Dissertations / Theses on the topic 'Malaria – Gene therapy'
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Bockstael, Olivier. "Evaluation of gene transfer strategies using recombinant adeno-associated viruses for Parkinson's disease cell and gene therapy." Doctoral thesis, Universite Libre de Bruxelles, 2010. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/210010.
Full textLes vecteurs dérivés des virus adéno-associés (rAAV) constituent des outils de choix pour le transfert de gènes dans les tissus cérébraux. Par ailleurs, de nombreuses applications nécessitent une régulation de l’expression du transgène. Nous disposons au laboratoire d’un vecteur rAAV inductible à la tétracycline (rAAV-TetON).
Nous décrivons dans ce travail :
i) le comportement du vecteur rAAV dérivé du sérotype 1 d’AAV utilisant la cassette d’expression TetON (rAAV2/1-TetON) comparé à celui du rAAV2/1 utilisant un promoteur constitutif pour l’expression du transgène (rAAV2/1-pCMV) dans le striatum et le mésencéphale (contenant la substance noire). A l’aide d’un vecteur rAAV2/1-TetON exprimant le GDNF, nous montrons que nous pouvons moduler le niveau d’expression du transgène dans le striatum par la dose d’inducteur administré aux animaux. Par ailleurs, nous montrons que le rAAV2/1-TetON présente dans le striatum une efficacité de transduction moindre que le rAAV2/1-pCMV mais qu’il présente un profil de biosécurité supérieur au rAAV2/1-pCMV car il limite fortement l’expression du transgène hors du striatum. De plus, le rAAV2/1-TetON n’entraîne pas de recrutement de lymphocytes T ni d’activation de la microglie dans le striatum. Lorsqu’il est injecté dans le mésencéphale, le vecteur rAAV2/1-TetON, contrairement au rAAV2/1-pCMV présente une expression préférentielle dans les neurones dopaminergiques de la SNpc et de l’aire tégmentale ventrale (VTA).
ii) le comportement des vecteurs rAAV2/1-pCMV et scAAV2/1-pCMV (vecteur « self-complémentaire » permettant une expression du transgène indépendamment de la synthèse du second brin du génome viral) dans la région neurogénique de la zone sous-ventriculaire (ZSV). Nous avons montré que les vecteurs rAAV2/1 infectent efficacement la ZSV et s’y expriment rapidement. Les vecteurs scAAV2/1 s’expriment plus rapidement dans la ZSV que les vecteurs rAAV2/1 (expression maximum à 24h et 48h, respectivement). De plus, les vecteurs rAAV2/1 présentent une efficacité de transfection importante pour les progéniteurs neuraux en prolifération (cellules C, transient amplifying progenitors) et les neuroblastes en migration (cellules A) mais pas pour les cellules souches neurales (cellules B). Nous observons, par ailleurs, que les rAAV2/1 induisent une baisse transitoire de la prolifération de la ZSV. Cet effet est indépendant de l’expression du génome et dépend donc probablement de la capside virale de nos vecteurs. De plus, cette baisse de prolifération n’induit pas d’apoptose. A long terme, nous observons des cellules exprimant le transgène dans la zone granulaire du bulbe olfactif, indiquant que la transduction des progéniteurs de la ZSV n’interfère pas avec leurs capacités de migration et de différenciation.
iii) l’efficacité de différents sérotypes de rAAV pour le transfert de gènes dans les cellules progénitrices neurales (NPC) in vitro. Nous avons montré que les rAAV peuvent transduire des NPC mais que l’efficacité spécifique des différents sérotypes testés varie en fonction de la région du cerveau fœtal et de l’espèce dont les NPC sont issues. Par ailleurs, les rAAV induisent une réduction drastique de la prolifération des cultures de NPC dépendante du sérotype de rAAV utilisé mais pas de l’origine fœtale des NPC ou de l’espèce dont elles sont issues.
Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished
Maire, Séverine. "Towards Trans-Splicing Gene Therapy for HD : Intronic Targets Identification in the Huntingtin Gene." Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLS054.
Full textHuntington’s disease (HD) is an autosomal dominant genetic disorder caused by the expansion of a CAG repeat encoding a polyglutamine tract in the first exon of the Huntingtin gene (HTT). This gene encode a ubiquitous protein in which mutation lead to severe motor, psychiatric and cognitive deficits and causes degeneration of specific neuronal populations, in particular the GABAergic medium spiny neurons of the striatum. We propose to use trans-splicing to develop a gene therapy vector that will significantly reduce or eliminate the expression of the mutant protein while restoring a physiological level of normal HTT in cells affected by the HD mutation. This technology is based on replacement of the mutated exon by a normal version during the mRNA maturation process. HTT mutation being dominant, therapeutic benefits necessitates a highly efficient trans-splicing reaction that would convert a significant proportion of mutant-HTT pre-mRNA into normal HTT mRNA. For this purpose, we developed a fluorescent reporter system enabling the detection of trans-splicing events in high content screening in order to identify the most potent trans-splicing sequences among hundreds of molecules. We validated our fluorescent screening strategy and implement trans-splicing screening on 3 HTT introns (3, 9 and 20), in which we demonstrated the presence of hotspot promoting trans-splicing reactions. A direct and absolute quantification method was also validated to accurately assess the correction rate. Overall, this work generated additional evidences of trans-splicing feasibility in HD
Costa, Verdera Helena. "Towards the development of a gene therapy for Pompe disease : Characterization of the immune phenotype in Pompe disease and comparison of the therapeutic potential of gene therapy with the current standard of care." Electronic Thesis or Diss., Sorbonne université, 2019. http://www.theses.fr/2019SORUS516.
