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Trimby, Christopher Matthew. "STRATEGIES FOR TARGETING LENTIVIRAL VECTORS." UKnowledge, 2011. http://uknowledge.uky.edu/gradschool_diss/157.
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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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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.
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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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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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Gelinas, Jean-Francois. "Enhancement of lentiviral vector production through alteration of virus-cell interactions." Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:9921b8b4-e2b5-4eec-9efc-6036765c8d55.
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Gene therapy is the introduction or alteration of genetic material with the intention to treat disease. To support this aim, viruses have been modified, with elements linked to viral pathogenicity removed from their genome and replaced by the genetic material to be delivered. Gene therapy vectors based on lentiviruses have many advantages, such as the ability to transduce non-dividing cells and to target specific cell types via pseudotyping. They have been successfully used in ex vivo clinical trials for several haematopoietic stem cell disorders. Lentiviral vectors, however, suffer from substantially lower titres than the more popular adeno-associated virus (AAV)-based vectors and therefore have limited applicability for in vivo gene therapy which requires much greater quantities of virus. The main aim of this thesis was to investigate strategies to improve lentiviral vector productivity during manufacture, in order to increase the likelihood of lentiviruses being adopted for disease treatment. Initial experiments were based on the lentiviral vector manufacturing process currently being developed by the United Kingdom Cystic Fibrosis Gene Therapy Consortium for the generation of highly concentrated, purified lentivirus for clinical use. Supplementation of FreeStyle 293 Expression Medium used during upstream processing was attempted, but none of the assessed supplements led to significant increases in lentiviral vector production. Investigation into intrinsic immunity to viral infection indicated that over-expression of the protein kinase RNA-activated (PKR) led to lower production titres, but over-expression of its inhibitors was not successful at increasing titres. The focus then shifted to reducing, or 'knocking-down', inhibitory factors present in the host cells, which could adversely affect viral titres. Investigation of the published HIV-1 literature revealed a possible 152 candidate inhibitory factors described as having a negative impact on HIV-1 replication in the late stages of the life cycle of the virus. A novel siRNA screen was developed to assess the effect of âknock-down' of inhibitory factors on lentiviral vector titre. Application of the screen to 89 candidate inhibitory factors identified nine genes which, when knocked-down, resulted in increased lentiviral vector production by more than 40%. Further work will be necessary to understand the role of the inhibitory factors in lentiviral vector production, but novel cell lines in which genes encoding these factors have been permanently deleted from producer cells could lead to higher titres, reducing costs in the manufacture of lentiviral vectors and making in vivo gene therapy more feasible from a health economics perspective.
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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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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/.
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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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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/.
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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.
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Oakland, Mayumi. "Improving lentiviral vector-mediated gene transfer by understanding cellular barriers." Diss., University of Iowa, 2013. https://ir.uiowa.edu/etd/4709.
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Cystic fibrosis (CF) is an autosomal recessive genetic disorder of which lung disease is the leading cause of morbidity and mortality. One attractive strategy for the treatment of CF lung disease is to directly deliver CF transmembrane conductance regulator gene to airway epithelia. Although promising results have been reported, barriers present in the lung make successful gene transfer to the respiratory tract difficult. In order to improve gene transfer strategies in the intrapulmonary airways, we need to identify the bottlenecks of transduction for the vector system. A previous study reported that feline immunodeficiency virus (FIV)-mediated gene transfer was more efficient in the nasal airways in mice than the intrapulmonary airways (Sinn, P.L. et al. 2008, J. Viol). Our first goal was to identify barriers to lentiviral gene transfer in the murine airways. We demonstrate that host immune response is not the major barrier preventing efficient FIV-mediated transduction in the intrapulmonary airways. We show that the FIV vector transduces murine primary nasal epithelial cell cultures with greater efficiency than murine primary tracheal epithelial cell cultures. In addition, GP64 pseudotyped vesicular stomatitis virus (VSV) transduces better in nasal epithelia compared to intrapulmonary airways in mice. On the other hand, we observed that VSVG glycoprotein-pseudotyped VSV transduces the intrapulmonary airway as well as nasal epithelia in mice with similar efficiency. Our results suggest that differentially expressed cellular factor(s) specific for GP64 or FIV vector may be the major barrier(s) for FIV vector-mediated gene transfer in the murine intrapulmonary airways.
The recent development of CF porcine models prompted us to investigate possible barriers for lentiviral vector-mediated gene transfer in porcine cells. Our preliminary results showed that HIV transduction was restricted in porcine but not human lung-derived cell lines. Porcine TRIM5 has sequences similar to restrictive bovine TRIM5 orthologs. Therefore, our second goal was to investigate the possible restriction of lentiviral vectors by porcine TRIM5. We demonstrate that transient overexpression or knockdown of endogenously expressed porcine TRIM5 does not affect HIV or FIV transduction.
Lastly, we characterized a mucin domain-deleted EBOV (EBOVΔO) glycoprotein mutant with increased transduction. This EBOVΔO 5-mer mutant was generated based on mutants with an increased transduction as identified by alanine scanning mutagenesis (Brindlay, M.A. et al. 2007. J. Viol). We show that VSV pseudotyped with the 5-mer mutant increased transduction both in vitro and in mice when compared to the wild-type EBOVΔO. Structural analysis demonstrated that 5 mutations were located proximal to the GP1-GP2 interface. Enhanced transduction likely results from a lower energy metastable state of the glycoprotein. FIV pseudotyped with 5-mer also shows increased transduction in multiple cell lines. Identification of barriers in intrapulmonary airways and improvements of vector systems will help the advancement of gene therapy for CF.
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Mekkaoui, Leila. "Lentiviral vector purification using genetically encoded biotin mimic in packaging cell." Thesis, University College London (University of London), 2018. http://discovery.ucl.ac.uk/10053191/.
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Lentiviral vectors (LVs) are powerful tools in gene therapy that have recently witnessed an increasing demand in both research and clinical applications. Current LVs purification represents the main bottle neck in their application as several methods are employed which are time consuming, cumbersome and yield low recoveries. The aim of this project was to develop a one-step method to specifically and efficiently purify LVs, with high vector yields and reduced levels of impurities, using the biotin-streptavidin system. Herein, packaging 293T cells were genetically engineered with biotin mimicking synthetic peptides and different cell membrane anchoring strategies for optimal streptavidin binding were tested. We have identified a flanked disulphide-constrained peptide, termed Ctag (ECHPQGPPCIEGRK), displayed on a CD8α stalk to be the most promising. LVs were modified with Ctag by its random incorporation onto viral surfaces during budding, without viral protein engineering or hindrance on infectivity. The expression of Ctag on LVs allowed complete capture of infectious particles by streptavidin magnetic beads. As Ctag binds streptavidin in the nanomolar range, we hypothesised that gentle elution from streptavidin matrix should occur by biotin’s competitive binding. Accordingly, addition of micromolar concentrations of biotin to captured LVs resulted in an overall yield of ≥60%. Analysis of eluted LVs revealed high purity levels, with a ≤3-log and 2-log reduction of DNA contamination and host cell proteins, respectively. This one-step purification was also tested for scalable vector processing using streptavidin monolith affinity chromatography and preliminary results were encouraging with 20% overall yield. In conclusion, we developed a single-step affinity chromatography which allows specific purification and concentration of infectious vectors modified with a biotin mimic. Based on intended usage, efficient LV purification can be achieved using both magnetic beads and column chromatography. This method will be of valuable use for both research and clinical applications of LVs.
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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.
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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.
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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.
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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, Xinyu. "Expression of polysialic acid mediated by lentiviral vector to promote axonal regeneration." Thesis, Queen Mary, University of London, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.443583.
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Langford-Smith, Alexander William Walker. "Lentiviral vector mediated haematopoietic stem cell gene therapy for mucopolysaccharidosis type IIIA." Thesis, University of Manchester, 2012. https://www.research.manchester.ac.uk/portal/en/theses/lentiviral-vector-mediated-haematopoietic-stem-cell-gene-therapy-for-mucopolysaccharidosis-type-iiia(89f8e108-58f3-42bb-8b80-0e0a1fe45fd7).html.
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Mucopolysaccharidosis type III (Sanfilippo) is comprised of four phenotypically similar lysosomal storage disorders (MPS IIIA-D) caused by the deficiency of enzymes that catabolise heparan sulphate (HS). Progressive accumulation of HS results in abnormal behaviour, progressive cognitive and motor impairment and death in mid-teens. There are currently no treatments for MPS III. To assess the effect of novel therapeutics in the mouse models of MPS III it is necessary to examine the effect on primary storage of HS, secondary storage and behaviour. The reported behaviour of MPS IIIA and B mice is conflicting therefore we developed a one-hour open field test, performed at the same time of day during a period of hyperactivity observed in a previous circadian rhythm study of MPS IIIB mice. At 8 months of age MPS IIIB mice were hyperactive, with increased rapid exploratory behaviour and a reduction in immobility time. The MPS IIIA mice presented with the same behavioural phenotype as the MPS IIIB mice and were significantly hyperactive at 4 and 6 months of age and also displayed a reduced sense of danger. The hyperactivity and reduced sense of danger observed in the mice is consistent with the patient phenotype. Whilst haematopoietic stem cell transplant (HSCT) is the standard therapy used to treat the similar HS storage disorder MPS I Hurler, it is ineffectual in MPS IIIA. We hypothesise that HSCT failure in MPS IIIA is due to insufficient enzyme production in the brain by donor-derived microglial cells. By increasing expression of N-sulphoglucosamine sulphohydrolase (SGSH) we may be able to treat MPS IIIA. Therefore we compared the effect of HSCT using normal haematopoietic stem cells (WT-HSCT) to lentiviral overexpression of SGSH in normal cells (LV-WT-HSCT) or MPS IIIA cells (LV-IIIA-HSCT) in MPS IIIA mice, using the behavioural tests developed.SGSH activity in the brain of MPS IIIA recipients was not significantly increased by WT-HSCT, but was significantly increased by LV-IIIA-HSCT and LV-WT-HSCT. HS was significantly reduced by all transplants but the best treatment was LV-WT-HSCT. Neuroinflammation, indicated by the number of microglia in the brain, was significantly reduced by all treatments but remains significantly elevated. GM2 gangliosides were significantly reduced by WT-HSCT and LV-WT-HSCT and were no longer significantly elevated, but LV-IIIA-HSCT had no significant effect. Critically LV-WT-HSCT corrected the behaviour at 4 and 6 months of age whilst the other treatments had no significant effect. LV-WT-HSCT and WT-HSCT reduced GM2 gangliosides and neuroinflammation equally but only LV-WT-HSCT corrected behaviour and primary HS storage, suggesting they are the important factors in MPS IIIA pathology. LV-WT-HSCT corrects the neurological phenotype in MPS IIIA mice and is a clinically viable approach to treat MPS IIIA and other neuropathic lysosomal storage disorders.
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SPINOZZI, GIULIO. "Anti-Cancer Drug Resistance Causal Modeling from Lentiviral-Vector Integration Site Studies." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2017. http://hdl.handle.net/10281/151647.
