Thèses : « Lysosomal storage diseases »

1

Roy, Elise. « Cell disorders in lysosomal storage diseases ». Phd thesis, Université René Descartes - Paris V, 2012. http://tel.archives-ouvertes.fr/tel-00683248.

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Mucopolysaccharidosis type IIIB (MPSIIIB) is a lysosomal storage disease (LSD) characterized by accumulation of heparan sulfate oligosaccharides (HSO), which results in progressive mental retardation, neurodegeneration and premature death in children. The underlying mechanisms are poorly understood. Coming to a better understanding of the pathophysiology of MPSIIIB has become a necessity to assess the efficacy of gene therapy treatment regarding loss of neuronal plasticity, and to define the best conditions for treatment. To address the link between HSO accumulation and downstream pathological events, new cell models of MPSIIIB were created. First, induced pluripotent stem cells (iPSc) were generated from fibroblasts of affected children, followed by differentiation of patient-derived iPSc into a neuronal progeny. Second, a HeLa cell model was created in which expression of shRNAs directed against a-N-acetylglucosaminidase (NAGLU), the deficient enzyme in MPSIIIB, is induced by tetracycline. Success in the isolation of these different models was pointed by the presence of cardinal features of MPSIIIB cell pathology. Studies in these models showed that: I) HSO excreted in the extracellular matrix modifies cell perception of environmental cues, affecting downstream signalling pathways with consequences on the Golgi morphology. II) Accumulation of intracellular storage vesicles, a hallmark of LSDs is due to overexpression of the cis-Golgi protein GM130 and subsequent Golgi alterations. It is likely that these vesicles are abnormal lysosomes formed in the cis- and medial-Golgi which are misrouted at an early step of lysosome biogenesis, giving rise to a dead-end compartment. III) Other cell functions controlled by GM130 are affected, including centrosome morphology and microtubule nucleation. These data point to possible consequences on cell polarization, cell migration and neuritogenesis.

2

Chen, Chun-Wu. « Defective iron homeostasis in lysosomal storage diseases ». Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:5127c241-be64-4990-bef5-70e15d391394.

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Niemann-Pick type Cl (NPC1) disease is a neurodegenerative lysosomal storage disorder characterized by the accumulation of multiple lipids in the late endosome/lysosomal system and reduced acidic store calcium levels. Since the lysosomal system is involved in regulating aspects of transition metal ion homeostasis and its intracellular compartmentalization, we have investigated whether there are metal ion metabolism defects and haematological abnormalities in NPC1 disease. We have identified multiple haematological changes, including decreased haematocrit, haemoglobin and mean corpuscular haemoglobin volume in mice.

3

Ross, Colin J. D. « Immuno-isolation gene therapy for lysosomal storage disease / ». *McMaster only, 2001.

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4

Rigal, Nathalie [Verfasser]. « Improving enzyme replacement therapy for lysosomal storage diseases / Nathalie Rigal ». Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2018. http://d-nb.info/115576093X/34.

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5

Lewis, Martin David. « Human lysosomal sulphate transport ». Title page, contents and abstract only, 2001. http://web4.library.adelaide.edu.au/theses/09PH/09phl6752.pdf.

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Addendum inserted at back Includes bibliographical references (leaves 266-287). 1. Introduction -- 2. Materials and general methods -- 3. Characterisation and partial purification of the lysosomal sulphate transporter -- 4. Identification of proteins involved in lysosomal sulphate transport -- 5. The relationship between a sulphate anion transporter family and the lysosomal sulphate transporter -- 6. Investigation of sulphate transport in human skin fibroblasts -- 7. Concluding remarks

6

Kanju, Patrick M. Suppiramaniam Vishnu. « Synaptic glutamate receptor dysfunction in tissue and animal models of Alzheimer's disease ». Auburn, Ala., 2005. http://repo.lib.auburn.edu/2005%20Summer/doctoral/KANJU_PATRICK_11.pdf.

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7

Champigny, Marc J. Igdoura Suleiman. « Transcriptional regulation of neu1 expression : Implications for lysosomal storage disease / ». *McMaster only, 2005.

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8

Maalouf, Katia Ghandour [Verfasser]. « Role of lipid rafts in the pathophysiology of lysosomal storage diseases / Katia Ghandour Maalouf ». Hannover : Technische Informationsbibliothek und Universitätsbibliothek Hannover (TIB), 2012. http://nbn-resolving.de/urn:nbn:de:gbv:089-7259318337.

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9

Ghandour, Maalouf Katia [Verfasser]. « Role of lipid rafts in the pathophysiology of lysosomal storage diseases / Katia Ghandour Maalouf ». Hannover : Technische Informationsbibliothek und Universitätsbibliothek Hannover (TIB), 2012. http://d-nb.info/1029515352/34.

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10

Gray, James Andrew Russell. « Modulating the heat-shock response : a potential therapy for lysosomal storage disorders ». Thesis, University of Oxford, 2014. https://ora.ox.ac.uk/objects/uuid:d9b746c9-9026-4a6e-97b5-00bb848100d7.

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Lysosomal storage disorders (LSDs) comprise a broad disease group of inherited metabolic disorders, the majority of which are associated with CNS pathology, significant disability and reductions in life expectancy. LSDs are caused by mutations in genes encoding proteins critical for the correct functioning of lysosomal homeostasis. The disruption of lysosomal homeostasis results in the abnormal accumulation of lysosomal content, initiating intracellular pathological events, including calcium dysregulation and lysosomal membrane permeablisation (LMP) affecting cell function and inducing cellular death mechanisms. These pathological events are particularly damaging within the CNS, due to its limited capacity for regeneration. Despite intensive scientific research into these disorders, and an increased understanding of the pathological events underlying these diseases, effective treatments are still lacking for most LSDs. Several therapeutic approaches have been investigated in the last 30 years, including enzyme replacement therapy, bone marrow transplantation, substrate reduction therapy, chemical chaperones and gene therapy. However, the CNS pathology in many of the LSDs remains unaddressed due to the restricted ability of many therapeutic agents to cross the blood-brain barrier. The heat-shock response (HSR) is an emerging element involved in the pathogenesis of a variety of disorders. The HSR is a physiological response to a wide range of cellular stresses. It functions to protect the cell from the aggregation of misfolded proteins and LMP. Of the HSR, several key players are integral to mounting a heat shock response, these include the heat-shock factor 1 (HSF-1) and HSP70. In this thesis, we provide proof-of-principle for the use of recombinant HSP70, and the small molecule up-regulator of the HSR, arimoclomol in treatment of a range of LSDs. We show that HSP70 is able to access the CNS, and increase the degradative capacity of lysosomal hydrolases. This provides differential behavioural, biochemical and survival effects in LSD models of Niemann-Pick type C, Sandhoff and Fabry disease. Additional studies using the HSF-1 upregulator arimoclomol, show a complex dose-response between the different models, possibly reflecting essential differences in the calcium dysregulation between these disease states.

11

Gliddon, Briony Lee. « Enzyme replacement therapy in a murine model of mucopolysaccharidosis type IIIA / ». Title page, contents and abstract only, 2002. http://web4.library.adelaide.edu.au/theses/09PH/09phg5595.pdf.

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12

Crawley, Allison Catherine. « Enzyme replacement therapy in a feline model of mucopolysaccharidosis type VI / ». Title page, contents and abstract only, 1998. http://web4.library.adelaide.edu.au/theses/09PH/09phc9107.pdf.

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13

Li, Yijun. « Detection of enzyme deficient genetic diseases by electrospray ionization mass spectrometry / ». Thesis, Connect to this title online ; UW restricted, 2004. http://hdl.handle.net/1773/11577.

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14

Kim, A. Rang M. S. « Discontinuing Enzyme Replacement Therapy in Patients with Lysosomal Storage Diseases due to Significant Clinical Decline ». University of Cincinnati / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1396522371.

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15

Yogalingam, Gouri. « Molecular characterisation of feline MPS VI and evaluation of gene therapy / ». Title page, contents and abstract only, 1998. http://web4.library.adelaide.edu.au/theses/09PH/09phy54.pdf.

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16

Freeman, Craig. « The lysosomal degradation of heparan sulphate : a comparative study of the physical and catalytic properties of the heparan sulphate degradative enzymes / ». Title page, contents and abstract only, 1991. http://web4.library.adelaide.edu.au/theses/09PH/09phf855.pdf.

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17

Zarrinkalam, Krystyna. « Characterisation of osteoblast function in a feline model of mucopolysaccharidosis type VI ». Title page, contents and introduction only, 2001. http://web4.library.adelaide.edu.au/theses/09PH/09phz38.pdf.

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Addenda slip inserted in back. Includes bibliographical references (leaves 178-231). To further the understanding of the molecular mechanisms that contribute to the skeletal pathology of mucopolysaccharidosis type VI and to investigate the production of organic matrix by mucopolysaccharidosis VI osteoblasts

18

Maghe, Clément. « Elucidating the Vulnerability of Glioblastoma Stem-like Cells to Lysosomal Dysfunctions ». Electronic Thesis or Diss., Nantes Université, 2023. http://www.theses.fr/2023NANU1035.

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Le glioblastome (GB) est le cancer du système nerveux central de l’adulte le plus commun et le plus meurtrier. Malgré un traitement invasif de résection chirurgicale suivi de séances de radio- et de chimiothérapies, la survie des patients atteints difficilement les 15 mois. Cette agressivité est considérée comme liée notamment à la présence de cellules souches cancéreuses appelées cellules de type souche de glioblastome, ou GSCs. Ces cellules, impliquées dans l’initiation, la croissance, et la récurrence du GB, sont considérées comme des cibles préférentielles. Les lysosomes jouent un rôle critique dans le maintien de l’homéostasie des GSCs. Ces organelles, agissant à la croisée des mécanismes d’anabolisme et de catabolisme, permettent la survie des GSCs hors de leur niche protectrice. Dans les GSCs, leur déstabilisation culmine en une mort spécifique, définissant ainsi les lysosomes comme un point de contrôle des décisions vie-et- mort dans ce contexte cellulaire. La paracaspase MALT1 a récemment été définie comme un médiateur crucial de l’homéostasie des lysosomes dans les GSCs. Cette protéase, initialement décrite comme impliquée dans les réponses immunitaires, restreint le compartiment lysosomal, son inhibition aboutissant en une mort lysosome-dépendante des GSCs, via un mécanisme impliquant la protéine de liaison à l’ARNm Quaking. Cependant, les événements engendrant la déstabilisation lysosomale ainsi que la mort des GSCs restaient incertains. Ainsi, mon travail de thèse a permis la cartographie des événements au niveau cellulaire et des organelles, participant à la déstabilisation lysosomale suivant le ciblage moléculaire et pharmacologique de la paracaspase MALT1
Glioblastoma (GB) is the deadliest and most prevalent primary tumor of the central nervous system (CNS) in adults. Despite invasive treatments of surgical resection followed by radio- and chemotherapy, the median survival of patients hardly reaches 15 months. This aggressiveness is thought to be in part linked to the presence of a subset of cancer stem cells termed glioblastoma stem-like cells (GSCs) within the tumor mass. Involved in the initiation, growth, and recurrence of GB tumors, these cells therefore represent a promising target. In this context, lysosomes are critical for the maintenance of GSCs homeostasis. These organelles, standing at the crossroad between anabolism and catabolism, permit the survival of GSCs in unfavorable conditions. Their destabilization culminates in the specific cell death of GSCs, defining lysosomes as a checkpoint for life-and-death decisions in this cellular context. The MALT1 paracaspase was recently defined as a crucial mediator of lysosomal homeostasis in GSCs. This protease, initially involved in immune responses, restrains the lysosomal compartment, its inhibition resulting in lysosomal-dependent cell death of GSCs through a mechanism involving the RNA binding protein Quaking. However, the events resulting in the lysosomal destabilization and cell death of GSCs remained unclear. In this context, my thesis work allowed the cartography of cellular and organellar events leading to GSC cell death upon MALT1 inhibition and silencing

19

Gomez, Grau Marta. « Models and therapeutic approaches for Niemann-Pick (A/B and C) and other lysosomal storage disorders ». Doctoral thesis, Universitat de Barcelona, 2015. http://hdl.handle.net/10803/385854.

