ABSTRACT
Mucopolysaccharidosis type I (MPS I), a lysosomal storage disease caused by a deficiency of α-L-iduronidase, leads to storage of the glycosaminoglycans, dermatan sulfate and heparan sulfate. Available therapies include enzyme replacement and hematopoietic stem cell transplantation. In the last two decades, newborn screening (NBS) has focused on early identification of the disorder, allowing early intervention and avoiding irreversible manifestations. Techniques developed and optimized for MPS I NBS include tandem mass-spectrometry, digital microfluidics, and glycosaminoglycan quantification. Several pilot studies have been conducted and screening programs have been implemented worldwide. NBS for MPS I has been established in Taiwan, the United States, Brazil, Mexico, and several European countries. All these programs measure α-L-iduronidase enzyme activity in dried blood spots, although there are differences in the analytical strategies employed. Screening algorithms based on published studies are discussed. However, some limitations remain: one is the high rate of false-positive results due to frequent pseudodeficiency alleles, which has been partially solved using post-analytical tools and second-tier tests; another involves the management of infants with late-onset forms or variants of uncertain significance. Nonetheless, the risk-benefit ratio is favorable. Furthermore, long-term follow-up of patients detected by neonatal screening will improve our knowledge of the natural history of the disease and inform better management.
Subject(s)
Mucopolysaccharidosis I , Heparitin Sulfate , Humans , Iduronidase/analysis , Infant , Infant, Newborn , Mucopolysaccharidosis I/diagnosis , Neonatal Screening/methods , Tandem Mass Spectrometry/methodsABSTRACT
Mucopolysaccharidoses type I (MPS I) is an inherited metabolic disease characterized by a malfunction of the α-l-iduronidase (IDUA) enzyme leading to the storage of glycosaminoglycans in the lysosomes. This disease has longtime been studied as a therapeutic target for those studying gene therapy and many studies have been done using various vectors to deliver the IDUA gene for corrective treatment. Many vectors have difficulties with efficacy and insertional mutagenesis concerns including adeno-associated viral (AAV) vectors. Studies of AAV vectors treating MPS I have seemed promising, but recent deaths in gene therapy clinical trials for other inherited diseases using AAV vectors have left questions about their safety. Additionally, the recent modifications to adenoviral vectors leading them to target the vascular endothelium minimizing the risk of hepatotoxicity could lead to them being a viable option for MPS I gene therapy when coupled with gene editing technologies like CRISPR/Cas9.
Subject(s)
Gene Editing/methods , Genetic Therapy/methods , Iduronidase/genetics , Mucopolysaccharidosis I/therapy , Animals , CRISPR-Cas Systems , Dependovirus/genetics , Disease Models, Animal , Gene Expression , Genetic Vectors/genetics , Glycosaminoglycans/urine , Humans , Iduronidase/analysis , Iduronidase/metabolism , Mucopolysaccharidosis I/pathologyABSTRACT
Objectives Mucopolysaccharidosis type I (MPS I) was added to our expanded screening panel in 2015. Since then, 127,869 newborns were screened by measuring α-L-iduronidase (IDUA) enzyme activity with liquid chromatography tandem mass spectrometry (LC-MS/MS). High false positives due to frequent pseudodeficiency alleles prompted us to develop a second-tier test to quantify glycosaminoglycan (GAG) levels in dried blood spot (DBS). Methods Heparan-sulfate (HS) and dermatan-sulfate (DS) were measured with LC-MS/MS after methanolysis. DBSs were incubated with methanolic-HCl 3 N at 65 °C for 45 min. Chromatographic separation used an amide column with a gradient of acetonitrile and water with 10 mM ammonium acetate in a 9-min run. The method was validated for specificity, linearity, lower limit of quantification (LOQ), accuracy and precision. Results Intra- and inter-day coefficients of variation were <15% for both metabolites. Reference values in 40 healthy newborns were: HS mean 1.0 mg/L, 0-3.2; DS mean 1.5 mg/L, 0.5-2.7). The two confirmed newborn MPS I patients had elevated HS (4.9-10.4 mg/L, n.v. <3.2) and DS (7.4-8.8 mg/L, n.v. <2.7). Since its introduction in February 2019, the second-tier test reduced the recall rate from 0.046% to 0.006%. Among 127,869 specimens screened, the incidence was 1:63,935 live births. Both patients started enzyme replacement therapy (ERT) within 15 days of birth and one of them received allogenic hematopoietic stem cell transplantation (HSCT) at ht age of 6 months. Conclusions GAGs in DBS increased the specificity of newborn screening for MPS I by reducing false-positives due to heterozygosity or pseudodeficiency. Early diagnosis and therapeutical approach has improved the outcome of our patients with MPS I.
