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At least 19 recordsLinked to original sources
PubMed2000

Long-term expression and transfer of arylsulfatase A into brain of arylsulfatase A-deficient mice transplanted with bone marrow expressing the arylsulfatase A cDNA from a retroviral vector.

A deficiency of arylsulfatase A (ASA) results in the lysosomal lipid storage disease metachromatic leukodystrophy. The disease mainly affects the central nervous system causing a progressive demyelination. A therapeutic effect will depend on the delivery of the deficient enzyme to the central nervous system. We have transplanted ASA-deficient mice with bone marrow transduced with a retroviral vector expressing the human ASA cDNA. All transplanted animals initially showed high serum levels of human ASA. In 50% of the recipients high ASA serum levels were sustained for 12 months after transplantation. In the remaining mice, serum levels decreased rapidly to low or undetectable levels. ASA activity and immunoreactivity was detectable in all organs of animals with continuous levels of ASA in serum. Most notably, substantial amounts of ASA activity were transferred into the brain, reaching up to 33% of the normal tissue level. In contrast to peripheral organs, the amount of enzyme delivered to the brain did not correlate with ASA serum levels as an indicator of overexpression. This reveals that enzyme transfer to the brain is not due to endocytosis of serum ASA by endothelial cells, but rather to bone marrow-derived cells migrated into the brain. Gene Therapy (2000) 7, 1250-1257.

Animals↗
PubMed1977

Arylsulfatase of human tissue. Studies on a form of arylsulfatase B found predominantly in brain.

The distribution of soluble arylsulfatase (aryl-sulfate sulfohydrolases, EC 3.1.6.1) in human tissues was investigated by DEAE-cellulose chromatography, All tissues examined contained arylsulfatase A and arylsulfatase B. In addition, brain singularly contained significant quantities (15-25% of total arylsulfatase) of a minor anionic arylsulfatase from designated arylsulfatase Bm, whereas only trace amounts of arylsulfatase Bm were found in liver, kidney, testis and placenta. Arylsulfatase B and arylsulfatase Bm had equal activity toward methyl-umbelliferyl sulfate, nitrocatechol sulfate and a physiological substrate UDP-N-acetylgalactosamine 4-sulfate, but both forms were inactive toward the arylsulfatase A substrates cerebroside sulfate and ascorbic acid 2-sulfate. Purified preparations of placental arylsulfatase B, brain arylsulfatase Bm, and urinary arylsulfatase A did not hydrolyze estrone sulfate, dehydroepiandrosterone sulfate or pregnenolone sulfate. The physico-chemical properties of arylsulfatase Band arylsulfatase Bm differed with respect to thermal lability, DEAE-cellulose chromatography, polyacrylamide gel electrophoresis and isoelectric focussing. In the latter technique, utilizing thin polyacrylamide slab gels, the isoelectric point for placental arylsulfatase B was 8.2, while brain arylsulfatase Bm resolved into 3 activity bands with pI values 6.8, 7.0 and 7.2. Although the physico-chemical properties differed, arylsulfatase B and arylsulfatase Bm appear to be functionally equivalent as well as generically related.

Brain↗
PubMed1980

Soluble arylsulfatases of human brain and some characteristics of the brain-specific arylsulfatase Bm.

The brain-specific arylsulfatase Bm (aryl-sulfate sulfohydrolase, EC 3.1.6.1) was demonstrable in human and monkey brain. Arylsulfatases A, B and Bm were separated employing DEAE-cellulose chromatography. There was a distinct difference in the proportion of the sulfatases in infant and adult human brain. Arylsulfatase Bm after concanavalin A-Sepharose chromatography showed the property of binding to Sephadex G-200 totally. Several dissociating agents failed to elute the enzyme from the bound form. Under similar conditions arylsulfatase A did not show any binding to Sephadex. On treatment with Escherichia coli alkaline phosphatase adult human brain arylsulfatase Bm but not arylsulfatase A was converted into a less acidic, presumably dephosphorylated form that did not bind to DEAE-cellulose. Monkey brain arylsulfatase Bm showed a similar susceptibility to E. coli phosphatase treatment. Inorganic phosphate and serine phosphate but not mannose 6-phosphate could inhibit this dephosphorylation. There were differences in the susceptibilities to alkaline phosphatase treatment of the arylsulfatase Bm from infant and adult human brain. Endogenous phosphatase also seemed to have a role on the phosphorylated state of arylsulfatase Bm.

