Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-06-16T14:06:04Z No. of bitstreams: 1 Cibele Gomes Bicalho Avaliação...2015.pdf: 1225744 bytes, checksum: 0e4067d0336141afabfd635be5750ec5 (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-06-16T14:06:14Z (GMT) No. of bitstreams: 1 Cibele Gomes Bicalho Avaliação...2015.pdf: 1225744 bytes, checksum: 0e4067d0336141afabfd635be5750ec5 (MD5) / Made available in DSpace on 2015-06-16T14:06:15Z (GMT). No. of bitstreams: 1 Cibele Gomes Bicalho Avaliação...2015.pdf: 1225744 bytes, checksum: 0e4067d0336141afabfd635be5750ec5 (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / Introdução: Mucopolissacaridose (MPS) é um conjunto de doenças raras causadas pela deficiência de enzimas lisossômicas levando ao acúmulo de glicosaminoglicanos (GAG) em órgãos e tecidos, responsáveis pelo quadro clínico multissistêmico, crônico e progressivo. O comprometimento auditivo é frequente. Objetivo: Avaliar manifestações auditivas de pacientes com MPS. Metodologia: Estudo descritivo, série de casos do comprometimento auditivo de pacientes com MPS. Foi realizada avaliação retrospectiva através de revisão de prontuário e avaliação prospectiva de dezembro de 2012 a outubro de 2014. Foram analisados a primeira e a última avaliação otorrinolaringológica (ORL) e audiológica realizada. Resultados: A principal queixa auditiva foi a hipoacusia. Aperdaauditiva estava presente em quase todos os pacientes, sendo que a perda auditiva condutiva foi a mais frequente, especialmente nos pacientes com MPS VI. Conclusão: A perda auditiva é muito frequente em pacientes com MPS, devendo o acompanhamento audiológicoser realizado precocemente. / Introduction: Mucopolysaccharidosis (MPS) is a set of rare diseases caused by deficiency of lysosomal enzymes leading to accumulation of glicosaminoglicanos (GAG) in tissues and organs responsible for the multisystemic clinical, chronic and progressive symptons. Objective: Todescribe the profile of otorhinolaryngological clinical examination and audiology tests of patients with MPS disease. Methods:Study of case series. The evaluation was performed, at the beginning, in 31 patients with MPS I, II, IIIA, IV and VI. Results: The most common hearing complaint was hearing loss and it was confirmed by audiology tests in almost 100% of patients, mostly with condutive hearing loss. Conclusions: It is important to evaluate the complaints, physical examination and audiology tests in MPS disease. Otorhinolaryngologist should be part of professional group that follow these patients in order to better monitor their hearing and provide early hearing rehabilitation.

Sleep disturbance and behavioural difficulties are both prevalent problems in the intellectual and developmental disability population and can have a significant impact on quality of life for the individual and their family. This thesis investigated sleep, behaviour and circadian rhythm functioning in children with intellectual and developmental disabilities, and is presented in three sections. The first two papers have been prepared in accordance with the author guidelines of the journals proposed for submission, excluding tables and figures for ease of reading. The first paper is a systematic review of the literature examining the relationship between sleep disturbance and challenging behaviour in children with intellectual and developmental disabilities. 15 studies were included in the review and overall there were consistent findings of an association between the presence of sleep disruption and increased behavioural difficulties. A causal relationship could not be inferred due to the cross-sectional methodology of studies. Other factors, such as parental wellbeing, child level of intellectual disability and comorbidity of physical health conditions, need to be considered to understand the complexity of this relationship. Children with the neurodevelopmental disorder mucopolysaccharidosis type III (MPS III or Sanfilippo syndrome) present with high rates of sleep disturbance and challenging behaviour. The second paper investigates circadian rhythm functioning and activity levels in children with MPS III, compared to typically developing controls. Objective measurement of circadian rhythm and activity levels was obtained through actigraphic recording for 7-10 days. Children with MPS III had increased fragmentation of circadian rhythm, less stability of rhythm in relation to external cues and a differential pattern of activity across the day compared to controls. Overall, results were indicative of a disruption of circadian rhythm function in children with MPS III. The implications for clinical practice and future research are discussed. The third paper provides a critical appraisal of the overall research process, including further consideration of the strengths and limitations, implications for clinical practice, wider context of the research and personal reflections. An account of the project that was originally proposed with the MPS III population is also presented, alongside reflections on its termination.