Full textPompe disease is a lysosomal storage disease caused by mutations on the enzyme acid α-glucosidase (GAA), responsible for degrading lysosomal glycogen. GAA deficiency causes the accumulation of glycogen in multiple tissues, particularly in muscles and central nervous system, leading to a complex neuromuscular disease with high morbidity and mortality. In addition to the known symptoms, our studies in Pompe disease patients and mice show that GAA deficiency affects the activation and function of immune cells, particularly of T cells, leading to a higher activation of effector cells and impaired induction and suppressive function of regulatory T cells (Tregs). Moreover, Pompe disease mice present tissue inflammation at early stages of the disease, altogether suggesting that alterations in the immune system could contribute to the disease pathophysiology. These findings could open new venues to investigate strategies that delay the progression of the disease. In a second set of experiments we show that liver-directed gene therapy with a secretable GAA transgene results in superior disease rescue in an immunodefficient Pompe disease mouse model, when compared to the current enzyme replacement therapy. Because Tregs play an essential role in liver-directed gene therapy, by inducing immune tolerance towards hepatic transgenes, future studies will have to evaluate the potential impact of immune alterations associated to Pompe disease on the efficacy of liver-targeted gene transfer
La enfermedad de Pompe es una enfermedad lisosomal (lysosomal storage disease, LSD) causada por mutaciones en la enzima α-glucosidasa ácida (GAA), que hidroliza el glucógeno en glucosa en los lisosomas. La disfunción de esta enzima causa la acumulación de glucógeno en múltiples tejidos, principalmente en células musculares y neuronas. Como resultado, los pacientes desarrollan hipertrofia cardíaca, debilidad muscular, insuficiencia respiratoria, alteraciones cognitivas y muerte prematura por paro cardiorrespiratorio. Actualmente la enfermedad de Pompe se trata con terapia de reemplazo enzimático (Enzyme replacement therapy, ERT) con GAA recombinante humana (rhGAA). Este tratamiento ha demostrado corregir la patología cardíaca y extender la esperanza de vida de los pacientes. Sin embargo, la eficacia de la ERT en los músculos respiratorios y esqueléticos es parcial, y nula en el sistema nervioso central (SNC). Además, la proteína rhGAA es altamente inmunogénica, por lo que el tratamiento no es efectivo en ciertos pacientes. Otros inconvenientes son el elevado coste de la ERT y la necesidad de infusiones continuadas a lo largo de la vida del paciente. En este estudio mostramos que la terapia génica dirigida al hígado mediante vectores adeno-asociados (AAV) expresando una versión modificada de la GAA revierte de forma significativa la enfermedad a nivel de la musculatura esquelética y del SNC, en un modelo animal de la enfermedad de Pompe. Además, mostramos que este tratamiento es superior en eficacia al tratamiento estándar por ERT, incluso a dosis reducidas de vector AAV. Con tal de comprender mejor los mecanismos de acción de estos dos tratamientos, hemos llevado a cabo un estudio farmacocinético de los niveles de GAA en circulación y en múltiples tejidos en los dos casos. Dicho estudio muestra que niveles reducidos pero constantes de GAA en circulación proporcionados por el hígado permiten una mayor acumulación de GAA en los tejidos en comparación a la ERT, mejorando así la eficacia del tratamiento. Debido a que las reacciones inmunes contra el vector AAV y el transgén son un obstáculo importante en la aplicación clínica de la terapia génica, y a que alteraciones en los lisosomas han demostrado tener un impacto sobre el sistema inmune en diferentes modelos, también hemos estudiado el sistema inmune en el caso de la enfermedad de Pompe. Mediante estos estudios, hemos observado que la acumulación de glucógeno en los lisosomas de células inmunes está asociada a una mayor activación de estas células, tanto en pacientes como en ratones con enfermedad de Pompe, y particularmente en las células T. Además, ratones con enfermedad de Pompe presentan inflamación en los tejidos ya en las primeras etapas de la enfermedad. Por otra parte, mostramos que el mayor estado de activación de las células T podría deberse a alteraciones en el metabolismo de estas células, como resultado de las alteraciones lisosómicas. Finalmente, los ratones con enfermedad de Pompe presentan un defecto en la inducción de células T reguladoras Foxp3+ (Tregs), y estas células tienen un menor potencial inhibidor en comparación con Tregs de ratones sanos. Alteraciones en el sistema inmunitario podrían contribuir a la fisiopatología de la enfermedad. Por lo tanto, estos hallazgos podrían abrir nuevos caminos para investigar estrategias que retrasen la progresión de la enfermedad. Además, las Tregs juegan un papel esencial en la terapia génica dirigida al hígado, mediante la inducción de tolerancia inmune hacia transgenes expresados por hepatocitos. Por lo tanto, futuros estudios deberán evaluar el impacto de las alteraciones inmunitarias asociadas a la enfermedad de Pompe sobre la eficacia de la terapia génica