Subject(s)
Glycosaminoglycans/analysis , Iduronidase/analysis , Mucopolysaccharidosis I/diagnosis , Chromatography, Liquid/methods , Glycosaminoglycans/blood , Humans , Iduronidase/blood , Infant, Newborn , Mucopolysaccharidosis I/blood , Neonatal Screening/methods , Reference Values , Tandem Mass Spectrometry/methodsABSTRACT
Cyclophellitol aziridines are potent irreversible inhibitors of retaining glycosidases and versatile intermediates in the synthesis of activity-based glycosidase probes (ABPs). Direct 3-amino-2-(trifluoromethyl)quinazolin-4(3H)-one-mediated aziridination of l-ido-configured cyclohexene has enabled the synthesis of new covalent inhibitors and ABPs of α-l-iduronidase, deficiency of which underlies the lysosomal storage disorder mucopolysaccharidosis typeâ I (MPSâ I). The iduronidase ABPs react covalently and irreversibly in an activity-based manner with human recombinant α-l-iduronidase (rIDUA, Aldurazyme® ). The structures of IDUA when complexed with the inhibitors in a non-covalent transition state mimicking form and a covalent enzyme-bound form provide insights into its conformational itinerary. Inhibitors 1-3 adopt a half-chair conformation in solution (4 H3 and 3 H4 ), as predicted by DFT calculations, which is different from the conformation of the Michaelis complex observed by crystallographic studies. Consequently, 1-3 may need to overcome an energy barrier in order to switch from the 4 H3 conformation to the transition state (2, 5 B) binding conformation before reacting and adopting a covalent 5 S1 conformation. rIDUA can be labeled with fluorescent Cy5 ABP 2, which allows monitoring of the delivery of therapeutic recombinant enzyme to lysosomes, as is intended in enzyme replacement therapy for the treatment of MPSâ I patients.
Subject(s)
Aziridines/chemistry , Cyclohexanols/chemistry , Enzyme Inhibitors/chemistry , Iduronidase/antagonists & inhibitors , Iduronidase/analysis , Chromatography, Liquid , Enzyme Assays , Fluorescent Dyes/chemistry , Humans , Microscopy, Fluorescence , Models, Molecular , Recombinant Proteins/analysis , Staining and Labeling , Tandem Mass SpectrometryABSTRACT
BACKGROUND: We developed an analytical method to measure α-L-iduronidase (IDUA) activity in dried blood spots. This was achieved by using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) with electrospray ionization in the positive ion mode. METHODS: Chromatographic separation was completed using mobile phase involving water-formic acid and acetonitrile-formic acid over 2.8 min of run time on a column with a Kinetex XB-C18 (Phenomenex, USA). The detection of column effluent was performed using a Xevo TQ-S triple quadrupole mass spectrometer (Waters, USA) in the multiple-reaction monitoring mode. This method was verified with blank and control samples at four activity levels: base, low, medium, and high. Control materials were provided from Centers for Disease Control and Prevention (CDC). RESULTS: Intra- and inter-day precisions were between 2.6% and 16.5% and between 7.9% and 17.0%, respectively. A correlative regression study on the IDUA activity in CDC-control samples performed to assess the validity of the developed method showed a highly significant linear association (r(2)=0.9976) between the calculated and CDC-reported values and an obvious difference in activity among the four levels. This reliable analytical method was applied to mucopolysaccharidosis I (Hurler) screening of patients under treatment (n=4) and in normal controls (n=129). IDUA activity ranged from 8.98 to 77.12 µmol/hr/L) in normal controls, and patients undergoing medical treatment showed low IDUA activity. CONCLUSIONS: This method had advantages of simplicity, rapid sample preparation, and liquid chromatographic separation, which efficiently inhibited ionization suppression induced by matrix effects in mass spectrometric detection.
