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Mucopolysaccharidoses (MPSs) are rare lysosomal storage diseases caused by the accumulation of undegraded glycosaminoglycans in cells and tissues. The effectiveness of early intervention for MPS has been reported. Multiple-assay formats using tandem mass spectrometry have been developed. Here, we developed a method for simultaneous preparation and better measurement of the activities of five enzymes involved in MPSs, i.e., MPS I, MPS II, MPS IIIB, MPS IVA, and MPS VI, which were validated using 672 dried blood spot samples obtained from healthy newborns and 23 patients with MPS. The mean values of the enzyme activities and standard deviations in controls were as follows: α-iduronidase (IDUA), 4.19 ± 1.53 µM/h; iduronate-2-sulfatase (I2S), 8.39 ± 2.82 µM/h; N-acetyl-α-glucosaminidase (NAGLU), 1.96 ± 0.57 µM/h; N-acetylgalactosamine-6-sulfatase (GALNS), 0.50 ± 0.20 µM/h; and N-acetylgalactosamine-4-sulfatase (ARSB), 2.64 ± 1.01 µM/h. All patients displayed absent or low enzyme activity. In MPS I, IIIB, and VI, each patient group was clearly separated from controls, whereas there was some overlap between the control and patient groups in MPS II and IVA, suggesting the occurrence of pseudo-deficiencies. Thus, we established a multiplex assay for newborn screening using liquid chromatography tandem mass spectrometry, allowing simultaneous pretreatment and measurement of five enzymes relevant to MPSs.

Significance Treatment of the lysosomal storage disease mucopolysaccharidosis type I (MPS I) is currently based on hematopoietic stem cell transplantation (HSCT) or weekly infusions of the deficient enzyme. To circumvent the morbidity and mortality associated with HSCT and the economic and quality of life costs of lifelong enzyme replacement therapy, we tested liver-directed gene therapy as a means of achieving endogenous enzyme expression in a feline model of MPS I. We found that hepatic gene transfer not only generated therapeutic levels of circulating enzyme, but in most cases also resulted in complete resolution of storage lesions in the cardiac valves, a tissue that is refractory to currently available therapies and responsible for much of the residual morbidity and mortality in treated patients. Patients with mucopolysaccharidosis type I (MPS I), a genetic deficiency of the lysosomal enzyme α-l-iduronidase (IDUA), exhibit accumulation of glycosaminoglycans in tissues, with resulting diverse clinical manifestations including neurological, ocular, skeletal, and cardiac disease. MPS I is currently treated with hematopoietic stem cell transplantation or weekly enzyme infusions, but these therapies have significant drawbacks for patient safety and quality of life and do not effectively address some of the most critical clinical sequelae, such as life-threatening cardiac valve involvement. Using the naturally occurring feline model of MPS I, we tested liver-directed gene therapy as a means of achieving long-term systemic IDUA reconstitution. We treated four MPS I cats at 3–5 mo of age with an adeno-associated virus serotype 8 vector expressing feline IDUA from a liver-specific promoter. We observed sustained serum enzyme activity for 6 mo at ∼30% of normal levels in one animal, and in excess of normal levels in three animals. Remarkably, treated animals not only demonstrated reductions in glycosaminoglycan storage in most tissues, but most also exhibited complete resolution of aortic valve lesions, an effect that has not been previously observed in this animal model or in MPS I patients treated with current therapies. These data point to clinically meaningful benefits of the robust enzyme expression achieved with hepatic gene transfer that extend beyond the economic and quality of life advantages over lifelong enzyme infusions.