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L'evoluzione nel cancro svolge un ruolo determinante attraverso l'accumulo di alterazioni genetiche, fornendo vantaggi selettivi ad una cellula tumorale, permettendo resistenza ai farmaci anti-cancro. Purtroppo, l'identificazione delle mutazioni driver e i meccanismi sottostanti la resistenza anti-tumorale ai farmaci (ACDR) rimangono ancora irrisolti. Abbiamo precedentemente dimostrato che vettori lentivirali, se adeguatamente modificati, possono integrare nei pressi di geni specifici, modificando la loro espressione e provocate il cancro o ACDR in vivo e in vitro. L'analisi delle integrazioni di questi vettori nei tumori o nelle cellule resistenti alle terapie, ha permesso di identificare geni responsabili di cancro al seno HER2+, su determinate linee cellulari, utilizzando un approccio statistico definito siti di inserzione comuni (CISs) che evidenziano regioni genomiche bersagliate a più alta frequenza di quanto previsto da una distribuzione normale. La ricostruzione della progressione del cancro accumulata dai geni (CISs) non è stata ancora affrontata in letteratura e può produrre reti causali di geni. Lo scopo di questo progetto è di studiare la resistenza anti-tumorale ai farmaci di geni esclusivi e concomitanti con la progressione cumulata del cancro rispetto ai CISs della linea cellulare e indagare il rapporto tra di loro.
Strumenti bioinformatici volti a inferire modelli di progressione del cancro, in termini di relazioni di vantaggio selettivo tra alterazioni genomiche, da dati trasversali (piattaforme di sequenziamento di nuova generazione), consentirebbero l'identificazione di specifiche combinazioni di farmaci mirati per superare l'insorgenza della resistenza in fase chemioterapica. In un nuovo contesto di siti di integrazione di vettori lentivirali, ho sviluppato un flusso di lavoro bioinformatico integrato, composto da: (i) una versione aggiornata e più accurata di VISPA (Vector Integration Site Parallel Analysis), una pipeline per l'identificazione automatica dei ISs e la loro annotazione sulla base di un ambiente distribuito con una interfaccia web semplice ed intuitiva; (ii) l'identificazione dei CISs con un approccio a finestra scorrevole; (iii) un nuovo strumento statistico, CAncer PRogression Inference (CAPRI), per dedurre le relazioni di vantaggio selettivo tra i vari eventi mutazionali nei genomi di cellule tumorali, per lo più in relazione con la resistenza ad un farmaco. Il modello si basa sul nesso di causalità probabilistica ed è in grado di ricostruire la progressione del cancro attraverso Direct Acyclic Graphs (DAG), che coinvolge i geni risultanti CISs. Con l'uso di GeneMANIA (http://www.genemania.org) e Enrichr (http://amp.pharm.mssm.edu/Enrichr), ho studiato l'interazione proteina-proteina, Gene Ontology e Pathway tra geni selezionati, raccogliendo e visualizzando i risultati in reti di geni. Applicando il nuovo metodo ai dati di integrazione già pubblicati delle due linee cellulari, sono stato in grado di generare modelli di progressione che coinvolgono i geni rilevanti (confermando che questi non sono geni che si escludono a vicenda, con Mutex). Questi sono in linea con i risultati precedentemente validati, a conferma del ruolo dei geni PIK3CA-ErbB2 in ACDR. Purtroppo, una delle due linee di cellule ha campioni a bassa qualità. Per questo motivo, CAPRI non è stati in grado di generare la progressione. Ho generato la progressione per l'altra linea cellulare, BT474, pre-trattamento e post-trattamento con lapatinib. L'ultimo passo è stato di indagare le relazioni tra geni, prodotte dal modello, cercando di trovare alcune nuove interazioni utili e conferme per gli studi di ACDR (SUMO1-ERBB2-PIK3CA-CSMD3). Nuovi dati di mutagenesi inserzionale da linee cellulari di cancro al polmone, volti a indurre ACDR in vivo e in vitro, sono in corso e permetteranno di validare e/o identificare nuovi modelli di progressione del cancro, così come possibili terapie combinatorie.Evolution plays a key role in Cancer as the result of the accumulation of genetic alterations, which provide selective advantages to a tumor cell, allowing resistance to anti-cancer drugs. Unfortunately, however, the identification of the driver mutations and thus the mechanisms underlying anti-cancer drug resistance (ACDR) still remains a challenge. We previously demonstrated that lentiviral vectors, when properly modified, might integrate near specific genes, alter their expression and induce cancer or ACDR in vivo and in vitro. The analysis of vector-cellular genomic junctions in tumor or ACDR cells allowed identifying causative genes of HER2+ breast cancer cell line using a statistical approach defined Common Insertion Sites (CISs) that highlight genomic regions targeted at significantly higher frequency than expected by a random distribution. The reconstruction of cumulative cancer progression from CISs genes has not been yet addressed and may produce causative gene networks. The aim of this project is studying anti-cancer drug resistance from exclusive and co-occurring genes using cumulative cancer progression from our cell line CISs genes and investigating the relation between them. Bioinformatics tools aimed at inferring cancer progression models, in terms of selective advantage relation among relevant genomic alteration from cross-sectional data (Next Generation Sequencing platforms), would allow identifying specific combinations of targeted drugs to overcome the occurrence of resistance. In a new context of vector Integration Sites (ISs), I developed an integrated bioinformatics workflow composed of: (i) an updated and more accurate version of VISPA (Vector Integration Site Parallel Analysis), a pipeline for automated ISs identification and annotation based on a distributed environment with a simple web based interface; (ii) identification of the CISs with a sliding window approach; (iii) a new statistical tool, CAncer PRogression Inference (CAPRI), to infer selective advantage relations among various mutational events in cancer cell genomes, mostly in relation with drug-resistance. The model is based on probabilistic causation and is able to reconstruct our cancer progression Direct Acyclic Graphs (DAGs), involving the CIS genes. With the use of GeneMANIA (http://www.genemania.org) and Enrichr (http://amp.pharm.mssm.edu/Enrichr), I studied the protein-protein interaction, Gene Ontology and Pathway relations between selected genes, collecting and visualizing results in gene networks. By applying our new method to the published ISs dataset from the two cell lines, I was able to generate progression models involving relevant genes (confirming that these are not mutually exclusive genes, by Mutex), which are consistent with previously validated results, confirming the role of PIK3CA-ERBB2 genes in ACDR. Unfortunately, one of the two cell line has a low quality samples. For this reason, CAPRI was not able to generate the progression DAG. I generated the progression DAG for the other cell line, BT474, pre-treatment and post-treatment with lapatinib respectively. The last step is to investigate the relations between genes, produced by the model, trying to find some useful new interactions and confirmations for ACDR studies (i.e. SUMO1-ERBB2-PIK3CA-CSMD3). New insertional mutagenesis data from lung cancer cell lines aimed to induce ACDR in vivo and in vitro are ongoing and will allow to validate and/or identify novel cancer progression models, as well as possible combinatorial therapies.
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Galvan, Laurie. "Étude de l'implication potentielle des marqueurs du striatum dans la maladie de Huntington." Thesis, Paris 11, 2011. http://www.theses.fr/2011PA11T024.
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La maladie de Huntington (MH) est une maladie neurodégénérative héréditaire, incurable.Elle est due à une mutation dans le gène HD codant l'huntingtine (htt). Cette mutation setraduit dans la protéine par une augmentation de l'expansion polyglutamine (polyGln) qui larend toxique. Bien que la htt soit ubiquitaire dans le système nerveux central, ladégénérescence touche préférentiellement le striatum. Un patron d'expression de gènesspécifiques du striatum pourrait expliquer cette vulnérabilité préférentielle. Nous avons étudiéles effets "modificateurs" de 5 gènes préférentiellement exprimés dans le striatum vis-à-visde la toxicité de la htt mutée par une approche lentivirale chez la souris. Nous avonscaractérisé les effets de ces marqueurs striataux sur la toxicité induite par la htt mutée pardifférentes approches histologiques. Les "modificateurs" de la MH ont été étudiés plus endétail. Nous avons examiné leur localisation et les mécanismes sous-jacents à leurs effetsneuroprotecteurs. Outre une meilleure compréhension du striatum, cette étude a permis ladécouverte de candidat neuroprotecteur qui pourrait permettre de développer de nouvellesthérapiesHuntington's disease (HD) is an incurable inherited neurodegenerative disease. HD iscaused by a mutation in the HD gene coding huntingtin (htt). This mutation leads to anexpanded polyglutamine tract (polyQ) in the protein which is toxic to neurons. Although thehtt is ubiquitously expressed in the central nervous system, the first area which degeneratesis the striatum. A pattern of genes selectively expressed into the striatum may confer itsvulnerability to mutated htt. We have studied the modifying effects of five newly identifiedstriatal markers against the toxicity induced by mutated htt using lentiviral strategy in miceand histological approaches. For one of these markers, Double Cortin Kinase Like 3(DCLK3), we have further determined their cellular localization and the potential mechanismsunderlying their neuroprotector effects. The present work led to a better understanding of thefunction of the newly identified markers in the striatum and their potential roles in thepreferential vulnerability of the striatum in HD
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Coleman, Jason Edward. "Efficient transduction and targeted expression of lentiviral vector transgenes in the developing retina." [Gainesville, Fla.]: University of Florida, 2003. http://purl.fcla.edu/fcla/etd/UFE0000665.
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Limberis, Maria. "A lentiviral gene transfer vector for the treatment of cystic fibrosis airway disease." Title page, synopsis and list of contents only, 2002. http://web4.library.adelaide.edu.au/theses/09PH/09phl735.pdf.
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"16th September 2002." Accompanying CD contains 2 MPEG clips with accompanying text, and a copy in PDF format of: Recovery of airway cystic fibrosis transmembrane conductance regulator function in mice with cystic fibrosis after single-dose lentivirus-mediated gene transfer / M. Limberis ... [et al.], published in Human gene therapy vol. 13 (2002). Bibliography: leaves xxix-li. This thesis focuses on modulating the physical barriers of the airway epithelium with mild detergents, so as to enhance gene transfer by a HIV-1 based lentivirus vector in vivo. The efficiency of the gene transfer was evaluated in the nasal airway of C57B1/6 mice using the Lac Z marker gene. This demonstration of lentivirus-mediated in vivo recovery of CFTR function in CF airway epithelium illustrated the potential of combining a pre-conditioning of the airway surface with a simple and brief HIV-1 based gene transfer vector exposure to produce therapeutic gene expression in the intact airway.
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Nilsson, S. M. "Process development of lentiviral vector expression, purification and formulation for gene therapy applications." Thesis, University College London (University of London), 2016. http://discovery.ucl.ac.uk/1485725/.