La malaltia de Pompe és una malaltia lisosomal (lysosomal storage disease, LSD) deguda a mutacions en l'enzim α-glucosidasa àcida (GAA), que hidrolitza el glicogen en glucosa als lisosomes. La disfunció d'aquest enzim causa l'acumulació de glicogen en múltiples teixits, principalment en cèl·lules musculars i neurones. Com a resultat, els pacients presenten hipertròfia cardíaca, debilitat muscular, insuficiència respiratòria, alteracions cognitives i mort prematura per aturada cardiorespiratòria. Actualment, la malaltia de Pompe és tractada amb teràpia de reemplaçament enzimàtic (Enzyme replacement therapy, ERT) amb GAA recombinant humana (rhGAA). Aquest tractament ha demostrat corregir la patologia cardíaca i estendre l'esperança de vida dels pacients. No obstant, l'eficàcia de l'ERT en els músculs respiratoris i esquelètics és parcial, i nul·la en el sistema nerviós central (SNC). A més, la proteïna rhGAA és altament immunogènica, de manera que el tractament no és efectiu en certs pacients. Altres inconvenients són l'elevat cost de l'ERT i la necessitat d'infusions continuades al llarg de la vida del pacient. En aquest estudi mostrem que la teràpia gènica dirigida al fetge mitjançant vectors adeno-associats (AAV) expressant una versió modificada de la GAA millora de forma significativa la malaltia a nivell de la musculatura esquelètica i del SNC, en un model animal de la malaltia de Pompe. A més, mostrem que aquest tractament és superior en eficàcia al tractament estàndard per ERT, fins i tot a dosis reduïdes de vector AAV. Per tal de comprendre millor els mecanismes d'acció d'aquests dos tractaments, hem dut a terme un estudi farmacocinètic dels nivells de GAA en circulació i en múltiples teixits en ambdós casos. Aquest estudi mostra que nivells reduïts però constants de GAA en circulació proporcionats pel fetge permeten una major acumulació de la GAA en els teixits en comparació a la ERT, millorant així l'eficàcia del tractament. Degut a que les reaccions immunes contra el vector AAV i el transgèn són un obstacle important en l'aplicació clínica de la teràpia gènica, i a que alteracions en els lisosomes han demostrat tenir un impacte sobre el sistema immune en differents models, també hem avaluat el sistema immune en el cas de la malaltia de Pompe. Mitjançant aquests estudis, hem observat que l'acumulació de glicogen en els lisosomes de les cèl·lules immunes està associada a una major activació d'aquestes cèl·lules en pacients i ratolins amb malaltia de Pompe, particularment en les cèl·lules T. A més, ratolins amb malaltia de Pompe presenten inflamació dels teixits ja en les primeres etapes de la malaltia. D’altra banda, hem observat que el major estat d'activació de les cèl·lules T podria ser degut a alteracions en el metabolisme d'aquestes cèl·lules, com a resultat de les alteracions lisosomals. Finalment, els ratolins amb malaltia de Pompe presenten un defecte en la inducció de cèl·lules T reguladores Foxp3+ (Tregs), i aquestes cèl·lules tenen un menor potencial inhibidor en comparació amb les Tregs de ratolins sans. Alteracions en el sistema immunitari podrien contribuir a la fisiopatologia de la malaltia. Per tant, aquestes observacions podrien obrir nous camins a l’hora d’investigar estratègies que retardin la progressió de la malaltia. A més, les Tregs juguen un paper essencial en la teràpia gènica dirigida al fetge, mitjançant la inducció de tolerància immune cap a transgens expressats per hepatòcits. Per tant, futurs estudis hauran d'avaluar l'impacte de les alteracions immunitàries associades a la malaltia de Pompe sobre l'eficàcia del tractament per teràpia gènica
Hajjar, Hélène. "Gene therapy approach on Charcot-Marie-Tooth type 1A rats." Thesis, Montpellier, 2018. http://www.theses.fr/2018MONTT027/document.
Full textMyelin, a tissue synthesized by Schwann cells, covers and protects nerves. If damaged, it causes many demyelinating diseases such as the inherited peripheral nervous system disorder Charcot Marie Tooth or CMT type 1. CMT neuropathies display a large variability from one patient to another. Nevertheless, the most common symptoms include muscle weakness, an awkward way of walking (gait), equilibrium problem and highly arched or very flat feet. The most common subtype of CMT is an autosomal dominant disorder known as CMT1A. CMT1A is caused by the duplication of the peripheral myelin protein 22 (PMP22) gene on the short arm of chromosome 17 (17p11.2) resulting in an excess of PMP22. This leads to demyelination. PMP22 is a small protein expressed by Schwann cells. There is still no cure for CMT diseases. One approach for a treatment is gene therapy. The aim of my thesis project was to deliver proof of principle for a gene therapy approach on a CMT1A rat model characterized by extra copies of mouse pmp22 gene (CMT1A rat). The treatment strategy consisted in reducing PMP22 overexpression in CMT1A rats with shRNA against PMP22. Viral vectors like adeno-associated virus (AAV having serotypes from1-10) are used to deliver shRNA in vivo so that they won’t be destroyed by the organism and for them to be long-lasting. Thus, we injected sciatic nerves of 6-7-day-old CMT1A rats with AAV9 expressing shRNA PMP22 with a GFP marker. We first confirmed that the virus highly transduced Schwann cells and that AAV9 shRNA PMP22 decreased PMP22 protein expression in CMT1A rats’ sciatic nerves. CMT1A rats treated with AAV9 shRNA PMP22 showed that they didn’t develop the motor phenotype seen in controls. Moreover, hypoalgesia observed in CMT1A rats was alleviated by treatment. In addition, gene therapy increased the reduced nerve conduction velocity found in CMT1A rats. Concerning safety, no viral off-targets were detected except in muscles close to the injection site (sciatic nerve) and in the dorsal root ganglions. Except for 2 rats, there was no immune response against AAV; no anti-AAV9 neutralizing factors. Consequently, this gene therapy could be used in clinical trials. Before moving to clinical studies, the minimal effective dosage should be very carefully defined because if PMP22 is completely deleted, another disease is caused: Hereditary Neuropathy with Pressure Palsies. It is also crucial to have a strong readout to evaluate the outcome of a treatment. However, no tool consistent enough exists for examining the peripheral nerve. Thus, we tested the label-free imaging technique Coherent Anti-stokes Raman Scattering (CARS) and successfully characterized myelination defects. Consequently, CARS could be used as a consistent outcome measure for developing new therapies for demyelinating peripheral neuropathies
Rouvière, Laura. "Transfert de gènes dans un modèle murin de la maladie de Sandhoff à l'aide d'un vecteur scAAV9 : intérêt d'une double voie d'administration ?" Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCB052/document.
