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1
FIRRITO, CLAUDIA. « Targeted Gene Correction and Reprogramming of SCID-X1 Fibroblasts to Rescue IL2RG Expression in iPSC-derived Hematopoietic Cells ». Doctoral thesis, Università degli Studi di Milano-Bicocca, 2015. http://hdl.handle.net/10281/94656.
Texte intégralRésumé :
La terapia genica basata sull’utilizzo di vettori integranti è stata già applicata con successo per la cura di varie malattie genetiche come le malattie da accumulo lisosomiale (LSD), la beta-talassemia (β-Thal) e le immunodeficienze primarie (PID). L’immunodeficienza combinata grave legata al cromosoma X (SCID-X1) è una malattia monogenica letale causata da mutazioni del gene codificante la catena comune gamma del recettore per l’interleuchina 2 (IL2RG). I primi studi clinici per la SCID-X1 hanno mostrato il potenziale terapeutico della terapia genica basata su vettori integranti, risultando nella ricostituzione del compartimento linfoide grazie al vantaggio selettivo delle cellule geneticamente modificate. D’altra parte, tali studi hanno evidenziato il rischio di mutagenesi inserzionale dovuto all’integrazione casuale del virus nel genoma della cellula ospite e all’espressione non regolata del transgene, sottolineando la necessità di sviluppare nuove strategie di terapia genica più sicure.
In questo lavoro, sfruttando la tecnologia delle Zinc-Finger Nucleasi (ZFN) per indurre una rottura del doppio filamento del DNA in maniera sito specifica e dei vettori lentivirali difettivi per l’integrazione (IDLV) per l’introduzione di un templato donatore, abbiamo impiegato il processo di riparazione del DNA guidata dall’omologia per la correzione delle mutazioni che causano la SCID-X1, ripristinando così la funzione genica e l’espressione fisiologica del gene IL2RG.
Mediante l’integrazione di un cDNA correttivo del gene IL2RG a valle del promotore endogeno sia in cellule B linfoblastodi, che esprimono costitutivamente la catena gamma comune, sia in linfociti T da donatori sani, che richiedono IL2RG per la loro sopravvivenza, abbiamo dimostrato la funzionalità e l’attività fisiologica del gene modificato. Abbiamo quindi accoppiato la correzione genica con la selezione delle cellule mediante l’inclusione di una cassetta excidibile di espressione della GFP o della resistenza alla puromicina (PuroR) a valle del cDNA correttivo, al fine di correggere fibroblasti, che normalmente non esprimono IL2R, derivati da pazienti SCID-X1. Abbiamo quindi ottenuto una popolazione di fibroblasti corretti che abbiamo “ riprogrammato” mediante un nuovo vettore di reprogramming che esprime i fattori di trascrizione (SOX2, OCT4, KLf4) e il microRNA cluster 367, generando così una fonte illimitata di cellule staminali pluripotenti indotte (iPSC) geneticamente corrette di interesse terapeutico.
L’espressione transiente della Cre-ricombinasi mediante IDLV ha inoltre permesso l’excisione del vettore di reprogramming e della cassetta di selezione, permettendo così l’ottenimento di cellule iPSC corrette, prive di vettore e con un normale cariotipo. Infine, attraverso il differenziamento delle cellule iPSC in progenitori T-linfoidi, un tipo cellulare assente nei pazienti SCID-X1, e l’osservazione di un vantaggio selettivo delle cellule linfoidi derivate dalle iPSC corrette, abbiamo dimostrato la correzione funzionale dell’allele IL2RG mutato.
In conclusione questi dati dimostrano la validità della nostra strategia di integrazione sito-specifica che, mediante la correzione e la riprogrammazione cellulare, consente di ottenere cellule iPSC geneticamente corrette, aprendo la strada a nuove opportunità terapeutiche più sicure per il trattamento della SCID-X1.Gene replacement by integrating vectors has been successfully used to treat several inherited diseases, such as Lysosomal Storage Disorders (LSD), Thalassemia and Primary Immunodeficiencies (PIDs). X-linked Combined Immunodeficiency (SCID-X1) is a fatal monogenic disorder, caused by mutation of the Interleukin 2 Receptor common γ-chain (IL2RG) gene. For SCID-X1, the early clinical studies have clearly shown the therapeutic potential of integrating vector based gene replacement therapy, which achieved efficient lymphoid reconstitution thanks to the selective growth advantage of the genetically modified cells. However, these studies also highlighted the potential risk of insertional mutagenesis due to random integration of the vector into the host cell genome and to unregulated transgene expression, thus calling for the development of safer gene therapy approaches. Here, by combining the Zinc Finger Nuclease (ZFNs) technology to induce site-specific DNA double-strand breaks (DSB) and of Integrase-Defective Lentiviral Vector (IDLV) to deliver a corrective donor template, we exploited Homology Driven Repair (HDR) to correct SCID-X1 mutation in situ, restoring both physiological expression and function of the IL2RG gene . By knocking-in a corrective IL2RG cDNA transgene downstream of its endogenous promoter in B-lymphoblastoid cells, which constitutively express IL2RG, and in primary T-lymphocytes, which requires IL2RG for their survival and growth, we provide evidence of physiologic activity of the gene-edited IL2RG gene. By including an excisable GFP- or a Puromycin Resistance (PuroR) expression cassette downstream of the corrective cDNA, we coupled correction with exogenous selection of corrected SCID-X1 primary fibroblasts, which do not physiologically express IL2RG, and obtained an enriched population of gene-corrected cells. We then reverted this population to pluripotency by using a novel reprogramming vector that expresses OCT4, SOX2, KLF4 and microRNA cluster 302-367 to obtain a potentially unlimited source of gene-corrected induced pluripotent stem cells (iPSC). We thus generated several gene-corrected bona-fide iPSCs, as confirmed by molecular analyses for targeted integration, which were characterized for their pluripotent state. IDLV-mediated transient delivery of the Cre-recombinase resulted in the co-excision of the reprogramming vector together with the selector cassette, thus allowing the generation of several gene-corrected, reprogramming-factor free iPSCs with normal karyotypes. Finally, by differentiating corrected iPSC to T-lymphoid progenitor cells, which are lacking in SCID-X1 patients, and showing a selective growth advantage of those derived from corrected iPSCs, we provide evidence of the functional correction of the IL2RG mutant allele. Overall these data demonstrate the feasibility of our targeted gene editing strategy, which couples gene correction with cell reprogramming to generate disease-free IPSC, thus paving the way for the development of novel and safer therapeutic approaches for SCID-X1.
2
CATTANEO, STEFANO. « Combinatorial gene therapy for epilepsy ». Doctoral thesis, Università Vita-Salute San Raffaele, 2022. http://hdl.handle.net/20.500.11768/128275.
Texte intégralRésumé :
Epilepsy is a neurological disease characterized by a persistent predisposition to generate seizures, that affects about 1% of the world population. About 30% of epileptic patients are drug-resistant, thus refractory to currently available anti-epileptic drugs (AEDs). Less than 10% of these drug-resistant patients are eligible for resective brain surgery, often due to generalized or multiple epileptic foci, or due to proximity of the epileptic focus to eloquent brain areas. Therefore, gene therapy may represent a doable approach for the unmet medical need of these patients.
Neuropeptide Y (NPY) can act as an endogenous anticonvulsant. NPY expression is increased both in rodent and human hippocampal sections from temporal lobe epilepsy surgical samples, despite the strong loss of hilar GABAergic interneurons. Therefore, NPY-based gene therapy may represent a novel approach for the treatment of focal epilepsies. Ideally, however, such vectors should contain multiple elements (at least NPY and Y2Rs driven by appropriate promoters).
In the past, great advancements in the field of viral vectors based on HSV-1 have been made by our laboratory. We therefore aimed at combining the potential of HSV vectors to accommodate large payloads with the complexity of the NPY system to create an “ideal” combinatorial therapeutic cassette. However, residual concerns on the safety and translatability of our new generation HSV-1 based vectors (named J∆NI8) let us first characterize their electrophysiological properties in primary neuronal culture, to assess both safety and efficacy profiles. Surprisingly and disappointingly, we show that mutations in the envelope glycoprotein B (gB), which is responsible for viral entry and cell fusion, might arise during viral vector production. In turn, mutated gB can increase firing frequency while reducing both input resistance and resting membrane potential of transduced neurons. Altogether, these data suggest that careful evaluation of envelope glycoproteins is needed to develop safe HSV-1 replication-defective vectors for the treatment of CNS disorders. We, therefore, decided to move to LV vectors, a more robustly characterized platform despite a more limited packaging capacity compared with HSV vectors.
To potentiate the protective effect of NPY, we developed a combinatorial gene therapy approach based on the expression of NPY together with its receptor (Y2). Since Y2 receptors act mainly pre-synaptically to reduce glutamate release by lowering Ca2+
influx, transgenes expression was driven by the minimal CamKII promoter, thereby biasing their expression in excitatory neurons. We characterized the ability of our lentiviral vectors to express NPY and its functional Y2 receptor in hippocampal neurons and mouse brains. Telemetry video-EEG monitoring was then used to assess the effect of the therapeutic genes on the epileptic phenotype of a genetic mouse model of epilepsy.
We found that the combined expression of NPY and Y2 is sufficient to reduce both the frequency and duration of seizures in the Synapsin triple-KO epilepsy model. These data further strengthen the hypothesis that strategies aimed at the delivery of NPY and Y2 may be successful for the treatment of epilepsy, particularly for pharmaco-resistant and genetic forms of the disease.L'epilessia è una malattia neurologica caratterizzata da una persistente predisposizione a generare crisi, che colpisce circa l'1% della popolazione mondiale. Circa il 30% dei pazienti epilettici sono resistenti ai farmaci, quindi refrattari ai farmaci antiepilettici attualmente disponibili (AED). Meno del 10% di questi pazienti resistenti ai farmaci sono eleggibili per la chirurgia, spesso a causa di foci epilettici generalizzati o multipli, o a causa della vicinanza del focus epilettico alle aree cerebrali eloquenti. Pertanto, la terapia genica può rappresentare un approccio fattibile. Il neuropeptide Y (NPY) può agire come un anticonvulsivo endogeno. L'espressione di NPY è aumentata sia nelle sezioni ippocampali di roditori che in quelle di campioni chirurgici umani di epilessia del lobo temporale, nonostante la forte perdita di interneuroni GABAergici a livello dell’ilo. Pertanto, la terapia genica basata su NPY può rappresentare un nuovo approccio per il trattamento delle epilessie focali. Idealmente, tuttavia, tali vettori dovrebbero contenere più elementi (almeno NPY e Y2R guidati da promotori appropriati). In passato, il nostro laboratorio ha fatto grandi progressi nel campo dei vettori virali basati su HSV-1. Abbiamo quindi mirato a combinare il potenziale dei vettori HSV di ospitare DNA di grandi dimensioni, e la complessità del sistema NPY, per creare una cassetta terapeutica combinatoria "ideale". Tuttavia, le preoccupazioni residue in merito alla sicurezza della nostra nuova generazione di vettori basati su HSV-1 (chiamati J∆NI8) ci hanno spinto a valutare i profili di sicurezza ed efficacia in vitro per valutare l’effetto dell’infezione sulle proprietà elettrofisiologiche in neuroni primari. Sorprendentemente e in maniera deludente, abbiamo dimostrato che mutazioni nella glicoproteina B dell'involucro (gB), che è responsabile dell'entrata virale e della fusione cellulare, potrebbero sorgere durante la produzione del vettore virale. A livello elettrofisiologico, abbiamo inoltre visto che la gB mutata può aumentare la frequenza di potenziali d’azione e contemporaneamente ridurre sia la resistenza di ingresso che il potenziale di riposo neuroni trasdotti. Complessivamente, questi dati suggeriscono che un'attenta valutazione delle glicoproteine dell'involucro è necessaria per sviluppare vettori sicuri non replicativi basati su HSV-1 per il trattamento dei disturbi del SNC. Abbiamo quindi deciso di passare ai vettori Lentivirali (LV), una piattaforma più robusta e caratterizzata nonostante una capacità di carico più limitata rispetto ai vettori HSV. Per potenziare l'effetto protettivo di NPY, abbiamo sviluppato un approccio combinatorio di terapia genica basato sull'espressione di NPY insieme al suo recettore (Y2). Poiché i recettori Y2 agiscono principalmente a livello pre-sinaptico per diminuire il rilascio di glutammato riducendo l’ingresso di Ca2+, l'espressione dei transgeni è stata guidata dal promotore minimal CamKII, orientando così la loro espressione selettivamente nei neuroni eccitatori. Abbiamo successivamente caratterizzato la capacità dei nostri vettori LV di esprimere NPY e il suo recettore funzionale Y2 nei neuroni ippocampali e nel cervello dei topi. In seguito, abbiamo utilizzato un sistema di monitoraggio video-EEG mediante telemetria per valutare l'effetto dei geni terapeutici sul fenotipo epilettico in un modello genetico di epilessia. Abbiamo scoperto che l'espressione combinata di NPY e Y2 è sufficiente a ridurre sia la frequenza che la durata delle crisi nel modello di epilessia Synapsin triple-KO. Questi dati rafforzano ulteriormente l'ipotesi che le strategie mirate all’utilizzo di NPY e Y2 possono avere successo per il trattamento dell'epilessia, in particolare per le forme resistenti ai farmaci ma anche per forme genetiche della malattia.
