No description available.

La myéline est une gaine formée par l’enroulement de la membrane plasmique de la cellule de Schwann autour de l’axone dans le nerf périphérique. Lorsque cette gaine est détruite, on parle de démyélinisation, cela provoque de nombreuses maladies, dont les maladies de Charcot Marie Tooth (CMT) de type 1. Les maladies CMT sont héréditaires et atteignent le système nerveux périphérique. Les symptômes communs incluent : une faiblesse musculaire, une démarche maladroite, des troubles de l’équilibre et des pieds très cambrés ou très plats. Le type le plus fréquent est la forme autosomique dominante CMT1A.Une duplication du bras court du chromosome 17 contenant le gène PMP22 (Peripheral Myelin Protein 22) induit la CMT1A. La PMP22, une petite protéine exprimée par les cellules de Schwann, est donc en excès et entraine une démyélinisation. Il existe un modèle de rats transgéniques PMP22 (ou rats CMT1A) mimant cette pathologie humaine. Les rats CMT1A surexpriment la pmp22 de souris de façon hétérozygote. Jusqu’à présent, aucun remède n’existe pour les maladies CMT. Un des traitements envisageables est la thérapie génique. Le but de mon projet de thèse était d’étudier la validité et l'efficacité de la thérapie génique chez les rats CMT1A. La stratégie consiste à réduire la surexpression de la protéine PMP22 chez le rat CMT1A à l’aide d’ARNsh anti-PMP22. Pour ne pas être détruits par l’organisme et maintenir une expression longue, ces ARN sh-PMP22 sont transférés chez le rat grâce à des vecteurs viraux dérivés de virus adéno-associés, ou AAV (pour adeno-associated virus). Nous avons donc injecté un des différents sérotypes d'AAV,l'AAV9 exprimant les ARN sh-PMP22 de souris ainsi que la GFP comme marqueur des cellules infectées dans les nerfs sciatiques de rats CMT1A à l’âge de 6 jours ou 7 jours.Nous avons d’abord confirmé que les virus thérapeutiques infectaient une très large proportion de cellules de Schwann dans le nerf sciatique de rat CMT1A et ensuite que l’infection de ces cellules par les virus exprimant les ARN sh-PMP22 induisait une diminution significative de l’expression de la protéine PMP22. L'analyse du phénotype moteur des rats CMT1A traités avec les AAV9 exprimant les ARN sh-PMP22 montre que les rats CMT1A traités ne développent pas la maladie observée dans les contrôles. Également, les rats CMT1A présentent une hypoalgésie, un phénotype qui n’apparait pas dans les CMT1A traités avec les vecteurs thérapeutiques. Le traitement par thérapie génique empêche la réduction de la vitesse de conduction nerveuse observé dans les rats malades. Concernant la biodistribution des virus, 2,5 mois après le traitement, en dehors des nerfs sciatiques ou les virus ont été injectés, le virus était présent dans les muscles qui entourent le nerf et aussi dans quelques ganglion dorsaux. Pour la réponse immunitaire,les rats injectés, à seulement 2 exceptions près, n’ont pas développé de facteurs neutralisants anti-AAV9. Cette thérapie génique pourrait être utilisée dans les essais cliniques.Avant de passer aux études cliniques pour le traitement de la maladie CMT1A à l’aide d’AAV9 exprimant des ARN sh-PMP22 humain, la dose d’expression de ce ARN sh-PMP22 doit être très soigneusement déterminée car si la PMP22 est trop réduite, une autre maladie peut se développer, la neuropathie héréditaire avec hypersensibilité à la pression. Il est aussi important d’avoir un outil bien adapté qui permet d’évaluer l’efficacité du traitement. Aucun existant n’est assez fiable pour mesurer la myéline du nerf périphérique. Pour remédier à ce manque, nous avons testé la technique d'imagerie Coherent Anti-stokes Raman Scattering (CARS) en caractérisant avec succès les défauts de la myéline. Par conséquent, le CARS est une technique prometteuse permettant d’évaluer l’avancement des maladies de la myéline et l’efficacité de nouvelles thérapies pour les neuropathies périphériques démyélinisantes. / Myelin, 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.

