Publications by authors named "Brian W Bigger"

56 Publications

Bile acid biosynthesis in Smith-Lemli-Opitz syndrome bypassing cholesterol: Potential importance of pathway intermediates.

J Steroid Biochem Mol Biol 2021 Feb 24;206:105794. Epub 2020 Nov 24.

Swansea University Medical School, ILS1 Building, Singleton Park, Swansea, SA2 8PP, Wales, UK. Electronic address:

Bile acids are the end products of cholesterol metabolism secreted into bile. They are essential for the absorption of lipids and lipid soluble compounds from the intestine. Here we have identified a series of unusual Δ-unsaturated bile acids in plasma and urine of patients with Smith-Lemli-Opitz syndrome (SLOS), a defect in cholesterol biosynthesis resulting in elevated levels of 7-dehydrocholesterol (7-DHC), an immediate precursor of cholesterol. Using liquid chromatography - mass spectrometry (LC-MS) we have uncovered a pathway of bile acid biosynthesis in SLOS avoiding cholesterol starting with 7-DHC and proceeding through 7-oxo and 7β-hydroxy intermediates. This pathway also occurs to a minor extent in healthy humans, but elevated levels of pathway intermediates could be responsible for some of the features SLOS. The pathway is also active in SLOS affected pregnancies as revealed by analysis of amniotic fluid. Importantly, intermediates in the pathway, 25-hydroxy-7-oxocholesterol, (25R)26-hydroxy-7-oxocholesterol, 3β-hydroxy-7-oxocholest-5-en-(25R)26-oic acid and the analogous 7β-hydroxysterols are modulators of the activity of Smoothened (Smo), an oncoprotein that mediates Hedgehog (Hh) signalling across membranes during embryogenesis and in the regeneration of postembryonic tissue. Computational docking of the 7-oxo and 7β-hydroxy compounds to the extracellular cysteine rich domain of Smo reveals that they bind in the same groove as both 20S-hydroxycholesterol and cholesterol, known activators of the Hh pathway.
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http://dx.doi.org/10.1016/j.jsbmb.2020.105794DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7816163PMC
February 2021

Dual Purpose Vectors for Rare Neurological Diseases.

Authors:
Brian W Bigger

Mol Ther 2020 10 18;28(10):2104-2105. Epub 2020 Sep 18.

3.721 Stopford Building, School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK. Electronic address:

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http://dx.doi.org/10.1016/j.ymthe.2020.09.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7545005PMC
October 2020

Actinomycin D downregulates Sox2 and improves survival in preclinical models of recurrent glioblastoma.

Neuro Oncol 2020 09;22(9):1289-1301

Brain Tumor Research Group, Stem Cell and Neurotherapies Laboratory, Division of Cell Matrix Biology and Regenerative Medicine, University of Manchester, Manchester, UK.

Background: Glioblastoma (GBM) has been extensively researched over the last few decades, yet despite aggressive multimodal treatment, recurrence is inevitable and second-line treatment options are limited. Here, we demonstrate how high-throughput screening (HTS) in multicellular spheroids can generate physiologically relevant patient chemosensitivity data using patient-derived cells in a rapid and cost-effective manner. Our HTS system identified actinomycin D (ACTD) to be highly cytotoxic over a panel of 12 patient-derived glioma stemlike cell (GSC) lines. ACTD is an antineoplastic antibiotic used in the treatment of childhood cancers. Here, we validate ACTD as a potential repurposed therapeutic for GBM in 3-dimensional GSC cultures and patient-derived xenograft models of recurrent glioblastoma.

Methods: Twelve patient-derived GSC lines were screened at 10 µM, as multicellular spheroids, in a 384-well serum-free assay with 133 FDA-approved compounds. GSCs were then treated in vitro with ACTD at established half-maximal inhibitory concentrations (IC50). Downregulation of sex determining region Y-box 2 (Sox2), a stem cell transcription factor, was investigated via western blot and through immunohistological assessment of murine brain tissue.

Results: Treatment with ACTD was shown to significantly reduce tumor growth in 2 recurrent GBM patient-derived models and significantly increased survival. ACTD is also shown to specifically downregulate the expression of Sox2 both in vitro and in vivo.

Conclusion: These findings indicate that, as predicted by our HTS, ACTD could deplete the cancer stem cell population within the tumor mass, ultimately leading to a delay in tumor progression.

Key Points: 1. High-throughput chemosensitivity data demonstrated the broad efficacy of actinomycin D, which was validated in 3 preclinical models of glioblastoma.2. Actinomycin D downregulated Sox2 in vitro and in vivo, indicating that this agent could target the stem cell population of GBM tumors.
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http://dx.doi.org/10.1093/neuonc/noaa051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7523458PMC
September 2020

Haematopoietic stem cell gene therapy with IL-1Ra rescues cognitive loss in mucopolysaccharidosis IIIA.

EMBO Mol Med 2020 03 14;12(3):e11185. Epub 2020 Feb 14.

Stem Cell and Neurotherapies, Division of Cell Matrix Biology & Regenerative Medicine, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK.

Mucopolysaccharidosis IIIA is a neuronopathic lysosomal storage disease, characterised by heparan sulphate and other substrates accumulating in the brain. Patients develop behavioural disturbances and cognitive decline, a possible consequence of neuroinflammation and abnormal substrate accumulation. Interleukin (IL)-1β and interleukin-1 receptor antagonist (IL-1Ra) expression were significantly increased in both murine models and human MPSIII patients. We identified pathogenic mechanisms of inflammasome activation, including that disease-specific 2-O-sulphated heparan sulphate was essential for priming an IL-1β response via the Toll-like receptor 4 complex. However, mucopolysaccharidosis IIIA primary and secondary storage substrates, such as amyloid beta, were both required to activate the NLRP3 inflammasome and initiate IL-1β secretion. IL-1 blockade in mucopolysaccharidosis IIIA mice using IL-1 receptor type 1 knockout or haematopoietic stem cell gene therapy over-expressing IL-1Ra reduced gliosis and completely prevented behavioural phenotypes. In conclusion, we demonstrate that IL-1 drives neuroinflammation, behavioural abnormality and cognitive decline in mucopolysaccharidosis IIIA, highlighting haematopoietic stem cell gene therapy treatment with IL-1Ra as a potential neuronopathic lysosomal disease treatment.
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http://dx.doi.org/10.15252/emmm.201911185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7059006PMC
March 2020

Hampering brain tumor proliferation and migration using peptide nanofiber:si/ complexes.

Nanomedicine (Lond) 2019 12;14(24):3127-3142

Nanomedicine Lab, Faculty of Biology, Medicine & Health, The University of Manchester, AV Hill Building, Manchester, M13 9PT, UK.

