Dissertationen zum Thema „Mucopolysaccharidosis Gene therapy“

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.

La Mucopolisacaridosis tipus II (MPSII), o síndrome de Hunter, és una malaltia d’acumulació lisosòmica d’herència recessiva lligada al cromosoma X i està causada per la deficiència de l’Iduronat-2-sulfatasa (IDS), enzim que actua en la via de degradació dels glicosaminoglicans (GAGs) heparan sulfat (HS) i dermatan sulfat (DS). Aquests GAGs no degradats s’acumulen als lisosomes de manera patològica, causant disfunció cel·lular. La forma més severa i també més prevalent de la MPSII es caracteritza per una neurodegeneració crònica i progressiva del sistema nerviós central (SNC) acompanyada de disfunció multisistèmica. Com a conseqüència, els pacients de MPSII solen morir durant la segona dècada de vida. Actualment, la única opció terapèutica disponible pels pacients amb Hunter és la teràpia de substitució enzimàtica (TSE), la qual consisteix en la infusió setmanal de l’enzim recombinant. No obstant, degut a la presència de la barrera hemato-encefàlica, la TSE no és efectiva en la correcció del deteriorament neurològic, a més de presentar altres inconvenients. Per tant, el desenvolupament d’una teràpia eficient pel tractament de la patologia neurodegenerativa característica de la MPSII és una necessitat mèdica no coberta. La teràpia gènica in vivo basada en l’administració de vectors virals derivats dels virus adeno-associat (AAV) representa una alternativa atractiva pel tractament d’aquesta malaltia, ja que ofereix la possibilitat d’obtenir benefici terapèutic de per vida després d’una única administració del producte terapèutic. Així doncs, la present tesi doctoral s’ha centrat en el desenvolupament d’una estratègia de teràpia gènica per a la MPSII basada en l’administració de vectors directe al líquid cefaloraquidi (LCR) amb la finalitat de tractar simultàniament tant la patologia neurologia com la somàtica de la malaltia. Mitjançant un procediment poc invasiu, es van administrar vectors AAV de serotip 9 (AAV9) que contenien el gen murí Ids (AAV9-Ids) al LCR de ratolins MPSII de 2 mesos d’edat, els quals ja presentaven una patologia ben establerta tant a nivell del SNC com a nivell somàtic. Transcorreguts 4 i 8 mesos després de l’administració, es va avaluar l’eficàcia del tractament en contrarestar la patologia de la MPSII. A nivell del SNC, l’augment d’activitat enzimàtica IDS obtinguda mitjançant el tractament va donar lloc a la completa correcció de les lesions lisosomals característiques a la MPSII. El tractament també va donar lloc a la correcció de la disfunció lisosomal del SNC, a la normalització de l’expressió gènica del cervell i a la eradicació de la neuroinflamació característica de la malaltia. A més, mitjançant l’administració al LCR, vectors AAV9-Ids també van transduir el fetge, convertit aquest òrgan en una font de la proteïna terapèutica a nivell perifèric. En conseqüència, l’enzim IDS produït al fetge de ratolins MPSII tractats va donar lloc a la correcció de la patologia somàtica. A més, la reversió de la patologia observada en aquells teixits somàtics no transduits pel vector AAV9-Ids va evidenciar el mecanisme de correcció creuada aconseguit mitjançant l’enzim IDS circulant. A banda d’aquests efectes, el tractament també va comportar una normalització de les alteracions de comportament característiques de la malaltia, així com també un augment significatiu de la supervivència dels ratolins MPSII. L’eficàcia obtinguda mitjançant l’administració de vectors AAV9 que contenien la seqüència codificant humana IDS també es va avaluar en ratolins MPSII. Després de 1,5 mesos de tractament, es va observar un increment en l’activitat IDS al cervell, fetge i sèrum, fet que va donar lloc a la correcció del contingut de GAGs tant a nivell del SNC com a nivell somàtic. Conjuntament, els resultats obtinguts en aquest treball recolzen la translació clínica de l’aproximació de teràpia gènica basada en l’administració al LCR de vectors AAV9-IDS pel tractament de pacients de Hunter amb afectació neurològica.
Mucopolysaccharidosis type II (MPSII), or Hunter syndrome, is an X-linked recessive lysosomal storage disease (LSD) caused by the deficiency in Iduronate-2-sulfatase (IDS), an enzyme involved in the stepwise degradation of the glycosaminoglycans (GAGs) heparan sulfate (HS) and dermatan sulfate (DS). The pathological accumulation of undegraded HS and DS in the lysosomes leads to cell dysfunction, causing severe neurologic and somatic disease. The most severe and most prevalent form of Hunter syndrome is characterized by chronic and progressive neurodegeneration of the central nervous system (CNS) and multisystem dysfunction; patients usually die during the second decade of life. To date, weekly intravenous enzyme replacement therapy (ERT) constitutes the only approved therapeutic option for MPSII. However, the inability of recombinant IDS to efficiently cross the blood-brain barrier (BBB) limits the efficacy of ERT in treating neurological symptoms. The therapy has several other drawbacks. Thus, an efficient therapy for the treatment of the neurodegeneration of MPSII disease represents a highly unmet medical need. In vivo gene therapy with adeno-associated vectors offers the possibility of lifelong therapeutic benefit following a single administration. Therefore, the present work was focused on the development of a new gene therapy approach for MPSII based on the delivery of vectors to the cerebrospinal fluid (CSF) and aimed at counteracting simultaneously the neurological and somatic pathology characteristic of the disease. Adeno-associated virus serotype 9 vectors (AAV9) containing the murine Ids gene were administered through a minimal invasive procedure to the CSF of 2-month-old MPSII mice, which already presented established pathology. The efficacy of AAV9-Ids vectors to counteract MPSII pathology after a single intra-CSF injection was evaluated 4 and 8 months after treatment. AAV9-mediated Ids gene transfer led to a significant increase in IDS activity throughout the encephalon, which resulted in full reversion of lysosomal storage lesions. In addition, correction of lysosomal dysfunction in the CNS, normalization of brain transcriptomic signature and disappearance of neuroinflammation were achieved after gene transfer. Moreover, after AAV9-Ids delivery to the CSF, vectors also transduced the liver, providing a peripheral source of the therapeutic protein that corrected storage pathology in visceral organs of treated MPSII mice. The reversion of the pathology in non-transduced somatic organs provided evidence of cross-correction by circulating enzyme. Importantly, AAV9-Ids treatment also resulted in normalization of behavioural deficits and considerably prolonged the survival of treated MPSII mice. The efficacy of the intra-CSF administration of AAV9 vectors containing the human IDS coding sequence was also evaluated in MPSII mice. One and a half months after gene transfer, a significant increase in IDS activity was documented throughout the encephalon, an in the liver and serum of treated MPSII mice. Consequently, pathological GAG content was reduced, or even normalized, in the CNS and in most somatic tissues of MPSII mice that received the vectors. Altogether, the results obtained in the present work provide a strong proof of concept that supports the clinical translation of the intra-CSF AAV9-IDS gene therapy for the treatment of Hunter patients with cognitive impairment.

