Restoration of arylsulphatase B activity in human mucopolysaccharidosis-type-VI fibroblasts by retroviral-vector-mediated gene transfer

Abstract

The Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI; MPS VI) is a lysosomal storage disease caused by deficiency of the enzyme arylsulphatase B (ASB). A human ASB cDNA has been subcloned into the retroviral vector pXT1 containing the bacterial neomycin-resistance gene and an internal thymidine kinase promoter for transcription of the inserted gene. Replication defective retrovirus was generated by transfecting the construct into the amphotropic packaging cell line PA317. Human MPS VI fibroblasts infected with recombinant retrovirus integrated the provirus into their genome and expressed retrovirus-encoded ASB mRNAs. In infected fibroblasts the level of ASB was up to 36-fold higher than in normal fibroblasts. Biosynthesis and processing of ASB in infected MPS VI fibroblasts was accomplished as in normal fibroblasts, and mature, enzymically active, ASB accumulated in dense lysosomes, indicating that the ASB deficiency in MPS VI fibroblasts was corrected by the retroviral gene transfer.

Multiple sulfatase deficiency: catalytically inactive sulfatases are expressed from retrovirally introduced sulfatase cDNAs.

“…We introduced the sulfatase cDNAs into human diploid fibroblasts by infection with recombinant retroviruses. cDNA fragments containing the coding sequences of human ASA, ASB, and STS were cloned in the retroviral expression vector pXT1 (10,11), yielding the plasmids pXT1ASA, pXT1ASB, and pXT1STS (Fig. 2).…”

“…The construction of the retroviral vectors containing the cDNAs of ASA and ASB were described previously (10,11). For the construct containing the coding part ofthe ASA pseudodeficiency allele (pdASA) a 1828-base-pair (bp) Xho I-Sal I DNA fragment was isolated from the eukaryotic expression vector pBEHpdASA (17).…”

“…According to the working hypothesis, sulfatase genes and their transcription should be normal in MSD, while expression of the endogenous genes and of the introduced cDNAs should yield sulfatase polypeptides that are inactive and/or unstable. On the other hand, expression of the same cDNAs in cells with single sulfatase deficiencies caused by mutations in the sulfatase genes should yield sulfatase polypeptides with normal catalytic properties and stability, as was shown recently (10,11).…”

Secondary Storage of Dermatan Sulfate in Sanfilippo Disease

“…As no fluorogenic substrate is available that is specific for only GalNAc-4-sulfatase, the activity of purified enzyme was tested using p-nitrocatechol sulfate as described previously (20). In some experiments, heparan sulfate (Neoparin), heparin (Neoparin), dermatan sulfate (Neoparin), or chondroitin sulfate (Sigma Aldrich) were added to reactions at indicated concentrations prior to activity analysis.…”

Arylsulfatase B activities and glycosaminoglycan levels in retrovirally transduced mucopolysaccharidosis type VI cells. Prospects for gene therapy.

“…Moreover, ASB retroviral vectors have been constructed and used to transduce skin fibroblasts from MPS VI patients (14,15). A surprising outcome of these latter experiments was that overexpression of ASB in human MPS VI skin fibroblasts did not reduce the level of 35 …”