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Mucopolysaccharidosis type IIIA (MPS IIIA, Sanfilippo A syndrome) is a lysosomal storage disease that causes a profound neurological deterioration. The disorder is caused by a deficiency of the lysosomal enzyme sulphamidase which is a requisite for the degradation of heparan sulphate. To facilitate the development of enzyme-replacement strategies for MPS IIIA patients, we have constructed a high-level expression system for recombinant human sulphamidase in Chinese hamster ovary (CHO) cells. An expression construct containing a methotrexate-resistant dihydrofolate reductase (DHFR) gene allowed amplification of expression levels from less than 1 mg of sulphamidase per litre of culture medium to approx. 15 mg/l. Unlike many cell lines made by gene amplification in DHFR-deficient CHO cells, and utilizing the normal DHFR gene, these cell lines appeared to be stable in the absence of selective pressure. Recombinant human sulphamidase was purified from unamplified and amplified cell lines. The native enzyme was found to be a dimer of 115 kDa. Denaturing and reducing SDS/PAGE revealed a subunit size of 62 kDa. Kinetic analysis demonstrated that the recombinant enzyme had broadly similar kinetic characteristics to sulphamidase purified from liver. Recombinant human sulphamidase was able to correct the storage phenotype of MPS IIIA fibroblasts after endocytosis via the mannose-6-phosphate receptor.

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.

Intrafamilial variability has not been reported previously in Hurler syndrome or Sanfilippo syndrome type A. We describe two families in which sibs with comparable deficiencies of alpha-iduronidase (Hurler) or sulfamidase (Sanfilippo type A) activities in vitro nonetheless have divergence in clinical severity and disease progression. These cases underscore the need for caution in counseling as well as the limitations of using sibs as controls in evaluating the outcome of treatment.

Unspecific immunostimulation by bacterial vaccines of a patient with mucopolysaccharidosis IIIA (Sanfilippo A) syndrome induces a marked increase in the urinary excretion of heparan sulfate and of uronate-containing oligosaccharides. This event is presumably linked to an increased vascular permeability and exocytosis of storage material, elicited by mediators of inflammation, as well as to enhanced degradation of stored polymers in activated macrophages and surrounding tissue.