Glycosaminoglycan Storage Research Articles

Proteomic analysis of mucopolysaccharidosis I mouse brain with two-dimensional polyacrylamide gel electrophoresis.

Mucopolysaccharidosis type I (MPS I) is due to deficiency of α-l-iduronidase (IDUA) and subsequent storage of undegraded glycosaminoglycans (GAG). The severe form of the disease, known as Hurler syndrome, is characterized by mental retardation and neurodegeneration of unknown etiology. To identify potential biomarkers and unveil the neuropathology mechanism of MPS I disease, two-dimensional polyacrylamide gel electrophoresis (PAGE) and nanoliquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) were applied to compare proteome profiling of brains from MPS I and control mice (5-month old). A total of 2055 spots were compared, and 25 spots (corresponding to 50 different proteins) with a fold change ≥3.5 and a p value <0.05 between MPS I and control mice were further analyzed by nanoLC-MS/MS. These altered proteins could be divided into three major groups based on Gene Ontology (GO) terms: proteins involved in metabolism, neurotransmission and cytoskeleton. Cytoskeletal proteins including ACTA1, ACTN4, TUBB4B and DNM1 were significantly downregulated. STXBP1, a regulator of synaptic vesicle fusion and docking was also downregulated, indicating impaired synaptic transmission. Additionally, proteins regulating Ca2+ and H+ homeostasis including ATP6V1B2 and RYR3 were downregulated, which may be related to disrupted autophagic and endocytotic pathways. Notably, there is no altered expression in proteins associated with cell death, ubiquitin or inflammation. These results for the first time highlight the important role of alterations in metabolism pathways, intracellular ionic homeostasis and the cytoskeleton in the neuropathology of MPS I disease. The proteins identified in this study would provide potential biomarkers for diagnostic and therapeutic studies of MPS I.

352. Functional Benefits of Systemic rAAV9-hIDS Gene Delivery in MPS II Mouse Model

Mucopolysaccharidosis (MPS) II is an X-linked recessive lysosomal storage disease caused by defect in iduronate-2-sulfatase (IDS), leading to in the accumulation of glycosaminoglycans (GAGs) in cells in the CNS and peripheral organs, and profound multisystem disorders in the majority of patients. No treatment is available for the neurological disorders of MPS II. In this study, we developed 2 self-complementary (sc) AAV9 vectors expressing human IDS and tested them in MPS II mice via systemic delivery. At a dose 5e12vg/kg, an IV injection of either scAAV9-U1a-hIDS or scAAV9-mCMV-IDS, led to rapid and persistent IDS expression and the correction of lysosomal GAG storage throughout the CNS, peripheral nervous system (PNS) and periphery tissues, as well as diminishing of astrocytosis in the CNS and PNS, in MPS II mice treated at 1mo, 3mo or 6mo of age. Importantly, we demonstrate significant improvement in cognitive and motor function, and extension of survival (ongoing) in the rAAV9-treated MPS II mice. These data demonstrate substantial functional benefits of a systemic scAAV9-hIDS gene delivery for treating MPS II at early and advanced disease stages, supporting the clinical potential of the approach.

348. Correction of CNS and Somatic Pathology by Intra-Cerebrospinal Fluid Gene Therapy for Mucopolysaccharidosis Type II

Mucopolysaccharidosis type II (MPSII), or Hunter Syndrome, is a X-linked recessive Lysosomal Storage Disease (LSD) caused by deficiency in Iduronate-2-sulfatase (IDS), an enzyme involved in the stepwise degradation of the glycosaminoglycans (GAGs) heparan sulfate (HS) and dermatan sulfate (DS). GAG storage builds up in the CNS and peripheral tissues causing severe neurologic and multisystemic somatic disease. Patients usually die during the second decade of life. Periodic intravenous enzyme replacement therapy (ERT) currently constitutes the only approved therapeutic option for MPSII. However, the inability of recombinant IDS to efficiently cross the blood-brain barrier limits the efficacy of the approach in treating neurological symptoms. Alternatively, the periodic infusion of ERT to the cerebrospinal fluid is under clinical testing, but serious adverse events associated with the use of intrathecal drug delivery devices have been reported. Here we report a gene therapy approach directly addressing the CNS pathology of MPSII. Through a minimally invasive procedure we delivered adeno-associated virus vectors of serotype 9 encoding IDS (AAV9-Ids) to the CSF of MPSII mice with already established disease. Four months after vector administration, treated mice showed a significant increase in IDS activity throughout the encephalon, with full resolution of lysosomal storage lesions, reversal of lysosomal dysfunction, normalization of the brain transcriptomic signature and disappearance of neuroinflammation. Moreover, our approach not only resulted in widespread distribution of vector in the CNS but also in liver transduction, providing a peripheral source of therapeutic protein that corrected the storage disease in visceral organs, with evidence of cross-correction of non-transduced organs by circulating enzyme. Importantly, MPSII mice treated with AAV9-Ids also showed normalization of behavioural deficits. These results provide a strong proof of concept for the clinical translation of our approach for the treatment of Hunter patients with cognitive impairment.

