Une transduction efficace des cellules souches hematopoïetiques est un préalable pour la thérapie génique des maladies génétiques comme la β‐thalassemie, l’Adrenoleucodystrophie et le Déficit Immunitaire Combiné Sévère. La petite molécule UM171 à été décrite comme étant une molécule capable de stimuler l’expansion in vitro des cellules souches hématopoïétiques humaines, permettant ainsi une plus large application des thérapies basées sur les cellules souches. Nous avons aussi conduit des études supplémetaires pour confirmer la capacité de UM171 à expandre les souches hématopoïétiques. Durant ce travail, nous avons découvert que UM171 pouvait aussi augmenter de maniére significative, l’efficience de la transduction lentivirale des cellules hématopoïetiques primitives dérivées de sang de cordon. En plus, nous avons montré que UM171 augmentait la transduction des cellules hématopoïeques ayant les phénotypes les plus immatures. Des études plus approfondies ont aussi révélé que UM171 pouvait aussi augmenter la transduction des cellules souches hématopoïétiques avec des lentivirus ayant diffèrent pseudotypes. Au total ces découvertes ont pour conséquence, une nette amélioration des protocoles d’expansion et de transduction des cellules souches hématopoïétiques à travers un meilleur rendement en cellules souches et des taux élevés de transfert de gène en utilisant des quantités réduites de particules virales / Efficient lentiviral gene transfer to hematopoietic stem cells is a prerequisite for theultimate goal of gene therapy for a range of major genetic diseases such as β‐thalassemia, Adrenoleucodystrophy and severe combined immnodeficiency. The small molecule UM171 was recently described as having potent ability to stimulate ex vivo expansion of human hematopoietic stem cells, another key to safer and wider application of stem cell mediated therapies. Here we have conducted additional studies to confirm the stem cell expansion properties of UM171 and in the course of this work discovered that it also has the ability to significantly enhance the efficiency of the lentiviral transduction of primitive hematopietic cells in human cord blood. Subsequent work confirmed that this enhancing effect extends importantly to the most primitive hematopoietic subset as assessed phenotypically and by functional readout in immunodeficient mouse xenografts. Further detailed characterization ofthis phenomenom revealed that UM171’s effects are manifest rapidly and extend to a range of lentiviral pseudotypes. Together these findingsprovide an avenue for improved protocols for hematopoietic stem cell transduction that achieve higher gene efficiency and stem cell recovery coupled with the potential for reduced viral titer requirements.

Cystic fibrosis (CF) is an autosomal recessive genetic disorder of which lung disease is the leading cause of morbidity and mortality. One attractive strategy for the treatment of CF lung disease is to directly deliver CF transmembrane conductance regulator gene to airway epithelia. Although promising results have been reported, barriers present in the lung make successful gene transfer to the respiratory tract difficult. In order to improve gene transfer strategies in the intrapulmonary airways, we need to identify the bottlenecks of transduction for the vector system. A previous study reported that feline immunodeficiency virus (FIV)-mediated gene transfer was more efficient in the nasal airways in mice than the intrapulmonary airways (Sinn, P.L. et al. 2008, J. Viol). Our first goal was to identify barriers to lentiviral gene transfer in the murine airways. We demonstrate that host immune response is not the major barrier preventing efficient FIV-mediated transduction in the intrapulmonary airways. We show that the FIV vector transduces murine primary nasal epithelial cell cultures with greater efficiency than murine primary tracheal epithelial cell cultures. In addition, GP64 pseudotyped vesicular stomatitis virus (VSV) transduces better in nasal epithelia compared to intrapulmonary airways in mice. On the other hand, we observed that VSVG glycoprotein-pseudotyped VSV transduces the intrapulmonary airway as well as nasal epithelia in mice with similar efficiency. Our results suggest that differentially expressed cellular factor(s) specific for GP64 or FIV vector may be the major barrier(s) for FIV vector-mediated gene transfer in the murine intrapulmonary airways. The recent development of CF porcine models prompted us to investigate possible barriers for lentiviral vector-mediated gene transfer in porcine cells. Our preliminary results showed that HIV transduction was restricted in porcine but not human lung-derived cell lines. Porcine TRIM5 has sequences similar to restrictive bovine TRIM5 orthologs. Therefore, our second goal was to investigate the possible restriction of lentiviral vectors by porcine TRIM5. We demonstrate that transient overexpression or knockdown of endogenously expressed porcine TRIM5 does not affect HIV or FIV transduction. Lastly, we characterized a mucin domain-deleted EBOV (EBOVΔO) glycoprotein mutant with increased transduction. This EBOVΔO 5-mer mutant was generated based on mutants with an increased transduction as identified by alanine scanning mutagenesis (Brindlay, M.A. et al. 2007. J. Viol). We show that VSV pseudotyped with the 5-mer mutant increased transduction both in vitro and in mice when compared to the wild-type EBOVΔO. Structural analysis demonstrated that 5 mutations were located proximal to the GP1-GP2 interface. Enhanced transduction likely results from a lower energy metastable state of the glycoprotein. FIV pseudotyped with 5-mer also shows increased transduction in multiple cell lines. Identification of barriers in intrapulmonary airways and improvements of vector systems will help the advancement of gene therapy for CF.

