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D S Anson

Publications and source records attributed to D S Anson.

At least 37 records · Page 2Linked to original sources
PubMed1997

In vitro correction of iduronate-2-sulfatase deficiency by adenovirus-mediated gene transfer.

Hunter syndrome is a lethal lysosomal storage disorder caused by the deficiency of iduronate-2-sulfatase and characterized by severe skeletal and neurological symptoms. Only symptomatic treatments are available and, although bone marrow transplantation has been suggested, no encouraging results have been obtained so far. Therefore, gene therapy might be a route to be pursued for treatment of the disease. In this respect, one major goal to achieve is the generation of an overexpressing vector able to correct, in particular, central nervous system (CNS) cells. Adenoviruses have been shown to infect CNS cells efficiently with minor or even absent immunological response. We describe the generation of a replication-defective adenoviral vector, AdRSVIDS, which is able to express in vitro high levels of iduronate-2-sulfatase. After infection, accumulation of mucopolysaccharides in treated Hunter cells was normalized. Furthermore, endocytosis of the transduced IDS did occur via the mannose-6-phosphate (M6P) receptor. Since no animal model for the disease is available, we developed a system based on the generation of derma-equivalents which enabled us to verify the expression of high levels of sulfatase up to 30 days after infection.

Adenoviridae↗
PubMed1997

Feline mucopolysaccharidosis type VI: correction of glycosaminoglycan storage in myoblasts by retrovirus-mediated transfer of the feline N-acetylgalactosamine 4-sulfatase gene.

Mucopolysaccharidosis type VI (MPS VI) is an autosomal recessive lysosomal storage disorder characterised by the deficiency of N-acetylgalactosamine 4-sulfatase (4S). MPS VI has also been described in the cat. As an initial step toward muscle-mediated gene therapy in the MPS VI cat, we have made two retroviral constructs (pLf4S and pLf4SSN) that transduce the feline 4S gene. Both constructs were designed to express the feline 4S sequence from the viral long terminal repeat promoter. In addition pLf4SSN expressed the neomycin resistance gene from the SV40 early promoter. Amphotrophic virus was produced for each construct and used to transduce feline MPS VI myoblasts. Lf4S- and Lf4SSN-transduced MPS VI feline myoblasts demonstrated correction of glycosaminoglycan storage and contained 55-fold and 3.5-fold elevated levels of 4S activity when compared with normal feline myoblasts respectively. Recombinant feline 4S (rf4S) secreted by Lf4S-transduced MPS VI myoblasts was shown to be endocytosed by MPS VI feline cells via the mannose-6-phosphate receptor system, leading to metabolic correction. The results from this study demonstrate that muscle-mediated gene replacement therapy may be a viable method for achieving circulating levels of recombinant f4S (rf4S) in the MPS VI cat.

Animals↗
PubMed1996

Feline mucopolysaccharidosis type VI. Characterization of recombinant N-acetylgalactosamine 4-sulfatase and identification of a mutation causing the disease.

Mucopolysaccharidosis type VI (MPS VI) is an autosomal recessive disease caused by a deficiency of N-acetylgalactosamine 4-sulfatase (4S) leading to the lysosomal accumulation and urinary excretion of dermatan sulfate. MPS VI has also been described in the Siamese cat. As an initial step toward enzyme replacement therapy with recombinant feline 4S (rf4S) in MPS VI cats, the feline 4S cDNA was isolated and expressed in CHO-KI cells and rf4S was immunopurified from the culture medium. SDS-polyacrylamide gel electrophoresis analysis showed that the precursor form of immunopurified rf4S was a 66-kDa polypeptide that underwent maturation to a 43-44-kDa polypeptide. Endocytosis of rf4S by cultured feline MPS VI myoblasts was predominantly mediated by a mannose 6-phosphate receptor and resulted in the correction of dermatan sulfate storage. The mutation causing feline MPS VI was identified as a base substitution at codon 476, altering a leucine codon to a proline (L476P). The L476P allele displayed no detectable 4S activity when expressed in CHO-KI cells and was observed only as a "precursor" polypeptide that was not secreted into the medium. Identification of the mutation has allowed the development of a rapid PCR-based screening method to genotype individuals within the cat colony.

