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Reliable behavioural tests in animal models of neurodegenerative diseases allow us to study the natural history of disease and evaluate the efficacy of novel therapies. Mucopolysaccharidosis IIIA (MPS IIIA or Sanfilippo A), is a severe, neurodegenerative lysosomal storage disorder caused by a deficiency in the heparan sulphate catabolising enzyme, sulfamidase. Undegraded heparan sulphate accumulates, resulting in lysosomal enlargement and cellular dysfunction. Patients suffer a progressive loss of motor and cognitive function with severe behavioural manifestations and premature death. There is currently no treatment. A spontaneously occurring mouse model of the disease has been described, that has approximately 3% of normal enzyme activity levels. Behavioural phenotyping of the MPS IIIA mouse has been previously reported, but the results are conflicting and variable, even after full backcrossing to the C57BL/6 background. Therefore we have independently backcrossed the MPS IIIA model onto the C57BL/6J background and evaluated the behaviour of male and female MPS IIIA mice at 4, 6 and 8 months of age using the open field test, elevated plus maze, inverted screen and horizontal bar crossing at the same circadian time point. Using a 60 minute open field, we have demonstrated that female MPS IIIA mice are hyperactive, have a longer path length, display rapid exploratory behaviour and spend less time immobile than WT mice. Female MPS IIIA mice also display a reduced sense of danger and spend more time in the centre of the open field. There were no significant differences found between male WT and MPS IIIA mice and no differences in neuromuscular strength were seen with either sex. The altered natural history of behaviour that we observe in the MPS IIIA mouse will allow more accurate evaluation of novel therapeutics for MPS IIIA and potentially other neurodegenerative disorders.

Mucopolysaccharide (MPS) diseases are characterized by accumulation of glycosaminoglycans (GAGs) due to deficiencies in lysosomal enzymes responsible for GAG breakdown. Using a murine model of MPSI Hurler (MPSIH), we have quantified the heparan sulfate (HS) accumulation resulting from α-l-iduronidase (Idua) deficiency. HS levels were significantly increased in liver and brain tissue from 12-week-old Idua(-/-) mice by 87- and 20-fold, respectively. In addition, HS chains were shown to contain significantly increased N-, 2-O-, and 6-O-sulfation. Disaccharide compositional analyses also uncovered an HS disaccharide uniquely enriched in MPSIH, representing the terminal iduronic acid residue capping the non-reducing end of the HS chain, where no further degradation can occur in the absence of Idua. Critically, we identified that excess HS, some of which is colocalized to the Golgi secretory pathway, acts as a positive regulator of HS-sulfation, increasing the N-sulfotransferase activity of HS-modifying N-deacetylase/N-sulfotransferase enzymes. This mechanism may have severe implications during disease progression but, now identified, could help direct improved therapeutic strategies.

Development of a human mitochondrial gene delivery vector is a critical step in the ability to treat diseases arising from mutations in mitochondrial DNA. Although we have previously cloned the mouse mitochondrial genome in its entirety and developed it as a mitochondrial gene therapy vector, the human mitochondrial genome has been dubbed unclonable in E. coli, due to regions of instability in the D-loop and tRNA(Thr) gene.We tested multi- and single-copy vector systems for cloning human mitochondrial DNA in E. coli and Saccharomyces cerevisiae, including transformation-associated recombination.Human mitochondrial DNA is unclonable in E. coli and cannot be retained in multi- or single-copy vectors under any conditions. It was, however, possible to clone and stably maintain the entire human mitochondrial genome in yeast as long as a single-copy centromeric plasmid was used. D-loop and tRNA(Thr) were both stable and unmutated.This is the first report of cloning the entire human mitochondrial genome and the first step in developing a gene delivery vehicle for human mitochondrial gene therapy.