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There is growing interest in the use of lentiviral vectors, particularly for cancer immunotherapy and the treatment of monogenic diseases. Manufacturing of these vectors is challenging primarily due to cytotoxic effects of vector components resulting in low cell culture titres and vector instability leading to low purification yields. In addition, currently used processes are typically not scalable as they rely on adherently cultured cells and unit operations such as batch centrifugation and gel filtration. To improve process scalability, suspension adaptation of a lentiviral vector packaging cell line was attempted, however, cell aggregation could not be prevented. For vector clarification it was found that membranes with pore sizes of 0.22 µm resulted in recoveries less than 50%, whereas the use of 0.45 µm membranes resulted in recoveries close to 100%. Successful vector concentration utilising centrifugal filters was possible with a membrane molecular weight cut-off (MWCO) of 100 kDa, whereas a 300 kDa MWCO led to low recoveries. Chromatography stationary phases that allow convective mass transfer, such as membranes and monoliths, are becoming increasingly popular for purification of large molecules. Lentiviral vector was found to bind monoliths with weak and strong anion exchangers over a wide range of conditions. Vector elution conditions determined for membrane- and monolith-based resins in high-throughput 96-well plate format were found to not be indicative of gradient elution conditions for 1 mL versions of these resins operated by a chromatography system. The thermolability of lentiviral vectors leads to a requirement for storage at less than -65°C. Inexpensive mixtures of sugars in combination with a glycine-derivative were studied for their ability to stabilise a lentiviral vector during freeze-drying and subsequent thermochallenge. The amorphous solid formed upon freeze-drying was able to stabilise the vector for up to 12 weeks at 4°C and eight weeks at 25°C. This is in comparison to formulation in phosphate buffered saline, where more than 90% of infectious titre was lost immediately upon freeze-drying.
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Hino, Shinjiro. "Sea Urchin Insulator Protects Lentiviral Vector from Silencing by Maintaining Active Chromatin Structure." Kyoto University, 2004. http://hdl.handle.net/2433/147507.
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Parker, Douglas George Anthony, and park0290@flinders edu au. "Lentivirus-mediated gene expression in corneal endothelium." Flinders University. Medicine, 2008. http://catalogue.flinders.edu.au./local/adt/public/adt-SFU20081204.094431.
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Modulation of corneal transplant rejection using gene therapy shows promise in experimental models but the most appropriate vector for gene transfer is yet to be determined. The overarching aim of the thesis was to evaluate the potential of a lentiviral vector for use in human corneal transplantation. Specific aims were: (i) to assess the ability of an HIV-1-based lentiviral vector to mediate expression of the enhanced yellow fluorescent protein (eYFP), and a model secreted protein interleukin-10 (IL10), in ovine and human corneal endothelium; and (ii) to examine the influence of lentivirus-mediated IL10 expression on the survival of ovine corneal allografts.
Four lentiviral vectors expressing eYFP under the control of different promoters, were tested: the simian virus type-40 (SV40) early promoter, the phosphoglycerate kinase (PGK) promoter, the elongation factor-1alpha (EF) promoter, and the cytomegalovirus (CMV) promoter. Two lentiviral vectors expressing IL10 were tested: one containing the SV40 promoter and another containing a steroid-inducible promoter (GRE5). Lentivirus-mediated expression in transduced ovine and human corneal endothelium was assessed by fluorescence microscopy, real-time quantitative RT-PCR and ELISA, following alterations of transduction period duration (224 hr) and vector dose, as well as in the presence or absence of polybrene or dexamethasone (GRE5 vector). It was also compared to expression mediated by adenoviral vectors. Orthotopic transplantation of ex vivo transduced donor corneas was performed in outbred sheep. Allografts were reviewed daily for vascularisation and signs of immunological rejection.
Lentivirus-mediated eYFP expression was delayed in ovine corneal endothelium compared to human. However, in both species the final transduction rate was greater than 80% and expression was stable for at least 14 d in vitro. Lentivirus-mediated expression in ovine and human corneal endothelium was higher with the viral promoters in comparison to the mammalian promoters. A 24 h transduction of ovine corneal endothelium with the lentiviral vector encoding IL10 resulted in expression levels which were increasing after 15 d of organ culture but logarithmically lower than those achieved by adenovirus. Shortening the lentiviral transduction period to 2 h led to a reduction in expression, but the addition of polybrene (40 micrograms / ml) to the transduction mixture restored expression to levels comparable to those attained after a 24 h transduction period. Lentivirus-mediated IL10 expression was higher and more rapid in human corneal endothelium compared to ovine corneas. Dexamethasone-responsive transgene expression was observed in both ovine and human corneal endothelium using the lentiviral vector containing the GRE5 promoter. Lentivirus-mediated expression in ovine corneal endothelium was stable for 28 d in vivo. A modest prolongation of ovine corneal allograft survival (median of 7 d) was achieved by transduction of donor corneas for 23 h with the lentivirus expressing IL10. Attempts to increase the expression of IL10 by the addition of polybrene (40 micrograms / ml) to the transduction mixture, resulted in a toxic effect on corneal allografts which abrogated the beneficial effect of IL10.
The lentiviral vector shows potential for the stable expression of therapeutic transgenes in human corneal transplantation. However, the mechanisms underlying the species-specific differences in HIV-1-mediated transgene expression will need to be elucidated and overcome if the ovine preclinical model is to provide justification for a clinical trial.
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McLean, Rebecca Kathryn. "Development of a novel lentiviral vaccine vector and characterisation of in vitro immune responses." Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/31135.
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Vaccines are a highly effective means of preventing infectious disease. However, for many diseases of livestock the available vaccines are ineffective or sub-optimal. This is partly due to challenges surrounding the specific targeting of antigen presenting cells (APCs). In order to improve the delivery of protective antigens to host APCs, a novel lentiviral vector derived from visna / maedi virus (VMV) has been developed. Initial characterisation using an enhanced green fluorescent protein (eGFP) reporter transgene found that the novel VMV vector efficiently transduced a wide range of cell lines including cells of ovine, human, murine, bovine and caprine origin. In addition, the VMV vector was found to elicit sustained transgene expression for at least 4 weeks in rapidly dividing cell lines. One of the most important factors for acceptable vaccines is their safety. Therefore, in order to increase the bio-safety of the VMV vector, integration-defective and self-inactivating forms were produced. Integration-defective VMV lentiviral vectors (IDLVs) were found to produce 1-LTR circular episomes favourably over integrated provirus following the transduction of target feline and ovine cell lines. This led to a decrease in transgene expression over time in dividing cells. In contrast, in non-dividing cells transgene expression was maintained at a similar level to integration-competent VMV vectors. Self-inactivating (SIN) VMV vectors were constructed and found to have a significant decrease in LTR activity. Transgene expression was maintained by the insertion of an internal promoter derived from human cytomegalovirus (CMV) acting directly on the transgene. When self-inactivating and integration-defective modifications were incorporated into the same vector particle, a 4-fold decrease in transduction relative to the parent vector was observed. Ovine monocyte-derived dendritic cells (MDDCs) and macrophages (MDMs) were found to be efficiently transduced by the VMV vector, whereas lentiviral vectors derived from HIV-1 poorly transduced both of these primary cell populations. Following this work, the ability to deliver pathogen genes into APCs was studied using the Chlamydia abortus (C. abortus) major outer membrane protein (MOMP) as the transgene. C. abortus is the most common infectious cause of ovine abortion worldwide and MOMP has previously been shown to stimulate strong antibody responses after vaccination. Unexpectedly, the VMV vector encoding either eGFP or MOMP was found to induce apoptosis in MDDCs and MDMs using Annexin V staining. Apoptotic cells were detectable as early as 6 hours post-transduction of cells. Furthermore, release of the pro-inflammatory cytokine IL-1β was associated with the formation of late apoptotic cells. Apoptotic bodies produced post-transduction were able to be phagocytosed by immature MDDCs and the transgene efficiently cross-presented to T-cells. The ability of the novel VMV vector to induce a suitable recall immune responses was investigated using an in vitro model. Here, an autologous population of MDDCs were cultured with the apoptotic bodies produced post-transduction before the addition of autologous PBMC. Proteins from the apoptotic bodies were presented by the MDDCs to PBMC leading to a strong, antigen specific recall immune response against C. abortus MOMP. This was proven by the detection of cytokines IFNγ and IL-10 in the co-culture supernatant from PBMC activated by the MOMP transgene cross-presented by MDDCs. No release of IL-4 or IL-17A could be detected. These data presented in this thesis show the potential for improving delivery of antigens in livestock vaccines by the use of lentiviral vectors. In addition, this vector system provides a strong base for the study of other potential protective antigens in vitro.
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Shinoda, Yasuhiko. "Efficient transduction of cytotoxic and anti-HIV-1 genes by a gene-regulatable lentiviral vector." Kyoto University, 2010. http://hdl.handle.net/2433/120584.
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Dresch, Christiane. "Antigen-specific tolerance induction by transcriptional targeting of dendritic cells with a novel lentiviral vector." Diss., kostenfrei, 2008. http://edoc.ub.uni-muenchen.de/9310/.
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Haynes, Soraya. "Construction of a novel bat coronavirus glycoprotein pseudotyped lentiviral vector and analysis of cell tropism." Thesis, Haynes, Soraya (2019) Construction of a novel bat coronavirus glycoprotein pseudotyped lentiviral vector and analysis of cell tropism. Honours thesis, Murdoch University, 2019. https://researchrepository.murdoch.edu.au/id/eprint/54215/.
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Since the SARS epidemic in 2003, and the subsequent identification of bats as the reservoir host, there has been a surge of interest in research into bat-borne viruses, with over 30 new bat coronaviruses alone being discovered. Investigating the cell tropism of these newly discovered coronaviruses deepens the understanding of their pathology and possible host ranges, as well as allowing assessments to be made on their zoonotic potential. Aside from biosafety concerns however, this is also reliant on isolation of the virus from its host, which can be difficult using conventional methods.
This project aimed to develop a second generation lentivirus pseudotyping system which would allow spike proteins, the main determinants of coronavirus cell entry, of a non-isolated coronavirus to be safely expressed on a lentiviral particle in order to assess its cell tropism. The system was developed and tested using the spike gene from CoV1087, a novel bat-borne coronavirus with a 70% similarity at both the coding RNA and amino acid level between its spike protein and that of porcine epidemic diarrhea virus strain CH-HNYF-14.
Overall this study resulted in the successful development of a lentiviral pseudotyping system which allows the efficient analysis of the cellular tropism of coronaviruses. Through the testing and development of this system, it was determined that CoV1087 could enter two human cell lines, 293T and CaCo-2 as well as one rabbit cell line, RK-13. This raises the possibility of the transfer of the virus to feral rabbits as well as the possibility of zoonotic human infections.
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Santoni, de Sio Francesca Romana. "Critical parameters and molecular analysis of lentiviral vector-mediated gene transfer into human haematopoietic stem cells." Thesis, Open University, 2006. http://oro.open.ac.uk/54821/.
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Camacho, Emely. "Optimization of Lentivirus Production for Cancer Therapy." Thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-164715.
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Vectors based on lentivirus backbones have revolutionized our ability to transfer genesinto many cell types. Lentiviral vectors integrate into the chromatin of target cells and do not transfer any viral genes causing vector replication. Both of these features arecommonly used in gene therapy and have been used clinically in individuals sufferingfrom cancer, infections and genetic diseases. It has been discovered that T-cells can be genetically modified to be used as effective weapons against cancer: therefore virus mustbe produced to deliver the gene of interest into the T-cells. In this project, lentiviralvectors have been produced to transfer the gene coding for a chimeric antigen receptor(CAR) which is directed to CD19 on B-cells. The vectors will, hence, be used to generateCD19 retargeted T-cells in purpose to kill CD19 cells such as B-cell lymphoma andleukemia. We have evaluated two production protocols to determine a feasible method toculture these vectors. We have also stimulate T-cells with two different antibodies (anti-CD3 and anti-CD28) and transduced T-cells. Our results demonstrate that theconcentration of virus was higher after prolonged incubation in 4˚C, which can not beexplained. The stimulation demonstrated that bound anti-CD3 was the best stimulator,and moreover the FACS-analysis showed that addition of anti-CD28 gave a highertransduction level. In conclusion, the viral vectors may be kept in 4˚C for two days beforeconcentrating the virus, and bound anti-CD3 is a better choice than soluble anti-CD3 forstimulation of T-cells.
