Macrophage migration inhibitory factor (MIF) has been implicated in mediating both innate and adaptive immune responses in inflammatory and infectious diseases. The sequence and structure of MIF is highly conserved across the avian phylogeny, which underlies high sequence homology and functional similarities between turkey and chicken MIFs. Turkey MIF (TkMIF) inhibited cell migration and promoted cell proliferation with production of inflammatory mediators, comparable to the biological properties of chicken MIF (ChMIF), thus indicating the biological cross-reactivity between turkey and chicken MIFs. This study identified the cell surface receptor(s) that could bind ChMIF and the biological roles triggered by such interactions. In addition to CD74, a previously identified receptor, CXCR4 also interacts with ChMIF. Moreover, the formation of receptor complexes was shown between CXCR4 and CD74. MIF signaling through CXCR4 and CD74 led to cell chemotaxis and proliferation activity as well as intracellular calcium influx. Intriguingly, Eimeria MIF (EMIF), a homologue secreted following parasitic infection, also interacted with CD74 leading to comparable biological functions to those of ChMIF. Given such observations, we hypothesized that CXCR4 and CD74 are receptors for ChMIF leading to the functional consequences similarly manifested by EMIF interaction with the corresponding receptors. EMIF, predominantly secreted from the invasive merozoite stage, may help the parasite exploit the host immune response by interacting with common ChMIF receptors. This may lead to functional mimicry thus provoking the question of whether EMIF would modulate the biological functions of ChMIF to manipulate the host defense that allows more efficient invasion of the host. To evaluate this concept, a transgenic E. tenella lacking MIF was generated by in vivo passage of E. tenella transfected with a CRISPR plasmid targeting EMIF. Although not fully disrupted, reduction of EMIF expression was observed in the transgenic E. tenella itself as well as in inoculated cells, which resulted in enhanced survival of host cells. Herein, we achieved a better characterization of the functional roles of both avian and parasite MIFs underlying the interaction with common host receptors, along with the essential role of parasite MIF promoting host cell death during parasitic infection. / PHD / When animals get infected or injured, their immune system senses invading pathogens or damaged tissues as danger signals, which often elicits the production of inflammatory mediators. These are chemical messengers secreted mostly by immune cells that initiate cellular communication and infiltration of immune cells to the infection/damaged site leading to inflammatory responses to eliminate the infectious agents and repair damaged tissues. Among many inflammatory mediators, macrophage migration inhibitory factor (MIF) is involved in inflammatory and immune response by regulating cell migration. Interestingly, MIF is secreted by Eimeria parasites (that cause the costly coccidiosis disease in poultry) as well as by chickens (host animal) after infection with this pathogen. Toward a better understanding of the impacts of both avian and parasite MIFs on the host immune response, three specific studies were completed. First, MIF displayed high degree of gene sequence identity and functional similarity between chicken and turkey, supporting the evolutionarily conservation of MIF across birds. The second study identified the MIF receptors and their complexes, which engage in the biological functions of chicken MIF. Through binding to these cell surface receptors, chicken MIF can regulate cell migration and proliferation with calcium release. Intriguingly, Eimeria MIF secreted after parasitic infection is able to bind the same receptors leading to comparable biological functions to those of chicken MIF. Lastly, the role of Eimeria MIF was further evaluated by disrupting its gene in the parasite. Although not fully disrupted in the transgenic parasites, its expression was decreased resulting in enhanced survival of host cells, thus suggesting a deleterious effect of Eimeria MIF on the host, as well as its potential as a therapeutic target to control coccidiosis in poultry.

As integrinas constituem uma família de receptores de membrana que tem como função primária a adesão de células a proteínas da matriz extracelular e alguns de seus membros estão envolvidos nos processos de diferenciação osteoblástica e formação óssea, eventos diretamente relacionados à osseointegração de implantes de titânio (Ti). Sabe-se que superfícies de Ti com nano e microtopografia podem favorecer a diferenciação osteoblástica e a mineralização da matriz extracelular. No entanto, os mecanismos celulares envolvidos nesses processos não são completamente entendidos. Neste contexto, os objetivos deste estudo foram: (1) caracterizar as superfícies de Ti com nano (Ti-Nano) e microtopografia (Ti-Micro), (2) investigar a participação da integrina V na diferenciação osteoblástica induzida pelo Ti-Nano e (3) investigar a participação da integrina β3 na diferenciação osteoblástica induzida por Ti-Nano e Ti-Micro. Para isso, discos de Ti-Nano e Ti-Micro foram preparados por ataque ácido com H2SO4/H2O2 ou com HNO3/H2SO4 / HCl, respectivamente, e caracterizados quanto à topografia, rugosidade e composição química de superfície. Discos de Ti usinados foram usados com controle (Ti-Controle) em alguns experimentos. Células-tronco mesenquimais derivadas de medula óssea de ratos foram cultivadas sobre as três superfícies de Ti e foi avaliada a expressão gênica de componentes envolvidos na via de sinalização das integrinas por PCR array. Com base nos resultados do PCR array, as