Mucopolysaccharidosis type IIIA (MPS-IIIA) is a severe neurodegenerative lysosomal storage disorder (LSD) inherited as an autosomal recessive trait and caused by sulfamidase deficiency. Using somatic gene transfer, we demonstrated therapeutic efficacy of a novel low-invasive gene therapy approach to treat the brain pathology in MPS-IIIA. The therapeutic strategy is based on a chimeric sulfamidase engineered with both the signal peptide (sp) from the highly secreted iduronate-2-sulfatase (IDS) linked to its N-terminal end and the blood-brain barrier (BBB)-binding domain of apolipoproteinB (ApoB-BO) linked to its C-terminal end. These modifications allow the enzyme (i) to be highly secreted from the liver and (ii) to efficiently cross the BBB. A single intravascular administration of vectors, based on adena-associated virus (AAV) serotype 8, was performed in one-month old MPS-IIIA mice to efficiently target the liver and convert it in a factory organ for sustained systemic release of high levels of the modified sulfamidase. We show that while the 10Ssp replacement results in higher enzyme secretion, the addition of the ApoB-BO allows efficient BBB transcytosis and restoration of sulfamidase activity in the brain of treated MPS-IIIA mice to ~ 12-15% of the normal levels. This, in turn, results in reduction of pathological vacuolization,

