins étaient moins performants chez les individus avec AF que sans. Nous avons montré que cette différence était en partie liée au poids d’autres déterminants génétiques et identifié un effet épistatique entre l’AF et le variant nul Duffy qui avait un effet très réduit en présence d’AF. Nous avons ensuite montré qu’un score polygénique simple pour l’Hb F expliquait une grande partie de sa variance et permettait d’améliorer la modélisation des crises de douleurs. Pour finir, à partir d’une réanalyse de la plus grande cohorte de patients avec AF ainsi que par randomisation mendélienne, nous avons montré l’effet causal protecteur de l’Hb F sur les accidents vasculaires cérébraux. Dans un second temps, nous avons analysé un crible génétique ciblé sur la densité du globule rouge, paramètre influençant la polymérisation de l’Hb S. Nous avons identifié de nombreux variants ayant un effet causal sur la densité à des fins de validation fonctionnelle. Pour finir, nous avons montré que l’AF influençait l’apparition d’une hématopoïèse clonale. Cette dernière était en effet détectée chez des patients plus jeunes et avait une prévalence plus élevée chez les individus avec AF que sans. En résumé, ce travail apporte un éclairage nouveau sur les conséquences de l’AF en montrant son influence sur l’expression de déterminants génétiques constitutionnels et somatiques. Ces déterminants représentent des outils pouvant permettre d’améliorer la prise en charge des patients avec AF. / Sickle cell disease (SCD) is a monogenic disorder leading to the polymerization of an abnormal hemoglobin (hemoglobin S, Hb S), that deforms the red cell, eventually leading to its destruction. SCD is associated with numerous complications with a great heterogeneity in its expression. Beside the important impact of fetal hemoglobin (Hb F), this variability is poorly understood, and few tools are available in the clinic to identify patients at high risk of complications. We used several approaches to study constitutional and somatic genetic determinants of SCD expression and their utility in patients’ management. First, we showed that polygenic scores for hematological traits were less performant in individuals with SCD than without. We showed that this difference was partly due to other genetic determinants and identified an epistatic effect between SCD and the Duffy antigen/chemokine receptor (DARC) null variant, which had a markedly reduced effect in SCD. We then showed that a simple polygenic score for Hb F explained a large part of the variance and improved modeling of pain crises. Based on the largest SCD cohort reanalysis and using Mendelian randomization, we showed that Hb F had a causal protective effect against stroke. Second, we analyzed a CRISPR-Cas9 targeted screening for red cell density, which influences Hb S polymerization. We identified many variants that influenced density as candidate to functional validations. Finally, we showed that SCD influenced the occurrence of clonal hematopoiesis. Clonal hematopoiesis was detected in younger patients and had an increased prevalence in individuals with SCD than without. In sum, this work shed light on a new category of consequences of SCD by showing its influence on the expression of constitutional and somatic determinants. These determinants are tools that can be used to improve the management of patients with SCD.

The development of the vertebrate eye is tightly controlled by precise genetic regulations. From a single ocular primordium to bilateral eyes with complex structures and cell types, it requires intensive proliferation and migration for cells in both the ectoderm and mesoderm to accomplish ocular morphogenesis, and during this process cell differentiation and interaction takes place to establish the complex composition of ocular cell types and cellular connections. Genetic defects can lead to severe abnormalities in eye morphogenesis and cell differentiation during ocular development. A tremendous amount of work has been done to identify both intrinsic and extrinsic factors that regulate ocular development. However, much more work is needed to fully understand this complex process. Sox4 is known as a transcription activator that regulates cell survival and differentiation in multiple embryonic tissues during development. Evidence of its requirement during ocular development has recently emerged, but the mechanism by which Sox4 regulates ocular development is far from elucidated. Chapter 1 of this dissertation provides an overview of different stages in embryonic eye development and known genetic interactions during each stage. It also reviews recent knowledge about SoxC proteins and their roles in ocular development. Chapter 2 presents data characterizing the expression profile of the zebrafish sox4 co-orthologs, sox4a and sox4b, in the developing eye. Additionally, it presents data from morpholino-mediated sox4 knockdown in zebrafish, which indicate that Sox4 deficiency leads to defects in choroid fissure closure through elevation in the Hedgehog (Hh) signaling pathway. Sox4 knockdown causes upregulation of the Hh ligand indian hedgehog b (ihhb), which alters the proximal-distal boundary of the optic vesicle and inhibits choroid fissure closure. Chapter 3 presents data reporting the generation of sox4 mutant zebrafish lines using the CRISPR/Cas9 genome editing system. Characterization of one sox4a maternal zygotic (MZ) mutant line confirms Sox4’s role in negative regulation of Hh signaling and reveals new evidence that maternal and zygotic sox4 are both critical for ocular development. Chapter 4 presents data demonstrating that sox4 is required for rod photoreceptor neurogenesis. Rod photoreceptor terminal differentiation is delayed in both sox4 morphants and sox4 CRISPR mutants, while rod progenitor and precursor cells are properly specified. In Chapter 5, the roles of Sox4 in regulating ocular development are summarized based on the results, and implications of the results are discussed to expand our understanding of the genetic regulation of ocular morphogenesis and retinal neurogenesis.