3
STARINIERI, FRANCESCO. « Investigating liver tissue dynamics to improve in vivo gene therapy ». Doctoral thesis, Università Vita-Salute San Raffaele, 2022. http://hdl.handle.net/20.500.11768/122896.
Texte intégralRésumé :
The liver is a relevant target organ for in vivo gene therapy, being involved in several coagulation disorders and metabolic diseases. Adeno-associated viral (AAV) vectors have been extensively used for liver gene therapy, obtaining successful results in clinical trials. Despite AAV vector genomes remain mostly episomal, the transgene has been shown to be maintained for several years in the adult liver. However, active hepatocyte proliferation during liver growth currently challenges application of AAV-vector gene therapy to young individuals. We have previously developed lentiviral vectors (LV) that integrate their genome into host DNA and achieve stable transgene expression in adult mice, dogs, and non-human primates. Here we performed and in-depth analysis of maintenance of LV-transduced hepatocytes following post-natal liver growth and homeostasis and of the age-dependent impact on liver-directed LV gene therapy in mice. We observed a high hepatocyte proliferation rate in newborn mice that decreased over time, with only 25% of hepatocytes contributing to liver growth, generating the vast majority of the adult liver. No major differences have been observed between proliferation of LV-transduced and non-transduced hepatocytes. We then observed a higher hepatocyte transduction efficiency in young (newborn and juvenile) mice compared to adults, paralleled by a lower uptake of LV by non-parenchymal cells. By intravenously (i.v.) administering LV expressing a human coagulation factor IX (FIX) transgene we observed the highest FIX output in mice treated as juvenile, which substantially dropped when mice were treated from the 4th week of age. Newborn-treated mice showed an intermediate level of transgene output. Young mice also showed a higher percentage of multiple-transduced hepatocytes, that might contribute to the differences in transgene output. We then investigated the distribution of transduced hepatocytes in the liver lobule and observed a preferential transduction in the peri-central area in young-treated mice and in the peri-portal area in adult-treated mice. We observed that also Kupffer cells shift from the central to the portal area during growth, however their depletion did not reduce the peri-portal transduction bias in adult mice, indicating that they are not determining the LV transduction bias. Overall, our work showed that i.v. LV administration to young mice results in higher hepatocytes transduction and transgene output than in adult-treated mice, with maintenance of the transgene following cell proliferation during liver growth. These findings inform further development of liver-directed LV gene therapy towards application to pediatric patients and shed light on mechanisms of post-natal liver growth in mice, which are relevant also for genome editing strategies.Il fegato è un importante organo bersaglio per la terapia genica in vivo, a causa del suo ruolo in diversi disordini della coagulazione e malattie metaboliche. I vettori adeno-associati (AAV) sono stati ampiamente usati per la terapia genica diretta al fegato, ottenendo significativi risultati terapeutici in diversi trial clinici. Nonostante il genoma dei vettori AAV rimane prevalentemente episomale, il transgene è mantenuto per diversi anni nel fegato adulto. Tuttavia, la proliferazione degli epatociti durante la crescita ostcola l’utilizzo dei vettori AAV in individui giovani. In passato abbiamo sviluppato vettori lentivirali (LV) che integrano il loro genoma in quello della cellula, e hanno mostrato espressione stabile in topi, cani e primati non umani adulti. Abbiamo effettuato un’analisi approfondita del mantenimento degli epatociti trasdotti con LV in seguito a crescita post-natale e omeostasi, e dell’impatto dell’età sulla terapiagenica con LV diretta al fegato nel topo. Abbiamo osservato una maggiore proliferazione degli epatociti in topi neonati, che decresce nel tempo fino, con solo il 25% degli epatociti che contribuisce alla crescita, generando la grande maggioranza del fegato adulto. Non abbiamo osservato differenze rilevanti tra la proliferazione di epatociti trasdotti e non trasdotti. Abbiamo poi osservato una maggiore efficienza di trasduzione degli epatociti in topi neonati o giovani rispetto agli adulti, in parallelo a un minor indirizzamento nelle cellule non parenchimali. Somministrando intravena (i.v.) un LV che esprime il fattore IX della coaglazione (FIX) umano abbiamo osservato la maggior produzione di FIX in topi trattai da giovani, che decresce sostanzialmente in quelli trattati dalla 4° settimana di vita. I topi neonati hanno mostrato invece un livello intermedio. I topi giovani hanno mostrato inoltre una percentuale più alta di epatociti trasdotti a multipla copia, che può contribuire alla differenza di secrezione. Abbiamo poi esaminato la distribuzione degli epatociti trasdotti nel lobulo epatico e abbiamo una preferenza di trasduzione per gli epatociti nell’area peri-centrale nei topi giovani e in quella peri-portale nei topi adulti. Queste differenze sono mantenute nel tempo, indicando che sono dovute a differenze di trasduzione e non causate da silenziamento del transgene. Abbiamo osservato che anche le cellule di Kupffer si spostano dalla zona centrale a quella portale durante la crescita, ma la loro deplezione aumenta la trasduzione della zona portale nei topi adulti, suggerendo che non determinano la distribuzine del LV nel lobulo. In conclusione, il nostro lavoro mostra che la somministrazione i.v. di LV in topi giovani porta a una maggiore trasduzione degli epatociti e secrezione del prodotto del transgene rispetto ai topi adulti, con mantenimento del transgene in seguito a proliferazione cellulare durante la crescita del fegato. Queste osservazioni forniscono informazioni sullo sviluppo della terapia genica con LV diretta al fegato verso l’applicazione in pazienti pediatrici, e gettano luce sui meccanismi di crescita post-natale del fegato nei topi, che sono rilevanti anche per strategie di modificazione sito-specifica del genoma.
4
Bobisse, Sara. « Ridirezionamento dell'immunità anti-tumorale in terapia cellulare adottiva : trasferimento genico del T-Cell Receptor mediato da vettori lentivirali ». Doctoral thesis, Università degli studi di Padova, 2008. http://hdl.handle.net/11577/3425463.
Texte intégralRésumé :
Genetic modification of T cells with genes encoding a tumor-specific T-cell Receptor (TCR) represents a novel strategy to obtain large quantities of tumor-reactive T lymphocytes to be employed in adoptive cell therapy protocols. As a transfer method, lentiviral vectors might represent an appealing alternative to the most widely used oncoretroviral vectors, because they do not require cell division for nuclear uptake.
We have characterized the TCR of a highly cytotoxic T lymphocyte (CTL) clone recognizing the HLA-A2-restricted Melan-A/MART-1 melanocyte differentiation antigen on both pulsed T2 cells and SK-23 MEL melanoma tumor cells. The ? (V?2.2) e ? (V?14) chains of the TCR were cloned and used to construct a lentiviral vector carrying a bidirectional promoter and capable of a robust and coordinated expression of the two transgenes.
Transduction of Jurkat T leukemia cells showed that more than 60% of cells could be transduced without selection at a low MOI, as assessed by antigen-specific tetramer staining. High expression Jurkat clones disclosed that the new assembled TCR was at high intensity, very stable over time, and fully functional, as demonstrated by intracellular signaling upon TCR triggering. Transduction of activated PBMC produced a highly expressed transgenic TCR in around 5-10% of cells. This initial low, but clearly detectable, fraction of transduced lymphocytes could be quickly expanded (4-6 weeks) upon subsequent antigen-specific in vitro restimulation. The resulting population had a 50-70% of transgenic TCR expression and mainly a CD8+ phenotype, specifically recognized antigen-expressing melanoma cells (cytokine production and cytotoxic activity), and exerted relevant therapeutic effects in vitro upon adoptive transfer in SK-23 MEL-bearing mice.
Our results indicate that LV constitute a valid tool for stable and high-intensity expression of transgenic TCR, and support further studies to address potential feasibility of this approach for clinical application.
5
CABRIOLU, ANNALISA. « Sviluppo di vettori virali per la terapia genica della β−Talassemia ». Doctoral thesis, Università degli Studi di Cagliari, 2012. http://hdl.handle.net/11584/266135.
Texte intégralRésumé :
Beta−thalassemia major is a severe congenital anemi for which there is presently no curative therapy other than allogeneic hematopoietic stem cell transplantation. This therapeutic option, however, applies only to the minority of thalassemia patients who have an HLA−matched bone marrow donor. Gene therapy by the delivery of a regulated globin gene to autologous hematopoietic stem cells is an attractive alternative approach as it is in principle applicable to all thalassemic subjects. Current vectors, althougheffective in correcting thalassemia in murine models still suffer some drawbacks in terms of safety and also in terms of low titer and expression. The aim of this study was to assemble globin vectors improved in both these aspects. Modifications of the globin cassette in the intron2 and in the LCR of the beta-‐globin gene can increase the expression of the globin gene without reducing the vector titer. We also examine the variegation in the expression among the different pools of transduce cells and we suppose that the presence of sequences with chromatin opening activity among the segments of b-‐globin IVS2
6
CASTELLANI, STEFANO. « Valutazione dell'efficienza, efficacia e sicurezza di vettori lentivirali nel trasferimento del gene CFTR in sistemi modello di epitelio respiratorio in fibrosi cistica ». Doctoral thesis, Università degli Studi di Cagliari, 2007. http://hdl.handle.net/11584/265953.
Texte intégralRésumé :
One of the possible strategies for therapy of Cystic Fibrosis is based on gene therapy. Gene therapy goal is to provide a normal copy of CFTR gene to defective tissues by using different gene transfer agents. HIV-1 derived vectors allow a prolonged expression of therapeutic gene and they are able to infect quiescent cells like respiratory epithelium cells.
Primary goal of the project is concerned with the realization of a lentivirus working live agent, transferring CFTR WT gene, or SHRNA molecules directed against ENAC subunits; ENAC is a sodium channel hyperactive in cystic fibrosis.
7
Martelli, Francesco. « Sviluppo di un approccio terapeutico basato su piccoli rna diretti verso i segmenti genomici codificanti la polimerasi del virus dell'influenza ». Doctoral thesis, Università degli studi di Padova, 2015. http://hdl.handle.net/11577/3423954.