Subject(s)
Chromatography, High Pressure Liquid , Dried Blood Spot Testing/instrumentation , Iduronidase/analysis , Mucopolysaccharidosis I/diagnosis , Tandem Mass Spectrometry , Humans , Iduronidase/metabolism , Mucopolysaccharidosis I/blood , Regression Analysis , Substrate SpecificityABSTRACT
BACKGROUND: We developed an analytical method to measure alpha-L-iduronidase (IDUA) activity in dried blood spots. This was achieved by using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) with electrospray ionization in the positive ion mode. METHODS: Chromatographic separation was completed using mobile phase involving water-formic acid and acetonitrile-formic acid over 2.8 min of run time on a column with a Kinetex XB-C18 (Phenomenex, USA). The detection of column effluent was performed using a Xevo TQ-S triple quadrupole mass spectrometer (Waters, USA) in the multiple-reaction monitoring mode. This method was verified with blank and control samples at four activity levels: base, low, medium, and high. Control materials were provided from Centers for Disease Control and Prevention (CDC). RESULTS: Intra- and inter-day precisions were between 2.6% and 16.5% and between 7.9% and 17.0%, respectively. A correlative regression study on the IDUA activity in CDC-control samples performed to assess the validity of the developed method showed a highly significant linear association (r2=0.9976) between the calculated and CDC-reported values and an obvious difference in activity among the four levels. This reliable analytical method was applied to mucopolysaccharidosis I (Hurler) screening of patients under treatment (n=4) and in normal controls (n=129). IDUA activity ranged from 8.98 to 77.12 micromol/hr/L) in normal controls, and patients undergoing medical treatment showed low IDUA activity. CONCLUSIONS: This method had advantages of simplicity, rapid sample preparation, and liquid chromatographic separation, which efficiently inhibited ionization suppression induced by matrix effects in mass spectrometric detection.
Subject(s)
Humans , Chromatography, High Pressure Liquid , Dried Blood Spot Testing/instrumentation , Iduronidase/analysis , Mucopolysaccharidosis I/blood , Regression Analysis , Substrate Specificity , Tandem Mass SpectrometryABSTRACT
The lack of methodological uniformity in enzyme assays has been a long-standing difficulty, a problem for bench researchers, for the interpretation of clinical diagnostic tests, and an issue for investigational drug review. Illustrative of the problem, α-L-iduronidase enzyme catalytic activity is frequently measured with the substrate 4-methylumbelliferyl-α-L-iduronide (4MU-iduronide); however, final substrate concentrations used in different assays vary greatly, ranging from 25 µM to 1425 µM (Km ≈ 180 µM) making it difficult to compare results between laboratories. In this study, α-L-iduronidase was assayed with 15 different substrate concentrations. The resulting activity levels from the same specimens varied greatly with different substrate concentrations but, as a group, obeyed the expectations of Michaelis-Menten kinetics. Therefore, for the sake of improved comparability, it is proposed that α-L-iduronidase enzyme assays should be conducted either (1) under substrate saturating conditions; or (2) when concentrations are significantly below substrate saturation, with results standardized by arithmetic adjustment that considers Michaelis-Menten kinetics. The approach can be generalized to many other enzyme assays.
Subject(s)
Enzyme Assays/standards , Hymecromone/analogs & derivatives , Iduronidase/analysis , Mucopolysaccharidosis I/enzymology , Calibration , Humans , Hymecromone/chemistry , Hymecromone/standards , Iduronidase/metabolism , Kinetics , Mucopolysaccharidosis I/diagnosis , Mucopolysaccharidosis I/pathology , Quality ControlABSTRACT
In recent years, new treatments have become available to treat some lysosomal storage disorders (LSDs) and many studies suggest that there is a benefit with starting therapy early. Newborn screening should detect diseases early enough for prompt treatment. Some countries include additional conditions, such as some LSDs, into their newborn screening panels. Mucopolysaccharidosis Type I (MPS I) is an autosomal recessive disorder caused by the deficiency of α-L-iduronidase (IDUA) activity. Currently, enzyme replacement therapy (ERT) or bone marrow transplantation is available and this has raised a growing interest for the development of a newborn screening test. In 2009, we reported a new fast and simplified tandem mass spectrometry-based method for quantifying five enzyme activities on dried blood spots. Here, we describe the inclusion of IDUA activity determination for the simultaneous detection of six lysosomal storage diseases. We have defined reference normal ranges by testing 680 healthy newborns and 240 adults. The assay was checked through three confirmed MPS I patients whose IDUA activity was below the normal range. Reproducibility of the assays has been established by assessing the intra-day and inter-day assay imprecisions. This quick assay has been devised to be implemented in newborn screening by liquid chromatography tandem mass spectrometry.