Background: Neurodegenerative metabolic disorders such as mucopolysaccharidosis IIIB (MPSIIIB or Sanfilippo disease) accumulate undegraded substrates in the brain and are often unresponsive to enzyme replacement treatments due to the impermeability of the blood brain barrier to enzyme. MPSIIIB is characterised by behavioural difficulties, cognitive and later motor decline, with death in the second decade of life. Most of these neurodegenerative lysosomal storage diseases lack effective treatments. We recently described significant reductions of accumulated heparan sulphate substrate in liver of a mouse model of MPSIIIB using the tyrosine kinase inhibitor genistein. Methodology/Principal Findings: We report here that high doses of genistein aglycone, given continuously over a 9 month period to MPSIIIB mice, significantly reduce lysosomal storage, heparan sulphate substrate and neuroinflammation in the cerebral cortex and hippocampus, resulting in correction of the behavioural defects observed. Improvements in synaptic vesicle protein expression and secondary storage in the cerebral cortex were also observed. Conclusions/Significance: Genistein may prove useful as a substrate reduction agent to delay clinical onset of MPSIIIB and, due to its multimodal action, may provide a treatment adjunct for several other neurodegenerative metabolic diseases. © 2010 Malinowska et al.

Mitochondrial DNA mutations are the direct cause of several physiological disorders and are also associated with the aging process. The modest progress made over the past two decades towards manipulating the mitochondrial genome and understanding its function within living mammalian cells means that cures for mitochondrial DNA mutations are still elusive. Here, we report that transformed mammalian cells internalize exogenous isolated mitochondria upon simple co-incubation. We first demonstrate the physical presence of internalized mitochondria within recipient cells using fluorescence microscopy. Second, we show that xenogenic transfer of murine mitochondria into human cells lacking functional mitochondria can functionally restore respiration in cells lacking mtDNA. Third, utilizing the natural competence of isolated mitochondria to take up linear DNA molecules, we demonstrate the feasibility of using cellular internalization of isolated exogenous mitochondria as a potential tool for studying mitochondrial genetics in living mammalian cells.

Emerging reports are conclusively demonstrating the mutagenic risks involved in using retroviral vectors for gene therapy. Animal studies, as well as cases from a human clinical trial, have proven the potential of insertional leukemogenesis caused by a retroviral vector. Here, we report the observation of six T-lymphoblastic leukemia cases arising during the course of a gene therapy study for hemophilia B after transplantation of ex vivo transduced hematopoietic stem cells (HSCs) by a lentivirus vector. Three of these animals comprised secondary recipients of the same donor and LAM-PCR was performed to identify the vector integration loci. We located integrations in repeat elements of known genes, including a candidate brain-tumor locus, but none of these clones could be tracked in the leukemic blasts. Although transduced clones with an intact proviral cassette were detected in the spleen of the leukemic animals, they comprised a very small proportion, not correlating to the levels of leukemic blasts. After propagation of the latter in NOD/SCID mice, we could no longer detect the proviral cassette suggesting that the leukemic blasts were untransduced. We did, however, detect increased levels of reverse transcriptase activity in the leukemic blasts which may suggest activation of endogenous retroviruses. This study demonstrates that tumors arising in these type of gene therapy protocols are not necessarily due to vector insertional mutagenesis and highlights the importance of careful functional studies to delineate the nature of tumorigenesis.

The mucopolysaccharidoses (MPSs) are a group of inherited, metabolic disorders characterized by progressive multisystem accumulation of partially degraded glycosaminoglycans. This manifests with multilevel airway obstruction, presenting with obstructive sleep apnea (OSA). We systematically reviewed the literature to determine the severity and prevalence of OSA in MPS based on polysomnography analysis. Fifteen studies with 294 participants met the inclusion criteria for review. The pretreatment prevalence of OSA in MPS was 81% with a mean apnea–hypopnea index (AHI) of 10.4. Patients with MPS I are most significantly affected, with 75% suffering with moderate to severe OSA (mean AHI, 16.6). Enzyme replacement therapy (ERT) results in an almost significant reduction in OSA in MPS I (P = .06), while adenotonsillar surgery significantly improves AHI (P = .002). Obstructive sleep apnea least affects MPS III. There is a lack of long-term post-ERT and hematopoietic stem cell transplant data relating to OSA outcom...