Texte intégralRésumé :
Influenza A viruses, capable of infecting humans and birds, are associated not only to seasonal epidemics, but also to pandemics, with an high impact on Public Health. The emergence of influenza viruses to the currently available drugs and the lack of universal vaccination strategies are main human and veterinarian concerns. Thus, the research of innovative targets for the development of therapeutic anti-influenza A virus strategies is still required. The aim of this study was to develop an innovative antiviral approach based on the delivery into eukaryotic cells susceptible to influenza virus infection of small RNA molecules targeting the highly conserved regions mapping at the 5’ ends of the 3 viral genomic segments encoding for the polymerase (PA, PB1, PB2), an enzyme essential for viral replication (Giannecchini S. et al., 2009- 2011). The target sequences represent the packaging signals of these 3 genomic segments and play an important role during the assembly and budding of the newly formed particles (Qinshan Gao et al., 2012).
To this end, we developed different lentiviral vectors expressing miRNAs [pLenti6/V5-GW/EmGFP-miR] or antisense-RNAs [pLentilox 3.7 GFP] targeting PA, PB1 and PB2 segments. Recombinant lentiviral particles, produced by transfecting human embryonic kidney cells (293T) with both vectors in combination with a packaging systems, were harvested and titrated on human alveolar adenocarcinoma cell line (A549) by FACS analysis. The lentiviral vectors transduction efficiency on different cell lines was evaluated by FACS assay and by quantitative Real-Time PCR. The transduced cells were infected with epidemiologically relevant human influenza viruses type A and B and avian viruses type A. The viral inhibitory activity was assessed by employing an infectivity assay.
We showed that recombinant lentiviral particles expressing miRNA or antisense-RNAs efficiently transduced A549 cell lines that are highly susceptible to influenza virus replication. More importantly, a reduction from 1 to 3 logatims of the viral titre was obtained in all tested cell lines infected with human and avian subtypes of influenza type A viruses. By contrast, no inhibition of influenza type B virus was observed. Furthermore, mutations within the target sequences abolished the antiviral effects of the developed vectors, thus confirming the specificity of the approach. Our study contributes to the demonstration that the expression of small RNAs targeting the packaging signal of the polymerases gene might represent an efficient strategy against the influenza A virus infection in human (especially in pandemic events) and birds.I virus dell'influenza di tipo A sono in grado di infettare gli esseri umani e gli uccelli ed essendo responsabili non solo di di epidemie stagionali, ma anche di pandemie, hanno un impatto importante per la Salute Pubblica. L' insorgenza di virus influenzali resistenti ai farmaci attualmente disponibili e la mancanza di una terapia vaccinale universale sono una fonte di costante preoccupazione tanto per la popolazione umana quanto per quella animale. Pertanto, la ricerca di nuovi bersagli per lo sviluppo di approcci terapeutici / preventivi nei confronti del virus influenzale di tipo A è ancora necessaria. Lo scopo di questo studio è stato quello di sviluppare e valutare una strategia antivirale innovativa basata sul delivery in cellule eucariotiche suscettibili d’infezione da parte del virus dell’influenza di tipo A di piccoli RNA. Questi ultimi hanno come bersaglio le regioni conservate presenti al 5’ dei 3 segmenti del genoma virale codificanti la polimerasi (PA, PB1, PB2), enzima essenziale perché il virus possa replicarsi (Giannecchini S. et al., 2009- 2011). Nelle regioni bersaglio si trovano i segnali di packaging, che svolgono un ruolo importante durante le fasi di assemblaggio e gemmazione delle particelle virali nascenti (Qinshan Gao et al., 2012). A tale scopo, abbiamo sviluppato diversi vettori lentivirali esprimenti miRNA [pLenti6/V5-GW/EmGFP-miR] o antisenso-RNA [pLentilox 3.7 GFP] diretti verso i segmenti genomici PA, PB1 e PB2. Le particelle lentivirali ricombinanti, sono state prodotte trasfettando cellule umane embrionali renali (293T) con entrambi i vettori insieme ad un appropriato sistema di packaging. Leparticelle lentivirali ricombinanti sono state utilizzate per trasdurre le cellule umane di derivazione alveolare (A549) che rappresentano un ottimo modello per lo studio di molecole in grado di interferire con la replicazione del virus dell’influenza di tipo A. Le cellule trasdotte sono state quindi infettate con virus dell'influenza di tipo A di origine umana e aviaria e con un virus di tipo B. L’effetto di inibizione sul titolo della progenie virale è stato valutato mediante un test di infettività. Abbiamo dimostrato che le particelle lentivirali ricombinanti che esprimono miRNA o antisenso-RNA, trasducendo in modo efficiente la linea cellulare A549, determinano una riduzione del titolo virale che varia dagli 1 a 3 logaritmi, a seconda del virus e del bersaglio molecolare preso in esame. Al contrario, non è stata osservato nessun effetto nel caso del virus dell'influenza di tipo B. Inoltre, mutazioni all'interno delle sequenze bersaglio fanno sì che non vi siano più effetti antivirali dei vettori sviluppati, confermando la specificità dell’approccio sviluppato. I nostri dati contribuiscono alla dimostrazione che il delivery intracellualre di piccoli RNA specifici per i segnali di packaging dei geni virali codificanti la polimerasi, possono rappresentare una valida strategia terapeutica nei confronti dei virus dell’influenza umani, specialmente nell’eventualità di insorgenza di virus pandemici, e aviari.
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Reis, Luiza Cunha Junqueira. « Geração de células de pluripotência induzida (iPS) humanas utilizando vetores lentivirais e determinação do perfil de integração lentiviral ». Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/60/60135/tde-26022013-092913/.
Texte intégralRésumé :
As células iPS surgiram com a promessa de contornar as limitações das células-tronco embrionárias, como questões éticas, segurança, compatibilidade e disponibilidade. Essas células podem ser obtidas a partir de células somáticas de indivíduos normais ou de pacientes com doenças genéticas, fazendo destas uma importante ferramenta para o screening de drogas, modelos de doenças e testes toxicológicos. Grandes avanços ocorreram na reprogramação de células diferenciadas pela expressão forçada de fatores de transcrição (FT), principalmente, através de vetores lentivirais (VL), que proporcionam uma reprogramação eficiente. Entretanto, a inserção lentiviral no genoma humano e sua influência na reprogramação é pouco conhecida. Neste trabalho, avaliamos o perfil de inserção dos VL utilizados na geração de iPS. As iPS foram geradas e caracterizadas por nosso grupo a partir de fibroblastos humanos transduzidos com VL contendo 3 FT [SOX2, TCL-1A e C-MYC (célula TSM)], e de células mesenquimais derivadas de tecido adiposo com um vetor lentiviral policistrônico contendo 4 FT [OCT4, SOX2, KLF4 e C-MYC (iPS 4FT)]. Cinco colônias isoladas de cada iPS foram mapeadas e analisadas quanto aos sítios de inserção pela técnica de LM-PCR. O DNA genômico digerido foi amplificado com um primer específico para o LTR viral e outro para um linker sintético. Os produtos foram clonados, sequenciados, e analisados em bancos de dados para identificar similaridades com o genoma humano, entre outras análises. Na célula TSM, 176 sequências, obtidas com a técnica de LM-PCR, apresentaram identidade com o genoma humano, sendo que cerca de 50% ocorreram em regiões gênicas com 94% destas em introns. Já nas iPS 4FT, 251 sequências apresentaram identidade, com cerca de 45% atingindo genes, 92% destas em introns. As inserções distribuíram-se por todos os cromossomos, com preferência pelos cromossomos 16, 17 e 20 para a TSM e pelos cromossomos 11, 15 e 17 para a iPS 4FT. Analisamos a distância da inserção ao sítio de início de transcrição (TSS), e inserções próximas a ilhas CpG, que em geral correspondem a regiões regulatórias. A maior proporção de inserção ocorreu a partir de ±30Kb de distância desses sítios. Os sítios frágeis e as regiões repetitivas do genoma foram atingidas, mas com uma frequência baixa. Os resultados mostraram uma preferência de inserção lentiviral por regiões gênicas nas iPS, indicando a possível participação de proteínas como LEDGF/p75 na integração nas células estudadas. Este trabalho mostrou que o local da integração pode contribuir para a reprogramação e, apesar de possíveis efeitos negativos das integrações, estas as células iPS ainda são uma ferramenta importante para estudos in vitro. E identificar fatores que influenciem a seleção do sítio de inserção é importante para determinar regiões cromossômicas \"seguras\" para a integração, aumentando a segurança no uso clínico.The induced pluripotent stem (iPS) cells came with the promise of circumvent some of the limitations in the use of embryonic stem cells, like ethical issues, biological safety, immune compatibility and availability. This cells can be generated from somatic cells of normal individuals or from patients with some genetic disease, making then an important tool for drug screening, construction of disease models and toxicological trials. Great advances have happened in reprogramming differentiated cells through the forced exogenous expression of transcription factors (TF), mostly by lentiviral vectors (LV), which provide an efficient reprogramming. However, the lentiviral insertion in the human genome and its influence in reprogramming is not well known. In this work, we evaluate the insertion profile of LV used to generate human iPS cells. The iPS cells were generated, by our group, from human fibroblasts transduced by LV containing 3 TF [SOX2, TCL-1A and C-MYC (TSM reprogrammed cell)], and from mesenchymal cells derived from human adipose tissue transduced by a polycistronic LV containing 4 TF [OCT4, SOX2, KLF4 and C-MYC (iPS 4TF)]. Five isolated colonies of each iPS cell were mapped and analyzed for the insertion sites through LM-PCR technique. The digested genomic DNA was amplified with a primer for the viral LTR e another for a synthetic linker. The products were cloned, sequenced and analyzed in database to identify similarities with the human genome, among other analyzes. In TSM cell, 176 sequences, derived from the LM-PCR technique, presented identity with the human genome, and about 50% of those occurred in genic regions with 94% in introns. In iPS 4TF, 251 sequences showed identity, with about 45% reaching genes, 92% of these in introns. The insertions were distributed on all chromosomes, with preference for the 16, 17 and 20 for the TSM cell, and for the 11, 15 and 17 for the iPS 4TF. We analyzed the distance of the insertion from de transcription start site, and insertions near CpG islands, which, overall, correspond to regulatory regions. The highest proportion of insertion occurred starting ±30Kb distance from these sites. The fragile sites and the repetitive regions of the genome were also reached, but with low frequency. The results showed a preference of lentiviral insertion for genic regions in iPS, indicating the potential participation of proteins like LEDGF/p75 in integration in the cells of this work. This work shows that the integration site may contribute to the reprogramming, and, despite possible negative effects of integration, these iPS cells are still an important tool for in vitro studies. Identify factors that influence the selection of insertion site is important for determination of \"safe\" chromosomal regions for the integration, increasing the safe in clinical use.
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Ngom, Mor. « Small molecule stimulators for enhanced yield of human hematopoietic stem cells ». Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCC320.
Texte intégralRésumé :
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 viralesEfficient 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
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Cordeil, Stéphanie. « Etude de la différence de susceptibilité des lentivirus de primates aux interférons de type I ». Thesis, Lyon, École normale supérieure, 2012. http://www.theses.fr/2012ENSL0781.
Texte intégralRésumé :
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 vivoType 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
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Coquin, Youna. « Caractérisation de vecteurs lentiviraux pseudotypés par les syncytines murines et de leurs cibles cellulaires in vitro et in vivo ». Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLE039.