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Lysosomal storage disorders (LSDs) are a group of more than 50 different genetic disorders due to the lack of degradation of substrates within lysosomes. Most of them are caused by mutations in genes coding for lysosomal hydrolases. LSDs are mainly inherited in an autosomal recessive manner. Although in the last 25 years there has been much effort and progress to develop therapies aiming the correction of metabolic defects for these diseases, yet there is no effective therapy for many of them, and patients are exclusively treated with supportive care. For several non-neurological LSDs, enzyme replacement therapy may be an option. However, this approach is not efficient, at the moment, for neurological patients. Thus, new therapeutic approaches need to be developed. One of these approaches is the use of drugs that are able to produce a read-through of premature stop codons. One advantage of this strategy is that, if successful, it can be applied to any disease, provided that the molecular cause is a nonsense mutation. The most extensively studied approach involves read-through by drugs affecting the ribosome-decoding site. Niemann-Pick A/B (NPA/B) and Niemann-Pick C (NPC) are two rare inherited monogenic diseases. Although initially were defined as types of the same disease, subsequently they have been considered independent diseases due to the fact that they have different biochemical and molecular features. NPA/B disease is caused by mutations in the SMPD1 gene, localized in chromosome 11, which codes for the enzyme acid sphingomyelinase, a soluble lysosome hydrolase. NPC disease is caused by mutations in the NPC1 gene, localized in chromosome 18, which codes for a lysosomal membrane protein, involved in cholesterol transport. NPC disease also may be caused by mutations in the NPC2 gene, localized in chromosome 14, which codes for a lysosomal soluble protein, involved in cholesterol transport too. The abnormal action of these proteins in patients promotes the accumulation of lipids, which differ in each disease, inside the lysosomes, causing their dysfunction. This thesis makes contributions to the field of LSDs study. Various aspects have been addressed: therapeutic approaches have been tested as a first step in the achievement of a successful therapy for those LSDs bearing nonsense mutations. Important steps in the generation of a new cellular model for NPA/B have been achieved. This model would be helpful to understand the molecular processes that contribute to the development of this pathology and would be a valuable tool for the search of successful treatments. Finally, two new mouse models for NPC have been generated and characterized while one dies few days after birth, the other mimics the main characteristics of the disease and will be useful to probe specific treatments.
Las enfermedades de acúmulo lisosómico (LSDs) son un grupo de más de 50 trastornos genéticos diferentes, debido a la falta de degradación de sustratos dentro de los lisosomas. La mayoría de ellas están causadas por mutaciones en los genes que codifican para las hidrolasas lisosomales. Las LSDs se heredan principalmente de forma autosómica recesiva. Aunque en los últimos 25 años ha habido un gran esfuerzo y progreso para desarrollar terapias dirigidas a la corrección de los defectos metabólicos de estas enfermedades, sin embargo, no hay un tratamiento eficaz para muchas de ellas, y los pacientes son tratados exclusivamente con terapias de soporte. Para muchas LSDs sin afectación neurológica, la terapia de reemplazo enzimático puede ser una opción. Sin embargo, este enfoque no es eficiente por el momento para los pacientes con afectación neurológica. Por lo tanto, nuevos aproximaciones terapéuticas han de ser desarrolladas. Una de estas aproximaciones es el uso de fármacos que son capaces de proseguir la lectura a través de los codones de parada prematuros. La ventaja de esta estrategia es que, si tiene éxito, se puede aplicar a cualquier enfermedad cuya causa molecular sea una mutación sin sentido. Niemann-Pick A/B (NPA/B) y Niemann-Pick C (NPC) son dos enfermedades raras, monogénicas y hereditarias. Aunque inicialmente se definieron como tipos de la misma enfermedad, posteriormente se han considerado enfermedades independientes debido al hecho de que tienen diferentes características bioquímicas y moleculares. La enfermedad de NPA/B está causada por mutaciones en el gen SMPD1, localizado en el cromosoma 11, que codifica para la enzima esfingomielinasa ácida, una hidrolasa soluble lisosomal. La enfermedad de NPC está causada por mutaciones en el gen NPC1, localizado en el cromosoma 18, que codifica para una proteína de membrana lisosomal, dicha proteína participa en el transporte del colesterol. La enfermedad de NPC también puede estar causada por mutaciones en el gen NPC2, localizado en el cromosoma 14, que codifica para una proteína soluble lisosomal, que también participa en el transporte del colesterol. La acción anormal de estas proteínas en los pacientes promueve la acumulación de lípidos, diferentes en cada enfermedad, el interior de los lisosomas, provocando su disfunción. Esta tesis realiza contribuciones en el campo de estudio de las LSDs. Se han abordado diversos aspectos. Varias aproximaciones terapéuticas han sido probadas como un primer paso en el logro de una terapia satisfactoria para aquellas LSDs causadas por mutaciones sin sentido. Se han alcanzado importantes avances en la generación de un nuevo modelo celular para NPA/B. Este modelo podría ser útil para entender los procesos moleculares que contribuyen al desarrollo de dicha patología y podría ser una valiosa herramienta para la búsqueda de tratamientos. Por último, se han generado dos nuevos modelos de ratón de NPC y se han caracterizado. Uno de ellos muere pocos días después de su nacimiento, el otro imita las principales características de la enfermedad y podría ser útil para la investigación de tratamientos específicos.

20

Harvey, John Steven. « Metachromatic leukodystrophy : the role of non-pathogenic sequence variants in the causation of disease / ». Title page, contents and abstract only, 1996. http://web4.library.adelaide.edu.au/theses/09PH/09phh341.pdf.

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21

Zaccariotto, Eva. « The blood-brain barrier and San Filippo Syndrome : a model for pathophisiology studies of CNS in lysosomal storage diseases ». Doctoral thesis, Università degli studi di Padova, 2009. http://hdl.handle.net/11577/3426008.

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The lysosomal storage diseases (LSDs) are a large and heterogeneous group of disorders resulting from defects in various aspects of lysosomal function. They commonly afflict infants and young children and mostly involve pathology of the brain which is currently untreatable when it’s present. All animals with a well developed central nervous system (CNS) have a blood-brain barrier (BBB) that largely isolates the brain from alterations in the composition of the blood stream and the continuous changes that take place in these general body fluids. This BBB also impedes the global CNS delivery of many therapeutic materials. Some studies in mouse models of lysosomal storage diseases, such as Batten and Sandhoff diseases and GM1 gangliosidosis, have also suggested that the BBB may be damaged as an integral part of the disease process. The aim of the present project was to determine whether similar changes to the BBB occur in Sanfilippo Syndrome. The in situ brain perfusion technique is the elective system for this study as it is not necessary to consider the effects of plasma binding, metabolism and other interactions within the body. Furthermore, it offers a superior sensitivity over other tracer based methods and can be used to precisely quantify the transport of solutes across the BBB. We developed a novel modification of the original technique of Takasato and Smith (1984) which ensures that all regions of the mouse brain are perfused rather than just the territory of a single carotid artery. This is important as in LSDs all brain regions are affected and in different mouse strains the circulus arteriosus cerebri exhibits different degrees of completeness (Ward et al. 1990). Thus the method allows full regional BBB function to be assessed, and the method can be applied to a comparison of genetically modified animals on differing genetic backgrounds. Several parameters, such as cerebral perfusion flow, brain vascular volume, and carrier-mediated transport of excitatory amino acids glutamic acid and glycine, were then investigated to determine whether the in situ brain perfusion method may be applied to the mouse without disturbance to the physical or functional integrity of the BBB. Electron microscopy studies with lanthanum nitrate were also performed to assess whether the tight junctions became leaky during the course of perfusion. Once the in situ brain perfusion technique was established as a real tool for assessing the penetrance of tracers across the BBB, this method was applied to determine if there were changes to the BBB in mouse models of two of the Sanfilippo Syndrome diseases (MPS IIIA and MPS IIIB) compared with their respective control strains of mouse. [14C]-sucrose and [3H]-inulin were used to assess vascular volume, but do not normally penetrate the BBB, unless it is defective. [14C]-diazepam was used as a marker of cerebral blood flow; and [3H]-glycine, [3H]-glutamic acid and [3H]-tyrosine as carrier-mediated substances with low brain penetrance. These are neuro-excitatory amino acids which can cause brain damage if their entry into brain is increased. Initial findings in Sanfilippo syndrome from in situ brain perfusion technique, though they need to be confirmed and examined more fully, showed the typical clinical heterogeneity of Sanfilippo patients and clearly highlight that some changes occurred in the BBB. Also the BBB permeability of [3H]- N-butyl-deoxynojirimycin (NB-DNJ, miglustat, Zavesca®) was assessed as it is currently employed for substrate-reduction therapy (SRT), is believed to penetrate the BBB and theoretically could be used to treat secondary storage in Sanfilippo Syndrome. From intraperitoneal injections of [3H]- NB-DNJ and evaluation of the unidirectional influx constant Kin for time intervals up to 60 minutes, a slow but progressive brain uptake of this small molecule was demonstrated. An improved understanding of the BBB and its function, both in health and disease, is absolutely and critically necessary for development of successful new and improved drugs that may repair the BBB and in addition are also capable of crossing the normal BBB in order to further treat early CNS manifestations of Sanfilippo Syndrome. These studies will produce information which will aid drug targeting in general to the CNS and will further advance the possibility of treating a wide range of neurodegenerative diseases.
Le patologie d’accumulo lisosomiale (LSD) rappresentano un grosso ed eterogeneo gruppo di malattie genetiche che derivano da difetti in diversi aspetti della biologia lisosomiale. Queste patologie interessano più comunemente i bambini, e per la maggior parte determinano coinvolgimento neurologico che, quando presente, non è trattabile. Tutti gli animali con un sistema nervoso centrale (SNC) ben sviluppato hanno una barriera emato-encefalica (BEE) che isola ampiamente il cervello dalle alterazioni nella composizione del flusso del sangue e dai continui cambiamenti che avvengono in generale in questi fluidi corporei. Questa barriera impedisce anche la somministrazione globale al SNC di molte sostanze terapeutiche. Diversi studi condotti in modelli murini delle malattie d’accumulo lisosomiale, come le patologie di Batten, Sandhoff e GM1 gangliosidosi, hanno inoltre suggerito che la BEE possa essere danneggiata come parte integrante del processo patologico. Lo scopo del presente progetto è stato quello di determinare se avvenissero simili cambiamenti nella BEE nella sindrome di Sanfilippo. La tecnica della perfusione cerebrale in situ è il sistema di elezione per questo studio in quanto per le molecole analizzate non è necessario considerare gli effetti dovuti ad eventuali legami con le proteine plasmatiche, metabolismo e altre interazioni all’interno del corpo. Inoltre, offre una sensibilità superiore rispetto ad altri metodi basati su tracciante e può essere usata per quantificare precisamente il trasporto di soluti attraverso la BEE. Abbiamo apportato una nuova modifica alla tecnica originale di Takasato e Smith (1984) che assicura che tutte le regioni del cervello del topo siano perfuse piuttosto che solo la zona di una singola carotide. Questo è importante poiché nelle LSD tutte le regioni cerebrali sono coinvolte e il circulus arteriosus cerebri presenta differenti gradi di completezza in diversi ceppi murini (Ward et al. 1990). Quindi il metodo permette che la funzione della BEE sia valutata in tutte le regioni, e può essere applicato per comparare animali modificati geneticamente di diversi background genetici. Diversi parametri, come il flusso della perfusione cerebrale, il volume vascolare del cervello, e il trasporto carrier-mediato degli amminoacidi acido glutammico e glicina, sono stati investigati per determinare se il metodo della perfusione cerebrale in situ possa essere applicato al topo senza disturbare l’integrità fisica e funzionale della BEE. Sono stati anche condotti studi con nitrato di lantano, e analizzati al microscopio elettronico, per valutare se le giunzioni occludenti subissero aperture durante il corso della perfusione. Una volta che la tecnica della perfusione cerebrale in situ è stata provata come strumento reale per la valutazione della penetrazione di traccianti attraverso la BEE, questo metodo è stato applicato per determinare se ci fossero cambiamenti nella BEE in modelli murini di due forme della sindrome di Sanfilippo (MPS IIIA e MPS IIIB) in confronto ai loro rispettivi ceppi murini di controllo. [14C]-saccarosio e [3H]-inulina sono stati impiegati per valutare il volume vascolare, ma normalmente non penetrano la membrana, a meno che non sia difettiva. [14C]-diazepam è stato utilizzato come marker del flusso sanguigno cerebrale; e [3H]-glicina, [3H]-acido glutammico e [3H]-tirosina come sostanze carrier-mediate a bassa penetrazione cerebrale. Questi sono amminoacidi neuro-eccitatori che possono causare danni al cervello se la loro entrata nel cervello è aumentata. Dati iniziali per la sindrome di Sanfilippo dalla tecnica della perfusione cerebrale in situ, sebbene necessitino di essere confermati e approfonditi, hanno dimostrato la tipica eterogeneità clinica dei pazienti di Sanfilippo ed evidenziano chiaramente che avvengono alcuni cambiamenti nella BEE. Anche la permeabilità di [3H]-N-butil-deossinojirimicina (NB-DNJ, miglustat, Zavesca®) alla BEE è stato valutata poichè è attualmente impiegata nella terapia di riduzione del substrato (SRT), si ritiene che penetri la BEE e teoricamente potrebbe essere usata per trattare l’accumulo secondario nella sindrome di Sanfilippo. Da iniezioni intraperitoneali di [3H]- NB-DNJ e valutazione della costante d’influsso unidirezionale Kin per intervalli di tempo fino a 60 minuti, un lento ma progressivo assorbimento di questa piccola molecola è stato dimostrato. Una comprensione maggiore della BEE e della sua funzione, sia in salute sia in malattia, è assolutamente e criticamente necessaria per lo sviluppo di farmaci nuovi e migliori che possano riparare la BEE e in più siano anche in grado di attraversare la BEE allo scopo di trattare manifestazioni precoci della sindrome di Sanfilippo nel SNC. Questi studi produrranno informazioni che aiuteranno la somministrazione di farmaci al SNC in generale e aumenteranno ulteriormente la possibilità di trattare un ampio numero di patologie neurodegenerative.