Animals↗
PubMed1996

Correction of Sanfilippo A skin fibroblasts by retroviral vector-mediated gene transfer.

The recent cloning of the sulfamidase gene has made possible the consideration of gene-based therapies for Sanfilippo A syndrome (mucopolysaccharidosis type IIIA), one of the most common of the mucopolysaccharidoses. In this paper, we present the construction of a retroviral vector in which a sulfamidase cDNA is under the transcriptional control of the Moloney murine leukemia virus long terminal repeat. This construct was used to make a high-titer (4 x 10(5) colony-forming units/ml) producer cell line, PA317/LNSSN#19, in the amphotropic packaging cell line PA317. This producer cell line was shown to be helper virus free using an assay for horizontal spread of virus. Virus supernatant from PA317/LNSSN#19 was used to transduce Sanfilippo A fibroblasts, resulting in complete correction of both the enzymatic defect and the storage phenotype as assessed by intracellular accumulation of 35SO4(-)-labeled material. Phenotypic correction was seen even when the levels of viral transduction were low. These results show that gene therapy of the Sanfilippo A syndrome is practicable, although the nature of the disorder suggests that careful consideration needs to be given to the choice of the cellular target for gene transfer.

3T3 Cells↗
PubMed1996

Structure and sequence of the human sulphamidase gene.

Sanfilippo A syndrome (MPS-IIIA) is a mucopolysaccharide lysosomal storage disorder caused by a deficiency in the lysosomal enzyme, sulphamidase (EC 3.10.1.1), which is required for the degradation of heparan sulphate. A genomic clone containing the entire sulphamidase gene was isolated from a chromosome 17-specific gridded cosmid library. The structure of the gene and the sequence of the exon/intron boundaries and the 5' promoter region were determined. The sulphamidase gene is split into 8 exons spanning approximately 11 kb.

Base Sequence↗
PubMed1996

Enzyme replacement therapy in a feline model of Maroteaux-Lamy syndrome.

We report studies that suggest enzyme replacement therapy will result in a significant reduction in disease progression and tissue pathology in patients with Maroteaux-Lamy syndrome (Mucopolysaccharidosis type VI, MPS VI). A feline model for MPS VI was used to evaluate tissue distribution and clinical efficacy of three forms of recombinant human N-acetylgalactosamine-4-sulfatase (rh4S, EC 3.1.6.1). Intravenously administered rh4S was rapidly cleared from circulation. The majority of rh4S was distributed to liver, but was also detected in most other tissues. Tissue half-life was approximately 2-4 d. Three MPS VI cats given regular intravenous infusions of rh4S for up to 20 mo showed variable reduction of storage vacuoles in Kupffer cells and connective tissues, however cartilage chondrocytes remained vacuolated. Vertebral bone mineral volume was improved in two MPS VI cats in which therapy was initiated before skeletal maturity, and increased bone volume appeared to correlate with earlier age of onset of therapy. One cat showed greater mobility in response to therapy.

Animals↗
PubMed1996

Long-term in vitro correction of alpha-L-iduronidase deficiency (Hurler syndrome) in human bone marrow.

Allogeneic bone marrow transplantation is the most effective treatment for Hurler syndrome but, since this therapy is not available to all patients, we have considered an alternative approach based on transfer and expression of the normal gene in autologous bone marrow. A retroviral vector carrying the full-length cDNA for alpha-L-iduronidase has been constructed and used to transduce bone marrow from patients with this disorder. Various gene-transfer protocols have been assessed including the effect of intensive schedules of exposure of bone marrow to viral supernatant and the influence of growth factors. With these protocols, we have demonstrated successful gene transfer into primitive CD34+ cells and subsequent enzyme expression in their maturing progeny. Also, by using long-term bone marrow cultures, we have demonstrated high levels of enzyme expression sustained for several months. The efficiency of gene transfer has been assessed by PCR analysis of hemopoietic colonies as 25-56%. No advantage has been demonstrated for the addition of growth factors or intensive viral exposure schedules. The enzyme is secreted into the medium and functional localization has been demonstrated by reversal of the phenotypic effects of lysosomal storage in macrophages. This work suggests that retroviral gene transfer into human bone marrow may offer the prospect for gene therapy of Hurler syndrome in young patients without a matched sibling donor.