Vectors based on lentivirus backbones have revolutionized our ability to transfer genesinto many cell types. Lentiviral vectors integrate into the chromatin of target cells and do not transfer any viral genes causing vector replication. Both of these features arecommonly used in gene therapy and have been used clinically in individuals sufferingfrom cancer, infections and genetic diseases. It has been discovered that T-cells can be genetically modified to be used as effective weapons against cancer: therefore virus mustbe produced to deliver the gene of interest into the T-cells. In this project, lentiviralvectors have been produced to transfer the gene coding for a chimeric antigen receptor(CAR) which is directed to CD19 on B-cells. The vectors will, hence, be used to generateCD19 retargeted T-cells in purpose to kill CD19 cells such as B-cell lymphoma andleukemia. We have evaluated two production protocols to determine a feasible method toculture these vectors. We have also stimulate T-cells with two different antibodies (anti-CD3 and anti-CD28) and transduced T-cells. Our results demonstrate that theconcentration of virus was higher after prolonged incubation in 4˚C, which can not beexplained. The stimulation demonstrated that bound anti-CD3 was the best stimulator,and moreover the FACS-analysis showed that addition of anti-CD28 gave a highertransduction level. In conclusion, the viral vectors may be kept in 4˚C for two days beforeconcentrating the virus, and bound anti-CD3 is a better choice than soluble anti-CD3 forstimulation of T-cells.

Sickle cell disease (SCD) is a severe hemoglobin disorder caused by co-inheritance of a single mutation in the β-globin gene of adult hemoglobin (HbA; α2β2). This alteration leads to the formation of sickle hemoglobin (HbS; α2βS2) and deformed, sickle-shaped red blood cells (RBCs). Sickle RBCs obstruct small blood vessels resulting in anemia, excruciating pain crises, organ damage, and stroke. For the millions of people affected by this disease, life expectancy is only 40-60 years of age. The only cure for SCD is hematopoietic stem cell (HSC, CD34+) transplantation, which requires a human leukocyte antigen (HLA)-matched donor. However, this option runs the risk of complications associated with graft versus host disease and infection. Before birth, individuals with SCD do well because their RBCs are filled with γ-globin containing fetal hemoglobin (HbF; α2γ2), which inhibits the formation of HbS. In fact, some SCD patients who co-inherit mutations that allow for high-level expression of HbF into adulthood are asymptomatic. This suggests that genetic modification of the patient’s own HSCs to permit HbF production would be a viable therapeutic alternative to HSC transplantation. Our work has focused on the use of lentiviral vectors to introduce an exogenous γ-globin gene or shRNA sequences designed to knockdown repressors of γ-globin, such as the zinc-finger transcription factor, BCL11A, to prevent silencing of the endogenous γ-globin genes allowing for persistent expression of HbF. Despite significant progress using both approaches, we have been unable to increase the level of HbF > 30%; a curative threshold for SCD patients who continue to produce HbF into adulthood. The goal of my project was to combine these approaches into a single lentiviral vector to achieve co-regulated, erythroid-specific expression and augmented levels of HbF. I successfully modified the insulated, erythroid-specific γ-globin vector (termed V5m3-400) to include microRNA (miR)-adapted shRNAs (or shmiRs) targeting BCL11A (based on miR-30 and miR-E architectures) in the first and second noncoding introns of the γ-globin genomic sequences. Inclusion of shmiRs had no appreciable effect on integrity of the integrated provirus or vector titer. Vector performance was initially tested using human K562 erythroleukemia cells expressing a flag-tagged version of BCL11A. In this cell line, BCL11A knockdown was significantly improved using miR-E-shRNAs due to a dramatic increase (up to 350-fold) in processing of mature shRNA sequences. The miR-E vectors also provided high-level expression of γ-globin. Erythroid-specific expression of the γ-globin transgene and BCL11A knockdown was confirmed in maturing erythroid cells derived from transduced CD34+ cells of a healthy donor resulting in a 50% increase in HbF levels compared with cells transduced with V5m3-400 as a control. While encouraging, I was unable to discriminate HbF derived from the vector-encoded versus endogenous γ-globin genes. To address this, I introduced a single base change in exon 2 of the γ-globin gene encoded by V5m3-400 such that threonine replaces isoleucine at amino acid 75 (I75T). This variant was successfully distinguished from endogenous γ-globin gene products by reverse phase high performance liquid chromatography (HPLC) in culture-differentiated erythroid cells. Based on these findings, I created compound γ-globin/shmiR-E vectors that include the I75T substitution (I75Tγ-globin/shmiR-E). Future studies will focus on testing this novel vector design in erythroid cells derived from transduced CD34+ cells of healthy donors and patients with SCD. I anticipate that this compound vector has the potential to maximize γ-globin expression and promote levels of HbF that are unlikely to be safely and effectively achieved by conventional globin gene addition or shRNA knockdown approaches alone.