CO RR EC TE D P RO OF insidious course as the accumulation of lysoGb3 progresses. Furthermore, in a study using late gadolinium enhancement magnetic resonance imaging of the heart, we found that approximately 1 in 3 males and 1 in 8 females with the IVS4 mutation, who were older than 40 years and who did not have hypertrophic cardiomyopathy, had already developed significant cardiac fibrosis. Endomyocardial biopsy of three of these cardiac fibrosis patients revealed significant pathological changes and Gb3 accumulation in their cardiomyocytes. Current treatment guidelines for cardiac-type Fabry disease recommend that ERT should only be started when significant hypertrophic cardiomyopathy has occurred; however, our findings indicate that this might be too late. We believe that early intervention is important to prevent the insidious and irreversible cardiac damage in these patients.

Background & Aims Bone marrow (BM) cells may transdifferentiate into or fuse with organ parenchymal cells. BM therapy shows promise in murine models of cirrhosis, and clinical trials of bone marrow stem cell therapy for organ healing are underway. However, the BM may contribute to scar-forming myofibroblasts in various organs including the liver. We have studied this axis of regeneration and scarring in murine models of cirrhosis, including an assessment of the temporal and functional contribution of the BM-derived myofibroblasts. Methods Female mice were lethally irradiated and received male BM transplants. Carbon tetrachloride or thioacetamide was used to induce cirrhosis. BM-derived cells were tracked through in situ hybridization for the Y chromosome. BM transplants from 2 strains of transgenic mice were used to detect intrahepatic collagen production. Results In the cirrhotic liver, the contribution of BM to parenchymal regeneration was minor (0.6%); by contrast, the BM contributed significantly to hepatic stellate cell (68%) and myofibroblast (70%) populations. These BM-derived cells were found to be active for collagen type 1 transcription in 2 independent assays and could influence the fibrotic response to organ injury. These BM-derived myofibroblasts did not occur through cell fusion between BM-derived cells and indigenous hepatic cells but, instead, originated largely from the BM's mesenchymal stem cells. Conclusions The BM contributes functionally and significantly to liver fibrosis and is a potential therapeutic target in liver fibrosis. Clinical trials of BM cell therapy for liver regeneration should be vigilant for the possibility of enhanced organ fibrosis.

The authors regret that in Table 2 on page 768, one of the insertion sites of the SMART 2 provirus vector identified using LAM-PCR as present on chromosome 5 positioned 32374 bp upstream of Cyp3a11 was incorrectly assigned to Mouse (tumour) 2 T1. This insertion site should be assigned to an independent mouse not listed in Table 2. This animal had only a single provirus insertion found by Southern and LAM-PCR analyses and should be labeled as mouse 7.

BackgroundMinicircle DNA is the non-replicating product of intramolecular site-specific recombination within a bacterial minicircle producer plasmid. Minicircle DNA can be engineered to contain predominantly human sequences which have a low content of CpG dinucleotides and thus reduced immunotoxicity for humans, whilst the immunogenic bacterial origin and antibiotic resistance marker gene sequences are entirely removed by site-specific recombination. This property makes minicircle DNA an excellent vector for non-viral gene therapy. Large-scale production of minicircle DNA requires a bacterial strain expressing tightly controlled site-specific recombinase, such asCrerecombinase. As recombinant plasmids tend to be more stable in RecA-deficient strains, we aimed to construct arecA-bacterial strain for generation of minicircle vector DNA with less chance of unwanted deletions.ResultsWe describe here the construction of the RecA-deficient minicircle DNA producerEscherichia coliHB101Cre with a chromosomally locatedCrerecombinase gene under the tight control of thearaCregulon. TheCregene expression cassette was inserted into the chromosomallacZgene by creating transient homologous recombination proficiency in therecA-strain HB101 using plasmid-bornrecETgenes and homology-mediated chromosomal "pop-in, pop-out" of the plasmid pBAD75Cre containing theCregene and a temperature sensitive replication origin. Favourably for theCregene placement, at the "pop-out" step, the observed frequency of RecET-led recombination between the proximal regions of homology was 10 times higher than between the distal regions. Using the minicircle producing plasmid pFIXluc containing mutantloxP66andloxP71sites, we isolated pure minicircle DNA from the obtainedrecA-producer strain HB101Cre. The minicircle DNA preparation consisted of monomeric and, unexpectedly, also multimeric minicircle DNA forms, all containing the hybridloxP66/71site 5'-TACCGTTCGT ATAATGTATG CTATACGAAC GGTA-3', which was previously shown to be an inefficient partner in Cre-mediated recombination.ConclusionUsing transient RecET-driven recombination we inserted a single copy of thearaCcontrolledCregene into thelacZgene on the chromosome ofE. coli recA-strain HB101. The resultantrecA-minicircle DNA producer strain HB101Cre was used to obtain pure minicircle DNA, consisting of monomeric and multimeric minicircle forms. The obtainedrecA-minicircle DNA producer strain is expected to decrease the risk of undesired deletions within minicircle producer plasmids and, therefore, to improve production of the therapeutic minicircle vectors.

