421. Liver Contains Sufficient Sulfatase Modifying Factor for 4-Sulfatase and Correction of MPS VI with Neonatal Retroviral Gene Therapy in Cats

Abstract

Sulfatases are enzymes that remove a sulfate from a substrate. 4-sulfatase (4S; also known as arylsulfatase B) is a lysosomal enzyme that removes a sulfate from the 4 position of N-acetylgalactosamine, a component of the glycosomaminoglycan (GAG) dermatan sulfate. Deficient activity of 4S results in mucopolysaccharidosis VI (MPS VI), a disorder that includes bone and joint abnormalities, corneal clouding, and cardiac disease, but does not usually involve the brain. The lack of neurological involvement makes this an excellent candidate for somatic therapies. All sulfatases are modified post-translationally by enzymes designated as sulfatase modifying factors (SUMF) that convert a cysteine in the active site to a C-alpha-formylglycine, which is absolutely essential for enzyme activity. SUMF is readily saturated after over expression of a sulfatase in fibroblasts in vitro, resulting in limitations in the amount of enzyme that can be produced and a reduction in activity of other sulfatases that require the same modification. It was, therefore, unclear if hepatocytes would contain sufficient SUMF activity to modify the 4S produced by hepatocytes after liver-directed gene therapy for MPS VI. A retroviral vector (RV) designated hAAT-f4S-WPRE expressing feline 4S was generated. 4S-deficient feline fibroblasts were transduced in vitro with increasing multiplicities of infection. The 4S enzyme activity (4MU-sulfate assay) and 4S protein levels (immunoblot) were directly proportional to the DNA copy number, which leveled off at about 2 copies per cell despite using higher multiplicities of infection. This likely reflects an inhibition of transduction when large amounts of vector are used, which has been described previously, suggesting that SUMF is not limiting when relatively low copy numbers are achieved. hAAT-f4S-WPRE (4.2-3.4X10E9 TU/kg) was injected IV into 6 newborn cats with MPS VI. They achieved stable expression at 109+/-49 U/ml in serum, which is 10-fold normal and has been maintained for up to 1.5 years in the oldest animals. Analysis of liver biopsies demonstrated that the 4S enzyme activity was directly proportional to the amount of 4S protein, suggesting that all of the enzyme was functional, and that modification by SUMF was not limiting. The cats have marked improvement in the clinical skeletal manifestations. The cats also have biochemical evidence of improvement, which includes high levels of 4S and a reduction in GAG and secondary lysosomal enzyme activity in other organs. We conclude that liver contains sufficient SUMF to modify 4S, and that liver-directed gene therapy can markedly improve the clinical manifestations of this disorder.