Gene therapy is a promising treatment option for cancer. Ideally, a therapeutic gene is delivered specifically into tumor cells sparing the neighboring normal cells. For this purpose gene delivery vectors are designed that can recognize structures, which are exclusively expressed on tumor cells (i.e. the tumor-associated antigens -TAA-). Retroviral vectors are commonly used for gene therapy by modifying the envelope protein responsible for the recognition of the target cell. The Vesicular Stomatitis Virus G protein (VSV-G) is a well-liked choice for pseudotyping the retroviral vectors since it confers on the viral particle stability to allow concentration to high titers necessary for the clinical applications. However, the main drawback of VSV-G, the ubiquitously expressed receptor and thus the broad target range, hinders the use of this protein for targeted gene therapy. In this thesis, we aimed to modify the VSV-G for targeted gene therapy against Prostate Stem Cell Antigen (PSCA) -expressing tumors. Therefore we followed two approaches. The first approach comprised of the fusion of a single-chain antibody fragment against PSCA to the N-terminus of VSV-G. In the second approach the VSV-G was modified by insertion of a small epitope. We could demonstrate that two positions in the N-terminal region of VSV-G protein permit insertion of a ten amino acid long epitope. These mutant VSV-G proteins were successfully assembled into retroviral particles. We demonstrated that the mutant retroviral particles can be used for targeting to PSCA-positive cells using nanobeads. The nanobeads were chemically coupled to antibodies against the epitope in the VSV-G protein and PSCA on the tumor cell. These bispecific nanobeads allowed the recruitment of mutant retroviral particles to the PSCApositive cells. Our results point out the potential of these mutant retroviral particles in targeted gene delivery. Further studies will be necessary to assess the efficiency of in vivo targeted gene therapy using these mutant retroviral particles.

Thesis (MSc (Genetics))--University of Stellenbosch, 2011. / Includes bibliography / ENGLISH ABSTRACT: This study investigated the use of antimicrobial peptides (AMPs) as possible source of resistance against a range of pathogens in grapevine. Whilst the ultimate aim would be to express AMPs in grapevine, the development of transgenic grapevine is time consuming and therefore pre-screening of potential AMPs is necessary. These small molecules, of less than 50 amino acids in length, are expressed by almost all organisms as part of their non-specific defence system. In vitro pre-screening of AMP activity is valuable but is limited since the activity on artificial media may differ from the AMP activity in planta. These tests are also restricted to pathogens which can be cultured in vitro. These limitations can be overcome by using transient expression systems to determine the in planta activity of AMPs against pathogens of interest. In this study transient systems were used to express AMPs in developed plant tissue to test their efficacy against grapevine pathogens such as Agrobacterium vitis, Xylophilus ampelinus and aster yellows phytoplasma. Aster yellows phytoplasma, which was recently discovered in local vineyards, is known to cause extensive damage and therefore pose a great threat to the South African grapevine industry. To study the in planta effect of AMPs against the abovementioned pathogens, transient expression vectors were constructed expressing either of the AMPs D4E1 or Vv-AMP1. D4E1 is a synthetically designed AMP known to be active against bacteria and fungi, while Vv-AMP1, isolated from grapevine berries, has already shown activity against fungi. In a transient approach in grapevine, the expression of foreign genes from viral and non-viral vectors was confirmed by expression of the marker genes β-glucuronidase and Green Fluorescent Protein, while tissue-printing immunoassays confirmed viral replication and systemic spread in Nicotiana benthamiana. The viral vectors were based on the phloem-limited virus grapevine virus A. Only Agrobacterium-mediated 35S transient expression vectors were used for AMP in planta activity screening since the viral-mediated expression in grapevine was insufficient for screening against A. vitis and X. ampelinus as it was restricted