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T cells modified for expression of Chimeric Antigen Receptors (CARs) were the first genemodified cell products approved for use in cancer immunotherapy. CAR-T cells engineered with gammaretroviral or lentiviral vectors (RVs/LVs) targeting B-cell lymphomas and leukemias have shown excellent clinical efficacy and no malignant transformation due to insertional mutagenesis to date. Large-scale production of RVs/ LVs under good-manufacturing practices for CAR-T cell manufacturing has soared in recent years. However, manufacturing of RVs/LVs remains complex and costly, representing a logistical bottleneck for CAR-T cell production. Emerging gene-editing technologies are fostering a new paradigm in synthetic biology for the engineering and production of CAR-T cells. Firstly, the generation of the modular reagents utilized for gene editing with the CRISPR-Cas systems can be scaled-up with high precision under good manufacturing practices, are interchangeable and can be more sustainable in the long-run through the lower material costs. Secondly, gene editing exploits the precise insertion of CARs into defined genomic loci and allows combinatorial gene knock-ins and knock-outs with exciting and dynamic perspectives for T cell engineering to improve their therapeutic efficacy. Thirdly, allogeneic edited CAR-effector cells could eventually become available as “off-the-shelf” products. This review addresses important points to consider regarding the status quo, pending needs and perspectives for the forthright evolution from the viral towards gene editing developments for CAR-T cells.

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Le stress oxydant joue un rôle majeur dans le développement et l’évolution des maladies inflammatoires de l’intestin. Le corps humain est doté d’une panoplie d’enzymes antioxydantes ayant pour fonction de protéger l’intégrité cellulaire. De nouvelles enzymes au fort potentiel antioxydant, les paraoxonases (PON) 1, 2 et 3, ont récemment été identifiées tout au long du tube digestif, mais leurs rôles y restent inconnus. Les cellules intestinales Caco-2/15, qui ont la capacité de se différencier et d’acquérir les caractéristiques physiologiques de l'intestin grêle, ont été utilisées dans le présent travail pour étudier la régulation des PON. Les cellules ont été traitées avec différents effecteurs physiologiques (cytokines, LPS, stress oxydant) et pharmacologiques (fibrates, thiazolidinédiones) et l’expression des leurs gènes et protéines a été évaluée. Les résultats ont mis en lumière la modulation distincte de l’expression des PON par le stress oxydant et l’inflammation. Ceci suggère que chaque PON peut jouer un rôle différent au niveau intestinal et être impliquée dans le maintien de l’homéostasie. La régulation de l’expression des PON a également été largement explorée dans un article de revue. Pour définir le rôle de PON2, celle-ci étant potentiellement la plus importante pour l’homéostasie intestinale, les cellules Caco-2/15 ont été infectées à l’aide de lentivirus contenant des ARN d’interférence, ce qui a fortement réduit l’expression de PON2. En l’absence de PON2, les cellules Caco-2/15 étaient plus susceptibles face à un stress oxydant, la réponse inflammatoire était exacerbée et la perméabilité cellulaire paraissait altérée. Toutes ces composantes sont majeures dans le développement des maladies inflammatoires de l’intestin chez l’humain. De plus, des cellules Caco-2/15 de la PON2, ce qui a renforcé la force de la défense antioxydante cellulaire. Les résultats suggèrent que les PON jouent un rôle dans le maintien de l’homéostasie intestinale et pourraient être impliquées dans l’étiologie et la pathogenèse des maladies inflammatoires de l’intestin. / Oxidative stress is a major part of the pathogenesis of inflammatory bowel disease (IBD). The endogenous antioxidative defence is formed of multiple enzymes that have to protect cellular integrity. Paraoxonases (PON1, 2 and 3) are antioxidant enzymes that have recently been identified throughout the digestive tract, but their roles remain unclear in the intestine. Intestinal Caco-2/15 cells, which have the capacity to differentiate and exhibit the functionality of the small intestine, were used to study PON’s regulation. Cells were treated with various physiological effectors (cytokines, lipopolysaccharides, oxidative stress) and pharmacological molecules (fibrates, thiazolidinediones) and gene and protein expression were determined. Results obtained showed that PONs are distinctly modulated especially by inflammation and oxidative stress and suggests that they could play different roles in the maintenance of intestinal homeostasis. PONs regulation has also been the main topic of a review article. Our results and the literature pointed to PON2 as the most important PON for intestinal health. To better define PON2 functions in the intestine, PON2 expression was knocked-down using lentiviral infection and plasmids containing anti-PON2 shRNA. In the relative absence of PON2, Caco-2/15 cells were more susceptible towards induction of oxidative stress, the inflammatory response was exacerbated and cell permeability seemed altered. All of these components are major players involved in the development of human IBD. Moreover, Caco-2/15 cells were treated with purified PON2, which increased their antioxidative defence. All of these results suggest that PONs are implicated in the antioxidative and anti-inflammatory response in intestinal epithelial cells and makes them potentially important players for the aetiology and pathogenesis of IBD.