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Frank, Sander B., Veronique V. Schulz, and Cindy K. Miranti. "A streamlined method for the design and cloning of shRNAs into an optimized Dox-inducible lentiviral vector." BIOMED CENTRAL LTD, 2017. http://hdl.handle.net/10150/623280.
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Background: Short hairpin RNA (shRNA) is an established and effective tool for stable knock down of gene expression. Lentiviral vectors can be used to deliver shRNAs, thereby providing the ability to infect most mammalian cell types with high efficiency, regardless of proliferation state. Furthermore, the use of inducible promoters to drive shRNA expression allows for more thorough investigations into the specific timing of gene function in a variety of cellular processes. Moreover, inducible knockdown allows the investigation of genes that would be lethal or otherwise poorly tolerated if constitutively knocked down. Lentiviral inducible shRNA vectors are readily available, but unfortunately the process of cloning, screening, and testing shRNAs can be time-consuming and expensive. Therefore, we sought to refine a popular vector (Tet-pLKO-Puro) and streamline the cloning process with efficient protocols so that researchers can more efficiently utilize this powerful tool. Methods: First, we modified the Tet-pLKO-Puro vector to make it easy("EZ") for molecular cloning (EZ-Tet-pLKO-Puro). Our primary modification was to shrink the stuffer region, which allows vector purification via polyethylene glycol precipitation thereby avoiding the need to purify DNA through agarose. In addition, we generated EZ-Tet-pLKO vectors with hygromycin or blasticidin resistance to provide greater flexibility in cell line engineering. Furthermore, we provide a detailed guide for utilizing these vectors, including shRNA design strategy and simplified screening methods. Results: Notably, we emphasize the importance of loop sequence design and demonstrate that the addition of a single mismatch in the loop stem can greatly improve shRNA efficiency. Lastly, we display the robustness of the system with a doxycycline titration and recovery time course and provide a cost/benefit analysis comparing our system with purchasing pre-designed shRNA vectors. Conclusions: Our aim was twofold: first, to take a very useful shRNA vector and make it more amenable for molecular cloning and, secondly, to provide a streamlined protocol and rationale for cost-effective design, cloning, and screening of shRNAs. With this knowledge, anyone can take advantage of this powerful tool to inducibly knockdown any gene of their choosing.
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CATTANEO, STEFANO. "Combinatorial gene therapy for epilepsy." Doctoral thesis, Università Vita-Salute San Raffaele, 2022. http://hdl.handle.net/20.500.11768/128275.
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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.
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Ravache, Thaís Terpins, Renata Simões, and Marcelo Demarchi Goissis. "Geração de animais transgênicos por inoculação de vetor viral em meio de cultura de óvulos." reponame:Repositório Institucional da UFABC, 2014.
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Orientador: Prof. Dr. Marcelo Augusto ChristoffoleteDissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biotecnociência, 2014.
Desde o século XV, animais fazem parte da rotina na área da pesquisa, principalmente para estudos de doenças, e hoje em dia o modelo animal mais utilizado para estes estudos é o camundongo, tendo uma participação em mais de 90% das pesquisas em todo o mundo, sendo considerado como uma primeira via para definir funções de genes em mamíferos. Os camundongos são considerados os principais modelos nas técnicas de transgenia animal, porém estas técnicas ainda estão em desenvolvimento, uma vez que as metodologias hoje utilizadas para a geração de animais transgênicos ainda se encontram com uma taxa de sucesso considerada baixa e são dispendiosas, necessitando de muitas etapas. Uma das dificuldades é o contato com a membrana do óvulo devido a zona pelúcida, que é considerada uma barreira física. Vetores virais estão em evidência nas técnicas de transgenia animal, sendo o lentivírus o mais utilizado. Portanto, o objetivo deste projeto é estabelecer um protocolo para a integração de DNA exógeno em óvulos por infecção lentiviral, anteriormente a fertilização in vitro juntamente com a técnica de dissecção parcial da zona pelúcida. Como vetor foi utilizado um lentivírus com GFP em sua construção. Para ocorrer a fertilização in vitro, foram feitas coletas de óvulos em camundongos fêmeas da linhagem C57BL/6, tratadas com injeções hormonais, e coletas de espermatozoides em machos desta mesma linhagem. Os óvulos obtidos foram divididos em grupos controle e com dissecção parcial da zona pelúcida, e estes foram subdivididos em grupos com e sem infecção lentiviral. Entre os grupos houve variação de 20% a 56,25% de embriões em estágio de duas células, e em alguns grupos foi possível alcançar o estágio de blastocisto eclodido. Porém não foi possível visualizar a emissão de fluorescência para confirmar a infecção lentiviral. Em conclusão as metodologias utilizadas tanto para a fertilização in vitro como para a dissecção parcial da zona pelúcida foram de sucesso. Porém a integração do DNA exógeno mostrou resultados não conclusivos, necessitando de estudos futuros.
Since the XV century, animals are used routinely in research, mainly for diseases studies, and nowadays the most used animal model is the mouse, which one has more than 90% of participation in researches around the world and it is considered the first track to define gene function in mammals. Mouse is the main model in transgenic techniques, however the methods available to generate transgenic animals still have a considerable low rate, and also it is expensive, requiring many degrees. An ordinary issue is the contact with the membrane of oocyte due zona pellucida that is considered a physical barrier. In transgenic animals technique, it is in evidence the utilization of viral vectors, and the most used are the lentiviruses. Therefore, the objective of this project is to establish a protocol for the integration of exogenous DNA by lentiviral infection into oocytes, before the in vitro fertilization, using the technique of partial dissection of the zona pellucida. It was used as a vector a lentivirus with GFP in your construction. For in vitro fertilization, were collected oocytes from C57Bl/6 mice, treated with hormones, and sperm from males of the same strain. The obtained oocytes were divided in control group and partial dissection of the zona pellucida group, and then subdivided in groups with and without lentiviral infection. Between the groups, was achieved 20% to 56,25% of two cells stage embryo, and hatched blastocysts stage were obtained at some groups. Therefore it was not possible to visualize florescence emission to confirm the lentiviral infection. In conclusion we have a practicable protocol for in vitro fertilization and partial dissection of the zona pellucida, reaching blastocysts stages in two groups. However the integration of exogenous DNA results were inconclusive, requiring further studies.
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Molina, Gil Alberto. "Lentiviral vector packaging cell line development using genome editing to target optimal loci discovered by high throughput DNA barcoding." Thesis, University College London (University of London), 2017. http://discovery.ucl.ac.uk/1573558/.
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Lentiviral vectors are increasingly used as delivery methods in gene therapy clinical trials due to their high efficiency transducing cells and stability of transgene expression. The development of packaging and producer cell lines for the production of lentiviral vectors has always been a labour-intensive and lengthy process. Sequential introduction of vector components, adaptability to suspension cultures, autotransduction and genetic, transcriptional or cell line growth instability are some of the limitations that cause significant drops in productivity. Improved transcription of self-inactivating vectors leading to high titers has been attempted in different ways with the intent to find a high stable producer clone. In this project, we studied the use of lentiviral vectors as a tool to target and identify high-transcribing loci in the genome of our host cells for lentiviral packaging cell line development. Third generation lentiviral vectors carrying eGFP under the control of an endogenous clinically-tested promoter (short EF1α) were produced, containing a variable DNA sequence tag (barcode) in their long terminal repeat (LTR). The aim of the barcode is to uniquely tag, identify and track a particular clone within the heterologous expressing population. Human embryonic kidney cell lines (HEK-293) were transduced with a barcoded lentiviral library at a low multiplicity of infection. We demonstrated that integration site analysis and next-generation sequencing of lentiviral barcoded vector junctions by ligation-mediated PCR (LM-PCR) coupled with RNA-Seq allows for quantification of the relative abundance of each barcode variant in each specific genomic position. Expression cassettes containing lentiviral vector components were then site-specifically integrated into these genomes sites using the CRISPR-Cas9 technology. The barcoding lentiviral system allows for rapid and high-resolution high-throughput screening of gene expression in a large number of genomic positions naturally targeted for optimal vector expression but also of lower expressing sites in order to meet lentiviral cytotoxicity and stoichiometric constraints.
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Chang, Chia-Wei. "Polycistronic lentiviral vector for hit and run reprogramming of mouse and human somatic cells to induced pluripotent stem cell." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2009. https://www.mhsl.uab.edu/dt/2009p/changc.pdf.
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Darbey, Annalucia Leigh. "Targeting and repair of adult testicular somatic cells through viral gene therapy." Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/33252.
