Restoration of arylsulphatase B activity in human mucopolysaccharidosis-type-VI fibroblasts by retroviral-vector-mediated gene transfer.
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ABSTRACT:
SUBMITTER: Peters C
PROVIDER: S-EPMC1151119 | biostudies-other | 1991 Jun
REPOSITORIES: biostudies-other
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Project description:Metachromatic leukodystrophy is a lysosomal storage disease caused by the deficiency of arylsulphatase A (ASA). A human ASA cDNA was subcloned into the retroviral vector pXT1. Replication-defective retrovirus was generated by transfection of the vector into the amphotropic packaging cell line PA317. Human fibroblasts from a patient suffering from metachromatic leucodystrophy was infected with the recombinant retrovirus. Infected fibroblasts expressed ten times more ASA compared with control fibroblasts from a normal individual. The ASA encoded by the integrated provirus was shown to be correctly transported into the lysosomes and to normalize the impaired degradation of cerebroside sulphate.
| S-EPMC1130570 | biostudies-other
Project description:Mucopolysaccharidosis type IIIB (MPS IIIB; or Sanfilippo syndrome type B) is a lysosomal disease, due to glycosaminoglycan storage caused by mutations on the alpha-N-acetylglucosaminidase (NAGLU) gene. The disease is characterized by neurological dysfunction but relatively mild somatic manifestations. No effective treatment is available for affected patients. In the present study, we evaluated the role of a lentiviral vector as the transducing agent of NAGLU cDNA in MPS IIIB fibroblasts. The vector expressed high transduction efficiency and high levels of enzymic activity, 20-fold above normal levels, persisting for at least 2 months. PCR experiments confirmed the integration of the viral vector into the target genome. The NAGLU activity restored by virus infection was sufficient to normalize glycosaminoglycan accumulation, which is directly responsible for the disease phenotype. Metabolic labelling experiments on transduced fibroblasts exhibited, in the medium and in cellular lysates, polypeptide forms of 84 and 80 kDa respectively related to the precursor and mature forms of the enzyme. The enzyme secreted by transduced MPS IIIB fibroblasts was endocytosed in deficient cells by the mannose 6-phosphate system. Thus we show that lentiviral vectors may provide a therapeutic approach for the treatment of MPS IIIB disease.
| S-EPMC1222624 | biostudies-other
Project description:Mucopolysaccharidosis (MPS) VI is due to a deficiency in the activity of N-acetylgalactosamine 4-sulfatase (4S), also known as arylsulfatase B. Previously, retroviral vector (RV)-mediated neonatal gene therapy reduced the clinical manifestations of MPS I and MPS VII in mice and dogs. However, sulfatases require post-translational modification by sulfatase-modifying factors. MPS VI cats were injected intravenously (i.v.) with a gamma RV-expressing feline 4S, resulting in 5 ± 3 copies of RV per 100 cells in liver. Liver and serum 4S activity were 1,450 ± 1,720 U/mg (26-fold normal) and 107 ± 60 U/ml (13-fold normal), respectively, and were directly proportional to the liver 4S protein levels for individual cats. This study suggests that sulfatase-modifying factor (SUMF) activity in liver was sufficient to result in active enzyme despite overexpression of 4S. RV-treated MPS VI cats achieved higher body weights and longer appendicular skeleton lengths, had reduced articular cartilage erosion, and reduced aortic valve thickening and aortic dilatation compared with untreated MPS VI cats, although cervical vertebral bone lengths were not improved. This demonstrates that therapeutic expression of a functional sulfatase protein can be achieved with neonatal gene therapy using a gamma RV, but some aspects of bone disease remain difficult to treat.