integrinas αV e β3 foram selecionadas e silenciadas por RNA de interferência (shRNA) ou CRISPR/Cas9, respectivamente, em células pré-osteoblásticas da linhagem MC3T3-E1 para investigarmos a participação dessas integrinas na diferenciação osteoblástica induzida por superfícies de Ti com diferentes topografias. Os resultados deste estudo mostraram que os tratamentos empregados foram eficientes para a produção de superfícies de Ti com topografias nas escalas nano e micrométrica. Além disso, foi demonstrado que o maior potencial osteogênico do Ti-Nano se deve, ao menos em parte, à integrina αV, uma vez que seu silenciamento reduziu a diferenciação osteoblástica induzida pela nanotopografia. Por fim, também demonstramos que a via de sinalização ativada pela integrina β3 exerce um papel fundamental no potencial osteogênico do Ti-Nano, mas não do Ti-Micro. O silenciamento da integrina β3 reduziu a diferenciação osteoblástica, concomitantemente com a regulação negativa da expressão de vários componentes das vias de sinalização de Wnt e de BMP, apenas nas células crescidas sobre a nanotopografia. Em conjunto, nossos resultados revelam um novo mecanismo para explicar a maior diferenciação osteoblástica induzida pelo Ti-Nano, que envolve uma complexa rede regulatória ativada pela maior expressão das integrinas αV e β3, esta última gerando ativação da transdução de sinal das vias de Wnt e de BMP / Integrins are a family of membrane receptors that primarily mediate cell adhesion to extracellular matrix proteins and some members are involved in the process of osteoblast differentiation and bone formation, key events of titanium (Ti) implant osseointegration. It is well known that Ti surfaces with nano and microtopography may favor osteoblast differentiation and matrix mineralization. However, the cellular mechanisms involved in this process are not entirely understood. In this context, the aims of this study were: (1) to characterize the Ti surfaces with nano (Ti-Nano) and microtopography (Ti-Micro), (2) to investigate the participation of integrin V on osteoblast differentiation induced by Ti-Nano and (3) to investigate the participation of integrin β3 on osteoblast differentiation induced by Ti-Nano and Ti-Micro. Discs of Ti-Nano and Ti-Micro were prepared with acid etching with H2SO4/H2O2 or with HNO3/H2SO4 / HCl, respectively, and characterized in terms of surface topography, roughness and chemical composition. Machined Ti discs (untreated) were used as control (Ti-Control) in some experiments. Mesenchymal stem cells from rat bone marrow were cultured on Ti discs with the three different surfaces and the gene expression of members of the integrin signaling pathway was evaluated by PCR array. Based on PCR array results, the integrins αV and β3 were selected and silenced using RNA interference (shRNA) or CRISPR-Cas9, respectively, in pre-osteoblastic cell line MC3T3-E1 to investigate the participation of these integrins in osteoblast differentiation induced by Ti with different surface topographies. The results showed that the treatments used were efficient to generate Ti surfaces with topographies at the nano and micrometric scales. We showed that the higher osteogenic potential of Ti-Nano may be, at least in part, due to the integrin &alphaV, since its silencing reduced the osteoblast differentiation induced by nanotopography. We also demonstrated that the signaling pathway triggered by integrin β3 plays a key role in the osteogenic potential of Ti-Nano, but not of Ti-Micro. The silencing of integrin β3 reduced the osteoblast differentiation concomitantly with the negative regulation of the gene expression of several Wnt and BMP signaling components only in cells grown on Ti-Nano. Taken together, our results uncover a novel mechanism to explain the higher osteoblast differentiation induced by Ti-Nano that involves a complex regulatory network triggered by integrins αV and β3 upregulation, with the integrin β3 activating the Wnt and BMP signal transductions

Mycoplasma hominis est un pathogène humain opportuniste responsable d’infections génitales et néo-natales. Modifier génétiquement cette bactérie est nécessaire afin de comprendre les mécanismes de virulence et d’infection de ce pathogène. Il n’existe à ce jour aucun outil moléculaire efficace permettant de manipuler le génome de M. hominis, limitant les recherches sur sa pathogénicité et son métabolisme particulier reposant sur l’arginine. De nouvelles technologies rassemblées sous le terme de Biologie de Synthèse (BS) ont récemment émergé, offrant des perspectives inédites pour l’étude des mycoplasmes en permettant de modifier leurs génomes à grande échelle et de produire des souches mutantes. Ces travaux menés au J. Craig Venter Institute (JCVI, USA) ont montré que le génome de mycoplasmes apparentés pouvait être cloné et manipulé dans la levure avant d’être transplanté dans une cellule receveuse. La levure sert d’hôte d’accueil temporaire pour modifier le génome de la bactérie. Cette approche novatrice ouvre de nombreuses perspectives dans le cadre du développement de la génomique fonctionnelle chez les mycoplasmes pour lesquels les outils génétiques efficaces sont peu nombreux. Le but de cette thèse a été d’adapter pour la première fois certains outils de BS à M. hominis dans le but de créer des mutants déficients pour une fonction donnée. Pour cela, le génome de la souche type de M. hominis PG21 (665 kb) a été cloné dans la levure Saccharomyces cerevisiae par « Transformation-Associated Recombination cloning » (TAR-cloning). Deux clones (B3-2 et B3-4) de levure possédant le génome complet de M. hominis ont été validés par analyse en PCR simplex, PCR multiplex et électrophorèse en champs pulsé (PFGE). Ces clones levures