Adult↗
PubMed1981

Arylsulfatases of human-lung tumors transplanted into athymic mice. Cancer-associated modification of arylsulfatase B variant.

The activities and properties of arylsulfatase A and B from human lung carcinoma transplanted into athymic mice were demonstrated. The activities of arylsulfatase A and B from transplanted carcinomas with four histological types were more than twofold higher as compared to those from surgical tumors, except for arylsulfatase A activity in blastoma. Arylsulfatase B in transplanted tumors was almost completely replaced, except for blastoma, by an anionic B variant (B1) which was a minor component of arylsulfatase B in surgical lung tumor and absent in normal human lung. The properties of arylsulfatases A and B from transplanted tumors were essentially identical, respectively, with those from normal lung or surgical tumors in respect of molecular weight, heat stability, pH optimum, isoelectric point (pI), Km, time course profile and substrate specificity. Arylsulfatase B1 showed the properties similar to B enzyme except for net charge. The cause of the negative charge of tumor B1 enzyme was investigated. By the action of phosphatase, which was added exogenously or had been persistently included in the partially purified enzyme preparation, B1 enzyme (pI 7.5) shifted to about pI 8.2. Treatment of B1 enzyme with neuraminidase, concomitant with the endogenous phosphatase, resulted in marked increase (pI 9.5) of the isoelectric point, identical to that of arylsulfatase B. Thus, it is most probable that tumor B1 enzyme is modified by additional sialic acid and phosphate bound to arylsulfate B.

Animals↗
PubMed1990

Deficient glycosylation of arylsulfatase A in pseudo arylsulfatase-A deficiency.

Deficient arylsulfatase-A activity is diagnostic of a neurodegenerative human lysosomal storage disease, metachromatic leukodystrophy. Paradoxically, similar enzyme deficiency also occurs in normal individuals, who are known as being pseudo arylsulfatase-A deficient. We showed previously that this phenotype is associated with a structural gene mutation that produces an exceptionally labile enzyme. We now report on the nature and consequence of this mutation. When the mutant arylsulfatase-A is deglycosylated by endoglycosidase H, only one smaller molecular species was generated, instead of the two from the normal enzyme. This is consistent with the loss of one of the two N-linked oligosaccharide side chains known to be present on the wild-type enzyme. Quantitative analysis of mannose and leucine incorporation showed that the mutant enzyme incorporated two- to tenfold less mannose than the normal enzyme on a molar basis. This deficient glycosylation was specific to arylsulfatase-A. Another lysosomal enzyme not affected in this mutation, beta-hexosaminidase, was glycosylated normally in the mutant cells. The remaining single oligosaccharide side chain released from the mutant arylsulfatase-A by pronase digestion was normally processed to complex and high-mannose forms. However, the high-mannose side chains contained 30% fewer phosphorylated residues than those of the normal enzyme. Nevertheless, this reduced level of phosphorylation did not prevent targeting of the mutant enzyme to the lysosomes, a process normally mediated through phosphorylated mannose residues. In conclusion, pseudo arylsulfatase-A deficiency is a unique human mutation associated with reduced glycosylation and phosphorylation of a lysosomal enzyme with the loss of one of the two carbohydrate side chains. The mutation results in greatly reduced enzyme stability, thus indicating a role for oligosaccharides in maintaining enzyme stability within the degradative environment of the lysosomes. However, the residual catalytic activity or subcellular targeting of the mutant enzyme was not affected. These properties probably account for the benign clinical presentation of pseudo arylsulfatase-A deficiency.

Acetylglucosaminidase↗
PubMed1995

Purification and characterization of the arylsulfatase synthesized by Pseudomonas aeruginosa PAO during growth in sulfate-free medium and cloning of the arylsulfatase gene (atsA).