Gene therapy by use of integrating vectors carrying therapeutic transgene sequences offers the potential for a permanent cure of genetic diseases due to the ability of these vectors to integrate in a stable manner into the patients’ chromosomes. Since three cases of T-cell leukaemia have been identified after retrovirus gene therapy for X-linked severe combined immune deficiency as being associated with the integrating vector used for gene therapy the need for animal models to test for vector safety has become of paramount importance. Our previous work has shown that a high frequency of hepatocellular carcinomas has occurred following in utero and neonatal injection with certain lentivirus vectors. It has been hypothesized that the woodchuck post regulatory element (WPRE) carried by the vectors used in this study could be implicated in the tumour development process. Our recent study using novel vectors with mutations in the WPRE shows that mice treated with these vectors still develop liver tumours. In this report we discuss these findings and preliminary data to support an alternative cause for tumorigenesis. We also discuss the fetal and neonatal system as a novel and sensitive in vivo model to test the effects and safety of integrating vectors under consideration for clinical applications.

Hemophilia B, also known as Christmas disease, arises from mutations in the factor IX (F9) gene. Its treatment in humans, by recombinant protein substitution, is expensive, thus limiting its application to intermittent treatment in bleeding episodes and prophylaxis during surgery; development of inhibitory antibodies is an associated hazard. This study demonstrates permanent therapeutic correction of his disease without development of immune reactions by introduction of an HIV-based lentiviral vector encoding the human factor IX protein into the fetal circulation of immunocompetent hemophiliac and normal outbred mice. Plasma factor IX antigen remained at around 9%, 13%, and 16% of normal in the 3 hemophilia B mice, respectively, until the last measurement at 14 months. Substantial improvement in blood coagulability as measured by coagulation assay was seen in all 3 mice and they rapidly stopped bleeding after venipuncture. No humoral or cellular immunity against the protein, elevation of serum liver enzymes, or vector spread to the germline or maternal circulation were detected.

Gene therapy for Duchenne muscular dystrophy has so far not been successful because of the difficulty in achieving efficient and permanent gene transfer to the large number of affected muscles and the development of immune reactions against vector and transgenic protein. In addition, the prenatal onset of disease complicates postnatal gene therapy. We have therefore proposed a fetal approach to overcome these barriers. We have applied beta-galactosidase expressing equine infectious anaemia virus (EIAV) lentiviruses pseudotyped with VSV-G by single or combined injection via different routes to the MF1 mouse fetus on day 15 of gestation and describe substantial gene delivery to the musculature. Highly efficient gene transfer to skeletal muscles, including the diaphragm and intercostal muscles, as well as to cardiac myocytes was observed and gene expression persisted for at least 15 months after administration of this integrating vector. These findings support the concept of in utero gene delivery for therapeutic and long-term prevention/correction of muscular dystrophies and pave the way for a future application in the clinic.