ont ensuite été propagés en milieu sélectif durant 180 générations (30 passages), afin d’évaluer la stabilité du génome bactérien dans son hôte. Cette expérience a montré que (i) si la taille du génome de M. hominis ne variait pas au cours des premiers passages, elle diminuait progressivement à partir du dixième passage (≈60 générations), et que (ii) les zones du génome enrichies en séquence répétées étaient préférentiellement perdues. En tenant compte de ces résultats, le génome de M. hominis a été modifié chez le clone B3-4 par la technique « Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 » (CRISPR/Cas9) lors de passages précoces. Des clones de S. cerevisiae possédant un génome de M. hominis PG21 complet délété du gène vaa, codant une protéine d’adhésion majeure, ont été ainsi produits. La dernière étape de cette approche consistait à transplanter le génome modifié dans une cellule receveuse de M. hominis ou de Mycoplasma arthritidis, espèce phylogénétiquement la plus proche de M. hominis. Aucun protocole de transformation de M. hominis n’étant disponible au début de nos travaux, cette étape constituait un verrou majeur dans la mise en place des outils de BS chez cette espèce. Ce verrou a été en partie levé puisqu’une méthode de transformation de M. hominis basée sur du polyéthylène glycol (PEG) et mettant en jeu le plasposon pMT85 (plasmide contenant un transposon conférant la résistance à la tétracycline) a été mise au point au laboratoire. Cette technique de transformation, développée pour la souche de référence M. hominis M132 (745 kb) reste encore peu efficace ; elle est néanmoins reproductible et a permis d’obtenir des mutants d’intérêt de M. hominis. Le transformant n°28-2 a, ainsi, été muté dans le gène Mhom132_2390, codant le précurseur de la protéine P75, une adhésine putative de M. hominis. Le séquençage des génomes complets d’autres transformants a révélé l’insertion de multiples copies du transposon et la présence d’évènements de duplication et d’inversion de larges fragments d’ADN dans au moins deux génomes de M. hominis. / Mycoplasma hominis is an opportunistic human pathogen responsible for genital and neonatal infections. Genetically modifying this bacterium is necessary to understand the virulence and infection mechanisms of this pathogen. There is currently no effective molecular tool to engineer the genome of this bacterium, limiting research on its pathogenicity and its peculiar metabolism based on arginine.New technologies have recently emerged in the field of Synthetic Biology (BS), offering new perspectives for the study of mycoplasmas by allowing large scale genome modifications and the production of mutant strains. Work at the J. Craig Venter Institute (JCVI, USA) has shown that the genome of related mycoplasmas can be cloned and manipulated in yeast before being transplanted into a recipient cell. The yeast serves as a temporary host to modify the genome of the bacterium. This innovative approach opens many perspectives in the development of functional genomics in mycoplasmas for which there are few effective genetic tools. The goal of this thesis was to adapt a number of BS tools to M. hominis for the first time, in order to create mutants deficient for a given function. To achieve this goal, the genome of the M. hominis type strain PG21 (665 kb) was cloned into the yeast Saccharomyces cerevisiae by Transformation-Associated Recombination cloning (TAR-cloning). Two yeast clones (B3-2 and B3-4) possessing the complete genome of M. hominis were validated by simplex PCR, multiplex PCR and Pulsed Field Gel Electrophoresis (PFGE) analyses. These yeast clones were then propagated in a selective medium for 180 generations (30 passages) to evaluate the stability of the bacterial genome in its host. This experiment showed that (i) the size of the genome of M. hominis did not change during the first passages, it decreased progressively from the tenth passage (≈60 generations), and (ii) the enriched genome areas in repeated sequence were preferentially lost. Thus, the genome of M. hominis was modified in the B3-4 clone at early passages using the Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR/Cas9) technology. Yeast clones with a complete M. hominis PG21 genome with a deleted vaa gene, encoding a major adhesion protein, were produced using this approach. The final step of this approach was to transplant the modified genome into a recipient cell of M. hominis or Mycoplasma arthritidis, the species phylogenetically closest to M. hominis. As no M. hominis transformation protocol was available at the beginning of our work, this step constituted a major obstacle in the implementation of BS tools in this species. This barrier has been partially lifted since a method of transformation of M. hominis based on polyethylene glycol (PEG) and involving the plasposon pMT85 (plasmid carrying a transposon conferring resistance to tetracycline) has been developed in the laboratory. This transformation technique, developed for the reference strain M. hominis M132 (745 kb) still remains not very efficient; it is nevertheless reproducible and allowed to obtain M. hominis mutants of interest. The Mhom132_2390 gene, encoding the precursor of the P75 protein, a putative adhesin of M. hominis, was effectively mutated in transformant No. 28-2. Complete genome sequencing of other transformants revealed the insertion of multiple copies of the transposon and the presence of duplication and inversion of large DNA fragments within at least two M. hominis genomes.In conclusion, this data has opened the way for the development and transposition of existing genetic modification approaches to M. hominis, previously considered as a genetically intractable bacterium.

