RESUMO
BACKGROUND: Sanfilippo syndrome (mucopolysaccharidosis type IIIA; MPS IIIA) is a childhood dementia caused by inherited mutations in the sulfamidase gene. At present, there is no treatment and children with classical disease generally die in their late teens. Intravenous or intra-cerebrospinal fluid (CSF) injection of AAV9-gene replacement is being examined in human clinical trials; evaluation of the impact on brain disease is an intense focus; however, MPS IIIA patients also experience profound, progressive photoreceptor loss, leading to night blindness. AIM: To compare the relative efficacy of the two therapeutic approaches on retinal degeneration in MPS IIIA mice. METHODS: Neonatal mice received i.v. or intra-CSF AAV9-sulfamidase or vehicle and after 20 weeks, biochemical and histological evaluation of neuroretina integrity was carried out. RESULTS: Both treatments improved central retinal thickness; however, in peripheral retina, outer nuclear layer thickness and photoreceptor cell length were only significantly improved by i.v. gene replacement. Further, normalization of endo-lysosomal compartment size and microglial morphology was only observed following intravenous gene delivery. CONCLUSIONS: Confirmatory studies are needed in adult mice; however, these data indicate that i.v. AAV9-sulfamidase infusion leads to superior outcomes in neuroretina, and cerebrospinal fluid-delivered AAV9 may need to be supplemented with another therapeutic approach for optimal patient quality of life.
Assuntos
Dependovirus , Terapia Genética , Mucopolissacaridose III , Retina , Animais , Mucopolissacaridose III/terapia , Mucopolissacaridose III/genética , Terapia Genética/métodos , Dependovirus/genética , Retina/patologia , Camundongos , Modelos Animais de Doenças , Hidrolases/genética , Animais Recém-Nascidos , Camundongos Endogâmicos C57BL , Demência/genética , Demência/terapia , Vetores Genéticos/administração & dosagem , Injeções IntravenosasRESUMO
Hampering assessment of treatment outcomes in gene therapy and other clinical trials in patients with childhood dementia is the lack of an objective, non-invasive measure of neurodegeneration. Optical coherence tomography (OCT) is a widely available, rapid, non-invasive, and quantitative method for examining the integrity of the neuroretina. Profound brain and retinal dysfunction occur in patients and animal models of childhood dementia, including Sanfilippo syndrome and we recently revealed a correlation between the age of onset and rate of progression of retinal and brain degeneration in sulfamidase-deficient Sanfilippo mice. The aim of the current study was to use OCT to visualise the discrete changes in retinal structure that occur during disease progression. A progressive decline in retinal thickness was readily observable in Sanfilippo mice using OCT, with differences seen in affected animals from 10-weeks of age. OCT applied to i.v. AAV9-sulfamidase-treated Sanfilippo mice enabled visualisation of improved retinal anatomy in living animals, an outcome confirmed via histology. Importantly, brain disease lesions were also ameliorated in treated Sanfilippo mice. The findings highlight the sensitivity, ease of repetitive use and quantitative capacity of OCT for detection of discrete changes in retinal structure and their prevention with a therapeutic. Combined with the knowledge that retinal and brain degeneration are correlated in Sanfilippo syndrome, OCT provides a window to the brain in this and potentially other childhood dementias.
Assuntos
Demência , Mucopolissacaridose III , Humanos , Camundongos , Animais , Mucopolissacaridose III/diagnóstico por imagem , Mucopolissacaridose III/genética , Mucopolissacaridose III/terapia , Retina/diagnóstico por imagem , Retina/patologia , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Terapia Genética , Demência/patologia , Modelos Animais de DoençasRESUMO
Acute neuronopathic (type II) Gaucher disease (GD) is a devastating, untreatable neurological disorder resulting from mutations in the glucocerebrosidase gene (GBA1), with subsequent accumulation of glucosylceramide and glucosylsphingosine. Patients experience progressive decline in neurological function, with onset typically within the first three-to-six months of life and premature death before two years. Mice and drosophila with GD have been described, however little is known about the brain pathology observed in the naturally occurring ovine model of GD. We have characterised pathological changes in GD lamb brain and compared the histological findings to those in GD patient post-mortem tissue, to determine the validity of the sheep as a model of this disease. Five GD and five age-matched unaffected lamb brains were examined. We observed significant expansion of the endo/lysosomal system in GD lamb cingulate gyrus however TPP1 and cathepsin D levels were unchanged or reduced. H&E staining revealed neurons with shrunken, hypereosinophilic cytoplasm and hyperchromatic or pyknotic nuclei (red neurons) that were also shrunken and deeply Nissl stain positive. Amoeboid microglia were noted throughout GD brain. Spheroidal inclusions reactive for TOMM20, ubiquitin and most strikingly, p-Tau were observed in many brain regions in GD lamb brain, potentially indicating disturbed axonal trafficking. Our findings suggest that the ovine model of GD exhibits similar pathological changes to human, mouse, and drosophila type II GD brain, and represents a model suitable for evaluating therapeutic intervention, particularly in utero-targeted approaches.