Enzyme replacement therapy with laronidase (Aldurazyme(®)) for treating mucopolysaccharidosis type I.

Mucopolysaccharidosis type I can be classified as three clinical sub-types; Hurler syndrome, Hurler-Scheie syndrome and Scheie syndrome, with the scale of severity being such that Hurler syndrome is the most severe and Scheie syndrome the least severe. It is a rare, autosomal recessive disorder caused by a deficiency of alpha-L-iduronidase. Deficiency of this enzyme results in the accumulation of glycosaminoglycans within the tissues. The clinical manifestations are facial dysmorphism, hepatosplenomegaly, upper airway obstruction, skeletal deformity and cardiomyopathy. If Hurler syndrome is left untreated, death ensues by adolescence. There are more attenuated variants termed Hurler-Scheie or Scheie syndrome, with those affected potentially not presenting until adulthood. Enzyme replacement therapy has been used for a number of years in the treatment of Hurler syndrome, although the current gold standard would be a haemopoietic stem cell transplant in those diagnosed by 2.5 years of age. This is an updated version of the original Cochrane review published in 2013. To evaluate the effectiveness and safety of treating mucopolysaccharidosis type I with laronidase enzyme replacement therapy as compared to placebo. We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group's Inborn Errors of Metabolism Trials Register, MEDLINE via OVID and Embase.Date of most recent search: 05 October 2015. Randomised and quasi-randomised controlled studies of laronidase enzyme replacement therapy compared to placebo. Two authors independently screened the identified studies. The authors then appraised and extracted data. One study of 45 patients met the inclusion criteria. This double-blind, placebo-controlled, randomised, multinational study looked at laronidase at a dose of 0.58 mg/kg/week versus placebo in patients with mucopolysaccharidosis type I. All primary outcomes listed in this review were studied in this study. The laronidase group achieved statistically significant improvements in per cent predicted forced vital capacity compared to placebo, MD 5.60 (95% confidence intervals 1.24 to 9.96) and in the six-minute-walk test (mean improvement of 38.1 metres in the laronidase group; P = 0.039, when using a prospectively planned analysis of covariance). The levels of urinary glycoaminoglycans were also significantly reduced. In addition, there were improvements in hepatomegaly, sleep apnoea and hypopnoea. Laronidase antibodies were detected in nearly all patients in the treatment group with no apparent clinical effect and titres were reducing by the end of the study. Infusion-related adverse reactions occurred in both groups but all were mild and none necessitated medical intervention or infusion cessation. The current evidence demonstrates that laronidase is effective when compared to placebo in the treatment of mucopolysaccharidosis type I. The included study was comprehensive and of good quality, although there were few participants. The study included all of the key outcome measures we wished to look at. It demonstrated that laronidase is efficacious in relation to reducing biochemical parameters (reduced urine glycosaminoglycan excretion) and improved functional capacity as assessed by forced vital capacity and the six-minute-walk test. In addition glycosaminoglycan storage was reduced as ascertained by a reduction in liver volume. Laronidase appeared to be safe and, while antibodies were generated, these titres were reducing by the end of the study. More studies are required to determine long-term effectiveness and safety and to assess the impact upon quality of life. Enzyme replacement therapy with laronidase can be used pre- and peri-haemopoietic stem cell transplant, which is now the gold standard treatment in those patients diagnosed under 2.5 years of age.

Immune-Mediated Inflammation May Contribute to the Pathogenesis of Cardiovascular Disease in Mucopolysaccharidosis Type I.