A produção de proteínas terapêuticas recombinantes compreende moléculas complexas e de alto valor agregado, incluindo a enzima glucocerebrosidase (GCase). Sua deficiência resulta na Doença de Gaucher, passível de tratamento por meio da terapia de reposição enzimática. A forma ativa da GCase recombinante usada na terapia apresenta resíduos terminais de manose expostos no seu perfil de glicosilação. Perfil este que espera-se ser reproduzido por meio da construção de uma linhagem de Pichia pastoris com um padrão de glicosilação humanizado, por meio da deleção de dois genes envolvidos no sistema de glicosilação da levedura: alg3 e och1, responsáveis pela posterior hiper-manosilação característica desse organismo. Assim, a expressão da GCase será usada como modelo no desenvolvimento desta linhagem de Pichia pastoris que permita a expressão de glicoproteínas com um perfil humanizado específico de glicosilação. Além da produção da linhagem mutante pela técnica de CRISPR/Cas9, propomos a construção de duas linhagens controle: uma expressando a proteína GCase para análise do seu padrão selvagem de glicosilação em P. pastoris e outra expressando a proteína Cas9 de Streptoccocus pyogenes (SpCas9). A linhagem P. pastoris/GCase foi construída testando-se duas sequências sinal de secreção diferentes: fosfatase alcalina (PHO1) e albumina humana (Alb). Resultados de western blot mostraram a GCase no lisado celular e baixos níveis de proteína secretada no sobrenadante de cultura, sendo mais expresso na linhagem contendo a sequência PHO1. A linhagem P. pastoris/SpCas9 foi construída e a enzima SpCas9 foi detectada via western blot no lisado celular após indução com metanol. Para a produção da linhagem com padrão de glicosilação humanizado propôs-se a deleção dos genes alg3 e och1 e a inserção, pela via de reparo por recombinação homóloga (HDR), de marcas de resistência aos antibióticos higromicina ou canamicina. Para tal, propusemos a construção de dois vetores finais de expressão do sistema CRISPR/Cas9 em P. pastoris, cada um contendo a enzima SpCas9 e os RNAs guia (gRNAs) para deleção do gene alg3 ou och1, e também a construção de dois fragmentos para HDR contendo o gene de resistência ao antibiótico flanqueado por regiões de 1Kb de homologia com a região de deleção do gene alg3 ou och1. A construção dos vetores e fragmentos para HDR foram inicialmente feitas por meio de técnicas de clonagem clássica. No entanto, apesar de inúmeras tentativas, resultados de PCR e sequenciamento mostraram o insucesso das construções. Partiu-se então para a técnica de Gibson Assembly®, através da qual os dois fragmentos para HDR foram construídos. Porém, os vetores de expressão contendo SpCas9 e os gRNAs ainda apresentam dificuldades na sua construção. Esforços ainda estão sendo feitos para a construção dos vetores e consequente tentativa de estabelecimento das linhagens mutantes. O sucesso no estabelecimento de um sistema de expressão de proteínas heterólogas com este padrão de glicosilação humano específico permitirá a obtenção e possível comercialização da GCase em sua forma terapêutica. Além disso, permitirá possíveis edições genômicas futuras para um padrão de maior complexidade de glicosilação humanizado, criando uma plataforma nacional para produção de outras glicoproteínas terapêuticas de interesse biotecnológico. / The production of therapeutic recombinant protein comprises complex and high valued molecules, including the glucocerebrosidase enzyme (GCase). Its deficiency results in Gaucher Disease, susceptible of treatment by enzymatic replacement therapy. The active form of recombinant GCase employed in therapy presents exposed terminal mannose residues in its glycosylation pattern. We hope to reproduce such pattern by constructing a Pichia pastoris strain with a specific human glycosylation pattern through the deletion of two genes involved in yeast glycosylation system, alg3 and och1, responsible for the final hyper-mannosylation characteristic of this organism. Therefore, the expression of GCase will be a case model for the development of the recombinant Pichia pastoris strain that could allow the expression of glycoproteins with a specific humanized glycosylation profile. Despite the establishment of the mutant strain using the CRISPR/Cas9 technique, we propose the construction of two control strains: one expressing the GCase protein for analysis of its wild type glycosylation pattern and another one expressing the Cas9 protein from Streptoccocus pyogenes (SpCas9). The P. pastoris/GCase strain was constructed testing two different secretion signal sequences: alkaline fosfatase (PHO1) and human albumin (Alb). Western blot results have shown GCase in cell lysate and in low expression levels in culture supernatant, being more expressed in the strain containing the PHO1 signal sequence. P. pastoris/SpCas9 strain was constructed and SpCas9 enzyme was detected via western blot in cell lysate after the induction with methanol. To produce the strain with the humanized glycosylation pattern, the deletion of alg3 and och1 genes was proposed along with the insertion, by homology directed repair pathway (HDR), of hygromycin and kanamycin antibiotics resistance marks. In order to do so, we have proposed the construction of two final expression vectors of the CRISPR/Cas9 system in P. pastoris, each one containing SpCas9 enzyme and the guide RNAs (gRNAs) for deletion of alg3 or och1, and also the construction of two fragments for HDR containing the antibiotics resistance gene flanked by 1Kb regions of homology with the deleted regions of alg3 or och1. Vectors and HDR fragments constructions were initially performed using classic cloning techniques. However, despite numerous tries, PCR and sequencing results have shown the failure of the constructions. Then, we moved on to the Gibson Assembly® technique, through which the two HDR fragments were built. Still, the expression vectors containing SpCas9 and the gRNAs presented difficulties in its assembly. Efforts continue to be made to successfully construct the remaining vectors and to establish the mutant lineage. Success in the establishment of a heterologous protein expression system with specific human glycosylation pattern will allow the obtainment and possible commercialization of the therapeutic form of GCase. Furthermore, it will also allow possible future genomic editing to a high complexity human glycosylation pattern, creating a national platform for the production of other therapeutic glycoproteins of biotechnological interest.