Deficiency in β-glucuronidase activity (EC 3.2.1.31) leads to the lysosomal storage disease mucopolysaccharidosis type VII not only in humans but also in a recently discovered murine mutant, the gus^mps/gus^mps mouse. Clinical and pathologic abnormalities common to the human and mouse phenotypes include shortened life span, dwarfism, dysmorphic facial features, skeletal deformities, corneal clouding, mental retardation and abnormal lysosomal storage material in the brain and peripheral organs. In the first part of this thesis, neonatal gus^mps/gus^mps mice and their normal littermates were transplanted with syngeneic normal bone marrow. Neurological function was then evaluated with two behavioral tests: the grooming test, a developmentally regulated and genetically based activity, and the Morris water maze test, which assessed spatial learning abilities. The results of these tests indicated that the behavioral deficits in the mutant mice were not restored to normal. Treated normal mice also showed significant functional deterioration, indicating the detrimental consequence of this therapy in the neonatal period. The second part of this thesis focused on a novel approach to somatic gene therapy using microcapsules. A non-autologous fibroblast cell line engineered to secrete high levels of β-glucuronidase was enclosed in perm-selective and immuno- protective microcapsules and implanted into the peritoneal cavity of gus^mps/gus^mps mice. During the 4 weeks of therapy, the biochemical and histological abnormalities of the mutant mice had significantly improved. β-Glucuronidase activity was restored to >50% of normal in the plasma and 11.3%-65.8% in the kidney, liver and spleen. No significant activity was found in the brain. As well, the secondary elevations of other lysosomal enzymes such as β-hexosaminidase and α-galactosidase had decreased in the kidney, liver, and spleen. Urinary glycosaminoglycan content had decreased in the treated mutants indicating that the β-glucuronidase was exerting a therapeutic effect. However, after three and a half weeks of therapy, the treated mutants became severely ill and developed haemorrhagic ascites. Since normal mice treated with similar microcapsules showed no adverse effects, we hypothesized that an immune response had been generated against the foreign protein (β-glucuronidase) by the mutants, leading to the high morbidity. Thus in spite of the biochemical and histological correction observed after bone marrow transplantation and somatic cell gene therapy, the long term efficacy of these treatments needs to be further evaluated. / Thesis / Master of Science (MS)

Introdução: Mucopolissacaridose IVA é uma doença lisossômica, autossômica recessiva, causada pela deficiência da enzima N-acetilgalactosamina-6-sulfatase. É uma doença rara e a incidência varia de 1:76.000 a 1:640.000 recém-nascidos vivos. Até o momento 319 diferentes mutações causadoras da doença já foram identificadas, o que demonstra a ampla variabilidade genética. Objetivo: Caracterizar o genótipo de pacientes com MPS IVA, analisar 6 polimorfismos intragênicos e identificar os haplótipos presentes em nossos pacientes, através do estudo molecular do gene GALNS. Métodos: O estudo foi realizado em 45 pacientes provenientes das regiões Nordeste, Sudeste, e Sul do Brasil, com diagnóstico bioquímico confirmado para MPS IVA. A análise molecular foi realizada através de PCR