Texte intégralRésumé :
Les vecteurs lentiviraux (LV) de thérapie génique sont des particules recombinantes qu'il est possible de pseudotyper avec diverses glycoprotéines d'enveloppe afin de modifier l'adressage cellulaire ou leurs propriétés immunogéniques. Mon travail de thèse a exploré l'hypothèse que la VSVG, la glycoprotéine d'enveloppe la plus utilisée pour pseudotyper les LV, puisse être remplacée par des protéines cellulaires afin d'obtenir des particules bien tolérées et administrables in vivo. J'ai utilisé les syncytines murines, qui sont des glycoprotéines d'origine rétrovirale endogène et qui ont des propriétés fusogéniques et un potentiel immunosuppressif. Mes résultats ont montré la possibilité de produire des LV pseudotypés par les syncytines murines A et B. Les particules ainsi produites (LV-SynA et LV-SynB) sont infectieuses en présence d'un adjuvant de transduction et présentent une forte capacité à transduire les lymphocytes B murins in vitro. Les vecteurs LV-Syn sont capables de transduire des lymphocytes B quiescents, ainsi que des cellules précurseurs des lymphocytes B issus de moelle osseuse de souris. L'administration de LV-SynA in vivo chez la souris par voie intraveineuse permet d'observer un transfert de gène stable. Un signal est observé dans la rate et plus particulièrement dans les lymphocytes B au niveau des centres germinatifs, en accord avec les résultats obtenus in vitro. Mes travaux confirment donc, et étendent, les observations préalables du laboratoire sur la transduction de lymphocytes B naïfs humains avec les syncytines humaines, et suggèrent un fort tropisme des syncytines en général pour les lymphocytes B. Par ailleurs, mes résultats montrent qu'in vivo, le poumon est également ciblé par les LV-SynA, en accord avec l'expression récemment identifiée du récepteur à la syncytine A, Ly6e, dans cet organe. Il semble que le transfert de gène puisse être obtenu dans l'épithélium alvéolaire et il est possible de transduire des cellules primaires épithéliales pulmonaires humaines avec les LV-Syn in vitro. Globalement, mes résultats montrent une nouvelle perspective d'interaction entre les syncytines et le système immunitaire à travers la transduction des lymphocytes B. Mes résultats permettent également d'envisager de nouvelles perspectives pour le transfert de gène dans le poumon in vivo et pour développer de nouvelles approches de thérapie génique par voie systémiqueLentiviral gene transfer vectors (LV) used in gene therapy are recombinant virus-like particles that can be pseudotyped with diverse envelope glycoproteins in order to modify their cellular targeting or their immunogenic properties. My thesis work explored the hypothesis that VSVG, the most-commonly-used viral envelope glycoproteins of LV, could by replaced by cellular glycoproteins to obtain well-tolerated particles that can be administered in vivo. I used murine syncytins which are glycoproteins of endogenous retroviral origin and which have fusogenic properties and immunosuppressive potential. My results showed the possibility of producing LV pseudotyped with murine syncytins A or B. The LV-SynA or LV-SynB particles produced were infectious in the presence of a transduction adjuvant and efficiently transduced murine B cells in vitro, including quiescent B cells and bone marrow B precursor cells. In vivo LV-SynA intravenous administration in mice enabled stable gene transfer. A positive signal was observed in the spleen, and especially in B cells at the level of the germinal centers, confirming in vitro observations. These results confirm and extend previous observations from the laboratory showing that human naive B cells can be transduced by LV pseudotyped with human syncytins, and emphasize the marked interactions between syncytins and B cells. Moreover, my results showed that in vivo in mice LV-SynA targets the lungs, possibly epithelial alveolar cells, in line with the recent findings of Syncytin A receptor Ly6e expression in the lung. Gene transfer was also obtained in human primary pulmonary epithelial cells in vitro with LV-Syn. Overall, my results reveal new interactions between syncytins and the immune system through the transduction of B cells. My results also provide novel perspectives for gene delivery in vivo in the lung and for designing new systemic gene therapy approaches
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Manic, Gwenola. « Impacts du design de vecteurs dérivés du VIH-1 et de la machinerie de réparation de l'ADN sur l'expression lentivirale ». Thesis, Paris 11, 2012. http://www.theses.fr/2012PA11T039/document.
Texte intégralRésumé :
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 hôteAn 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
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Trimby, Christopher Matthew. « STRATEGIES FOR TARGETING LENTIVIRAL VECTORS ». UKnowledge, 2011. http://uknowledge.uky.edu/gradschool_diss/157.
Texte intégralRésumé :
Lentiviral gene therapy has held great promise for treating a wide range of neurological disorders due to its ability to stably integrate into the genome of nondividing cells like neurons, in addition to dividing cells. The nervous system is a complex and highly heterogeneous system, and while a therapeutic intervention may have beneficial effects in one population of cells it may have severe side effects in another. For this reason, specific targeting of lentiviral vectors is crucial for their ultimate utility for research and clinical research use.
Two different approaches for focusing the targeting of lentiviral vectors were employed in these studies. The first method involved assessing the effects of vector production strategies on the resulting virus’s tropism both in vivo and in vitro. The changes in vector transduction were determined via flow cytometry on cells in culture and immunohistochemistry following brain injections. Results from these experiments suggest that while the production conditions do impact the vectors efficacy, there is not a distinct effect on their tropism.
A unique characteristic of retroviral and lentiviral vectors is their capacity for being pseudotyped, conferring a new tropism on the vector. Native tropisms are generally not specific beyond very broad cell types, which may not be sufficient for all applications. In this case, chimeric targeting molecules can provide an even more refined targeting profile compared to native pseudotypes.
The second approach utilizes novel chimeric glycoproteins made from nerve growth factor and the vesicular stomatitis virus glycoprotein. These chimeras are designed to pseudotype lentiviral vectors to target nociceptive sensory neurons for a variety of disorders. While these chimeras were successfully produced as protein, they were misfolded and sequestered in the endoplasmic reticulum and therefore unavailable to produce lentivirus.
While neither strategy was completely successful, they do provide interesting information for the design and creation of lentiviral vectors. This research shows that small differences in the steps followed as part of a lentivirus production protocol can greatly impact the resulting vectors efficacy. It also shows that while VSV has been used to create chimeric glycoproteins, not all targeting molecules are suitable for this purpose.
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Knight, S. B. « Lentiviral vectors for gene therapy ». Thesis, University College London (University of London), 2012. http://discovery.ucl.ac.uk/1346459/.
Texte intégralRésumé :
Lentiviral vectors, derived from HIV-1, are promising tools for gene therapy. Recent clinical trials have demonstrated the translation of their effectiveness in laboratory studies to clinical trials. However there are still limitations, relating to vector safety and efficiency of production, that could confine their future use. I investigated the ability of lentiviral vectors to perturb cellular gene expression by insertional mutagenesis (IM), using an in vitro model that detects aberrant splicing from lentiviral vectors to the growth hormone receptor gene (Ghr). The lentiviral vector pHV with full long terminal repeats (LTRs) and an internal spleen focus forming virus promoter (SFFV), was previously found to activate Ghr expression by a fusion mRNA transcript initiated in the HIV LTR (46). I extended this discovery to show that the SFFV promoter was enhancing expression from the HIV LTR, leading to IM (269). Application of our in vitro IM assay to potential clinical lentiviral vectors revealed that the novel ‘UCOE’ (ubiquitously acting chromatin opening element) promoter, within a selfinactivating (SIN) lentiviral vector, could drive UCOE-Ghr mRNA transcripts, causing IM. Mutation of splice donor sites in UCOE alone was insufficient in abrogating IM, however internal deletions around these splice donor sites were more successful. In other work, I made a packaging cell line for lentiviral vectors by stably expressing rev and a modified RD114 env (RDpro) in a cell line expressing HIV gag-pol. This led to the isolation of 57R10E, a cell line that made a titer of over 104 infectious units per ml when a SIN lentiviral vector was transiently or stably expressed. Taken together, this work will broaden the application of lentiviral vectors in clinical gene therapy by reducing both the chances of adverse events and the costs associated with vector production.
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Dietrich, Isabelle. « Feline restriction factors to lentiviral replication ». Thesis, University of Glasgow, 2013. http://theses.gla.ac.uk/4066/.
Texte intégralRésumé :
Strong adaptive evolutionary forces shape the interactions between pathogens and their hosts and typically lead to a stable co-existence. In this process of co-evolution, mammals have developed restriction factors that limit retrovirus infectivity, replication or assembly and narrow the spectrum of potential host species. These restriction factors are either constitutively expressed, such as APOBEC3 proteins, cytidine deaminases that interfere with reverse transcription, or form part of the type I interferon-induced innate immunity, such as TRIM5, a member of the tripartite motif protein family that induces degradation of retroviral capsid, blocks reverse transcription, or tetherin (BST-2, CD317), which inhibits release of nascent viral particles from infected cells. Conversely, viruses have evolved antagonists of restriction factors or proteins that limit IFN-induced gene expression, thus evading immune surveillance. The interaction between host and viral components is delicately balanced and has a significant impact on disease outcome. Feline immunodeficiency virus (FIV), a lentivirus closely related to human immunodeficiency virus (HIV), is a recent introduction into domestic cats and causes an immunodeficiency syndrome analogous to human AIDS. Interestingly, non-domestic cats such as lion or pumas have co-existed with lentiviruses for prolonged periods of time and FIV infections are largely benign. Although plasma viral and proviral loads are high in both domestic and non-domestic cats, in vitro studies have shown that FIV infection of non-domestic cat T lymphocytes is significantly less efficient than that of domestic cat T cells. Thus, this thesis tests the hypothesis that the differential disease outcome of FIV infections in felids is caused by differences in lentiviral restriction factor activities or their sensitivities to FIV restriction factor antagonists. Data presented in this study show for the first time that feline APOBEC3 proteins are expressed in tissues and cell types relevant for FIV infection. The APOBEC3 proteins A3H and A3CH exhibited a high antiviral activity against FIV lacking the APOBEC3 antagonist Vif in single-cycle replication assays, with no difference in activity being detected between domestic and non-domestic cat proteins. However, domestic cat A3CH was significantly more sensitive to antagonism by FIV Vif than lion or puma A3CH, which would allow efficient viral replication in domestic cat T lymphocytes and subsequently lead to T cell loss and immunodeficiency. Furthermore, this thesis provides evidence that felid tetherins can prevent FIV particle release from producer cells in single-cycle replication assays; however, stable expression of domestic and non-domestic cat tetherins in feline cell lines did not abrogate FIV replication. Indeed, syncytium formation indicative of viral cell-to-cell spread was significantly enhanced in type I interferon-treated feline cells infected with CD134-independent strains of FIV which often arise in chronic (late) stages of FIV infections in vivo. Finally, this work reports the generation of a synthetic domestic cat TRIM5α-cyclophilin A fusion protein which was highly efficient at preventing FIV pseudotype and productive infection. This novel feline restriction factor represents a potent antiviral defence agent with very low potential for toxicity and could in future be used in gene therapy approaches to treat FIV-infected cats.
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Byland, Rahel. « Trafficking of primate lentiviral envelope proteins ». Thesis, University College London (University of London), 2006. http://discovery.ucl.ac.uk/1445339/.
Texte intégralRésumé :
The assembly of enveloped viruses requires the correct trafficking of the viral envelope or membrane proteins to the site of virus assembly. To understand the molecular and cellular mechanisms in human immunodeficiency virus envelope protein (Env) trafficking, I analysed the activities of putative endocytosis signals in the cytoplasmic domain of Env. Morphological and biochemical analysis of HxB2 Env as well as CD4-Env chimeras revealed two functional endocytosis motifs, the membrane proximal GY712XX0 motif and the C-terminal dileucine motif. Both of these motifs work with equivalent efficiency and show no obvious additive effect. RNAi knock down experiments show that endocytosis mediated by both signals is at least partly dependent on the clathrin pathway and the clathrin adaptor AP-2. Motifs of the Yxx0 type have been implicated in transport to the late endosome and HIV has been reported to assemble on membranes of this compartment. I studied the intracellular itineraries of HIV Env and found clear evidence of trafficking through early endosomes. However, no obvious colocalisation with CD63 or LAMP-1 was observed. Further analysis suggested that Env is transported to an endosomal compartment positive for the tetraspanin CD81, a compartment that has recently been recognised as the site of HIV assembly in macrophages. Env was targeted to this compartment independently of other viral components in HeLa cells as well as in macrophages. In order to identify other cellular components interacting with Env and the machinery responsible for Env incorporation into virions I performed yeasts- hybrid assays. I found that Vps37B, a component of ESCRT-I, which is required for HIV assembly, can interact with both HIV and SIV Env cytoplasmic tails, and that the ubiquitin E3-ligase WWP2 binds SIV Env. These findings may enable us to establish a molecular mechanism for the incorporation of Env into budding HIV particles.