Assuntos
Doença de Gaucher , Doenças do Sistema Nervoso , Humanos , Animais , Ovinos , Camundongos , Doença de Gaucher/genética , Doença de Gaucher/patologia , Glucosilceramidase/genética , Encéfalo/patologia , Doenças do Sistema Nervoso/patologia , DrosophilaRESUMO
Lysosomal dysfunction may be an important factor in the pathogenesis of neurodegenerative disorders such as Parkinson's disease (PD). Heterozygous mutations in the gene encoding the lysosomal enzyme glucocerebrosidase (GBA1) have been found in PD patients, and some but not all mutations in other lysosomal enzyme genes, for example, NPC1 and MCOLN1 have been associated with PD. We have examined the behaviour and brain structure of mice carrying a D31N mutation in the sulphamidase (Sgsh) gene which encodes a lysosomal sulphatase. Female heterozygotes and wildtype mice aged 12-, 15-, 18- and 21-months of age underwent motor phenotyping and the brain was comprehensively evaluated for disease-associated lesions. Heterozygous mice exhibited impaired performance in the negative geotaxis test when compared with wildtype mice. Whilst the brain of Sgsh heterozygotes aged up to 21-months did not exhibit any of the gross features of PD, Alzheimer's disease or the neurodegenerative lysosomal storage disorders, for example, loss of striatal dopamine, reduced GBA activity, α-synuclein-positive inclusions, perturbation of lipid synthesis, or cerebellar Purkinje cell drop-out, we noted discrete structural aberrations in the dendritic tree of cortical pyramidal neurons in 21-month old animals. The overt disease lesions and resultant phenotypic changes previously described in individuals with heterozygous mutations in lysosomal enzyme genes such as glucocerebrosidase may be enzyme dependent. By better understanding why deficiency in, or mutant forms of some but not all lysosomal proteins leads to heightened risk or earlier onset of classical neurodegenerative disorders, novel disease-causing mechanisms may be identified.
Assuntos
Glucosilceramidase/metabolismo , Heterozigoto , Hidrolases/genética , Doença de Parkinson/genética , Fatores Etários , Animais , Comportamento Animal , Modelos Animais de Doenças , Dopamina/metabolismo , Feminino , Camundongos , Mutação , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Fatores de Risco , alfa-Sinucleína/metabolismoRESUMO
Sanfilippo syndrome is an untreatable form of childhood-onset dementia. Whilst several therapeutic strategies are being evaluated in human clinical trials including i.v. delivery of AAV9-based gene therapy, an urgent unmet need is the availability of non-invasive, quantitative measures of neurodegeneration. We hypothesise that as part of the central nervous system, the retina may provide a window through which to 'visualise' degenerative lesions in brain and amelioration of them following treatment. This is reliant on the age of onset and the rate of disease progression being equivalent in retina and brain. For the first time we have assessed in parallel, the nature, age of onset and rate of retinal and brain degeneration in a mouse model of Sanfilippo syndrome. Significant accumulation of heparan sulphate and expansion of the endo/lysosomal system was observed in both retina and brain pre-symptomatically (by 3 weeks of age). Robust and early activation of micro- and macroglia was also observed in both tissues. There was substantial thinning of retina and loss of rod and cone photoreceptors by ~ 12 weeks of age, a time at which cognitive symptoms are noted. Intravenous delivery of a clinically relevant AAV9-human sulphamidase vector to neonatal mice prevented disease lesion appearance in retina and most areas of brain when assessed 6 weeks later. Collectively, the findings highlight the previously unrecognised early and significant involvement of retina in the Sanfilippo disease process, lesions that are preventable by neonatal treatment with AAV9-sulphamidase. Critically, our data demonstrate for the first time that the advancement of retinal disease parallels that occurring in brain in Sanfilippo syndrome, thus retina may provide an easily accessible neural tissue via which brain disease development and its amelioration with treatment can be monitored.