22

Litjens, Tom. « The molecular genetics of mucopolysaccharidosis type VI / ». Title page, contents and abstract only, 1994. http://web4.library.adelaide.edu.au/theses/09PH/09phl776.pdf.

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23

White, Elaine Joanna. « Evaluation of receptor-mediated gene transfer using an integrin-targeting vector as a potential form of therapy for lysosomal storage diseases ». Thesis, University College London (University of London), 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.340643.

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24

Gerber, Scott Anthony. « Direct profiling of multiple enzymes in human cell lysates by affinity chromatography/electrospray ionization mass spectrometry : application to clinical enzymology / ». Thesis, Connect to this title online ; UW restricted, 2001. http://hdl.handle.net/1773/8490.

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25

Weismann, Cara M. « Approaches and Considerations Towards a Safe and Effective Adeno-Associated Virus Mediated Therapeutic Intervention for GM1-Gangliosidosis : A Dissertation ». eScholarship@UMMS, 2014. http://escholarship.umassmed.edu/gsbs_diss/767.

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GM1 gangliosidosis is a lysosomal storage disorder caused by a deficiency in the catabolizing enzyme β-galactosidase (βgal). This leads to accumulation of GM1-ganglioside (GM1) in the lysosome inducing ER stress and cell death. GM1 gangliosidosis is primarily a disorder of the central nervous system (CNS) with peripheral organ involvement. In this work we report two major findings, 1) systemic treatment of GM1 gangliosidosis with an adenoassociated virus (AAV9) encoding mouse-βgal (mβgal) in a GM1 gangliosidosis mouse model (βGal-/-), and 2) an investigation into an intracranial injection of a therapeutic AAVrh8 encoding mβgal. Systemic treatment of GM1 gangliosidosis with AAV9 resulted in a moderate expression of enzyme in the CNS, reduction of GM1 storage, significant retention of motor function and a significant increase in lifespan. Interestingly, the therapeutic effect was more robust in females. Intracranial injections of AAVrh8 vector expressing high levels of βgal resulted in enzyme spread throughout the brain, significant retention of motor function and a significant increase in lifespan. Histological alterations were also found at the injection site in both βGal-/- and normal animals. We constructed a series of vectors with a range of decreasing enzyme expression levels to investigate the cause for the unanticipated result. Microarrays were performed on the injection site and we showed that a lower expressing AAVrh8-mβgal vector mitigated the negative response. Intracranial injection of this newly developed vector was shown to clear lysosomal storage throughout the CNS of βGal-/- mice. Taken together, these studies indicate that a combined systemic and fine-tuned intracranial approach may be the most effective in clearing lysosomal storage completely in the CNS while providing therapeutic benefit to the periphery.

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Da, Silva Afitz. « Glycovecteurs pour le ciblage thérapeutique d'une maladie rare lysosomale : la maladie de Pompe ». Thesis, Montpellier, 2017. http://www.theses.fr/2017MONTT001.

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Sur la cinquantaine de maladies rares lysosomales, seules 8 peuvent être traitées par enzymothérapie substitutive avec plus ou moins d’efficacité. Il y a donc un réel besoin de développer de nouveaux traitements mais aussi de mieux caractériser les causes de ces maladies. Durant cette thèse nous nous sommes intéressés à la maladie de Pompe qui résulte de l’absence ou de la carence en enzyme lysosomale alpha-glucosidase acide (GAA) responsable de la dégradation du glycogène en glucose dans de nombreux tissus. Actuellement seule la forme infantile de cette maladie peut être traitée alors que la forme juvénile/adulte est faiblement améliorée par le traitement Myozyme®. Cette thèse a eu pour but de développer une nouvelle enzymothérapie qui, à terme, permettrait d’empêcher la progression de la maladie et de soigner, de manière satisfaisante, les formes juvéniles et adultes de la maladie de Pompe. Dans ce but, nous avons utilisé des dérivés monosaccharidiques « Analogues du Mannose-6-phosphate (M6P) Fonctionnalisés sur l’Aglycone (AMFA) », qui sont ensuite greffés sur la GAA recombinante humaine (rhGAA) afin d’améliorer son adressage aux lysosomes obtenant la rhGAA-AMFA.Une première étude in vitro, sur des fibroblastes de patients atteints de la forme adulte de la maladie, a démontré que le greffage des AMFA sur la rhGAA produite en cellules d’insectes Sf9 améliorait significativement l’affinité pour le récepteur du M6P (RM6P), l’internalisation et l’activité de l’enzyme et lui conférait une efficacité sur les souris GAA-/-, modèles de la maladie de Pompe, par rapport au traitement actuel (Article 1). Puis nous avons pu démontrer, pour la première fois, l’efficacité de la rhGAA-AMFA produite en cellules CHO sur des souris GAA-/- âgées. Ces résultats suggèrent, ainsi, la possibilité d’utiliser cette néo-enzyme dans le traitement de la forme adulte de la maladie (Article2). Enfin, le greffage des AMFA permet d’obtenir une maturation intracellulaire complète de la rhGAA sous forme active dans des myoblastes et myotubes de patients adultes et dans les quadriceps de souris âgées modèles Pompe, ce qui n’a pas été observé pour Myozyme® (Article 3). Lors de cette thèse, nous avons également démontré que de nouveaux analogues disaccharidiques, ayant une meilleure affinité que les monosaccharides pour le RM6P, peuvent efficacement cibler la rhGAA pour le traitement de la maladie de Pompe. Un brevet a été déposé sur ces résultats (Brevet PCT/FR2016/052339).En conclusion, ce travail a permis de développer une nouvelle technologie de ciblage plus efficace des enzymes lysosomales par des analogues synthétiques. La désignation de médicament orphelin pour l’alpha glucosidase acide conjuguée aux analogues du mannose-6-phosphate a été obtenue suite à ces travaux auprès de l’Agence Européenne du Médicament pour le traitement de la maladie de Pompe (EMA/OD/098/16).Mots clés: maladies lysosomales, maladie de Pompe, enzymothérapie, récepteur du mannose 6-phosphate
On 53 known rare lysosomal diseases, only 8 can be treated by enzyme replacement therapy with more or less efficiency. There is therefore a need to develop new treatments but also to better characterize these diseases. During this thesis, we focused on Pompe disease which results from the absence or deficiency of the lysosomal enzyme alpha-glucosidase acid (GAA), responsible for the degradation of glycogen in glucose in many tissues. Currently only the infantile form of this disease can be treated while the juvenile/adult form is slightly improved by Myozyme® treatment. This thesis aimed to devel a new enzyme replacement therapy which could prevent the progression of the disease and satisfactorily treat the late onset form of the disease. To do that, we used monosaccharide derivatives “Mannose-6-phosphate analogues (M6P) Functionalized on Aglycone (AMFA)”, which were grafted onto human recombinant GAA (rhGAA) in order to improve its lysosome addressing obtaining the rhGAA-AMFA.A first in vitro study on adult patient fibroblasts showed that the addition of AMFA to rhGAA, produced in Sf9 insect cells, significantly improved its affinity for the M6P receptor (RM6P), its internalization and activity. It was also more efficient on the GAA-/- Pompe mouse model compared to current treatment (Article 1). Then, we demonstrated for the first time the efficiency of rhGAA-AMFA produced in CHO cells in aged mice model. These results suggest the possibility to use this neo-enzyme in the treatment of the adult form that still resists to treatment (Article 2). Finally, the addition of AMFA allows a complete maturation of rhGAA into its active form in myoblasts and myotubes of adult patients and in the quadriceps of aged mice Pompe model. This was not observed for Myozyme® (Article 3). In this thesis we have also demonstrated that novel disaccharide analogues with a better affinity than monosaccharides for RM6P can efficiently target GAA for the treatment of Pompe disease. A patent has been filed on these results (Patent PCT / FR2016 / 052339).In conclusion, this work has led to the development of a new technology more efficient in targeting lysosomal enzymes by mean of new synthetic analogues. An orphan drug designation for the recombinant human acid alpha-glucosidase conjugated with mannose-6-phosphate analogues was obtained on the basis of this work at the European Medicines Agency for the treatment of Pompe disease (EMA/OD/098/16).Key words: lysosomal diseases, Pompe disease, enzyme replacement therapy, mannose 6-phosphate receptor

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Leishman, Alison Jane. « Harnessing the immunomodulatory capacity of dendritic cells differentiated from human induced pluripotent stem cells and the therapeutic potential of dendritic cell-derived exosomes for the treatment of lysosomal storage diseases ». Thesis, University of Oxford, 2015. https://ora.ox.ac.uk/objects/uuid:97f6791f-ff69-4645-9a3d-2ff23ce69529.