To develop a nonviral tool for the delivery of siRNA to brain tumor cells using peptide nanofibers (PNFs). Uptake of PNFs was evaluated by confocal microscopy and flow cytometry. Gene silencing was determined by RT-qPCR and cell invasion assay. PNFs enter phagocytic (BV-2) and nonphagocytic (U-87 MG) cells via endocytosis and passive translocation. si delivered using PNFs reduced the expression of polo-like kinase 1 mRNA and induced cell death in a panel of immortalized and glioblastoma-derived stem cells. Moreover, targeting MMP2 using PNF:si reduced the invasion capacity of U-87 MG cells. We show that stereotactic intra-tumoral administration of PNF:si significantly extends the survival of tumor bearing mice comparing with the untreated tumor bearing animals. Our results suggest that this nanomedicine-based RNA interference approach deserves further investigation as a potential brain tumor therapeutic tool.
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http://dx.doi.org/10.2217/nnm-2019-0298DOI Listing
December 2019

Post-transplant laronidase augmentation for children with Hurler syndrome: biochemical outcomes.

Sci Rep 2019 Oct 1;9(1):14105. Epub 2019 Oct 1.

University of Minnesota, Division of Pediatric Blood and Marrow Transplant, Minneapolis, 55455, USA.

Allogeneic hematopoietic cell transplantation (HCT) benefits children with Hurler syndrome (MPS-IH). However, survivors remain burdened by substantial MPS-IH related residual disease. We studied the feasibility, safety and biochemical impact of augmentative recombinant intravenous enzyme replacement therapy (IV-ERT) post transplantation. Ten children with MPS-IH and ≥2 years from successful HCT underwent IV-ERT for 2 years' duration. Patients were monitored for anti-drug antibody (ADA) development, including inhibitory capacity and changes in urinary excretion of glycosaminoglycans (uGAG). Three patients demonstrated low-level ADA at baseline, though all children tolerated IV-ERT well. Eight patients developed ADA over the 2-year study, with 3 (38%) meeting criteria for an inhibitory ADA response. The aggregate cohort experienced a reduction in uGAG from baseline to study end, which was enhanced in children with low or no ADA response. Conversely, children with inhibitory ADA showed increase in uGAG over time. IV-ERT in previously transplanted children with MPS-IH appears safe and can reduce uGAG, although this is reversed by the presence of inhibitory ADA. These data show a biochemical change after initiation of post-HCT IV-ERT, but the occurrence of ADA and inhibitory antibodies are a concern and should be monitored in future efficacy trials. This trial was registered at www.clinicaltrials.gov , NCT01173016, 07/30/2010.
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http://dx.doi.org/10.1038/s41598-019-50595-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6773848PMC
October 2019

Pre-clinical Safety and Efficacy of Lentiviral Vector-Mediated Stem Cell Gene Therapy for the Treatment of Mucopolysaccharidosis IIIA.

Mol Ther Methods Clin Dev 2019 Jun 6;13:399-413. Epub 2019 Apr 6.

Stem Cell & Neurotherapies Group, School of Biological Sciences, Faculty of Biology Medicine and Health, Division of Cell Matrix Biology and Regenerative Medicine, University of Manchester, Manchester M13 9PT, UK.

Hematopoietic stem cell gene therapy is a promising therapeutic strategy for the treatment of neurological disorders, since transplanted gene-corrected cells can traffic to the brain, bypassing the blood-brain barrier, to deliver therapeutic protein to the CNS. We have developed this approach for the treatment of Mucopolysaccharidosis type IIIA (MPSIIIA), a devastating lysosomal storage disease that causes progressive cognitive decline, leading to death in early adulthood. In a previous pre-clinical proof-of-concept study, we demonstrated neurological correction of MPSIIIA utilizing hematopoietic stem cell gene therapy via a lentiviral vector encoding the gene. Prior to moving to clinical trial, we have undertaken further studies to evaluate the efficiency of gene transfer into human cells and also safety studies of biodistribution and genotoxicity. Here, we have optimized hCD34 cell transduction with clinical grade SGSH vector to provide improved pharmacodynamics and cell viability and validated effective scale-up and cryopreservation to generate an investigational medicinal product. Utilizing a humanized NSG mouse model, we demonstrate effective engraftment and biodistribution, with no vector shedding or transmission to germline cells. SGSH vector genotoxicity assessment demonstrated low transformation potential, comparable to other lentiviral vectors in the clinic. This data establishes pre-clinical safety and efficacy of HSCGT for MPSIIIA.
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http://dx.doi.org/10.1016/j.omtm.2019.04.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6479204PMC
June 2019

An Improved Adeno-Associated Virus Vector for Neurological Correction of the Mouse Model of Mucopolysaccharidosis IIIA.

Hum Gene Ther 2019 09 4;30(9):1052-1066. Epub 2019 Jun 4.

Stem Cell and Neurotherapies, Division of Cell Matrix Biology and Regenerative Medicine, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, Manchester, United Kingdom.

Patients with the lysosomal storage disease mucopolysaccharidosis IIIA (MPSIIIA) lack the lysosomal enzyme N-sulfoglucosamine sulfohydrolase (SGSH), one of the many enzymes involved in degradation of heparan sulfate. Build-up of un-degraded heparan sulfate results in severe progressive neurodegeneration for which there is currently no treatment. Experimental gene therapies based on gene addition are currently being explored. Following preclinical evaluation in MPSIIIA mice, an adeno-associated virus vector of serotype rh10 designed to deliver SGSH and sulfatase modifying factor 1 (SAF301) was trialed in four MPSIIIA patients, showing good tolerance and absence of adverse events with some improvements in neurocognitive measures. This study aimed to improve SAF301 further by removing sulfatase modifying factor 1 (SUMF1) and assessing if expression of this gene is needed to increase the SGSH enzyme activity (SAF301b). Second, the murine phosphoglycerate kinase (PGK) promotor was exchanged with a chicken beta actin/CMV composite (CAG) promotor (SAF302) to see if SGSH expression levels could be boosted further. The three different vectors were administered to MPSIIIA mice via intracranial injection, and SGSH expression levels were compared 4 weeks post treatment. Removal of SUMF1 resulted in marginal reductions in enzyme activity. However, promotor exchange significantly increased the amount of SGSH expressed in the brain, leading to superior therapeutic correction with SAF302. Biodistribution of SAF302 was further assessed using green fluorescent protein (GFP), indicating that vector spread was limited to the area around the injection tract. Further modification of the injection strategy to a single depth with higher injection volume increased vector distribution, leading to more widespread GFP distribution and sustained expression, suggesting this approach should be adopted in future trials.
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http://dx.doi.org/10.1089/hum.2018.189DOI Listing
September 2019

Metabolism of Non-Enzymatically Derived Oxysterols: Clues from sterol metabolic disorders.