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Androgens are essential for the maintenance of male health and wellbeing. A disturbance in androgen signalling has been associated with a number of clinically relevant disorders such as cardiovascular disease, diabetes and metabolic disorders as well as infertility. Primarily produced in the testis in males, the actions of androgens are mediated through binding to androgen receptor (AR), a member of the nuclear receptor superfamily of ligand-activated transcription factors. The somatic cells of the testis are known to have a number of key roles in both testis function and development and the Sertoli, Leydig and Peritubular Myoid cells are known to express AR in adulthood. It is through AR that some testicular functions are mediated; for example, the Sertoli cells support of complete spermatogenesis with Sertoli cell androgen receptor knockout (SCARKO) testis demonstrating a halt of spermatogenesis before meiosis. However, how androgen signalling is impacting testicular function through each of the somatic cell types is not yet fully understood. Currently, treatments for male reproductive disorders such as hypogonadism (low androgens) and infertility are limited to treatment of the symptoms; using androgen replacement therapy and in vitro fertilisation techniques. This has been, up until recently, a result of a lack of understanding of the causes of these conditions and a lack of resources able to treat them, with research suggesting that a genetic component may be responsible in a number of cases. However, due to the limited genetic investigation diagnosis of men with male reproductive disorders, the wider understanding of the genetics underpinning male hypogonadism and infertility is incomplete. Developments in technology for the investigation and editing of the genetic code are triggering a surge in the exploration of genetic disorders and, in parallel, into the fields of gene delivery vectors and editing technologies. These technologies will allow an expansion into the knowledge and understanding of genetic disorders whilst simultaneously affording the opportunity to exploit this understanding for the development of therapeutics. There have been a small handful of previous studies using technologies such as viral vectors to target the testicular somatic cells and deliver exogenous transgenes with the purpose of both gene editing and repair, all with varying degrees of success. Here, techniques to introduce and target the Leydig and Sertoli cells were investigated to determine the most appropriate methodology for gene delivery to and manipulation of the testis. Refinement of injections into the interstitial compartment were carried out before introducing lentiviral vectors and targeting of Leydig cells was validated and optimised. Lentiviral vectors are able to permanently integrate into the host cell. Surprisingly, analysis of testis post lentiviral injection determined that the lentiviral targeted Leydig cells began to undergo apoptosis one week post injection and were subsequently cleared from the testis after ten days. Contrastingly, this was not the case when adenoviral vectors were introduced into the interstitial compartment, with Leydig cells continuing to express the delivered reporter transgene and, importantly, not expressing markers of apoptosis, ten days post injection. This would suggest that using adenoviral vectors to target the Leydig cell population in the adult testis would be more appropriate than using lentiviral vectors. Previous studies have successfully used lentiviral vectors to target the Sertoli cells in the adult testis via the introduction of the particles through the efferent duct. However, this can result in damage to efferent duct, resulting in blockages and subsequently the seminiferous tubules. To circumvent this, introduction of the lentiviral particles through the rete compartment of the testis at a range of lower injection pressures was examined and injecting at a lower pressure through the rete testis was found to reduce the likelihood of introducing negative impacts on testicular histology when targeting the seminiferous tubules. Using these refined methods of introducing lentiviral vectors, targeted Sertoli cells stably expressed the delivered transgene for up to one year post injection. Using viral vector delivered transgenes for both the investigation of testicular genetic disorders and for the development of therapeutics has great potential. To explore this potential, we first generated a mouse model in which AR was ablated from both the Leydig and Sertoli cells using Cre/LoxP technology, termed the SC-LC-ARKO. Alongside providing a potential model to 'repair' with viral vectors, the SC-LC-ARKO model also provided an additional model for comparison with other models exhibiting ablation of AR from both single somatic cell types and double somatic cell types. This further enabled a characterisation of the roles of AR in adult testicular function, with results suggesting that loss of AR from more than one cell type results in an additive phenotype when compared to single cell knock outs. Despite providing further insight into the roles of AR in the testis, further analysis of the Cre line used to generate the SC-LC-ARKO model indicated that a small number of Leydig cells were expressing the Cre recombinase, resulting in only a small population of Leydig cells with ablated AR. Considering this, to explore the potential of rescuing Sertoli cell AR using lentiviral vectors, we then utilised an already well characterised Sertoli Cell AR knockout (SCARKO) model. Lentiviral vectors expressing mouse AR and monomeric GFP (moeGFP) downstream of a CMV promoter were generated and injected into the rete testis of WT and SCARKO adult (day 100) males at low pressure. The contralateral testis was injected with a lentiviral vector expressing moeGFP alone (also downstream of a CMV promoter) using the same technique. Analysis of testis sections revealed a reintroduction of AR to Sertoli cells in 100% of SCARKO testis injected with lentivirus expressing mouse AR. As a result of this re-expression of AR in Sertoli cells, 66% of the testis injected with lentivirus expressing mouse AR had evidence of morphologically mature elongated spermatids, indicative of ongoing spermatogenesis. These results suggest that a rescue of the infertility phenotype reported in previous studies of SCARKO testis. Also demonstrated is the reversal of the SCARKO testicular phenotype in tubules targeted by the mAR expressing lentiviral vector. This suggests that absence Sertoli cell AR throughout development does not have a permanent impact on the Sertoli cells capacity to support spermatogenesis in adulthood following rescue of SC AR expression in adulthood. In summary, the results of these studies have provided a refinement in the methodologies for targeting the Sertoli and Leydig cells of the adult testis with viral vectors as well as demonstrating successful rescue of a previously reported mouse model exhibiting infertility through reintroduction of a functional gene. Alongside this, comparisons of AR knockout models have afforded insight into maintenance of testis function through AR.
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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.
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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.
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Hiraragi, Hajime. "Study of lentiviral vector for in utero gene transfer and functional analysis of human T-lymphotropic virus type p13(II)." Connect to resource, 2005. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1116532636.
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Thesis (Ph. D.)--Ohio State University, 2005.Title from first page of PDF file. Document formatted into pages; contains xvii, 230 p.; also includes graphics. Includes bibliographical references (p. 200-230). Available online via OhioLINK's ETD Center
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Holder, Maxine Virginia. "Development of a lentiviral vector system based on equine infectious anaemia virus for use in liver directed fetal gene therapy." Thesis, Imperial College London, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.428014.
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Moussa, Maha. "Immunité et protection induites par un lentivecteur ADN innovant chez les modèles animaux de vaccination VIH-1." Thesis, Université Grenoble Alpes (ComUE), 2015. http://www.theses.fr/2015GREAV029/document.
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Nous avons récemment développé un prototype lentivecteur ADN non intégratif vaccinal contre VIH-1/SIDA que nous avons testé chez des modèles animaux. L'immunisation avec une dose unique de ce vaccin (CAL-SHIV-IN-) a permis la mise en place rapide de réponses immunes spécifiques contre tous les antigènes exprimés par le vaccin chez tous les animaux vaccinés. Les analyses longitudinales ont démontré la mise en place de réponses cellulaires et humorales spécifiques et persistantes sur une durée de plus de 74 semaines en absence de réintroduction d'antigènes chez tous les macaques vaccinés. La caractérisation de ces réponses a révélé la présence de cellules T CD4+ et CD8+ polyfonctionnelles composées de fractions de cellules effectrices mémoires à fonction immédiate (EM), de cellules centrales mémoires (CM) et de cellules précurseurs mémoires ayant une haute capacité de prolifération (PHPC). Ces réponses corrèlent, chez tous les macaques vaccinés (6/6), avec un contrôle du virus d'épreuve hautement hétérologue et pathogénique (SIVmac251) inoculé à petites doses répétées par la voie mucosale rectale. Cette protection est maintenue durant toute la période d'un an de suivi après l'infection avec une différence statistiquement significative de la charge virale plasmatique des groupes contrôles et vaccinés au moins jusqu'à 18 semaines post-infection. Par ailleurs, le contrôle du virus d'épreuve est maintenu plus de 10 mois (correspondant au temps d'arrêt de l'étude) après l'infection. Parmi les corrélats immunologiques de protection nous avons identifié la présence de cellules de type PHPC spécifiques des antigènes du vaccin et qui sont dotées d'une capacité importante de prolifération ex vivo en présence des signaux antigéniques et homéostatiques. Nous avons démontré que ces PHPC contiennent une fraction de cellules T souches mémoires « TSCM » spécifiques du vaccin. Ces TSCM récemment identifiées constitueraient un atout majeur en faveur de notre vecteur et notre stratégie vaccinale du fait de leur haute capacité d'auto-régénération/maintien en absence d'antigène et leur capacité à se différencier en d'autres cellules mémoires TCM et TEMWe recently developed an innovative prototype non-integrative lentivector DNA vaccine against HIV-1 /AIDS that we tested in pilot studies using animal models of HIV vaccine. We found that a single immunization with our prototype vaccine (CAL-SHIV-IN-) allowed the implementation of potent humoral and cellular responses in all immunized macaques. In addition, both types of responses persisted over a period of 74 weeks post-immunization in absence of antigenic boost. The characterization of the above revealed that vaccine specific T cell responses included polyfunctional CD4+ and CD8+ T cells against all antigens expressed by the vaccine. Detailed phenotypic and functional examinations of these cells showed that they were composed of effector (EM) and central memory (CM) T cells. More importantly they also contained a fraction of precursor memory T cells with high proliferative capacity (PHPC). Immune responses primed by our vaccine regiment correlated with protection in all vaccinated macaques (6/6). As expected our vaccine-induced immune responses did not prevent from infection acquisition but controlled the replication of the highly pathogenic and heterologous SIVmac251 challenge given as repeated low dose by the intrarectal mucosal route. All vaccinated animals (6/6) controlled their viremia to undetectable level using conventional PCR during at least 10 months post infection (end of the experiment). We further focused on PHPC responses associated with viral control and found that these cells vigorously proliferate upon ex vivo stimulation with specific antigens in presence of the homeostatic IL-7 and IL-15 cytokines. Proliferating antigen specific cells contained a type of stem cell-like memory T cells (TSCM). These latter (TSCM) might be a major asset in favor of our lentivector and vaccination strategy due to their high capacity for self-regeneration/maintenance in absence of antigen source
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Kitowski, Katherine Anne. "A LENTIVIRAL VECTOR CONFERRING COREGULATED, ERYTHROID-SPECIFIC EXPRESSION OF γ-GLOBIN AND shRNA SEQUENCES TO BCL11A FOR THE TREATMENT OF SICKLE CELL DISEASE." OpenSIUC, 2016. https://opensiuc.lib.siu.edu/theses/1995.
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Sickle cell disease (SCD) is a severe hemoglobin disorder caused by co-inheritance of a single mutation in the β-globin gene of adult hemoglobin (HbA; α2β2). This alteration leads to the formation of sickle hemoglobin (HbS; α2βS2) and deformed, sickle-shaped red blood cells (RBCs). Sickle RBCs obstruct small blood vessels resulting in anemia, excruciating pain crises, organ damage, and stroke. For the millions of people affected by this disease, life expectancy is only 40-60 years of age. The only cure for SCD is hematopoietic stem cell (HSC, CD34+) transplantation, which requires a human leukocyte antigen (HLA)-matched donor. However, this option runs the risk of complications associated with graft versus host disease and infection. Before birth, individuals with SCD do well because their RBCs are filled with γ-globin containing fetal hemoglobin (HbF; α2γ2), which inhibits the formation of HbS. In fact, some SCD patients who co-inherit mutations that allow for high-level expression of HbF into adulthood are asymptomatic. This suggests that genetic modification of the patient’s own HSCs to permit HbF production would be a viable therapeutic alternative to HSC transplantation. Our work has focused on the use of lentiviral vectors to introduce an exogenous γ-globin gene or shRNA sequences designed to knockdown repressors of γ-globin, such as the zinc-finger transcription factor, BCL11A, to prevent silencing of the endogenous γ-globin genes allowing for persistent expression of HbF. Despite significant progress using both approaches, we have been unable to increase the level of HbF > 30%; a curative threshold for SCD patients who continue to produce HbF into adulthood. The goal of my project was to combine these approaches into a single lentiviral vector to achieve co-regulated, erythroid-specific expression and augmented levels of HbF. I successfully modified the insulated, erythroid-specific γ-globin vector (termed V5m3-400) to include microRNA (miR)-adapted shRNAs (or shmiRs) targeting BCL11A (based on miR-30 and miR-E architectures) in the first and second noncoding introns of the γ-globin genomic sequences. Inclusion of shmiRs had no appreciable effect on integrity of the integrated provirus or vector titer. Vector performance was initially tested using human K562 erythroleukemia cells expressing a flag-tagged version of BCL11A. In this cell line, BCL11A knockdown was significantly improved using miR-E-shRNAs due to a dramatic increase (up to 350-fold) in processing of mature shRNA sequences. The miR-E vectors also provided high-level expression of γ-globin. Erythroid-specific expression of the γ-globin transgene and BCL11A knockdown was confirmed in maturing erythroid cells derived from transduced CD34+ cells of a healthy donor resulting in a 50% increase in HbF levels compared with cells transduced with V5m3-400 as a control. While encouraging, I was unable to discriminate HbF derived from the vector-encoded versus endogenous γ-globin genes. To address this, I introduced a single base change in exon 2 of the γ-globin gene encoded by V5m3-400 such that threonine replaces isoleucine at amino acid 75 (I75T). This variant was successfully distinguished from endogenous γ-globin gene products by reverse phase high performance liquid chromatography (HPLC) in culture-differentiated erythroid cells. Based on these findings, I created compound γ-globin/shmiR-E vectors that include the I75T substitution (I75Tγ-globin/shmiR-E). Future studies will focus on testing this novel vector design in erythroid cells derived from transduced CD34+ cells of healthy donors and patients with SCD. I anticipate that this compound vector has the potential to maximize γ-globin expression and promote levels of HbF that are unlikely to be safely and effectively achieved by conventional globin gene addition or shRNA knockdown approaches alone.