Monitoring of therapeutic response in mucopolysaccharidosis (MPS) patients is problematic as most biomarkers are specific for either disease complications or specific organ system involvement. Recent studies have indicated that serum heparin-cofactor II-thrombin complex (HCII-T) may serve as an important biomarker in the group of MPSs where dermatan sulphate is stored. This complex forms when blood coagulates in the presence of glycosaminoglycans (GAGs) where the ultimate amount of HCII-T that forms reflects the concentration of circulating GAGs. We have studied serum HCII-T levels in 9 MPS I and 11 MPS II treated patients and have compared values to studies of urinary GAGs. In severe MPS I patients treated with either transplantation or enzyme replacement therapy (ERT), serum HCII-T levels never reach the range of normal despite normalization of uGAGs in some patients. Some attenuated MPS I patients have normalization of HCII-T but require a protracted exposure time relative to the drop in urinary GAGs. Treated MPS II patients show a clear correlation of serum HCII-T levels with the presence of antibodies to Idursulfase, with antibody positive patients showing an early drop in HCII-T levels with eventual increases in levels often to levels above those seen at baseline. This is contrasted by a robust and persistent drop in uGAGs. Antibody negative MPS II patients show a drop in HCII-T levels on treatment but levels never normalize despite normalization of uGAGs. This study highlights the utility and biologic relevance of serum HCII-T levels in monitoring therapy in these disorders.

Busulfan is an alkylating agent routinely used in conditioning regimens prior to allogeneic hematopoietic cell transplantation (HCT) for various nonmalignant disorders, including inborn errors of metabolism. The combination of model-based dosing and therapeutic drug monitoring (TDM) of busulfan pharmacokinetics (PK) to a lower exposure target has the potential to reduce the regimen-related toxicity while opening marrow niches sufficient for engraftment in diseases such as mucopolysaccharidosis type I (MPS I). We present four cases of the severe form of MPS I or Hurler syndrome, demonstrating successful and stable CD14/15 donor chimerism following the prospective application of model-based dosing and TDM aimed to achieve lower busulfan exposure. All patients received a busulfan-based conditioning regimen with a median cumulative area-under-the-curve (cAUC) target of 63.7 mg h/L (range, 62.4 to 65.0) in protocol-specific combination of chemotherapeutic regimen. The donor source was unrelated umbilical cord blood for three patients and matched sibling donor bone marrow for one patient. The observed median busulfan cAUC was 66.1 mg h/L (range, 65.2 to 70.6) and was within 10% of the intended target. Stable, full donor myeloid chimerism was achieved for three patients, while one patient achieved a stable mixed chimerism (76% donor CD14/15 at 53 months) without a recurring need for enzyme replacement. The normalization of α-L-iduronidase enzyme levels followed the attainment of successful donor myeloid chimerism in all patients. Regimen-related toxicity remained low with no evidence of acute graft-versus-host disease (GVHD) grades II to IV and chronic GVHD.

Mucopolysaccharidosis type I (MPS I) is a progressive disorder caused by deficiency of α-L-iduronidase (IDUA), which leads to storage of heparan and dermatan sulphate. It is suggested that early enzyme replacement therapy (ERT) leads to better outcomes, although many patients are diagnosed late and don't receive immediate treatment. This study aims to evaluate the effects of late onset ERT in a MPS I murine model. MPS I mice received treatment from 6 to 8 months of age (ERT 6-8mo) with 1.2mg laronidase/kg every 2 weeks and were compared to 8 months-old wild-type (Normal) and untreated animals (MPS I). ERT was effective in reducing urinary and visceral GAG to normal levels. Heart GAG levels and left ventricular (LV) shortening fraction were normalized but cardiac function was not completely improved. While no significant improvements were found on aortic wall width, treatment was able to significantly reduce heart valves thickening. High variability was found in behavior tests, with treated animals presenting intermediate results between normal and affected mice, without correlation with cerebral cortex GAG levels. Cathepsin D activity in cerebral cortex also did not correlate with behavior heterogeneity. All treated animals developed anti-laronidase antibodies but no correlation was found with any parameters analyzed. However, intermediary results from locomotion parameters analyzed are in accordance with intermediary levels of heart function, cathepsin D, activated glia and reduction of TNF-α expression in the cerebral cortex. In conclusion, even if started late, ERT can have beneficial effects on many aspects of the disease and should be considered whenever possible.