Free Radic Biol Med 2019 11 19;144:124-133. Epub 2019 Apr 19.

Institute of Life Science, Swansea University Medical School, Singleton Park, Swansea, SA2 8PP, UK. Electronic address:

Cholestane-3β,5α,6β-triol (3β,5α,6β-triol) is formed from cholestan-5,6-epoxide (5,6-EC) in a reaction catalysed by cholesterol epoxide hydrolase, following formation of 5,6-EC through free radical oxidation of cholesterol. 7-Oxocholesterol (7-OC) and 7β-hydroxycholesterol (7β-HC) can also be formed by free radical oxidation of cholesterol. Here we investigate how 3β,5α,6β-triol, 7-OC and 7β-HC are metabolised to bile acids. We show, by monitoring oxysterol metabolites in plasma samples rich in 3β,5α,6β-triol, 7-OC and 7β-HC, that these three oxysterols fall into novel branches of the acidic pathway of bile acid biosynthesis becoming (25R)26-hydroxylated then carboxylated, 24-hydroxylated and side-chain shortened to give the final products 3β,5α,6β-trihydroxycholanoic, 3β-hydroxy-7-oxochol-5-enoic and 3β,7β-dihydroxychol-5-enoic acids, respectively. The intermediates in these pathways may be causative of some phenotypical features of, and/or have diagnostic value for, the lysosomal storage diseases, Niemann Pick types C and B and lysosomal acid lipase deficiency. Free radical derived oxysterols are metabolised in human to unusual bile acids via novel branches of the acidic pathway, intermediates in these pathways are observed in plasma.
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http://dx.doi.org/10.1016/j.freeradbiomed.2019.04.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6863434PMC
November 2019

Strategies for the Induction of Immune Tolerance to Enzyme Replacement Therapy in Mucopolysaccharidosis Type I.

Mol Ther Methods Clin Dev 2019 Jun 2;13:321-333. Epub 2019 Mar 2.

Stem Cell and Neurotherapies, Faculty of Medical and Human Sciences, University of Manchester, Manchester, UK.

Enzyme replacement therapy with laronidase is an established treatment for Mucopolysaccharidosis type I (MPS I), but its efficacy may be limited by the development of anti-drug antibodies, which inhibit cellular uptake of the enzyme. In a related disorder, infantile Pompe disease, immune tolerance induction with low-dose, short-course methotrexate appears to reduce antibody formation. We investigated a similar regimen using oral methotrexate in three MPS I patients. All patients developed anti-laronidase immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies, and they had clinically relevant levels of cellular uptake inhibition. We then explored several immune tolerance induction strategies in MPS I mice: (1) methotrexate, (2) combination of non-depleting anti-CD4 and anti-CD8 monoclonal antibodies, (3) methotrexate with anti-CD4 and anti-CD8 monoclonals, (4) anti-CD4 monoclonal, and (5) anti-CD8 monoclonal. Treated mice received 10 weekly laronidase injections, and laronidase was delivered with adjuvant on day 49 to further challenge the immune system. Most regimens were only partially effective at reducing antibody responses, but two courses of non-depleting anti-CD4 monoclonal antibody (mAb) ablated immune responses to laronidase in seven of eight MPS I mice (87.5%), even after adjuvant stimulation. Immune tolerance induction with methotrexate does not appear to be effective in MPS I patients, but use of non-depleting anti-CD4 monoclonal is a promising strategy.
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http://dx.doi.org/10.1016/j.omtm.2019.02.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6441787PMC
June 2019

Non-myeloablative busulfan chimeric mouse models are less pro-inflammatory than head-shielded irradiation for studying immune cell interactions in brain tumours.

J Neuroinflammation 2019 Feb 5;16(1):25. Epub 2019 Feb 5.

Stem Cell and Neurotherapies Laboratory, Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK.

Background: Chimeric mouse models generated via adoptive bone marrow transfer are the foundation for immune cell tracking in neuroinflammation. Chimeras that exhibit low chimerism levels, blood-brain barrier disruption and pro-inflammatory effects prior to the progression of the pathological phenotype, make it difficult to distinguish the role of immune cells in neuroinflammatory conditions. Head-shielded irradiation overcomes many of the issues described and replaces the recipient bone marrow system with donor haematopoietic cells expressing a reporter gene or different pan-leukocyte antigen, whilst leaving the blood-brain barrier intact. However, our previous work with full body irradiation suggests that this may generate a pro-inflammatory peripheral environment which could impact on the brain's immune microenvironment. Our aim was to compare non-myeloablative busulfan conditioning against head-shielded irradiation bone marrow chimeras prior to implantation of glioblastoma, a malignant brain tumour with a pro-inflammatory phenotype.

Methods: Recipient wild-type/CD45.1 mice received non-myeloablative busulfan conditioning (25 mg/kg), full intensity head-shielded irradiation, full intensity busulfan conditioning (125 mg/kg) prior to transplant with whole bone marrow from CD45.2 donors and were compared against untransplanted controls. Half the mice from each group were orthotopically implanted with syngeneic GL-261 glioblastoma cells. We assessed peripheral blood, bone marrow and spleen chimerism, multi-organ pro-inflammatory cytokine profiles at 12 weeks and brain chimerism and immune cell infiltration by whole brain flow cytometry before and after implantation of glioblastoma at 12 and 14 weeks respectively.

Results: Both non-myeloablative conditioning and head-shielded irradiation achieve equivalent blood and spleen chimerism of approximately 80%, although bone marrow engraftment is higher in the head-shielded irradiation group and highest in the fully conditioned group. Head-shielded irradiation stimulated pro-inflammatory cytokines in the blood and spleen but not in the brain, suggesting a systemic response to irradiation, whilst non-myeloablative conditioning showed no cytokine elevation. Non-myeloablative conditioning achieved higher donor chimerism in the brain after glioblastoma implantation than head-shielded irradiation with an altered immune cell profile.

Conclusion: Our data suggest that non-myeloablative conditioning generates a more homeostatic peripheral inflammatory environment than head-shielded irradiation to allow a more consistent evaluation of immune cells in glioblastoma and can be used to investigate the roles of peripheral immune cells and bone marrow-derived subsets in other neurological diseases.
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http://dx.doi.org/10.1186/s12974-019-1410-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6362590PMC
February 2019

The role of innate immunity in mucopolysaccharide diseases.

J Neurochem 2019 03 13;148(5):639-651. Epub 2018 Dec 13.