to phloem tissues after whole-leaf infiltration. No phytoplasma-infected material could be established and as a result AMP activity screening was only performed against the A. vitis and X. ampelinus. Quantification of the bacteria was performed by qPCR. Vv-AMP1 did not show activity against either of the two bacteria in planta while D4E1 was found to be active against both. The observed in planta activity of D4E1 correlated with the in vitro activity as measured in an AMP plate bioassay. In contrast to in vitro screenings, the in planta AMP activity screening might give a more accurate representation of the potential antimicrobial activity of the peptide in a transgenic plant environment. This study proved that transient expression systems can be used as a pre-screening method of AMP activity in planta against grapevine pathogens, allowing the screening of various AMPs in a relatively short period of time before committing to transgenic grapevine development. / AFRIKAANSE OPSOMMING: Hierdie studie het die gebruik van antimikrobiese peptiede (AMPe) as 'n moontlik bron van weerstand teen 'n reeks van patogene in wingerd ondersoek. Alhoewel die uiteindelike doel sal wees om AMPe uit te druk in wingerd, is transgeniese wingerd ontwikkeling tydrowend en daarom is vooraf evaluering van potensiële AMPe nodig. Hierdie klein molekules, van minder as 50 aminosure in lengte, word uitgedruk deur amper alle organismes as deel van hul nie-spesifieke verdedigingsisteem. In vitro vooraf evaluering van AMP aktiwiteit is van waarde, maar is beperk aangesien die aktiwiteit op kunsmatige media mag verskil van die AMP-aktiwiteit in planta. Hierdie toetse is ook beperk tot patogene wat in vitro gekweek kan word. Hierdie beperkinge kan oorkom word deur gebruik te maak van tydelike uitdrukkingsisteme om die in planta aktiwiteit van AMPe te bepaal teen patogene van belang. In hierdie studie is tydelike uitdrukkingsisteme gebruik om AMPe uit te druk in ontwikkelde plantweefsel om hul effektiwiteite te toets teen wingerdpatogene soos Agrobacterium vitis, Xylophilus ampelinus en aster yellows fitoplasma. Aster yellows fitoplasmas, wat onlangs in plaaslike wingerde ontdek is, is bekend vir die uitgebreide skade wat hul aanrig en hou daarom 'n groot bedreiging in vir die Suid-Afrikaanse wingerd industrie. Om die in planta effek van AMPe teen die bogenoemde patogene te bestudeer is tydelike uitdrukkingsvektore ontwikkel wat die AMPe D4E1 of Vv-AMP1 uitdruk. D4E1 is 'n sinteties-ontwerpte AMP wat aktief is teen bakterieë en fungi, terwyl Vv-AMP1, wat uit druiwekorrels geïsoleer is, alreeds aktiwiteit teen fungi getoon het. In 'n tydelike uitdrukkingsbenadering in wingerd is die uitdrukking van transgene, vanaf virus of nie-virus gebaseerde vektore, bevestig deur die uitdrukking van die merker gene β-glukuronidase en die Groen Fluoresserende Proteïen, terwyl weefsel afdrukkings-immunotoetse virus replisering en sistemiese beweging in Nicotiana benthamiana bevestig het. Die virusvektore was gebaseer op die floëem-beperkte virus, wingerdvirus A. Slegs Agrobacterium-bemiddelde 35S tydelike uitdrukkingsvektore is gebruik om die AMP in planta aktiwiteit te bepaal aangesien die virus-bemiddelde uitdrukking in wingerd onvoldoende was vir evaluering teen A. vitis en X. ampelinus weens die beperking tot die floëem weefsel na infiltrering van die totale blaar. Geen fitoplasma geïnfekteerde materiaal kon gevestig word nie, en daarom is AMP aktiwiteitsevaluering slegs teen A. vitis en X. ampelinus uitgevoer. Kwantifisering van die bakterieë is deur middel van qPCR uitgevoer. Vv-AMP1 het geen aktiwiteit getoon teen enige van die bakterieë in planta nie, terwyl D4E1 aktief was teen beide. Die waargenome in planta aktiwiteit van D4E1 het ooreengestem met die in vitro aktiwiteit soos bepaal deur 'n AMP plaat bio-toets. In kontras tot in vitro evaluering kan die in planta AMP-aktiwiteit evaluering 'n meer akkurate voorspelling bied van die potensiële antimikrobiese aktiwiteite van die peptied in 'n transgeniese plant omgewing. Hierdie studie het bewys dat tydelike uitdrukkingsisteme gebruik kan word as 'n voorafgaande evalueringsmetode vir AMP in planta aktiwiteit teen wingerdpatogene, wat die evaluering van 'n verskeidenheid AMPe in 'n relatiewe kort tydperk toelaat voor verbintenis tot die ontwikkeling van transgeniese wingerd.