Nous avons récemment développé un prototype lentivecteur ADN non intégratif vaccinal contre VIH-1/SIDA que nous avons testé chez des modèles animaux. L'immunisation avec une dose unique de ce vaccin (CAL-SHIV-IN-) a permis la mise en place rapide de réponses immunes spécifiques contre tous les antigènes exprimés par le vaccin chez tous les animaux vaccinés. Les analyses longitudinales ont démontré la mise en place de réponses cellulaires et humorales spécifiques et persistantes sur une durée de plus de 74 semaines en absence de réintroduction d'antigènes chez tous les macaques vaccinés. La caractérisation de ces réponses a révélé la présence de cellules T CD4+ et CD8+ polyfonctionnelles composées de fractions de cellules effectrices mémoires à fonction immédiate (EM), de cellules centrales mémoires (CM) et de cellules précurseurs mémoires ayant une haute capacité de prolifération (PHPC). Ces réponses corrèlent, chez tous les macaques vaccinés (6/6), avec un contrôle du virus d'épreuve hautement hétérologue et pathogénique (SIVmac251) inoculé à petites doses répétées par la voie mucosale rectale. Cette protection est maintenue durant toute la période d'un an de suivi après l'infection avec une différence statistiquement significative de la charge virale plasmatique des groupes contrôles et vaccinés au moins jusqu'à 18 semaines post-infection. Par ailleurs, le contrôle du virus d'épreuve est maintenu plus de 10 mois (correspondant au temps d'arrêt de l'étude) après l'infection. Parmi les corrélats immunologiques de protection nous avons identifié la présence de cellules de type PHPC spécifiques des antigènes du vaccin et qui sont dotées d'une capacité importante de prolifération ex vivo en présence des signaux antigéniques et homéostatiques. Nous avons démontré que ces PHPC contiennent une fraction de cellules T souches mémoires « TSCM » spécifiques du vaccin. Ces TSCM récemment identifiées constitueraient un atout majeur en faveur de notre vecteur et notre stratégie vaccinale du fait de leur haute capacité d'auto-régénération/maintien en absence d'antigène et leur capacité à se différencier en d'autres cellules mémoires TCM et TEM. / We recently developed an innovative prototype non-integrative lentivector DNA vaccine against HIV-1 /AIDS that we tested in pilot studies using animal models of HIV vaccine. We found that a single immunization with our prototype vaccine (CAL-SHIV-IN-) allowed the implementation of potent humoral and cellular responses in all immunized macaques. In addition, both types of responses persisted over a period of 74 weeks post-immunization in absence of antigenic boost. The characterization of the above revealed that vaccine specific T cell responses included polyfunctional CD4+ and CD8+ T cells against all antigens expressed by the vaccine. Detailed phenotypic and functional examinations of these cells showed that they were composed of effector (EM) and central memory (CM) T cells. More importantly they also contained a fraction of precursor memory T cells with high proliferative capacity (PHPC). Immune responses primed by our vaccine regiment correlated with protection in all vaccinated macaques (6/6). As expected our vaccine-induced immune responses did not prevent from infection acquisition but controlled the replication of the highly pathogenic and heterologous SIVmac251 challenge given as repeated low dose by the intrarectal mucosal route. All vaccinated animals (6/6) controlled their viremia to undetectable level using conventional PCR during at least 10 months post infection (end of the experiment). We further focused on PHPC responses associated with viral control and found that these cells vigorously proliferate upon ex vivo stimulation with specific antigens in presence of the homeostatic IL-7 and IL-15 cytokines. Proliferating antigen specific cells contained a type of stem cell-like memory T cells (TSCM). These latter (TSCM) might be a major asset in favor of our lentivector and vaccination strategy due to their high capacity for self-regeneration/maintenance in absence of antigen source.