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Lysosomal storage diseases (LSDs) are a collection of disorders that feature the pathological accumulation of substrate frequently due to an enzymatic defect within the lysosomes. The most effective treatment regime for LSDs is enzyme replacement therapy. However, this treatment has faced two main challenges which have limited its treatment efficacy and clinical impact. One challenge constitutes the potential immunogenicity of the replaced enzyme, which can lead to the induction of an antibody response that prevents its effective targeting. Therefore, this thesis investigated the potential to derive patient-specific autologous dendritic cells (ipDCs) from fibroblasts which were obtained from a healthy donor and from a patient diagnosed with infantile-onset Pompe disease and were reprogrammed into induced pluripotent stem cells (iPSCs). This study demonstrated the feasibility of differentiating these iPSCs into ipDCs and investigated the potential to modulate their immunogenicity using a variety of agents. Using IL-10, this work was able to show the feasibility of generating patient-specific ipDCs with pro-tolerogenic characteristics which may be exploited for the induction of tolerance towards therapeutic enzymes. Secondly, the delivery of therapeutic enzymes to the central nervous system (CNS), which is frequently involved in disease pathogenesis, is limited by the selective-permeability of the blood brain barrier. As specifically-labelled exosomes have been shown capable of targeting to the CNS for the delivery of therapeutic molecules, this study has shown the possibility of harvesting exosomes from ipDC cultures. The potential of exploiting the endocytic capacity of dendritic cells for the loading of enzyme into exosomes was explored. Furthermore, this study has found that the administration of syngeneic and allogeneic exosomes from mouse bone-marrow derived DCs and ipDCs elicited an antibody-mediated immune response which may limit the clinical application of exosomes further highlighting the need for tolerance induction. Altogether, this study constitutes a first step towards potential improvements in the treatment of LSDs.

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Leishman, Alison Jane. « Harnessing the immonomodulatory capacity of dendritic cells differentiated from human induced pluripotent stem cells and the therapeutic potential of dendritic cell-derived exosomes for the treatment of lysosomal storage diseases ». Thesis, University of Oxford, 2014. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.711748.

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Wang, Ding. « Application of mass spectrometry in enzyme deficiency assay for newborn screening purpose / ». Thesis, Connect to this title online ; UW restricted, 2006. http://hdl.handle.net/1773/11557.

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Bender, Fernanda. « Triagem neonatal para mucopolissacaridose tipo VI (Síndrome de Maroteux-Lamy) em uma região com alta incidência da doença ». reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2011. http://hdl.handle.net/10183/48987.

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A mucopolissacaridose tipo VI (MPS VI) ou Síndrome de Maroteaux-Lamy, é uma doença autossômica recessiva causada pela deficiência da enzima lisossomal Nacetilgalactosamina- 4-sulfatase (ARSB), a qual resulta no armazenamento lisossômico de dermatan sufato em vários tecidos e órgãos, dando origem a uma condição clínica de espectro variável, desde formas mais graves até formas mais atenuadas. O acúmulo de substrato não degradado causa um importante comprometimento ósseo, problemas respiratórios, baixa estatura e outros problemas, afetando os olhos, o coração e outros órgãos. Embora a síndrome de Maroteaux-Lamy não tenha uma incidência definida no Brasil, é reconhecido que, no nosso meio, é muito mais freqüente do que em outros países e regiões. Ela é particularmente freqüente no município de Monte Santo (Bahia), de aproximadamente 50.000 habitantes e onde já foram registrados 13 casos da doença. O diagnóstico é importante porque existe hoje um tratamento específico para a doença, a terapia de reposição enzimática (TRE), que vem mostrando bons resultados, especialmente quando iniciada em idade precoce. Descrevemos neste trabalho uma adaptação para microplacas da medida fluorimétrica da atividade de ARSB, e uma nova metodologia de análise molecular, ambas padronizados para sangue total impregnado em papel-filtro (STIPF). Essas técnicas foram desenvolvidas para incluir um teste de triagem neonatal para MPS VI, realizado nas amostras coletadas para o “teste do pezinho” nos neonatos do município de Monte Santo. Esses métodos permitem a detecção de pacientes com MPS VI e dos portadores da mutação específica que parece ser responsável pela alta incidência de MPS VI nessa localidade, uma vez que todos os pacientes lá diagnosticados apresentavam a mesma mutação (p.H178L) em homozigose. O trabalho foi desenvolvido em três etapas: na primeira foi realizada a padronização das técnicas em 100 amostras de STIPF; na segunda foi feito um teste-piloto com amostras de neonatos de Monte Santo, para avaliação das técnicas padronizadas e para o estudo de termoestabilidade em controles hígidos; na terceira foram analisadas amostras de STIPF de neonatos provenientes de Monte Santo pelos dois métodos (bioquímico e molecular). A padronização para realização da medida fluorimétrica da atividade enzimática de ARSB em microplacas indicou que o método é sensível, permitiu diferenciar os valores da população normal dos valores dos pacientes afetados e possibilitou a identificação segura de pacientes com MPS VI. Nas padronizações da análise molecular da mutação p.H178L em STIPF foi possível diferenciar os indivíduos normais, heterozigotos e homozigotos. Os resultados preliminares disponíveis indicam que o protocolo de triagem neonatal para MPS VI desenvolvido no presente trabalho poderá ser facilmente incorporado por laboratórios de referência, contribuindo para a detecção e tratamento precoce dos pacientes afetados por MPS VI.
Mucopolysaccharidosis type VI (MPS VI) or Maroteux-Lamy syndrome, is an autosomal recessive disorder caused by deficiency of the lysosomal enzyme Nacetylgalactosamine- 4-sulfatase (ARSB), which results in lysosomal storage of dermatan sufate in various tissues and organs and leads to a variable clinical spectrum, including more severe and attenuated forms. The accumulation of undegraded substrate causes bone involvement, respiratory problems and short stature, among other signs and symptoms, affecting the eyes, heart and other organs. Although the Maroteaux-Lamy syndrome does not have a defined incidence in Brazil, it is recognized that in our environment it is much more frequent than in other countries and regions. It is particularly frequent in the municipality of Monte Santo (Bahia) approximately 50,000 inhabitants and where there have already been 13 cases of the disease. The diagnosis is important because today there is a specific treatment for the disease, enzyme replacement therapy (ERT) which has shown good results, especially when started at an early age. We describe herein the standardization of the microplate fluorometric method for the ARSB test and a new methodology of molecular analysis, both adapted for dried blood spots (DBS) samples. These techniques were developed for inclusion of MPS VI in the newborn screening program that already tests the neonates of the city of Monte Santo, Bahia, Brasil for metabolic diseases. The methods were developed to detect patients with MPS VI and also for carriers, once the disease seems to have a high incidence (around 1:5.000) at this location. Also, all patients that have already been diagnosed in this city presented the same mutation (p.H178L) in homozygosis. The study was conducted in three stages: in the first was performed in 100 DBS samples an standardization of the techniques; in the second was done a pilot test with samples of newborns of Monte Santo, for the evaluation of standardized techniques and for the thermostability study in healthy controls; in the third were analyzed newborns samples from Monte Santo for both biochemical and molecular methods. Standardization on microplate for fluorimetric enzyme activity of the ARSB showed the assay sensitivity, differentiating values between normal and affected and allowing a reliable detection of patients with MPS VI. On the standardization for molecular analysis in DBS it was possible to differentiate the results for normal individuals, heterozygous and affected for the mutation p.H178L. The preliminary results available indicate that the protocol of neonatal screening for MPS VI developed in this work can be easily incorporated by reference laboratories, contributing to the detection and premature treatment of MPS VI affected patients.

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SAMARANI, MAURA. « CELL DAMAGE INDUCED BY LYSOSOMAL IMPAIRMENT : STUDY OF THE ROLE OF PLASMA MEMBRANE SPHINGOLIPIDS ». Doctoral thesis, Università degli Studi di Milano, 2017. http://hdl.handle.net/2434/482301.

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Lysosomes are the principal site of the catabolism of sphingolipids, a class of bioactive lipids mainly associated with the external leaflet of cell plasma membrane. Several lines of evidence support a direct correlation between modifications in sphingolipid pattern and the activation of specific signaling pathways, including apoptosis and autophagy. Loss-of-function mutations in genes coding for lysosomal enzymes involved in sphingolipid catabolism result in severe clinical manifestations called sphingolipidoses. These pathologies belong to the group of Lysosomal Storage Diseases and are characterized by the accumulation of undegraded materials leading to lysosomal impairment and consequent cell damage. Until now, the molecular mechanisms by which the perturbation of lysosomal homeostasis affects cell functionality and viability are unknown. To investigate this issue, I used an artificial in vitro model of lysosomal impairment obtained by loading human fibroblasts with 88 mM sucrose for 14 days. In these experimental conditions, the absence of invertase induces sucrose accumulation into lysosomes. I found that sucrose loaded fibroblasts are characterized by a growth slowdown and by the activation of both apoptosis and autophagy. By RNA-sequencing, approximately a thousand of genes were found to be dysregulated after sucrose loading. In particular, 56 cell cycle-related genes are downregulated, whereas 37 lysosomal-related genes are upregulated. Using biochemical approaches, I found that sucrose loading activates lysosomal biogenesis although sucrose storage inhibits lysosomal functionality. In particular, in sucrose loaded cells lipid catabolism is blocked and complex lipids, such as phospholipids, cholesterol, glycosphingolipids, and gangliosides are accumulated. Moreover, I found that sucrose loading induces the nuclear translocation of the Transcription Factor EB (TFEB), a master-gene regulator of lysosomal function, which in turn promotes the increased fusion between lysosomes and the plasma membrane. This last event leads to higher levels of sphingolipid hydrolases at the cell surface resulting in the alteration of the plasma membrane sphingolipid composition and the consequent ectopic production of pro-apoptotic and pro-autophagic ceramide. Interestingly, in sucrose loaded fibroblasts the blocking of glycosphingolipid hydrolysis at the plasma membrane results in a reduction of autophagy and apoptosis. Similar results were also obtained in response to sphingomyelin accumulation in Niemann-Pick Type A disease (NPA). NPA is a sphingolipidosis caused by acid sphingomyelinase deficiency which leads to sphingomyelin storage. Interestingly, using NPA-derived human fibroblasts loaded with 50 µM exogenous sphingomyelin for 30 days, I found that the lysosomal impairment caused by sphingomyelin accumulation activates the same molecular pathways described in healthy fibroblasts subjected to sucrose loading. A pathogenic role of TFEB has also been suggested by biochemical analysis on brains from Acid Sphingomyelinase Knockout (ASMKO) mice. In fact, ASMKO mouse brains are characterized by TFEB nuclear translocation, increased lysosomal biogenesis, increased glycohydrolytic activities and onset of apoptosis and autophagy. Collectively, these data suggest the existence of a cross-talk among lysosomes and the cell plasma membrane. In this context, the lysosomal impairment caused by the accumulation of uncatabolized substrates leads to an altered composition of plasma membrane sphingolipids resulting in the ectopic production of ceramide which in turn is responsible for the onset of cell damage.