Full textSandhoff disease (SD) is a genetic disorder due to mutations in the HEXB gene. It is characterized by a double Hex A (αβ) and B (ββ) deficiency, responsible for a GM2 accumulation, mainly in the central nervous system (CNS). Clinically, SD begins in the first months of life and culminates in death around 3 years of age. So far, no specific treatment is available for Sandhoff disease. The murine model obtained by invalidation of the Hexb gene is a useful tool for the development of therapeutic approaches, as it exhibits a phenotype quite close to the human disease. The main aim of my PhD project was to explore a gene transfer approach in Sandhoff mice using a specific scAAV9. This vector has the particularity to cross the blood-brain barrier after intravenous (IV) administration and to transduce brain. A vector encoding the hexosaminidases β chain, called scAAV9-Hexb, has been previously IV injected in neonatal Hexb-/- mice with a dose of 3.5 x 1013 vg/kg. I participated to the long-term analysis of the scAAV9-Hexb treated mice using behavioral tests and analysis of tissues at 24 months post-injection. Mice had a survival similar to normal mice (>700 days) without neurological sign and peripheral damage by comparison with naïve Sandhoff mice (death around 120 days). At 4 months post-treatment, lipid analysis using HPTLC showed that GM2 storage was absent in brain, but it was only decreased in cerebellum of treated mice. Even if no symptom was associated with this residual storage in mice at 2 years, we wondered if it could possibly be pathogenic at longer-term if extrapolated to patients. Therefore, we decide to test a combined way of administration i.e. intravenous (IV) + intracerebroventricular (ICV) using the same vector with the same final dose. Two groups of mice were injected using different doses in both compartments and treatment efficacy was evaluated at short- and long-term. In the cerebrum, at short-term, enzymatic activities were partially but significantly restored, GM2 accumulation was completely prevented and disease biomarkers corrected. In the cerebellum, a significant increase of enzymatic activity was only obtained for the group treated with the highest dose in the ICV compartment. Regarding GM2 analysis and long-term behavioral analysis, we confirmed that this dose is required to cure cerebellum. In liver, our results suggest that IV minimal dose is needed to obtain a decrease of lipid accumulation. Our results showed that minimal doses are required in ICV and IV to obtain a good efficacy in each compartments, and that combined administration permit a widespread correction in the CNS. These data will permit to treat adult mice with the optimal treatment. The other goal of my project was to explore signaling defects and cellular pathophysiology in Sandhoff disease using in vivo and in vitro studies. For in vitro studies, fibroblasts from Tay-Sachs and Sandhoff patients were analyzed and mouse embryonic fibroblasts (MEF) were obtained from the Hexb-/- murine model, lysosomal storage was confirmed. mTOR (mammalian target of rapamycin) pathway was studied showing signaling deregulation. Autophagy was analyzed in vitro and in vivo, as defect in this pathway has been reported in other lysosomal storage disorders. An increase of autophagosomes number was observed in Hexb-/- subjects suggesting a defect in autophagy. These results offer novel biomarkers of Sandhoff pathology which can be useful to test the efficacy of therapeutic approaches. They can also provide new therapeutic targets that could be tested in combination with gene transfer
Sánchez, Moreno Xavier. "Desenvolupament d'una aproximació de teràpia gènica pel tractament de la malaltia de Niemann-Pick tipus C2." Doctoral thesis, Universitat Autònoma de Barcelona, 2020. http://hdl.handle.net/10803/670821.
Full textLa enfermedad de Niemann-Pick tipo C2 (NPC2) es una enfermedad minoritaria de acumulación lisosomal (LSD, Lysosomal Storage Disease) de herencia autosómica recesiva, causada por la deficiencia de la NPC2. La proteína NPC2 interviene en la salida del colesterol no esterificado del compartimento endo-lisosómico. Su deficiencia resulta en la acumulación patológica del colesterol no esterificado los lisosomas, que conduce a la disfunción y muerte celular. Como consecuencia, los pacientes desarrollan una patología neurológica severa y de carácter progresivo, que incluye la ataxia cerebelosa, la disartria, la disfagia y la demencia; así como una patología en los tejidos somáticos relativamente más leve. Los pacientes suelen morir durante la primera y la segunda década de vida. Actualmente, el tratamiento con miglustat, una terapia de reducción de sustrato es el único aprobado para el tratamiento de la NPC2, que presenta una eficacia limitada. Así pues, es necesario desarrollar nuevas estrategias terapéuticas más eficaces para el tratamiento de esta enfermedad. La terapia génica in vivo basada en la administración de los vectores virales adeno-asociados (AAV, Adeno-Associated Virus) representa una alternativa prometedora, ya que una única administración del tratamiento permite conseguir una eficacia terapéutica a largo plazo. Por lo tanto, el objetivo de esta tesis doctoral fue el desarrollo de una aproximación de terapia génica basada en la administración de vectores AAV de serotipo 9 (AAV9) en líquido cefalorraquídeo (LCR) para el tratamiento de la enfermedad de NPC2. En primer lugar, se caracterizó un nuevo ratón modelo de la enfermedad, generado a partir de la disrupción del gen NPC2. Este modelo recapituló las principales alteraciones patológicas de la enfermedad. En la segunda parte de esta tesis doctoral, se administró los vectores AAV9 codificante para la proteína NPC2 murina directamente al LCR los ratones NPC2-/- de 18 y 30 días de edad, cuando la patología ya estaba establecida. La transferencia génica mediante el vector AAV9-Npc2 condujo a una marcada reducción de la acumulación del colesterol no esterificado y de la patología lisosomal al sistema nervioso central. Consecuentemente, se logró una mejora de la mielinización, así como también una reducción de la neurodegeneración y la neuroinflamación. Además, parte del vector administrado al LCR también fue capaz de transducir el hígado, el cual actuó como una bomba secretora de la proteína y permitió corregir las alteraciones patológicas en el resto de órganos periféricos. Finalmente, el tratamiento con AAV9-Npc2 condujo a la normalización de los problemas locomotores, a una mejora del peso corporal y un incremento muy marcado de la esperanza de vida. En conjunto, estos resultados podrían ser la base para la futura traslación clínica de la aproximación de terapia génica mediante la administración intra-LCR del vector AAV9-Npc2 para el tratamiento de la enfermedad de NPC2.