Mucopolysaccharidosis type IIIA (MPS IIIA) is caused by mutations in the N-sulphoglucosamine sulphohydrolase (SGSH) gene, leading to cellular accumulation of heparan sulphate and progressive neurodegeneration in patients. One of the proposed treatment methods is haematopoietic stem cell (HSC) gene therapy, which should result in an excess of SGSH produced in the peripheral organs and brain. The pre-clinical feasibility of this approach was demonstrated by our group in a mouse model of MPS IIIA. However, the overall efficiency of this method was limited and a number of approaches to solving these issues were addressed in this project in order to bring this therapy closer to clinical application. Our first aim was to optimise transduction of HSCs using cytokines, bovine serum albumin (BSA), and chemicals, such as MG132, genistein and valproic acid. Addition of BSA with cytokines improved cell viability, addition of MG132/ BSA/ cytokines improved transduction, but also caused cellular toxicity, while addition of genistein was inefficient. Addition of valproic acid with cytokines resulted in increased number of colony forming units. Next, we generated clinically applicable third generation pCCL lentiviral vector backbones with the eGFP reporter gene driven by one of ubiquitous hPGK or myeloid specific hCD11b and hCD18 internal human promoters, and optimised production of lentiviral vectors to increase titre and reduce production cost. These lentiviral vectors were used to transduce lineage depleted HSCs and transplanted into WT mice. Full chimerism and over 80% transduction were achieved with an average of 5 vector copy numbers/ cell. The hCD11b promoter resulted in the highest eGFP expression in monocytes and B cells in blood, but was weaker than the hPGK in T cells. The hCD18 promoter was more monocyte-specific but weak. Significant numbers of GFP-positive microglial cells were present in the brain from all groups, with an average of 25% transduced CD11b-positive cells in perfused mice. We subsequently codon-optimised (CO) the SGSH gene significantly improving enzyme activity, and transduced lineage depleted WT cells with one of hCD18.SGSH-CO, hCD11b.SGSH-CO, or hPGK.SGSH-CO lentiviral vectors, or MPS IIIA cells with either hCD11b.SGSH-CO or hPGK.SGSH-CO lentiviral vectors. These transduced cells were transplanted into MPS IIIA mice and outcomes were measured 6 months later. Only treatment with the hCD11b.SGSH-CO-LV transduced WT or MPS IIIA HSCs corrected abnormal behaviour of MPS IIIA mice. However, all treatments resulted in complete GAG storage clearance in the periphery and brain, and significantly elevated enzyme activity in the brain, liver and spleen to 7-11%, 60-75%, and 170-250% of WT enzyme activity respectively. A fine threshold of over 8.6% brain enzyme activity appeared to be required for behavioural correction in MPS IIIA mice. Further assessment of treated mice for the amount of secondary storage, HS sulphation patterning, neuroinflammation and longevity are still required for complete therapeutic assessment. However, it appears that neurological correction of the MPS IIIA mouse using MPS IIIA cells is feasible using a clinically-relevant pCCL vector with the hCD11b promoter and the codon-optimised SGSH gene.

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.