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Mason, Lyndel Ann. « Expression variation in lysosomal storage disorder genes ». Thesis, Queensland University of Technology, 2006. https://eprints.qut.edu.au/16240/1/Lyndel_Mason_Thesis.pdf.

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Metachromatic leukodystrophy (MLD) and Gaucher disease (GD) are caused by a deficiency of arylsulphatase A (ASA) and b-glucocerebrosidase (GBA), respectively. They are lysosomal storage disorders with a heterogeneous clinical spectrum encompassing visceral, skeletal and neurologic involvement resulting in high morbidity and mortality. The overall aim of this study is to elucidate the genetic component/s of high ASA and GBA enzyme activity in normal healthy individuals with the ultimate goal of using this information to produce greater protein activity from a recombinant protein. A wide variation in ASA and GBA enzyme activity levels has been observed in the normal population. The first objective of this project was to identify and characterise single nucleotide polymorphisms (SNPs) in the arylsulphatase A (ARSA) and glucocerebrosidase (GBA) genes that are responsible for determining the levels of expressed enzyme activity in the normal population. The second objective was to assess the contribution of transcriptional regulation and TCP80 mediated translational control to normal enzyme variation. TCP80, a translational control protein that interacts with the GBA coding region, is a splice variant of the interleukin binding factor 3 (ILF3) gene. Ten samples from individuals with high ASA activity and twenty samples from individuals with high GBA activity were screened for polymorphisms via denaturing high pressure liquid chromatography (dHPLC) and sequencing. The frequency of these polymorphisms in the normal population was determined using dot-blot hybridisation. Fifteen ARSA polymorphisms (4 promoter, 5 coding, 5 intronic and 1 poly(A) signal) and two GBA polymorphisms (1 intronic and 1 in 3¢-UTR) were identified. Two low frequency ASA polymorphisms (2723A > G, W193C) were found to be correlated with low activity, while another low frequency ASA polymorphism (1101+123C > T) was found to be correlated with high activity in a population of 113 individuals. Real time PCR was used to measure mRNA levels of GBA, ASA and LF3 along with enzyme activity levels of GBA and ASA in two cell types (leucocytes and skin fibroblasts) from four healthy individuals and seven cell lines (HL60, THP1, Huh7, U118, SW1353, Hep G2, and B-cells). Transcriptional control was evident for all three genes with GBA mRNA levels varying over 30 fold, ASA mRNA levels varying over seven fold and ILF3 levels varying more than 24 fold. The 5¢-flanking region of GBA was investigated for the cis-elements responsible for tissue-specific expression. However, it was not possible to demonstrate that the cis-element region was influencing GBA expression. Translational efficiency was measured using the magnitude of the mRNA:enzyme activity ratio as an indicator. GBA translational inefficiency was most pronounced in B cells which require four times more mRNA molecules than hepatocytes (Hep G2) and over 25 times more mRNA molecules than chondrocytes (SW1353) to produce one unit of GBA enzyme activity. Except in B-cells, GBA translational efficiency appears to increase as ILF3 mRNA levels decrease. The tissue-specific variation observed in the protein levels of the ILF3 splice variants, TCP80 and DRBP76, may play a role. The correlation of several low frequency SNPs with low ASA enzyme activity or high ASA activity indicates a role in determining the distribution of enzyme activity levels in the normal population. However, there do not appear to be any common high activity polymorphisms. Knowledge of the exact mechanisms responsible for the observed transcriptional and translational control of these lysosomal genes will greatly enhance the understanding of genotype-phenotype correlation and the contribution of genetic variants to natural variation.

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Mason, Lyndel Ann. « Expression variation in lysosomal storage disorder genes ». Queensland University of Technology, 2006. http://eprints.qut.edu.au/16240/.

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Metachromatic leukodystrophy (MLD) and Gaucher disease (GD) are caused by a deficiency of arylsulphatase A (ASA) and b-glucocerebrosidase (GBA), respectively. They are lysosomal storage disorders with a heterogeneous clinical spectrum encompassing visceral, skeletal and neurologic involvement resulting in high morbidity and mortality. The overall aim of this study is to elucidate the genetic component/s of high ASA and GBA enzyme activity in normal healthy individuals with the ultimate goal of using this information to produce greater protein activity from a recombinant protein. A wide variation in ASA and GBA enzyme activity levels has been observed in the normal population. The first objective of this project was to identify and characterise single nucleotide polymorphisms (SNPs) in the arylsulphatase A (ARSA) and glucocerebrosidase (GBA) genes that are responsible for determining the levels of expressed enzyme activity in the normal population. The second objective was to assess the contribution of transcriptional regulation and TCP80 mediated translational control to normal enzyme variation. TCP80, a translational control protein that interacts with the GBA coding region, is a splice variant of the interleukin binding factor 3 (ILF3) gene. Ten samples from individuals with high ASA activity and twenty samples from individuals with high GBA activity were screened for polymorphisms via denaturing high pressure liquid chromatography (dHPLC) and sequencing. The frequency of these polymorphisms in the normal population was determined using dot-blot hybridisation. Fifteen ARSA polymorphisms (4 promoter, 5 coding, 5 intronic and 1 poly(A) signal) and two GBA polymorphisms (1 intronic and 1 in 3¢-UTR) were identified. Two low frequency ASA polymorphisms (2723A > G, W193C) were found to be correlated with low activity, while another low frequency ASA polymorphism (1101+123C > T) was found to be correlated with high activity in a population of 113 individuals. Real time PCR was used to measure mRNA levels of GBA, ASA and LF3 along with enzyme activity levels of GBA and ASA in two cell types (leucocytes and skin fibroblasts) from four healthy individuals and seven cell lines (HL60, THP1, Huh7, U118, SW1353, Hep G2, and B-cells). Transcriptional control was evident for all three genes with GBA mRNA levels varying over 30 fold, ASA mRNA levels varying over seven fold and ILF3 levels varying more than 24 fold. The 5¢-flanking region of GBA was investigated for the cis-elements responsible for tissue-specific expression. However, it was not possible to demonstrate that the cis-element region was influencing GBA expression. Translational efficiency was measured using the magnitude of the mRNA:enzyme activity ratio as an indicator. GBA translational inefficiency was most pronounced in B cells which require four times more mRNA molecules than hepatocytes (Hep G2) and over 25 times more mRNA molecules than chondrocytes (SW1353) to produce one unit of GBA enzyme activity. Except in B-cells, GBA translational efficiency appears to increase as ILF3 mRNA levels decrease. The tissue-specific variation observed in the protein levels of the ILF3 splice variants, TCP80 and DRBP76, may play a role. The correlation of several low frequency SNPs with low ASA enzyme activity or high ASA activity indicates a role in determining the distribution of enzyme activity levels in the normal population. However, there do not appear to be any common high activity polymorphisms. Knowledge of the exact mechanisms responsible for the observed transcriptional and translational control of these lysosomal genes will greatly enhance the understanding of genotype-phenotype correlation and the contribution of genetic variants to natural variation.

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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.

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La maladie de Sandhoff est une maladie génétique rare due à des mutations du gène HEXB. Elle se caractérise par un double déficit en hexosaminidase A (αβ) et B (ββ), responsable d’une accumulation de ganglioside GM2 essentiellement dans le système nerveux central (SNC). Cliniquement, la maladie débute dès les premiers mois de vie et le décès survient vers l’âge de 3 ans. A ce jour, aucun traitement n’est disponible pour cette maladie. Le modèle murin obtenu par invalidation du gène Hexb est un bon outil pour le développement d’approches thérapeutiques, car il présente un phénotype proche de la maladie humaine. Le but principal de mon projet de thèse était d’explorer une approche de transfert de gène dans le modèle murin de la maladie de Sandhoff en utilisant un vecteur scAAV9. Ce vecteur a la particularité de pouvoir traverser la barrière hématoencéphalique et de transduire le SNC après administration intraveineuse (IV). Un vecteur codant la chaîne β des hexosaminidases, appelé scAAV9-Hexb, a précédemment été administré par voie IV à des souris en période néonatale à une dose de 3,5 x 1013 vg/kg. Les souris traitées ont survécu comme les souris normales (>700 jours) sans développer d’atteinte neurologique, ni périphérique alors que les souris Sandhoff non traitées sont décédées vers l’âge de 4 mois. J’ai réalisé toutes les analyses à long terme des souris traitées en utilisant des tests de comportements, ainsi que des analyses tissulaires 24 mois après le traitement. Une analyse lipidique par HPTLC a montré que la surcharge en ganglioside GM2 est totalement absente au niveau du cerveau (4 mois après l'injection), alors que dans le cervelet cette accumulation est non significative, mais pas totalement absente. Aucun symptôme lié à cette surcharge n’a été mis en évidence chez les souris à 24 mois, mais nous nous sommes posé la question d’un possible effet délétère à long terme en cas d’extrapolation à la clinique. Nous avons donc décidé de tester une double administration IV + ICV (intracérébroventriculaire) en utilisant le même vecteur et la même dose globale de façon à mieux corriger le cervelet. Deux groupes de souris ont été injectés en période néonatale en utilisant des doses différentes dans les deux compartiments. Les analyses ont montré que dans le cerveau, à court terme, la restauration de l’activité enzymatique est partielle, mais significative. Par ailleurs, il existe une absence totale de surcharge en GM2, ainsi qu’une correction des biomarqueurs associés à la maladie. Dans le cervelet, l’efficacité du traitement a été montrée seulement pour le groupe traité avec la dose la plus importante en ICV, ce qui suggère qu’une dose minimale en ICV est nécessaire pour atteindre de manière globale le SNC. Ces résultats ont été confirmés par l’analyse à long terme. Concernant le foie, nos résultats ont montré qu’une dose IV minimale est nécessaire pour obtenir une baisse de l’accumulation lipidique. Ce travail a permis de définir les doses minimales nécessaires dans chaque compartiment (IV et ICV) et il montre que la double administration peut être avantageuse pour traiter toutes les régions du SNC et notamment les plus atteintes, comme le cervelet. Il va maintenant nous permettre de traiter de façon optimale les souris adultes. L’autre but de mon projet était d’explorer les défauts de signalisation et la physiopathologie cellulaire dans la maladie de Sandhoff en utilisant des études in vivo et in vitro. Les études in vitro ont été réalisées sur des fibroblastes de patients et des cellules embryonnaires murines (MEF) obtenues à partir des souris Hexb-/- et la surcharge lysosomale a été confirmée dans ces cellules. La voie mTOR (mammalian target of rapamycin) a été analysée et nous avons montré qu’elle était dérégulée. L’activité autophagique a aussi été étudiée et nous avons mis en évidence une augmentation du nombre d’autophagosomes chez les souris Hexb-/- suggérant un défaut de cette voie. (...)
Sandhoff 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

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Salgues, Frédéric. « Ciblage des lysosomes pour la thérapie enzymatique substitutive ou pour la thérapie photodynamique ». Thesis, Montpellier 2, 2011. http://www.theses.fr/2011MON20148.