Niemann-Pick type C2 (NPC2) disease is a rare autosomal recessive lysosomal storage disease caused by deficiency of the NPC2. NPC2 protein is involved in the egress of unesterified cholesterol from the endo-lysosomal compartment to other parts of the cell. Its deficiency leads to pathologic accumulation of cholesterol in lysosomes, which results in cellular damage and cell death. As a consequence, patients develop a severe, progressive neurological disorder, including cerebellar ataxia, dysarthria, dysphagia, and progressive dementia; as well as a relatively mild somatic pathology. Death usually occurs during the first or second decade of life. To date, only substrate reduction therapy with miglustat is approved for the treatment of NPC2, with limited efficacy. Thus, an efficient therapy for the treatment of NPC2 disease represents a highly unmet medical need. In vivo gene therapy with adeno-associated vectors (AAV) offers the possibility of lifetime treatment following a single administration. Therefore, the present work was focused on the development of a new gene therapy approach based on the delivery of AAV serotype 9 (AAV9) into the cerebrospinal fluid (CSF) for the treatment of the NPC2 disease. To this aim, the first part of this work consisted in the characterization of a new mouse model of the disease, generated by targeted disruption of the Npc2 gene, which recapitulated the main hallmarks of the disease. The second part of this work consisted in the assessment of efficacy of a single intra-CSF delivery of AAV9-Npc2 vectors to counteract NPC2 pathology in Npc2-/- mice treated at 18 or 30 days of age, when they already presented an established pathology. AAV9-mediated Npc2 gene transfer led to a significant decrease in unesterified cholesterol storage and lysosomal pathology in the central nervous system. Consequently, an increase of myelination, as well as a reduction in neurodegeneration and neuroinflammation was achieved. Moreover, after AAV9-Npc2 delivery into the CSF, vectors also transduced the liver, providing a peripheral source of the therapeutic protein that corrected cholesterol storage and lysosomal pathology in visceral organs of treated mice. Finally, AAV9-Npc2 treatment also resulted in normalization of locomotor deficits, improved body weight and considerably prolonged the survival of treated Npc2-/- mice. Altogether, the results obtained in the present work supports the clinical translation of the intra-CSF AAV9-Npc2 gene therapy for the treatment of NPC2 disease.
Universitat Autònoma de Barcelona. Programa de Doctorat en Bioquímica, Biologia Molecular i Biomedicina
Rodríguez, Aguilar Ester. "Caracterització i anàlisi del potencial terapèutic de l’Adenovirus 5/40 quimèric de tropisme específic intestinal com a vector de teràpia gènica per al tractament de la malaltia inflamatòria intestinal." Doctoral thesis, Universitat Autònoma de Barcelona, 2015. http://hdl.handle.net/10803/377475.
Full textInflammatory Bowel Disease (IBD) comprises a group of diseases (such as CD and UC) characterized by a chronic inflammation of the digestive tract. The IBD requires a personalized medical and pharmacological approach, and although currently there are different therapies to improve the quality of life of patients, none of these has healed the disease. Gene therapy is one of the most promising biomedical disciplines for the treatment of genetic diseases, but also for other diseases in which the pathology is not determined by a single gene, but there is an alteration of the homeostasis of the system, such as cancer, cardiovascular disease and neurological diseases. Therefore, gene therapy seems a promising alternative to administer local therapy in the intestine for IBD. Within the family of adenovirus vectors, the most used vectors in gene therapy, there is subgroup F adenovirus (Ad40 and Ad41) with intestinal tropism. These adenoviruses are characterized by the presence of two different size fibers and its poor growth in vitro. By pseudotyping technique, you can replace the genes encoding the Ad5 fiber protein, one of the most used adenoviral vector, for the genes encoding the protein of Ad40 short fiber. Thus, besides changing the natural tropism of Ad5 tropism by the new fiber, it improves production and simplify the cloning of therapeutic genes. Therefore, we decided to analyse and characterize the therapeutic potential of chimeric adenovirus 5/40 specific intestinal tropism for future use as gene therapy vectors for Inflammatory Bowel Disease. In the first part of this work, we focused on characterizing the chimeric vector to determine the real possibilities to become gene therapy vectors. Firstly, we optimize the protocol for the vectors production, increasing the efficiency from 2-4 times to 20 and 25 times per cycle, and we studied what new features the Ad40 short fiber transferred to Ad5 particles. In the second part, we evaluate the ability of chimeric vectors as gene therapy vectors in the intestine. We found that the presence of fiber increased vectors resistance to acidic pH (but not to proteases) and we studied the biodistribution and biosafety of these vectors in mice by three different routes: oral, rectal and intravenous by quantifying the expression of β-gal luminometry and by histology and immunohistochemistry analysis of adenoviral transfection. The results showed that the chimeric adenovirus administered by rectal route were more efficient than Ad5, transfecting intestinal epithelial cells in villi and crypts, and enteroendocrine cells and local macrophages in intestinal mucosa. After evaluating the ability of chimeric adenovirus to infect the intestines of healthy mice, we proceeded to assess their infectivity capacity in a MMI animal model of MMI, choosing the model of DSS-induced colitis mice. In the third part of this work, we generate new chimeric vectors that allow easy and fast cloning of any therapeutic gene. In summary, this work shows that the short fiber of the Ad40 pseudotipying transferred many of the unique characteristics of enteric adenovirus, and it proposes an optimized protocol for production levels equivalent to the Ad5. Our results also suggest that the chimeric adenovirus F/40S is an excellent candidate as a gene therapy vector for for the local administration of therapeutic genes in the gut
Vila, Julià Ferran. "Avenços en la teràpia gènica per al MNGIE amb vectors adenoassociats: validació en un model millorat de la malaltia i optimització de seqüència del gen terapèutic." Doctoral thesis, Universitat Autònoma de Barcelona, 2021. http://hdl.handle.net/10803/671813.