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Estève, Julie. "Transfert de gènes dans les cellules souches pluripotentes induites : application à la thérapie génique de l'hyperoxalurie primitive de type 1." Thesis, Bordeaux, 2018. http://www.theses.fr/2018BORD0280/document.
Full textAbstract:
L’hyperoxalurie primitive de type 1 (ou HP1) est une maladie héréditaire du métabolisme liée à un déficit en enzyme hépatocytaire AGT (alanine:glyoxylate aminotransférase), codée par le gène AGXT. Ce déficit entraîne, chez les patients atteints d’HP1, une excrétion hépatique accrue d’oxalate ; celui-ci est ensuite éliminé dans les urines où il se complexe avec le calcium pour former des néphrolithiases oxalo-calciques massives, pouvant conduire à une insuffisance rénale chronique. Le seul traitement curatif disponible pour cette pathologie est la greffe allogénique combinée hépatorénale, actuellement limitée par la disponibilité des donneurs de greffons, une morbi-mortalité significative et la nécessité d’un traitement immunosuppresseur au long cours. L’objectif du projet de recherche est de développer une thérapie génique de l’HP1 par greffe de cellules hépatiques autologues génétiquement corrigées. La faible disponibilité et la difficulté d’amplification in vitro des hépatocytes adultes nous a conduit à explorer la piste des cellules souches pluripotentes induites (iPSCs) pour produire des cellules hépatiques humaines utilisables en médecine régénérative. Nous avons dérivé et caractérisé des lignées de cellules iPSCs à partir de fibroblastes de patients atteints d’HP1, après expression transitoire des facteurs de reprogrammation par des vecteurs Sendai. Nous avons développé deux stratégies de thérapie génique additive par insertion d’un minigène codant une séquence optimisée de l’ADNc AGXT au moyen (1) d’un vecteur lentiviral à expression hépato-spécifique et (2) d’un processus de recombinaison homologue au locus AAVS1 facilité par le système de clivage ciblé de l’ADN « CRISPR/Cas9 ». Enfin, nous avons mis en évidence l’expression de la cassette thérapeutique après différenciation hépatocytaire des iPSCs génétiquement corrigées. Ces résultats ouvrent de nouvelles perspectives de médecine régénérative pour l’HP1 par transplantation de cellules hépatocytaires autologues génétiquement corrigées dérivées d’iPSCs de patientsPrimary hyperoxaluria type 1 (or PH1) is an inherited metabolic disorder related to the deficiency of the hepatic AGT enzyme (alanine:glyoxylate aminotransferase), which is encoded by the AGXT gene. In PH1 patients, this deficiency leads to oxalate overexcretion by liver, followed by urine filtration and complexation with calcium to form massive calcium-oxalate nephrolithiasis potentially leading to chronic renal failure. The only available curative treatment is combined hepatorenal allogeneic engraftment, which is currently limited by the availability of transplant donors, significant morbidity and mortality, and the need for long-term immunosuppressive treatment. The aim of our research project is to develop gene therapy for PH1, consisting in engraftment of genetically corrected autologous liver cells. Considering that adult hepatocytes are hardly available and expandable in vitro, we chose to explore the use of induced pluripotent stem cells (iPSCs) to produce human liver cells for application in regenerative medicine. We derived and characterized iPSC lines from PH1 patient fibroblasts after transient expression of reprogramming factors delivered by Sendai virus vectors. We developed two additive gene therapy strategies by inserting a minigene encoding an optimized AGXT cDNA sequence using (1) a lentiviral vector designed for liver-specific expression and (2) homologous recombination process at the AAVS1 locus favoured by the targeted DNA cutting system “CRISPR/Cas9”. Finally, we highlighted therapeutic cassette expression after hepatic differentiation of genetically corrected iPSCs. These results pave the way for regenerative medicine for PH1 by transplantation of genetically modified autologous hepatocyte-like cells derived from patient-specific iPSCs
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Lin, Yuan. "In Vivo Imaging of Engraftment and Enrichment of Lentiviral Transduced Hematopoietic Bone Marrow Cells Under MGMT-P140K Mediated Selection." Case Western Reserve University School of Graduate Studies / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=case1295039430.
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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.
Full textAbstract:
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
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Facchinello, Nicola. "Conditional inactivation of Emilin1 and Col6a1 genes." Doctoral thesis, Università degli studi di Padova, 2010. http://hdl.handle.net/11577/3422723.
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Conditional gene expression methods are important approaches for examining the function of particular genes in development and disease. In particular, a lentiviral vector system for RNAi- mediated in vivo silencing of Col6a1 and a Cre-loxP based procedure for conditional-inducible knockout (KO) of Emilin1 were used in this work.
The use of lentiviral vectors can accelerate the generation of animals with substantial suppression of gene expression in an inducible way. Some authors have observed limitations of the technique due to low efficiency of transgenesis and mosaicism in transgenic mice. We have generated several transgenic mouse lines expressing siRNAs that target the α1(VI) mRNA. Characterization of the different lines and comparison of the phenotypes with that of Col6a1 knockout mice have allowed a systematic evaluation of the different factors affecting silencing of Col6a1, a gene of the extracellular matrix with a complex pattern of tissue-specific expression. The results, obtained with vectors pLVTHM and pLVPT-rtTRKRAB, point out three parameters as major determinants of the efficiency of interference: the choice of interfering sequence, the number of proviral copies integrated into the mouse genome and the site of integration of the provirus. A lentiviral vector (pLVPT-rtTRKRAB) with doxycycline inducible production of shRNA was also tested. Control of expression by the drug was stringent in many tissues; however, in some tissues turning off of shRNA synthesis was not complete. The data support the application of the lentiviral vectors used here in transgenesis (Frka, Facchinello, et al., 2009).
Emilin1 is a gene coding for a protein, Emilin-1, of the elastic extracellular matrix expressed in interstitial connective tissue and in the cardiovascular system starting form early stages of embryonic development to adulthood. Emilin1 null mice display reduced diameter of blood vessels and arterial hypertension. The protein regulates the bioavailability of TGF-β, a cytokine with major effects on the cardiovascular system. Specifically, it has been shown that Emilin1 inhibits proteolysis of the proTGF-β precursor to LAP/TGF-β, a complex from which the growth factor can be subsequently released for receptor binding. In the absence of Emilin1, the amount of active TGF-β is increased, reducing the proliferation rate of smooth muscle cells and the diameter of blood vessels. To establish whether the Emilin1-/- phenotype is the result of a developmental defect or the function of the protein is required for the regulation of blood pressure and arterial structure also in the adult, a conditional gene targeting procedure was used to inactivate Emilin1 in a tissue and time-specific manner. The genetic set up of the transgenic mouse model included the use of floxed Emilin1, CreERT2 (a tamoxifen inducible Cre recombinase) tissue-specific drivers and Rosa26R-lacZ, an inducible reporter for histological visualization of gene rearrangement.
A targeting vector was synthesized in which exons 1-2 of the Emilin1 gene were flanked by loxP sites (floxed allele). The correct integration of the targeting construct in embryonic stem (ES) cell clones was confirmed by Southern blot and PCR analyses. Four of the ES cell clones were subsequently injected into blastocysts resulting in chimeric mice with 10-100% chimerism.
The CreERT2 gene was driven by the Emilin1 or the smooth muscle myosin heavy chain (SMMHC) promoters in order to be expressed in cells active in Emilin1 synthesis or in smooth muscle cells respectively. After tamoxifen administration, activity of both promoters was evident in vascular and visceral smooth muscle cells, where SMMHC-CreERT2 induced recombination more strongly than Emilin1-CreERT2. PCR and RT-PCR analyses confirmed that the SMMHC-CreERT2 was expressed and efficiently excised the loxP flanked sequences in the Emilin1flox/wt locus sufficiently to reduce Emilin1 mRNA expression.
Emilin1flox/flox mice appeared and bred normally and showed no difference in Emilin1 mRNA expression and in blood pressure levels as compared to controls, confirming that introduction of the loxP sites did not interfere with regulation of the gene.