seguida de sequenciamento, pelo método de Sanger, a fim de identificar as mutações causadoras da doença. Para o estudo de haplótipos foram analisados 6 polimorfismos intragênicos através de PCR em Tempo Real, pelo método Taqman, em pacientes e controles. Resultados: A análise do gene GALNS, nos 45 pacientes, permitiu a identificação de 18 diferentes mutações, e a caracterização de 6 haplótipos distintos. Das 18 mutações encontradas, 5 apresentaram uma alta frequência (p.Ser341Arg, p.Arg386Cys, p.Gly301Cys, p.Arg94Leu e p.Gly116Ser), além disso, foram encontradas 4 novas mutações em outros três pacientes (p.Gly115Arg, p.Asn45Gly, p.Thr394Ala e c.759-2A>G). Dentre as mutações encontradas com maior frequência, a mutação p.Ser341Arg foi identificada em um maior número de pacientes, sendo a maioria proveniente da região Nordeste. Além disso, todos os pacientes com esta mutação apresentaram um único haplótipo. Conclusão: Os resultados obtidos permitiram a identificação de 18 mutações dentre elas 4 novas mutações. A alta frequência da mutação p.Ser341Arg no Nordeste do Brasil, principalmente no estado da Paraíba nos leva a inferir um possível efeito fundador da doença. Esta mutação foi observada somente na população brasileira e todos os pacientes com mutação em homozigose apresentaramum único haplótipo. Estas análises são importantes para identificar portadores nas famílias, para diagnóstico pré-natal, e também como forma de identificar uma origem comum em mutações frequentes em determinadas populações. / Background: Mucopolysaccharidosis IVA is an autosomal recessive lysosomal disease, caused by deficiency of N-acetilgalactosamina-6-sulfatase. It is a rare disease and the incidence ranges from 1: 76,000 to 1:640,000 live births. To date 319 mutations have been identified in this gene, demonstrating the wide variability of disease causing mutations. Objective: Analyze and characterize the genotype of patients with MPS IVA, through molecular analysis of GALNS. Methods: Molecular analysis of 45 patients with confirmed biochemical diagnosis for MPS IVA was performed. Mutation analysis was performed by PCR followed by Sanger sequencing. Haplotype analysis was performed using 6 intragenic polymorphisms by Real-Time PCR. Results: In this study we found 18 different mutations among 45 Brazilian patients and identified 5 common mutations (p.Ser341Arg, p.Arg386Cys, p.Gly301Cys, p.Arg94Leu e p.Gly116Ser). Four novel mutations were also identified through molecular analysis, including: p.Gly115Arg, p.Asn45Gly, p.Thr394Ala e c.759-2A>G. Patients are distributed in Northeast, Southeast and South regions of Brazil. Six different haplotypes were identified among patients. The p.Ser341Arg mutation showed the highest frequency, and most patients are located in the Northeast, additionally, all patients with this mutation show the same haplotype.Conclusion: These analyzes are important to identify carriers in families, for prenatal diagnosis, and in order to identify the mutation origin when certain recurrent mutation is associated with the same haplotype. In this study, we observed a high frequency of p.Ser341Arg mutation in Northeast, mainly in the state of Paraíba. This mutation was detected with higher frequency among patients, and showed only a haplotype. This mutation is unique for the Brazilian population and thus, we could suggest that a possible founder effect for this mutation could exist.