Assuntos
Encéfalo/patologia , Mucopolissacaridose III/patologia , Doenças Neurodegenerativas/patologia , Retina/patologia , Degeneração Retiniana/patologia , Animais , Doenças Assintomáticas , Modelos Animais de Doenças , Endossomos/patologia , Terapia Genética , Heparitina Sulfato/metabolismo , Humanos , Hidrolases/genética , Lisossomos/patologia , Camundongos , Microglia/patologia , Mucopolissacaridose III/genética , Mucopolissacaridose III/metabolismo , Doenças Neurodegenerativas/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Degeneração Retiniana/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/patologiaRESUMO
Patients with mucopolysaccharidosis type IIIA (MPS IIIA) lack the lysosomal enzyme sulfamidase (SGSH), which is responsible for the degradation of heparan sulfate (HS). Build-up of undegraded HS results in severe progressive neurodegeneration for which there is currently no treatment. The ability of the vector adeno-associated virus (AAV)rh.10-CAG-SGSH (LYS-SAF302) to correct disease pathology was evaluated in a mouse model for MPS IIIA. LYS-SAF302 was administered to 5-week-old MPS IIIA mice at three different doses (8.6E+08, 4.1E+10, and 9.0E+10 vector genomes [vg]/animal) injected into the caudate putamen/striatum and thalamus. LYS-SAF302 was able to dose-dependently correct or significantly reduce HS storage, secondary accumulation of GM2 and GM3 gangliosides, ubiquitin-reactive axonal spheroid lesions, lysosomal expansion, and neuroinflammation at 12 weeks and 25 weeks post-dosing. To study SGSH distribution in the brain of large animals, LYS-SAF302 was injected into the subcortical white matter of dogs (1.0E+12 or 2.0E+12 vg/animal) and cynomolgus monkeys (7.2E+11 vg/animal). Increases of SGSH enzyme activity of at least 20% above endogenous levels were detected in 78% (dogs 4 weeks after injection) and 97% (monkeys 6 weeks after injection) of the total brain volume. Taken together, these data validate intraparenchymal AAV administration as a promising method to achieve widespread enzyme distribution and correction of disease pathology in MPS IIIA.
RESUMO
Membranes are an important barrier used in recycled water treatment plants for pathogen removal. Understanding performance over operational life is important to inform membrane replacement. In this study, full scale virus challenge testing was conducted on newly commissioned membranes to validate virus log removal values for accreditation. After six years of operation, the membrane integrity was repeated to ensure compliance with the state regulatory health authority and gain an understanding of the asset's condition. Membrane performance was assessed using a combination of complementary tests including membrane autopsy and chemical tolerance testing to assess individual modules and selected membrane fibres, followed by a full scale virus challenge for whole of unit assessment. The results demonstrated that the aged membrane fibres were intact and had not been affected by long-term exposure to chlorine, which provides valuable information for membrane asset replacement strategies.
Assuntos
Membranas Artificiais , Reciclagem , Eliminação de Resíduos Líquidos/métodos , Purificação da Água/métodos , Cloro , Poluentes Químicos da Água/químicaRESUMO
Axonal dystrophy has been described as an early pathological feature of neurodegenerative disorders including Alzheimer's disease and amyotrophic lateral sclerosis. Axonal inclusions have also been reported to occur in several neurodegenerative lysosomal storage disorders including Mucopolysaccharidosis type IIIA (MPS IIIA; Sanfilippo syndrome). This disorder results from a mutation in the gene encoding the lysosomal sulphatase sulphamidase, and as a consequence heparan sulphate accumulates, accompanied by secondarily-stored gangliosides. The precise basis of symptom generation in MPS IIIA has not been elucidated, however axonal dystrophy may conceivably lead to impaired vesicular trafficking, neuronal dysfunction and/or death. We have utilised a faithful murine model of MPS IIIA to determine the spatio-temporal profile of neuronal inclusion formation and determine the effect of restoring normal lysosomal function. Dopaminergic (tyrosine hydroxylase-positive), cholinergic (choline acetyltransferase-positive) and GABAergic (glutamic acid decarboxylase65/67-positive) neurons were found to exhibit axonal dystrophy in MPS IIIA mouse brain. Axonal lesions present by ~seven weeks of age were Rab5-positive but lysosomal integral membrane protein-2 negative, suggesting early endosomal involvement. By 9-12-weeks of age, immunoreactivity for the autophagosome-related proteins LC3 and p62 and the proteasomal subunit 19S was noted in the spheroidal structures, together with wildtype α-synuclein, phosphorylated Thr-181 Tau and amyloid precursor protein, indicative of impaired axonal trafficking. Sulphamidase replacement reduced but did not abrogate the axonal lesions. Therefore, if axonal dystrophy impairs neuronal activity and ultimately, neuronal function, its incomplete resolution warrants further investigation.