L’adrénoleucodystrophie liée à l’X (X-ALD) est une maladie neurodégénérative sévère caractérisée par une accumulation d’acides gras à très longue chaîne (AGTLC), conséquence d’un défaut de β-oxydation peroxysomale. La maladie est associée à l’absence de la protéine ABCD1, transporteur ABC du peroxysome qui, tout comme son homologue le plus proche, ABCD2, participe à l’import des AGTLC-CoA au sein du peroxysome, l’unique site de leur dégradation par β-oxydation. La compréhension des mécanismes physiopathologiques est aujourd’hui limitée par le manque de modèles expérimentaux pertinents, cellulaires ou animaux. Puisque le défaut peroxysomal dans la microglie apparait comme un événement pathogénique majeur, nous avons généré des lignées de cellules microgliales incapable de transporter et/ou β-oxyder les AGTLC au sein du peroxysome. Quatre lignées cellulaires microgliales BV-2 déficientes en ABCD1, ABCD2, ABCD1 et ABCD2 ou ACOX1 (l’enzyme limitante de la β-oxydation peroxysomale) ont ainsi été générées par édition génique par CRISPR-Cas9. Ces cellules déficientes présentent d’importants défauts biochimiques, une accumulation d’AGTLC mais aussi des changements des contenus en acides gras et cholestérol. Les analyses ultrastructurales effectuées démontrent l’existence d’importantes inclusions lipidiques et indiquent également une augmentation du nombre de peroxysomes et mitochondries dans ces cellules. Les profils transcriptomiques signalent des altérations de la plasticité de ces cellules microgliales et de leur capacité de reprogrammation métabolique en réponse à un stimulus inflammatoire. Les fonctions de phagocytose ou de présentation antigénique des cellules microgliales semblent être affectées par le défaut peroxysomal. Enfin, les résultats obtenus à l’aide de ces modèles suggèrent que l’altération du métabolisme lipidique peroxysomal modifie l’organisation des membranes cellulaires. Ces lignées cellulaires apparaissent donc comme des modèles prometteurs, d’un grand intérêt pour la compréhension de la physiopathologie et l’identification de cibles thérapeutiques de cette maladie neurodégénérative complexe. / X-linked adrenoleukodystrophy (X-ALD) is a severe neurodegenerative disorder characterized by very-long-chain fatty acid (VLCFA) accumulation resulting from a peroxisomal β-oxidation defect. The disease is caused by mutations in the ABCD1 gene, which encodes for a peroxisomal half ABC transporter predicted, like its closest homologue ABCD2, to participate in the entry of VLCFA-CoA into the peroxisome, the unique site of their β-oxidation. Progress in understanding the physiopathogenesis of X-ALD suffers from the lack of appropriate cell and animal models. Since peroxisomal defects in microglia seem to be a key element of the onset of the disease, we generated four microglial cell lines unable to transport and/or β-oxidize VLCFA into the peroxisome. BV-2 microglial cells were engineered with CRISPR-Cas9 to generate four microglial cell lines deficient in ABCD1, ABCD2, both ABCD1 and ABCD2 or ACOX-1 (the first rate-limiting enzyme of the peroxisomal β-oxidation system). Biochemical defects and lipid content changes associated with VLCFA accumulation but also fatty acids and cholesterol changes were identified in deficient microglia. Ultrastructural investigations confirmed cytosolic lipid inclusions and an increased number of peroxisome and mitochondria. Transcriptomic profiles of deficient microglia are indicative of an impaired plasticity and an impaired capacity to operate the metabolic shift required upon an inflammatory stimulation. Peroxisomal defect is likely to affect phagocytosis and antigen presentation capacity of microglia. Peroxisomal lipid metabolism defect is also suggested to modify cell membranes organization. Altogether, these novel mutant cell lines represent a promising model that should permit identification of new therapeutic targets for this complex neurodegenerative disease.

Les β-hémoglobinopathies (β-thalassémies et drépanocytose) sont des anémies génétiques qui touchent des milliers de nouveaux nés chaque année dans le monde. Ces maladies sont causées par des mutations affectant l'expression de l'hémoglobine chez l'adulte. Le seul traitement disponible est la transfusion sanguine à vie, associée à une chélation du fer. Pour les patients les plus touchés, la greffe de cellule souche hématopoïétique (CSH) demeure le seul traitement curatif. Néanmoins, la transplantation autologue de cellules souches génétiquement corrigées représente une alternative thérapeutique pour les patients dépourvus de donneur compatible. Certaines délétions naturelles comprenant les gènes de la β- et δ- globine dans le locus de l'hémoglobine sont corrélées à une persistance de l'expression de l'hémoglobine fœtale (HPFH) à l'âge adulte. Ainsi il a été démontré que un taux élevé d'hémoglobine fœtale (HbF) améliore l'évolution clinique de ces deux pathologies. Afin d'identifier les régions régulatrices potentielles de la γ-globine, nous avons combiné les données issues d'analyses de mutations rencontrées chez des patients HPFH avec les sites d'hybridation de facteur de transcription. Sur la base de cette analyse, en ayant recours à la technologie CRISPR/CAS9, nous avons développé un protocole permettant de générer: (i) la délétion d'un potentiel suppresseur de l'HbF situé entre les gènes des globines δ et γ, ciblé par le répresseur de l’HbF BCL11A chez les érythroblastes adultes; (ii) la plus courte délétion associée à des taux élevés d’HbF (délétion Corfu) chez les patients β-thalassemiques; (iii) une délétion de 13.6-kb rencontrée fréquemment chez les patients HPFH et incluant les gènes des globines β et δ ainsi que le