Le déclin de la fonction respiratoire dans l’asthme est associé à une augmentation de la massedu muscle lisse bronchique. La cellule musculaire lisse (CML) bronchique joue un rôle central dans la physiopathologie de l'asthme. Elle participe à l'inflammation et constitue une composante importante du remodelage des voies aériennes. Récemment, le rôle de la chitinaseYKL-40 comme bio marqueur de ce remodelage dans l'asthme a été évoqué. Dans ce contexte, la question centrale ayant motivé une partie de ce travail de thèse, a été d'étudier les effets d'YKL-40 sur différentes propriétés des CML. Nous nous sommes également intéressés au récepteur activé par les protéases, de type 2 (PAR-2), potentiel récepteur d'YKL-40, mais aussi acteur principal de l’inflammation bronchique.Ce travail a permis d'établir qu'YKL-40 est plus qu'un simple bio marqueur dans l'asthme puisqu’elle altère les propriétés physiologiques de la CML, et semble jouer un rôle dans le remodelage musculaire lisse bronchique. Par ailleurs, ce travail a également permis de mettre en évidence une surexpression du PAR-2 dans les CML d’asthmatiques ainsi qu’une augmentation de son expression et de la prolifération cellulaire, en réponse à une stimulation chronique. Ce travail a enfin permis d’optimiser la technique de l’interférence ARN lentivirale dans les CML bronchiques humaines. / The decline in lung function in asthma is associated with an increased bronchial smooth muscle (BSM) mass. BSM cells play a central role in the pathophysiology of asthma. They are involved in inflammation and are an important component of airway remodelling. Recently, the role of the chitinase YKL-40 as biomarker of this remodelling in asthma has been evoked. In this context, the central question that motivated part of this thesis was to study the effects of YKL-40 on various properties of BSM cells. We also studied the protease activated receptor 2 (PAR-2), as a potential receptor of YKL-40, as well as an important actor of airway inflammation.This work has established that YKL-40 is more than just a biomarker for asthma since YKL-40 alters physiological properties of BSM cells and appears to play a role in BSM remodelling. Moreover, this work has also highlighted an overexpression of PAR-2 in asthmatic BSM cells, as well as an increase of both PAR-2 expression and BSM cell proliferation in response to chronic stimulation. This work has finally allowed us to optimize lentiviral RNA interference in human BSM cells.

A ce jour, un certain nombre d'adénomes hypophysaires somatotropes échappent encore au contrôle de la maladie. Il reste nécessaire d'étudier plus finement les différentes voies de contrôle de la sécrétion hormonale et de la prolifération tumorale afin d'identifier des traitements alternatifs susceptibles de contrôler ces symptômes tumoraux. De même, un certain nombre de patients porteurs de prolactinomes (environ 10%), à l'origine de dysfonctionnement gonadique et sexuels, échappent au contrôle de l'hypersécrétion de prolactine par les traitements actuels. Pour ces deux types de tumeurs, s'ajoutent des complications neurologiques consécutives à l'accroissement des volumes tumoraux. Dans une perspective de contrôle de l'hypersécrétion tumorale nous nous sommes intéressés à deux stratégies faisant intervenir un transfert de gènes dans les cellules. POU1F1 est un facteur de transcription crucial pour le développement et le maintien des lignages somatotrope, lactotrope et thyréotrope et son expression est retrouvée dans les adénomes hypophysaires. De plus, des mutations de ce gène, notamment le mutant dominant négatif R271W, ont été identifiés chez des patients présentant des déficits hypophysaires combinés en GH, en PRL et en TSH. Une première partie de ma thèse a donc porté sur l'étude du transfert du mutant dominant négatif de POU1F1 par un vecteur lentiviral dans les tumeurs hypophysaires somatotropes et lactotropes afin d'évaluer son impact sur la sécrétion hormonale et sur la viabilité cellulaire. Nous avons montré que l'inactivation de ce facteur de transcription entrainait une diminution de la viabilité cellulaire et de la sécrétion de toutes les tumeurs testées. / To this date, some somatotroph adenomas do not respond to the current treatment of pituitary tumors. It still necessary to study the different ways to control the hormonal secretion and the tumor progression to identify alternate therapy capable to manage these symptoms. Likewise, some patient with prolactinomas (about 10%) responsible of gonad and sexual dysfunction do not respond to the control of prolactin hypersecretion. In addition, some neurological complications are finding due to the neighboring structures compression for both of these tumors. To better control the hormonal secretion we investigate two different strategies involving a gene transfer in the cells. POU1F1 is a major transcription factor involved in the pituitary development and the management of somatotroph, thyreotroph and lactotroph cell lines. Its expression is found in pituitary adenomas belonging of these 3 cell lines. Moreover some mutations like the R271W mutant are identified in patients with combined pituitary hormonal deficiency (CPHD). In the first part of my thesis we transfer this dominant negative mutant via a lentiviral transfer in the somatotroph and lactotroph adenomas to evaluate its impact on hormonal secretion and cell viability. We show an inhibitory effect of the POU1F1 inactivation on the secretion and the cell viability of all our tumors. Somatostatin analogs are the major medical therapy for somatotroph adenomas and control GH secretion in 60% of these patients. Octreotide resistance is highly correlated to a low expression of the sst2 receptor.