BackgroundCardiovascular disease, a progressive manifestation of α-L-iduronidase deficiency or mucopolysaccharidosis type I, continues in patients both untreated and treated with hematopoietic stem cell transplantation or intravenous enzyme replacement. Few studies have examined the effects of α-L-iduronidase deficiency and subsequent glycosaminoglycan storage upon arterial gene expression to understand the pathogenesis of cardiovascular disease.MethodsGene expression in carotid artery, ascending, and descending aortas from four non-tolerized, non-enzyme treated 19 month-old mucopolysaccharidosis type I dogs was compared with expression in corresponding vascular segments from three normal, age-matched dogs. Data were analyzed using R and whole genome network correlation analysis, a bias-free method of categorizing expression level and significance into discrete modules. Genes were further categorized based on module-trait relationships. Expression of clusterin, a protein implicated in other etiologies of cardiovascular disease, was assessed in canine and murine mucopolysaccharidosis type I aortas via Western blot and in situ immunohistochemistry.ResultsGene families with more than two-fold, significant increased expression involved lysosomal function, proteasome function, and immune regulation. Significantly downregulated genes were related to cellular adhesion, cytoskeletal elements, and calcium regulation. Clusterin gene overexpression (9-fold) and protein overexpression (1.3 to 1.62-fold) was confirmed and located specifically in arterial plaques of mucopolysaccharidosis-affected dogs and mice.ConclusionsOverexpression of lysosomal and proteasomal-related genes are expected responses to cellular stress induced by lysosomal storage in mucopolysaccharidosis type I. Upregulation of immunity-related genes implicates the potential involvement of glycosaminoglycan-induced inflammation in the pathogenesis of mucopolysaccharidosis-related arterial disease, for which clusterin represents a potential biomarker.

Fundus Autofluorescence and Enhanced Depth Imaging Spectral-Domain Optical Coherence Tomography in Hunter Syndrome-New Insights

Introduction: Hunter syndrome or mucopolysaccharidosis type II is a rare progressive multi-systemic disorder, caused by an abnormal storage of glycosaminoglycans (GAGs) in almost every cell type, including most ocular tissues [1,2]. Patients have a short life expectancy and ocular manifestations can be present early in the course of disease [1,2]. Purpose: To report the fundus autofluorescence and tomographic ocular findings in Hunter syndrome. Methods: A 18-year-old male patient with Hunter syndrome with progressive nyctalopia was submitted to color fundus photography, blue fundus autofluorescence (FAF), fluorescein angiography (FA) and spectral domain optical coherence tomography with enhanced-depth imaging (EDI-SD OCT). Results and discussion: Fundus examination and wide-field fluorescein angiogram revealed normal optic discs and bilateral pigmentary atrophic changes at the mid periphery with macular sparing. SD OCT revealed a retinal thinning due to external retinal atrophy affecting the photoreceptor layer beyond the parafoveal area. Although a prominent central external limiting membrane (ELM) was present, both the ellipsoid zone band and ELM could not be tracked beyond the central 2-mm and 2. 5 mm diameter ring, respectively. EDI-SD OCT revealed a highly irregular choroid, especially in its outer boundary, probably due to GAG scleral deposition. Blue FAF presented a symmetric hyperautofluorescent parafoveal ring that corresponded to the area where the ELM was present in the absence of the ellipsoid band. A mottled hyper/hypofluorescent pattern was present at the mid-peripheral retina. Scarce hyperautofluorescent dots were found in the left optic disc resulting from disc drusen which could result from axoplasmic flow disturbances due to GAGs scleral deposition. Conclusions: To our knowledge this is the first report of fundus auto-fluorescence imaging in Hunter syndrome. We also report, for the first time, optic disc drusen in this disease. New imaging techniques can provide new insights for the understanding of this disease, and could potentially provide valuable biomarkers for treatment guidance.

Functional correction of neurological and somatic disorders at later stages of disease in MPS IIIA mice by systemic scAAV9-hSGSH gene delivery

The reversibility of neuropathic lysosomal storage diseases, including MPS IIIA, is a major goal in therapeutic development, due to typically late diagnoses and a large population of untreated patients. We used self-complementary adeno-associated virus (scAAV) serotype 9 vector expressing human N-sulfoglucosamine sulfohydrolase (SGSH) to test the efficacy of treatment at later stages of the disease. We treated MPS IIIA mice at 1, 2, 3, 6, and 9 months of age with an intravenous injection of scAAV9-U1a-hSGSH vector, leading to restoration of SGSH activity and reduction of glycosaminoglycans (GAG) throughout the central nervous system (CNS) and somatic tissues at a dose of 5E12 vg/kg. Treatment up to 3 months age improved learning ability in the Morris water maze at 7.5 months, and lifespan was normalized. In mice treated at 6 months age, behavioral performance was impaired at 7.5 months, but did not decline further when retested at 12 months, and lifespan was increased, but not normalized. Treatment at 9 months did not increase life-span, though the GAG storage pathology in the CNS was improved. The study suggests that there is potential for gene therapy intervention in MPS IIIA at intermediate stages of the disease, and extends the clinical relevance of our systemic scAAV9-hSGSH gene delivery approach.