Assuntos
Axônios/patologia , Encéfalo/patologia , Mucopolissacaridose III/patologia , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Feminino , Hidrolases/genética , Imuno-Histoquímica , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucopolissacaridose III/diagnóstico por imagem , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismoRESUMO
Mucopolysaccharidosis (MPS) type IIIA, or Sanfilippo syndrome, is a neurodegenerative lysosomal storage disorder caused by a deficiency of the lysosomal enzyme N-sulfoglucosamine sulfohydrolase (SGSH), involved in the catabolism of heparan sulfate. The clinical spectrum is broad and the age of symptom onset and the degree of preservation of cognitive and motor functions appears greatly influenced by genotype. To explore this further, we generated a conditional knockout (Sgsh KO ) mouse model with ubiquitous Sgsh deletion, and compared the clinical and pathological phenotype with that of the spontaneous Sgsh D31N MPS-IIIA mouse model. Phenotypic deficits were noted in Sgsh KO mice prior to Sgsh D31N mice, however these outcomes did not correlate with any shift in the time of appearance nor rate of accumulation of primary (heparan sulfate) or secondary substrates (GM2/GM3 gangliosides). Other disease lesions (elevations in lysosomal integral membrane protein-II expression, reactive astrocytosis and appearance of ubiquitin-positive inclusions) were also comparable between affected mouse strains. This suggests that gross substrate storage and these neuropathological markers are neither primary determinants, nor good biomarkers/indicators of symptom generation, confirming similar observations made recently in MPS-IIIA patients. The Sgsh KO mouse will be a useful tool for elucidation of the neurological basis of disease and assessment of the clinical efficacy of new treatments for Sanfilippo syndrome.
Assuntos
Hidrolases/metabolismo , Mucopolissacaridose III/metabolismo , Mucopolissacaridose III/patologia , Animais , Biomarcadores/metabolismo , Modelos Animais de Doenças , Genótipo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , FenótipoRESUMO
Mucopolysaccharidosis type IIIA (MPS IIIA) is predominantly a disorder of the central nervous system, caused by a deficiency of sulfamidase (SGSH) with subsequent storage of heparan sulfate-derived oligosaccharides. No widely available therapy exists, and for this reason, a mouse model has been utilized to carry out a preclinical assessment of the benefit of intraparenchymal administration of a gene vector (AAVrh10-SGSH-IRES-SUMF1) into presymptomatic MPS IIIA mice. The outcome has been assessed with time, measuring primary and secondary storage material, neuroinflammation, and intracellular inclusions, all of which appear as the disease progresses. The vector resulted in predominantly ipsilateral distribution of SGSH, with substantially less detected in the contralateral hemisphere. Vector-derived SGSH enzyme improved heparan sulfate catabolism, reduced microglial activation, and, after a time delay, ameliorated GM3 ganglioside accumulation and halted ubiquitin-positive lesion formation in regions local to, or connected by projections to, the injection site. Improvements were not observed in regions of the brain distant from, or lacking connections with, the injection site. Intraparenchymal gene vector administration therefore has therapeutic potential provided that multiple brain regions are targeted with vector, in order to achieve widespread enzyme distribution and correction of disease pathology.