potentiel suppresseur de l'HbF. Notre travail a montré que la délétion de la région génomique de 13.6-kb entraîne une forte production de HbF et une réduction concomitante de l'expression de la β-globine soit dans des lignées cellulaires érythroïdes humaines soit dans des érythroblastes primaires dérivées des cellules souches et progéniteurs hématopoïétiques (CSPH). Par ailleurs, nous avons montré que la génération de cette délétion sur des CSPHs issus de patients drépanocytaires entraîne une augmentation de la transcription de la γ-globine dans une proportion significative d'érythroblastes, conduisant à une amélioration du phénotype drépanocytaire. Enfin, nous avons exploré le mécanisme menant à la réactivation de l'expression de la γ-globine. Nous avons évalué des changements dans la conformation de la chromatine et des modifications épigénétiques dans le locus de la β-globine lors de la délétion ou de l'inversion de la région de 13.6 kb. Dans l'ensemble, cette étude contribue à la connaissance des mécanismes favorisant l'échange de l'hémoglobine fœtale à l'adulte et fournit des indices pour une approche d'édition du génome dans le traitement de la β-thalassémies et de la drépanocytose. / Β-hemoglobinopathies (β-thalassemias and sickle cell disease) are genetic anemias affecting thousands of newborns annually worldwide. β-thalassemias and sickle cell disease (SCD) are caused by mutations affecting the adult hemoglobin expression and are currently treated by red blood cell transfusion and iron chelation regiments. For patients affected by severe β-hemoglobinopathies, allogenic hematopoietic stem cell (HSCs) transplantation is the only definitive therapy. However, transplantation of autologous, genetically corrected HSCs represents an alternative therapy for patients lacking a suitable HSC donor. Naturally occurring large deletions encompassing β- and δ-globin genes in the β-globin gene cluster, defined as Hereditary Persistence of Fetal Hemoglobin (HPFH) traits, lead to increased fetal hemoglobin (HbF) expression ameliorating both thalassemic and SCD clinical phenotypes. In this study, we integrated transcription factor binding site analysis and HPFH genetic data to identify potential HbF silencers in the β-globin locus. Based on this analysis, we designed a CRISPR/Cas9 strategy disrupting: (i) a putative δγ-intergenic HbF silencer targeted by the HbF repressor BCL11A in adult erythroblasts; (ii) the shortest deletion associated with elevated HbF levels (“Corfu” deletion) in β-thalassemic patients, encompassing the putative δγ-intergenic HbF silencer; (iii) a 13.6-kb genomic region including the δ- and β-globin genes and the putative intergenic HbF silencer. Targeting the 13.6-kb region, but not the Corfu and the putative δγ-intergenic regions, caused a robust HbF re-activation and a concomitant reduction in β-globin expression in an adult erythroid cell line and in healthy donor hematopoietic stem/progenitor cells (HSPC)-derived erythroblasts. We provided a proof of principle of this potential therapeutic strategy: disruption of the 13.6-kb region in HSPCs from SCD donors favored the β-to-γ globin switching in a significant proportion of HSPC-derived erythroblasts, leading to the amelioration of the SCD cell phenotype. Finally, we dissected the mechanisms leading to HbF de-repression demonstrating changes in the chromatin conformation and epigenetic modifications within the β-globin locus upon deletion or inversion of the 13.6-kb region. Overall, this study contributes to the knowledge of the mechanisms underlying fetal to adult hemoglobin switching, and provides clues for a genome editing approach to the treatment of SCD and β-thalassemia.

A produção de proteínas terapêuticas recombinantes compreende moléculas complexas e de alto valor agregado, incluindo a enzima glucocerebrosidase (GCase). Sua deficiência resulta na Doença de Gaucher, passível de tratamento por meio da terapia de reposição enzimática. A forma ativa da GCase recombinante usada na terapia apresenta resíduos terminais de manose expostos no seu perfil de glicosilação. Perfil este que espera-se ser reproduzido por meio da construção de uma linhagem de Pichia pastoris com um padrão de glicosilação humanizado, por meio da deleção de dois genes envolvidos no sistema de glicosilação da levedura: alg3 e och1, responsáveis pela posterior hiper-manosilação característica desse organismo. Assim, a expressão da GCase será usada como modelo no desenvolvimento desta linhagem de Pichia pastoris que permita a expressão de glicoproteínas com um perfil humanizado específico de glicosilação. Além da produção da linhagem mutante pela técnica de CRISPR/Cas9, propomos a construção de duas linhagens controle: uma expressando a proteína GCase para análise do seu padrão selvagem de glicosilação em P. pastoris e outra expressando a proteína Cas9 de Streptoccocus pyogenes (SpCas9). A linhagem P. pastoris/GCase foi construída testando-se duas sequências sinal de secreção diferentes: fosfatase alcalina (PHO1) e albumina humana (Alb). Resultados de western blot mostraram a GCase no lisado celular e baixos níveis de proteína secretada no sobrenadante de cultura, sendo mais expresso na linhagem contendo a sequência PHO1. A linhagem P. pastoris/SpCas9 foi construída e a enzima SpCas9 foi detectada via western blot no lisado celular após indução com metanol. Para a produção da linhagem com padrão de glicosilação humanizado propôs-se a deleção dos genes alg3 e och1 e a inserção, pela via de reparo por recombinação homóloga (HDR), de marcas de resistência aos antibióticos higromicina ou canamicina. Para tal, propusemos a construção de dois vetores finais de expressão do sistema CRISPR/Cas9 