L’infection par le virus de l’hépatite B (VHB) reste un problème de santé publique avec plus de 880 000 décès chaque année dans le monde. Au stade chronique de l’infection virale B, des complications peuvent survenir comme la fibrose, la cirrhose et le carcinome hépatocellulaire. L'implication du VHB, de ses protéines ou de sa variabilité génétique dans la fibrose hépatique reste à élucider. Toutefois, des méta-analyses semblent montrer un lien statistiquement significatif entre la présence de la double mutation A1762T/G1764A dans le promoteur basal du core (PBC) du VHB et la fibrose sévère. C’est pourquoi nous avons orienté nos travaux de recherche selon deux axes : l’implication des mutations du PBC et de la région précore (PC) dans la sévérité des lésions hépatiques ainsi que le rôle des protéines HBc et HBe du VHB dans l’induction de la fibrogénèse. Dans un premier temps, deux études cliniques ont permis de confirmer l’association significative du double mutant PBC avec la fibrose sévère indépendamment du génotype viral. Nous avons également démontré un effet antagoniste de la mutation G1899A située dans la région PC vis-à-vis du double mutant PBC dans la sévérité de la fibrose. La deuxième partie de nos travaux a consisté à développer une technique innovante de production de protéines en utilisant la transduction de la lignée HepaRG par des vecteurs lentiviraux contenant la séquence de la protéine GFP (green fluorescent protein). En parallèle, nous avons synthétisé des particules lentivirales contenant les séquences sauvages et mutées du PBC et de la région PC, avec pour objectif de produire les protéines HBc et HBe dans les HepaRG. / The hepatitis B virus (HBV) infection remains a significant public health problem with more than 880 000 deaths every year worldwide. At the stage of chronic HBV infection, complications can occur such as fibrosis, cirrhosis and hepatocellular carcinoma. The role of HBV, its protein and its genomic variability in fibrosis are still unclear. Meta-analysis seems to indicate a strong link between the double mutation A1762T/G1764A detection in the basal core promotor (BCP) of HBV and the development of fibrosis. In this context, our work aimed to explore: i. the implication of BCP or precore (PC) regions mutations on the severity of fibrosis, and ii. the role of HBc and HBe proteins in fibrosis induction. For the first approach, our studies confirmed the association between the presence of the BCP double mutation and severe fibrosis, independently of the viral genotype. We also showed that the G1899A mutation in the PC region presents an antagonist effect regarding the double BCP mutant for fibrosis severity. In the second part of our work, we developed an innovative technology to produce protein via the transduction of HepaRG cells using lentiviral technology with a plasmid vector containing GFP (greenfluorescent protein) sequence. We also obtained lentiviral particles containing the wild and mutated sequences for the BCP and PC regions, in order to produce HBc and HBeprotein in HepaRG cells and to explore of the pathogenic role of BCP and PC mutants.

