Evaluation of 4 - Sulfatase Activity in Fibroblasts
Modified with a Gene Therapy Vector

Mucopolysaccharidosis VI (MPS VI) is a lysosomal storage disease caused by a deficiency of N-acetylgalactosomine-4-sulfatase (4-sulfatase), an enzyme that breaks down glycosaminoglycans (GAGs). This deficiency results in accumulation of GAGs in the lysosomes leading to lysosomal storage throughout the body of the affected organism and causing skeletal deformities, heart disorders, corneal clouding, and lung disease.

Two modifications to the enzyme are necessary for 4-sulfatase to function. One is the addition of mannose-6-phosphate to N-linked oligosaccharides, which occurs in the Golgi. This targets most of the enzyme produced by the cell to the lysosome, although approximately 10% of the enzyme produced is appropriately modified but gets secreted. The other modification is conversion of cysteine at amino acid 91 in the active site to C-±-formylglycine, which occurs in the endoplasmic reticulum. The C-±-formylglycine binds the sulfate of the substrate and is necessary for 4-sulfatase to function. This modification is performed by the sulfatase modifying factor (SUMF1).

The approach to treating MPS VI that has been studied in Dr. Ponder's lab is one of neonatal gene therapy. A retroviral vector (RV) was generated that contained the feline 4-sulfatase gene and a liver specific promoter and was injected into newborn MPS VI cats. This approach was effective in modifying liver cells and correcting disease in the liver. However, the amount of 4-sulfatase in blood was low, suggesting that this enzyme might be poorly secreted from liver cells.

The goal of these experiments was to 1) determine the efficiency of secretion of 4-sulfatase, to ascertain if low secretion is the cause of low serum activity; and 2) determine if the sulfatase modifying factor becomes saturated at high multiplicities of infection. MPS VI cat fibroblasts were infected with the RV expressing 4-sulfatase at various multiplicities of infection, from 0 to 100. Both the activity of 4-sulfatase in the media and intracellular activity of these cells were measured using a fluorometric assay. It was found that approximately 50% of the 4-sulfatase produced in the cells was secreted, indicating that low secretion is probably not the cause of low activity in the serum. It was also found that enzyme activity increased linearly up to a multiplicity of infection of about 10 but beyond that point there was little increase in activity. This indicates that SUMF1 is probably being saturated by such high over expression of the enzyme. Since the multiplicity of infection used in the gene therapy technique for treating MPS VI is seldom more than 1, one can conclude that this approach will probably not saturate SUMF1.