La Mucopolisacaridosi tipus IIIB (MPSIIIB) és una malaltia minoritària d’acumulació lisosòmica, d’herència autosòmica recessiva, causada per la deficiència de l’enzim lisosòmic α-N-acetilglucosaminidasa (NAGLU), la qual cosa provoca l’acumulació de formes parcialment degradades del glicosaminoglicà (GAG) heparan sulfat (HS) a l’interior dels lisosomes. Aquesta acumulació sostinguda en el temps genera disfunció cel·lular i la posterior mort de les cèl·lules. La MPSIIIB presenta una profunda afectació progressiva del Sistema Nerviós Central (SNC) caracteritzada per neurodegeneració i neuroinflamació, acompanyades d’alteracions perifèriques moderades. A més, amb l’edat, els pacients presenten també afectacions altament invalidants de les funcions auditiva i visual, que provoquen una severa hipoacúsia i una progressiva pèrdua de la visió. Actualment, no existeix cap teràpia efectiva aprovada pel tractament de la MPSIIIB. Les teràpies disponibles se centren en pal·liar la simptomatologia i millorar la qualitat de vida dels pacients i de les seves famílies. Per tant, és fa necessària la cerca d’una teràpia eficaç que permeti revertir les alteracions derivades de la MPSIIIB. La teràpia gènica in vivo basada en l’administració de vectors virals adenoasociados (AAV) representa una alternativa atractiva, ja que una única administració del vector permetria una eficàcia terapèutica a llarg termini. Prèviament, en el nostre laboratori, es va desenvolupar una aproximació de teràpia gènica basada en una única administració al Líquid Cefalorraquidi (LCR) de vectors AAV9 codificants per a l’enzim NAGLU murí (AAV9-Naglu) a la dosi de 3.0x1010 genomes virals (vg)/ratolí a ratolins model de la malaltia. Aquest estudi va donar lloc a la correcció de les alteracions del SNC i perifèriques de la malaltia, així com també a la reversió de les alteracions conductuals i a un augment de l’esperança de vida del model murí de la MPSIIIB. Per a la translació a la clínica de la qualsevol aproximació de teràpia gènica amb vectors AAV és necessari realitzar estudis per tal de minimitzar la dosi de vector administrada, preservant la màxima eficàcia terapèutica. Per aquesta raó, es va realitzar un estudi basat en l’administració IC del vector AAV9-Naglu a ratolins MPSIIIB, a 4 dosis diferents equidistants entre sí, amb l’objectiu de determinar la dosi "mínima terapèutica". Finalment, es va poder assignar la dosi de 9.3x109 vg/ratolí com la mínima terapèutica, ja que donava lloc a la correcció de les principals alteracions del SNC i perifèriques, així com també conductuals del model murí de la MPSIIIB. Aquesta dosi va ser aproximadament 3 vegades menor que la utilitzada al treball anterior del nostre laboratori. A continuació, es va avaluar l’eficàcia del tractament IC amb el vector AAV9-Naglu a la dosi de 9.3x109 vg/ratolí sobre les alteracions auditives del ratolí model de la MPSIIIB. Es va demostrar la capacitat d’aquest tractament per recuperar els nivells d’activitat NAGLU i de normalitzar el contingut de GAGs a la còclea, preservant la citoarquitectura coclear i l’audició després de 4 mesos de tractament. També es va poder observar que aquest tractament donava lloc a l’augment de l’activitat NAGLU de l’ull i a la correcció de la distensió lisosòmica de la retina, la qual cosa es va traduir en la prevenció de la degeneració de la retina i a la conservació de l’agudesa visual, després de 10 mesos de tractament a llarg termini. En conjunt, aquests resultats demostren l’eficàcia terapèutica d’una única administració al LCR del vector AAV9-Naglu en ratolins MPSIIIB joves sobre la preservació de les afectacions auditives i visuals.
La Mucopolisacaridosis tipo IIIB (MPSIIIB) es una enfermedad rara de acúmulo lisosomal autosómica recesiva causada por la deficiencia en la enzima lisosomal α-N-acetilglucosaminidasa (NAGLU). La deficiencia de esta enzima provoca la acumulación de formas parcialmente degradadas del glucosaminoglicano (GAG) heparán sulfato (HS) en el interior de los lisosomas. Esta acumulación anómala sostenida en el tiempo genera la disfuncionalidad celular y la posterior muerte de las células. La MPSIIIB presenta una profunda afectación del Sistema Nervioso Central (SNC) caracterizada por la neurodegenración y la neuroinflamación. Alteraciones periféricas moderadas también están presentes en esta enfermedad. Además, con la edad, los pacientes presentan afectaciones altamente invalidantes de las funciones auditiva y visual, que provocan una severa hipoacusia y una progresiva pérdida de la visión. Actualmente, no existe ninguna terapia efectiva aprobada para tratar la MPSIIIB. Los tratamientos disponibles se centran en paliar la sintomatología y mejorar la calidad de vida de los pacientes y sus familias. Por tanto, es necesaria la búsqueda de una terapia eficaz que permita revertir las alteraciones derivadas de la MPSIIIB. La terapia génica in vivo basada en la administración de vectores virales adenoasociados (AAV: Adeno-Associated Virus) representa una alternativa prometedora, ya que una única administración del tratamiento permitiría una eficacia terapéutica a largo plazo. Previamente, en nuestro laboratorio, se desarrolló una aproximación de terapia génica basada en una única administración al Líquido