Subject(s)
Chromatography, Liquid/methods , Dried Blood Spot Testing/methods , Mass Spectrometry/methods , Mucopolysaccharidosis I/diagnosis , Neonatal Screening/methods , Chromatography, Liquid/standards , Dried Blood Spot Testing/standards , Humans , Iduronidase/analysis , Iduronidase/blood , Iduronidase/chemistry , Infant, Newborn , Mass Spectrometry/standards , Reproducibility of ResultsABSTRACT
In vivo tracking of the delivery of therapeutic proteins is a useful tool for preclinical studies. However, many labels are too large to use without disrupting the normal uptake, function, or other properties of the protein. Low-molecular-weight fluorescent labels allow in vivo and ex vivo tracking of the distribution of therapeutic proteins, and should not alter the protein's characteristics. We tested the in vitro properties of fluorescent-labeled recombinant human alpha-l-iduronidase (rhIDU, the enzyme deficient in Hurler syndrome) and compared labeled to unlabeled proteins. Labeled rhIDU retained full enzymatic activity and showed similar kinetics to nonlabeled rhIDU. Uptake of labeled rhIDU into human Hurler fibroblasts, measured by activity assay, was equivalent to unlabeled rhIDU enzyme and showed an uptake constant of 0.72 nM. Labeled rhIDU was also able to enter cells via the mannose 6-phospate receptor pathway and reduce glycosaminoglycan storage in Hurler fibroblasts. Subcellular localization was verified within lysosomes by confocal microscopy. These findings suggest that fluorescent labeling does not significantly interfere with enzymatic activity, stability, or uptake, and validates this method as a way to track exogenously administered enzyme.
Subject(s)
Iduronidase/analysis , Iduronidase/metabolism , Lysosomes/enzymology , Cells, Cultured , Fibroblasts/metabolism , Fluorescent Dyes/chemistry , Glycosaminoglycans/metabolism , Humans , Iduronidase/chemistry , Molecular Weight , Mucopolysaccharidosis I/enzymology , Receptor, IGF Type 2/metabolism , Recombinant Proteins/analysis , Recombinant Proteins/chemistry , Recombinant Proteins/metabolismABSTRACT
BACKGROUND: Mutations in the alpha-l-iduronidase A (IDUA) gene cause mucopolysaccharidosis type I (MPS I), a progressive multisystem disorder with features ranging over a continuum from mild to severe which is inherited in an autosomal recessive manner. To date over 100 mutations are known, nonetheless genotype-phenotype prediction is complicated and hampered due to attenuating polymorphisms, rare sequence variants, varied genetic backgrounds and environmental effects. METHODS: In this study we report the first development of a denaturating high performance liquid chromatography (dHPLC) protocol for the rapid and accurate detection of recently described mutations in the IDUA gene. Optimal PCR running and dHPLC partial denaturing conditions for mutation detection were established for each PCR amplicon corresponding to 14 IDUA exons and their adjacent intronic/flanking sequences. RESULTS: A total of 12 different mutations, 5 nonsense, 4 missense, 1 deletion, and 2 splice site (intron), in 10 MPS I patients were screened. All mutations revealed a distinct dHPLC pattern thus enabling their accurate detection. CONCLUSIONS: A dHPLC screening method was developed for the detection of mutations and sequence variants in the IDUA gene and the results presented in this study revealed that this promising method proved to be robust, automated, economical and above all, highly sensitive. Costs for the detection of mutations causing MPS I disease should be reduced by using this method as a pre-analytical tool followed by sequencing of aberrant heteroduplex-forming amplicons.