Antigens, CD34↗
PubMed1996

Isolation of the canine alpha-L-fucosidase cDNA and definition of the fucosidosis mutation in English Springer Spaniels.

Fucosidosis is a lysosomal storage disorder caused by deficiency of alpha-L-fucosidase. A biochemically and clinically well characterized canine model of fucosidosis exists in a colony of English Springer Spaniels. To facilitate its use as a model for gene therapy and enzyme replacement therapy in lysosomal storage disorders displaying neurological symptoms, isolation of the canine alpha-L-fucosidase cDNA was undertaken. Both the nucleotide sequence and the predicted amino acid sequence of canine fucosidase show high levels of identity with the human and rat sequences. Fucosidosis dogs were found to have a greatly reduced level of alpha-L-fucosidase mRNA when compared with normal dogs by Northern blot analysis. Direct PCR sequencing of products generated from cDNA demonstrated a 14-bp deletion in mRNA from affected dogs. This deletion creates a frameshift mutation and introduces a premature translation termination codon at amino acid position 152 and was shown to correspond to a deletion of the last 14 base pairs of exon 1 of the canine alpha-L-fucosidase gene. Rapid PCR-based screening for the mutation has now been performed on genomic DNA from dogs within the colony, enabling detection of both carriers and homozygotes.

Amino Acid Sequence↗
PubMed1995

Isolation and characterisation of a recombinant, precursor form of lysosomal acid alpha-glucosidase.

Glycogenosis type II (GSD II, Pompe disease) is an autosomal recessive lysosomal storage disease that results from a deficiency of acid alpha-glucosidase (GAA). Patients with this disorder are unable to break down lysosomal glycogen, which consequently accumulates in the lysosome. To evaluate enzyme replacement therapy for GSD II patients, we have expressed human GAA cDNA in Chinese hamster ovary-K1 cells utilising a vector that places the cDNA under the transcriptional control of the human polypeptide chain elongation factor 1 alpha gene promoter. A clonal cell line that secreted precursor recombinant GAA at approximately 18 mg.l-1.day-1 was identified. The precursor recombinant GAA was purified to homogeneity, had a molecular mass of 110 kDa as measured by SDS/PAGE, and was shown to have pH optima and kinetic parameters similar to those of GAA purified from human tissues. The partial N-terminal amino acid sequence of recombinant GAA conformed to that derived from the nucleotide sequence of the cloned cDNA. The recombinant enzyme was taken up by cultured fibroblasts and skeletal muscle cells from GSD II patients, and was shown to correct the storage phenotype. Endocytosed GAA was localised to the lysosome and showed evidence of intracellular processing to a more mature form. Activity levels increased up to twice the normal value and uptake was prevented if cells were cultured in the presence of mannose 6-phosphate.

Amino Acid Sequence↗
PubMed1995

Expression, purification and characterization of recombinant human N-acetylgalactosamine-6-sulphatase.

Full-length cDNA sequences encoding human N-acetylgalactosamine-6-sulphatase were stably expressed in Chinese hamster ovary cells under the transcriptional control of the human polypeptide chain elongation factor 1 alpha gene promoter. A clonal cell line overexpressing recombinant N-acetylgalactosamine-6-sulphatase to a level of approx. 3 mg/l of culture medium was isolated. The secreted precursor enzyme was purified to homogeneity by a two-column procedure with an overall yield of 53% of the activity. The physical and catalytic parameters of the recombinant enzyme were similar to those of the mature form isolated from liver. On SDS/PAGE and gel filtration, recombinant N-acetylgalactosamine-6-sulphatase had a native molecular mass of 58-60 kDa. Recombinant N-acetylgalactosamine-6-sulphatase was endocytosed by mucopolysaccharidosis IVA fibroblasts via the mannose-6-phosphate receptor-mediated pathway and was efficiently localized to lysosomes.