Stem Cell and Neurotherapies, Division of Cell Matrix Biology and Regenerative Medicine, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK.

Mucopolysaccharidoses are lysosomal storage disorders characterised by accumulation of abnormal pathological glycosaminoglycans, cellular dysfunction and widespread inflammation, resulting in progressive cognitive and motor decline. Lysosomes are important mediators of immune cell function, and therefore accumulation of glycosaminoglycans (GAGs) and other abnormal substrates could affect immune function and directly impact on disease pathogenesis. This review summarises current knowledge with regard to inflammation in mucopolysaccharidosis, with an emphasis on the brain and outlines a potential role for GAGs in induction of inflammation. We propose a model by which the accumulation of GAGs and other factors may impact on innate immune signalling with particular focus on the Toll-like receptor 4 pathway. Innate immunity appears to have a dominating role in mucopolysaccharidosis; however, furthering understanding of innate immune signalling would have significant impact on highlighting novel anti-inflammatory therapeutics for use in mucopolysaccharide diseases. This article is part of the Special Issue "Lysosomal Storage Disorders".
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http://dx.doi.org/10.1111/jnc.14632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6590380PMC
March 2019

Substrate accumulation and extracellular matrix remodelling promote persistent upper airway disease in mucopolysaccharidosis patients on enzyme replacement therapy.

PLoS One 2018 18;13(9):e0203216. Epub 2018 Sep 18.

Stem Cell and Neurotherapies, Division of Cell Matrix Biology & Regenerative Medicine, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, United Kingdom.

Introduction: Mucopolysaccharide diseases are a group of lysosomal storage disorders caused by deficiencies of hydrolase enzymes, leading to pathological glycosaminoglycan accumulation. A number of mucopolysaccharidosis (MPS) types are characterised by severe airway disease, the aetiology of which is poorly understood. There is ongoing evidence of significant clinical disease in the long-term despite disease modifying therapeutic strategies, including enzyme-replacement therapy (ERT). To provide a better understanding of this aspect of disease, we have characterised extracellular matrix (ECM) and inflammatory alterations in adenotonsillar tissue samples from 8 MPS patients.

Methods: Adenotonsillar samples from MPS I, IVA and VI ERT treated patients and from a single enzyme naïve MPS IIIA individual were compared to non-affected control samples using quantitative immunohistochemistry, qPCR and biochemical analysis.

Results: Significantly increased lysosomal compartment size and total sulphated glycosaminoglycan (p = 0.0007, 0.02) were identified in patient samples despite ERT. Heparan sulphate glycosaminoglycan was significantly elevated in MPS I and IIIA (p = 0.002), confirming incomplete reversal of disease. Collagen IV and laminin α-5 (p = 0.002, 0.0004) staining demonstrated increased ECM deposition within the reticular and capillary network of MPS samples. No significant change in the expression of the pro-inflammatory cytokines IL-1α, IL-6 or TNF-α was seen compared to control.

Conclusion: This study suggests a role for ECM remodelling contributing to the obstructive phenotype of airway disease in MPS. Current therapeutic strategies with ERT fail to normalise these pathological alterations within adenotonsillar samples. Our findings lend novel insight into the pathological cascade of events, with primarily structural rather than inflammatory changes contributing to the continuing phenotype seen in patients despite current therapeutic regimes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0203216PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6143186PMC
February 2019

Anatomical changes and pathophysiology of the brain in mucopolysaccharidosis disorders.

Mol Genet Metab 2018 12 10;125(4):322-331. Epub 2018 Aug 10.

Departments of Pediatrics and Biochemistry, CHU Sainte-Justine, Research Center, University of Montreal, Montreal, QC, Canada.

Mucopolysaccharidosis (MPS) disorders are caused by deficiencies in lysosomal enzymes, leading to impaired glycosaminoglycan (GAG) degradation. The resulting GAG accumulation in cells and connective tissues ultimately results in widespread tissue and organ dysfunction. The seven MPS types currently described are heterogeneous and progressive disorders, with somatic and neurological manifestations depending on the type of accumulating GAG. Heparan sulfate (HS) is one of the GAGs stored in patients with MPS I, II, and VII and the main GAG stored in patients with MPS III. These disorders are associated with significant central nervous system (CNS) abnormalities that can manifest as impaired cognition, hyperactive and/or aggressive behavior, epilepsy, hydrocephalus, and sleeping problems. This review discusses the anatomical and pathophysiological CNS changes accompanying HS accumulation as well as the mechanisms believed to cause CNS abnormalities in MPS patients. The content of this review is based on presentations and discussions on these topics during a meeting on the brain in MPS attended by an international group of MPS experts.
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http://dx.doi.org/10.1016/j.ymgme.2018.08.003DOI Listing
December 2018

Delivering Hematopoietic Stem Cell Gene Therapy Treatments for Neurological Lysosomal Diseases.

ACS Chem Neurosci 2019 01 23;10(1):18-20. Epub 2018 Aug 23.

Stem Cell and Neurotherapies, Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health , University of Manchester , Manchester M13 9PT , U.K.

Neurological lysosomal storage diseases are rare, inherited conditions resulting mainly from lysosomal enzyme deficiencies. Current treatments, such as enzyme replacement therapy and hematopoietic stem cell transplantation, fail to effectively treat neurological disease due to insufficient brain delivery of the missing enzyme. Ex vivo gene therapy approaches to overexpress the missing enzyme in hematopoietic stem cells prior to transplant are an emerging technology that has the potential to offer a viable therapy for patients with these debilitating diseases.
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http://dx.doi.org/10.1021/acschemneuro.8b00408DOI Listing
January 2019

Brain-targeted stem cell gene therapy corrects mucopolysaccharidosis type II via multiple mechanisms.

EMBO Mol Med 2018 07;10(7)

Stem Cell and Neurotherapies, Division of Cell Matrix Biology & Regenerative Medicine, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, UK

The pediatric lysosomal storage disorder mucopolysaccharidosis type II is caused by mutations in IDS, resulting in accumulation of heparan and dermatan sulfate, causing severe neurodegeneration, skeletal disease, and cardiorespiratory disease. Most patients manifest with cognitive symptoms, which cannot be treated with enzyme replacement therapy, as native IDS does not cross the blood-brain barrier. We tested a brain-targeted hematopoietic stem cell gene therapy approach using lentiviral IDS fused to ApoEII (IDS.ApoEII) compared to a lentivirus expressing normal IDS or a normal bone marrow transplant. In mucopolysaccharidosis II mice, all treatments corrected peripheral disease, but only IDS.ApoEII mediated complete normalization of brain pathology and behavior, providing significantly enhanced correction compared to IDS. A normal bone marrow transplant achieved no brain correction. Whilst corrected macrophages traffic to the brain, secreting IDS/IDS.ApoEII enzyme for cross-correction, IDS.ApoEII was additionally more active in plasma and was taken up and transcytosed across brain endothelia significantly better than IDS via both heparan sulfate/ApoE-dependent receptors and mannose-6-phosphate receptors. Brain-targeted hematopoietic stem cell gene therapy provides a promising therapy for MPS II patients.
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http://dx.doi.org/10.15252/emmm.201708730DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6034129PMC
July 2018

A novel adeno-associated virus capsid with enhanced neurotropism corrects a lysosomal transmembrane enzyme deficiency.