em P. pastoris, cada um contendo a enzima SpCas9 e os RNAs guia (gRNAs) para deleção do gene alg3 ou och1, e também a construção de dois fragmentos para HDR contendo o gene de resistência ao antibiótico flanqueado por regiões de 1Kb de homologia com a região de deleção do gene alg3 ou och1. A construção dos vetores e fragmentos para HDR foram inicialmente feitas por meio de técnicas de clonagem clássica. No entanto, apesar de inúmeras tentativas, resultados de PCR e sequenciamento mostraram o insucesso das construções. Partiu-se então para a técnica de Gibson Assembly®, através da qual os dois fragmentos para HDR foram construídos. Porém, os vetores de expressão contendo SpCas9 e os gRNAs ainda apresentam dificuldades na sua construção. Esforços ainda estão sendo feitos para a construção dos vetores e consequente tentativa de estabelecimento das linhagens mutantes. O sucesso no estabelecimento de um sistema de expressão de proteínas heterólogas com este padrão de glicosilação humano específico permitirá a obtenção e possível comercialização da GCase em sua forma terapêutica. Além disso, permitirá possíveis edições genômicas futuras para um padrão de maior complexidade de glicosilação humanizado, criando uma plataforma nacional para produção de outras glicoproteínas terapêuticas de interesse biotecnológico. / The production of therapeutic recombinant protein comprises complex and high valued molecules, including the glucocerebrosidase enzyme (GCase). Its deficiency results in Gaucher Disease, susceptible of treatment by enzymatic replacement therapy. The active form of recombinant GCase employed in therapy presents exposed terminal mannose residues in its glycosylation pattern. We hope to reproduce such pattern by constructing a Pichia pastoris strain with a specific human glycosylation pattern through the deletion of two genes involved in yeast glycosylation system, alg3 and och1, responsible for the final hyper-mannosylation characteristic of this organism. Therefore, the expression of GCase will be a case model for the development of the recombinant Pichia pastoris strain that could allow the expression of glycoproteins with a specific humanized glycosylation profile. Despite the establishment of the mutant strain using the CRISPR/Cas9 technique, we propose the construction of two control strains: one expressing the GCase protein for analysis of its wild type glycosylation pattern and another one expressing the Cas9 protein from Streptoccocus pyogenes (SpCas9). The P. pastoris/GCase strain was constructed testing two different secretion signal sequences: alkaline fosfatase (PHO1) and human albumin (Alb). Western blot results have shown GCase in cell lysate and in low expression levels in culture supernatant, being more expressed in the strain containing the PHO1 signal sequence. P. pastoris/SpCas9 strain was constructed and SpCas9 enzyme was detected via western blot in cell lysate after the induction with methanol. To produce the strain with the humanized glycosylation pattern, the deletion of alg3 and och1 genes was proposed along with the insertion, by homology directed repair pathway (HDR), of hygromycin and kanamycin antibiotics resistance marks. In order to do so, we have proposed the construction of two final expression vectors of the CRISPR/Cas9 system in P. pastoris, each one containing SpCas9 enzyme and the guide RNAs (gRNAs) for deletion of alg3 or och1, and also the construction of two fragments for HDR containing the antibiotics resistance gene flanked by 1Kb regions of homology with the deleted regions of alg3 or och1. Vectors and HDR fragments constructions were initially performed using classic cloning techniques. However, despite numerous tries, PCR and sequencing results have shown the failure of the constructions. Then, we moved on to the Gibson Assembly® technique, through which the two HDR fragments were built. Still, the expression vectors containing SpCas9 and the gRNAs presented difficulties in its assembly. Efforts continue to be made to successfully construct the remaining vectors and to establish the mutant lineage. Success in the establishment of a heterologous protein expression system with specific human glycosylation pattern will allow the obtainment and possible commercialization of the therapeutic form of GCase. Furthermore, it will also allow possible future genomic editing to a high complexity human glycosylation pattern, creating a national platform for the production of other therapeutic glycoproteins of biotechnological interest.

The CRISPR-Cas9 gene editing system gives researchers the ability to manipulate and edit DNA with unprecedented ease and precision. It was discovered in bacteria as part of their adaptive immune system, but has been reengineered to target any double stranded DNA. This burgeoning molecular tool has created great excitement as scientists are rapidly adopting it to study fields including human gene therapy, disease modeling, agriculture, gene drive in mosquitos, and many others. This paper will explore the potential impact of CRISPR-Cas9 in human therapeutics. Specifically, the potential of CRISPR-Cas9 to treat Duchenne Muscular Dystrophy will be examined. In several ways, this debilitating degenerative disease is an ideal candidate for gene-editing with CRISPR-Cas9. Recent progress in the lab has demonstrated the gene editing system’s ability to rescue dystrophin protein levels in vivo. Although CRISPR-Cas9 holds great promise for previously incurable diseases, there are still many limitations that must be overcome before the gene editing system can be used in patients. This paper will discuss these barriers as well as recent advancements to overcome them.