Assuntos
Dependovirus/genética , Terapia Genética , Vetores Genéticos/genética , Hidrolases/genética , Mucopolissacaridose III/genética , Animais , Anticorpos Neutralizantes/imunologia , Autofagia , Biomarcadores , Encéfalo/metabolismo , Proteínas de Ligação a DNA , Dependovirus/classificação , Modelos Animais de Doenças , Endossomos/metabolismo , Ativação Enzimática , Feminino , Gangliosídeo G(M3)/metabolismo , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/efeitos adversos , Vetores Genéticos/normas , Proteína Glial Fibrilar Ácida/metabolismo , Heparitina Sulfato/metabolismo , Proteínas de Grupo de Alta Mobilidade , Humanos , Hidrolases/imunologia , Hidrolases/metabolismo , Lisossomos/metabolismo , Masculino , Camundongos , Mucopolissacaridose III/metabolismo , Mucopolissacaridose III/terapia , Proteínas de Saccharomyces cerevisiae , Transdução GenéticaRESUMO
Repeated replacement of sulphamidase via cerebrospinal fluid injection is an effective treatment for pathological changes in the brain in mice and dogs with the lysosomal storage disorder, mucopolysaccharidosis type IIIA (MPS IIIA). Investigational trials of this approach are underway in children with this condition, however, infusions require attendance at a specialist medical facility. We sought to comprehensively evaluate the effectiveness of sustained-release (osmotic pump-delivered) enzyme replacement therapy in murine MPS IIIA as this method, if applied to humans, would require only subcutaneous administration of enzyme once the pump was installed. Six-week-old MPS IIIA and unaffected mice were implanted with subcutaneous mini-osmotic pumps connected to an infusion cannula directed at the right lateral ventricle. Either recombinant human sulphamidase or vehicle were infused over the course of 7 weeks, with pumps replaced part-way through the experimental period. We observed near-normalisation of primarily stored substrate (heparan sulphate) in both hemispheres of the MPS IIIA brain and cervical spinal cord, as determined using tandem mass spectrometry. Immunohistochemistry indicated a reduction in secondarily stored GM 3 ganglioside and neuroinflammatory markers. A bias towards the infusion side was seen in some, but not all outcomes. The recombinant enzyme appears stable under pump-like conditions for at least 1 month. Given that infusion pumps are in clinical use in other nervous system disorders, e.g. for treatment of spasticity or brain tumours, this treatment method warrants consideration for testing in large animal models of MPS IIIA and other lysosomal storage disorders that affect the brain. Clinical trials of repeated injection of replacement enzyme into CSF are underway in patients with the inherited neurodegenerative disorder mucopolysaccharidosis type IIIA. In this pre-clinical study, we examined an alternative approach - slow, continual infusion of enzyme using pumps. We observed significant reductions in substrate accumulation and other disease-based lesions in treated mouse brain. Thus, the strategy warrants consideration for testing in large animal models of MPS IIIA and also in other neurodegenerative lysosomal storage disorders.
Assuntos
Encéfalo/patologia , Terapia de Reposição de Enzimas/métodos , Hidrolases/uso terapêutico , Mucopolissacaridose III/tratamento farmacológico , Mucopolissacaridose III/patologia , Animais , Biomarcadores/metabolismo , Química Encefálica , Gliose/tratamento farmacológico , Gliose/patologia , Heparitina Sulfato/metabolismo , Humanos , Hidrolases/administração & dosagem , Bombas de Infusão Implantáveis , Ventrículos Laterais , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/uso terapêutico , Medula Espinal/metabolismoRESUMO
Mucopolysaccharidosis IIIA (MPS IIIA) is a neurodegenerative lysosomal storage disorder characterised by progressive loss of learned skills, sleep disturbance and behavioural problems. Reduced activity of lysosomal sulfamidase results in accumulation of heparan sulfate and secondary storage of glycolipids in the brain. Intra-cisternal sulfamidase infusions reduce disease-related neuropathology; however, repeated injections may subject patients to the risk of infection and tissue damage so alternative approaches are required. We undertook a proof-of-principle study comparing the ability of slow/continual or repeat/bolus infusion to ameliorate neuropathology in MPS IIIA mouse brain. Six-week-old MPS IIIA mice were implanted with subcutaneously located mini-osmotic pumps filled with recombinant human sulfamidase (rhSGSH) or vehicle, connected to lateral ventricle-directed cannulae. Pumps were replaced at 8 weeks of age. Additional MPS IIIA mice received intra-cisternal bolus infusions of the same amount of rhSGSH (or vehicle), at 6 and 8 weeks of age. Unaffected mice received vehicle via each strategy. All mice were euthanised at 10 weeks of age and the brain was harvested to assess the effect of treatment on neuropathology. Mice receiving pump-delivered rhSGSH exhibited highly significant reductions in lysosomal storage markers (lysosomal integral membrane protein-2, GM3 ganglioside and filipin-positive lipids) and neuroinflammation (isolectin B4-positive microglia, glial fibrillary acidic protein-positive astroglia). MPS IIIA mice receiving rhSGSH via bolus infusion displayed reductions in these markers, but the effectiveness of the strategy was inferior to that seen with slow/pump-based delivery. Continual low-dose infusion may therefore be a more effective strategy for enzyme delivery in MPS IIIA.