Brain 2018 07;141(7):2014-2031

Department of Infectious Diseases, School of Immunology and Microbial Sciences, King's College London, London, UK.

Recombinant adeno-associated viruses (AAVs) are popular in vivo gene transfer vehicles. However, vector doses needed to achieve therapeutic effect are high and some target tissues in the central nervous system remain difficult to transduce. Gene therapy trials using AAV for the treatment of neurological disorders have seldom led to demonstrated clinical efficacy. Important contributing factors are low transduction rates and inefficient distribution of the vector. To overcome these hurdles, a variety of capsid engineering methods have been utilized to generate capsids with improved transduction properties. Here we describe an alternative approach to capsid engineering, which draws on the natural evolution of the virus and aims to yield capsids that are better suited to infect human tissues. We generated an AAV capsid to include amino acids that are conserved among natural AAV2 isolates and tested its biodistribution properties in mice and rats. Intriguingly, this novel variant, AAV-TT, demonstrates strong neurotropism in rodents and displays significantly improved distribution throughout the central nervous system as compared to AAV2. Additionally, sub-retinal injections in mice revealed markedly enhanced transduction of photoreceptor cells when compared to AAV2. Importantly, AAV-TT exceeds the distribution abilities of benchmark neurotropic serotypes AAV9 and AAVrh10 in the central nervous system of mice, and is the only virus, when administered at low dose, that is able to correct the neurological phenotype in a mouse model of mucopolysaccharidosis IIIC, a transmembrane enzyme lysosomal storage disease, which requires delivery to every cell for biochemical correction. These data represent unprecedented correction of a lysosomal transmembrane enzyme deficiency in mice and suggest that AAV-TT-based gene therapies may be suitable for treatment of human neurological diseases such as mucopolysaccharidosis IIIC, which is characterized by global neuropathology.
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http://dx.doi.org/10.1093/brain/awy126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6037107PMC
July 2018

Identification of unusual oxysterols and bile acids with 7-oxo or 3β,5α,6β-trihydroxy functions in human plasma by charge-tagging mass spectrometry with multistage fragmentation.

J Lipid Res 2018 06 6;59(6):1058-1070. Epub 2018 Apr 6.

Swansea University Medical School, Swansea SA2 8PP, Wales, United Kingdom

7-Oxocholesterol (7-OC), 5,6-epoxycholesterol (5,6-EC), and its hydrolysis product cholestane-3β,5α,6β-triol (3β,5α,6β-triol) are normally minor oxysterols in human samples; however, in disease, their levels may be greatly elevated. This is the case in plasma from patients suffering from some lysosomal storage disorders, e.g., Niemann-Pick disease type C, or the inborn errors of sterol metabolism, e.g., Smith-Lemli-Opitz syndrome and cerebrotendinous xanthomatosis. A complication in the analysis of 7-OC and 5,6-EC is that they can also be formed ex vivo from cholesterol during sample handling in air, causing confusion with molecules formed in vivo. When formed endogenously, 7-OC, 5,6-EC, and 3β,5α,6β-triol can be converted to bile acids. Here, we describe methodology based on chemical derivatization and LC/MS with multistage fragmentation (MS) to identify the necessary intermediates in the conversion of 7-OC to 3β-hydroxy-7-oxochol-5-enoic acid and 5,6-EC and 3β,5α,6β-triol to 3β,5α,6β-trihydroxycholanoic acid. Identification of intermediate metabolites is facilitated by their unusual MS fragmentation patterns. Semiquantitative measurements are possible, but absolute values await the synthesis of isotope-labeled standards.
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http://dx.doi.org/10.1194/jlr.D083246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983402PMC
June 2018

High content screening of patient-derived cell lines highlights the potential of non-standard chemotherapeutic agents for the treatment of glioblastoma.

PLoS One 2018 2;13(3):e0193694. Epub 2018 Mar 2.

Brain Tumour Research Group, Stem Cell and Neurotherapies Laboratory, Division of Cell Matrix Biology & Regenerative Medicine, University of Manchester, Manchester, United Kingdom.

Background: Glioblastoma (GBM) is the most common primary brain malignancy in adults, yet survival outcomes remain poor. First line treatment is well established, however disease invariably recurs and improving prognosis is challenging. With the aim of personalizing therapy at recurrence, we have established a high content screening (HCS) platform to analyze the sensitivity profile of seven patient-derived cancer stem cell lines to 83 FDA-approved chemotherapy drugs, with and without irradiation.

Methods: Seven cancer stem cell lines were derived from patients with GBM and, along with the established cell line U87-MG, each patient-derived line was cultured in tandem in serum-free conditions as adherent monolayers and three-dimensional neurospheres. Chemotherapeutics were screened at multiple concentrations and cells double-stained to observe their effect on both cell death and proliferation. Sensitivity was classified using high-throughput algorithmic image analysis.

Results: Cell line specific drug responses were observed across the seven patient-derived cell lines. Few agents were seen to have radio-sensitizing effects, yet some drug classes showed a marked difference in efficacy between monolayers and neurospheres. In vivo validation of six drugs suggested that cell death readout in a three-dimensional culture scenario is a more physiologically relevant screening model and could be used effectively to assess the chemosensitivity of patient-derived GBM lines.

Conclusion: The study puts forward a number of non-standard chemotherapeutics that could be useful in the treatment of recurrent GBM, namely mitoxantrone, bortezomib and actinomycin D, whilst demonstrating the potential of HCS to be used for personalized treatment based on the chemosensitivity profile of patient tumor cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0193694PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5834163PMC
June 2018

Macrophage enzyme and reduced inflammation drive brain correction of mucopolysaccharidosis IIIB by stem cell gene therapy.

Brain 2018 01;141(1):99-116

Stem Cell and Neurotherapies, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK.