CRISPR-Cas9 är ett effektivt genredigeringsverktyg som har upptäckts på senare år. Verktyget härstammar från ett adaptivt immunförsvar hos prokaryoter. Tekniken används för att modifiera DNA hos växter, djur och människor på ett enkelt och billigt sätt. CRISPR-Cas9 har visat sig ha stor potential vid bekämpning av olika sjukdomar däribland cancer som idag är ett globalt hälsoproblem. Inom cancerforskningen ses CRISPR-Cas9 som ett lovande verktyg vid cancerterapi och läkemedelsutveckling. I denna studie sammanställer vi aktuella metoder och användningsområden med CRISPR-Cas9 inom cancerforskning. Dessutom undersöker vi hur denna form av genteknik kan lyftas upp och tillämpas i biologiundervisningen. / CRISPR-Cas9 has recently emerged as an effective genome editing tool. The tool derives from an adaptive immune system in prokaryotes. The technology is used for modification of DNA in plants, animals and humans in a simple and inexpensive way. CRISPR-Cas9 has shown great potential in fighting different diseases like cancer which today is a global health issue. It is seen as a promising tool for cancer research when it comes to cancer therapy and drug development. Here we summarize current methods and applications of CRISPR-Cas9 for cancer research. Furthermore, we explore the possibilities of introducing and applying this kind of genetic engineering in biology teaching. / <p>Framläggning, opponering och respondering skedde skriftligt till följd av covid19.</p>

L'intérêt de l’utilisation des vecteurs viraux comme le Adeno-Associated Virus recombinant (rAAV) dans la recherche pour le traitement des maladies génétiques a conduit à une évolution rapide des méthodes de production d'AAV au cours des deux dernières décennies (Ayuso et al., 2010). Leur large biodisponibilité in vivo et leur efficacité à long terme dans les tissus postmitotiques en font de bons candidats pour de nombreuses applications de transfert de gènes. En plus, la spécificité du traitement peut être augmentée lorsque le sérotype correct est choisi pour cibler un tissu spécifique. Parmi les méthodes de production actuellement utilisées, la tri-transfection de cellules embryonnaires humaines rénales 293 (HEK293) reste la plus populaire pour l'échelle de recherche; Et la production de rAAV médiée par des baculovirus pour des échelles plus importantes. L'importance croissante des vecteurs viraux dans l'application pratique de la thérapie génique exige l'amélioration des processus de production, en particulier en ce qui concerne les rendements et la pureté du produit final. Mon travail au cours de ces quatre années a été axé sur deux points principaux: (1) améliorer les processus biotechnologiques employés dans la production de rAAV pour la recherche et les échelles d'étude préclinique et (2) tester in vitro et in vivo les applications pour le rAAV dans le l’édition de genome. L'édition de gènes médiée par des nucléases spécialement conçues offre de nouveaux espoirs pour le traitement de plusieurs maladies héréditaires monogéniques. Récemment découvert, le système CRISPR Cas9 (Clustered Regular Interspaced Short Palindromic Repeats) fournit des outils importants nécessaires pour corriger les mutations par homologie. Notre modèle canonique est la souris mdx, un modèle animal naturel de la dystrophie musculaire de Duchenne (DMD). Les mutations DMD, qui conduisent à l'absence de protéine dystrophine, entraînent une myopathie progressive et fatale. Plusieurs stratégies, allant des stratégies pharmacologiques aux stratégies de saut-d’éxon, ont tenté de renverser le phénotype et ralentisser la progression de la maladie, mais les résultats ne sont pas encore satisfaisants. Ce nouvel et puissant outil d'édition de génome peut être vectorisé par rAAV. Les résultats de la première partie ont été publiés en 2015 et 2016 et seront présentés sous la forme d'articles et pour la deuxième partie, je présenterai les résultats préliminaires et les perspectives du travail qui se poursuivra dans le laboratoire. / The interest of recombinant Adeno-Associated Virus (rAAV) vectors for research and clinical purposes in the treatment of genetic diseases have led to the rapid evolution of methods for AAV production in the last two decades (Ayuso et al., 2010). Their broad in vivo biodistribution and long-term efficacy in postmitotic tissues make them good candidates for numerous gene transfer applications. In addition, the specificity of the treatment can be increased when the right serotype is chosen to target a specific tissue. Among the production methods currently in use, tri-transfection of human embryonic kidney 293 (HEK293) cells remains the most popular for research scale; and rAAV production mediated by baculoviruses for larger scales. The increasing importance of viral vectors in the practical application of gene therapy demands the improvement of production processes, especially when it concerns the yields and purity of the final product. My work during these four years was focused in two main points: (1) improve biotechnological processes employed in rAAV production for research and pre-clinical study scales and (2) test in vitro and in vivo the applications for rAAV in the field of genome editing. Gene-editing mediated by engineered nucleases offers new hopes for the treatment of several monogenic inherited diseases. Recently discovered, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) Cas9 system provides important tools needed to correct by homology-directed repair mutations. Our canonical model is the mdx mouse, a naturally occurring animal model of Duchenne Muscular Dystrophy (DMD). DMD mutations, which lead to the absence of the protein dystrophin, results in a progressive and fatal myopathy. Several strategies, from pharmacological to exon-skipping strategies, have attempt to revert the phenotype and slow down the disease progress, however results are not yet satisfactory. This new and powerful genome editing tool can be vectorized by rAAV. Results for the first part were published in 2015 and 2016 and will be presented in the form of articles and for the second part I will present preliminary results and perspectives for the work that will be continued in the lab.