Subject(s)
Iduronidase/analysis , Mucopolysaccharidosis I/genetics , Chromatography, High Pressure Liquid/economics , Cost-Benefit Analysis , DNA Mutational Analysis/economics , Genetic Variation/genetics , Humans , Iduronidase/metabolism , Mutation , Nucleic Acid Denaturation , Polymerase Chain Reaction/economicsABSTRACT
Inherited deficiencies in critical components of metabolic pathways are the primary cause of many liver and lysosomal disorders, most of which are incurable. Stem cell transplantation may offer a new type of treatment for these diseases. We have isolated hepatocyte precursors from human fetal livers. These cells were highly proliferative in vitro in media with or without serum. Expanded hepatocyte precursors expressed endoderm and early hepatocyte markers. The precursors synthesized a large number of molecules related to human metabolic diseases and released some of them into the environment. In a homing test, these cells migrated preferentially into the liver. When transplanted into fetal sheep liver, they incorporated into the liver tissue and differentiated into hepatocytes. Transplantation of the liver precursors to alpha-l-iduronidase-deficient mice partially corrected the enzyme deficiency. Data from these studies suggest that in vitro expanded human liver precursor cells are a potential cell source for the treatment of liver- and lysosome-related disorders.
Subject(s)
Fetal Tissue Transplantation , Liver Diseases/therapy , Liver Transplantation/methods , Liver/cytology , Stem Cells/cytology , Animals , Biomarkers/analysis , Biomarkers/metabolism , Cell Culture Techniques , Cells, Cultured , Hepatocytes/cytology , Hepatocytes/transplantation , Humans , Iduronidase/analysis , Iduronidase/genetics , Iduronidase/metabolism , Immunohistochemistry , Liver/embryology , Mice , Mice, Knockout , Mice, SCID , Stem Cell TransplantationABSTRACT
As an initial step to develop plants as systems to produce enzymes for the treatment of lysosomal storage disorders, Arabidopsis thaliana wild-type (Col-0) plants were transformed with a construct to express human alpha-l-iduronidase (IDUA; EC 3.2.1.76) in seeds using the promoter and other regulatory sequences of the Phaseolus vulgaris arcelin 5-I gene. IDUA protein was easily detected on Western blots of extracts from the T(2) seeds, and extracts contained IDUA activity as high as 2.9 nmol 4-methylumbelliferone (4 MU)/min/mg total soluble protein (TSP), corresponding to approximately 0.06 microg IDUA/mg TSP. The purified protein reacted with an antibody specific for xylose-containing plant complex glycans, indicating its transit through the Golgi complex. In an attempt to avoid maturation of the N-linked glycans of IDUA, the same IDUA transgene was introduced into the Arabidopsis cgl background, which is deficient in the activity of N-acetylglucosaminyl transferase I (EC 2.4.1.101), the first enzyme in the pathway of complex glycan biosynthesis. IDUA activity and protein levels were significantly higher in transgenic cgl vs. wild-type seeds (e.g. maximum levels were 820 nmol 4 MU/min/mg TSP, or 18 microg IDUA/mg TSP). Affinity-purified IDUA derived from cgl mutant seeds showed a markedly reduced reaction with the antibody specific for plant complex glycans, despite transit of the protein to the apoplast. Furthermore, gel mobility changes indicated that a greater proportion of its N-linked glycans were susceptible to digestion by Streptomyces endoglycosidase H, as compared to IDUA derived from seeds of wild-type Arabidopsis plants. The combined results indicate that IDUA produced in cgl mutant seeds contains glycans primarily in the high-mannose form. This work clearly supports the viability of using plants for the production of human therapeutics with high-mannose glycans.
Subject(s)
Arabidopsis/genetics , Iduronidase/metabolism , Plants, Genetically Modified/enzymology , Golgi Apparatus/enzymology , Humans , Iduronidase/analysis , Iduronidase/genetics , Mannose/metabolism , Mutation , N-Acetylglucosaminyltransferases/genetics , N-Acetylglucosaminyltransferases/metabolism , Polysaccharides/metabolism , Protein Transport , Recombinant Fusion Proteins/metabolism , Seeds/enzymology , Seeds/genetics , Transformation, GeneticABSTRACT
Mucopolysaccharidosis type I (MPS I) is caused by an inherited deficiency of alpha-L-iduronidase (IDUA). The result is a progressive, lysosomal storage disease with central nervous system (CNS) as well as systemic involvement. To target gene therapy to the CNS, recombinant adeno-associated virus (AAV) vectors carrying IDUA sequence were administered to MPS I mice via injection into cerebrospinal fluid. In contrast to intravenous administration, this intrathecal administration was effective in generating widespread IDUA activity in the brain, with the cerebellum and olfactory bulbs having highest activities. In general, IDUA levels correlated with vector dose, although this correlation was obscured in cerebellum by particularly high variability. High doses of vector (4 x 10(10) particles) provided IDUA levels approaching or exceeding normal levels in the brain. Histopathology indicated that the number of cells with storage vacuoles was reduced extensively or was eliminated entirely. Elimination of storage material in Purkinje cells was particularly dramatic. A lower vector dose (2 x 10(9) particles) reduced both the number of storage cells and the extent of storage per cell, but the effect was not complete. Some perivascular cells with storage persisted, and this cell type appeared to be more resistant to treatment than neurons or glial cells. We conclude that intrathecal administration of AAV-IDUA delivers vector to brain cells, and that this route of administration is both minimally invasive and effective.