Moreover preliminary data showed that adult animals of the double transgenic lines Emilin1flox/flox and SMMHC-Cre-ERT2 treated with tamoxifen displayed increased blood pressure. This result suggests that hypertension in Emilin1flox/flox mice is not due to a developmental defect of blood vessels, but to the lack of a continuous effect of Emilin-1 on blood pressure regulation even in the adult.Tecniche che prevedono il controllo dell’espressione genica tramite inattivazione condizionale sono di particolare utilità nello studio della funzione di geni durante lo sviluppo e nel determinare patologie. In questo lavoro sono stati utilizzati un sistema di silenziamento in vivo del gene Col6a1 mediante RNAi utlizzando vettori lentivirali e un sistema “Cre/loxP” per generare un modello murino di knockout condizionale inducibile. L’uso di vettori lentivirali può accelerare la generazione di animali transgenici con una consistente riduzione dell’espressione genica e in modo inducibile, anche se in letteratura alla tecnica sono state attribuite alcune limitazioni dovute alla bassa efficienza di transgenesi e alla presenza di mosaicismo nei topi così ottenuti. In questo lavoro abbiamo prodotto diverse linee di animali transgenici con diminuiti livelli di messaggero per il gene Col6a1 grazie alla produzione di siRNA. La caratterizzazione delle varie linee e la comparazione del fenotipo con quello dei topi knockout per lo stesso gene ha permesso l’identificazione dei diversi fattori che sono in grado di influire sul processo di silenziamento di Col6a1, un gene codificante per una proteina della matrice extracellulare con un complesso pattern di espressione tessuto specifica. I risultati ottenuti con i vettori pLVTHM e pLVPT-rtTRKRAB, hanno permesso di definire tre importanti fattori come maggiori determinanti nell’efficienza dell’interferenza: la scelta della sequenza interferente, il numero di copie provirali integrate nel genoma e il sito di integrazione degli stessi. É stato inoltre utilizzato un vettore lentivirale (pLVPT-rtTRKRAB) per la produzione inducibile di shRNA mediante somministrazione di doxyciclina. Il controllo dell’espressione dopo doxycicilina è risultato essere stringente in molti tessuti, anche se in alcuni la inattivazione della produzione di shRNA non è stata completa. I risultati ottenuti dimostrano l’applicabilità di vettori lentivirali nella generazione di animali transgenici (Frka, Facchinello, et al., 2009). Emilina1 è una proteina della matrice extracellulare presente nei tessuti connettivi interstiziali e nel sistema cardiovascolare a partire da stadi precoci di sviluppo embrionale e nell’adulto. I topi mancanti di Emilina1 sono ipertesi e mostrano un diametro ridotto dei vasi. Emilina1 svolge la sua funzione regolando la biodisponibilità di TGF-β, una citochina che svolge importanti funzioni nel sistema cardiovascolare. In particolare, è stato dimostrato che Emilina1 inibisce la proteolisi del precursore pro-TGF-β a LAP/TGF-β, un complesso dal quale il fattore di crescita attivo deve essere successivamente rilasciato per permetterne il legame con il recettore. In assenza di Emilina1, la quantità di TGF-β attivo è aumentata, riducendo la proliferazione delle cellule muscolari lisce e quindi il diametro del vaso. Per stabilire se il fenotipo dei topi Emilina1-/- è il risultato di un’alterata morfogenesi dei vasi o se la funzione della proteina è richiesta nella regolazione della pressione e nella struttura del vaso anche nell’animale adulto, è stato necessario produrre un knockout condizionale del gene di Emilin1 in modo da indurre l’assenza della proteina con un preciso controllo temporale e spaziale. Il modello murino prevede la presenza di siti loxP posti nel gene di Emilin1 in modo da produrre l’inattivazione genica, una Cre-ERT2 (cioè una Cre ricombinasi inducibile tramite tamoxifen) e il locus Rosa26R-lacZ (un gene reporter inducibile per la rilevazione istologica delle cellule nelle quali si è avuto il riarrangiamento genetico). É stato preparato un costrutto dove l’esone 1 e 2 del gene di Emilina1 sono fiancheggiati da due siti loxP. Mediante Southern Blot e PCR, è stata verificata la corretta integrazione del costrutto nelle cellule embrionali staminali di topo. 4 cloni ricombinanti omologhi così identificati sono stati trasferiti in vivo mediante microiniezione in blastocisti ottenendo topi chimerici con un grado di chimerismo compreso tra il 10 e il 100%. L’espressione del gene Cre-ERT2 è stato posto sotto il controllo delle sequenze promotoriali proprie di Emilina1 o, alternativamente, del gene per la catena pesante della miosina di muscolo liscio, in modo da essere attivamente espressa dalle cellule che producono Emilina1 o da quelle muscolari lisce rispettivamente. Dopo la somministrazione di tamoxifen, l’attività di entrambi i promotori è evidente sia nelle cellule muscolare lisce vascolari e viscerali, dove SMMHC-CreERT2 induce una ricombinazione più efficiente rispetto al costrutto Emilina1-CreERT2. Le analisi mediante PCR e RT-PCR confermano che la cre ricombinasi sotto il promotore della miosina di muscolo liscio induce efficientemente la ricombinazione dei siti loxP nel locus di Emilina1, permettendo in questo modo una riduzione consistente dei livelli di messaggero. I topi Emilina1flox/flox generati sono fertili e presentano un fenotipo normale e inoltre comparati con degli animali di controllo non mostrano differenze per quanto riguarda l’espressione del messaggero di Emilina1 e nei livelli di pressione sanguigna, confermando che l’introduzione dei siti loxP non interferisce con la regolazione dell’espressione del gene. Risultati preliminari inoltre indicano un aumento della pressione sanguigna nelle doppie linee transgeniche Emilina1flox/flox e SMMHC-Cre-ERT2 in seguito a trattamento con tamoxifen. Questo risultato suggerisce che l’ipertensione non è dovuta ad un’alterata morfogenesi dei vasi sanguigni, ma al ruolo continuativo di Emilina-1 nella regolazione della pressione sanguigna anche nell’adulto.
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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.
Full textAbstract:
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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Gagnepain, Anaïs. "Évaluation de nouveaux pseudotypes de vecteurs lentiviraux pour le transfert de gènes dans les cellules hématopoiétiques." Thesis, Lyon, École normale supérieure, 2014. http://www.theses.fr/2014ENSL0939/document.
Full textAbstract:
Le transfert de gènes dans les cellules souches hématopoïétiques par des vecteurs lentiviraux s’inscrit dans les protocoles actuels de traitement par thérapie génique de plusieurs maladies monogéniques (B-thalassémie, Adrénoleucodystrophie, SCID…). De même, le transfert de gènes dans les lymphocytes T et B ouvre des perspectives tant au niveau de la thérapie génique que pour l’immunothérapie. Nous avons mis au point des vecteurs lentiviraux pseudotypés par des glycoprotéines chimérique (BaEV/TR) et mutante (BaEVRLess) du rétrovirus endogène de babouin. Nous avons montré que ces nouveaux vecteurs peuvent transduire de manière plus efficace les cellules souches hématopoïétiques stimulées et quiescentes que les vecteurs pseudotypés par la glycoprotéine du virus de la stomatite vésiculaire (VSV-G). Il en est de même pour les vecteurs développés récemment et pseudotypés par les Glycoprotéines H et F du virus de la rougeole. Nous avons aussi comparé la capacité de ces derniers vecteurs à ceux pseudotypés par les glycoprotéines BaEV/TR et BaEVRLess dans le transfert de gènes dans les lymphocytes B et T ainsi que dans l’ensemble des cellules de la lignée T. Nous sommes désormais en mesure de proposer des vecteurs adaptés au transfert de gènes à chaque étape de la différenciation des cellules CD34+ en thymocytes ainsi qu’en lymphocytes T matures. Ceci pourrait permettre de proposer de nouveaux protocoles cliniques en thérapie génique avec une co-transplantation de cellules souches génétiquement modifiées et de cellules T différenciées à partir de ces cellules. Ceci permettrait notamment de réduire les phases d’aplasie actuellement nécessaires pour la greffe de cellules souchesLentiviral vectors and their ability to transfer gene into hematopoietic stem cells are currently evaluated for the cure of several single-gene diseases (eg : B-thalassemia, Adrenoleucodystrophy, SCID). Likewise, gene transfer into B and T lymphocytes is of major interest in gene therapy and immunotherapy. We engineered new lentiviral vectors pseudotyped by some chimeric (BaEV/TR) and mutant (BaEVRLess) glycoproteins from the baboon endogenous retrovirus. We demonstrated that these new vectors can transduce more efficiently resting and mild stimulated hematopoietic stem cells than obtained with lentivectors pseudotyped by the glycoprotein G from the vesicular stomatitis virus (VSV-G). It is the same with the recently developed lentiviral vectors pseudotyped by the H and F glycoprotein from measles virus (H/F-LVs). We also compared the ability of the H/F-LVs with the BaEV/TR and BaEVRLess lentiviral vector pseudotype to transfer genes into B and T lymphocytes and into the whole T lineage. From now on, we are able to propose adapted vectors for gene transfer at each stage of differentiation from CD34+ cells to thymocytes and mature T cells. This could allow us to propose some new clinical protocols in gene therapy with a co-transplantation of genetically modified stem cells and their differentiated T progenitors in order to reduce the aplasia stage induced by current transplantation protocols
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Vogiatzis, Stefania. "Generation of lentiviral vectors expressing chimeric NEDD4 ubiquitin ligases specifically targeting alpha-synuclein: a tool for studying Parkinson's disease pathogenesis and for the development of innovative therapeutic approaches." Doctoral thesis, Università degli studi di Padova, 2019. http://hdl.handle.net/11577/3424860.
Full textAbstract:
Background. The main pathological features of Parkinson's disease (PD) are the death of dopaminergic neurons and the diffuse accumulation of alpha-synuclein (aS) aggregates in neurons. The NEDD4 E3 ubiquitin ligase has been shown to promote aS degradation by the endosomal/lysosomal route. Interestingly, NEDD4 is protective against human aS toxicity in evolutionary distant models. Furthermore, a small molecule able to activate NEDD4 functions, was neuroprotective in evolutionary distant models of aS toxicity. While activation of E3s cannot be easily obtained pharmacologically, their flexibility and the lack of consensus motifs for ubiquitin (Ub) conjugation allow the development of engineered Ub-ligases able to target proteins of interest. The aim of our study was to exploit chimeric Ub-ligases, named ubiquibodies, specifically targeting aS to prove the protective role of aS degradation pathway towards the development of innovative strategies and rescue the normal physiology of neurons.
Methods. To this end, we have developed lentiviral vectors encoding well characterized human scFvs fused in frame to the NEDD4 catalytic domain, in order to obtain enzymes that specifically ubiquitinate aS either in its monomeric and/or oligomeric form. We also generated two lentiviral vectors expressing wild type aS and a mutant form (A53T aS), known to play a role in PD pathogenesis, either alone or fused in frame with the reporter protein EGFP.
Furthermore, we adopted different in vitro models including human and murine dopaminergic cell lines and three different lines of human induced pluripotent stem cells (hiPSCs) derived from a healthy donor and from Parkinson's patients, transduced with lentiviral particles expressing ubiquibodies, with the aim of analyzing their ability to rescue of hiPSCs-derived dopaminergic neurons physiological features.
Results And Conclusions. We are able to demonstrated that: i) the recombinant proteins are expressed in human embryonic 293T cells, as well as in the human and mouse dopaminergic neuroblastoma, SH-SY5Y and MN9D in cell line respectively; ii) recombinant lentiviral particles transduce not only 293T, MN9D and SH-SY5Y cells, but also in human hiPSCs, neural stem cells (NSCs) and NSC-derived dopaminergic neurons; iii) ubiquibodies interacts in a specific manner with overexpressed aS also in dopaminergic cell lines; iv) a specific degradation of aS takes place in 293T cells transduced with one of the developed the chimeric Ub-ligases (Nac32HECTWT) that was selected based on the achieved results on its characteristics. Finally, experiment conducted in hiPSCs-derived NSCs indicate that aS overexpression and mutation have a negative effect on NSCs differentiation into dopaminergic neurons with an increase in cell mortality. The phenotype can be rescued by Nac32HECTWT expression.
The results achieved so far represent a starting point strongly supporting the validity of the strategy we intend to adopt in order to obtain a specific degradation of aS.
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ANNONI, ANDREA. "Strategies for tolerance induction to gene therapy derived products." Doctoral thesis, Università degli Studi di Roma "Tor Vergata", 2009. http://hdl.handle.net/2108/763.
Full textAbstract:
I vettori lentivirali (LV) rappresentano una concreta realtà per lo sviluppo di protocolli di terapia genica. I LV, infatti, hanno la capacità di trasdurre ed integrare stabilmente il proprio genoma anche in cellule che non sono in attivo stato di replicazione. Tuttavia, uno dei maggiori ostacoli al successo degli approcci di terapia genica che prevedono l’utilizzo di vettori virali è rappresentato dalla risposta immune innata ed adattativa da essi indotta. Le risposte immuni cellulo-mediate ed anticorpali dirette verso antigeni associati alle particelle virali e alla proteina derivante dal transgene eliminano le cellule geneticamente modificate, rendendo inefficacie la terapia genica.
Nello studio qui descritto, abbiamo sviluppato un modello murino di terapia genica, che prevede somministrazione sistemica in topi immunocompetenti di LV codificante per una proteina fluorescente. Tale modello sperimentale è funzionale alla caratterizzazione della cinetica d’espressione del transgene e della risposta immunitaria contro di esso e allo stesso tempo è utile per lo sviluppo di nuovi approcci per la modulazione di tale risposta.
Diverse strategie sono state esplorate per la modulazione della risposta al transgene, come l’immunosoppressione e l’utilizzo di vie di somministrazione meno immunogeniche. In questo studio abbiamo indagato la possibilità di ristabilire la tolleranza immunologica nei confronti del prodotto di terapia genica attraverso due differenti modalità: una terapia cellulare da associare al trasferimento genico o l’utilizzo di LV con una regolazione d’espressione dipendente da micro-RNA endogeni.