Diagnostic and treatment strategies in mucopolysaccharidosis VI.

Mucopolysaccharidosis VI (MPS VI) is a very rare autosomal recessive disorder caused by mutations in the ARSB gene, which lead to deficient activity of the lysosomal enzyme ASB. This enzyme is important for the breakdown of the glycosaminoglycans (GAGs) dermatan sulfate and chondroitin sulfate, which accumulate in body tissues and organs of MPS VI patients. The storage of GAGs (especially dermatan sulfate) causes bone dysplasia, joint restriction, organomegaly, heart disease, and corneal clouding, among several other problems, and reduced life span. Despite the fact that most cases are severe, there is a spectrum of severity and some cases are so attenuated that diagnosis is made late in life. Although the analysis of urinary GAGs and/or the measurement of enzyme activity in dried blood spots are useful screening methods, the diagnosis is based in the demonstration of the enzyme deficiency in leucocytes or fibroblasts, and/or in the identification of pathogenic mutations in the ARSB gene. Specific treatment with enzyme replacement has been available since 2005. It is safe and effective, bringing measurable benefits and increased survival to patients. As several evidences indicate that early initiation of therapy may lead to a better outcome, newborn screening is being considered for this condition, and it is already in place in selected areas where the incidence of MPS VI is increased. However, as enzyme replacement therapy is not curative, associated therapies should be considered, and research on innovative therapies continues. The management of affected patients by a multidisciplinary team with experience in MPS diseases is highly recommended.

EXTL2 and EXTL3 inhibition with siRNAs as a promising substrate reduction therapy for Sanfilippo C syndrome.

Sanfilippo syndrome is a rare lysosomal storage disorder caused by an impaired degradation of heparan sulfate (HS). It presents severe and progressive neurodegeneration and currently there is no effective treatment. Substrate reduction therapy (SRT) may be a useful option for neurological disorders of this kind, and several approaches have been tested to date. Here we use different siRNAs targeting EXTL2 and EXTL3 genes, which are important for HS synthesis, as SRT in Sanfilippo C patients’ fibroblasts in order to decrease glycosaminoglycan (GAG) storage inside the lysosomes. The results show a high inhibition of the EXTL gene mRNAs (around 90%), a decrease in GAG synthesis after three days (30–60%) and a decrease in GAG storage after 14 days (up to 24%). Moreover, immunocytochemistry analyses showed a clear reversion of the phenotype after treatment. The in vitro inhibition of HS synthesis genes using siRNAs shown here is a first step in the development of a future therapeutic option for Sanfilippo C syndrome.

Dimethylmethylene blue: a tool for MPS screening

Mucopolysaccharidoses are a group of lysosomal diseases characterized by storage of glycosaminoglycans (GAGs) in tissue and as a consequence in urine. GAGs consist of N- and /or O-sulfate groups and are covalently linked to core proteins to form proteoglycans. During catabolism, GAGs are internalized in lysosome and degraded into their monomolecular constituents by lysosomal endoglycosidases, exoglycosidases, and exosulfatases. In Mucopolysaccharidoses (MPS) one of the hydrolytic lysosomal enzymes is deficient, which results in the accumulation of GAGs in the lysosomes and the resulting syndrome depends on which enzyme is deficient. The present spectrophotometric procedure based on the color reaction with Dimethyl Methylene Blue can be performed directly on untimed urine sample without prior precipitation. We have tested urine samples without preservative of 21 patients with various Mucopolysaccharidoses (eight hunters, four Hurler, two morquio, two scheie, three sanfilippo A and two sanfilippo C) through Dimethyl Methylene Blue assay and compared the result with Cetypyridinium Chloride (CPC) turbidity assay at pH: 4.8 (Citrate buffered) and at pH: 7.0 (Tris buffered). A total 72 normal urine samples were also tested with the CPC turbidity assay and the DMB assays to establish a reference range. GAG content is measured at absorbance 520 nm. Condroitin-4- sulfate standard were used to prepare calibration curves. In Normal subjects the GAG content in the younger age groups was more than the older one. Glycosaminoglycans level in the DMB assay were increased in 21 of 21 patients; whereas GAG’s level in the turbidity test at pH 7.0, value were increased in 19 of the 21 patients and at pH 4.0, GAG’s content was increased in only 15 of the 21 patients. The Dimethyl Methylene Blue assay procedure is a rapid, sensitive and reliable screening procedure to determine concentration of GAG’s for various MPS.