Subject(s)
Brain/metabolism , Dependovirus/genetics , Genetic Therapy/methods , Genetic Vectors/administration & dosage , Iduronidase/genetics , Mucopolysaccharidosis I/therapy , Animals , Brain/ultrastructure , Fluorescent Antibody Technique, Indirect , Gene Expression , Iduronidase/analysis , Iduronidase/metabolism , Injections, Spinal , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Confocal , Mucopolysaccharidosis I/metabolism , Mucopolysaccharidosis I/pathologyABSTRACT
Murine models of lysosomal storage diseases provide an opportunity to evaluate the potential for gene therapy to prevent systemic manifestations of the disease. To determine the potential for treatment of mucopolysaccharidosis type I using a gene delivery approach, a recombinant adeno-associated virus (AAV) vector, vTRCA1, transducing the human iduronidase (IDUA) gene was constructed and 1 x 10(10) particles were injected intravenously into 1-day-old Idua(-/-) mice. High levels of IDUA activity were present in the plasma of vTRCA1-treated animals that persisted for the 5-month duration of the study, with heart and lung of this group demonstrating the highest tissue levels of gene transfer and enzyme activity overall. vTRCA1-treated Idua(-/-) animals with measurable plasma IDUA activity exhibited histopathological evidence of reduced lysosomal storage in a number of tissues and were normalized with respect to urinary GAG excretion, craniofacial bony parameters, and body weight. In an open field test, vTRCA1-treated Idua(-/-) animals exhibited a significant reduction in total squares covered and a trend toward normalization in rearing events and grooming time compared to control-treated Idua(-/-) animals. We conclude that AAV-mediated transduction of the IDUA gene in newborn Idua(-/-) mice was sufficient to have a major curative impact on several of the most important parameters of the disease.
Subject(s)
Craniofacial Abnormalities/therapy , Dependovirus/genetics , Genetic Therapy/methods , Iduronidase/genetics , Mucopolysaccharidosis I/therapy , Animals , Craniofacial Abnormalities/pathology , Gene Expression , Genetic Vectors/genetics , Glycosaminoglycans/urine , Habituation, Psychophysiologic , Humans , Iduronidase/analysis , Iduronidase/metabolism , Lysosomes/metabolism , Mice , Mice, Knockout , Mucopolysaccharidosis I/pathology , Nervous System Malformations/pathology , Nervous System Malformations/therapy , Tissue Distribution , Transduction, GeneticABSTRACT
To optimize a gene transfer system for hematopoietic stem cell gene therapy of patients with mucopolysaccharidosis (MPS) type I, 10 retroviral vectors were constructed to express the human alpha-L-iduronidase (IDUA) cDNA. These vectors were designed to evaluate the potential effects of specific promoters, the addition of selectable markers, and the use of multiple promoters versus an internal ribosome entry site for expression of IDUA and selectable maker genes. The effect of vector design was investigated in primary patient fibroblasts (F(MPS)) or murine fibroblast cell lines; while overall comparison of transgene expression was determined in patients' peripheral blood lymphocytes (PBL(MPS)) and CD34+ progenitors (PBPC(MPS)). We observed that the human PGK promoter introduced the highest IDUA activity per 1% relative transgene frequency in F(MPS). Use of the same promoter to separately regulate both the therapeutic gene and a drug-resistance gene resulted in decreased expression of the unselected gene. Co-selection using bicistronic vectors not only increased the number of transductants, but also elevated transgene expression under selective pressure in transgene-positive progenitors. Bicistronic vector LP1CD overcame down-regulation and practically introduced the highest IDUA level in unselected PBL(MPS) and an intermediate level in PBPC(MPS). These studies provide a better understanding of factors contributing to efficient gene expression in hematopoietic cells.