An arylsulfatase (EC 3.1.6.1) was extracted from Pseudomonas aeruginosa PAO1 and purified 2700-fold to homogeneity. Synthesis of this enzyme was repressed when sulfate, cysteine or thiocyanate was supplied as the sole sulfur source for growth, but derepressed with all other sulfur sources tested. The apparent molecular mass was determined by SDS/PAGE to be 57 kDa, and the enzyme was presumed to be a monomer after gel filtration chromatography. The arylsulfatase showed maximal activity at 57 degrees C and pH 8.9, and a Km of 105 microM for 4-nitrocatecholsulfate. Despite previous reports that both inducible and derepressible forms of arylsulfatase exist in P. aeruginosa, we found only one enzyme under a variety of growth conditions: a sulfate-repressed enzyme with a native isoelectric point of 4.76. The gene encoding this enzyme (atsA) was isolated by complementation of a Tn5-751 mutant of P. aeruginosa PAO1. Sequencing revealed a 1602-bp reading frame encoding a 534-amino-acid protein with sequence similarity to known bacterial and eukaryotic arylsulfatases (30-40% and 25-30% identity, respectively), but lacking the signal peptide which is present in all known sequences. The lack of this signal peptide suggests that the P. aeruginosa arylsulfatase is neither periplasmic nor membrane-associated, unlike other known arylsulfatases. The atsA gene was located at 15-17' on the P. aeruginosa genome by Southern hybridization. Only a single copy was observed under moderate stringency conditions.

Amino Acid Sequence↗
PubMed1978

Comparative immunological studies on arylsulfatase in bacteria of the family Enterobacteriaceae: occurrence of latent arylsulfatase protein regulated by sulfur compounds and tyramine.

The arylsulfatases of 21 strains of the family Enterobacteriaceae were compared by measuring their enzymatic activities and immunological reactivities. Enzyme formation under repressing, nonrepressing, and derepressing conditions was tested. Antiserum prepared against pure arylsulfatase from Klebsiella aerobgenes W70 was tested against the enzyme extracts from the strains using double diffusion, quantitative precipitation, and immunoelectrophoresis. No close relationship was found between arylsulfatase activity and immunological cross-reactionship was found between arylsulfatase activity and immunological cross-reactivity. The strains in the family Enterobacteriaceae could be divided into two groups on the basis of the immunological properties of their enzyme. Antisera formed a precipitin band with both active and inactive enzyme proteins from Escherichia, Citrobacter, Salmonella, Klebsiella, and Enterobacter, but not with the proteins from Serratia, Proteus, and Erwinia, even though some strains of these species had enzyme activity. It was also found that the formation of arylsulfatase proteins, irrespective of whether they had enzyme activity, were under regulation by sulfur compounds and tyramine.

Arylsulfatases↗
PubMed1989

Evidence for the presence of a very high concentration of arylsulfatase A in the pig thyroid: identification of arylsulfatase A subunits as the two major glycoproteins in purified thyroid lysosomes.

In addition to their general function in cellular homeostasis, thyroid lysosomes play an essential role in the biosynthesis of thyroid hormones by cleaving the macromolecular prohormone, thyroglobulin. In the present work, we have attempted to determine whether the enzyme composition of thyroid lysosomes differs from that of lysosomes from other tissues. Lysosomal enzymes, cathepsin D, beta-D-galactosidase, beta-D-glucosidase, alpha-D-mannosidase, alpha-L-fucosidase, hexosaminidase, and arylsulfatase A and B, were assayed in crude fractions from various pig tissues, heart, brain, liver, kidney, thyroid, adrenals, ovary, and spleen. It appeared that the specific activity of arylsulfatase A was at least 20 times higher in the thyroid than in most other tissues. Thyroid lysosomes purified by isopycnic centrifugation on Percoll gradients contained two major polypeptides with apparent molecular weights of 58,000 and 54,000 representing about 30% of the total protein. These polypeptides were glycosylated and were exclusively found in the intralysosomal soluble fraction obtained by osmotic pressure-dependent lysis. By fractionating intralysosomal soluble proteins by velocity sedimentation on sucrose gradients or gel permeation chromatography we identified a thyroid arylsulfatase A holoenzyme which corresponds to a 120,000 Mr species. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses of the gradient or column fractions showed that the 120-kDa protein peak with arylsulfatase A activity essentially contained the 58- and 54-kDa polypeptides in equivalent amounts. In conclusion, arylsulfatase A, a heterodimer of 120 kDa composed of two nonidentical subunits, is the major protein component of thyroid lysosomes. The superabundance of this protein in purified thyroid lysosomes is related to the very high specific activity of the enzyme in the thyroid as compared to other tissues.