L’hyperoxalurie primitive de type 1 (ou HP1) est une maladie héréditaire du métabolisme liée à un déficit en enzyme hépatocytaire AGT (alanine:glyoxylate aminotransférase), codée par le gène AGXT. Ce déficit entraîne, chez les patients atteints d’HP1, une excrétion hépatique accrue d’oxalate ; celui-ci est ensuite éliminé dans les urines où il se complexe avec le calcium pour former des néphrolithiases oxalo-calciques massives, pouvant conduire à une insuffisance rénale chronique. Le seul traitement curatif disponible pour cette pathologie est la greffe allogénique combinée hépatorénale, actuellement limitée par la disponibilité des donneurs de greffons, une morbi-mortalité significative et la nécessité d’un traitement immunosuppresseur au long cours. L’objectif du projet de recherche est de développer une thérapie génique de l’HP1 par greffe de cellules hépatiques autologues génétiquement corrigées. La faible disponibilité et la difficulté d’amplification in vitro des hépatocytes adultes nous a conduit à explorer la piste des cellules souches pluripotentes induites (iPSCs) pour produire des cellules hépatiques humaines utilisables en médecine régénérative. Nous avons dérivé et caractérisé des lignées de cellules iPSCs à partir de fibroblastes de patients atteints d’HP1, après expression transitoire des facteurs de reprogrammation par des vecteurs Sendai. Nous avons développé deux stratégies de thérapie génique additive par insertion d’un minigène codant une séquence optimisée de l’ADNc AGXT au moyen (1) d’un vecteur lentiviral à expression hépato-spécifique et (2) d’un processus de recombinaison homologue au locus AAVS1 facilité par le système de clivage ciblé de l’ADN « CRISPR/Cas9 ». Enfin, nous avons mis en évidence l’expression de la cassette thérapeutique après différenciation hépatocytaire des iPSCs génétiquement corrigées. Ces résultats ouvrent de nouvelles perspectives de médecine régénérative pour l’HP1 par transplantation de cellules hépatocytaires autologues génétiquement corrigées dérivées d’iPSCs de patients. / Primary hyperoxaluria type 1 (or PH1) is an inherited metabolic disorder related to the deficiency of the hepatic AGT enzyme (alanine:glyoxylate aminotransferase), which is encoded by the AGXT gene. In PH1 patients, this deficiency leads to oxalate overexcretion by liver, followed by urine filtration and complexation with calcium to form massive calcium-oxalate nephrolithiasis potentially leading to chronic renal failure. The only available curative treatment is combined hepatorenal allogeneic engraftment, which is currently limited by the availability of transplant donors, significant morbidity and mortality, and the need for long-term immunosuppressive treatment. The aim of our research project is to develop gene therapy for PH1, consisting in engraftment of genetically corrected autologous liver cells. Considering that adult hepatocytes are hardly available and expandable in vitro, we chose to explore the use of induced pluripotent stem cells (iPSCs) to produce human liver cells for application in regenerative medicine. We derived and characterized iPSC lines from PH1 patient fibroblasts after transient expression of reprogramming factors delivered by Sendai virus vectors. We developed two additive gene therapy strategies by inserting a minigene encoding an optimized AGXT cDNA sequence using (1) a lentiviral vector designed for liver-specific expression and (2) homologous recombination process at the AAVS1 locus favoured by the targeted DNA cutting system “CRISPR/Cas9”. Finally, we highlighted therapeutic cassette expression after hepatic differentiation of genetically corrected iPSCs. These results pave the way for regenerative medicine for PH1 by transplantation of genetically modified autologous hepatocyte-like cells derived from patient-specific iPSCs.