Made available in DSpace on 2014-12-17T14:05:18Z (GMT). No. of bitstreams: 1 LourenaMF_TESE_Capa_ate_pag79.pdf: 4572586 bytes, checksum: 2630047160ff526cc964f166fb86ab33 (MD5) Previous issue date: 2011-03-14 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Gene therapy is one of the major challenges of the post-genomic research and it is based on the transfer of genetic material into a cell, tissue or organ in order to cure or improve the patient s clinical status. In general, gene therapy consists in the insertion of functional genes aiming substitute, complement or inhibit defective genes. The achievement of a foreigner DNA expression into a population of cells requires its transfer to the target. Therefore, a key issue is to create systems, vectors, able to transfer and protect the DNA until it reaches the target. The disadvantages related to the use of viral vectors have encouraged efforts to develop emulsions as non-viral vectors. In fact, they are easy to produce, present suitable stability and enable transfection. The aim of this work was to evaluate two different non-viral vectors, cationic liposomes and nanoemulsions, and the possibility of their use in gene therapy. For the two systems, cationic lipids and helper lipids were used. Nanoemulsions were prepared using sonication method and were composed of Captex? 355; Tween? 80; Spam? 80; cationic lipid, Stearylamine (SA) or 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) and water (Milli-Q?). These systems were characterized by average droplet size, Polidispersion Index (PI) and Zeta Potential. The stability of the systems; as well as the DNA compaction capacity; their cytotoxicity and the cytotoxicity of the isolated components; and their transfection capacity; were also evaluated. Liposomes were made by hydration film method and were composed of DOTAP; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), containing or not Rhodaminephosphatidylethanolamine (PE- Rhodamine) and the conjugate Hyaluronic Acid DOPE (HA-DOPE). These systems were also characterized as nanoemulsions. Stability of the systems and the influence of time, size of plasmid and presence or absence of endotoxin in the formation of lipoplexes were also analyzed. Besides, the ophthalmic biodistribution of PE-Rhodamine containing liposomes was studied after intravitreal injection. The obtained results show that these systems are promising non-viral vector for further utilization in gene therapy and that this field seems to be very important in the clinical practice in this century. However, from the possibility to the practice, there is still a long way / A terapia g?nica ? um dos maiores desafios propostos pela pesquisa p?s-gen?mica e se baseia na transfer?ncia de material gen?tico a uma c?lula, tecido ou ?rg?o com o intuito de curar ou melhorar o estado cl?nico do paciente. Em sua forma mais simples, a terapia g?nica consiste na inser??o de genes funcionais em c?lulas com genes defeituosos objetivando substituir, complementar ou inibir esses genes causadores de doen?as. Para que o DNA ex?geno seja expresso em uma popula??o celular faz-se necess?ria a sua transfer?ncia at? o local de a??o. Assim, ? necess?rio criar ve?culos, que transportem e protejam o DNA at? que este chegue a uma popula??o celular alvo. Os obst?culos encontrados com a utiliza??o de vetores virais t?m proporcionado o interesse no desenvolvimento de vetores n?o-virais, por serem f?ceis de produzir, apresentarem estabilidade control?vel e facilitarem a transfec??o g?nica. O objetivo deste trabalho foi avaliar dois diferentes vetores n?o virais, lipossomas e nanoemuls?es cati?nicos, e sua poss?vel utiliza??o na terapia g?nica. Para isso, foram utilizados lip?deos cati?nicos e co-tensoativos na produ??o dos dois sistemas. As nanoemuls?es foram produzidas pelo m?todo de sonica??o e compostas por Captex? 355; Tween? 