Full textEl MNGIE (Mitochondrial NeuroGastroIntestinal Encephalomyopathy) es una enfermedad mitocondrial de herencia autosómica recesiva causada por mutaciones en el gen TYMP, que codifica la enzima timidina fosforilasa (TP). La TP cataliza la degradación de timidina (dThd) y desoxiuridina (dUrd), y su ausencia en pacientes causa la acumulación sistémica de estos metabolitos, tóxica para la función mitocondrial. Hoy en día, las únicas terapias efectives son el trasplante de células madre hematopoyéticas y el trasplante de hígado. No obstante, los trasplantes están limitados por la necesidad de un donante compatible y tienen una tasa de mortalidad y morbididad asociada especialmente elevada en pacientes de MNGIE. Para superar estos inconvenientes, hace tiempo propusimos la terapia génica mediante vectores adenoasociados (rAAV) dirigidos al hígado como alternativa terapéutica, y demostramos su viabilidad en el modelo animal de MNGIE, el ratón doble knockout (dKO) Tymp-/-Upp1-/-. Pero este modelo solo presenta fenotipo bioquímico, de forma que solo pudimos demostrar la corrección de este fenotipo. En 2014 se describió que aumentando el desequilibrio metabólico mediante la administración oral de dThd y dUrd al modelo dKO durante toda su vida provocaba la aparición de algunos rasgos que reproducían la sintomatología clínica de los pacientes. En esta tesis hemos estudiado el uso de la terapia génica en este modelo mejorado, mediante el tratamiento con tres rAAVs que expresan la secuencia codificante de TYMP bajo el control de tres promotores hepáticos a distintas dosis. El tratamiento con rAAV incrementó la actividad TP hepática y disminuyo la concentración sistémica de nucleósidos de los ratones dKO, (sin tratamiento eren 30 veces superiores a los valores normales). A nivel fenotípico, en la mayoría de los ratones, el tratamiento también previno el aumento del volumen de los ventrículos cerebrales y el deterioro motor observados en los ratones no tratados. Cuando comparamos los tres vectores utilitzados, el rAAV con el promotor de la alfa-1-antitripsina (AAT) fue el más eficaz. Estos resultados confirman que la terapia génica por rAAV dirigida al hígado restaura la homeóstasis bioquímica y demuestran la prevención de la aparición del fenotipo clínico del modelo animal mejorado de MNGIE. Otro aspecto importante para la translación de la terapia a la práctica clínica es optimizar el vector para reducir la dosis efectiva. En este sentido, hemos testado dos aproximaciones: la modificación de la secuencia codificante (ADNc) del gen según la frecuencia de uso de codón para aumentar su expresión, y la eliminación de los dinucleótidos CpG del ADNc del gen para reducir la immunogenicidad del vector. Diseñamos cuatro secuencies optimizadas del ADNc de TYMP utilitzando 4 algoritmos diferentes. Generamos vectores lentivirales para transducir 4 líneas celulares humanas y determinar la eficiencia de expresión de cada secuencia comparada con la secuencia natural, teniendo en cuenta el grado de actividad TP, el número de copias del vector y los niveles relativos de ARNm. De todos los experimentos, solo una secuencia optimizada mejoró el grado de expresión de TYMP comparado con el de la secuencia natural, en la línea celular hepática Huh7. En cuanto a la reducción de la immunogenicidad del vector, eliminamos los dinucleótidos CpG de las secuencias diseñadas y analizamos el nivel de expresión de TYMP. Solo la secuencia natural sin dinucleótidos CpG se acercó a la expresión de la secuencia natural. Aunque se observa una reducción de expresión aproximada del 20%, se compensa con la ventaja que aporta en términos de reducción de la respuesta inmunológica de cara al uso clínico del vector. En conclusión, entre las opciones testadas, recomendamos el rAAV que contiene el ADNc natural de TYMP sin dinucleótidos CpG bajo el control del promotor AAT para un uso eventual en la práctica clínica.
MNGIE (Mitochondrial NeuroGastroIntestinal Encephalomyopathy) is an autosomal recessive disease caused by mutations in TYMP, which encodes for the enzyme thymidine phosphorylase (TP). TP catalyses the first step of the catabolism of the nucleosides thymidine (dThd) and deoxyuridine (dUrd). The lack of TP activity causes systemic nucleoside accumulation which is toxic for mitochondrial function. Nowadays, the only available therapies for MNGIE are allogeneic hematopoetic stem cell transplantation or liver transplantation. However, these treatments are limited by the need of a compatible donor and are associated to high mortality and morbidity rates in MNGIE patients. To overcome these limitations, our laboratory proposed adenoassociated virus (rAAV) mediated gene therapy targeted to the liver for MNGIE and demonstrated its feasibility in the Tymp-/- Upp1-/- (dKO) mouse model of the disease. However, dKO mice show biochemical phenotype only, therefore we could only demonstrate the efficacy of our approach at the biochemical level. In 2014 it was reported that increasing the biochemical imbalances of the dKO model by chronic oral administration of dThd and dUrd to dKO mice over their entire life induced some features that recapitulate part of the clinical manifestations observed in patients. In this thesis we have studied the effect of gene therapy in this enhanced model, by treating the animals with three rAAVs expressing the human TYMP coding sequence under the control of three different liver-specific promoters. rAAV treatment increased liver TP activity and limited the systemic nucleoside concentration present in the dKO enhanced model, which was 30-fold higher as compared with non-exposed wild-type mice. AAV-treatment also prevented, in most animals, the enlarged brain ventricles and the motor impairment observed in the exposed dKO mice. When we compared the three promoters tested, the rAAV containing the AAT promoter showed the best efficacy. These results confirm that AAV-mediated gene therapy restores the biochemical homeostasis and demonstrate that this treatment prevents the clinical phenotype developed by the enhanced murine model of MNGIE. Another key point for the clinical translation of gene therapy is the optimization of the therapeutic gene expression to reduce the vector dose. For this reason, we tested two approaches: modification of the coding sequence of TYMP based on the codon use frequency to increase the expression of the gene, and CpG dinucleotide removal from the coding sequence to reduce immunogenicity of the vector. We designed four different codon-optimized sequences of the TYMP coding sequence, using four different algorithms. We cloned each optimized sequence in a lentiviral vector and transduced 4 different human cell lines. We analysed the degree of expression achieved with each sequence, as compared with the non-optimized wild-type TYMP sequence through evaluation of TP activity, vector copy number, and mRNA levels in the cell lines. Among all the sequences studied, only one optimized sequence resulted in higher TP activity when expressed per vector copy number in the hepatic cell line Huh7. To reduce the immunogenicity of the vector we removed the CpG dinucleotides from the wild-type coding TYMP sequence and two codon-optimized TYMP sequences. The wild-type sequence without CpG was the only one showing expression levels similar to those of the wild-type sequence. Even though the CpG free sequence shows a reduced expression of about 20% of that observed in the wild-type sequence, it is compensated by the advantages associated to the absence of CpG sites in terms of reduction of the immunological response when considering the vector for clinical use. In conclusion, among the different options tested, we recommend the rAAV vector containing the wild-type coding TYMP CpG-free sequence under the control of the AAT promoter for its use in the clinical practice.