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Ward, N. J. « Lentiviral vectors for treatment of haemophilia ». Thesis, University College London (University of London), 2010. http://discovery.ucl.ac.uk/19905/.
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Haemophilia A and B are X‐linked recessive disorders caused by defects in coagulation factors (F) VIII and IX, respectively. Severe cases of haemophilia are characterised by episodes of spontaneous bleeding, predominantly into the joints and muscles, and can result in permanent disability and even mortality if left untreated. The haemophilias are compelling candidates for treatment with gene therapy as therapeutic benefit only requires a modest increase in the endogenous coagulation factor level, response to treatment can be easily monitored, and factor expression can be mediated by many cell types in vivo. Integration deficient lentiviral vectors (IDLVs) offer marked advantages over currently used integrating lentiviral vectors (ILVs) as side effects caused by insertional mutagenesis are potentially minimised. Previous work has shown that efficient and sustained transgene expression in non‐dividing cells, such as brain and muscle tissue, using IDLVs can be achieved. ILVs have previously been used to mediate long term expression of coagulation factors in vivo. In this study, we investigated the use of IDLVs as treatment for haemophilia with muscle and liver tissue (primarily nondividing hepatocytes) as principal targets. Transduction efficiency and relative transgene expression in vivo from ILVs and IDLVs were assessed in both tissues, and a number of strategies, including pseudotyping and tissue specific promoters, were utilised to improve targeted expression. Overall, despite achieving sustained transgene expression from IDLVs, in comparison to ILVs, the levels obtained were significantly lower and IDLVs were unable to mediate expression of human FIX at therapeutic levels in liver. Finally, the expression of bioengineered forms of human factor VIII (hFVIII) was assessed using ILVs in vivo after neonatal delivery. Long‐term expression was achieved and a 20‐fold increase in expression was observed after codon optimisation of the hFVIII cDNA sequence. In conclusion, IDLVs can mediate sustained transgene expression in vivo, however, vectors may need to be further optimised for increased expression to achieve clinical benefit for haemophilia patients.
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Bomfim, Aline de Sousa. « Clonagem e expressão de fator IX recombinante em células 293T e SK-Hep-1 e caracterização das células produtoras ». Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/60/60135/tde-13122013-111826/.
Texte intégralRésumé :
O fator IX (FIX) da coagulação sanguínea é uma proteína dependente de vitamina K de grande valor farmacêutico no tratamento da Hemofilia B, o qual é baseado na administração do fator de coagulação derivado de plasma humano ou da proteína recombinante produzida em células murinas. A terapia baseada nestas abordagens apresenta alto custo e está associada às contaminações com vírus e príons, além do desenvolvimento de inibidores de FIX. Esses efeitos aumentam o risco de morbidade e mortalidade relacionadas às hemorragias. Neste trabalho, clonamos o cDNA do FIX em um vetor lentiviral e avaliamos a expressão da proteína recombinante em duas linhagens celulares humanas. A clonagem do cDNA do FIXh no vetor de expressão lentiviral 1054 foi confirmada através da análise com enzimas de restrição específicas obtendo-se as bandas esperadas de 1407 pb e 10054 pb visualizadas em gel de agarose. As linhagens celulares 293T e SK-Hep-1 foram transduzidas com o vetor lentiviral 1054-FIX gerado em nosso laboratório e as células que apresentaram maior expressão de EGFP foram selecionadas e separadas por citometria de fluxo. A quantificação da expressão de FIXrh foi realizada por ensaios de ELISA e cromogênico. A quantificação de FIXrh total foi de 500 ng/106 células para a linhagem 293T e 803 ng/106 células para a linhagem SK-Hep-1. A atividade biológica específica de FIXh nas células 293T e SK-Hep-1 foi 0,047 UI/106 células e 0,186 UI/106 células, respectivamente. Com o intuito de avaliar o perfil de produção de FIXrh ativo ao longo do tempo, foi realizado um acompanhamento de 180 dias, no qual foi observado que a linhagem SK-Hep-1 cessou a expressão de FIX, enquanto as células 293T mantiveram a expressão durante o período. O FIXrh foi caracterizado por western blot confirmando a presença de uma banda imunoreativa esperada de 57 kDa. As linhagens 293T e SK-Hep-1 apresentaram 7,67 e 17 cópias do vetor inserido/célula, respectivamente. Considerando a importância do processo de ?-carboxilação, foi realizada uma análise da expressão gênica dos genes envolvidos neste processo, tais como o VKORC1, ?-carboxilase e o inibidor calumenina, nas linhagens celulares. Os resultados demonstraram razões elevadas entre os genes VKORC1 e calumenina e VKORC1 e ?-carboxilase nas duas linhagens. A cinética de crescimento das células foi realizada por um período de 7 dias apresentando diferenças significativas entre as células SK-Hep-1 transduzidas e não transduzidas, enquanto que as células 293T não presentaram diferenças estatísticas no crescimento celular. A suplementação do meio de cultura com íons Ca+2 e Mg+2 foi testada para avaliar sua influência na expressão de FIXrh ativo. As células 293T apresentaram melhor desempenho nas concentrações de 0,5 mmol/L de Ca+2 e 1,0 mmol/L de Mg+2 e as células SK-Hep-1 no meio de cultura não suplementado. Nossos dados indicam que a linhagem hepática SK-Hep-1 é a melhor produtora de FIXrh funcional e as comparações realizadas entre os dois tipos celulares são importantes na caracterização do comportamento de linhagens geneticamente modificadas voltadas para a expressão de proteínas recombinantes heterólogas e abre novos caminhos para futuros estudos que visam o melhoramento da produção desse tipo de proteína.Blood coagulation factor IX is a vitamin K-dependent protein, and it has become a valuable pharmaceutical in the treatment of Hemophilia B which is based on the plasma-derived coagulation factors or recombinant protein produced in murine cells. Coagulation therapy based on these approaches has high costs and is closely associated with prion and virus contamination besides the FIX inhibitors development. These effects increase the risk for bleeding-related morbidity and mortality. The purpose of this study was to clone hFIX into a lentiviral vector and evaluate the expression of the recombinant protein in two human cell lines. The cloning of the hFIX cDNA into 1054 lentiviral expression vector was confirmed by enzymatic restriction obtaining the expected 1407 bp and 10054 bp bands in agarose gel. The 293T and SK-Hep-1 cell lines have been stable transduced with 1054-FIX lentiviral vector generated in our laboratory and the cells with higher expression of EGFP were selected and separated by flow cytometry. The quantification of the expression of rhFIX was performed by ELISA and chromogenic assays. The concentration of total rhFIX was 500 ng/106 cells in 293T cell line and 803 ng/106 cells in SK-Hep-1 cell line. The biological activity of FIX secreted by 293T and SK-Hep-1 was 0,047 UI/106 cells and 0,186 UI/106 cells, respectively. In order to evaluate the active rhFIX production profile over time, we conducted a monitoring of 180 days, which was noted that the SK-Hep-1 cell line ceased FIX expression, while 293T cells maintained the expression during this period. rhFIX was characterized by western blot analysis confirming the presence of a expected 57 kDa immunereactive band. The 293T and SK-Hep-1 cell lines showed 7.67 and 17 integrated vector copies/cell, respectively. Considering the importance of the ?-carboxylation process, we performed a gene expression analysis of genes involved in this process, such as VKORC1, ?-carboxylase and calumenin, in cell lines. The results showed high ratios among the genes VKORC1 and calumenin and among VKORC1 and ?-carboxylase in both cell lines. The cell growth kinetics was performed by a 7-day period, showed significant differences between SK-Hep-1 transduced cells and non-transduced cells, whereas 293T cells showed no difference in cell growth. Enrichment of culture medium with Ca +2 and Mg +2 ions was tested to evaluate its influence on the expression of active FIX. 293T cells showed better performance in 0.5 mmol/L Ca+2 and 1.0 mmol/L Mg +2 concentrations and SK-Hep-1 cells in culture medium control. Our data indicate that transduced SK-Hep-1 cells are the best producer of functional rhFIX, and comparisons between these two cell lines are important in characterizing the behavior of genetically modified cell lines focused on the heterologous expression of recombinant proteins and opens new avenues for future studies aimed at improving the production of this type of protein.
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Godfrey, Andrew Robert. « Lentiviral vectors as tools for gene manipulation ». Thesis, University College London (University of London), 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.427691.
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Van, Tulleken C. R. « Characterisation of lentiviral Vpr function and mechanism ». Thesis, University College London (University of London), 2017. http://discovery.ucl.ac.uk/1567969/.
Texte intégralRésumé :
Genetic conflict between viruses and their hosts has driven an ‘arms race’, forcing the evolution of both immune defencesdefenses and multiple viral strategies to counteract and evade them. In the case of HIV many of these processes are well characterised – the virus carries with it a set of accessory proteins which target specific host restriction factors. Of these accessory proteins Vpr is the least well understood, with no described role that adequately explains its conservation across all known primate lentiviruses. Unpublished data from our lab indicate that Vpr is able to rescue infection in macrophages from addition of cGAMP, a second messenger protein produced by the cytosolic DNA sensor cGAS which activates antiviral immune signalling pathways. This study first sought to test the hypothesis that Vpr has evolved to counteract cGAS/STING mediated cytosolic DNA sensing using a co-transfection assay to test Vpr proteins from all groups of primate lentiviruses. Initial observations appeared to demonstrate specific degradation of innate immune signalling proteins. It was subsequently shown that HIV-1 M Vpr antagonises expression from all tested co-transfected plasmids. This phenotype was demonstrated to be species specific, and to correlate with both the history of zoonotic transmission and localisation of Vpr to the nuclear rim. Additionally, it was shown that Vpr antagonises NFB signalling activated by TNF, independent of an effect on expression from transfected plasmids, but with the same dependence on nuclear localisation, putatively by the same mechanism. Next, this study characterised an observation that the Vpr from the lentivirus infecting a mona monkey (SIVmon) stimulates NFB signalling. It was hypothesised that the SIVmon Vpr might have molecular binding partners in common with the HIV-1 M Vpr and conditions were optimised for proteomics studies to determine these binding partners. This study provides insights into the role of Vpr in antagonising innate immune sensing. Additionally, overexpression assays have been used widely in the literature describing Vpr. The data presented here indicate that observations using these assays, apparently demonstrating specific degradation of host cellular proteins, should be interpreted cautiously.
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Bichel, Katsiaryna. « Understanding post-entry pre-integration lentiviral biology ». Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.648287.
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Perro, M. « Lentiviral TCR gene transfer for tumour immunotherapy ». Thesis, University College London (University of London), 2010. http://discovery.ucl.ac.uk/134272/.