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Le récepteur du mannose-6-phosphate cation indépendant (RM6P-CI) permet l'endocytose puis le transfert de molécules porteuses du marqueur M6P vers les lysosomes. Pour améliorer à la fois l'affinité pour le RM6P-CI et la stabilité du résidu M6P, nous avons procédé à la synthèse d'analogues isostères stables et fonctionnalisés en position anomère pour permettre un couplage efficace à des molécules d'intérêt thérapeutique. Tout d'abord un couplage à des enzymes recombinantes humaines a été réalisé. Le remodelage de la partie oligosaccharidique de l'enzyme lysosomale GAA, dont la déficience est responsable de la maladie de Pompe, a permis de mettre en évidence que la néoglycoGAA est reconnue efficacement par les RM6P-CI et que son activité enzymatique est totalement conservée. Deuxièmement, le couplage de ces analogues du M6P à des porphyrines en vue de la thérapie photodynamique des cancers est envisagé. Le modèle mis au point en série du mannose a permis de valider notre approche de ligation de saccharides à des photosensibilisateurs par des méthodes évitant après couplage les étapes classiques de déprotection des saccharides. L'étude biologique menée sur les porphyrines glycosylées préparées a démontré leur cytotoxicité photoinduite
The cation independent mannose-6-phosphate receptor (CI-M6PR) allows the endocytosis and the transfer of molecules bearing the M6P marker to lysosomes. To improve both the affinity for the CI-M6PR and stability of the M6P residue, we carried out the synthesis of isosteric M6P analogues functionalized at the anomeric position to allow efficient coupling to molecules of therapeutic interest. First, the coupling on human recombinant enzymes was performed. The remodelling of the oligosaccharide part of the lysosomal enzyme GAA, whose deficiency is responsible for Pompe disease, helped to highlight the neoglycoGAA is recognized efficiently by CI-M6PR and its enzymatic activity is completely preserved. Second, the coupling of these analogues of M6P to porphyrins for photodynamic therapy of cancer was considered. The model developed in the mannose series has validated our strategy of ligation of saccharides to photosensitizers. The employed methods avoid the conventional steps of deprotection of saccharides after coupling. The biological study with the prepared glycosylporphyrins demonstrated the photoinduced cytotoxicity

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Munõz, Rojas Maria Verônica. « Tratamento inovador da compressão medular com reposição enzimática intratecal nas mucopolissacaridoses tipos I e VI : relato de uma série de casos ». reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2010. http://hdl.handle.net/10183/29034.

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As mucopolissacaridoses apresentam uma história natural progressiva, causada por defeitos no metabolismo dos glicosaminoglicanos. Frequentemente graves, as mucopolissacaridoses encurtam de forma considerável a expectativa de vida do paciente. Apesar de que em muitos casos a função intelectual é normal, morbidade neurológica considerável pode ser causada por compressão medular secundária ao acúmulo de glicosaminoglicanos nas meninges. O tratamento deste problema pode requerer a descompressão medular através de laminectomia cervical. A terapia de reposição enzimática endovenosa, para o tratamento de mucopolissacaridose, reduz o acúmulo lisossômico e alivia muitos dos sintomas da doença, porém não oferece benefício direto para o sistema nervoso central uma vez que não atravessa a barreira hemato-encefálica. Esta limitação da reposição enzimática endovenosa levou alguns pesquisadores a trabalhar com uma nova opção de via de liberação medicamentosa de alcance direto no sistema nervoso central, aproveitando o extenso contato que existe entre o líquido cefaloraquidiano e as meninges e com as granulações aracnoideas, para o tratamento de algumas doenças de depósito lisossomal. Estudos em modelos animais têm sido conduzidos e com resultados promissores. Este trabalho se propõe a estudar uma nova via de administração da enzima recombinante, diretamente no espaço liquórico que foi utilizada em dois pacientes com MPS I e em um paciente com MPS VI, com acesso a esta terapêutica através de uso compassivo individual aprovado pelo Comitê de Ética, no Hospital de Clínicas de Porto Alegre. Até então, apenas estudos animais haviam sido realizados abordando esta via de acesso em doenças de depósito lisossomal e estes pacientes foram os primeiros indivíduos com MPS no mundo a receber terapia de reposição intratecal para o tratamento de compressão medular sintomática por depósito de glicosaminoglicanos. Em 2005 um paciente adulto com MPS I apresentando compressão medular foi incluído em um protocolo de terapia de reposição enzimática intratecal por uso compassivo no Hospital de Clínicas de Porto Alegre. Em 2006 uma menina com MPS I apresentando compressão cervical medular sintomática também recebeu terapia de reposição enzimática intratecal por uso compassivo no Hospital de Clínicas de Porto Alegre. Em 2007 um menino com MPS VI e com compressão medular cervical também foi tratado através de reposição enzimática intratecal por uso compassivo neste mesmo hospital.
The mucopolysaccharidoses present a progressive natural course caused by defects on glycosaminoglycan degradation pathways. Usually severe, the mucopolysaccharidoses considerably shorten patient lifespan. Although in many cases the cognitive function is preserved, considerable neurological morbidity can be present due to spinal cord compression which is secondary to glycosaminoglycan storage in the meninges. Treatment for this complication usually requires surgical intervention with cervical laminectomy for thickened meninges removal. Enzyme replacement therapy used for the treatment of mucopolysaccharidoses reduces lysosomal storage and ameliorates many somatic symptoms but does not provide any direct benefit to central nervous system as the enzyme does not cross the blood-brain-barrier. Due to this limitation of intravenous enzyme replacement therapy some researchers have been working with an alternative option of enzyme delivery with direct action on central nervous system through the extensive close contact provided by cefalo-spinal fluid and meniniges and arachnoid villosities, to the treatment of some lysosomal disorders. Animal model studies have been conducted and some promising results have been achieved. This study intends to present an alternative route for the administration of a recombinant enzyme, directly in the cefalo-spinal fluid, which was used in two patients with mucopolysaccharidosis I and one patient with mucopolysaccharidois VI. These patients gained access to this therapy by individual compassionate use enrollment approved by local Ethics Board at Hospital de Clínicas de Porto Alegre. So far, only animal model trials had been conducted with the use of this administration route in lysosomal storage diseases, and these were the first three patients with mucopolysaccharidoses and cord compression to receive intrathecal enzyme replacement therapy in the world. In 2005, an adult mucopolysaccharidosis I patient presenting cervical cord compression was enrolled in a compassionate use trial of intrathecal enzyme replacement therapy, at the Hospital de Clínicas de Porto Alegre. In 2006, a girl with mucopolysaccharidosis I presenting spinal cord compression was also enrolled in a compassionate use trial of intrathecal enzyme replacement therapy, at the Hospital de Clínicas de Porto Alegre. In 2007, a boy with mucopolysaccharidosis VI and cord compression was enrolled in compassionate use trial of intrathecal enzyme replacement therapy in the same hospital.

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Manwaring, V. J. « The identification of potential diagnostic biomarkers amd disease mechanisms in lysosomal storage disorders ». Thesis, University College London (University of London), 2014. http://discovery.ucl.ac.uk/1427920/.

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Fabry disease (FD) is an X-linked lysosomal storage disorder caused by a deficiency of the enzyme alpha-galactosidase A. The resultant progressive intracellular accumulation of the glycosphingolipids globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3) are the source of a variety of clinical consequences. Biomarkers capable of detecting FD at an early stage of disease progression and that enable monitoring of response to treatment are required urgently. Using label-free quantitative proteomics two potential biomarkers, proactivator polypeptide and ganglioside GM2 activator protein, were identified in the urine of paediatric FD patients. An ultra-performance liquid chromatography-tandem mass spectrometry assay was then developed for validation purposes. Subsequently a second, larger multiplexed assay was developed to identify biomarkers capable of detecting and monitoring pre-symptomatic kidney disease in those patients most at risk. A complementary metabolomic approach was also used to identify and evaluate new Gb3-related biomarkers in the plasma of FD patients. This aspect of the study revealed five novel Gb3-related biomarkers as well as existing Gb3-related analogues and isoforms providing a metabolic profile in these patients. Potential disease mechanisms involved in FD were investigated by studying the interactions between proteins and those molecules involved in the glycosphingolipid pathway, with particular interest to Gb3. A number of mitochondrial proteins were found to interact with Gb3 resulting in the investigation of the effect of glycosphingolipids and their deacylated counterparts on ATP synthase activity. Increasing concentrations of Gb3 and lyso-Gb3 were shown to increase ATP synthase activity. Subsequently the activities of the enzymes preceding ATP synthase, the mitochondrial respiratory chain enzymes, were investigated in a Fabry mouse model. In this aspect of the study complex II/III activity was found to be significantly decreased in kidney tissues. Finally, assessment of pain thresholds in mice has demonstrated significant increases in sensitivity to applied mechanical stimuli following exposure to Gb3 and lyso-Gb3.

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Boomkamp, Stephanie. « Lysosomal storage and pathogenesis in a novel in vitro cellular model of Sandhoff disease ». Thesis, University of Oxford, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.496832.

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Rockwell, Hannah. « AAV-Mediated Gene Delivery Corrects CNS Lysosomal Storage in Cats with Juvenile Sandhoff Disease ». Thesis, Boston College, 2013. http://hdl.handle.net/2345/3929.

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Thesis advisor: Thomas N. Seyfried
Sandhoff Disease (SD) is an autosomal recessive neurodegenerative disease caused by a mutation in the Hexb gene for the β-subunit of β-hexosaminidase A, resulting in the inability to catabolize ganglioside GM2 within the lysosomes. SD presents with an accumulation of GM2 and its asialo derivative GA2 primarily in the CNS. Myelin-enriched glycolipids, cerebrosides and sulfatides, are also decreased in SD corresponding with dysmyelination. At present, no treatment exists for SD. Previous studies have shown the therapeutic benefit of using adeno-associated virus (AAV) vector-mediated gene therapy in the treatment of SD in murine and feline models. In this study, CNS tissue was evaluated from SD cats (4-6 week old) treated with bilateral injections of AAVrh8 expressing feline β-hexosaminidase α and β into the thalamus and deep cerebellar nuclei (Thal/DCN) or into the thalamus combined with intracerebroventricular injections (Thal/ICV). Both groups of treated animals had previously shown improved quality of life and absence of whole-body tremors. The activity of β-hexosaminidase was significantly elevated whereas the content of GM2 and GA2 was significantly decreased in tissue samples taken from the cerebral cortex, cerebellum, thalamus, and cervical intumescence. Treatment also increased levels of myelin-enriched cerebrosides and sulfatides in the cortex and thalamus. This study demonstrates the therapeutic benefits of AAV treatment for feline SD and suggests a similar potential for human SD patients
Thesis (MS) — Boston College, 2013
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Biology

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Haslett, Luke. « Lysosomal storage disorders and neurodegenerative disease : related mechanisms of pathogenesis and identification of novel therapeutic targets ». Thesis, Cardiff University, 2015. http://orca.cf.ac.uk/89191/.