| S-EPMC3345988 | biostudies-literature
Project description:Mucopolysaccharidosis type VI (MPS VI) is a rare, autosomal recessive lysosomal storage disorder caused by deficient enzymatic activity of N-acetyl galactosamine-4-sulphatase resulting from mutations in the arylsulphatase B (ARSB) gene. The ARSB gene is located on chromosome 5q11-q13 and is composed of eight exons. More than hundred ARSB mutations have been reported so far, but the mutation spectrum of MPS VI in India is still unknown. Hence, the aim of the present study was to identify the mutational spectrum in patients with MPS VI in India and to study the genotype-phenotype association and functional outcomes of these mutations.Molecular characterization of the ARSB gene by Sanger sequencing was done for 15 patients (aged 15 months to 11 yr) who were enzymatically confirmed to have MPS VI. Age of onset, clinical progression and enzyme activity levels in each patient were studied to look for genotype-phenotype association. Haplotype analysis performed for unrelated patients with the recurring mutation W450C, was suggestive of a founder effect. Sequence and structural analyses of the ARSB protein using standard software were carried out to determine the impact of detected mutations on the function of the ARSB protein.A total of 12 mutations were identified, of which nine were novel mutations namely, p.D53N, p.L98R, p.Y103SfsX9, p.W353X, p.H393R, p.F166fsX18, p.I220fsX5, p.W450L, and p.W450C, and three were known mutations (p.D54N, p.A237D and p.S320R). The nine novel sequence variants were confirmed not to be polymorphic variants by performing sequencing in 50 unaffected individuals from the same ethnic population.Nine novel mutations were identified in MPS VI cases from India in the present study. The study also provides some insights into the genotype-phenotype association in MPS VI.
| S-EPMC4683826 | biostudies-literature
Project description:AimTo investigate and characterise an inborn error of metabolism in a dog with skeletal and ocular abnormalities.MethodsA 2.5-year-old small male Miniature Poodle-like dog was presented with gross joint laxity and bilateral corneal opacities. Clinical examination was augmented by routine haematology, serum chemistry, radiographs, pathology, enzymology and molecular genetic studies. Euthanasia was requested when the dog was 3 years of age because of progressively decreasing quality of life.ResultsRadiology revealed generalised epiphyseal dysplasia, malformed vertebral bodies, luxation/subluxation of appendicular and lumbosacral joints with hypoplasia of the odontoid process and hyoid apparatus. These clinical and radiographic findings, together with a positive urinary Berry spot test for mucopolysaccharides, and metachromatic granules in leucocytes, were indicative of a mucopolysaccharidosis (MPS), a lysosomal storage disease. Histological lesions included vacuolation of stromal cells of the cornea, fibroblasts, chondrocytes, macrophages and renal cells. The brain was essentially normal except for moderate secondary Wallerian-type degeneration in motor and sensory tracts of the hind brain. Dermatan sulphate-uria was present and enzymology revealed negligible activity of N-acetylgalactosamine-4-sulphatase, also known as arylsulphatase B, in cultured fibroblasts and liver tissue. A novel homozygous 22 base pair (bp) deletion in exon 1 of this enzyme's gene was identified (c.103_124del), which caused aframe-shift and subsequent premature stop codon. The "Wisdom pure breed-mixed breed" test reported the dog as a cross between a Miniature and Toy Poodle.ConclusionsThe clinicopathological features are similar to those of MPS type VI as previously described in dogs, cats and other species, and this clinical diagnosis was confirmed by enzymology and molecular genetic studies. This is an autosomal recessively inherited lysosomal storage disease.Clinical relevanceThe prevalence of MPS VI in Miniature or Toy Poodles in New Zealand and elsewhere is currently unknown. Due to the congenital nature of the disorder, malformed pups may be subject to euthanasia without investigation and the potential genetic problem in the breed may not be fully recognised. The establishment of a molecular genetic test now permits screening for this mutation as a basis to an informed breeding policy.