E’ stato ampiamente dimostrato che le cellule CD4+CD25+ T regolatorie (Tregs) rappresentano una sottopopolazione linfocitaria con la capacità di controllare le risposte immunitarie in vivo garantendo la tolleranza agli antigeni “self”. Con l’ausilio del nostro modello animale abbiamo testato se dette cellule fossero in grado di sopprimere la risposta al transgene. Abbiamo così definito che il trasferimento di Tregs, derivanti da topi wild type (wt) o da topi GFP-transgenici (GFP-tg), tolleranti per GFP, non è in grado di controllare la risposta al transgene. Contrariamente, il trasferimento di un’ alta dose di cellule presentanti l’antigene (APC), derivate da topi GFP-tg, sono risultate efficaci nel controllo della risposta immune estendendo significativamente i tempi di espressione del transgene.
La risposta immune verso il transgene si genera poiché, dopo la somministrazione del vettore lentivirale, una frazione delle APC esprime il transgene e ne presenta epitopi con modalità immunogenica attivando le cellule T. Promotori tessuto-specifici, come quello per l’albumina, sono stati testati per ridurre l’espressione del transgene nelle APC, restringendola ai soli epatociti. Recentemente, Brown et al. (Nat. Med. 2006; 12:585-591) hanno dimostrato che inserendo ripetute sequenze riconosciute da un micro-RNA, specifico della linea ematopoietica, mir142-3p, nel vettore codificante per GFP (LV.PGK.GFP.mir142-3pT) l’espressione del transgene era abrogata in tutte le cellule di derivazione ematopoietica ma era stabile e persistente nel fegato sia negli epatociti che nelle cellule endoteliali (LSEC). Così, abbiamo studiato come il sistema immunitario reagisce alla somministrazione di tale LV micro-RNA regolato. I dati ottenuti indicano che si genera uno stato di tolleranza verso il transgene, mediata da cellule Tregs.
Questi risultati nel loro insieme potranno avere importanti sviluppi per le future applicazioni di protocolli di terapia genica.Due to their ability to transduce non-dividing cells and stably integrate, lentiviral vectors (LV) are a candidate system for therapeutic gene transfer in a number of genetic diseases. However, the use of LV may be hampered by their ability to trigger innate and adaptive immunity. Immune responses against genetically modified cells represent a major obstacle to the success of gene therapy. Cellular and humoral immune responses to vector associated and transgene-derived proteins lead to prompt elimination of transgene expressing cells abrogating, the benefits of gene transfer. A non-therapeutic murine model of gene therapy has been established. LV, encoding for green fluorescent protein (GFP) under the control of an ubiquitous promoter, was intravenously administrated into immunocompetent mice in order to define the kinetics of transgene expression, characterize the anti-transgene immune response and develop different approaches to overcome the anti-transgene immunity and achieve long term transgene expression. Several strategies have been used to limit immune response following gene transfer, including immunosuppression, and the use of less immunogenic routes of administration. In the present work we focused our attention in inducing immunological tolerance to transgene expressing cells as first approach, by a cellular therapy, and second, through the use micro-RNA (mir) regulated lentiviral vectors. Regulatory T cells (Tregs), which have been demonstrated to control immune responses in vivo, were tested for their ability to suppress anti-transgene response leading to stable long-term gene expression. Adoptive transfer of naturally occurring CD4+CD25+ Tregs (nTregs) isolated from wt mice or from transgene tolerant transgenic (tg) mice did not suppress the anti-transgene immune response after LV delivery. These data demonstrate that neither increasing the endogenous pool of nTregs nor transferring nTregs selected in a transgene expressing thymus can modulate the immune response and mediate sustained transgene expression. Conversely, adoptive transfer of antigen presenting cells (APC) isolated from transgene tolerant tg mice efficiently reduced the immune response leading to stable LV-encoded protein expression in vivo. Most of the immunogenicity of the LV-based gene transfer protocols depends on the transduction and transgene expression by professional APC that prime T cells inducing an anti-transgene immune response. The use of tissue specific promoters, such as the albumin promoter, has been explored in order to target transgene expression in hepatocytes and drastically reduce transgene expression in APC. Recently, in the same direction, Brown et al. (Nat. Med. 2006; 12:585-591) demonstrated that addition of target sequences for an hematopoietic-specific micro-RNA, mir-142-3p, into LV encoding for GFP under transcriptional control of the ubiquitous PGK promoter (LV.PGK.GFP.142-3pT) prevented transgene expression in all hematopoietic lineages and led to stable transgene expression in the liver. Here, we set out to elucidate the immunological events that enabled stable gene transfer with this microRNA-regulated LV. Results demonstrate that systemic gene transfer by the mir-142-regulated LV can provide robust tolerance to a specific antigen which is mediated by CD4+ Tregs. These findings will have important implications for developing future gene therapy strategies.
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PASINI, SILVIA. "Role of activated transcription factor 4 (ATF4) in learning and memory." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2011. http://hdl.handle.net/10281/27132.
Full textAbstract:
The aim of this study is to understand the role of Activated Transcription Factor 4 (ATF4) in the processes of learning and memory. The topic of learning and memory has always aroused great interest from time immemorial and although a lot of researches have been focused on this subject for a long time, many mechanisms have not yet been fully understood.
Identifying the players and the mechanisms involved in learning and memory is of utmost importance because deficits in these cognitive functions are symptoms of common neurological diseases like stoke, depression, dementia and Alzheimer’s disease, one of the most wide spread neurodegenerative disease.
It has already been established that new gene expression and protein synthesis are required for long term memory, providing the basis to think that transcription factors may play a key role in these processes. Several studies have demonstrated the involvement of different transcription factors in memory formation such as cAMP response element binding protein (CREB), CCAAT enhancer binding protein (C/EBP), activated protein 1 (AP1), early growth response factor (Egr) and Rel/nuclear factor kB (Rel/NFkB).
Very little is known about the involvement of another transcription factor, Activated Transcription Factor 4. ATF4 is a member of the activated transcription factor (ATF)/cyclic AMP response element binding protein (CREB) family. It was originally described as a repressor of CRE-dependent gene transcription but recent studies have shown it to be a transcriptional activator. It is also a stress responsive gene, regulating the adaptation of cells to stress stimuli such as anoxia, endoplasmic reticulum stress and oxidative stress. ATF4 plays an essential role in development, and is particularly required for proper skeletal and eye development and is also involved in tumor progression and metastasis.
ATF4 has always been reported as a memory repressor that blocks new gene expression required for memory formation but no study has ever investigated it in a specific and direct way. The aim of this thesis is to study, in a specific and direct manner, the role of ATF4 in the processes of learning and memory. To reach this goal, ATF4 expression was modified in mouse hippocampi, the brain region mainly involved in learning and memory, with the injection of lentivirus carrying ATF4 gene, for the gain-of-function analysis, and lentivirus carrying shATF4, for the loss-of-function studies.
Before starting the experiments of ATF4 overexpression and downregulation, preliminary experiments were conducted to set up the injection coordinates to target the mouse hippocampi, to verify the lentiviral tropism and most importantly to evaluate the lentiviral spread, within the hippocampus, after the injection.
The consequence of ATF4 gain- and loss-of-function was then studied in the performance of standard behavioral tests such as Water Maze tests and Fear Conditioning, widely used to assess spatial and associative memory respectively. The behavioral test results showed that ATF4 protein overexpression enhances spatial memory, under the weak training paradigm in the Morris Water Maze test, and associative memory while ATF4 downregulation impairs spatial memory under the standard training condition.
After completing the behavioral tests, ATF4 overexpressed and downregulated mice were subjected to electrophysiological and neuronal spine analysis to verify if the alteration in cognitive functions, as a result of ATF4 modification, is supported by changes in synaptic potentiation and spine density and morphology.
Long Term Potentiation (LTP) is a long lasting enhancement in neuronal transmission and is widely considered as a cellular model of learning and memory in the central nervous system. The long-term memory impairment of ATF4 downregulated mice is supported by electrophysiological analysis, in which ATF4 downregulated slices showed an impairment in LTP. Unexpectedly, LTP impairment was also found in ATF4 overexpressed slices, maybe due to the difference in the time between the injection and the behavioral tests or the electrophysiological recordings.
Most of the intracellular pathways responsible for LTP require new gene expression and protein synthesis. This, in turn, leads to morphological changes required to sustain the enhancement of signal transmission. One of these morphological changes is the modification of the density and the morphology of dendritic spines. ATF4 up- and downregulation in hippocampal neurons does not affect spine density but ATF4 overexpression causes a significant increase in the percentage of mushroom spines as compared to that found after ATF4 downregulation. Mushroom spines with a large head are the most stable neuronal spines and contribute to strong synaptic connections, hence it has been hypothesized that they represent the “memory spines”.
Collectively, these results support the hypothesis that the transcription factor ATF4 plays a positive role in synaptic plasticity and memory formation. Further studies need to be done to understand the molecular mechanisms through which ATF4 acts.
This thesis represents only a step on the road towards understanding the complicate mechanisms of learning and memory, not forgetting that the most important discoveries were the result of small knowledge acquired step by step.
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Zanatta, Daniela Bertolini. "Sequenciamento de um código de barras como ferramenta para quantificação de alterações na dinâmica de populações celulares transduzidas com vetores lentivirais." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-21092012-102415/.
Full textAbstract:
Os vetores retrovirais representam uma das melhores opções para transferência e terapia gênica, pois fornecem expressão do transgene em longo prazo. Entretanto, a inserção do provírus pode causar mutagênese insercional, induzindo proto-oncogenes. Eventos deste tipo têm sido descritos em protocolos clínicos para o tratamento de SCID-X1, doença granulomatosa crônica e talessemia beta, quando vetores retrovirais (oncorretrovirus) foram utilizados. Atualmente, existem poucos métodos simples e rápidos para revelar e quantificar a expansão clonal. Assim, descrevemos a construção uma biblioteca de vetores contendo uma marcação aleatória, denominada código de barras. O sequenciamento do código de barras permitirá revelar, caracterizar e até quantificar a expansão clonal de uma população de células transduzidas. Esta metodologia ajudará a testar novos arranjos de promotores e genes terapêuticos, para o desenvolvimento de vetores mais seguros contribuindo para a redução da probabilidade de um evento de proliferação clonal desencadeado pela mutagênese insercional.Retroviral vectors represent one of the best options for gene transfer and therapy, where long-term transgene expression is required. However, insertion of the provirus can cause insertional mutagenesis, which may have adverse consequences, such as induction of proto-oncogenes. Such events have been described in clinical trials for the treatment of SCID-X1, chronic granulomatous disease and beta thalessemia with some retroviral vectors. Currently, there are few simple and quick methods that can reveal and quantify clonal expansion. Thus, we describe the construction of a vector library containing random markers, called \"barcodes\". The sequencing of the barcode could reveal, characterize and quantify the clonal expansion of a transduced cells population. This methodology will be valuable to test new arrangements of promoters and therapeutic genes, allowing the development of safer vectors, helping to reduce the probability of clonal proliferation events triggered by insertional mutagenesis.
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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/.
Full textAbstract:
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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Ward, N. J. "Lentiviral vectors for treatment of haemophilia." Thesis, University College London (University of London), 2010. http://discovery.ucl.ac.uk/19905/.
Full textAbstract:
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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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/.
Full textAbstract:
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.