Animals↗
PubMed1987

Pseudo arylsulfatase A deficiency. Biosynthesis of an abnormal arylsulfatase A.

Pseudo arylsulfatase A deficiency, an asymptomatic condition, and metachromatic leukodystrophy, a severe neurodegenerative disease, are both associated with profound reductions of arylsulfatase A activity in man. We now report that with metabolic labelling, cultured pseudo deficient cells synthesized about 20% of the normal amount of arylsulfatase A at a reduced rate of apparent synthesis and increased rate of degradation. However, in the presence of ammonium chloride which stimulated secretion of lysosomal enzymes, these cells synthesized about 80% of the normal amount of enzyme protein. Hence, the defect in pseudo arylsulfatase A deficiency is associated with labile arylsulfatase A molecules which can be stabilized if they are diverted from intracellular storage.

Ammonium Chloride↗
PubMed1979

Arylsulfatase of sea urchin sperm--distribution of arylsulfatase in the gonads and gametes of echinoderms.

1. Fairly high activities of arylsulfatase are found in the sperm and mature testes of all the sea urchins studied; Strongylocentrotus intermedius, Strongylocentrotus nudus, Hemicentrotus pulcherrimus and Anthocidaris crassispina, whereas the activities in the ovaries and eggs of these animals are low. 2. Neither the sand dollar, Clypeaster japonicus nor the starfishes, Asterias amurensis and Asterina pectinifera prove to have considerable activities of the enzyme in their gonads and gametes. 3. Most of the activity of arylsulfatase in the sperm of S. intermedius is found in the seminal plasma, but the significant activity is bound to the spermatozoa. 4. Part, if not all, of the spermatozoa-borne arylsulfatase is suggested to exist on the surface of spermatozoa or in the acrosome or both. 5. The ubiquitous distribution of sperm arylsulfatase in sea urchins on the contrary to its absence in starfish or sand dollar is discussed in connection with the penetration of sperm through egg investments.

Animals↗
PubMed1990

Phylogenetic conservation of arylsulfatases. cDNA cloning and expression of human arylsulfatase B.

A 2.2-kilobase cDNA clone for human arylsulfatase B (ASB) and several genomic clones were isolated and sequenced. The deduced amino acid sequence of 533 amino acids contains a 41-amino acid N-terminal signal peptide and a mature polypeptide of 492 amino acid residues. Overexpression of ASB in transfected baby hamster kidney (BHK) cells resulted in up to 68-fold higher ASB activity than in untransfected BHK cells. Pulse-chase labeling showed that ASB was synthesized and secreted as a 64-kDa precursor and processed to a 47-kDa mature form in BHK cells. The 47-kDa ASB form was located in dense lysosomes. Transport of ASB to the lysosomes was accomplished in a mannose 6-phosphate receptor-dependent manner. The ASB cDNA clone hybridizes to 4.8-, 2.5-, and 1.8-kilobase species of RNA from human fibroblasts. The same pattern was observed in RNA from fibroblasts of three Maroteaux-Lamy patients who were deficient in ASB activity, as well as in RNA from fibroblasts of three patients with multiple sulfatase deficiency, in which all known sulfatases were markedly diminished. Deduced amino acid sequences of human arylsulfatase A, human ASB, human steroid sulfatase, human glucosamine-6-sulfatase, and an arylsulfatase from sea urchin showed a substantial degree of similarity suggesting that they arose from a common ancestral gene and are members of an arylsulfatase gene family.