Base teórica: A Mucopolissacaridose II (MPS II) é uma doença genética recessiva ligada ao X causada por mutações no gene IDS. Como consequência, há acúmulo dos glicosaminoglicanos (GAGs) no lisossomo, fato que é responsável pelo fenótipo de MPS II. Anormalidades articulares e ósseas são conhecidas nos pacientes com MPS II e os tratamentos existentes não são eficientes para sanar tais anormalidades, portanto, realizamos este estudo de caracterização da doença articular e óssea, buscando evidenciar possíveis mecanismos responsáveis pela progressão da doença. Objetivo: Avaliar a progressão das alterações osteoarticulares em animais com MPS II dos dois aos oito meses de idade. Métodos: Foram utilizados camundongos nocaute B6N.Cg-Idstm1Muen/J, adquiridos do Jackson’s Lab. Os machos foram genotipados para compor o grupo controle (normal) ou o grupo de animais com MPS II. Ambos foram avaliados aos 2, 4, 6 e 8 meses de idade. Foi realizada análise histológica da articulação tíbio-femural, avaliando presença de infiltrado inflamatório, reabsorção óssea, reabsorção cartilaginosa e proliferação fibrocartilaginosa. Também foi realizada a mensuração do tamanho total da placa de crescimento e suas zonas e avaliação de anormalidades ósseas mediante exame de imagem por Raio-X dos ossos fêmur e zigomático. Resultados: Nos animais MPS II foi observado que o focinho era menos afilado (mais arredondado) e, em comparação com os animais controle, os animais MPS II apresentaram peso significativamente maior a partir dos 4 meses de idade. O escore histológico teve como principal característica a presença de reabsorção cartilaginosa, presente em 80% (4/5 animais) dos animais aos 8 meses, outras anormalidades encontradas neste tempo foram presença de infiltrado inflamatório (2/5 animais aos 8 meses) e proliferação fibrocartilaginosa (1/5 animais) Não houve diferença significativa entre animais normais e MPS II no tamanho das zonas de cartilagem da placa de crescimento ósseo. As medidas em diâmetro do osso zigomático apresentaram-se significativamente superiores nos animais MPS II aos 4, 6 e 8 meses. Quanto ao comprimento do fêmur não houve diferença significativa entre os grupos. Já, na medida da espessura do fêmur, os animais MPS II do grupo de 6 meses de idade mostraram diferença significativa. Conclusões: Anormalidades na articulação tíbio-femural foram detectadas nos animais aos 8 meses de idade. Não foram encontradas anormalidades óbvias na estrutura da placa de crescimento. Foi observado aumento na espessura do fêmur e do zigomático nos animais MPS II, sem alterações do tamanho do fêmur. / Background: Mucopolysaccharidosis II (MPS II) is a recessive X-linked genetic disease caused by mutations in the IDS gene. Consequently, there is an accumulation of glycosaminoglycans (GAGs) in the lysosome, fact that is responsible by the MPS II phenotype. Joints and bone abnormalities are known in MPS II patients and existing treatments are not efficient in correcting these abnormalities. Therefore, this study was performed to evidence bone and joint disease description in the animal model, searching for mechanisms responsible for disease progression. Objective: To evaluate the progression of osteoarticular changes in animals with MPS II from two to eight months of age. Methods: Male animals from the MPS II colony (B6N.Cg-Idstm1Muen/J) were genotyped to form the control (normal) group or MPS II group. Both groups were evaluated at the 2, 4, 6 or 8 months. Histological analysis of knee joint (presence of inflammatory infiltrate, bone resorption, cartilaginous reabsorption and fibrocartilaginous proliferation), measurement of total growth plate size and its zones, and evaluation of bone abnormalities by X-ray imaging of the femur and zygomatic bones were performed. Results: MPS II mice presented progressive abnormal features, such as a more rounded snout and a significative increased weight from 4 months of age. The main histological alteration was the presence of cartilage reabsorption, present in 80% (4/5 animals) of the eighth-month old animals. Other abnormalities found at this period were the presence of inflammatory infiltrate (2/5 animals at the eighth months old) and fibrocartilaginous proliferation (1/5 animals). There was no significant difference in the growth plate between normal and MPS II animals. The zygomatic bone diameter was increased in MPS II at fourth, sixth and eighth months There were no significant differences in femur length between groups. Thickness of the femur was increased in MPS II at six months. Conclusions: Abnormalities in the joint were detected in the animals at 8 months of age. No obvious abnormalities were found in the growth plate structure. An increase in femur and zygomatic thickness was observed in MPS II animals, with no changes in femur size.