RESUMO
MPS IIIA is an inherited neurodegenerative lysosomal storage disorder characterized by cognitive impairment, sleep-wake cycle disturbance, speech difficulties, eventual mental regression and early death. Neuropathological changes include accumulation of heparan sulfate and glycolipids, neuroinflammation and degeneration. Pre-clinical animal studies indicate that replacement of the deficient enzyme, sulfamidase, via intra-cerebrospinal fluid (CSF) injection is a clinically-relevant treatment approach, reducing neuropathological changes and improving symptoms. Given that there are several routes of administration of enzyme into the CSF (intrathecal lumbar, cisternal and ventricular), determining the effectiveness of each injection strategy is crucial in order to provide the best outcome for patients. We delivered recombinant human sulfamidase (rhSGSH) to a congenic mouse model of MPS IIIA via each of the three routes. Mice were euthanized 24h or one-week post-injection; the distribution of enzyme within the brain and spinal cord parenchyma was investigated, and the impact on primary substrate levels and other pathological lesions determined. Both ventricular and cisternal injection of rhSGSH enable enzyme delivery to brain and spinal cord regions, with the former mediating large, statistically significant decreases in substrate levels and reducing microglial activation. The single lumbar CSF infusion permitted more restricted enzyme delivery, with no reduction in substrate levels and little change in other disease-related lesions in brain tissue. While the ventricular route is the most invasive of the three methods, this strategy may enable the widest distribution of enzyme within the brain, and thus requires further exploration.
Assuntos
Vias de Administração de Medicamentos , Terapia de Reposição de Enzimas/métodos , Hidrolases/administração & dosagem , Hidrolases/líquido cefalorraquidiano , Mucopolissacaridose III/tratamento farmacológico , Mucopolissacaridose III/genética , Animais , Encéfalo/patologia , Cisterna Magna , Modelos Animais de Doenças , Heparitina Sulfato/líquido cefalorraquidiano , Humanos , Infusões Intraventriculares , Infusão Espinal , Injeções , Injeções Intraventriculares , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/líquido cefalorraquidianoRESUMO
Intracerebrospinal fluid (CSF) infusion of replacement enzyme is under evaluation for amelioration of disease-related symptoms and biomarker changes in patients with the lysosomal storage disorder mucopolysaccharidosis type IIIA (MPS IIIA; www.clinicaltrials.gov ; NCT#01155778; #01299727). Determining the optimal dose/dose-frequency is important, given the invasive method for chronically supplying recombinant protein to the brain, the main site of symptom generation. To examine these variables, we utilised MPS IIIA Huntaway dogs, providing recombinant human sulphamidase (rhSGSH) to young pre-symptomatic dogs from an age when MPS IIIA dog brain exhibits significant accumulation of primary (heparan sulphate) and secondary (glycolipid) substrates. Enzyme was infused into CSF via the cisterna magna at one of two doses (3 mg or 15 mg/infusion), with the higher dose supplied at two different intervals; fortnightly or monthly. Euthanasia was carried out 24 h after the final injection. Dose- and frequency-dependent reductions in heparan sulphate were observed in CSF and deeper layers of cerebral cortex. When we examined the amount of immunostaining of the general endo/lysosomal marker, LIMP-2, or quantified activated microglia, the higher fortnightly dose resulted in superior outcomes in affected dogs. Secondary lesions such as accumulation of GM3 ganglioside and development of GAD-reactive axonal spheroids were treated to a similar degree by both rhSGSH doses and dose frequencies. Our findings indicate that the lower fortnightly dose is sub-optimal for ameliorating existing and preventing further development of disease-related pathology in young MPS IIIA dog brain; however, increasing the dose fivefold but halving the frequency of administration enabled near normalisation of disease-related biomarkers.