Animals↗
PubMed1995

Cloning and sequence analysis of caprine N-acetylglucosamine 6-sulfatase cDNA.

Mucopolysaccharidosis IIID results from the deficiency of N-acetylglucosamine 6-sulfatase activity. A Nubian goat with this lysosomal storage disease has been identified. As a first step in developing this animal model for testing treatment methods, we cloned and sequenced the caprine N-acetylglucosamine 6-sulfatase cDNA coding region. Overall there is 88% nucleotide homology between the goat and human sequence and 94% homology of the deduced amino acid sequence. The human and two ruminant species differ by the presence of an imperfect trinucleotide (CCG) repeat in the ruminant signal sequence.

Amino Acid Sequence↗
PubMed1995

Two site-directed mutations abrogate enzyme activity but have different effects on the conformation and cellular content of the N-acetylgalactosamine 4-sulphatase protein.

The sulphatase family of enzymes have regions of sequence similarity, but relatively little is known about either the structure-function relationships of sulphatases, or the role of highly conserved amino acids. The sequence of amino acids CTPSR at position 91-95 of 4-sulphatase has been shown to be highly conserved in all of the sequenced sulphatase enzymes. The cysteine at amino acid 91 of 4-sulphatase was selected for mutation analysis due to its potential role in either the active site, substrate-binding site or part of a key structural domain of 4-sulphatase and due to the absence of naturally occurring mutations in this residue in mucopolysaccharidosis type VI (MPS VI) patients. Two mutations, C91S and C91T, altering amino acid 91 of 4-sulphatase were generated and expressed in Chinese hamster ovary cells. Biochemical analysis of protein from a C91S cell line demonstrated no detectable 4-sulphatase enzyme activity but a relatively normal level of 4-sulphatase polypeptide (180% of the wild-type control protein level). Epitope detection, using a panel of ten monoclonal antibodies, demonstrated that the C91S polypeptide had a similar immunoreactivity to wild-type 4-sulphatase, suggesting that the C91S substitution does not induce a major structural change in the protein. Reduced catalytic activity associated with normal levels of 4-sulphatase protein have not been observed in any of the MPS VI patients tested and all show evidence of structural modification of 4-sulphatase protein with the same panel of antibodies [Brooks, McCourt, Gibson, Ashton, Shutter and Hopwood (1991) Am. J. Hum. Genet. 48, 710-719]. The loss of enzyme activity without a detectable protein conformation change suggests that Cys-91 may be a critical residue in the catalytic process. In contrast, analysis of protein from a C91T cell line revealed low levels of catalytically inactive 4-sulphatase polypeptide (0.37% of the wild-type control protein level) which had missing or masked epitopes, suggesting an altered protein structure or conformation. Subcellular fractionation studies of the C91T cell line demonstrated a high proportion of 4-sulphatase polypeptide content in organelles characteristic of microsomes. The aberrant intracellular localization and the reduced cellular content of 4-sulphatase polypeptide was consistent with the observed structural modification leading to retention and degradation of the protein within an early vacuolar compartment.

Animals↗
PubMed1994

Recombinant alpha-L-iduronidase: characterization of the purified enzyme and correction of mucopolysaccharidosis type I fibroblasts.