The liver is normally proliferatively quiescent, but hepatocyte loss through partial hepatectomy, uncomplicated by virus infection or inflammation, invokes a rapid regenerative response from all cell types in the liver to perfectly restore liver mass. Moreover, hepatocyte transplants in animals have shown that a certain proportion of hepatocytes in foetal and adult liver can clonally expand, suggesting that hepatoblasts/hepatocytes are themselves the functional stem cells of the liver. More severe liver injury can activate a potential stem cell compartment located within the intrahepatic biliary tree, giving rise to cords of bipotential transit amplifying cells (oval cells), that can ultimately differentiate into hepatocytes and biliary epithelial cells. A third population of stem cells with hepatic potential resides in the bone marrow; these haematopoietic stem cells may contribute to the albeit low renewal rate of hepatocytes, but can make a more significant contribution to regeneration under a very strong positive selection pressure. In such instances, cell fusion rather than transdifferentiation appears to be the underlying mechanism by which the haematopoietic genome becomes reprogrammed.

Inefficient gene transfer, inaccessibility of stem cell compartments, transient gene expression, and adverse immune and inflammatory reactions to vector and transgenic protein are major barriers to successful in vivo application of gene therapy for most genetic diseases. Prenatal gene therapy with integrating vectors may overcome these problems and prevent early irreparable organ damage. To this end, high-dose attenuated VSV-G pseudotyped equine infectious anaemia virus (EIAV) encoding beta-galactosidase under the CMV promoter was injected into the fetal circulation of immuno-competent MF1 mice. We saw prolonged, extensive gene expression in the liver, heart, brain and muscle, and to a lesser extent in the kidney and lung of postnatal mice. Progressive clustered hepatocyte staining suggests clonal expansion of cells stably transduced. We thus provide proof of principle for efficient gene delivery and persistent transgene expression after prenatal application of the EIAV vector and its potential for permanent correction of genetic diseases.

Abstract 4010 The primary axis of migration for transplanted hematopoietic stem and progenitor cells (HSPC) is CXCL12/CXCR4. Heparan sulphate (HS) is required for CXCL12 presentation and receptor binding, but the functional role of HS is poorly defined. The alpha-L-iduronidase knockout mouse (Idua−/−) accumulates HS and dermatan sulphate, recapitulating the neurodegenerative lysosomal storage disease Mucopolysaccharidosis I Hurler (MPSIH). MPSIH is primarily treated with HSPC transplant, but clinical experience suggests a historical engraftment defect in patients. We show significantly reduced HSPC migration in Idua−/− recipients and under limiting engraftment conditions we show a significant haematopoietic engraftment defect in Idua−/− recipients. No significant donor cell effect was observed. Bone marrow but not peripheral blood CXCL12 levels are slightly elevated in Idua−/− mice. CFU frequency in BM is unchanged between genotypes but reduced significantly in peripheral blood of Idua−/− mice. In whole bone marrow, and on mesenchymal stem cells from Idua−/− mice, HS is present in significant excess, particularly in extracellular matrix, and cell surface locations, with significant increases in all sulphation modifications, especially 2-O-sulphation. Finally we show that excess HS, and particularly HS with increased 2-O -sulphation, functionally inhibit haematopoietic progenitor cell migration in vitro. These data provide novel insight into the influence of highly sulphated HS in CXCL12 mediated haematopoietic progenitor cell migration and help to explain why HSCT engraftment has been historically low in MPSIH. Disclosures: No relevant conflicts of interest to declare.

Stem cell gene delivery offers a potential cure for blood related disorders such as haemophilia B and degenerative diseases. We have shown permanent phenotypic correction of a murine model of haemophilia B for over a year by lentiviral delivery of the human factor IX gene to haematopoietic stem cells using a myeloablative conditioning regimen. In addition we were able to show full haematopoietic chimerism and robust tolerance, even after challenge with factor IX.

Top of pageAbstract Gene therapy for Duchenne muscular dystrophy has so far not been successful because of the difficulty in achieving efficient and permanent gene transfer to the large number of affected muscles and the development of immune reactions against vector and transgenic protein. In addition, the prenatal onset of disease complicates postnatal gene therapy. We have therefore proposed a fetal approach to overcome these barriers. We have applied b-galactosidase expressing EIAV lentiviruses by single or combined injection via different routes to the MF1 mouse fetus on day 15 of gestation and describe substantial gene delivery to the musculature. Highly efficient gene transfer to skeletal muscles, including the diaphragm and intercostal muscles, as well as to cardiac myocytes was observed and gene expression persisted for at least five months after administration of this integrating vector. Using alternative envelope glycoproteins to pseudotype the EIAV vector also appears to provide improved gene targeting not only to muscle fibres but also muscle satellite cells important for muscle regeneration. These findings support the concept of in utero gene delivery for therapeutic and long term prevention/correction of muscular dystrophies and pave the way for a future application in the clinic.