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Lysosomal storage disorders (LSDs) are rare diseases caused by inherited mutations in genes coding for proteins of the endolysosomal system. The lysosome is an organelle responsible for the degradation of dysfunctional organelles and for the catabolism, and subsequent recycling, of macromolecules within the cell. When this process becomes defective the substrates of lysosomal catabolism accumulate; these can include lipids, proteins, polysaccharides, nucleotides and diverse combinations of all three. The phenotypic spectrum of these diseases in isolation, and even more so as a group, is extremely broad but an almost universal consequence of lysosomal dysfunction is severe, early onset neurodegeneration. Neurodegenerative diseases of ageing such as Alzheimer’s disease, Parkinson’s disease and Huntington’s disease represent a major challenge to the provision of human healthcare in light of an ageing global population. Whilst some commonalities exist between these three diseases a myriad of hypotheses for the onset of pathology has been proposed. There is growing evidence for involvement of the lysosome in all three of these diseases. We have been looking at specific lysosomal pathologies such as lipid storage, endocytosis and Ca2+ dysregulation in forms of these three neurodegenerative diseases of ageing whilst using LSDs as models to inform our study. We have found that lysosomal alkalisation in familial models of Alzheimer’s disease results in changes to lipid and Ca2+ homeostasis in this compartment and identified a lysosomal ion channel, transient receptor potential cation channel, mucolipin subfamily, member 1 (TRPML1), as a key constituent of this process. Our study of models of Huntington’s disease have implicated the Niemann-Pick type C1 protein (NPC1) in the pathogenesis of this disease and identified ways in which this could be therapeutically targeted. Finally, we have found evidence of Ca2+ dyshomeostasis throughout the cell in genetic models of Parkinson’s disease which have defects in lysosomal proteins. Taken together, these studies strengthen the evidence for lysosomal involvement in neurodegenerative diseases of ageing, albeit with different mechanisms in each case, whilst expanding on the molecular basis for these processes. Accordingly, our understanding of the mechanisms underlying the pathogenesis of these diseases has improved and new therapeutic targets have been identified by these studies.

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Hermans, Monique Maria Petra. « Structural and functional analysis of lysosomal [alpha]-glucosidase in relation to glycogen storage disease type II ». [S.l.] : Rotterdam : [The Author] ; Erasmus University [Host], 1993. http://hdl.handle.net/1765/13746.

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Wolf, Heike [Verfasser], Torben [Akademischer Betreuer] Lübke et von Mollard Gabriele [Akademischer Betreuer] Fischer. « The lysosomal storage disease fucosidosis : towards enzyme replacement therapy / Heike Wolf ; Torben Lübke, Gabriele Fischer von Mollard ». Bielefeld : Universitätsbibliothek Bielefeld, 2016. http://d-nb.info/1118688295/34.

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Fletcher, Jessica Louise. « Pathophysiology of canine fucosidosis ». Thesis, The University of Sydney, 2013. http://hdl.handle.net/2123/10420.

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Pathophysiology of Canine Fucosidosis by Jessica L Fletcher Understanding processes involved in the onset and progression of fucosidosis, a childhood neurodegenerative lysosomal storage disease, is critical to the development of effective treatment. Fucosidosis is caused by deficiency of the lysosomal hydrolase α-L-fucosidase due to inherited mutations in the FUCA1 gene. Canine fucosidosis, the only preserved animal model, was used to characterise preclinical pathophysiology, to establish potential markers of disease progression and test their capacity to measure responses to early therapy. Statistical interrogation of clinical data revealed that clinical signs are consistent from 12 months, and that rapid decline is signalled by sensory dysfunction from 18 months of age. Regression analysis against existing neuropathological data identified apoptosis and neuroinflammation as significant contributors to clinical dysfunction. Gene expression profiling of the frontal cortex at 16 weeks identified neuroinflammation to be prominent in fucosidosis pathogenesis (33% of differentially expressed genes) with both neuroinflammatory and mitochondrial-mediated pathways contributing to apoptotic cell death. Myelin genes were significantly downregulated, prompting investigation of oligodendrocyte populations in preclinical disease. Mature oligodendrocytes were reduced by 45-67%. Intracisternal enzyme replacement therapy from 8-16 weeks improved oligodendrocyte survival by 21%, and 29% of Purkinje neurons demonstrated reduced vulnerability to apoptosis. Neuroinflammatory cytokines and chemokines were investigated in cerebrospinal fluid to identify candidate biomarkers. MCP-1/CCL2 and KC/CXCL1 demonstrated potential, with significant increases with disease progression, and reductions with enzyme replacement therapy. Overall, these findings provide tools for disease management and a revised model of pathophysiology which can be used as a framework for future investigations into fucosidosis pathogenesis and therapy.

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Mütze, Ulrike, Friederike Bürger, Jessica Hoffmann, Helmut Tegetmeyer, Jens Heichel, Petra Nickel, Johannes R. Lemke, Steffen Syrbe et Skadi Beblo. « Multigene panel next generation sequencing in a patient with cherry red macular spot ». Universitätsbibliothek Leipzig, 2017. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-217944.

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Background: Lysosomal storage diseases (LSD) often manifest with cherry red macular spots. Diagnosis is based on clinical features and specific biochemical and enzymatic patterns. In uncertain cases, genetic testing with next generation sequencing can establish a diagnosis, especially in milder or atypical phenotypes. We report on the diagnostic work-up in a boy with sialidosis type I, presenting initially with marked cherry red macular spots but non-specific urinary oligosaccharide patterns and unusually mild excretion of bound sialic acid. Methods: Biochemical, enzymatic and genetic tests were performed in the patient. The clinical and electrophysiological data was reviewed and a genotype-phenotype analysis was performed. In addition a systematic literature review was carried out. Case report and results: Cherry red macular spotswere first noted at 6 years of age after routine screening myopia. Physical examination, psychometric testing, laboratory investigations aswell as cerebralMRIwere unremarkable at 9 years of age. So far no clinical myoclonic seizures occurred, but EEG displays generalized epileptic discharges and visual evoked potentials are prolonged bilaterally. Urine thin layer chromatography showed an oligosaccharide pattern compatible with different LSD including sialidosis, galactosialidosis, GM1 gangliosidosis or mucopolysaccharidosis type IV B. Urinary bound sialic acid excretion was mildly elevated in spontaneous and 24 h urine samples. In cultured fibroblasts, α-sialidase activity was markedly decreased to b1%; however, bound and free sialic acid were within normal range. Diagnosis was eventually established by multigene panel next generation sequencing of genes associated to LSD, identifying two novel, compound heterozygous variants in NEU1 gene (c.699CNA, p.S233R in exon 4 and c.803ANG; p.Y268C in Exon 5 in NEU1 transcriptNM_000434.3), leading to amino acid changes predicted to impair protein function. Discussion: Sialidosis should be suspected in patients with cherry red macular spots, even with non-significant urinary sialic acid excretion. Multigene panel next generation sequencing can establish a definite diagnosis, allowing for counseling of the patient and family.

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Del, Grosso Ambra. « Nanoparticle-mediated enzyme replacement therapy and autophagy modulation in Krabbe disease ». Doctoral thesis, Scuola Normale Superiore, 2020. http://hdl.handle.net/11384/85899.

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Zancan, Ilaria. « Understanding bone alterations in Gaucher disease using the zebrafish animal model : development of a novel pathogenetic paradigm for lysosomal storage disorders ». Doctoral thesis, Università degli studi di Padova, 2015. http://hdl.handle.net/11577/3424231.