Mucopolysaccharidosis-like phenotype in feline Sandhoff disease and partial correction after AAV gene therapy.

Sandhoff disease (SD) is a fatal neurodegenerative disease caused by a mutation in the enzyme β-N-acetylhexosaminidase. Children with infantile onset SD develop seizures, loss of motor tone and swallowing problems, eventually reaching a vegetative state with death typically by 4years of age. Other symptoms include vertebral gibbus and cardiac abnormalities strikingly similar to those of the mucopolysaccharidoses. Isolated fibroblasts from SD patients have impaired catabolism of glycosaminoglycans (GAGs). To evaluate mucopolysaccharidosis-like features of the feline SD model, we utilized radiography, MRI, echocardiography, histopathology and GAG quantification of both central nervous system and peripheral tissues/fluids. The feline SD model exhibits cardiac valvular and structural abnormalities, skeletal changes and spinal cord compression that are consistent with accumulation of GAGs, but are much less prominent than the severe neurologic disease that defines the humane endpoint (4.5±0.5months). Sixteen weeks after intracranial AAV gene therapy, GAG storage was cleared in the SD cat cerebral cortex and liver, but not in the heart, lung, skeletal muscle, kidney, spleen, pancreas, small intestine, skin, or urine. GAG storage worsens with time and therefore may become a significant source of pathology in humans whose lives are substantially lengthened by gene therapy or other novel treatments for the primary, neurologic disease.

373. Expression of Human Iduronidase from Sleeping Beauty Engineered Human B Lymphocytes as a Cellular Therapy for Mucopolysaccharidosis Type I

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disorder cause by absence of the glycosidase alpha-L-idurondase (IDUA), resulting in the systemic accumulation of glycosaminoglycan storage materials heparan sulfate and dermatan sulfate. Affected individuals exhibit a range of manifestations including hepatosplenomegaly, skeletal dysplasias, and cardiopulmonary obstruction. The severe form of the disease (Hurler syndrome) is associated with progressive neurologic dysfunction and death by age 10. Current treatments for MPS I include enzyme replacement (ERT), and hematopoietic stem cell transplantation (HSCT). However, these treatments are expensive, insufficiently effective, and allogeneic HSCT is associated with considerable morbidity and mortality.As an alternate approach to MPS I, Immusoft Corporation is developing as a cellular therapeutic primary B cells that have been genetically engineered for long-term expression and secretion of IDUA. At the core of the technology is the generation of genetically engineered autologous human B cells, which are isolated and expanded from patient blood. Mature B cells (specifically long-lived plasma cells) are the ideal cell type to produce protein since they live for long periods of time, occupy diverse tissues throughout the body and are capable of secreting very high levels of protein. In collaboration with Discovery Genomics, Inc., the University of Minnesota and the Fred Hutchinson Cancer Research Center, Immusoft has developed a treatment for Mucopolysaccharidosis type I (MPS I). In initial tests, Sleeping Beauty (SB) transposons expressing the human iduronidase gene were introduced into cultured B lymphoblastoid cells with and without a source of SB transposase, demonstrating long-term, high level expression of IDUA activity in vitro. SB transposons expressing both IDUA and a methotrexate-resistant human dihydrofolate reductase allowed for a >10-fold expansion of IDUA-expressing cells in the presence of methotrexate, as determined by intracellular staining for human IDUA and flow cytometry. Overall IDUA expression was also increased >10-fold in the methotrexate-selected cell population. IDUA encoding SB transposons were also nucleofected along with a source of SB transposase into primary human memory B cells, demonstrating expression of IDUA at levels more than 100 times greater than that observed in cultures of untreated primary B cells. These data support the development of a clinical trial to test SB-engineered B cells in the treatment of MPS I, with implications for improved treatment of other lysosomal storage disorders as well.