80; Spam? 80; lip?deo cati?nico, Estearilamina (EA) ou N-[1-(2,3-Dioleoiloxi)propil]-N,N,Ntrimetilamonio metilsulfato (DOTAP); e ?gua ultra-pura (Milli-Q?). Estes sistemas foram caracterizados quanto ao tamanho m?dio de got?cula, ?ndice de polidispers?o (PI) e potencial zeta. Avaliou-se ainda a estabilidade dos sistemas e suas capacidades de compacta??o do material gen?tico. Os lipossomas foram preparados a partir do m?todo de hidrata??o do filme e compostos por DOTAP, Dioleilfosfatidiletanolamina (DOPE), na presen?a ou aus?ncia de Rodaminafosfatidiletanolamina (PE-Rodamina) e do conjugado ?cido Hialur?nico DOPE (HA-DOPE). Estes sistemas foram caracterizados da mesma forma que as nanoemuls?es e tamb?m foram avaliados estabilidade, influ?ncia do tempo, tamanho de material gen?tico e presen?a ou aus?ncia de endotoxinas na forma??o dos lipoplexos. Os resultados obtidos permitem afirmar que os sistemas s?o promissores para posterior utiliza??o na terapia g?nica e que esta ?rea promete ser uma ?rea f?rtil de pesquisa cient?fica e cl?nica por muitos anos, e provavelmente se tornar? uma pr?tica cl?nica importante neste s?culo. No entanto, da possibilidade ? pr?tica existe um longo caminho a percorrer

Les dendrimères sont une famille de macromolécules utilisées dans de nombreux domaines d’applications. Parmi ceux-ci, le domaine biomédical concentre une grande partie de l’intérêt de recherche. En effet, la structure tridimensionnelle parfaitement définie et monodispersée des dendrimères en font de parfaits candidats pour une application en médecine. Initialement utilisés en tant que mimes de protéines comme cela fût le cas lors du développement de la première famille de dendrimères, les poly(amidoamines) (PAMAM), de nombreuses études sur la capacité de transfection de ces molécules ont été réalisées, avec des résultats extrêmement encourageants. Afin d’améliorer l’efficacité et la biocompatibilité de ces vecteurs non viraux, nous avons orienté nos recherches sur le développement de nouveaux dendrimères poly(aminoesters) potentiellement biodégradables par hydrolyse enzymatique ou par variation de pH.Compte tenu des résultats précédemment obtenus au laboratoire, concernant la synthèse en solution de ces dendrimères, nous avons envisagé de les synthétiser en deux parties, à savoir un coeur central fonctionnalisé et des dendrons comportant la fonctionnalité appropriée. Notre choix s’est plus particulièrement porté sur la chimie « Click », en l’occurrence la cycloaddition 1,3-dipolaire de Huisgen entre un azoture et un alcyne catalysée par du cuivre. D’autre part, nous avons également envisagé de créer de nouveaux dendrons à l’aide de la chimie supportée. En effet, cette méthodologie de synthèse basée sur deux étapes répétitives d’addition de Michael et d’estérification, semble très prometteuse pour obtenir des dendrons de plus hautes générations. / Dendrimers are a special family of synthetic macromolecules with myriad applications, in particular biomedical implementation. The tridimensional, monodispersed and well defined structure of dendrimers give to them a unique position in medicine applications. Initially used as a mimic of proteins, poly(amidoamine) dendrimers (PAMAM) are also very efficient for nucleic acid delivery. With the aim to improve the biocompatibility and delivery efficiency of these non viral vectors, we designed and synthesized new poly(aminoester) dendrimers as potential biodegradable dendrimers sensitive to enzymatic hydrolysis or pH variations.On the basis of our previous results for the solution-phase synthesis of poly(aminoester) dendrimers, we decided to construct our dendrimers using a multi functionalized core and dendrons with complementary functions. These building units will be connected together at the end of the synthesis by a Huisgen dipolar cycloaddition through a copper-catalysed azide-alkyne cycloaddition (CuAAC) well known as « Click » reaction. In order to obtain higher generation dendrimers, we explore the supported chemistry using both soluble and solid supports. The solid-phase synthesis based on two iterative steps, Michael addition and esterification, seems to be very promising.