Mucopolysaccharidosis IIIB is a paediatric lysosomal storage disease caused by deficiency of the enzyme α-N-acetylglucosaminidase (NAGLU), involved in the degradation of the glycosaminoglycan heparan sulphate. Absence of NAGLU leads to accumulation of partially degraded heparan sulphate within lysosomes and the extracellular matrix, giving rise to severe CNS degeneration with progressive cognitive impairment and behavioural problems. There are no therapies. Haematopoietic stem cell transplant shows great efficacy in the related disease mucopolysaccharidosis I, where donor-derived monocytes can transmigrate into the brain following bone marrow engraftment, secrete the missing enzyme and cross-correct neighbouring cells. However, little neurological correction is achieved in patients with mucopolysaccharidosis IIIB. We have therefore developed an ex vivo haematopoietic stem cell gene therapy approach in a mouse model of mucopolysaccharidosis IIIB, using a high-titre lentiviral vector and the myeloid-specific CD11b promoter, driving the expression of NAGLU (LV.NAGLU). To understand the mechanism of correction we also compared this with a poorly secreted version of NAGLU containing a C-terminal fusion to IGFII (LV.NAGLU-IGFII). Mucopolysaccharidosis IIIB haematopoietic stem cells were transduced with vector, transplanted into myeloablated mucopolysaccharidosis IIIB mice and compared at 8 months of age with mice receiving a wild-type transplant. As the disease is characterized by increased inflammation, we also tested the anti-inflammatory steroidal agent prednisolone alone, or in combination with LV.NAGLU, to understand the importance of inflammation on behaviour. NAGLU enzyme was substantially increased in the brain of LV.NAGLU and LV.NAGLU-IGFII-treated mice, with little expression in wild-type bone marrow transplanted mice. LV.NAGLU treatment led to behavioural correction, normalization of heparan sulphate and sulphation patterning, reduced inflammatory cytokine expression and correction of astrocytosis, microgliosis and lysosomal compartment size throughout the brain. The addition of prednisolone improved inflammatory aspects further. Substantial correction of lysosomal storage in neurons and astrocytes was also achieved in LV.NAGLU-IGFII-treated mice, despite limited enzyme secretion from engrafted macrophages in the brain. Interestingly both wild-type bone marrow transplant and prednisolone treatment alone corrected behaviour, despite having little effect on brain neuropathology. This was attributed to a decrease in peripheral inflammatory cytokines. Here we show significant neurological disease correction is achieved using haematopoietic stem cell gene therapy, suggesting this therapy alone or in combination with anti-inflammatories may improve neurological function in patients.
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http://dx.doi.org/10.1093/brain/awx311DOI Listing
January 2018

Recommendations on clinical trial design for treatment of Mucopolysaccharidosis Type III.

Orphanet J Rare Dis 2017 06 26;12(1):117. Epub 2017 Jun 26.

Willink Biochemical Genetics Unit, Manchester Centre For Genomic Medicine, Manchester Academic Health Science Centre, Central Manchester University Hospitals NHS Foundation Trust, Manchester, UK.

Background: Mucopolysaccharidosis type III is a progressive, neurodegenerative lysosomal storage disorder for which there is currently no effective therapy. Though numerous potential therapies are in development, there are several challenges to conducting clinical research in this area. We seek to make recommendations on the approach to clinical research in MPS III, including the selection of outcome measures and trial endpoints, in order to improve the quality and impact of research in this area.

Results: An international workshop involving academic researchers, clinical experts and industry groups was held in June 2015, with presentations and discussions on disease pathophysiology, biomarkers, potential therapies and clinical outcome measures. A set of recommendations was subsequently prepared by a working group and reviewed by all delegates. We present a series of 11 recommendations regarding the conduct of clinical research, outcome measures and management of natural history data in Mucopolysaccharidosis type III.

Conclusions: Improving the quality of clinical research in Mucopolysaccharidosis type III will require an open, collaborative and systematic approach between academic researchers, clinicians and industry. Natural history data should be published as soon as possible and ideally collated in a central repository. There should be agreement on outcome measures and instruments for evaluation of clinical outcomes to maximise the effectiveness of current and future clinical research.
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http://dx.doi.org/10.1186/s13023-017-0675-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5485703PMC
June 2017

Identification of age-dependent motor and neuropsychological behavioural abnormalities in a mouse model of Mucopolysaccharidosis Type II.

PLoS One 2017 16;12(2):e0172435. Epub 2017 Feb 16.

Stem Cell & Neurotherapies, Division of Cell Matrix Biology & Regenerative Medicine, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, United Kingdom.

Severe mucopolysaccharidosis type II (MPS II) is a progressive lysosomal storage disease caused by mutations in the IDS gene, leading to a deficiency in the iduronate-2-sulfatase enzyme that is involved in heparan sulphate and dermatan sulphate catabolism. In constitutive form, MPS II is a multi-system disease characterised by progressive neurocognitive decline, severe skeletal abnormalities and hepatosplenomegaly. Although enzyme replacement therapy has been approved for treatment of peripheral organs, no therapy effectively treats the cognitive symptoms of the disease and novel therapies are in development to remediate this. Therapeutic efficacy and subsequent validation can be assessed using a variety of outcome measures that are translatable to clinical practice, such as behavioural measures. We sought to consolidate current knowledge of the cognitive, skeletal and motor abnormalities present in the MPS II mouse model by performing time course behavioural examinations of working memory, anxiety, activity levels, sociability and coordination and balance, up to 8 months of age. Cognitive decline associated with alterations in spatial working memory is detectable at 8 months of age in MPS II mice using spontaneous alternation, together with an altered response to novel environments and anxiolytic behaviour in the open-field. Coordination and balance on the accelerating rotarod were also significantly worse at 8 months, and may be associated with skeletal changes seen in MPS II mice. We demonstrate that the progressive nature of MPS II disease is also seen in the mouse model, and that cognitive and motor differences are detectable at 8 months of age using spontaneous alternation, the accelerating rotarod and the open-field tests. This study establishes neurological, motor and skeletal measures for use in pre-clinical studies to develop therapeutic approaches in MPS II.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0172435PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5313159PMC
September 2017

Sterols and oxysterols in plasma from Smith-Lemli-Opitz syndrome patients.

J Steroid Biochem Mol Biol 2017 05 11;169:77-87. Epub 2016 Mar 11.