| S-EPMC3401909 | biostudies-literature
Project description:Mucopolysaccharidosis VI (MPS VI) is caused by deficient arylsulfatase B (ARSB) activity resulting in lysosomal storage of glycosaminoglycans (GAGs). MPS VI is characterized by dysostosis multiplex, organomegaly, corneal clouding, and heart valve thickening. Gene transfer to a factory organ like liver may provide a lifetime source of secreted ARSB. We show that intravascular administration of adeno-associated viral vectors (AAV) 2/8-TBG-felineARSB in MPS VI cats resulted in ARSB expression up to 1 year, the last time point of the study. In newborn cats, normal circulating ARSB activity was achieved following delivery of high vector doses (6 × 10(13) genome copies (gc)/kg) whereas delivery of AAV2/8 vector doses as low as 2 × 10(12) gc/kg resulted in higher than normal serum ARSB levels in juvenile MPS VI cats. In MPS VI cats showing high serum ARSB levels, independent of the age at treatment, we observed: (i) clearance of GAG storage, (ii) improvement of long bone length, (iii) reduction of heart valve thickness, and (iv) improvement in spontaneous mobility. Thus, AAV2/ 8-mediated liver gene transfer represents a promising therapeutic strategy for MPS VI patients.
| S-EPMC3048181 | biostudies-literature
Project description:A full-length human N-acetylgalactosamine-4-sulphatase (4-sulphatase) cDNA clone was constructed and expressed in CHO-DK1 cells under the transcriptional control of the Rous sarcoma virus long terminal repeat. A clonal cell line expressing high activities of human 4-sulphatase was isolated. The maturation and processing of the human enzyme in this transfected CHO cell line showed it to be identical with that seen in normal human skin fibroblasts. The high-uptake precursor form of the recombinant enzyme was purified from the medium of the transfected cells treated with NH4Cl and was shown to be efficiently endocytosed by control fibroblasts and by fibroblasts from a mucopolysaccharidosis type-VI (MPS VI) patient. Enzyme uptake was inhibitable by mannose 6-phosphate. After uptake, the enzyme was processed normally in both normal and MPS VI fibroblasts and was shown both to correct the enzymic defect and to initiate degradation of [35S]sulphated dermatan sulphate in MPS VI fibroblasts. The stabilities of the recombinant enzyme and enzyme from human fibroblasts appeared to be similar after uptake. However, endocytosed enzyme has a significantly shorter half-life than endogenous human enzyme. The purified precursor 4-sulphatase had a similar pH optimum and catalytic parameters to the mature form of 4-sulphatase isolated from human liver.
| S-EPMC1132608 | biostudies-other
Project description:Mucopolysaccharidosis I (MPS I) is a lysosomal storage disease due to ?-L-iduronidase (IDUA) deficiency that results in the accumulation of glycosaminoglycans (GAG). Systemic gene therapy to MPS I mice can reduce lysosomal storage in the brain, but few data are available regarding the effect upon behavioral function. We investigated the effect of gene therapy with a long-terminal-repeat (LTR)-intact retroviral vector or a self-inactivating (SIN) vector on behavioral function in MPS I mice. The LTR vector was injected intravenously to 6-week-old MPS I mice, and the SIN vector was given to neonatal or 6-week-old mice. Adult-LTR, neonatal-SIN, and adult-SIN-treated mice achieved serum IDUA activity of 235 ± 20 (84-fold normal), 127 ± 10, and 71 ± 7 U/ml, respectively. All groups had reduction in histochemical evidence of lysosomal storage in the brain, with the adult-LTR group showing the best response, while adult-LTR mice had reductions in lysosomal storage in the cristae of the vestibular system. Behavioral evaluation was performed at 8 months. Untreated MPS I mice had a markedly reduced ability to hold onto an inverted screen or climb down a pole. LTR-vector-treated mice had marked improvements on both of these tests, whereas neonatal-SIN mice showed improvement in the pole test. We conclude that both vectors can reduce brain disease in MPS I mice, with the LTR vector achieving higher serum IDUA levels and better correction. Vestibular abnormalities may contribute to mobility problems in patients with MPS I, and gene therapy may reduce symptoms.