Haemophilia B arises from mutations in the factor IX gene. Its treatment in humans, by recombinant protein substitution, is expensive thus limiting its application to intermittent treatment in bleeding episodes and prophylaxis during surgery; development of inhibitory antibodies is an associated hazard. This study demonstrates permanent therapeutic correction of this disease without development of immune reactions by introduction of HIV-based lentiviral vector encoding the human factor IX protein into the fetal circulation of immunocompetent haemophiliac and normal outbred mice. Plasma factor IX persists at therapeutic concentrations in all treated mice to date (ten months). Functional normalisation of the blood clotting has been achieved in two haemophiliac mice and significant correction in a third. In comparison, administration of an adenoviral vector encoding human factor IX resulted in transient and diminishing factor IX expression which was nearly undetectable by 8 months. Following lentiviral administration, no anti-factor IX antibodies were detected by ELISA and no cellular immunity was detected by immunohistochemical staining of macrophages, neutrophils, CD4-positive or CD8-positive cells. There was no elevation of serum liver enzymes or evidence of vector spread to the maternal circulation. No vector spread to sperm was detected by TaqMan analysis. This is the first demonstration of complete phenotypic correction of a severe genetic disease by in utero gene therapy with no evidence of toxicity.

Bile acids, bile alcohols, and hormonal steroids represent the ultimate biologically active products of cholesterol metabolism in vertebrates. However, intermediates in their formation, including oxysterols and cholestenoic acids, also possess known, e.g., as ligands to nuclear and G-protein-coupled receptors, and unknown regulatory activities. The potential diversity of molecules originating from the cholesterol structure is very broad and their abundance in biological materials ranges over several orders of magnitude. Here we describe the application of enzyme-assisted derivatization for sterol analysis (EADSA) in combination with liquid chromatography–electrospray ionization–mass spectrometry to define the oxysterol and cholestenoic acid metabolomes of human plasma. Quantitative profiling of adult plasma using EADSA leads to the detection of over 30 metabolites derived from cholesterol, some of which are ligands to the nuclear receptors LXR, FXR, and pregnane X receptor or the G-protein-coupled receptor Epstein–Barr virus-induced gene 2. The potential of the EADSA technique in screening for inborn errors of cholesterol metabolism and biosynthesis is demonstrated by the unique plasma profile of patients suffering from cerebrotendinous xanthomatosis. The analytical methods described are easily adapted to the analysis of other biological fluids, including cerebrospinal fluid, and also tissues, e.g., brain, in which nuclear and G-protein-coupled receptors may have important regulatory roles.

Smith-Lemli-Opitz syndrome (SLOS) is a severe autosomal recessive disorder resulting from defects in the cholesterol synthesising enzyme 7-dehydrocholesterol reductase (Δ7-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).

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.

Background Sleep disturbance is part of the behavioural phenotype of the rare genetic condition mucopolysaccharidosis (MPS) type III. A growing body of evidence suggests that underlying disturbance in circadian rhythm functioning may explain sleep problems within the MPS III population. Methods Actigraphic data were recorded in eight children with MPS III over 7–10 days and compared to age-matched typically developing controls. Parameters of circadian rhythmicity and activity levels across a 24-h period were analysed. Results Statistically and clinically significant differences between the two groups were noted. Analysis indicated that children with MPS III showed significantly increased fragmentation of circadian rhythm and reduced stability with external cues (zeitgebers), compared to controls. Average times of activity onset and offset were indicative of a phase delayed sleep-wake cycle for some children in the MPS III group. Children with MPS III had significantly higher activity levels during the early morning hours (midnight–6 am) compared to controls. Conclusions Results are consistent with previous research into MPS III and suggest that there is an impairment in circadian rhythm functioning in children with this condition. Implications for clinical practice and the management of sleep difficulties are discussed.