Subject(s)
Genetic Therapy/methods , Genetic Vectors/genetics , Hematopoietic Stem Cells/enzymology , Iduronidase/genetics , Mucopolysaccharidosis I/therapy , Animals , Cell Line , Drug Resistance/genetics , Fibroblasts/enzymology , Gene Expression , Genetic Engineering , Humans , Iduronidase/analysis , Methotrexate , Mice , Phosphoglycerate Kinase/genetics , Polymerase Chain Reaction , Promoter Regions, Genetic , Retroviridae/genetics , TransfectionABSTRACT
Fibroblasts from 16 patients with known alpha-L-iduronidase gene mutations and different clinical phenotypes of mucopolysaccharidosis type I (MPS I) were investigated in order to establish genotype/phenotype correlations. Enzyme kinetic studies were performed using the specific alpha-L-iduronidase substrate iduronosyl anhydro[1-3H]mannitol-6-sulfate. Specific residual enzyme activities were estimated using the kinetic parameters and an immunoquantification assay which determines levels of alpha-L-iduronidase protein. Cells were cultured in the presence of [35S]sulfate and the in vivo degradation of accumulated labelled glycosaminoglycans measured after different chase times. Residual enzyme activity and different amounts of residual enzyme protein were present in extracts from 9 of 16 cell lines covering a wide spectrum of clinical severity. Catalytic capacity, calculated as the product of kcat/Km and ng iduronidase protein per mg cell protein, was shown in most cases to be directly related to the severity of clinical phenotype, with up to 7% of normal values for patients with the attenuated form of MPS I (Scheie) and less than 0.13% for severely affected patients (Hurler) In vitro turnover studies allowed further refinement of correlations between genotype and phenotype. Scheie disease compared to Hurler disease patients were shown to accumulate smaller amounts of glycosaminoglycans that were also turned over faster. A combination of turnover and residual enzyme data established a correlation between the genotype, the biochemical phenotype and the clinical course of this lysosomal storage disorder.
Subject(s)
Iduronidase/genetics , Mucopolysaccharidosis I/genetics , Mutation , Cell Line , Fibroblasts/enzymology , Genotype , Glycosaminoglycans/metabolism , Humans , Iduronidase/analysis , Kinetics , Mucopolysaccharidosis I/enzymology , PhenotypeABSTRACT
Mucopolysaccharidosis type I (MPS I: McKusick 25280) is a clinically heterogenous lysosomal storage disorder which is caused by a variable deficiency in alpha-L-iduronidase activity (alpha-L-iduronide iduronohydrolase, EC 3.2.1.76). Cultured fibroblasts from an MPS I patient (cell line 2827) with a severe clinical phenotype (Hurler syndrome) have been characterized using immunochemical and biochemical techniques. Using a specific immunoquantification assay, we have demonstrated that cell line 2827 had an alpha-L-iduronidase protein content (189 ng/mg of extracted cell protein) at least six times greater than the mean level found in normal control fibroblasts (30 ng/mg of extracted cell protein). This was the only MPS I cell line, from a group of 23 MPS I patients, that contained greater than 7% of the mean level of alpha-L-iduronidase protein detected in normal controls. Cell line 2827 had very low alpha-L-iduronidase activity toward the fluorogenic substrate 4-methylumbelliferyl-alpha-L-iduronide, and a radiolabeled disaccharide substrate derived from heparin. Maturation studies of alpha-L-iduronidase in cell line 2827 showed apparently normal levels of alpha-L-iduronidase synthesis with delayed processing to the mature form. Subcellular fractionation experiments demonstrated alpha-L-iduronidase protein in lysosomal-enriched fractions isolated from cell line 2827, suggesting a normal cell distribution and supporting the proposed delayed processing. It is proposed that the MPS I patient described has an alpha-L-iduronidase gene mutation which affects both the active site and post-translational processing of the enzyme. This mutation must be structurally conservative because it does not result in instability either during maturation or in the lysosome.