A mucopolissacaridose tipo I (MPS I) é causada pela deficiência de alfa-L-iduronidase (IDUA), responsável pelo catabolismo de glicosaminoglicanos (GAGs), levando ao acúmulo multissistêmico de sulfato de heparano e dermatano. Este estudo tem por objetivo avaliar o potencial de sistemas lipídicos nanoestruturados como carreadores do plasmídeo do sistema CRISPR/Cas9 e um vetor doador da sequência do gene IDUA/Idua para edição gênica em fibroblastos de pacientes e em modelo murino de MPS I. Foram produzidos lipossomas (DOTAP, DOPE e DSPE-PEG) e nanoemulsões (e TCM) por homogeneização à alta pressão e microfluidização. O DNA foi associado às formulações por adsorção, ou por encapsulamento dos complexos pré-formados DNA/DOTAP no núcleo oleoso da nanoemulsão. A eficiência de transfecção dos complexos foi avaliada em fibroblastos de pacientes MPS I e ocorreu um aumento significativo da atividade de IDUA em 2, 15 e 30 dias após os tratamentos, que promoveu uma redução na quantidade de lisossomos nos fibroblastos tratados. A caracterização físico-química de formulações produzidas por microfluidização complexadas a somente um plasmídeo ou juntamente com um oligonucleotídeo foi verificada e pode-se afirmar que a capacidade de complexação e transfecção depende diretamente do tipo celular e da relação de cargas, e não há implicações quanto ao tamanho das sequências de ácidos nucleicos. Camundongos MPS I receberam os complexos lipossomais por injeção hidrodinâmica e sua biodistribuição foi detectada principalmente no pulmão, coração e fígado. A atividade sérica de IDUA normal aumentou em cerca de 6% e foi mantida por seis meses. A atividade aumentada no pulmão, coração, fígado e rim após eutanásia promoveu redução dos GAGs na urina e nos mesmos tecidos, corroborando com as análises histológicas. Em um estudo em andamento, foi realizada uma investigação mais aprofundada do efeito do tratamento lipossomal na morfologia óssea, sistemas cardiovascular e respiratório, e funções cerebrais dos animais tratados. A análise ecocardiográfica demonstrou uma melhora na hipertrofia e contratilidade do coração, porém não houve melhora na espessura das válvulas. O diâmetro da aorta foi similar ao de animais normais, porém as quebras de elastina ficaram entre o grupo normal e o não tratado. A morfologia facial dos animais tratados foi intermediária, assim como a espessura do osso zigomático. Entretanto, o osso femoral demonstrou espessura comparável ao normal. Já a resistência pulmonar apresentou uma tendência de redução nos animais tratados em relação aos animais MPS I. O conjunto de resultados demonstra o potencial das nanoestruturas lipídicas co-complexadas com o plasmídeo CRISPR/Cas9 e um vetor doador da sequência IDUA/Idua para terapia gênica da MPS I. / Mucopolysaccharidosis type I (MPS I) is caused by the deficiency of alpha-L-iduronidase (IDUA), responsible for the catabolism of glycosaminoglycans (GAGs), leading to multisystemic accumulation of heparan and dermatan sulfate. This study aims to evaluate the potential of lipid-based nanostructures as carriers of the CRISPR/Cas9 plasmid and a vector donor of the IDUA/Idua sequence for gene editing in patients’ fibroblasts and in a murine model of MPS I. Liposomes (DOTAP, DOPE, and DSPE-PEG) and nanoemulsions (also MCT) were produced through high-pressure homogenization or microfluidization. DNA was associated with liposomes and nanoemulsions by adsorption or by encapsulation of DNA/DOTAP preformed complexes in the oil core of nanoemulsions. The transfection efficiency of complexes was evaluated in fibroblasts from MPS I patients and a significant increase in IDUA activity was demonstrated at 2, 15, and 30 days after treatments. It was also possible to observe a significant reduction in lysosomal amount in treated fibroblasts. The physicochemical characterization of liposomes and nanoemulsions produced through microfluidization complexed with a single plasmid or along with an oligonucleotide has been verified and it can be stated that the complexing and transfection capacity of the complexes depends directly on the cell type and the charge ratio, and there are no implications of the size of the nucleic acid sequences. MPS I mice received the liposomal complexes by hydrodynamic injection and their immediate biodistribution was