La maladie de Huntington (MH) est une maladie neurodégénérative causée par une extension de répétitions du codon CAG dans le gène de la Huntingtine (Htt). Cette maladie est caractérisée par la mort des neurones striataux et la présence d’agrégats de Htt mutée (mHtt). De plus, au cours de la MH, les astrocytes, qui sont essentiels au bon fonctionnement neuronal, changent d’état et deviennent réactifs. La réactivité astrocytaire est caractérisée par des changements morphologiques et transcriptomiques mais l’impact fonctionnel de cette réactivité reste peu compris.Afin d’étudier le rôle des astrocytes réactifs dans la MH, nous avons utilisé des vecteurs viraux récemment développés par notre équipe, qui induisent ou bloquent la réactivité astrocytaire in vivo en ciblant la voie JAK2-STAT3. Nous avons montré que les astrocytes réactifs diminuent le nombre et la taille des agrégats de mHtt majoritairement présents dans les neurones. Ceci est associé à l’amélioration de plusieurs altérations neuronales observées dans ces modèles. Une analyse transcriptomique réalisée sur des astrocytes réactifs révèle des changements majeurs d’expression de gènes liés aux systèmes de protéostasie. De plus, l’activité du lysosome et du protéasome est augmentée dans les astrocytes réactifs de souris modèles de la MH. Nous montrons également que les astrocytes réactifs éliminent plus efficacement leurs propres agrégats de mHtt, suggérant qu’au cours de la MH, ces cellules pourraient dégrader plus efficacement la mHtt provenant des neurones. De plus, certaines protéines chaperonnes sont induites dans les astrocytes réactifs. En particulier, la co-chaperonne DNAJB1/Hsp40 est surexprimée dans les astrocytes réactifs et est retrouvée dans les exosomes isolés à partir de striata de souris MH. Des expériences de gain et perte de fonction suggèrent que cette chaperonne est impliquée dans les effets bénéfiques des astrocytes réactifs sur l’agrégation de la mHtt et l’état des neurones. Les astrocytes réactifs pourraient donc libérer des protéines anti-agrégantes qui favorise l’élimination de la mHtt dans les neurones.Notre étude montre que les astrocytes peuvent, en devenant réactifs au cours de la MH, acquérir des propriétés bénéfiques pour les neurones et favoriser, via un dialogue complexe avec les neurones, l’élimination des agrégats de mHtt. / Huntington’s disease (HD) is a hereditary neurodegenerative disease caused by an expansion of CAG codons in the Huntingtin gene. It is characterized by the death of striatal neurons and the presence of mutant Huntingtin (mHtt) aggregates. In pathological conditions, as in HD, astrocytes change and become reactive. Astrocyte reactivity is characterized by morphological and significant transcriptomic changes. Astrocytes are essential for the proper functioning of neurons but the functional changes associated with reactivity are still unclear.To better understand the roles played by reactive astrocytes in HD, we took advantage of our recently developed viral vectors that infect selectively astrocytes in vivo and either block or induce reactivity, through manipulation of the JAK2-STAT3 pathway. We used these vectors in two complementary mouse models of HD and found that reactive astrocytes decrease the number and the size of mHtt aggregates that mainly form in neurons. Reduced mHtt aggregation was associated with improvement of neuronal alterations observed in our mouse models of HD. A genome-wide transcriptomic analysis was performed on acutely sorted reactive astrocytes and revealed an enrichment in genes linked to proteolysis. Lysosomal and proteosomal activities were also increased in reactive astrocytes in HD mice. Moreover, we show that reactive astrocytes degrade more efficiently their own mHtt aggregates, suggesting that these cells could siphon mHtt away from neurons. Alternatively, several chaperones were induced in reactive astrocytes. In particular, the co-chaperone DNAJB1/Hsp40 was upregulated in reactive astrocytes and was present in exosomal fraction from HD mouse striatum. Loss and gain of function experiments suggest that this chaperone is involved in the beneficial effects of reactive astrocytes on mHtt aggregation and neuronal status. Therefore, reactive astrocytes could release anti-aggregation proteins that could promote mHtt clearance in neurons.Overall, our data show that astrocytes, by becoming reactive in HD, develop a protective response that involves complex bidirectional signaling with neurons to reduce mHtt aggregation.