Amino Acid Sequence↗
PubMed1990

Human arylsulfatase B: MOPAC cloning, nucleotide sequence of a full-length cDNA, and regions of amino acid identity with arylsulfatases A and C.

cDNAs encoding the human lysosomal hydrolase, arylsulfatase B (ASB; N-acetylgalactosamine-4-sulfatase, EC 3.1.6.1), were isolated from a hepatoma cell cDNA library using an ASB-specific oligonucleotide generated by the MOPAC (mixed oligonucleotide primed amplification of cDNA) technique. To facilitate cDNA cloning, human ASB was purified to apparent homogeneity and a total of 112 amino acid residues were microsequenced from the N-terminus and four internal tryptic peptides of the 47-kDa subunit. Based on the ASB N-terminal amino acid sequence, two oligonucleotide mixtures containing inosines to reduce the mixture complexity were constructed and used as primers to amplify an ASB-specific product from human placental cDNA by the polymerase chain reaction. DNA sequencing of this MOPAC product demonstrated colinearity with 21 N-terminal ASB amino acids. Based on this sequence and on codon usage for the adjacent conserved amino acids in human arylsulfatases A and C, a unique 66-mer was synthesized and used to screen a human hepatoma cell cDNA library. Four putative positive cDNA clones were isolated, and the largest insert (pASB-1) was sequenced in both orientations. The 1834-bp pASB-1 insert had a 1278-bp open reading frame encoding 425 amino acids that was colinear with 85 microsequenced amino acids of the purified enzyme, demonstrating its authenticity. Using the pASB-1 cDNA as a probe, a full-length cDNA clone, pASB-4, was isolated from a human testes library and sequenced in both orientations. pASB-4 had a 2811-bp insert containing a 559-bp 5' untranslated sequence, a 1602-bp open reading frame encoding 533 amino acids (six potential N-glycosylation sites), a 641-bp 3' untranslated sequence, and a 9-bp poly(A) tract. Comparison of the predicted amino acid sequences of arylsulfatases A, B, and C revealed regions of identity, particularly in their N-termini.

Amino Acid Sequence↗
PubMed1986

Attenuated activities and structural alterations of arylsulfatase A in tissues from subjects with pseudo arylsulfatase A deficiency.

It had been shown previously that arylsulfatase A activity was attenuated in pseudo arylsulfatase A deficiency fibroblasts and that subunits of the enzyme were smaller than subunits of the enzyme in normal fibroblasts. Attenuated enzyme activity has now been affirmed in other tissues. Subunits of the enzyme from these sources were also found to be smaller with apparent molecular size 59 and 56 kdaltons. Subunits of enzyme in corresponding control tissues were larger and there was heterogeneity in apparent molecular size as follows: fibroblasts, 63 and 59 kdaltons; liver, 63 and 59 kdaltons; kidney, 63 and 58 kdaltons; spleen, 63 and 58 kdaltons; placenta, 62 and 58 kdaltons; and urine, 61 and 57 kdaltons. Attenuated enzyme activity and structurally altered enzyme in pseudo arylsulfatase A deficiency appears to be systemic. However, the reason for reduced amounts of structurally altered enzyme with normal catalytic activity is unresolved.

Cerebroside-Sulfatase↗
PubMed1979

Lysosomal arylsulfatase deficiencies in humans: chromosome assignments for arylsulfatase A and B.

Genetics of human lysosomal arylsulfatases A and B (aryl-sulfate sulfohydrolase, EC 3.1.6.1), associated with childhood disease, has been studied with human-rodent somatic cell hybrids. Deficiency of arylsulfatase A (ARS(A)) in humans results in a progressive neurodegenerative disease, metachromatic leukodystrophy. Deficiency of arylsulfatase B (ARS(B)) is associated with skeletal and growth malformations, termed the Maroteaux-Lamy syndrome. Simultaneous deficiency of both enzymes is associated with the multiple sulfatase deficiency disease, suggesting a common relationship for ARS(A) and ARS(B). The genetic and structural relationships of human ARS(A) and ARS(B) have been determined by the use of human-Chinese hamster somatic cell hybrids. Independent enzyme segregation in cell hybrids demonstrated different chromosome assignments for the structural genes, ARS(A) and ARS(B), coding for the two lysosomal enzymes. ARS(A) activity showed concordant segregation with mitochondrial aconitase encoded by a gene assigned to chromosome 22. ARS(B) segregated with beta-hexosaminidase B encoded by a gene assigned to chromosome 5. These assignments were confirmed by chromosome analyses. The subunit structures of ARS(A) and ARS(B) were determined by their electrophoretic patterns in cell hybrids; a dimeric structure was demonstrated for ARS(A) and a monomeric structure for ARS(B). Although the multiple sulfatase deficiency disorder suggests a shared relationship between ARS(A) and ARS(B), independent segregation of these enzymes in cell hybrids did not support a common polypeptide subunit or structural gene assignment. The evidence demonstrates the assignment of ARS(A) to chromosome 22 and ARS(B) to chromosome 5. A third gene that affects ARS(A) and ARS(B) activity is suggested by the multiple sulfatase deficiency disorder.