280. Combination of Low-Dose Gene Therapy and Monthly Enzyme Replacement Therapy Improves the Phenotype of a Mouse Model of Lysosomal Storage Disease

Enzyme replacement therapy (ERT) is the current standard of care for Mucopolysaccharidosis type VI (MPS VI) that is caused by deficiency of arylsulfatase B (ARSB), which results in widespread accumulation and excretion of toxic glycosaminoglycans (GAGs). However, ERT is associated with inconvenient multiple and costly administrations and fails to ameliorate cardiac, visual and bone abnormalities. To overcome ERT limitations, we developed a successful gene therapy approach based on a single administration of AAV2/8 that targets liver of MPS VI animal models. Importantly, we showed that a single systemic administration of AAV2/8 at high doses (2×10e12 gc/kg) is at least as effective as the current ERT therapeutic regimen based on weekly infusions of recombinant human ARSB (rhARSB). If this translates to humans, gene therapy could replace ERT for MPS VI. However, the administration of high doses of AAV2/8 requires a challenging and costly production process, and results in cell-mediated immune responses that eliminate transduced hepatocytes. Similarly, weekly ERT infusions are costly and require high patient compliance. We therefore evaluated in a mouse model of MPS VI whether the combination of low doses of AAV2/8 at 6×10e11 or 2×10e11 gc/kg with a rarified ERT schedule (1mg/kg once a month) may be as effective as the single treatments at high doses or frequent regimen. Significant increase of ARSB activity was found in liver of all treated mice. Detectable but low activity was variably observed in spleen and kidney and was associated with significant reduction of tissue GAGs, regardless of treatment and ARSB activity levels, similarly to what observed in mice treated with high doses of AAV2/8 or weekly ERT. This supports previous data indicating that low enzymatic levels improve MPS VI visceral phenotype. Evaluation of GAG storage in myocardium and heart valves is in progress. Urinary GAG, which are a sensitive biomarker of systemic clearance of lysosomal storage and, thus of therapeutic efficacy, are slightly reduced in mice treated with either monthly ERT or 2×10e11 gc/kg of AAV2/8. The reduction is more consistent at 6×10e11 gc/kg. Importantly, urinary GAG decreased more in mice receiving the combined therapy than in those receiving single treatments. In particular, urinary GAG reduction in mice treated with both 6×10e11 gc/kg of AAV2/8 and monthly ERT was comparable to that obtained following administration of either high doses of AAV2/8, i.e 2×10e12 gc/kg, or weekly ERT. The data collected so far show similar efficacy between low-dose gene therapy combined with rarified ERT and the corresponding single treatments at high doses or frequent regimen. This should increase the safety and reduce the risks and costs associated with both therapeutic approaches.

706. Non-Invasive Intranasal Administration of AAV9-Iduronidase Prevents Emergence of Neurologic Disease and Neurocognitive Dysfunction in a Murine Model of Mucopolysaccharidosis Type I

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive storage disease caused by deficiency of alpha-L-iduronidase (IDUA), resulting in accumulation of glycosaminoglycans (GAGs). In the severe form of the disease (Hurler syndrome), death results by age 10. Current treatments for this disease include hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT). However, ERT is ineffective in treating CNS disease due to the inability of lysosomal enzymes to traverse the blood-brain barrier, and while there is neurologic benefit to HSCT the procedure is associated with significant morbidity and mortality.We have taken a novel approach to treat neurologic disease associated with Hurler syndrome, using intranasal administration of an IDUA-encoding AAV9 vector. A CAGS regulated AAV9-IDUA vector was infused intranasally into adult mice (2-3 months of age) that had been immunotolerized at birth with Aldurazyme to prevent anti-IDUA immune response. Mice sacrificed at 3 months post-infusion exhibited IDUA enzyme activity levels that were 100-fold that of wild type in the olfactory bulb, with wild type levels of enzyme restored in all other parts of the brain. Intranasal treatment with AAV9-IDUA also resulted in clearance of tissue GAG storage materials in all parts of the brain. QPCR analysis of vector genomes indicated only background levels in all portions of the brain. There was strong IDUA immunofluorescence staining of tissue sections observed in the nasal epithelium and olfactory bulb but there was no evidence for the presence of transduced cells in other portions of the brain. This indicates that clearing of storage materials most likely occurred as a result of enzyme diffusion from the olfactory bulb and the nasal epithelium into deeper areas of the brain. At 6 months of age, intranasally treated animals along with age-matched heterozygote and IDUA-deficient control animals were subjected to neurocognitive testing using the Barnes maze. Unaffected heterozygote animals exhibited improved performance in this test while MPS I mice displayed a deficit in locating the escape. Remarkably, MPS I mice treated intranasally with AAV9-IDUA exhibited behavior similar to the heterozygote controls, demonstrating prevention of the neurocognitive deficit seen in the untreated MPS I animals. There was no significant difference between heterozygote animals and treated animals, while latency to escape was significantly different between these two groups and MPS I deficient animals (P<0.001). This novel, non-invasive strategy for intranasal AAVIDUA administration could potentially be used to treat CNS manifestations of several disorders, including lysosomal storage diseases.