| S-EPMC3548941 | biostudies-literature
Project description:High-titre stocks of an amphotropic retrovirus, constructed so as to express a full-length cDNA encoding the human lysosomal enzyme N-acetylgalactosamine-4-sulphatase (4-sulphatase) from the cytomegalovirus immediate early promoter, were used to infect skin fibroblasts from a clinically severe mucopolysaccharidosis type VI (MPS VI) patient. The infected MPS VI cells showed correction of the enzymic defect with the enzyme being expressed at high levels and in the correct subcellular compartment. Surprisingly this did not result in correction of glycosaminoglycan turnover as measured by accumulation of 35S in metabolically labelled cells. We demonstrate that this is apparently caused by an induced reduction of the activities of other lysosomal sulphatases, presumably due to competition for a sulphatase-specific processing mechanism by the over-expressed 4-sulphatase. The level of steroid sulphatase, which is a microsomal sulphatase, was also reduced. Infection of skin fibroblasts from a second, clinically mildly affected, MPS VI patient with the same virus also resulted in no significant change in the level of glycosaminoglycan storage. However, in this case the cause of the observed phenomenon was less clear. These results are of obvious practical importance when considering gene therapy for a sulphatase deficiency such as MPS VI and also provide possible new avenues for exploration of the processes involved in sulphatase synthesis and genetically determined multiple sulphatase deficiency.
| S-EPMC1134512 | biostudies-other
Project description:Mucopolysaccharidosis VI (MPS VI) is a lysosomal storage disease with progressive multisystem involvement, associated with a deficiency of arylsulfatase B leading to the accumulation of dermatan sulfate. Birth prevalence is between 1 in 43,261 and 1 in 1,505,160 live births. The disorder shows a wide spectrum of symptoms from slowly to rapidly progressing forms. The characteristic skeletal dysplasia includes short stature, dysostosis multiplex and degenerative joint disease. Rapidly progressing forms may have onset from birth, elevated urinary glycosaminoglycans (generally >100 microg/mg creatinine), severe dysostosis multiplex, short stature, and death before the 2nd or 3rd decades. A more slowly progressing form has been described as having later onset, mildly elevated glycosaminoglycans (generally <100 microg/mg creatinine), mild dysostosis multiplex, with death in the 4th or 5th decades. Other clinical findings may include cardiac valve disease, reduced pulmonary function, hepatosplenomegaly, sinusitis, otitis media, hearing loss, sleep apnea, corneal clouding, carpal tunnel disease, and inguinal or umbilical hernia. Although intellectual deficit is generally absent in MPS VI, central nervous system findings may include cervical cord compression caused by cervical spinal instability, meningeal thickening and/or bony stenosis, communicating hydrocephalus, optic nerve atrophy and blindness. The disorder is transmitted in an autosomal recessive manner and is caused by mutations in the ARSB gene, located in chromosome 5 (5q13-5q14). Over 130 ARSB mutations have been reported, causing absent or reduced arylsulfatase B (N-acetylgalactosamine 4-sulfatase) activity and interrupted dermatan sulfate and chondroitin sulfate degradation. Diagnosis generally requires evidence of clinical phenotype, arylsulfatase B enzyme activity <10% of the lower limit of normal in cultured fibroblasts or isolated leukocytes, and demonstration of a normal activity of a different sulfatase enzyme (to exclude multiple sulfatase deficiency). The finding of elevated urinary dermatan sulfate with the absence of heparan sulfate is supportive. In addition to multiple sulfatase deficiency, the differential diagnosis should also include other forms of MPS (MPS I, II IVA, VII), sialidosis and mucolipidosis. Before enzyme replacement therapy (ERT) with galsulfase (Naglazyme), clinical management was limited to supportive care and hematopoietic stem cell transplantation. Galsulfase is now widely available and is a specific therapy providing improved endurance with an acceptable safety profile. Prognosis is variable depending on the age of onset, rate of disease progression, age at initiation of ERT and on the quality of the medical care provided.
| S-EPMC2873242 | biostudies-literature