detected mainly in the lung, heart, and liver. An increase of about 6% in normal serum IDUA activity was maintained for six months, in addition to increased lung, heart, liver, and kidney activity after euthanasia. The enhanced enzymatic activity promoted a significant GAGs reduction in urine and in the same tissues, corroborating with histological analysis. In an ongoing study, a deeper investigation was carried out on the effect of liposomal treatment on bone morphology, cardiovascular and respiratory systems, and brain function. The echocardiographic analysis showed an improvement in the parameters of hypertrophy and contractility of the heart, but there was no improvement in heart valves. Aorta diameter was similar to that of normal animals, but elastin breaks were between the normal and untreated groups. Facial morphology of treated animals was intermediate, as well as the analysis of zygomatic bone thickness. However, femoral bone showed thickness comparable to normal animals. Lung resistance, on the other hand, showed a tendency to reduction in treated animals when compared to MPS I. The set of results demonstrates the potential of the co-complexed lipid nanostructures with the CRISPR/Cas9 plasmid and a donor vector of the IDUA/Idua sequence for MPS I gene therapy.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / As Mucopolissacaridoses (MPS) constituem um grupo de doenÃas raras, caracterizadas por deficiÃncias enzimÃticas que resultam em bloqueio na degradaÃÃo dos glicosaminoglicanos. O objetivo deste estudo foi avaliar a situaÃÃo periodontal de todos os pacientes com Mucopolissacaridose, atendidos no Hospital Infantil Albert Sabin e no Hospital Geral CÃsar Cals, FortalezaâCE. Foram avaliados 18 pacientes, por um cirurgiÃo-dentista previamente calibrado, que registrou os Ãndice de Pernus, Ãndice de Placa Bacteriana VisÃvel, Ãndice de Sangramento Gengival, PSR e Ãndice CPO-D/ceo-d. Em relaÃÃo ao volume gengival, observou-se que 72,2% dos pacientes apresentaram Ãndice de Pernus nos escores 1, 2 ou 3, que corresponde à presenÃa de hiperplasia gengival. Quando se avaliaram os cuidados com relaÃÃo à higiene oral dos pacientes, observou-se que quanto ao Ãndice de Sangramento Gengival 77,8% dos pacientes apresentaram Ãndice maior que 10%, 66,6% dos pacientes apresentaram Ãndice de Placa Bacteriana VisÃvel maior que 40%. Com relaÃÃo ao PSR 94,4% dos pacientes apresentou escore 1, correspondendo a sangramento, e 5,6% escore 2, correspondendo à presenÃa de cÃlculo alÃm de sangramento, e quanto ao CPO-D/ceo-d 61,1% apresentou Ãndice maior que 5. De uma maneira geral, observou-se que a situaÃÃo periodontal dos pacientes portadores de mucopolissacaridose à uma situaÃÃo que inspira maior cuidado, mostrando caracterÃsticas prevalentes como o aumento gengival, alÃm de altos Ãndices de Sangramento Gengival, Placa Bacteriana VisÃvel e CPO-D/ceo-d. / The Mucopolysaccharidosis (MPS) are a group of rare diseases characterized by enzyme deficiency that results in nondegradation of the glicosaminoglicans (GAGs). The aim of this study was to evaluate the periodontal status of all the patiente with MPS that are treated at the Hospital Infantil Albert Sabin and at the Hospital Geral CÃsar Cals, in Fortaleza, CearÃ, Brazil. A number of 18 patients were evaluated by a dentist that was previously calibrated, which registered the Pernus Index (IP), Visible Bacterium Plaque Index (IPB-V), Gingival Bleeding Index (ISG), PSR and Index that shows number of teeth that showed cavities, tooth loss or restoration (CPO-D/ceo-d). In relation to the gingival volume it was observed that 72,2% of the patients showed that the gingival was not in itÂs regular size. On the evaluation of dental hygiene, it was observed that 77,8% of the patients showed ISG greater than 10% and 66,6% of the patients showed IPB-V greater than 40%. In relation to PSR, 94,4% of the patients showed scored 1 which means gingival bleeding and 5,6% showed score 2 which means calculus besides gingival bleeding. And in relation to CPO-D/ceo-d 61,1% of the patients showed score greater than 5. In a general way it was observed that the periodontal status of the patients is a situation that requires more care, showing characteristics like gingival hyperplasias besides high scores of ISG, IPB-V and CPO-D/ceo-d.