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Les IFN-I (interférons de type I), principalement IFN et , constituent un mécanisme de défense primordial de l’hôte contre les pathogènes. Pourtant, dans le cas du VIH-1 (virus de l’immunodéficience humaine), la relation entre les IFN-I et la réplication virale apparaît plus complexe. En effet, si les IFN-I inhibent la réplication du VIH-1 ex vivo, un état d’hyperactivation permanent de la réponse IFN-I a été récemment associé à la progression vers le SIDA ainsi qu’à une forte virémie chez les patients infectés par le VIH-1. De même, la dérégulation de la réponse IFN-I est un critère déterminant dans l’issue pathogénique de certains modèles d’infection virale chez le singe. Si l’hypothèse du rôle pathogénique des IFN-I s’avère correcte, le VIH-1 pourrait avoir évolué afin de se répliquer même en présence d’une telle réponse, qui semble être au final, plus délétère pour l’hôte que pour le virus. L’objectif de ce travail a été d’évaluer la résistance du VIH-1 aux IFN. Dans ce contexte, le VIH-1 a été comparé au VIH-2 et au SIVmac (virus de l’immunodéficience simienne), virus phylogénétiquement proches mais peu ou pas pathogènes pour l’homme, lors de l’infection de plusieurs types cellulaires tels que des lymphocytes, des macrophages et des cellules dendritiques. En accord avec l’hypothèse initiale de travail, les expériences réalisées ont montré que le VIH-1 est capable de se répliquer dans les cellules primaires prétraitées avec des doses d’IFN comparables à celles mesurées in vivo, alors que la réplication des virus VIH-2/SIVmac est complètement bloquée, même à des concentrations très faibles d’IFN. Ce travail a permis de démontrer que le blocage induit par l’IFN s’exerce au niveau des phases précoces de l’infection et plus précisément à l’étape de la transcription inverse. En effet, les données obtenues suggèrent que l’IFN induit l’expression d’un effecteur cellulaire qui affecte différentiellement la stabilité des complexes viraux, ce qui se traduit par un défaut d’accumulation de l’ADN viral plus important pour le VIH-2 et le SIVmac, que pour le VIH-1. La différence de susceptibilité des lentivirus de primates aux IFN-I pourrait ainsi expliquer en partie, les différents niveaux de réplication de ces virus, associés à leurs degrés de pathogénicité in vivo. / Type I Interferons (IFN-α/β, herein IFNs) provide an important mechanism of defense against pathogens and regulate in a paracrine and autocrine manner both intrinsic and adaptive immune responses. In the case of HIV-1 however, the relationship between IFNs and viral replication appears more complex. Indeed, if IFNs have been described to interfere with HIV-1 at basically all phases of its life cycle ex vivo, an IFN-induced state is linked to AIDS progression and to high viral loads in HIV-1 infected individuals. Similarly, a deregulated and prolonged IFN production/state seems one of the main distinguishing features between pathogenic and non-pathogenic SIV infection in primate animal models, suggesting that a deregulated IFN-state may be more detrimental to the host than to the virus itself in vivo.If this hypothesis is correct and if HIV-1 plays an active role in the perpetration of this antiviral state, it is possible that HIV-1 may have overall evolved to cope with this environment, remaining able to replicate despite it.To determine whether HIV-1 was better armed to replicate in the presence of an IFN-state environment than other primate lentiviruses, we compared HIV-1 to SIVmac and more importantly to HIV-2 that albeit capable of inducing AIDS in humans does so in a much less aggressive manner. In agreement with the initial hypothesis, our results indicate that HIV-1 is better fit to replicate in primary cells in the presence of amounts of IFN comparable to the ones measured in vivo, while the replication of HIV-2/SIVmac viruses is completely blocked even in the presence of low levels of IFN. By decorticating the effects of IFNs on the early and late phases of the viral life cycle in primary macrophages, we show here that the main target of the differential action of IFNs are the early phases of infection. More specifically, with time kinetics that we determine herein, IFNs induce cellular factor/s that differentially affect the stability of pre-reverse transcription complexes of HIV-2, but not of HIV-1. Our results could underlie a different evolutionary adaptation of primate lentiviruses to interferons that might be responsible for their different pathogenicity in vivo.