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The ability to manipulate the immune system to induce protection against tumour, is one of the most fascinating challenges in immunology. In this regard, TCR gene transfer is an attractive and powerful strategy to generate high numbers of tumour antigen-specific T cells for adoptive transfer treatment. This thesis describes the optimization of lentiviral vectors for TCR gene transfer in the absence of polyclonal activation of the transduced T cells, which may improve subsequent adoptive T cell therapy. The murinised and codon optimised chains of an HLA-A*0201-restricted TCR specific for Wilms` tumour antigen 1 were cloned in lentiviral vector constructs improved with a Leader sequence and the WPRE elements for redirecting T cells specificity. The effects of common gamma chain receptor cytokines IL-2, IL-7, IL-15 and IL-21 were investigated using WT1 TCR-transduced T cells for transduction efficiency, proliferative potential, phenotype and functional activity in response to cognate antigen. Although all cytokines tested allowed transduction, stimulations with IL-15 and IL-15 with IL-7 or with IL-21 promoted a higher efficiency. Expression analysis of CD28 and CD62L showed an important role of IL-21 in maintenance of a naïve phenotype. In addition, all cytokines promoted maintenance of “quality” of T cells as shown by co-expression of IL- 2, IFN-γ and TFN-α after specific stimulation. To further sustain the in vitro results, several in vivo models were tested. Consistently, using F5 transgenic mice recognizing the NP peptide presented on EL4-NP cell line, IL-15 with IL- 21 exposed CD8+ T cells were able to efficiently protect against tumour and to persist longer in tumour bearing mice compared to IL-2 treated T cells. Because previous reports demonstrated that efficient LN homing of T cell correlates with efficient tumour protection in vivo, an imaging approach to study the molecular signalling in vivo during T cell activation in the LN has been developed. In conclusion, in this thesis, it is demonstrated that lentiviral transduction of cytokine exposed T cell can improve gene therapy approach of adoptive therapy.
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Ingrao, Dina. « Etude de l'étape d'entrée des vecteurs lentiviraux dérivés du VIH-1 dans les cellules hématopoïétiques humaines ». Thesis, Evry-Val d'Essonne, 2013. http://www.theses.fr/2013EVRY0021/document.
Texte intégralRésumé :
Les vecteurs lentiviraux (LV) sont des outils efficaces de transfert de gène, largement utilisés en thérapie génique, en particulier pour la transduction ex vivo de cellules souches et progénitrices hématopoïétiques (CSPH). Afin d’étudier simultanément la fusion et la transduction dans les CSPH avec les LV, nous avons adapté une méthode basée sur latechnologie du transfert d’énergie entre deux molécules fluorescentes (FRET). Pour mettre en place cette technique, des LV capables d’incorporer spécifiquement une enzyme, la bétalactamase (BLAM-LV) et de coder une forme tronquée du récepteur au facteur de croissance nerveuse (DELTA-NGFR), sont produits. Nos résultats montrent que les LV sont soumis à une restriction post-entrée forte dans les cellules hématopoïétiques, que ce soit dans des lymphocytes T immortalisés ou bien des CSH CD34+. Nous montrons également que cette inhibition post-entrée peut être partiellement saturée après une forte augmentation de la multiplicité d’infection ou en présence d’additifs de culture, comme la Vectofusin-1® ou laRetronectin®. De plus, nous avons montré lors de la transduction de CSPH avec des vecteurs BLAM-LV que la Vectofusin-1® agit sur l’étape d’entrée en augmentant l’adhésion et la fusion entre les membranes virale et cellulaire. Cette technique représente donc un nouvel outil sensible et efficace pour étudier de façon concomitante l’étape de fusion et le niveau de transduction dans les cellules cibles. A terme, ce travail permettra une meilleure compréhension de la biologie des LV mais pourra également conduire à l’élaboration de protocoles de transduction lentivirale plus efficacesLentiviral vectors (LV) are used for various gene transfer applications, notably for hematopoietic gene therapy, but methods are lacking to precisely evaluate parameters that control the efficiency of transduction in relation with the entry of vectors into target cells. We adapted a fluorescence resonance energy transfer (FRET)-based HIV-1 fusion assay to measure the entry of non-replicative recombinant LV in various cell types, including primary human hematopoietic stem and progenitor cells, and to quantify the level of transduction of he same initially-infected cells. The assay utilizes recombinant LV containing betalactamase (BLAM)-Vpr chimeric proteins (BLAM-LV) and encoding a truncated form of thelow affinity nerve growth factor receptor (DELTA-NGFR). This LV-based fusion/transduction assay is a dynamic and versatile tool, revealing for instance the extent of lentiviral post-entry restrictions occuring in cells of hematopoietic origin. The assay also shows that transduction enhancers like Vectofusin®-1 or Retronectin® can partially relieve this post-entry block but their effects differ in the way to promote LV entry. Furthermore, our results show that Vectofusin®-1 acts at the entry step by promoting the adhesion and the fusion between lentiviral and cellular membranes. In conclusion, one such assay should be useful to study hematopoietic post-entry restrictions directed against LV and should allow improvements in various LV-based gene therapy protocols
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Lucke, Susann. « Reduktion der Vektormobilisierung durch RRE-defiziente lentivirale Vektoren ». [S.l.] : [s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=975691090.
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Moço, Pablo Diego. « Estabelecimento de uma plataforma para produção de vetores lentivirais para a modificação de linfócitos T com CAR anti-CD19 ». Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/17/17153/tde-13092018-153935/.
Texte intégralRésumé :
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.
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Ewerling, Sonja. « Evaluation of laser assisted lentiviral transgenesis in bovine ». Diss., [S.l.] : [s.n.], 2006. http://edoc.ub.uni-muenchen.de/archive/00006070.
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Aitchison, K. L. « Lentiviral vectors for gene therapy of Gaucher disease ». Thesis, University College London (University of London), 2015. http://discovery.ucl.ac.uk/1465062/.
Texte intégralRésumé :
Gaucher disease (GD), a recessive disorder characterised by hepatosplenomegaly, pancytopenia and skeletal complications, is caused by deficiency of the enzyme glucocerebrosidase (GC). GD leads to the accumulation of glucocerebrosides within macrophages, particularly in the liver and spleen. Current treatment is limited to enzyme replacement therapy (ERT) which is effective for most symptoms however skeletal problems are slow to respond. Treatment also has significant cost and impact on quality of life as infusions must be administered every two weeks. GD is a candidate for gene therapy as bone marrow transplantation has been shown to be curative which serves as a proof-of-concept that correction of haematopoietic stem cells (HSCs) can alleviate disease. This project produced lentiviral vectors carrying a range of constructs. GC was modified to contain a protein transduction domain (PTD) which could facilitate cross-correction of untreated cells in vivo. Recombinant vectors carrying PTD-GBA cDNA corrected the metabolic defect in patient-derived fibroblasts with levels of enzyme activity restored to within the healthy range. Transduced cells secreted active protein, uptake of which by untransduced cells was mediated by fusion of a PTD to the C- but not the N-terminus of the enzyme. The skeletal complications of GD are likely to be caused by enzyme deficiency in the osteoclast, a cell of haematopoietic origin. Therefore it is possible that by transducing HSCs we will be able to alleviate skeletal symptoms. To this end it is shown that modification of HSCs does not affect their ability to generate osteoclasts. It is also demonstrated that osteoclasts derived in vitro from the neuronopathic GD mouse model have increased activity and this could be a useful model for osteoclast correction when treating GD. In conclusion, this project generated lentiviral vectors for use in treating Gaucher disease. Further work should include correction of the osteoclast phenotype and further investigation of the potential for cross-correction in vivo.
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Stephanou, Coralea. « Advancing lentiviral gene therapy vectors for β-thalassaemia ». Thesis, King's College London (University of London), 2015. http://kclpure.kcl.ac.uk/portal/en/theses/advancing-lentiviral-gene-therapy-vectors-for-thalassaemia(5e010667-da35-449e-98f8-12229b28a523).html.
Texte intégralRésumé :
Background: β-thalassaemia is a potentially lethal hereditary anaemia, caused by reduced or absent expression of HBB polypeptide chains of adult haemoglobin (HbA: α2β2). Current curative treatment options are limited to few patients, while alternative, chronic palliative therapy consisting of frequent transfusions coupled with iron chelation therapy, is costly and reduces patients’ quality of life. Increased levels of fetal haemoglobin (HbF: α2γ2) were shown to lessen the severity of β-thalassaemia, highlighting the therapeutic potential of a gene-therapy-mediated increase in HBG1 and HBG2 (HBG) expression. Aims: The aim of this project was to develop a new generation of lentiviral vectors (LVs) in order to deliver a combinatorial gene therapy for β-thalassaemia (and sickle cell disease). LVs were designed to enhance HbF levels by engineering the HBB-expressing GLOBE LV to co-express short-hairpin RNAs (shRNAs) that will post-transcriptionally knockdown repressors of HBG expression, such as BCL11A. Conceptually, it was envisaged that the combination of LV-derived HBB plus increased endogenous HBG chains would achieve levels of total haemoglobin that would reproducibly be in the range (~50% of normal values) to be curative for β-thalassaemia major (and sickle cell disease). Methodology/Results: A series of modified LVs with simple shRNAs or microRNA-adapted shRNAs (shRNA-miRs) inserted within the second intron of HBB (βIVS-II) within GLOBE, were produced and functionally tested in erythroid tissue culture cell lines (K562, HEL, MEL) and in a human primary erythroid cell culture model system. Early experiments were conducted using shRNA-bearing vectors targeting mRNA of the GFP reporter to rapidly establish proof-of-principle for the vector design. Technical difficulties with the execution of experiments in GFP-expressing transgenic cell lines prompted the parallel use of BCL11A-targeting vectors aiming to induce HBG expression. The cell line work did not produce concrete findings, with every indication that these systems were unsuitable platforms for the intended analyses. Lastly, vectors were functionally tested in primary erythroid cells derived from CD34+ haematopoietic stem cells from both normal individuals and patients with β-thalassaemia. To this end, extraction protocols of CD34+ cells from peripheral blood samples were optimised, based on density-gradient centrifugation and simple ACK lysis of red blood cells. The effect of these protocols on cell viability, proliferation and differentiation was compared in standardised small-scale assays on semi-solid methylcellulose media. Standardising liquid cultures for large-scale CD34+ cell expansion and differentiation, however, posed unexpected challenges and prompted the evaluation of three separate protocols before suitable cell numbers and differentiation levels were achieved. Using the established systems, the RNAi competency of stem-loop sequences in shRNAs was functionally validated by expression from the RNA-polymerase-III U6 promoter of control vectors. Vectors carrying the same basic shRNA cassettes in the GLOBE-encoded HBB βIVS-II showed no discernible effect on target genes. In contrast, subsequent preliminary analyses of second-generation shRNA-miR vectors demonstrated their RNAi activity, making them the subject of further investigation for combinatorial gene therapy for the haemoglobinopathies.
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Neil, Stuart John Douglas. « Lentiviral mediated gene delivery to human antigen presenting cells ». Thesis, University College London (University of London), 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.251820.
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Hofmann, Andreas. « Entwicklung lentiviraler Transgenese in höheren Säugetieren ». [S.l.] : [s.n.], 2006. http://edoc.ub.uni-muenchen.de/archive/00005196.
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Hofmann, Andreas. « Entwicklung lentiviraler Transgenese in höheren Säugetieren ». Diss., lmu, 2006. http://nbn-resolving.de/urn:nbn:de:bvb:19-51961.
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Wen-Hsin, S. L. « Lentiviral-based RNA interference of genes in leukaemic cells ». Thesis, University College London (University of London), 2006. http://discovery.ucl.ac.uk/1445137/.