Cell Line↗
PubMed2005

Galsulfase: arylsulfatase B, BM 102, recombinant human arylsulfatase B, recombinant human N-acetylgalactosamine-4-sulfatase, rhASB.

Galsulfase [Aryplase, arylsulfatase B, BM 102, Naglazyme, rhASB, recombinant human N-acetylgalactosamine-4-sulfatase, recombinant human arylsulfatase B] is under development with BioMarin Pharmaceutical as an enzyme replacement therapy for the treatment of mucopolysaccharidosis (MPS) VI. MPS VI (also known as Maroteaux-Lamy syndrome) is a progressive, debilitating genetic disease resulting in early death. Patients with MPS VI have a deficiency in the arylsulfatase B (ASB) enzyme that is essential for the progressive breakdown of certain complex carbohydrates. The deficiency in ASB results in the build-up of carbohydrate residues in the lysosomes in all cells of the body. Patients are usually diagnosed at 6-24 months of age, and the symptoms include deceleration of growth, enlarged liver and spleen, skeletal and joint deformities, and upper airway obstruction. Patients do not survive past 20-30 years of age in the more severe cases, but may live longer with the milder cases, but with significant medical problems. While the symptoms of MPS VI are similar to those of MPS I, mental retardation associated with the severe forms of MPS I had not been reported for patients with MPS VI. For some patients, bone marrow transplantation is a treatment, albeit risky, option. MPS VI afflicts approximately 1100 patients in the world. In November 2004, BioMarin announced that it has filed a Biologics License Application (BLA) with the the US FDA for galsulfase for the treatment of MPS VI. The company has requested a priority review as part of the BLA submission, which, if granted, is expected to be completed within 6 months of submission. The FDA accepted the filing of the BLA for galsulfase for MPS VI in February 2005, and granted it a 6-month priority review period. The FDA's decision is due on 31 May 2005. The FDA has granted galsulfase orphan drug status and fast-track designation. Orphan drug status will provide BioMarin Pharmaceutical with 7 years of marketing exclusivity for galsulfase in the US providing that galsulfase is the first agent to gain approval in the US for MPS VI. BioMarin received an orphan drug designation from the EC for galsulfase for the treatment of MPS VI. Following positive safety and efficacy results from the phase I study with galsulfase, BioMarin Pharmaceutical commenced and successfully completed a phase II trial with rhASB in ten patients with MPS VI. This 24-week, open-label, multicentre trial was conducted at two sites, in the US and Australia (at the Lysosomal Diseases Research Unit, Women's and Children's Hospital, Adelaide, Australia, by Dr John Hopwood), and evaluated the safety, efficacy and pharmacokinetics of weekly intravenous infusions of galsulfase at a dose of 1.0 mg/kg. BioMarin Pharmaceutical completed a phase I/II clinical trial of galsulfase in six patients with MPS VI in the Children's Hospital, Oakland, CA, USA, with Dr Paul Harmatz as a principal investigator. This randomised, double-blind study evaluated the safety and efficacy of two doses of galsulfase administered by weekly intravenous infusions for 24 weeks. Five patients from the phase I study had completed the 24-week, open-label extension study. Data from this study confirmed safety and good tolerability of both doses of galsulfase with the 1.0 mg/kg dose producing greater sustained effects. The patients will continue receiving therapy in the future. Seven preclinical trials with galsulfase were conducted in a naturally occurring feline model of MPS VI disease at the Lysosomal Diseases Research Unit, Women's and Children's Hospital, Adelaide, Australia, by Dr John Hopwood. The company manufactures galsulfase at a GMP facility licensed from the State of California.

Animals↗
PubMed2000

Measurements from normal umbilical cord blood of four lysosomal enzymatic activities: alpha-L-iduronidase (Hurler), galactocerebrosidase (globoid cell leukodystrophy), arylsulfatase A (metachromatic leukodystrophy), arylsulfatase B (Maroteaux-Lamy).