Cefalorraquídeo (LCR) de ratones modelo de la enfermedad de vectores AAV9 codificantes para la NAGLU murina (AAV9-Naglu) a la dosis de 3.0x1010 genomas virales (vg)/ratón. Este estudió demostró la eficacia del tratamiento en la corrección de las alteraciones periféricas y del SNC de la enfermedad, así como una reversión de las alteraciones conductuales y un aumento en la esperanza de vida del modelo murino de la MPSIIIB. Para la traslacionalidad clínica de la cualquier aproximación de terapia génica con vectores AAV es crucial minimizar la dosis de vector administrada preservando la máxima eficacia terapéutica. Por ello, se realizó un estudio con el objetivo de establecer una dosis del vector AAV9-Nalgu más baja que se pudiera designar como la "mínima terapéutica". En este trabajo se observó que, tras la evaluación de 4 dosis diferentes equidistantes entre sí, se pudo designar la de 9.3x109 vg/ratón como la mínima terapéutica, pues era 3 veces menor q la empleada en el estudio anterior y además corregía tanto las alteraciones del SNC y periféricas, como las alteraciones del comportamiento. Además, se evaluó la eficacia del tratamiento a esta dosis sobre las alteraciones auditivas del ratón modelo de la MPSIIIB y se demostró su capacidad de recuperar los niveles de actividad NAGLU en la cóclea y de normalizar el contenido de GAGs en este tejido, preservando la citoarquitectura coclear y de la audición tras 4 meses de tratamiento. Además, también se observó que este tratamiento repercutía en la normalización de la actividad NAGLU en el ojo y la corrección de la distensión lisosomal en la retina. Ello contribuyó a la prevención de la degeneración retiniana, conllevando a la conservación de la agudeza visual a largo plazo. En conjunto, estos resultados demuestran la eficacia terapéutica de una única administración al LCR del vector AAV9-Naglu en ratones MPSIIIB jóvenes sobre la preservación de las afectaciones auditivas y visuales.
Mucopolysaccharidosis type IIIB (MPSIIIB) is a rare autosomal recessive lysosomal storage disease caused by the deficiency of a lysosomal enzyme called α-N-acetylglucosaminidase (NAGLU). This deficiency causes the accumulation of partially degraded forms of the glycosaminoglycan (GAG) heparan sulfate (HS) within the lysosomes. This abnormal accumulation sustained over time generates cellular dysfunction and subsequent cell death. MPSIIIB presents a profound involvement of the central nervous system (CNS) characterized by neurodegeneration and neuroinflammation. Moderate peripheral alterations are also present in this disease. In addition, with age, patients present highly disabling impairments of the auditory and visual functions, which cause severe hearing loss and progressive loss of vision. Currently, there is no effective treatment approved for MPSIIIB. The available treatments are focused on alleviating the symptoms and improving the quality of life of patients and their families. Therefore, it is necessary to develop an effective therapy that allows the correction of the alterations derived from MPSIIIB. In vivo gene therapy based on the administration of adeno-associated viral vectors (AAV) represents a promising alternative, since a single administration of the treatment would allow long-term therapeutic efficacy. Previously, in our laboratory, a gene therapy approach was developed based on a single administration to the cerebrospinal fluid (CSF) of AAV9 vectors encoding murine NAGLU (AAV9-Naglu) at the dose of 3 x 1010 viral genomes (vg)/mouse on the MPSIIIB mouse model. This study demonstrated the efficacy of the treatment in correcting the peripheral and CNS pathology, as well as a reversing the behavioural alterations and increasing the life expectancy of the MPSIIIB mouse model. For the clinical translation of any AAV-mediated gene therapy approach, it is crucial to minimize the vector dose administered while preserving maximum therapeutic efficacy. Therefore, a study was conducted with the aim of establishing a lower dose of the AAV9-Naglu vector that could be designated as the “minimum therapeutic dose”. Among 4 different equidistant doses, it was possible to designate 9.3 x 109 vg/mouse as the minimum therapeutical dose. Even though it was 3 times lower than the one used in the previous study, it was able to correct the behaviour and the CNS and peripheral alterations. In addition, the therapeutic efficacy of this dose in the hearing alterations of the MPSIIIB mice model was evaluated, resulting in the recovery of NAGLU activity and normalization of GAG accumulation in the cochlea. Cochlear cytoarchitecture and hearing function were preserved in MPSIIIB mice 4 months after treatment. Furthermore, we also observed that this treatment was able to normalize the NAGLU activity in the eye, thus correcting the lysosomal pathology in the retina. This contributed to the prevention of retinal degeneration, leading to long-term preservation of visual acuity. Overall, these results demonstrate the therapeutic efficacy of a single CSF administration of the AAV9-Naglu vector in young MPSIIIB mice on the preservation of auditory and visual impairments.
Universitat Autònoma de Barcelona. Programa de Doctorat en Bioquímica, Biologia Molecular i Biomedicina

Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, Discipline of Paediatrics, 2007.
"June 2007" Includes Addenda attached to back page. Bibliography: leaves 215-274. Also available in print form.

Bibliography: leaves 269-297.
xvii, 297, [10] leaves, [31] leaves of plates : ill. (some col.) ; 30 cm.
Evaluates the efficacy of enzyme replacement therapy (ERT) with artifically produced recombinant human 4S (rh4S) in feline mucopolysaccharidosis Type VI (MPS VI) and tests the hypothesis that this form of therapy would reverse or alter the disease course, particularly the bone dysplasia and connective tissue pathologics.
Thesis (Ph.D.)--University of Adelaide, Dept. of Paediatrics, 1998

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

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.
Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2007

Addenda page on inside back cover.
Bibliography: leaves 153-176.
xiii, 176 leaves ; ill. (some col.) ; 30 cm
Mucopolysaccharideosis type IIIA (MPS IIIA, Sanfilippo A syndrome) is an autosomal recessive lysosomal storage disease, with a prevalence in Australia of 1 in 114,000. MPS IIIA is caused by a deficiency of the lysosomal enzyme sulphamidase which is needed together with other exohydrolases and a N-acetyltransferase to break down the glycosaminoglycan heparan sulphate to sulphate and monosaccharides. Patients are characterised by severe central nervous systems degeneration together with mild somatic involvement; this disproportionate correlation is unique amongst the mucopolysaccharidoses. Features include severe behavioural disturbances, such as hyperactivity and aggressiveness, coarse hair and mild hepatosplenomegaly. Death is usually in the mid- to late-teenage years. Enzyme replacement therapy by intravenous administration of recombinant human NS (rhNS) has been proposed as a potential therapy for MPS IIIA. This thesis suggests that rhNS, entering the brain in the first few weeks of life, is able to retard the behaviour and learning difficulties in MPS IIIA mice.
Thesis (Ph.D.)--University of Adelaide, Dept. of Paediatrics, 2003

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive disorder that is characterised by a deficiency in the α-L-iduronidase (IDUA) enzyme, resulting in the accumulation of undegraded heparan sulphate and dermatan sulphate glycosaminoglycans (gags) within the lysosome of nearly every cell. MPS I is a multi-tissue and organ disease, presenting with profound mental retardation and skeletal abnormalities. Haematopoietic stem cell (HSC) transplant and enzyme replacement therapy, two clinically available forms of treatment, are able to correct the soft tissue aspects of MPS disease, but have had a limited effect on the more complex skeletal and neurological symptoms. Stem cell therapy utilizing mesenchymal stem cells (MSC) has the potential to overcome these limitations due to their ability to differentiate into cells that are the major sites of MPS pathology. MSCs naturally produce and secrete significantly higher levels of multiple MPS enzymes than HSCs in vitro and can be engineered to over-express multiple MPS enzymes using a lentiviral system. MSCs were found to secrete up to 5,559 fold greater IDUA enzyme after lentiviral transduction in vitro, suggesting a greater potential to cross-correct MPS pathology than HSCs. Lentiviral transduction was stable and persistent in vitro, and over-expression of MPS enzyme did not affect MSC in vitro differentiation down osteogenic, adipogenic, chondrogenic or neurogenic lineages. Systemically administered human derived MSCs distribute widely, to multiple MPS I affected organs, including the brain, and persist in vivo for at least two months post administration. Significantly elevated brain and serum IDUA activity was observed two and six months post administration, respectively, and was associated with sustained functional improvements in neuromuscular strength, motor control, coordination and spatial learning. MSCs were found to limit astroglial activation and modulate brain inflammatory gene expression of Cd68, Gfap and Tnf in vivo. Vertebral body width also returned towards normal. However, no improvement in gag storage or elevations of IDUA were observed in other tissues. For the first time, this thesis has investigated the biochemical and behavioural changes due to i.v. administered hMSCs in MPS I mice. This thesis demonstrates that MSCs can exert added neurological improvements in MPS I pathology through exhibiting a combined effect between their superior enzyme secretion and anti-inflammatory effects. While minimal changes were noted in MPS I associated somatic pathology, MSCs could be administered in combination with already implemented ERT and/or BMT, which have both shown resolution in patients stored gag, therefore providing additional clinical benefits to MPS I children.
Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2015.