Texte intégralRésumé :
Childhood leukemia is a common paediatric cancer in the developed world and the biologically diverse subtypes of this disease are characterised by specific chromosomal translocations that alter the normal proliferative and survival signals of haematopoietic cells. Despite of greatly improved cure rate of childhood leukaemias over the past years, younger patients with acute leukaemias involving E2A-HLF and MLL-ENL translocations still confer a poor prognosis that is associated with a very unfavourable outcome. The incidence of relapse after complete remission seems to crucially depend on a small population of leukaemic stem cells that survive from the initial therapy and sustain the disease. So far it has been unsuccessful to induce long-term gene silencing using siRNA technology in the primary haematopoietic cell lines and leukaemic stem cells. Thus, this project aimed to optimise the current 2nd generation of miR30-based shRNA lentiviral vector to achieve this. The silencing cassettes were delivered to the cells of interest by lentivirus and long-term expression was seen. The result revealed that effective E2A-HLF and MLL-ENL gene silencing was achieved while LM02 gene expression was not significantly knocked down by the predicted LM02 shRNA constructs. The most efficient lentiviral vectors against specific genes will then be used to infect leukaemic cells to test the effect on aspects of the leukaemic phenotype. Understanding of these fusion genes and identification of their downstream target genes in initiating and maintaining transformation events in the leukaemic stem cells may aid the development of revolutionary therapeutics that specifically target leukaemic stem cells. In conjunction with standard therapies, this approach could be more effective in treating MLL-ENL and E2A-HLF patients who tend to have an extremely unfavourable prognosis.
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Heilmann, Jessica [Verfasser]. « Lentiviral Nef-mediated CD4 and CD28 downmodulation / Jessica Heilmann ». Ulm : Universität Ulm. Medizinische Fakultät, 2014. http://d-nb.info/104689014X/34.
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Bokhoven, Marieke Christina. « Measurement of insertional mutagenesis by retroviral and lentiviral vectors ». Thesis, University College London (University of London), 2008. http://discovery.ucl.ac.uk/1444113/.
Texte intégralRésumé :
Retroviral vectors have been successfully used in the clinic for the correction of inherited immunodeficiencies. However, in 2 recent gene therapy trials for X-linked Severe Combined Immunodeficiency, 5 patients developed leukaemia. This was associated with vector integration near cellular proto-oncogenes, leading to their activation a process known as insertional mutagenesis. This thesis describes the development of a cell line assay that allows us to quantify insertional mutagenesis by retroviral and lentiviral vectors and to analyse the mechanisms by which this occurs. The interleukin-3 (IL-3) dependent cell line BCL15 is derived from the mouse bone marrow cell line BAF3 and over- expresses human Bcl2. The frequency at which IL-3 independent mutants are obtained following vector transduction is measured. Lentiviral and retroviral vectors transform BCL15 cells at similar integrant frequencies of 4.3 x 108 and 1.2 x 107, respectively. However, they cause insertional mutagenesis in this assay by different mechanisms. The human immunodeficiency virus-1 (HIV-1)- derived lentiviral vector HV transforms BCL15 cells by insertional activation of the growth hormone receptor (Ghr) gene. An HIV-Ghr fusion transcript was detected. It originates from the HIV-1 5'-LTR it then splices from the HIV-1 major splice donor to the splice acceptor of Ghr exon 2. The mutants express GHR and grow in response to bovine growth hormone in the foetal calf serum of the culture medium. Deletion of the HIV-1 enhancer/promoter in a self-inactivating vector prevents this mechanism of transformation. Retroviral vector transformation of the BCL15 cell line does not occur via Ghr gene activation. Retroviral vectors up- regulate expression of the cytokine IL-3, either by insertion into the IL-3 gene or by insertion into other genes, which may act as upstream activators of IL-3 expression. This assay is a general method to quantitate insertional mutagenesis. It may inform the design of safer vectors and can be used in initial safety testing of (pre-) clinical vectors.
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Thomas, Joan Helen. « Studies in gene transfer using pseudotyped lentiviral vector systems ». Thesis, University of Cambridge, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.621818.
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Cheeks, Matthew Christopher. « Intensified processing of recombinant lentiviral vectors with structured adsorbents ». Thesis, University of Cambridge, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.611765.
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Guy, H. M. « Microscale characterisation of a manufacturing route for lentiviral vectors ». Thesis, University College London (University of London), 2015. http://discovery.ucl.ac.uk/1461139/.
Texte intégralRésumé :
Lentiviral vectors used in clinical trials are currently produced by transient transfection of adherent human embryonic kidney (HEK)293(T) cells. However, this approach is not scalable and for commercialisation the development of alternative strategies based on suspension-adapted producer cell lines, that have all genes for vector production stably integrated, is desired. To assist progress in this area, the aim of this thesis was to establish a microscale cell culture platform that enables key bioprocess design data to be acquired rapidly and cost-effectively. ProSavin®, an equine infectious anaemia virus (EIAV)-derived lentiviral vector developed for the treatment of Parkinson’s disease (Palfi et al., 2014) was used as a model system. First, the suitability of a shaken 24-well plate system for the suspension culture of HEK293T-derived producer cells was established. This system was shown to support equivalent cell growth and ProSavin® titres to conventional shake flasks while providing substantially greater opportunity for parallelisation. Second, the utility of the microscale platform when combined with statistical Design of Experiments (DoE) techniques for optimising titres and informing the design of a scale-up strategy was demonstrated. An initial screening experiment identified three parameters as having a critical influence on ProSavin® titres, which were post-induction period, liquid fill volume and concentration of doxycycline (inducer compound). Subsequent optimisation experiments defined operating ranges for these parameters. Third, the insights obtained during the microwell investigations were shown to aid successful scale-up of the ProSavin® process to a single-use 2 L WAVE bioreactor. Fourth, with a view to informing further improvements in process design, the half-life of ProSavin® and other EIAV-based lentiviral vectors was determined, and approaches to moderate the rate of decay during upstream processing trialled. Overall, it was concluded that the microwell platform may be viewed as an effective tool for future use in the development of lentiviral vector bioprocesses.
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Agnew, Kelly Kathleen. « Butterfly oviposition behavior, pika biogeography, and lentiviral sequence evolution / ». Digital version accessible at:, 1999. http://wwwlib.umi.com/cr/utexas/main.
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Jármy, Gergely. « Herstellung und Anwendungen eines replikationsinkompetenten lentiviralen Vektorsystems ». [S.l.] : [s.n.], 2001. http://deposit.ddb.de/cgi-bin/dokserv?idn=964527359.
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Messow, Diana. « Lentivirale Vektoren und RNAi für den Einsatz in der Xenotransplantation ». Diss., Ludwig-Maximilians-Universität München, 2014. http://nbn-resolving.de/urn:nbn:de:bvb:19-164826.
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Henriot, Dorothée. « Vectorisation lentivirale transitoire pour la thérapie génique et la transgénèse ». Paris 7, 2010. http://www.theses.fr/2010PA077233.
Texte intégralRésumé :
Le transfert de gène est un outil puissant pour la transgénèse et la thérapie génique. Les lentivirus sont parmi les vecteurs de transfert les plus prometteurs. Mais leur capacité d'intégration pose un problème de biosécurité. L'intégration ciblée nécessite elle-même la vectorisation sécurisée et transitoire des facteurs de recombinaison en raison de leur toxicité en cas d'expression stable. Nous avons donc développé plusieurs systèmes de vecteurs lentiviraux permettant l'expression transitoire d'un transgène dans des cellules en arrêt de division ou en division. Le premier système développé est un vecteur non-intégratif. Nous avons montré que l'adjonction de séquences MAR ne permet pas d'améliorer le niveau d'expression du transgène. Puis nous avons comparé des vecteurs portant différentes mutations de l'intégrase et avons ainsi déterminé que les mutations D64V et LQ permettent une expression plus efficace du transgène que la mutation N. Nous avons également mis au point des systèmes de vectorisation lentivirale reposant sur des substrats protéiques ou ARN. La vectorisation protéique repose sur la fusion de la protéine d'intérêt à VPR ou à l'intégrase. Nous avons montré l'encapsidation de la recombinase PhiC31 fusionnée à VPR. Les systèmes de vectorisation sous forme ARN reposent sur l'inhibition du mécanisme de rétro-transcription par délétion du PBS ou mutation de la rétro-transcriptase. Nous avons réalisé pour les deux systèmes la vectorisation de transgènes rapporteurs et d'une enzyme de recombinaison Cre. Nous avons prouvés que les événements observés sont dus à l'ARN viral, que les vecteurs sont dépourvus de rétro-transcription et d'intégration résiduelleGene transfer is a powerful tool for both transgenesis and gene therapy. Lentivirus are among the most promising gene transfer vectors. However, the issue of their integrative properties is crucial in terms of biosafety. Targeted-integration protocols usually imply the use of recombination factors that should themselves be vectorized in a safe and transitory manner because of their potential toxicity when stably expressed. Thus, we developed several Systems of lentiviral vectors allowing transient expression of a transgene in arrested or dividing cells. The first System consists of a non-integrating lentiviral vector. In a first step, we demonstrated that the addition of MAR sequences do not improve the transgene expression level. Then we compared vectors bearing various mutations of the integrase (D64V, N and LQ) and demonstrated that thé D64V and LQ mutations allow for higher expression than the N mutation. In a second step, we developed lentivectors based on proteic or ribonucleic substrates. The proteic vectorization Systems are based on the fusion of the protein of interest to VPR or Integrase. We demonstrated that PhiC31 recombinase fused to VPR can be encapsidated into lentiviral virions. The ribonucleic vectorization is based on the inhibition of the retrotranscription mechanism of retroviruses by deletion of the PBS or mutation of the - retrotranscriptase. We showed for both Systems their ability to vectorize reporter as well as the Cre recombination enzyme. In parallel, we demonstrated that the observed recombination events were due to the viral RNA and that the vectors are lacking retrotranscription and integration activities
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Berger, Grégory. « Etude de la restriction des cellules myeloïdes à l'infection lentivirale ». Thesis, Lyon, École normale supérieure, 2011. http://www.theses.fr/2011ENSL0699.
Texte intégralRésumé :
Les cellules de la lignée myéloïde jouent un rôle majeur dans la pathogénèse du VIH, servant à la fois de réservoir viral et permettant la transmission du virus aux cellules T. Cependant, ces cellules sont relativement résistantes à l’infection lentivirale par comparaison aux cellules provenant d’autres lignées. Des études provenant de divers laboratoires, dont le notre, ont montré que les étapes précoces de l’infection semblent se dérouler beaucoup moins efficacement dans ces cellules. Nous avons donc cherché à identifier des facteurs spécifiquement exprimés dans ces cellules pouvant être à l’origine de ce blocage. Ainsi, nous avons pu identifier APOBEC3A (A3A), un membre de la famille des APOBEC3s. A3A est spécifiquement exprimée dans les cellules de la lignée myéloïde mais n’était pas connue pour bloquer la réplication du VIH. Nous avons pu montrer que le pool d’A3A présent dans les cellules cibles est capable de cibler les particules entrantes d’une manière dépendante de son activité enzymatique. Sa déplétion, au moyen d’ARNs interférents, permet d’augmenter l’accumulation de l’ADN viral. Nos données suggèrent donc que A3A induit la dégradation des génomes viraux et que son activité antivirale est dirigée plus généralement contre les lentivirus de Primates. Cependant, la protéine Vpx des membres de la famille VIH-2/SIVSM permet de protéger contre l’action de A3A en induisant sa dégradation via le protéasome.Nous avons mis à jour un nouveau rôle de A3A lors de l’infection lentivirale des cellules myéloïdes. Ces données remettent en question le mode de fonctionnement des membres de la famille des APOBECS et ouvrent de nouvelles questions sur leurs modes d’action et leurs régulations dans les cellules primairesMyeloid cells are important for HIV pathogenesis both for viral transmission to T cells and as a viral reservoir. Nonetheless, these cells are quite restrictive to HIV infection compared to established cell lines or primary T cells. Studies from our group as well as other laboratories suggest thatthe early steps of infection are particularly inefficient in these cells . We tried to identify cellular factors specifically expressed in myeloid cells that could be responsible for this block. We identified APOBEC3A as one of such factors. A3A is a member of the APOBEC3 family and is the sole specifically expressed in myeloid cells. We showed that A3A blocks HIV incoming viral particles and more generally primates lentiviruses specifically in myeloid cells in a cytidine deaminase dependant manner. Our data suggest that A3A decreases viral DNA accumulation by inducing the degradation of newly synthesized genome most probably after deamination. Among the proteins coded by primate lentivirus, the HIV-2/SIVsm Vpx protein interacts and degrades A3A thus providing partial protection against A3A.Overall, our data reveal a novel role for A3A in the infection of myeloid cells and raises important questions about the regulation of the cell type specific antiviral role of A3A in primary myeloid cells
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Giorgi, Marie. « Optimisation de la stratégie de thérapie génique par vecteur lentiviral pour la β-thalassémie ». Thesis, Sorbonne Paris Cité, 2019. http://www.theses.fr/2019USPCC047.