Introduction Mucopolysaccharidosis type I (MPS I) results in a defective breakdown of the glycosaminoglycans (GAGs) heparan sulfate and dermatan sulfate, which leads to a progressive disease. Enzyme replacement therapy (ERT) results in clearance of these GAGs from a range of tissues and can significantly ameliorate several symptoms. The biochemical efficacy of ERT is generally assessed by the determination of the total urinary excretion of GAGs. However, this has limitations. We studied the concentrations of heparan sulfate and dermatan sulfate derived disaccharides (HS and DS, respectively) in the plasma and urine of seven patients and compared these levels with total urinary GAGs (uGAGs) levels. Methods Plasma and urine samples were collected at different time points relative to the weekly ERT for three non-consecutive weeks in seven MPS I patients who had been treated with ERT for at least 2.5 years. Heparan and dermatan sulfate in plasma and urine were enzymatically digested into disaccharides, and HS and DS levels were determined by HPLC-MS/MS analysis. uGAGs were measured by the DMB test. Results The levels of HS and DS were markedly decreased compared with the levels before the initiation of ERT. However, the concentrations of DS in plasma and of both HS and DS in urine remained significantly elevated in all studied patients, while in six patients the level of total uGAGs had normalized. The concentrations of plasma and urinary HS during the weekly ERT followed a U-shaped curve. However, the effect size is small. The concentrations of plasma and urinary DS and uGAGs appeared to be in a steady state. Conclusions HS and DS are sensitive biomarkers for monitoring the biochemical treatment efficacy of ERT and remain elevated despite long-term treatment. This finding may be related to the labeled dose or antibody status of the patient. The timing of the sample collection is not relevant, at least at the current dose of 100 IU/kg/weekly.

We sought to develop a tandem mass spectrometry assay in which the enzymatic activities of 3 lysosomal enzymes (α-glucosidase, α-galactosidase A, and α-l-iduronidase) could be quantified in dried blood spots by using a single assay buffer. A 3-mm dried blood spot punch was incubated in a single assay buffer with 3 different substrates and internal standards. The sample was processed by a simple liquid-liquid extraction by using ethyl acetate. The extract was dried down and resuspended in solvent for injection into the tandem mass spectrometer. Products and internal standards were monitored by multiple reaction monitoring. Assay for the 3 lysosomal enzymes was successfully achieved with acceptable statistics. The assay can be performed by using a minimal quantity of disposable supplies and equipment. The entire procedure fits into a 48-h cycle including data analysis. Data from 5990 anonymous newborn dried blood spots showed an approximate bell-shaped distribution of enzymatic activities (mean values of 19.0, 11.5, and 3.5 μmol · h(-1) · (L blood)(-1) for α-glucosidase, α-galactosidase A, and α-l-iduronidase, respectively. Blank values obtained in the absence of blood were 0.13, 0.24, and 0.45 μmol · h(-1) · (L blood)(-1), respectively). By assaying 3 enzymes at once, problematic samples are spotted for reanalysis if enzyme activity values are low for all enzymes (for example, if insufficient blood is present in the assay). This method demonstrates that a triplex assay in a single buffer and with minimal supplies and labor can be adapted to a high-throughput newborn screening laboratory for the analysis of Pompe, Fabry, and mucopolysaccharidosis-I (Hurler) diseases.