A mucopolissacaridose tipo I (MPS I) é uma doença autossômica recessiva causada pela deficiência da enzima α-L-iduronidade (IDUA). Essa deficiência resulta no acúmulo de glicosaminoglicanos levando a diversas manifestações clínicas. A microencapsulação de células recombinantes que superexpressam IDUA tem sido considerada uma estratégia promissora para o tratamento de MPS I. Neste contexto, o presente estudo teve por objetivo a otimização da encapsulação de células BHK (Baby Hamster Kidney) superexpressando IDUA em microcápsulas de alginato revestidas com poli-L-lisina (PLL) utilizando-se um extrusor eletrostático. Em uma primeira etapa, um estudo de otimização das microcápsulas de alginato (MC-A) foi realizado por meio de um desenho experimental do tipo Box-Behnken (software Mini-Tab®) que permitiu avaliar simultaneamente a influência da voltagem (kV), fluxo alginato/células (mL/h) e concentração de alginato (%) sobre o tamanho das microcápsulas e a atividade de IDUA. Após, as microcápsulas foram revestidas sequencialmente com PLL e alginato (MC-APA) com o objetivo de aumentar a sua estabilidade. Nas condições experimentais empregadas, MC-A e MC-APA apresentaram-se monodispersas (span < 1,22) com um diâmetro médio inferior a 350 μm, determinado por difração a laser. O revestimento alterou a morfologia das microcápsulas (microscopia eletrônica de varredura) e a sua resistência mecânica (analisador de textura), sendo observado um aumento de cerca de 6 vezes na força necessária para compressão das mesmas. O revestimento final pelo alginato (MC-APA) parece ter sido parcial de acordo com as análises de infravermelho por transformada de Fourier com refletância atenuada. Em uma última etapa, a atividade enzimática foi avaliada em modelo murino MPS I após implante subcutâneo de MC-APA. Foi observado um aumento significativo da atividade de IDUA na pele, após 30 dias de tratamento. Nas análises histológicas foi observado um infiltrado inflamatório no local da aplicação que não impediu a liberação da enzima nas condições avaliadas. No seu conjunto, esse estudo demonstra a potencialidade das MC-APA para a liberação local de IDUA. / Mucopolysaccharidosis type I (MPS I) is an autosomal recessive disorder caused by the deficiency of α-L-iduronidase (IDUA). This deficiency results in the accumulation of glycosaminoglycans leading to various clinical manifestations. The microencapsulation of recombinant cells overexpressing IDUA has been considered as a promising strategy for the treatment of MPS I. In this context, the present study aimed to optimize the encapsulation of BHK cells overexpressing IDUA in poly-L-lysine (PLL) coated alginate microcapsules using an electrostatic extruder. In a first step, a Box-Behnken experimental design (Mini-Tab® software) was carried out for the optimization of the alginate microcapsules (MC-A), which allowed to evaluate simultaneously the influence of voltage (kV), alginate/cell flow (mL/h) and alginate concentration (%) on the size of the microcapsules and IDUA activity. Thereafter, the microcapsules were sequentially coated with PLL and alginate (MC-APA) in order to increase their stability. In the experimental conditions used, MC-A and MC-APA were monodisperse (span <1.22) with an average diameter of less than 350 μm, determined by laser diffraction. The coating modified microcapsules morphology (scanning electron microscopy) and their mechanical resistance (texture analyzer), being observed a six-fold increase in the required force for their compression. The final alginate coating (MC-APA) appears to have only partially coated the microcapsules, according to the attenuated total reflectance Fourier transform infrared spectroscopy analyses. In a final step, the enzymatic activity was evaluated in a MPS I murine model after subcutaneous implantation of MC-APA. A significant increase in IDUA activity was observed in the skin at 30 days after treatment. Histological analszes revealed an inflammatory infiltrate at the application site, which did not prevent the release of the enzyme under the evaluated conditions. Overall, this study demonstrates the potentiality of MC-APA for the local release of IDUA.