A imunoterapia utilizando linfócitos T modificados com receptor quimérico de antígenos (CAR) tem se mostrado eficaz no tratamento de leucemia e linfomas resistentes à quimioterapia. A proteína CD19 é considerada um alvo ideal porque é expressa na maioria dos tumores de linfócitos B e linfócitos B normais, mas não em outras células. Estudos clínicos recentes mostraram excelentes respostas de linfócitos T-CAR em uma variedade de tumores de células B. Os vetores lentivirais são o método mais comumente utilizado para modificação genética em ensaios clínicos. Este estudo teve como objetivo desenvolver uma plataforma eficiente para a produção de lentivírus e testar a funcionalidade desses vetores para que possam ser usados para modificar geneticamente linfócitos T. A transfecção transiente de céulas HEK293T com plasmídeos na proporção de 3:1:1:1 (transgene:gag-pol:VSV-G:rev) utilizando lipossomos catiônicos e 5 mM de butirato de sódio resultou nos títulos virais mais elevados. Isso representa um aumento de 17 vezes no título viral da transfecção com polietilenoimina (PEI). Três métodos para concentracao lentiviral foram utilzados nesse trabalho, ultracentrifugação, filtração tangencial e ultrafiltração. A ultrafiltração sobre membrana com corte de peso molecular (MWCO) de 100 kDa resultou na maior taxa de recuperação de partículas virais viáveis, aproximadamente 82%. As partículas virais produzidas por este processo demonstraram ser funcionais para a transdução de linfócitos T. Além disso, o receptor quimérico (CAR) se mostrou específico contra o antígeno CD19 de células B, resultando na ativação dos linfócitos T-CAR e gerando citotoxicidade contra células CD19+ in vitro. Houve uma redução de aproximadamente 87% das células alvo, quando analisado por citometria de fluxo e uma citotoxicidade média de 50% foi observada por ensaios colorimétricos. / Immunotherapy using T cells modified with chimeric antigen receptor (CAR) has been proven effective in the treatment of leukemia and lymphomas resistant to chemotherapy. CD19 protein has been shown to be an ideal target because it is expressed on most B-cell tumors and normal B cells, but not in other cells. Recent clinical studies have shown excellent responses of CAR T-cells in a variety of B-cell tumors. Lentiviral vectors are the most commonly used method for genetic modification in clinical trials. This study aimed to develop an efficient platform for lentiviral production and to test the functionality of those vectors so that they can be used in to genetically modify T cells. Transient transfection of HEK293T cells with plasmids in a 3:1:1:1 ratio (transgene:gag-pol:VSV-G:rev) using cationic liposomes and 5 mM sodium butyrate resulted in the highest viral titers. That represents a 17-fold increase in viral titer from polyethylenimine (PEI) transfection. Three methods for lentiviral concentration were used in this work, ultracentrifugation, tangential filtration and ultrafiltration. Membrane ultrafiltration with 100 kDa molecular weight cutoff (MWCO) resulted in the highest recovery rate of viable viral particles, approximately 82%. The viral particles produced by this process have been shown to be functional for the transduction of T cells. In addition, the chimeric receptor (CAR) was shown to be specific against the B cell antigen CD19, resulting in the activation of CAR-T cells and generating cytotoxicity against CD19+ cells in vitro. There was a reduction of approximately 87% of the target cells when analyzed by flow cytometry and an average cytotoxicity of 50% was observed by colorimetric assays.

Os vetores retrovirais representam uma das melhores opções para transferência e terapia gênica, pois fornecem expressão do transgene em longo prazo. Entretanto, a inserção do provírus pode causar mutagênese insercional, induzindo proto-oncogenes. Eventos deste tipo têm sido descritos em protocolos clínicos para o tratamento de SCID-X1, doença granulomatosa crônica e talessemia beta, quando vetores retrovirais (oncorretrovirus) foram utilizados. Atualmente, existem poucos métodos simples e rápidos para revelar e quantificar a expansão clonal. Assim, descrevemos a construção uma biblioteca de vetores contendo uma marcação aleatória, denominada código de barras. O sequenciamento do código de barras permitirá revelar, caracterizar e até quantificar a expansão clonal de uma população de células transduzidas. Esta metodologia ajudará a testar novos arranjos de promotores e genes terapêuticos, para o desenvolvimento de vetores mais seguros contribuindo para a redução da probabilidade de um evento de proliferação clonal desencadeado pela mutagênese insercional. / Retroviral vectors represent one of the best options for gene transfer and therapy, where long-term transgene expression is required. However, insertion of the provirus can cause insertional mutagenesis, which may have adverse consequences, such as induction of proto-oncogenes. Such events have been described in clinical trials for the treatment of SCID-X1, chronic granulomatous disease and beta thalessemia with some retroviral vectors. Currently, there are few simple and quick methods that can reveal and quantify clonal expansion. Thus, we describe the construction of a vector library containing random markers, called \"barcodes\". The sequencing of the barcode could reveal, characterize and quantify the clonal expansion of a transduced cells population. This methodology will be valuable to test new arrangements of promoters and therapeutic genes, allowing the development of safer vectors, helping to reduce the probability of clonal proliferation events triggered by insertional mutagenesis.