College of Medicine, Grove Building, Swansea University, Singleton Park, Swansea SA2 8PP, UK. Electronic address:

Smith-Lemli-Opitz syndrome (SLOS) is a severe autosomal recessive disorder resulting from defects in the cholesterol synthesising enzyme 7-dehydrocholesterol reductase (Δ-sterol reductase, DHCR7, EC 1.3.1.21) leading to a build-up of the cholesterol precursor 7-dehydrocholesterol (7-DHC) in tissues and blood plasma. Although the underling enzyme deficiency associated with SLOS is clear there are likely to be multiple mechanisms responsible for SLOS pathology. In an effort to learn more of the aetiology of SLOS we have analysed plasma from SLOS patients to search for metabolites derived from 7-DHC which may be responsible for some of the pathology. We have identified a novel hydroxy-8-dehydrocholesterol, which is either 24- or 25-hydroxy-8-dehydrocholesterol and also the known metabolites 26-hydroxy-8-dehydrocholesterol, 4-hydroxy-7-dehydrocholesterol, 3β,5α-dihydroxycholest-7-en-6-one and 7α,8α-epoxycholesterol. None of these metabolites are detected in control plasma at quantifiable levels (0.5ng/mL).
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http://dx.doi.org/10.1016/j.jsbmb.2016.03.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5018427PMC
May 2017

Enzyme replacement therapy prior to haematopoietic stem cell transplantation in Mucopolysaccharidosis Type I: 10 year combined experience of 2 centres.

Mol Genet Metab 2016 Mar 27;117(3):373-7. Epub 2016 Jan 27.

Bone Marrow Transplantation Unit, Royal Manchester Children's Hospital, Oxford Road, Manchester M13 9WL, UK. Electronic address:

Haematopoietic stem cell transplantation is the treatment of choice for the severe form of Mucopolysaccharidosis Type I, or Hurler syndrome. In many centres standard practice is to deliver enzyme replacement therapy alongside haematopoietic stem cell transplantation to improve the condition of the patient prior to transplant. We report the combined 10 year experience of this approach in two paediatric metabolic and transplant centres. Of 81 patients who underwent a first transplant procedure for Hurler, 88% (71/81) survived and 81% (66/81) were alive and engrafted at a median follow-up of 46 months (range 3-124 months). The incidence of grade II-IV acute and any chronic graft versus host disease was 17% and 11% respectively. Urinary glycosaminoglycans were significantly reduced after a period of enzyme replacement therapy, and further reductions were seen at 13-24 months and 25+months after transplantation. In several individuals with decreased cardiac contractility, an improvement of their condition during enzyme replacement therapy enabled them to undergo transplantation, with one individual receiving full intensity conditioning.
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http://dx.doi.org/10.1016/j.ymgme.2016.01.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5177791PMC
March 2016

The role of antibodies in enzyme treatments and therapeutic strategies.

Best Pract Res Clin Endocrinol Metab 2015 Mar 24;29(2):183-94. Epub 2015 Jan 24.

Department of Endocrinology and Metabolism, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Electronic address:

Substitution of the defective lysosomal enzyme in lysosomal storage disorders (LSDs) often elicits antibody formation towards the infused protein. Aside from Gaucher disease, antibodies often lead to infusion associated reactions and a reduced biochemical response. In Pompe disease, antibody titer is predictive of clinical outcome, but this is less apparent in other LSDs and warrants further study. Few laboratories are capable of enzyme-antibody determination: often physicians need to rely on the enzyme manufacturer for analysis. Currently, laboratories employ different antibody assays which hamper comparisons between cohorts or treatment regimens. Assay standardisation, including measurement of antibody-related enzyme inhibition, is therefore urgently needed. Successful immunomodulation has been reported in Pompe and in Gaucher disease, with variable success. Immunomodulation regimens that contain temporary depletion of B-cells (anti-CD20) are most used. Bone marrow transplantation in MPS-I results in disappearance of antibodies. No other clinical studies have been conducted in humans with immunomodulation in other LSDs.
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http://dx.doi.org/10.1016/j.beem.2015.01.006DOI Listing
March 2015

Sleep disordered breathing in mucopolysaccharidosis I: a multivariate analysis of patient, therapeutic and metabolic correlators modifying long term clinical outcome.

Orphanet J Rare Dis 2015 Apr 10;10:42. Epub 2015 Apr 10.

Stem Cell & Neurotherapies, Faculty of Medical and Human Sciences, University of Manchester, Manchester, UK.

Background: The lysosomal storage disorder, mucopolysaccharidosis I (MPS I), commonly manifests with upper airway obstruction and sleep disordered breathing (SDB). The success of current therapies, including haematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT) may be influenced by a number of factors and monitored using biomarkers of metabolic correction. We describe the pattern of SDB seen in the largest MPS I cohort described to date and determine therapies and biomarkers influencing the severity of long-term airway disease.

Methods: Therapeutic, clinical and biomarker data, including longitudinal outcome parameters from 150 sleep oximetry studies were collected in 61 MPS I (44 Hurler, 17 attenuated) patients between 6 months pre to 16 years post-treatment (median follow-up 22 months). The presence and functional nature of an immune response to ERT was determined using ELISA and a cellular uptake inhibition assay. Multivariate analysis was performed to determine significant correlators of airway disease.

Results: The incidence of SDB in our cohort is 68%, while 16% require therapeutic intervention for airway obstruction. A greater rate of progression (73%) and requirement for intervention is seen amongst ERT patients in contrast to HSCT treated individuals (24%). Multivariate analysis identifies poorer metabolic clearance, as measured by a rise in the biomarker urinary dermatan sulphate: chondroitin sulphate (DS:CS) ratio, as a significant correlator of increased presence and severity of SDB in MPS I patients (p = 0.0017, 0.008). Amongst transplanted Hurler patients, delivered enzyme (leukocyte iduronidase) at one year is significantly raised in those without SDB (p = 0.004). Cellular uptake inhibitory antibodies in ERT treated patients correlate with reduced substrate clearance and occurrence of severe SDB (p = 0.001).

Conclusion: We have identified biochemical and therapeutic factors modifying airway disease across the phenotypic spectrum in MPS I. Interventions maximising substrate reduction correlate with improved long-term SDB, while inhibitory antibodies impact on biochemical and clinical outcomes. Monitoring and tolerisation strategies should be re-evaluated to improve detection and minimise the inhibitory antibody response to ERT in MPS I and other lysosomal storage diseases. Future studies should consider the use of sleep disordered breathing as an objective parameter of clinical and metabolic improvement.
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http://dx.doi.org/10.1186/s13023-015-0255-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4450482PMC
April 2015

An investigation of the middle and late behavioural phenotypes of Mucopolysaccharidosis Type-III.

J Neurodev Disord 2014 31;6(1):46. Epub 2014 Dec 31.

Section for Clinical and Health Psychology, School of Psychological Sciences, University of Manchester, Zochonis Building, Brunswick Street, Manchester, M13 9PL UK.

Background: Mucopolysaccharidosis type-III (MPS III) is an autosomal recessive lysosomal storage disorder. It causes progressive physical and cognitive decline and has been linked to increased incidences of behavioural problems.