Subject(s)
Iduronidase/analysis , Mucopolysaccharidosis I/metabolism , Proteins/analysis , Cells, Cultured , Child, Preschool , Fibroblasts/metabolism , Humans , Lysosomes/enzymology , Male , Subcellular Fractions/metabolismABSTRACT
alpha-L-Iduronidase activity is deficient in mucopolysaccharidosis type I (MPS I; Hurler syndrome, Scheie syndrome) patients and results in the disruption of the sequential degradation of the glycosaminoglycans dermatan sulfate and heparan sulfate. A monoclonal antibody-based immunoquantification assay has been developed for alpha-L-iduronidase, which enables the detection of at least 16 pg alpha-L-iduronidase protein. Cultured human skin fibroblasts from 12 normal controls contained 17-54 ng alpha-L-iduronidase protein/mg extracted cell protein. Fibroblasts from 23 MPS I patients were assayed for alpha-L-iduronidase protein content. Fibroblast extracts from one MPS I patient contained at least six times the level of alpha-L-iduronidase protein for normal controls--but contained no associated enzyme activity--and is proposed to represent a mutation affecting the active site of the enzyme. Fibroblast extracts from 11 MPS I patients contained 0.05-2.03 ng alpha-L-iduronidase protein/mg extracted cell protein, whereas immunodetectable protein could not be detected in the other 11 patients. Four fibroblast extracts with no immunodetectable alpha-L-iduronidase protein had residual alpha-L-iduronidase activity, suggesting that the mutant alpha-L-iduronidase in cultured cells from these MPS I patients has been modified to mask or remove the epitopes detected by two monoclonal antibodies used in the quantification assay. Both the absence of immunoreactivity in a mild MPS I patient and high protein level in a severe MPS I patient present limitations to the use of immunoquantification analysis as a sole measure of patient phenotype. Enzyme kinetic analysis of alpha-L-iduronidase from MPS I fibroblasts revealed a number of patients with either abnormal substrate binding or catalytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Iduronidase/analysis , Mucopolysaccharidosis I/enzymology , Antibodies, Monoclonal , Cells, Cultured , Fibroblasts/enzymology , Humans , Iduronidase/immunologyABSTRACT
Twenty-four pregnancies at risk for Hurler disease (MPS I) were monitored by measurement of alpha-iduronidase in chorionic villi. Adequate samples were obtained for direct assay of the villi in 22 pregnancies. Five were found to be affected and the pregnancies were terminated. In another pregnancy an equivocal result was obtained on direct assay but analysis of the cultured chorionic cells showed the fetus to be affected. In one pregnancy where an exceptionally small biopsy was obtained, direct assay indicated the fetus to be unaffected. Following amniocentesis this result was shown to be incorrect. These results confirm that, provided an adequate sample is obtained, an accurate diagnosis can be made by direct assay of chorionic villi in pregnancies at risk for Hurler disease.
Subject(s)
Chorionic Villi/enzymology , Iduronidase/analysis , Mucopolysaccharidosis I/diagnosis , Prenatal Diagnosis/methods , Amniotic Fluid/chemistry , Cells, Cultured , Female , Fetus , Fibroblasts/enzymology , Glycosaminoglycans/analysis , Humans , Mucopolysaccharidosis I/enzymology , PregnancyABSTRACT
We investigated the alpha-L-iduronidase activity of various pig tissues. Furthermore, we examined the tissues using antibody, enzyme immunoassay (EIA), and immunohistochemical methods. The amounts of enzyme measured by the EIA method in the various tissues were proportional to their enzyme activities and also to their immunohistochemical characteristics. The tissues could thus be classified into three groups: a high enzyme activity group composed of the liver, kidney, and spleen; a moderate activity group comprising the lung, lymph nodes, stomach, ileum, colon, and pancreas; and a low activity group consisting of the heart, diaphragm, iliopsoas muscle, cerebrum, cerebellum, and skin. The molecular weight of the enzyme in each tissue did not reveal any heterogeneity, having two components of 70 KD and 62 KD by Western blot analysis. Immunohistochemically, alpha-L-iduronidase was strongly detected in the lysosomal membranes of cells of the mononuclear phagocyte system, epithelial cells of the proximal tubules in the kidney, and some blastic cells, whereas hepatocytes revealed weak positive reactions. The tissue and cellular distribution of the enzyme appeared to have a close relation to tissues that manifest or are affected by alpha-L-iduronidase deficiency.