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

Mucopolysaccharidosis type IIIA (MPS IIIA) is an autosomal-recessively inherited disorder caused by the deficiency of lysosomal sulphamidase (NS) enzyme activity, resulting in the accumulation of the glycosaminoglycan (GAG) heparan sulphate (HS). MPS IIIA patients experience progressive and severe neurological deterioration with death usually occurring in the mid-late teenage years. A naturally-occurring mouse model of MPS IIIA has been characterised and the biochemical, histological and behavioural changes closely parallel the human condition. In order to treat the neurological effects of MPS IIIA, it is anticipated that a continual supply of replacement enzyme to affected cells will be required. Consequently, this study aimed to evaluate the efficacy, longevity and safety of gene therapy as a potential treatment for MPS IIIA. Canine adenoviral vectors (CAV-2) were selected on the basis of several important properties. They are non-integrating, are predicted to be less immunogenic in humans than human-derived viral vectors and mediate transgene expression for at least 1 year in vivo. An E1-deleted (∆E1) CAV-2 vector, CAV-NS, co-expressing recombinant human NS (rhNS) and Green Fluorescent Protein (GFP) was constructed and purified. In vitro testing revealed rhNS produced by CAV-NS significantly decreased sulphated GAG storage in human MPS IIIA fibroblasts in a mannose-6-phosphate-dependent manner. Preliminary studies in young adult guinea pigs with CAV-GFP demonstrated widespread GFP expression in the absence of a humoral response. In contrast, minimal GFP expression was found in CAV-injected adult mice due to formation of neutralising antibodies against the CAV-2 capsid. Consequently, intraventricular delivery of CAV-NS was evaluated in newborn mice at various doses. Widespread and dose-dependent GFP expression was observed and the optimal dose for large-scale studies was determined to be 109 CAV-NS particles/hemisphere. Antibodies against CAV-2, rhNS or GFP were not detected. Concurrently, the cognitive function and anxiety-related behaviours of unaffected and MPS IIIA mice were evaluated. MPS IIIA mice had significantly impaired memory and spatial learning in the Morris Water Maze (16-wks) and reduced anxiety in the Elevated Plus Maze (18-wks) when compared to unaffected animals. In a large therapeutic assessment trial, newborn MPS IIIA or unaffected mice received 109 particles of CAV-NS, saline or remained uninjected. GFP expression was visualised for at least 20-wks post-injection. Reductions in the vacuolation of ependymal and choroidal cells of the lateral ventricle and the cerebral cortex of treated MPS IIIA animals were observed in some GFP-positive (and presumably rhNS-expressing) regions. Furthermore, improvements in reactive astrogliosis, but not in the number of activated microglia, were measured in CAVNS- treated MPS IIIA mice. However, insufficient CAV-NS-mediated rhNS expression was generated to improve functional changes as assessed by a behavioural test battery (motor function, open field activity, Elevated Plus Maze, Morris Water Maze), potentially due to chronic inflammatory responses against the CAV-2 vector. Collectively, these data suggest that early intervention with ∆E1 CAV-NS gene therapy was able to improve several components of neuropathology in MPS IIIA animals but was unable to significantly alter the clinical progression of murine MPS IIIA. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1295758 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2007