Enzymatic replacement therapy for Hunter disease: Up to 9 years experience with 17 patients

Hunter disease is an X-linked lysosomal storage disorder characterized by progressive storage of glycosaminoglycans (GAGs) and multi-organ impairment. The central nervous system (CNS) is involved in at least 50% of cases. Since 2006, the enzymatic replacement therapy (ERT) is available but with no effect on the cognitive impairment, as the present formulation does not cross the blood–brain barrier. Here we report the outcome of 17 Hunter patients treated in a single center. Most of them (11) started ERT in 2006, 3 had started it earlier in 2004, enrolled in the phase III trial, and 3 after 2006, as soon as the diagnosis was made. The liver and spleen sizes and urinary GAGs significantly decreased and normalized throughout the treatment. Heart parameters improved, in particular the left ventricular mass index/m2 decreased significantly. Amelioration of hearing was seen in many patients. Joint range of motion improved in all patients. However, no improvement on respiratory function, eye, skeletal and CNS disease was found. The developmental quotient of patients with a CNS involvement showed a fast decline. These patients were no more testable after 6years of age and, albeit the benefits drawn from ERT, their quality of life worsened throughout the years. The whole group of patients showed a consistent residual disease burden mainly represented by persistent skeletal disease and frequent need of surgery. This study suggests that early diagnosis and treatment and other different therapies which are able to cross the blood–brain barrier, might in the future improve the MPS II outcome.

Broad Functional Correction of Molecular Impairments by Systemic Delivery of scAAVrh74-hSGSH Gene Delivery in MPS IIIA Mice

Mucopolysaccharidosis (MPS) IIIA is a neuropathic lysosomal storage disease caused by deficiency in N-sulfoglucosamine sulfohydrolase (SGSH). Genome-wide gene expression microarrays in MPS IIIA mice detected broad molecular abnormalities (greater than or equal to twofold, false discovery rate ≤10) in numerous transcripts (314) in the brain and blood (397). Importantly, 22 dysregulated blood transcripts are known to be enriched in the brain and linked to broad neuronal functions. To target the root cause, we used a self-complementary AAVrh74 vector to deliver the human SGSH gene into 4-6 weeks old MPS IIIA mice by an intravenous injection. The treatment resulted in global central nervous system (CNS) and widespread somatic restoration of SGSH activity, clearance of CNS and somatic glycosaminoglycan storage, improved behavior performance, and significantly extended survival. The scAAVrh74-hSGSH treatment also led to the correction of the majority of the transcriptional abnormalities in the brain (95.9%) and blood (97.7%), of which 182 and 290 transcripts were normalized in the brain and blood, respectively. These results demonstrate that a single systemic scAAVrh74-hSGSH delivery mediated efficient restoration of SGSH activity and resulted in a near complete correction of MPS IIIA molecular pathology. This study also demonstrates that blood transcriptional profiles reflect the biopathological status of MPS IIIA, and also respond well to effective treatments.

Enzyme Replacement Therapy With Elosulfase Alfa Decreases Storage of Glycosaminoglycan in White Blood Cells of Patients With Morquio A Syndrome

Mucopolysaccharidosis IVA (MPS IVA; Morquio A syndrome) is a lysosomal storage disorder caused by a deficient N-acetylgalactosamine-6-sulfate sulfatase activity, leading to cellular storage of undegraded keratan sulfate. Recently enzyme replacement therapy (ERT) was approved for MPS IVA, but some of ERT effects are still unknown. In the present study, we aimed to evaluate the efficacy of elosulfase alfa upon glycosaminoglycan (GAG) storage in peripheral blood white blood cells of patients with MPS IVA treated for 6 months, comparing samples from patients who received weekly infusions of enzyme (ERT-W) versus infusions every other week (ERT-EOW) versus placebo. A significant reduction in GAG storage was observed in both ERT-treated groups, with weekly ERT showing slightly better performance than ERT-EOW.