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Lysosomal storage disorders (LSDs) are rare inherited metabolic disorders due to a deficiency of specific lysosomal enzymes or transporters. Almost 50 genetic disorders caused by deficiencies of lysosomal and non-lysosomal proteins are known nowadays, collectively with an incidence of ~1/7000 newborns in the worldwide population. Dysfunctions of such lysosomal enzymes or transporters can lead to several consequences that include incomplete degradation and/or recycling of intracellular molecules. Despite lysosomal proteins are present in almost all type of cells, the accumulation of undegraded substrates in LSDs affected patients is generally limited to cells, tissues and organs in which substrate turnover is high, leading to a wide spectrum of phenotypes and affected organs for different LSDs. Among these LSDs, the most common is the Gaucher disease (GD) with an incidence of 1 in 200.000 newborn in the worldwide population, a rate that increases to 1 in 850 in the population of Ashkenazi Jewish (Eastern Europen). This disorder is due to a deficiency of the lysosomal enzyme β-glucocerebrosidase (GBA) that in pathological conditions is not able to degrade its substrate, glucosylceramide, into glucose and ceramide. A hallmark of the disease is the presence of “Gaucher cells”, which are lipid laden-macrophages, in different tissues. Together with this characteristic, GD patients manifest hepatosplenomegaly, anemia, thrombocytopenia and severe bone disfunction such as osteonecrosis, osteopenia, bone crisis and fracture of long bones. GD patients can be classified in three different clinical subtype, based upon the presence and severity of neurological defects. Type I GD, also referred to as non-neuronophatic form, is the most frequent subtype, the symptoms manifest in adulthood and patients do not present neurological involvement. Type II GD patients, manifest severe neurological defects since early life stages and death occurs in childhood. Type III GD patients manifest less severe neurological defects when compared to type II GD and generally symptoms occur during childhood. This classification of the disease based on the absence or presence and severity of neurological defects is oversimplified. Nowadays, the broad spectrum of phenotypes and the recognition of overlap among these GD subtypes has led to the concept that this disorder is a continuum of phenotypes, ranging from the less severe (GD 1) to the more severe forms (GD2 and GD3). The most effective and well tolerated treatment available for this disorders is the enzyme replacement therapy (ERT), that consists in the administration of a recombinant enzyme able to restore the β-glucocerebrosidase functions. Despite its effectiveness on hepatosplenomegaly, anemia and thrombocytopenia symptoms, this therapy has a very limited effect on the recovery of the skeletal and neurological defects. Different GD murine model have been developed through the years to understand the pathogenetic mechanism behind these broad spectrum of phenotype. Despite the availability of all mice models mimicking differents Gaucher disease phenotypes, a completely reliable animal model does not exist and the pathogenic alterations occuring during early life stages can not be explored and elucidated yet. The aim of my PhD project was to investigate the bone pathogenetic mechanisms of Gaucher disease using a new animal model. To address this purpose, I’ve used a zebrafish model due to its easy manipulation and the transparency of the embryos that allow to follow all the early developmental stages. Using a morpholino-mediated knockdown approach and a stable genetic mutant line, I could investigate what was the effect of deficiency of the enzyme Gba1 during early stages of embryonic development. Moreover, the defects observed in these zebrafish models resemble the most common GD phenotypes, like hepatosplenomegaly, anemia and skeletal deformity, making them good models to study the molecular mechanisms of the bone phenotype. By analyzing the main molecular markers involved in bone development, as col10a1, runx2b and osx, I could point out that bone defects observed in these models are determined by an alteration in the osteoblasts differentiation process. Also, using zebrafish transgenic lines in which fluorescent proteins such as GFP are expressed under the control of specific promoters for major molecular signaling pathways, allowed me to identify alterations of Wnt and BMP due to deficiency of the enzyme β-glucocerebrosidase. In this work, characterization of a novel animal model for the study of Gaucher disease, highlighted that dysfunction of the lysosomal enzyme β-glucocerebrosidase can lead to alteration of major molecular signaling involved in the embryonic development, such as Wnt and BMP. Both these pathways have an important role in the formation and maintenance of osteoblasts lineage and early defects in these signal during embryogenesis could lead to defect in the differentiation program of mesenchymal stem cells progenitors. The results showed in this doctoral thesis, highlight for the first time the early involvement of two pathways, the Wnt and BMP signaling, behind the bone pathogenesis of Gaucher disease.
Le patologie da accumulo lisosomiale sono malattie metaboliche rare a carattere ereditario determinate da carenze di specifici enzimi o trasportatori lisosomiali, che hanno complessivamente un’incidenza di ~1/7000 nuovi nati nella popolazione mondiale. Al giorno d’oggi, almeno 50 disordini genetici sono causati da difetti in enzimi lisosomiali, che determinano l’incompleta degradazione e/o il riciclaggio di molecole a livello intracellulare con conseguente accumulo all’interno del lisosoma dei substrati enzimatici. Nonostante la presenza di proteine lisosomiali in quasi tutti i tessuti ed organi del corpo, l’accumulo del materiale non digerito è generalmente limitato solo a quelle cellule, tessuti od organi nel quale il ricambio del substrato enzimatico è molto elevato. Questa caratteristica determina differenti fenotipi per le varie patologie da accumulo lisosomiale, in quanto diversi organi o cellule possono essere coinvolti. Tra queste patologie, la malattia di Gaucher è la più frequente con un’incidenza di 1 su 200.000 nati vivi nella popolazione mondiale. La frequenza di questa patologia, aumenta drasticamente a 1 su 850 all’interno della popolazione degli ebrei Ashkenazi (Europa dell’Est). Questa malattia è causata da mutazioni a carico del gene che codifica l’enzima lisosomiale β-glucocerebrosidase (GBA). Tali mutazioni determinano l’incorretto ripiegamento della proteina enzimatica che, di conseguenza, non è in grado di degradare il suo substrato, la glucosilceramide, che si accumula nel lisosoma. Una delle caratteristiche di questa patologia è la presenza delle così dette “cellule di Gaucher”, ovvero macrofagi ad elevato contenuto di substrato non degradato, in differenti tessuti. Insieme alla presenza di questi macrofagi alterati, pazienti affetti dalla malattia di Gaucher presentano ingrossamento di fegato e milza (epatosplenomegalia), anemia, trombocitopenia e gravi disfunzioni a carico del sistema scheletrico quali osteonecrosi, riduzione della densità ossea, dolori cronici e frequenti fratture a carico delle ossa lunghe. Si possono distinguere tre sottocategorie di pazienti affetti da GD, generalmente classificati sulla base della presenza e gravità dei difetti a carico del sistema nervoso centrale (SNC). I pazienti affetti da GD di tipo I sono i più frequenti, hanno un’insorgenza della patologia in età tardiva ma non presentano coinvolgimento del SNC. I pazienti affetti da GD di tipo II, invece, manifestano i primi sintomi della malattia fin nei primi anni di vita e spesso i gravi difetti a carico del sistema nervoso possono portare alla morte del paziente. La terza categoria di pazienti, GD tipo III, manifestano i sintomi durante l’età infantile e i difetti neurologici sono meno gravi rispetto a quelli dei pazienti di tipo II. Al giorno d’oggi, questa classificazione basata sulla presenza di difetti neurologici è poco credibile a causa della presenza di fenotipi diversificati all’interno della stessa sottocategoria di pazienti. Il concetto di uno spettro continuo di fenotipi che variano dal meno grave (GD tipo I) al più severo (GD tipo II e III) è più appropriato per descrivere questa patologia. La terapia maggiormente utilizzata per il trattamento della sintomatologia di questa malattia è la terapia enzimatica sostitutiva (ERT), che consiste nella somministrazione di un enzima ricombinante in grado si sopperire alla mancanza della β-glucocerebrosidasi. Nonostante sia ben tollerata dalla maggioranza dei pazienti e sia in grado di far regredire l’ingrossamento di fegato e milza, l’anemia e la trombocitopenia, tale terapia ha effetti davvero limitati sui difetti scheletrici e neurologici. Nel corso degli anni, diversi modelli murini sono stati sviluppati per cercare di comprendere quali siano i meccanismi patogenetici della malattia che inducono questo ampio spettro di fenotipi. Sfortunatamente, la maggior parte di questi modelli animali non sono vitali o non manifestano tutti i difetti della malattia. Lo scopo del mio progetto di dottorato è stato quello di generare un nuovo modello animale per comprendere i meccanismi patogenetici a monte dei difetti ossei della malattia di Gaucher. A tal fine, mi sono avvalsa dell’uso dello zebrafish per la sua facilità di manipolazione e la trasparenza delle uova che permettono di seguire lo sviluppo embrionale fin dalle prime fasi. Utilizzando la tecnica del morfolino e avvalendomi di un modello genetico mutante stabile in zebrafish, ho potuto studiare quale fosse l’effetto della mancanza dell’enzima Gba1 fin dalle prime fasi dello sviluppo embrionale. Questi modelli, inoltre, manifestano insieme ai principali difetti di questa patologia, come l’ingrossamento di milza e fegato e l’anemia, anche i difetti a carico del sistema scheletrico, rendendoli dei buoni modelli per studiare i meccanismi molecolari a monte del fenotipo osseo. Analizzando i principali marcatori molecolari coinvolti nello sviluppo osseo, come col10a1, runx2b e osx, ho potuto evidenziare che i difetti ossei osservati in questi modelli sono determinati da un difetto nel processo di differenziamento degli osteoblasti. Inoltre, l’utilizzo di linee transgeniche di zebrafish nelle quali proteine fluorescenti, come la GFP, sono espresse sotto il controllo di promotori specifici per le principali vie di segnale molecolari, mi ha permesso di individuare alterazioni a carico delle vie di segnale Wnt e BMP in conseguenza alla carenza dell’enzima β-glucocerebrosidasi. Con questo lavoro di dottorato, la caratterizzazione di un nuovo modello animale per lo studio della malattia di Gaucher ha permesso di evidenziare che, disfunzioni a carico di un enzima lisosomiale come la β-glucocerebrosidasi, può determinare alterazioni in segnali molecolari molto importanti per lo sviluppo embrionale, quali il Wnt ed il BMP. Entrambe queste vie molecolari svolgono ruoli importanti nel processo di formazione e mantenimento degli osteoblasti e alterazioni precoci di questi segnali durante l’embriogenesi possono determinare difetti nel processo di differenziamento cellulare da progenitori mesenchimali staminali. I risultati ottenuti durante questo lavoro di dottorato, hanno evidenziato per la prima volta il precoce coinvolgimento di due vie di segnale molecolari, il Wnt e il BMP, nella patogenesi ossea della malattia di Gaucher.

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Al, Eisa Nada. « Evaluation of new therapies in Niemann-Pick type C disease ». Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:1538a0d6-b08e-444c-900d-de3ea3834ca5.

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Peterneva, Ksenia. « Determining the mechanism of pathogenesis of mucolipidosis type IV and related lysosomal storage disorders for development of novel therapies ». Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:321b1da6-0033-4230-b047-b643e5ea3e60.

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Mucolipidosis type IV (MLIV) is a rare, autosomal recessive, neurodegenerative, lysosomal storage disorder. MLIV is caused by mutations in a gene (MCOLN1) encoding a TRP channel family member known as Mucolipin 1 or TRPML1. TRPML1 is a lysosomal transmembrane protein that appears to be required for normal lysosomal pH regulation, recycling of molecules and membrane reorganisation including lysosomal biogenesis, fusion and exocytosis. The exact function of the channel is unknown but it is permeable to multiple ions including Ca²⁺, Na⁺ and K⁺, possibly also Fe²⁺ and Zn²⁺. How normal TRPML1 function regulates lysosomal processes is not clearly understood. Mutations in the MCOLN1 gene can lead to complete loss of TRPML1 function, partial loss of function or mislocalisation, all of which lead to lysosomal dysfunction, lysosomal lipid storage and ultimately neurodegeneration. The disease processes that lead to neurodegeneration are poorly understood and at present no therapy exists for MLIV. We have discovered that TRPML1 results in regulating lysosomal Ca²⁺ homeostasis that is the opposite of the Ca²⁺ dysregulation associated with Niemann-Pick type C disease (NPC). Our findings indicate that disrupted function of TRPML1 leads to enhanced Ca²⁺ release via the NAADP receptor, recently shown to be the lysosomal two-pore channel TPC2. This indicates that TRPML1 is not the NAADP receptor as suggested by others, indeed NAADP mediated Ca²⁺ release is enhanced with multiple NAADP induced lysosomal Ca²⁺ release events occurring in TRPML1 null cells compared to single releases in normal cells. This phenotype appears to be responsible for the cellular dysfunction associated with MLIV disease cells, enhanced lysosomal fusion, defective endocytosis and potentially even altered lysosomal pH. Several of these phenotypes are normalised by the NAADP receptor specific antagonist Ned-19. These findings illustrate that the NAADP receptor is central to MLIV disease pathology and may be a novel candidate for disease therapy.

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Mütze, Ulrike, Friederike Bürger, Jessica Hoffmann, Helmut Tegetmeyer, Jens Heichel, Petra Nickel, Johannes R. Lemke, Steffen Syrbe et Skadi Beblo. « Multigene panel next generation sequencing in a patient with cherry red macular spot : identification of two novelmutations in NEU1 gene causing sialidosis type I associated with mild to unspecific biochemical and enzymatic findings ». Molecular Genetics and Metabolism Reports 10 (2017) 1–4 doi:10.1016/j.ymgmr.2016.11.004, 2016. https://ul.qucosa.de/id/qucosa%3A15254.

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Background: Lysosomal storage diseases (LSD) often manifest with cherry red macular spots. Diagnosis is based on clinical features and specific biochemical and enzymatic patterns. In uncertain cases, genetic testing with next generation sequencing can establish a diagnosis, especially in milder or atypical phenotypes. We report on the diagnostic work-up in a boy with sialidosis type I, presenting initially with marked cherry red macular spots but non-specific urinary oligosaccharide patterns and unusually mild excretion of bound sialic acid. Methods: Biochemical, enzymatic and genetic tests were performed in the patient. The clinical and electrophysiological data was reviewed and a genotype-phenotype analysis was performed. In addition a systematic literature review was carried out. Case report and results: Cherry red macular spotswere first noted at 6 years of age after routine screening myopia. Physical examination, psychometric testing, laboratory investigations aswell as cerebralMRIwere unremarkable at 9 years of age. So far no clinical myoclonic seizures occurred, but EEG displays generalized epileptic discharges and visual evoked potentials are prolonged bilaterally. Urine thin layer chromatography showed an oligosaccharide pattern compatible with different LSD including sialidosis, galactosialidosis, GM1 gangliosidosis or mucopolysaccharidosis type IV B. Urinary bound sialic acid excretion was mildly elevated in spontaneous and 24 h urine samples. In cultured fibroblasts, α-sialidase activity was markedly decreased to b1%; however, bound and free sialic acid were within normal range. Diagnosis was eventually established by multigene panel next generation sequencing of genes associated to LSD, identifying two novel, compound heterozygous variants in NEU1 gene (c.699CNA, p.S233R in exon 4 and c.803ANG; p.Y268C in Exon 5 in NEU1 transcriptNM_000434.3), leading to amino acid changes predicted to impair protein function. Discussion: Sialidosis should be suspected in patients with cherry red macular spots, even with non-significant urinary sialic acid excretion. Multigene panel next generation sequencing can establish a definite diagnosis, allowing for counseling of the patient and family.

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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.

Thèses sur le sujet « Lysosomal storage diseases »

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