Methods: Data on the behaviour and adaptive skills of 20 children with MPS III and 25 children with intellectual disability (ID) (17 included in analysis) were gathered via parental report questionnaire. The frequencies of different types of behaviour displayed by children with MPS III and children with ID were compared across two age categories.

Results: The total frequency of challenging behaviours displayed by children aged 2-9 years with MPS III and ID was not significantly different. Behaviours associated with hyperactivity, orality, unusual body movements and inattention were seen significantly more frequently in 2-9 year olds with MPS III than in those with ID. Children aged 10-15 years with MPS III showed significantly fewer problem behaviours than a contrasting group with ID. The frequency of challenging behaviours displayed by children with MPS III and their adaptive skills was found to decrease with age.

Conclusions: Behaviours relating to hyperactivity, orality, unusual body movements and inattention are part of the behavioural phenotype of the middle phase of MPS III. The late phase of MPS III is associated with low rates of problem behaviour and loss of adaptive skills. Therefore, families with a child with MPS III may benefit from a different type of clinical service when the child is aged 2-9 years, than when aged 10-15 years.
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http://dx.doi.org/10.1186/1866-1955-6-46DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4318134PMC
February 2015

Neuroinflammation, mitochondrial defects and neurodegeneration in mucopolysaccharidosis III type C mouse model.

Brain 2015 Feb 6;138(Pt 2):336-55. Epub 2015 Jan 6.

1 CHU Ste-Justine, University of Montreal, Montreal, QC, Canada

Severe progressive neurological paediatric disease mucopolysaccharidosis III type C is caused by mutations in the HGSNAT gene leading to deficiency of acetyl-CoA: α-glucosaminide N-acetyltransferase involved in the lysosomal catabolism of heparan sulphate. To understand the pathophysiology of the disease we generated a mouse model of mucopolysaccharidosis III type C by germline inactivation of the Hgsnat gene. At 6-8 months mice showed hyperactivity, and reduced anxiety. Cognitive memory decline was detected at 10 months and at 12-13 months mice showed signs of unbalanced hesitant walk and urinary retention. Lysosomal accumulation of heparan sulphate was observed in hepatocytes, splenic sinus endothelium, cerebral microglia, liver Kupffer cells, fibroblasts and pericytes. Starting from 5 months, brain neurons showed enlarged, structurally abnormal mitochondria, impaired mitochondrial energy metabolism, and storage of densely packed autofluorescent material, gangliosides, lysozyme, phosphorylated tau, and amyloid-β. Taken together, our data demonstrate for the first time that deficiency of acetyl-CoA: α-glucosaminide N-acetyltransferase causes lysosomal accumulation of heparan sulphate in microglial cells followed by their activation and cytokine release. They also show mitochondrial dysfunction in the neurons and neuronal loss explaining why mucopolysaccharidosis III type C manifests primarily as a neurodegenerative disease.
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http://dx.doi.org/10.1093/brain/awu355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4306821PMC
February 2015

Quantitative charge-tags for sterol and oxysterol analysis.

Clin Chem 2015 Feb 15;61(2):400-11. Epub 2014 Dec 15.

College of Medicine and

Background: Global sterol analysis is challenging owing to the extreme diversity of sterol natural products, the tendency of cholesterol to dominate in abundance over all other sterols, and the structural lack of a strong chromophore or readily ionized functional group. We developed a method to overcome these challenges by using different isotope-labeled versions of the Girard P reagent (GP) as quantitative charge-tags for the LC-MS analysis of sterols including oxysterols.

Methods: Sterols/oxysterols in plasma were extracted in ethanol containing deuterated internal standards, separated by C18 solid-phase extraction, and derivatized with GP, with or without prior oxidation of 3β-hydroxy to 3-oxo groups.

Results: By use of different isotope-labeled GPs, it was possible to analyze in a single LC-MS analysis both sterols/oxysterols that naturally possess a 3-oxo group and those with a 3β-hydroxy group. Intra- and interassay CVs were <15%, and recoveries for representative oxysterols and cholestenoic acids were 85%-108%. By adopting a multiplex approach to isotope labeling, we analyzed up to 4 different samples in a single run. Using plasma samples, we could demonstrate the diagnosis of inborn errors of metabolism and also the export of oxysterols from brain via the jugular vein.

Conclusions: This method allows the profiling of the widest range of sterols/oxysterols in a single analytical run and can be used to identify inborn errors of cholesterol synthesis and metabolism.
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http://dx.doi.org/10.1373/clinchem.2014.231332DOI Listing
February 2015

Biomarker responses correlate with antibody status in mucopolysaccharidosis type I patients on long-term enzyme replacement therapy.

Mol Genet Metab 2015 Feb 29;114(2):129-37. Epub 2014 Oct 29.

Stem Cell & Neurotherapies, Centre for Genomic Medicine, University of Manchester, Manchester, UK.

Background: Antibody formation can interfere with effects of enzyme replacement therapy (ERT) in lysosomal storage diseases. Biomarkers are used as surrogate marker for disease burden in MPS I, but large systematic studies evaluating the response of biomarkers to ERT are lacking. We, for the first time, investigated the response of a large panel of biomarkers to long term ERT in MPS I patients and correlate these responses with antibody formation and antibody mediated cellular uptake inhibition.

Methods: A total of 428 blood and urine samples were collected during long-term ERT in 24 MPS I patients and an extensive set of biomarkers was analyzed, including heparan sulfate (HS) and dermatan sulfate (DS) derived disaccharides; total urinary GAGs (DMBu); urinary DS:CS ratio and serum heparin co-factor II thrombin levels (HCII-T). IgG antibody titers and the effect of antibodies on cellular uptake of the enzyme were determined for 23 patients.

Results: Median follow-up was 2.3 years. In blood, HS reached normal levels more frequently than DS (50% vs 12.5%, p=0.001), though normalization could take several years. DMBu normalized more rapidly than disaccharide levels in urine (p=0.02). Nineteen patients (83%) developed high antibody titers. Significant antibody-mediated inhibition of enzyme uptake was observed in 8 patients (35%), and this correlated strongly with a poorer biomarker response for HS and DS in blood and urine as well as for DMBu, DS:CS-ratio and HCII-T (all p<0.006).

Conclusions: This study shows that, despite a response of all studied biomarkers to initiation of ERT, some biomarkers were less responsive than others, suggesting residual disease activity. In addition, the correlation of cellular uptake inhibitory antibodies with a decreased biomarker response demonstrates a functional role of these antibodies which may have important clinical consequences.
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http://dx.doi.org/10.1016/j.ymgme.2014.10.012DOI Listing
February 2015