Texte intégralRésumé :
La β-thalassémie est l’une des maladies génétiques les plus répandues au monde. Elle résulte d’un déséquilibre fort entre les chaînes alpha et beta de l’hémoglobine. La thérapie génique des cellules souches hématopoïétiques est une stratégie innovante et prometteuse pour le traitement des patients -thalassémiques. En l’occurrence, l’utilisation d’un vecteur lentiviral délivrant la chaîne β-globine, testé chez dix-huit individus, a fait ses preuves chez la majorité d’entre eux. Nous avons entrepris d’améliorer la balance bénéfice risque de cette stratégie de différentes manières. Grâce à un système de sélection alliant le gène de résistance à la puromycine et le transgène thérapeutique, nous avons réussi à augmenter le pourcentage de cellules souches hématopoïétiques génétiquement modifiées. Par l’introduction d’un shARNmir dirigé contre l’α-globine, nous avons rendu plus efficace le rééquilibre des chaînes alpha et beta-globines. Enfin nous avons élaboré et testé des stratégies visant à réduire les phénomènes de mutagénèse insertionnelle associés à l’utilisation des vecteurs lentiviraux. Sans obtenir les résultats escomptés, nous avons modifié le profil d’intégration des vecteurs lentiviraux. Au total, ces travaux contriburont à l’amélioration de l’efficacité de la procédure de traitement des patients β-thalassémiques par thérapie génique lentiviraleΒ-thalassemia is one of the most frequent genetic disease in the world. It results from the defective production of -globin and a major disequilibrium between beta and alpha globin chains. Gene therapy of hematopoietic stem cells is an innovative and promising strategy for the patient recovery. A lentiviral vector delivering the β-globin chain had recently proved its efficacy upon clinical trials. We have worked to improve this strategy using several approaches. Thanks to a selection system, combining the puromycin resistant gene and the therapeutic transgene, we have succeeded in extending the proportion of genetically modified cells. Through the introduction of a shRNAmir targeting the α-globin chain in the lentiviral vector, we have observed a high improvement of the beta to alpha globin ratios, in fetal and in patient’s erythroid cells. In addition, we have developed and analyzed several strategies in order to reduce the insertional mutagenesis linked to the use of lentiviral vectors. We did not obtain convincing results to target lenviral vector into expected harbors but the vector insertion profile was successfully modified. In summary, this work will contribute to the enhancement of the efficacy of the gene therapy treatment strategy of β-thalassemic patients
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Zhang, Bing. « Lentiviral vector-mediated gene transfer in vitro and in vivo / ». St. Lucia, Qld, 2004. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe18024.pdf.
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Til, Nico Peter van. « Lentiviral gene therapy for the treatment of inherited liver disease ». [S.l. : Amsterdam : s.n.] ; Universiteit van Amsterdam [Host], 2007. http://dare.uva.nl/document/49087.
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Belzile, Jean-Philippe. « Redirecting lentiviral integration : a study of human immunodeficiency virus integrase ». Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=97906.
Texte intégralRésumé :
Despite great advances in our understanding of the human immunodeficiency virus (HIV) life cycle, the mechanisms that underlie the progression of HIV from cellular entry of the viral core to stable integration of the provirus are poorly understood. Sites of integration of the HIV provirus are distributed along the full length of actively transcribed genes and appear to be determined through protein-protein interactions between the viral integrase and cellular proteins.Two cellular proteins have been proposed to perform integration targeting roles, the chromatin-remodeling factor integrase interactor 1 (INI1/hSNF5/BAF47) and the lens epithelium-derived growth factor/transcriptional co-activator (LEDGF). Here, we report the initiation of two novel integration assays to study the contribution of INI1 and LEDGF in target site selection. Elucidating these molecular determinants and their functional implications is also of particular interest to anti-HIV therapy and could have major impact on the safety of gene therapy protocols.
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Booth, C. A. « Lentiviral vector mediated gene therapy for X-linked lymphoproliferative disease ». Thesis, University College London (University of London), 2012. http://discovery.ucl.ac.uk/1356299/.
Texte intégralRésumé :
X-linked lymphoproliferative disease (XLP) is a rare primary immunodeficiency characterised by severe immune dysregulation and is caused by mutations in the SH2D1A gene. Clinical manifestations vary and include haemophagocytic lymphohistiocytosis (HLH), lymphoma and dysgammaglobulinaemia, often triggered by Epstein-Barr virus (EBV) infection. SLAM-associated protein (SAP) is a key regulator of immune function in T, NK, and NKT cells and defects in this protein lead to the cellular and humoral immune defects described in patients. Treatment options for XLP are limited and currently haematopoietic stem cell transplant (HSCT) is the only curative option. Results are variable and dependent on a good donor match and absence of active infection at transplant. Somatic gene therapy is now successfully used to correct certain severe immunodeficiencies and offers a potential cure in XLP. The use of self-inactivating (SIN) lentiviral vectors with transgene expression driven by non-viral promoters has improved the biosafety profile of haematopoietic stem cell gene therapy procedures. In this study we have successfully corrected both cellular and humoral defects in a SAP deficient murine model using a SIN lentiviral vector with a codon optimised SAP transgene under the transcriptional control of the elongation factor 1α short form (EFS) promoter. Initial attempts with a non-codon optimised version of SAP led to insufficient protein expression levels to restore immune function. We also assessed the CD2 locus control region (LCR) to evaluate any lymphoid specificity to permit more regulated SAP expression but were unable to demonstrate any benefit with this regulatory element. The results presented here provide proof of concept for the development of gene therapy for XLP and further work is warranted to improve the efficiency of gene transfer, secure engraftment of long term repopulating haematopoietic stem cell progenitors and additional characterisation of immune reconstitution after gene therapy.
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Ward, E. M. « Novel fusion protein-expressing lentiviral vectors ameliorate collagen induced arthritis ». Thesis, University College London (University of London), 2010. http://discovery.ucl.ac.uk/20480/.
Texte intégralRésumé :
Collagen induced arthritis (CIA) is a mouse model of autoimmunity that closely resembles human rheumatoid arthritis (RA), a debilitating disease with no cure. This study has been undertaken to generate antigen-specific tolerance to an autoantigen implicated in RA and immunodominant in animal models. Gene therapy protocols for RA that have gone to clinical trial have been designed to drive expression of therapeutic molecules at the site of inflammation, but not to modulate the immune response to key autoantigens. This study has shown that lentiviral vectors (lvv) expressing fusion proteins (FP-lvv) confer antigen-specific tolerance in CIA. Fusion proteins comprised of an endosomal-targeting domain coupled to the immunodominant CII259-273 peptide and an eGFP tag, were expressed in APCs. Confocal microscopy revealed substantial colocalisation with endosomes and lysosomes. Expression of the fusion proteins in APCs resulted in MHCII-presentation of the immunodominant CII259-273 peptide to CII259-273-reactive CD4+ T cell hybridomas. Furthermore, co-transduction with a lvv expressing the enzyme lysyl-hydroxylase 3 enhanced glycosylation of the expressed CII construct. Administering mice iv with FP-lvv one month prior to disease induction reduces by half the arthritic score during the first two weeks of clinical symptoms in CIA, providing partial but significant protection. The use of suitable controls showed that this effect is antigen-specific and measurements of α-CII IgG show a significantly lower titre in treated animals. This study provides evidence that lvv-mediated MHCII-presentation can be tolerogenic and hence, this approach could form an important part of future treatments for autoimmune diseases.
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Chan, E. « Lentiviral gene therapy for HIV using TRIM-cyclophilin restriction factors ». Thesis, University College London (University of London), 2012. http://discovery.ucl.ac.uk/1362851/.
Texte intégralRésumé :
Lentiviral vector delivery of anti-HIV elements could provide the basis of alternative therapies against HIV, potentially providing long term protection after a single intervention. Some primate species have evolved restriction factors formed by the fusion of TRIM5α and Cyclophilin A (TRIM5Cyp) following retrotransposition of CypA cDNA into the TRIM5 gene, which provide potent resistance against certain lentiviruses. We have designed humanised versions of these proteins combining both TRIM5 and TRIM21 with CypA, and investigated their potential for use in gene therapy against HIV-1. Both TRIM5- and TRIM21-Cyp fusion proteins provided strong restriction of HIV-1 in all of the systems tested, including primary human T cells. However, TRIM5Cyp was shown to disrupt the antiretroviral effect of endogenous TRIM5α and rescue murine retrovirus infection, whereas TRIM21Cyp caused no interference. In contrast, neither TRIM5CypA nor TRIM21CypA expression affected the antiviral activity of endogenous TRIM21. In addition to TRIMCyp restriction factors, a second anti-HIV strategy was investigated using zinc finger nucleases (ZFNs) to knockout the HIV-1 co-receptor, CCR5. ZFNs introduce a double stranded break into the CCR5 gene, which can be restored by homology directed repair. Provision of a green fluorescent protein (GFP) or TRIM21Cyp donor template exploits this repair mechanism to allow site specific integration at the CCR5 locus, although at low efficiency. Using integrating vectors, we have shown that TRIMCyp mediated restriction is so potent that no additional inhibition was conferred by CCR5 knockout. In conclusion, delivery of TRIMCyp genes using lentiviral vectors could form the basis of an intracellular vaccination strategy against HIV-1, with TRIM21Cyp having benefits by maintaining endogenous TRIM function. With further optimisation to improve efficiency, this could be combined with ZFNs for site specific integration of the transgene and knockout of CCR5 to provide a dual method of HIV-1 inhibition.
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Macdonald, D. « Lentiviral vector vaccines for T-cell-mediated protection against influenza ». Thesis, University College London (University of London), 2014. http://discovery.ucl.ac.uk/1417882/.
Texte intégralRésumé :
Vaccines that induce T cells which recognize conserved viral proteins could confer cross-strain protection against pathogens with fast-mutating B cell epitopes. Influenza is an example of such a pathogen for which there is a pressing need for a universal vaccine. Lentiviral vectors are a counterintuitive choice as vaccines since they have low inherent immunogenicity. However, their efficient transduction of non-dividing cells and high capacity permits transduction of antigen presenting cells with not only antigen but also molecular adjuvants that directly or indirectly enhance the T cell response. We therefore investigated the potential of two such adjuvants: viral flice-like inhibitor protein, which activates dendritic cells through nuclear factor kappa-B, and 4-1BB ligand, which activates T cells directly through 4-1BB. By co-encoding these with influenza nucleoprotein, we have shown that the influenza-specific T cell response to lentiviral vector vaccination is significantly enhanced in mice. Furthermore, we have demonstrated that intranasally delivered lentiviral vectors transduce alveolar macrophages with high efficiency, recalling and expanding large and sustained populations of nucleoprotein-specific CD8+ T cells in the lung and airway in mice that have been primed subcutaneously or previously exposed to influenza. These lung-resident T cell populations persist for at least 4 months and are sufficiently abundant to rapidly control a mouse-adapted lethal influenza challenge without invocation of a secondary cytokine response, weight loss or lung injury. Furthermore, dendritic cells expressing 4-1BBL potently trans-activate bystander dendritic cells, both in vitro and in vivo, demonstrating an indirect mechanism by which the 4-1BBL:4-1BB signaling axis can enhance T cell responses.