Gene therapy is a promising treatment option for cancer. Ideally, a therapeutic gene is delivered specifically into tumor cells sparing the neighboring normal cells. For this purpose gene delivery vectors are designed that can recognize structures, which are exclusively expressed on tumor cells (i.e. the tumor-associated antigens -TAA-). Retroviral vectors are commonly used for gene therapy by modifying the envelope protein responsible for the recognition of the target cell. The Vesicular Stomatitis Virus G protein (VSV-G) is a well-liked choice for pseudotyping the retroviral vectors since it confers on the viral particle stability to allow concentration to high titers necessary for the clinical applications. However, the main drawback of VSV-G, the ubiquitously expressed receptor and thus the broad target range, hinders the use of this protein for targeted gene therapy. In this thesis, we aimed to modify the VSV-G for targeted gene therapy against Prostate Stem Cell Antigen (PSCA) -expressing tumors. Therefore we followed two approaches. The first approach comprised of the fusion of a single-chain antibody fragment against PSCA to the N-terminus of VSV-G. In the second approach the VSV-G was modified by insertion of a small epitope. We could demonstrate that two positions in the N-terminal region of VSV-G protein permit insertion of a ten amino acid long epitope. These mutant VSV-G proteins were successfully assembled into retroviral particles. We demonstrated that the mutant retroviral particles can be used for targeting to PSCA-positive cells using nanobeads. The nanobeads were chemically coupled to antibodies against the epitope in the VSV-G protein and PSCA on the tumor cell. These bispecific nanobeads allowed the recruitment of mutant retroviral particles to the PSCApositive cells. Our results point out the potential of these mutant retroviral particles in targeted gene delivery. Further studies will be necessary to assess the efficiency of in vivo targeted gene therapy using these mutant retroviral particles.

Ziel der Arbeit war, zu untersuchen, ob der gezielte Einsatz gentherapeutischer Methoden zu einer Verhinderung der Abstoßung allogener Transplantate bzw. zu einer Verhinderung der Induktion des Ischämie-/Reperfusionsschadens in verschiedenen Transplantationsmodellen der Ratte beitragen kann. Dabei wurden zwei Schwerpunkte gesetzt: Zum einen wurde auf den ex-vivo Gentransfer von therapeutischen Molekülen direkt in das Transplantat mit Hilfe von rekombinanten Adenoviren fokussiert. Zum anderen wurde das Potenzial von retroviral modifizierten, allospezifischen T-Zellen als Träger therapeutischer Gene zur Verhinderung der Transplantatrejektion untersucht. Diese Habilitationsschrift umfasst dreizehn Originalartikel in internationalen Zeitschriften, sechs Übersichtsartikel (Reviews) und vier Manuskripte, die bereits zur Veröffentlichung eingereicht sind. / The aim of the research was to investigate, whether the specific use of gene therapeutic methods can play a role in the prevention of allogeneic graft rejection or in the prevention of the induction of ischemia/reperfusion damage in various rat transplantation models. Doing this there were to main focusses: First we concentrated on the ex-vivo gene transfer of therapeutic molecules directly into the graft, which was done using recombinant adenoviruses. Then we investigated the potential of retrovirally modified, allospecific T-cells as carriers for therapeutic genes for the prevention of graft rejection. This publication consists of thirteen original papers in international journals, six reviews and four manuscripts that have been submitted for publication.