No description available.

No description available.

Macrophages belong to the mononuclear phagocyte system and they perform fundamental roles to maintain homeostasis in the organism. Macrophage development, survival, proliferation and functionality depend upon the colony stimulating factor 1 (CSF1) and interleukin-34 (IL-34), which signal through the CSF1 receptor (CSF1R). CSF1R is a type III tyrosine kinase receptor that is present in the plasma membrane of monocytes and macrophages. Mutations in Csf1r in mice produce the loss of many tissue macrophage populations and multiple developmental abnormalities. In humans, abnormal enhancement of CSF1R expression has been correlated to adverse prognosis in a subset of carcinomas; and mutations in the human CSF1R are associated with an autosomal-dominant neurodegenerative disease. CSF1R is encoded by the c-fms proto-oncogene and its expression is partially controlled by the fms-intronic regulatory element (FIRE). The FIRE sequence is highly conserved across species and contains binding motifs for multiple transcription factors, which are relevant for haematopoiesis. Previous results from murine Csf1r transgenes showed that FIRE is essential for driving Csf1r expression, and that interactions between FIRE and multiple myeloid transcription factors contribute to maximal regulatory activity. This project aimed to study the role of FIRE in its normal chromatin context, in vivo. A FIRE knockout (FIRE-/-) mouse model was generated using the CRISPR/Cas9 technology in mouse embryonic stem cells (ESCs) and in mice. In ESCs, the deletion severely compromised the differentiation of macrophages from embryoid bodies generated in vitro. In mice, the frequency of the FIRE- /- genotype in the progeny does not follow a Mendelian distribution and about 5% of the offspring developed hydrocephalus. Unlike Csf1r -/-mice, which die before weaning, most surviving FIRE-/- mice grew normally and were fertile. The impact of the mutation on macrophage populations is selective. FIRE-/- mice are not monocyte deficient (identified as F4/80+ Csf1r+ cells in peripheral blood), although these cells have reduced levels of Csf1r mRNA and do not bind porcine CSF1 Fc fusion protein. The development of peritoneal macrophages and Iba-1+ microglia was abolished, but Adgre1+ (F4/80+) macrophage populations in liver and spleen were unaffected. Csf1r was greatly reduced in bone marrow progenitors, but about 30% of these cells were able to differentiate into macrophages in vitro, upon exposure to recombinant human CSF1 (rhCSF1). This study shows that FIRE is essential for the development of a subset of tissue-resident macrophage populations. In FIRE-/- mice, potential compensation from additional regulatory elements within Csf1r might underlie the development of unaffected tissue-resident macrophages.

abstract: Biomarkers find a wide variety of applications in oncology from risk assessment to diagnosis and predicting and monitoring recurrence and response to therapy. Developing clinically useful biomarkers for cancer is faced with several challenges, including cancer heterogeneity and factors related to assay development and biomarker performance. Circulating biomarkers offer a rapid, cost-effective, and minimally-invasive window to disease and are ideal for population-based screening. Circulating immune biomarkers are stable, measurable, and can betray the underlying antigen when present below detection levels or even no longer present. This dissertation aims to investigate potential circulating immune biomarkers with applications in cancer detection and novel therapies. Over 600,000 cancers each year are attributed to the human papillomavirus (HPV), including cervical, anogenital and oropharyngeal cancers. A key challenge in understanding HPV immunobiology and developing immune biomarkers is the diversity of HPV types and the need for multiplexed display of HPV antigens. In Project 1, nucleic acid programmable protein arrays displaying the proteomes of 12 HPV types were developed and used for serum immunoprofiling of women with cervical lesions or invasive cervical cancer. These arrays provide a valuable high-throughput tool for measuring the breadth, specificity, heterogeneity, and cross-reactivity of the serologic response to HPV. Project 2 investigates potential biomarkers of immunity to the bacterial CRISPR/Cas9 system that is currently in clinical trials for cancer. Pre-existing B cell and T cell immune responses to Cas9 were detected in humans and Cas9 was modified to eliminate immunodominant epitopes while preserving its function and specificity. This dissertation broadens our understanding of the immunobiology of cervical cancer and provides insights into the immune profiles that could serve as biomarkers of various applications in cancer. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2018