Universitat Autònoma de Barcelona. Programa de Doctorat en Bioquímica, Biologia Molecular i Biomedicina
Sargent, Dorian. "Étude moléculaire de la propagation de l’agrégation de l’alpha-synucléine dans un modèle transgénique de la maladie de Parkinson : impact de la surexpression de la beta-synucléine à l’aide de vecteurs viraux adéno-associés (AAV)." Thesis, Lyon, 2018. http://www.theses.fr/2018LYSE1071.
Full textThe aggregation of α-synuclein (α-syn) is central in the pathological process observed in Parkinson’s disease or multiple system atrophy. Β synuclein (β-syn) shares some similarities with α-syn but may have a neuroprotective effect by inhibiting the aggregation of α-syn. The goal of this thesis was to develop and to test a therapeutical strategy potentially able to inhibit the aggregation of α-syn by expressing human β-syn using adeno-associated vectors (AAV). First, we characterized a model of synucleinopathy, the M83 transgenic mouse. These mice express the human A53T mutated α-syn and develop a severe disease associated with the aggregation of α-syn. Our results confirm the ability of the aggregation of α-syn to spread through the central nervous system following a peripheral injection of aggregates of α-syn. Furthermore, they show that the accumulation of pathological α-syn in sick M83 mice depends on the inoculum used to accelerate their disease and the expression level of human A53T mutated α-syn in these mice. Gene therapy trials realized in the M83 model suggest that, regardless the strategy used to inject AAV vector, β syn did not have a protective effect against the aggregation of α-syn. A lasting expression of the human β-syn protein was however detected in sick M83 mice injected with AAV vector carrying human β-syn gene. Nevertheless, β-syn could have aggregated in our specific experimental conditions, as this has already been described, which might explain the absence of protective effect detected
Fol, Romain. "Conséquences de la surexpression des formes solubles de l’APP dans les mécanismes de mémoire : application à la maladie d'Alzheimer." Thesis, Sorbonne Paris Cité, 2016. http://www.theses.fr/2016USPCB047/document.
Full textOne of the main characteristic of Alzheimer’s Disease (AD) is the intracerebral accumulation of the neurotoxic Amyloid β peptide (Aβ) either as oligomeric or aggregated forms known as the amyloid plaques. This peptide is produced via the Amyloid Precursor Protein (APP) processing following the amyloidogenic pathway, pathological pathway overactivated in AD. Most of the research performed during the last 25 years focused on pathogenic consequences of this dysregulation, deprioritizing the understanding of the APP physiological functions. Nonetheless, numerous studies show that these physiological functions might be mediated via APP soluble forms (APPs). In the physiological APP processing pathway, the non-amyloidogenic pathway, APP is cleaved by the α secretase, releasing the APPsα which display neuroprotective and synaptotrophic properties, essential for brain normal functions. In the context of AD, the amyloidogenic pathway overactivation leads to APPsβ overproduction at the expense of APPsα. Therefore, AD harmful consequences could be due to the decrease of APPsα concentration associated with an increase of APPsβ. My thesis project aimed to characterize mnemonic and functional properties following the overexpression of these two soluble forms of APP and their therapeutic potential in AD. We firstly overexpressed APPsα in hippocampal neurons of APP/PS1ΔE9 mice, animal model of AD, which display cognitive and synaptic deficits. The continual expression of APPsα, mediated via AAV viruses, enabled restoration of spatial memory, long-term potentiation and dendritic spines density in the hippocampus. This phenotypic rescue was accompanied with the decrease of both Aβ levels and amyloid plaques. This might be due to the activation of microglia, cell type able to internalize and degrade Aβ. In a second hand, I studied the involvement of APPsβ in AD, which remains poorly known. Its overexpression in APP/PS1ΔE9 did not induce neither LTP nor spatial memory restoration. However, APPsβ injection lead to the decrease of Aβ levels without reducing amyloid plaques. This default might be due to the lack of microglial activation. In conclusion, my thesis work show that, unlike APPsβ, APPsα overexpression might overcome the AD inevitable evolution by reducing synaptic and memory alterations, typical of AD. These results reinforce a new way of treatment to cope with AD progression. The use of APPsα as therapeutic agent might be an important tool for future AD therapies
Perdomini, Morgane. "Développement d'une thérapie génique dans le modèle murin cardiaque de l'ataxie de Friedreich en utilisant le vecteur adéno-associé rAAVrh10." Thesis, Strasbourg, 2013. http://www.theses.fr/2013STRAJ105.
Full textFriedreich ataxia (FRDA) is a mitochondrial disease with neurodegeneration, hypertrophic cardiomyopathy and diabetes. FRDA is caused by reduced level of frataxine (FXN), an essential mitochondrial protein involved in iron-sulfur cluster biogenesis and mitochondrial homeostasis. Cardiac failure is the most common cause of mortality in FRDA. To date, no treatment exists for FRDA cardiomyopathy. During my PhD, we showed that an adeno-associated vector (AAV) rh10 expressing human FXN injected intravenously not only prevented the onset of the cardiac disease in a faithful FRDA cardiac mouse model, but also, when administered in animals with cardiac failure, reversed rapidly and completely cardiac remodeling and insufficiency. Our results demonstrate the capacity of defective cardiomyocytes with severe energy failure and ultrastructure disorganization to be rapidly corrected and remodeled by gene therapy. Thus, we showed that gene therapy may be a relevant therapeutical approach for FRDA
De, Montigny Charline. "Compréhension de la neurophysiopathologie de l'ataxie de Friedreich et développement d'une approche de thérapie génique dans un nouveau modèle murin." Thesis, Strasbourg, 2018. http://www.theses.fr/2018STRAJ051/document.