Mucopolysaccharidosis type I (MPS I, Hurler and Scheie syndromes) is an autosomal recessive lysosomal storage disorder that results from a deficiency of the hydrolase alpha-L-iduronidase (IDUA) which is involved in the lysosomal degradation of both heparan sulphate (HS) and dermatan sulphate (DS). Patients with MPS I store and excrete large amounts of partially degraded HS and DS. In order to evaluate enzyme replacement therapy for MPS I patients we have expressed human IDUA cDNA in Chinese Hamster Ovary (CHO)-K1 cells utilizing a plasmid vector that places the cDNA under the transcriptional control of the human polypeptide-chain-elongation factor I alpha gene promoter. A clonal cell-line that secreted recombinant IDUA in a precursor form at approximately 2.2 micrograms/10(6) cells per day was identified. This enzyme was shown to be endocytosed into cultured MPS I fibroblasts via mannose-6-phosphate receptors and to correct the storage phenotype of these cells by enabling the lysosomal-digestion of accumulated sulphated glycosaminoglycans. The recombinant IDUA had on SDS/PAGE a molecular mass of 85 kDa and was processed to 74 kDa and smaller forms following its uptake by fibroblasts. Milligram quantities of the recombinant IDUA were immunopurified and the enzyme was shown to have pH optimum and kinetic parameters differing from those of the mature enzyme purified from human liver. The specific activity of the recombinant enzyme was shown to increase on dilution and on incubation with reducing agents. This was in contrast to the mature IDUA form (74 kDa) which did not have its activity stimulated by reducing agents or dilution.

Base Sequence↗
PubMed1994

Transcriptional activity of the CD45 gene promoter in retroviral vector constructs.

Both specific and housekeeping transcriptional control elements have been successfully used to generate retroviral vectors that express in a stable tissue specific manner. However, no such vectors have been developed specifically for expression in haematopoietic cells. This paper describes the construction of retroviral vectors incorporating sequences from the promoter region of the murine CD45 (leucocyte common antigen) gene and using the human alpha-L-iduronidase cDNA as a reporter gene. These vectors were tested for expression of the alpha-L-iduronidase gene in skin fibroblasts and a lymphoblastoid cell line which is permissive for CD45 expression, derived from patients with mucopolysaccharidosis type I (MPS I, alpha-L-iduronidase deficiency). The results obtained demonstrate that, in the context of a retroviral construct, the activity of the CD45 gene promoter in lymphocytes and fibroblasts parallels the activity of the retroviral long terminal repeat (LTR). These results suggest that additional transcriptional control elements from the CD45 gene, or other haematopoietic specific genes, will be necessary for the construction of a haematopoietic specific vector.

Animals↗
PubMed1993

Overexpression of N-acetylgalactosamine-4-sulphatase induces a multiple sulphatase deficiency in mucopolysaccharidosis-type-VI fibroblasts.

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.

Chondro-4-Sulfatase↗
PubMed1993

Recombinant human iduronate-2-sulphatase: correction of mucopolysaccharidosis-type II fibroblasts and characterization of the purified enzyme.

Mucopolysaccharidosis type II (MPS II, Hunter syndrome) is an X-chromosome-linked recessive lysosomal storage disorder that results from a deficiency of iduronate-2-sulphatase (12S). Patients with MPS II store and excrete large amounts of partially degraded heparan sulphate and dermatan sulphate. In order to evaluate enzyme-replacement therapy for MPS II we have expressed a chimaeric I2S cDNA in CHO (Chinese-hamster ovary)-K1 cells utilizing a plasmid vector that places the cDNA under the transcriptional control of the human polypeptide-chain-elongation factor-1 alpha gene promoter. A clonal cell line that accumulated recombinant I2S at greater than 10 mg/ml in conditioned medium was identified. Enzyme secreted from this cell line grown in the presence of NH4Cl was shown to be endocytosed into MPS II fibroblasts via the mannose 6-phosphate receptor and localized to the lysosomal compartment, resulting in correction of the storage phenotype of these cells. Milligram quantities of the recombinant I2S were purified, and the enzyme was shown to have a pH optimum and kinetic parameters similar to those for the mature form of I2S purified from human liver. The recombinant I2S had a molecular mass of approx. 90 kDa; this was reduced to 60 kDa by endoglycosidase treatment.

Animals↗
PubMed1992

Correction of human mucopolysaccharidosis type-VI fibroblasts with recombinant N-acetylgalactosamine-4-sulphatase.

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.

Alkaline Phosphatase↗