Une meilleure connaissance des déterminants viraux et cellulaires impliqués dans la régulation de l’expression lentivirale est essentielle pour (i) maitriser l’expression d’un transgène dans le cadre d’un transfert de gène et (ii) mieux comprendre l’absence/la perte de l’expression du virus de l’immunodéficience humaine de type 1 (VIH-1) observé en cas de latence virale après infection. Dans ce contexte, les facteurs cellulaires impliqués dans la reconnaissance et la réparation des cassures de l'ADN, activés dès les étapes précoces du cycle viral, pourraient participer au contrôle de l’expression rétrovirale. Dans la première partie de cette étude, nous avons généré une collection de vecteurs lentiviraux codant le transgène green fluorescent protein (gfp) avec des design variés : promoteur du VIH-1 [long terminal repeat (LTRs)] ou d’origine hétérologue [e.g., promoteur viral CMV (cytomegalovirus), ou humain PGK (phosphoglycerate kinase)], intégrase native ou mutée, LTRs natifs ou self inactivating (SIN). En particulier, nous avons caractérisé l’impact de l’insertion de différentes séquences hétérologues au sein des SIN-LTRs sur l’expression du transgène gfp au cours du temps dans un contexte compétent ou déficient pour l’intégration. Nous avons mis en évidence un phénomène de modulation du niveau d’expression du transgène (d’un facteur 0,3 à 1,8; comparé aux vecteurs sans insertion) qui est dépendant de la séquence insérée et de son orientation. L’expression transgénique résulterait donc d’une balance coûts/bénéfices associés à l’insertion d’éléments aux extrémités des vecteurs qui pourrait déterminer le niveau d’expression du transgène à partir de vecteurs modifiés. Dans la seconde partie, nous avons réalisé une analyse systématique et comparative de l’expression lentivirale au sein des cellules humaines de carcinome de côlon HCT 116 déplétées pour le complexe Ku. Ce complexe, qui est essentiel à la survie chez l’homme et est impliqué dans les processus de réparation de l’ADN, a été identifié comme une cible anti-VIH potentielle. Nous avons mis en évidence que la déplétion en Ku induit une diminution de l’expression précoce du VIH-1 de façon spécifique du LTR et indépendante de Tat. Même si l’action de Ku nécessite l’intégration du VIH-1, sa déplétion ne modifie pas l’efficacité d’intégration virale mais agit plutôt au niveau transcriptionnel. Da façon importante, la réactivation de l’expression du transgène après traitement par l’activateur de NF-κB (Nuclear Factor κB), le TNFα (tumor necrosis factor α) ou un inhibiteur des déacétylases d’histones, la trichostatine A est favorisée par la déplétion en Ku. A l’inverse, en présence d’un niveau normal en Ku, les cellules exprimant le VIH-1 seraient contre-sélectionnées dans le temps. Ainsi, la déplétion en Ku pourrait promouvoir l’établissement d’un état (reactivable) de latence transcriptionelle associé à une moindre contre-sélection des cellules transduites. Les résultats issus de ce travail de thèse démontrent que l’expression lentivirale varie en fonction de nombreux paramètres, dont (i) le design des vecteurs, (ii) le type cellulaire transduit et son fond génétique, et (iii) le temps écoulé depuis la transduction, reflétant ainsi des interactions différentielles entre le vecteur et son hôte. / An improved knowledge of the viral and cellular determinants implicated in the regulation of the lentiviral gene expression is crucial (i) for a better control of transgene expression in strategies designed for gene transfer and (ii) for uncovering the mechanisms of viral latency observed after infection with the human immunodeficiency virus type 1 (HIV-1) and accounting for the absence/loss of HIV-1 expression. Cellular factors involved in the mechanism of detecting/repairing DNA lesions are largely activated during the initial steps of viral cycle and, thus, may participate in the control of lentiviral expression. In the first part of this study, we generated a set of lentiviral vectors encoding for the transgene green fluorescent protein (gfp) with various design: the original promoter [long terminal repeat (LTRs)] or of heterologuous origins (e.g., the viral cytomegalovirus, CMV or the human phosphoglycerate kinase, PGK), wild-type or mutated integrase and wild-type or self inactivating (SIN) LTRs. By taking advantage of these constructs, we characterized the impact of the insertion of distinct heterologuous sequences within SIN-LTRs on the expression of gfp over the time