Umbilical cord blood (UCB) has received increasing attention as a source of unrelated hematopoietic stem cells for transplantation. Lysosomal diseases have been effectively treated and normal enzymatic activity has occurred subsequent to engraftment using UCB. The use of donor cells with normal amounts of enzyme, rather than those from carriers whose level may be 50% or less, is an obvious goal. The frequency of such heterozygotes varies from 1:10 to 1:140 or lower depending upon the disease at issue. We assayed the levels of lysosomal enzymes in normal UCB in random samples as well as those used for transplantation. We measured the following enzymatic activities: alpha-l-iduronidase (Hurler), galactocerebrosidase (globoid cell leuko- dystrophy) and arylsulfatase A (metachromatic leukodystrophy). For the latter, levels of activity in UCB are comparable to those found in adult blood. In the case of arylsulfatase B (Maroteaux-Lamy) a level lower than adult level was found. An informed choice by the transplanting physician based on the activity of the relevant enzyme in the UCB donor will provide a better opportunity for an improved prognosis for more complete correction of the recipient's primary disease. Bone Marrow Transplantation (2000) 25, 541-544.

Adult↗
PubMed2000

Retrovirally expressed human arylsulfatase A corrects the metabolic defect of arylsulfatase A-deficient mouse cells.

A deficiency of arylsulfatase A (ASA) causes the lysosomal storage disease metachromatic leukodystrophy (MLD) which is characterized primarily by demyelination of the central nervous system. ASA-deficient mice develop a disease which resembles MLD in many respects and thus serve as an appropriate animal model for this disease. To establish gene therapy protocols for ASA-deficient mice, we constructed two retroviral vectors based on the murine stem cell virus. Both vectors harbor the human ASA cDNA controlled by the retroviral promoter/enhancer element, but differ by the presence or absence of a neomycin resistance gene driven by an internal promoter. A comparative analysis of the one- versus the two-gene vector and an amphotropic versus an ecotropic producer cell line revealed that the amphotropic producer cell line for the one-gene vector transfers ASA overexpression to the target cells most efficiently. The human ASA encoded by this vector is correctly expressed in heterologous mouse cells and corrects the metabolic defect of transduced ASA-deficient murine cells. The constructed one-gene vector might thus be a potentially useful tool for the development of a gene-based therapy for ASA-deficient mice. Gene Therapy (2000) 7, 805-812.

Animals↗
PubMed2001

Bone marrow stem cell gene therapy of arylsulfatase A-deficient mice, using an arylsulfatase A mutant that is hypersecreted from retrovirally transduced donor-type cells.

Arylsulfatase A (ASA)-deficient mice represent an animal model for the fatal lysosomal storage disease metachromatic leukodystrophy, which is characterized by widespread intralysosomal deposition of sulfatide. Bone marrow stem cell gene therapy in mice, using a retroviral vector mediating expression of wild-type human ASA, has the potential to ameliorate the visceral pathology, but improves the prevailing brain disease and neurologic symptoms only marginally. One factor that influences the efficacy of bone marrow transplantation therapy in lysosomal storage diseases is the secretion level of the therapeutic enzyme from donor-type cells. Here we test the potential of a hypersecreted glycosylation variant of ASA. Although this mutant lacks mannose 6-phosphate residues it is taken up by cells by a mannose 6-phosphate receptor-independent pathway and causes partial metabolic correction of ASA-deficient mouse cells. Retrovirally mediated transfer of the mutant cDNA into ASA-deficient mice results in the sustained expression of the transgene. Serum levels argue for an increased secretion of the glycosylation mutant also in vivo. Tissue levels were reduced to 2% in liver and up to 40% in kidney compared with animals treated with the wild-type enzyme, indicating reduced endocytosis. Thus, the limited uptake of the variant enzyme outweighs the putative advantageous effect of improved supply. Although the mutant enzyme is able to correct the metabolic defect partially, histological examinations did not reveal any reduction of sulfatide storage in treated animals. Surprisingly, analysis of neurologic symptoms indicated a significant improvement of the gait pattern.

3T3 Cells↗