Elosulfase alfa decreases glycosaminoglycan storage in white blood cells from Morquio syndrome type A patients undergoing enzyme replacement

Elosulfase alfa decreases glycosaminoglycan storage in white blood cells from Morquio syndrome type A patients undergoing enzyme replacement

Shotgun proteomics reveals possible mechanisms for cognitive impairment in Mucopolysaccharidosis I mice.

Mucopolysaccharidosis type I (MPS I) is due to deficient alpha-L-iduronidase (IDUA) which leads to storage of undegraded glycosaminoglycans (GAG). The severe form of the disease is characterized by mental retardation of unknown etiology. Trying to unveil the mechanisms that lead to cognitive impairment in MPS I, we studied alterations in the proteome from MPS I mouse hippocampus. Eight-month old mice presented increased LAMP-1 expression, GAG storage in neurons and glial cells, and impaired aversive and non-aversive memory. Shotgun proteomics was performed and 297 proteins were identified. Of those, 32 were differentially expressed. We found elevation in proteins such as cathepsins B and D; however their increase did not lead to cell death in MPS I brains. Glial fibrillary acid protein (GFAP) was markedly elevated, and immunohistochemistry confirmed a neuroinflammatory process that could be responsible for neuronal dysfunction. We didn't observe any differences in ubiquitin expression, as well as in other proteins related to protein folding, suggesting that the ubiquitin system is working properly. Finally, we observed alterations in several proteins involved in synaptic plasticity, including overexpression of post synaptic density-95 (PSD95) and reduction of microtubule-associated proteins 1A and 1B. These results together suggest that the cognitive impairment in MPS I mice is not due to massive cell death, but rather to neuronal dysfunction caused by multiple processes, including neuroinflammation and alterations in synaptic plasticity.

Extracellular matrix disruption is an early event in the pathogenesis of skeletal disease in mucopolysaccharidosis I

Progressive skeletal and connective tissue disease represents a significant clinical burden in all of the mucopolysaccharidoses. Despite the introduction of enzyme replacement strategies for many of the mucopolysaccharidoses, symptomatology related to bone and joint disease appears to be recalcitrant to current therapies. In order to address these unmet medical needs a clearer understanding of skeletal and connective tissue disease pathogenesis is required. Historically the pathogenesis of the mucopolysaccharidoses has been assumed to directly relate to progressive storage of glycosaminoglycans. It is now apparent for many lysosomal storage disorders that more complex pathogenic mechanisms underlie patients' clinical symptoms. We have used proteomic and genome wide expression studies in the murine mucopolysaccharidosis I model to identify early pathogenic events occurring in micro-dissected growth plate tissue. Studies were conducted using 3 and 5-week-old mice thus representing a time at which no obvious morphological changes of bone or joints have taken place. An unbiased iTRAQ differential proteomic approach was used to identify candidates followed by validation with multiple reaction monitoring mass spectrometry and immunohistochemistry. These studies reveal significant decreases in six key structural and signaling extracellular matrix proteins; biglycan, fibromodulin, PRELP, type I collagen, lactotransferrin, and SERPINF1. Genome-wide expression studies in embryonic day 13.5 limb cartilage and 5week growth plate cartilage followed by specific gene candidate qPCR studies in the 5week growth plate identified fourteen significantly deregulated mRNAs (Adamts12, Aspn, Chad, Col2a1, Col9a1, Hapln4, Lum, Matn1, Mmp3, Ogn, Omd, P4ha2, Prelp, and Rab32). The involvement of biglycan, PRELP and fibromodulin; all members of the small leucine repeat proteoglycan family is intriguing, as this protein family is implicated in the pathogenesis of late onset osteoarthritis. Taken as a whole, our data indicates that alteration of the extracellular matrix represents a very early event in the pathogenesis of the mucopolysaccharidoses and implies that biomechanical failure of chondro-osseous tissue may underlie progressive bone and joint disease symptoms. These findings have important therapeutic implications.