Τα επισωματικά αυτο-αναπαραγώμενα συστήματα αποτελούν υποσχόμενα εναλλακτικά οχήματα γονιδιακής μεταφοράς για εφαρμογές της γονιδιακής θεραπείας. Η πρόσφατη κατανόηση της ικανότητας των αλληλουχιών S/MAR να διαμεσολαβούν την επισωματική διατήρηση γενετικών στοιχείων επέτρεψε την ανάπτυξη ενός πρότυπου κυκλικού επισωματικού φορέα που λειτουργεί χωρίς να κωδικοποιεί πρωτεΐνες ιϊκής προέλευσης. Σε αυτή τη μελέτη, διερευνήθηκε για πρώτη φορά η δυνατότητα αυτού του φορέα, pEPI-eGFP, να μεσολαβεί γονιδιακή μεταφορά σε κυτταρικές σειρές προγονικών αιμοποιητικών κυττάρων καθώς και σε πρωτογενή ανθρώπινα κύτταρα και, κυρίως, σε ανθρώπινα προγονικά αιμοποιητικά κύτταρα. Δείχνουμε ότι ο φορέας pEPI-eGFP διατηρείται επισωματικά και υποστηρίζει παρατεταμένη έκφραση του γονιδίου αναφοράς eGFP, ακόμα και χωρίς πίεση επιλογής, στην ανθρώπινη κυτταρική σειρά K562, καθώς και σε πρωτογενείς ανθρώπινους ινοβλάστες. Αντίθετα, στην κυτταρική σειρά ερυθρολευχαιμίας ποντικού MEL, η έκφραση της eGFP αποσιωπάται μέσω αποακετυλίωσης ιστονών, παρά την επισωματική διατήρηση του φορέα. Προγονικά αιμοποιητικά κύτταρα με κλωνογόνο ικανότητα, προερχόμενα από αίμα ομφάλιου λώρου, διαμολύνονται αποτελεσματικά με το φορέα μέσω ηλεκτροδιάτρησης. Ημιστερεές αποικίες προερχόμενες από διαμολυσμένα CD34+ κύτταρα διατηρούν το φορέα και εκφράζουν eGFP. Μετά από 4 εβδομάδες ο φορέας διατηρείται επισωματικά σε περίπου 1% των θυγατρικών κυττάρων. Τα αποτελέσματά μας αποδεικνύουν για πρώτη φορά ότι ένα πλασμίδιο βασιζόμενο σε μια αλληλουχία S/MAR μπορεί να λειτουργεί ως σταθερό επιίσωμα σε πρωτογενή ανθρώπινα κύτταρα και, ιδιαίτερα, σε προγονικά αιμοποιητικά κύτταρα, υποστηρίζοντας παρατεταμένη έκφραση του διαγονιδίου. Η μελέτη αυτή αναδεικνύει τη χρησιμότητα του συστήματος αυτού για τους σκοπούς της γονιδιακής θεραπείας. Παράλληλα, καταδεικνύει τους στόχους στους οποίους πρέπει να επικεντρωθεί η μελλοντική έρευνα προς την κατεύθυνση της βελτίωσής του. / Episomally maintained self-replicating systems present attractive alternative vehicles for gene therapy applications. Recent insights into the ability of chromosomal scaffold/matrix attachment regions (S/MARs) to mediate episomal maintenance of genetic elements cloned in cis allowed the development of a small circular episomal vector that functions independently of virally encoded proteins. In this study, we investigated the potential of this vector, pEPI-eGFP, to mediate gene transfer in hematopoietic progenitor cell lines as well as in primary human cells and, importantly, in human hematopoietic progenitor cells. pEPI-eGFP was episomally maintained and conferred sustained eGFP expression even in nonselective conditions in the human cell line, K562, as well as in primary human fibroblast-like cells. In contrast, in the murine erythroleukemia cell line, MEL, transgene expression was silenced through histone deacetylation, despite the vector’s episomal persistence. Hematopoietic semisolid cell colonies derived from transfected human cord blood retained the vector and expressed eGFP. After 4 weeks, the vector was maintained in approximately 1% of progeny cells. Our results provide the first evidence that a S/MAR-based plasmid can function as a stable episome in primary human cells, supporting long-term transgene expression. The present study constitutes a proof of principle for the utility of this system in gene therapy applications and points at targets for future improvements.