in conditions of proficiency or deficiency for the integration. We put in evidence a phenomenon of modulation of the level of the transgene expression due to the insertion (by a factor of 0.3 to 1.8, as compared to vectors without insert) that was dependant from the nature and/or the orientation of the insert. We speculate that a balance between the costs and the benefits associated to insertion at the extremities of lentiviral vectors may dictates the level expression of transgene from this engineered construct.In the second part, we performed a systematic and comparative analysis of the lentiviral expression on human HCT 116 colon carcinoma cells depleted from the complex Ku. This complex, which is essential for the survival in humans and has a described role in DNA repair process, has been previously identified as a potential target against HIV-1. Here, we showed that Ku depletion induced a decrease of the HIV-1 early expression in a fashion that was specific for LTR and independent from Tat. Although Ku action needed HIV-1 integration to host genome, its depletion did not modify the viral integration efficiency but rather acted at transcriptional level. Importantly, the reactivation of transgene expression by administering either the NF-κB (Nuclear Factor κB) activator, tumor necrosis factor α (TNFα) or the histone deacetylase inhibitor named trichostatin A was favored in a condition of Ku depletion. On the contrary, in presence of normal level of Ku, cells expressing HIV-1 displayed a high level of counter-selection over the time. Thus, our observations pleased to favor the hypothesis that Ku depletion promotes the establishment of a state of (reactivable) transcriptional latency associated to a lesser counter-selection of transduced cells. Altogether, the results obtained during this thesis demonstrate that lentiviral expression vary depending on (i) specific vector design, (ii) the transduced cell line and its genetic backbone, and (iii) the time elapse from transduction, as a consequence of modified interactions between the vector and its host.

Cellular therapies rely on the delivery of therapeutic cells into patients, but their safety can be compromised by the manipulation of cells ex vivo or their placement outside of their natural context in vivo. Cell Fate Control Gene Therapy (CFCGT) offers the possibility of establishing pharmacological controls over gene-modified cells (GMCs) with regards to their proliferation, differentiation, or function. In its simplest form, 'suicide' gene therapy (SGT), stable introduction of a 'suicide' gene that can activate a non-toxic prodrug establishes control over the survival of GMCs. Current SGT modalities are sub-optimal in clinical setting. To overcome the many limitation of current strategies, we have developed a next-generation CFCGT approach based on the active site-engineered variants of human deoxyCytidine Kinase (dCK), which enable robust activation of multiple Nucleoside Analogue (NA)-based prodrugs, act early in the pathway enabling rapid accumulation of activated NAs in target cells, and also provide the capabilities for the direct imaging of GMCs. Stable introduction of dCK variants into target cells by means of Lentiviral (LV) gene transfer significantly increases their sensitivity to multiple prodrugs. Our dCK variant with only two active site amino acid substitutions is expected to be non-immunogenic yet capable of specifically activating deoxythymidine- and deoxyuridine-based NAs that are not substrates for the wild-type enzyme, such as bromovinyldeoxyuridine (BVdU) and L-deoxythymidine (LdT). We show here that dCK can be used for controlling the survival of GMCs, in cell lines and primary cells in vitro and in a murine xenogeneic transplant models in vivo. To characterize dCK/prodrug-mediated killing mechanisms in GMCs, we have examined the levels of active metabolites in cells and the cellular pathways they antagonize. We describe here the experimental basis for the application of this novel CFCGT in bone marrow transplantation for management of Graft-versus-Host Disease (GvHD) and in enhancing chemotherapy in direct treatment of tumors. In summary, we have developed a novel and robust strategy for effective CFCGT that addresses the many shortcomings of existing modalities. Future studies will validate this novel system in a variety of primary cells and animal disease models, including models of hematopoietic transplantation and ES/iPS-based cell therapies.