Mucopolysaccharidosis I (MPS I) is an autosomal recessive disorder caused by deficiency of α-L-iduronidase leading to accumulation of its catabolic substrates, dermatan sulfate (DS) and heparan sulfate (HS), in lysosomes. This results in progressive multiorgan dysfunction and death in early childhood. The recent success of enzyme replacement therapy (ERT) for MPS I highlights the need for biomarkers that reflect response to such therapy. To determine which biochemical markers are better, we determined serum and urine DS and HS levels by liquid chromatography tandem mass spectrometry in ERT-treated MPS I patients. The group included one Hurler, 11 Hurler/Scheie, and two Scheie patients. Seven patients were treated from week 1, whereas the other seven were treated from week 26. Serum and urine DS (ΔDi-4S/6S) and HS (ΔDiHS-0S, ΔDiHS-NS) were measured at baseline, week 26, and week 72. Serum ΔDi-4S/6S, ΔDiHS-0S, and ΔDiHS-NS levels decreased by 72%, 56%, and 56%, respectively, from baseline at week 72. Urinary glycosaminoglycan level decreased by 61.2%, whereas urine ΔDi-4S/6S, ΔDiHS-0S, and ΔDiHS-NS decreased by 66.8%, 71.8%, and 71%, respectively. Regardless of age and clinical severity, all patients showed marked decrease of DS and HS in blood and urine samples. We also evaluated serum DS and HS from dried blood-spot samples of three MPS I newborn patients, showing marked elevation of DS and HS levels compared with those in control newborns. In conclusion, blood and urine levels of DS and HS provide an intrinsic monitoring and screening tool for MPS I patients.

Mucopolysaccharidosis type I (MPSI) is an autosomal recessive disease that leads to systemic lysosomal storage, which is caused by the absence of α-L-iduronidase (IDUA). Enzyme replacement therapy is recognized as the best therapeutic option for MPSI; however, high titers of anti-IDUA antibody have frequently been observed. Due to the immunosuppressant properties of MSC, we hypothesized that MSC modified with the IDUA gene would be able to produce IDUA for a long period of time. Sleeping Beauty transposon vectors were used to modify MSC because these are basically less-immunogenic plasmids. For cell transplantation, 4×10(6) MSC-KO-IDUA cells (MSC from KO mice modified with IDUA) were injected into the peritoneum of KO-mice three times over intervals of more than one month. The total IDUA activities from MSC-KO-IDUA before cell transplantation were 9.6, 120 and 179 U for the first, second and third injections, respectively. Only after the second cell transplantation, more than one unit of IDUA activity was detected in the blood of 3 mice for 2 days. After the third cell transplantation, a high titer of anti-IDUA antibody was detected in all of the treated mice. Anti-IDUA antibody response was also detected in C57Bl/6 mice treated with MSC-WT-IDUA. The antibody titers were high and comparable to mice that were immunized by electroporation. MSC-transplanted mice had high levels of TNF-alpha and infiltrates in the renal glomeruli. The spreading of the transplanted MSC into the peritoneum of other organs was confirmed after injection of 111In-labeled MSC. In conclusion, the antibody response against IDUA could not be avoided by MSC. On the contrary, these cells worked as an adjuvant that favored IDUA immunization. Therefore, the humoral immunosuppressant property of MSC is questionable and indicates the danger of using MSC as a source for the production of exogenous proteins to treat monogenic diseases.

Enzyme-replacement therapy has been assessed as a treatment for patients who have mucopolysaccharidosis I (α-L-iduronidase deficiency). We aimed to investigate the humoral immune response to recombinant human α-L-iduronidase among these patients. We characterised the antibody titres and specific linear sequence epitope reactivity of serum antibodies to α-L-iduronidase for ten patients with mucopolysaccharidosis I, at the start of treatment and after 6, 12, 26, 52, and 104 weeks. We compared the values for patients' samples with those for samples from normal human controls. Before enzyme-replacement therapy, all patients had low serum antibody titres to recombinant human α-L-iduronidase that were within the control range. Five of the ten patients produced higher-than-normal titres of antibody to the replacement protein during the treatment course (serum antibody titres 130 000–500 000 and high-affinity epitope reactivity). However, by week 26, antibody reactivity was reduced, and by week 104 all patients had low antibody titres and only low-affinity epitope reactivity. Patients who had mucopolysaccharidosis I with antibody titres within the normal range at 6–12 weeks did not subsequently develop immune responses. After 2 years of treatment, patients who initially had an immune reaction developed immune tolerance to α-L-iduronidase. This finding has positive implications for long-term enzyme-replacement therapy in patients who have mucopolysaccharidosis I.