Assuntos
Encéfalo/efeitos dos fármacos , Terapia de Reposição de Enzimas , Hidrolases/administração & dosagem , Mucopolissacaridose III/tratamento farmacológico , Animais , Biomarcadores/metabolismo , Encéfalo/enzimologia , Encéfalo/patologia , Modelos Animais de Doenças , Cães , Esquema de Medicação , Cálculos da Dosagem de Medicamento , Glicolipídeos/metabolismo , Heparitina Sulfato/metabolismo , Infusões Intraventriculares , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Mucopolissacaridose III/enzimologia , Mucopolissacaridose III/genética , Mucopolissacaridose III/patologia , Proteínas Recombinantes/administração & dosagem , Fatores de TempoRESUMO
Injection of lysosomal enzyme into cisternal or ventricular cerebrospinal fluid (CSF) has been carried out in 11 lysosomal storage disorder models, with each study demonstrating reductions in primary substrate and secondary neuropathological changes, and several reports of improved neurological function. Whilst acute studies in mucopolysaccharidosis (MPS) type II mice revealed that intrathecally-delivered enzyme (into thoraco-lumbar CSF) accesses the brain, the impact of longer-term treatment of affected subjects via this route is unknown. This approach is presently being utilized to treat children with MPS types I, II and III. Our aim was to determine the efficacy of repeated intrathecal injection of recombinant human sulfamidase (rhSGSH) on pathological changes in the MPS IIIA dog brain. The outcomes were compared with those in dogs treated via intra-cisternal or ventricular routes. Control dogs received buffer or no treatment. Significant reductions in primary/secondary substrate levels in brain were observed in dogs treated via all routes, although the extent of the reduction differed regionally. Treatment via all CSF access points resulted in large reductions in microgliosis in superficial cerebral cortex, but only ventricular injection enabled amelioration in deep cerebral cortex. Formation of glutamic acid decarboxylase-positive axonal spheroids in deep cerebellar nuclei was prevented by treatment delivered via any route. Anti-rhSGSH antibodies in the sera of some dogs did not reduce therapeutic efficacy. Our data indicates the capacity of intra-spinal CSF-injected rhSGSH to circulate within CSF-filled spaces, penetrate into brain and mediate a significant reduction in substrate accumulation and secondary pathology in the MPS IIIA dog brain.
Assuntos
Hidrolases/administração & dosagem , Mucopolissacaridoses/patologia , Animais , Cromatografia Líquida , Modelos Animais de Doenças , Cães , Ensaio de Imunoadsorção Enzimática , Heparitina Sulfato/análise , Humanos , Imuno-Histoquímica , Injeções Espinhais , Espectrometria de Massas , Proteínas Recombinantes/administração & dosagemRESUMO
Lysosomal storage disorders are a large group of inherited metabolic conditions resulting from the deficiency of proteins involved in lysosomal catabolism, with resulting accumulation of substrates inside the cell. Two-thirds of these disorders are associated with a neurodegenerative phenotype and, although few therapeutic options are available to patients at present, clinical trials of several treatments including lysosomal enzyme replacement are underway. Although animal studies indicate the efficacy of presymptomatic treatment, it is largely unknown whether symptomatic disease-related pathology and functional deficits are reversible. To begin to address this, we used a naturally-occurring mouse model with Sanfilippo syndrome (mucopolysaccharidosis type IIIA) to examine the effectiveness of intracisternal cerebrospinal fluid enzyme replacement in early, mid- and symptomatic disease stage mice. We observed a disease-stage-dependent treatment effect, with the most significant reductions in primary and secondary substrate accumulation, astrogliosis and protein aggregate accumulation seen in mucopolysaccharidosis type IIIA mice treated very early in the disease course. Affected mice treated at a symptomatic age exhibited little change in these neuropathological markers in the time-frame of the study. Microgliosis was refractory to treatment regardless of the age at which treatment was instigated. Although longer-term studies are warranted, these findings indicate the importance of early intervention in this condition.
Assuntos
Terapia de Reposição de Enzimas/métodos , Hidrolases/uso terapêutico , Mucopolissacaridose III/tratamento farmacológico , Proteínas Recombinantes/uso terapêutico , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Astrócitos/fisiologia , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Encéfalo/fisiopatologia , Modelos Animais de Doenças , Progressão da Doença , Feminino , Gliose/tratamento farmacológico , Gliose/patologia , Gliose/fisiopatologia , Humanos , Masculino , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/patologia , Microglia/fisiologia , Mucopolissacaridose III/patologia , Mucopolissacaridose III/fisiopatologia , Doenças Neurodegenerativas , Medula Espinal/efeitos dos fármacos , Medula Espinal/patologia , Medula Espinal/fisiopatologiaRESUMO
Mucopolysaccharidosis type IIIA (MPS-IIIA) is a severe neurodegenerative lysosomal storage disorder caused by a deficiency of N-sulfoglucosamine sulfohydrolase (SGSH) activity with subsequent accumulation of partially-degraded heparan sulfate and other glycolipids. In this study, we have evaluated a gene therapy approach using a helper-dependent canine adenovirus vector that expresses human SGSH as a means of delivering sustained transgene expression to the brain. Initial testing in a mixed neural cell culture model demonstrated that the vector could significantly increase SGSH activity in transduced cells, resulting in near-normalization of heparan sulfate-derived fragments. While administration of vector by direct injection into the brain of adult MPS-IIIA mice enabled transgene expression for at least 8.5 months post-treatment, it was only in discrete areas of brain. Heparan sulfate storage was reduced in some regions following treatment, however there was no improvement in secondary neuropathological changes. These data demonstrate that helper-dependent canine adenovirus vectors are capable of neural transduction and mediate long-term transgene expression, but increased SGSH expression throughout the brain is likely to be required in order to effectively treat all aspects of the MPS-IIIA phenotype.