Full textFriedreich ataxia (FA) is a rare mitochondrial disease characterized by sensory and spinocerebellar ataxia, hypertrophic cardiomyopathy, and diabetes, for which there is no treatment. FA is caused by reduced levels of frataxin (FXN), an essential mitochondrial protein involved in the biosynthesis of Fe-S clusters. To date, FA precise neuropathophysiology is not identified and despite significant progresses in recent the years, there was no good model to develop therapeutic approaches in order to stop or reverse the sensory ataxia associated to the FA. Thus, we have generated a new neuronal mouse model that recapitulates the sensory ataxia and the neuropathy associated to FXN deficiency. Several molecular mechanisms dysregulated in the absence of FXN in the proprioceptive neurons, primarily affected in FA, were identified. Furthermore, we have demonstrated the efficacy of a gene therapy (GT) approach, based on the delivery of adeno-associated vectors (AAV) expressing the human FXN, to reverse the sensitive neuropathy, thus establishing the preclinical proof of concept for the potential of GT in treating FA sensitive neuropathy
Monlleó, Mas Ester. "Aminocyclitol and iminosugar derivatives related to Gauche disease." Doctoral thesis, Universitat de Barcelona, 2016. http://hdl.handle.net/10803/400871.
Full textLa malaltia de Gaucher és una de les malalties d’emmagatzematge lisosomal més freqüent. Una mutació al gen gba1 provoca un incorrecte plegament de l’enzim Glucocerebrosidasa (GCase) que impedeix el seu transport del reticle endoplasmàtic (ER) fins al lisosoma, on té lloc la hidròlisi de la glucosilceramida (GluCer). Això provoca una acumulació d’aquest substrat, originant els símptomes clínics. En aquesta tesi s’ha sintetitzat una petita col·lecció de compostos per tal estudiar la influència del pKa de diferents inhibidors en la seva potencial activitat chaperona, és a dir, estudiar la seva capacitat de unir-se a la GCase mal plegada del reticle per facilitar-ne el correcte plegament i permetre el seu transport cap a lisosoma. Per aquest motiu, es buscaven compostos que presentessin una alta afinitat per la GCase en les condicions neutres del reticle endoplasmàtic, però que tinguessin baixa afinitat per aquest enzim a pHs lleugerament àcids com el del lisosoma (pH 5.2), per tal que no bloquegés l’activitat hidrolasa d’aquest enzim al lisosoma. Desprès d’analitzar els compostos amb diferents assajos, es va posar de manifest la importància de les condicions d’assaig a l’hora d’estudiar la inhibició d’un enzim, ja que diferents assajos poden conduir a conclusions diferents. Malgrat que no es va poder establir cap correlació directa entre el pKa dels inhibidors i la diferència d’activitat segons el pH de l’assaig, es va descobrir un dels derivats de DNJ amb millor potència inhibitòria de imiglucerasa (Gcase recombinant) descrit fins el moment. Per altra banda, es va analitzar la capacitat d’inhibició de GCS dels compostos sintetitzats, descobrint-se alguns inhibidors d’aquest enzim més potents que la NB-DNJ, compost que s’utilitza actualment com a inhibidor de GCS per al tractament de la malaltia de Gaucher, i possible comportament dual (inhibidors de GCS i chaperones per GCase).
Lainas, Panagiotis. "Transplantation d'hépatocytes génétiquement modifiés : régénération hépatique et moyens d'amélioration de la prise de greffe hépatocytaire." Phd thesis, Université Paris Sud - Paris XI, 2012. http://tel.archives-ouvertes.fr/tel-00746653.
Full textKalaani, Joanna. "Molecular guidance of dopaminergic cells transplanted in a mouse model of Parkinson's disease." Thesis, Poitiers, 2016. http://www.theses.fr/2016POIT2252/document.
Full textParkinson's disease (PD) is characterised by the degeneration of the dopaminergic nigrostriatal pathway. Cell therapy using intranigral transplantation of foetal ventral mesencephalon (VM) cells in a mouse model of PD results in anatomical and functional reconstruction of the pathway. This suggests a role for axon guidance molecules (GMs) in reconnecting transplanted cells to their striatal target. To test this hypothesis, we studied the expression of axon GMs in the intact adult brain, on cells used for transplantation and in a mouse model of PD after cell therapy. In the intact brain, we showed that GMs as semaphorin7A (Sema7A) and Sema3A and their corresponding receptors, plexinC1 and neuropilin1, retain an expression at the protein level, therefore showing a possible role for these guidance cues in the adult brain. Moreover, using microarray, we studied GM receptor expression profiles in two types of cells used for transplantation and exhibiting different functional ameliorations. Robo2, neuropilin1, neuropilin2, EphA5 and DCC receptors showed differential expression between the two cellular populations, indicating their possible contribution to the different functional outcomes observed. In the lesioned mouse brain, we observed, using RT-qPCR, variations of mRNA expression of these axon GMs after intranigral transplantation of foetal VM derived cells, thus suggesting the implication of Sema3A, Sema3F, and Sema7A in the reconstruction of the pathway. Overall, this work highlights particular importance of semaphorins in the nigrostriatal pathway reconstruction. Integrating these cues in transplantation procedures can possibly optimize cell therapy for PD patients
Katrib, Marilyn School of Biotechnology & Biomolecular Science UNSW. "The malarial carbamoyl phosphate synthetase II gene as a target for DNAzyme therapy." 2007. http://handle.unsw.edu.au/1959.4/40660.
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