Assuntos
Adenovirus Caninos/genética , Hidrolases/genética , Mucopolissacaridose III/terapia , Animais , Encéfalo/metabolismo , Células Cultivadas , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos , Vírus Auxiliares/genética , TransgenesRESUMO
There is no treatment for the progressive neurodegenerative lysosomal storage disorder mucopolysaccharidosis type IIIA (MPS IIIA), which occurs due to a deficiency of functional N-sulfoglucosamine sulfohydrolase (SGSH), with subsequent accumulation of partially-degraded heparan sulfate and secondarily-stored compounds including GM2 and GM3 gangliosides and unesterified cholesterol. The brain is a major site of pathology and affected children exhibit progressive cognitive decline and early death. In the present study, six MPS IIIA dogs received intravenous recombinant human SGSH (rhSGSH) from birth to either 8 or 12 weeks of age (1 mg/kg, up to 5 mg), with subsequent intra-cerebrospinal fluid injection of 3 or 15 mg rhSGSH (or vehicle) on a weekly or fortnightly basis to 23 weeks of age. All dogs completed the protocol without incident, and there was no clinically-relevant cellular or humoral immune response to rhSGSH delivery. Immunohistochemistry demonstrated rhSGSH delivery to widespread regions of the brain, and tandem mass spectrometry revealed an apparent dose-dependent decrease in the relative level of a heparan sulfate-derived disaccharide, with near normalization of substrate in many brain regions at the higher dose. Secondarily-stored GM3 ganglioside and unesterified cholesterol, determined using histological methods, were also reduced in a dose-dependent manner, as was the number of activated microglia. We have demonstrated that pre-symptomatic treatment of this progressive neurodegenerative disorder via intra-cerebrospinal fluid injection of rhSGSH mediates highly significant reductions in neuropathology in this MPS IIIA model and clinical trials of this treatment approach in MPS IIIA patients are therefore indicated.
Assuntos
Terapia de Reposição de Enzimas/métodos , Hidrolases/farmacologia , Mucopolissacaridose III/tratamento farmacológico , Mucopolissacaridose III/enzimologia , Degeneração Neural/tratamento farmacológico , Degeneração Neural/enzimologia , Animais , Modelos Animais de Doenças , Cães , Humanos , Hidrolases/uso terapêutico , Injeções Intraventriculares , Mucopolissacaridose III/genética , Degeneração Neural/genética , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/uso terapêuticoRESUMO
Mucopolysaccharidosis type IIIA (MPS IIIA) is a neurodegenerative lysosomal storage disorder that results from a deficiency of sulfamidase (N-sulfoglucosamine sulfohydrolase), with consequential accumulation of its substrate, partially degraded heparan sulfate. Conventional doses (e.g. 1mg/kg) of intravenously delivered recombinant human sulfamidase (rhSGSH) do not improve neuropathology in MPS IIIA mice due to an inability to traverse the blood-brain barrier; however high-dose treatment or administration of enzyme that has been chemically modified to remove mannose-6-phosphate glycans has been shown to reduce neuropathology in related animal models. We have combined these approaches to evaluate the ability of 1, 5, 10 or 20mg/kg of similarly chemically modified or unmodified rhSGSH to reduce neuropathology following repeated intravenous delivery to adult MPS IIIA mice. rhSGSH was detected in brain homogenates from mice treated with all doses of modified rhSGSH and those receiving the two higher doses of unmodified rhSGSH, albeit at significantly lower levels. Immunohistochemically, rhSGSH visualized in the brain was localized to the endothelium, meninges and choroid plexus, with no convincing punctate intra-neuronal staining seen. This presumably underlies the failure of the treatment to reduce the relative level of a heparan sulfate-derived oligosaccharide (GlcNS-UA), or secondarily stored substrates that accumulate in MPS IIIA brain cells. However, modification of rhSGSH significantly increased its effectiveness in degrading GlcNS-UA in non-CNS tissues, potentially as a result of its reduced plasma clearance. If this observation is generally applicable, chemical modification may permit the use of significantly lower doses of lysosomal enzymes in patients currently receiving intravenous enzyme replacement therapy.