Enzyme Replacement in a Human Model of Mucopolysaccharidosis IVA In Vitro and Its Biodistribution in the Cartilage of Wild Type Mice

Abstract

Mucopolysaccharidosis IVA (MPS IVA; Morquio A syndrome) is a lysosomal storage disorder caused by deficiency of N-acetylgalactosamine-6-sulfatase (GALNS), an enzyme that degrades keratan sulfate (KS). Currently no therapy for MPS IVA is available. We produced recombinant human (rh)GALNS as a potential enzyme replacement therapy for MPS IVA. Chinese hamster ovary cells stably overexpressing GALNS and sulfatase modifying factor-1 were used to produce active (∼2 U/mg) and pure (≥97%) rhGALNS. The recombinant enzyme was phosphorylated and was dose-dependently taken up by mannose-6-phosphate receptor (Kuptake = 2.5 nM), thereby restoring enzyme activity in MPS IVA fibroblasts. In the absence of an animal model with a skeletal phenotype, we established chondrocytes isolated from two MPS IVA patients as a disease model in vitro. MPS IVA chondrocyte GALNS activity was not detectable and the cells exhibited KS storage up to 11-fold higher than unaffected chondrocytes. MPS IVA chondrocytes internalized rhGALNS into lysosomes, resulting in normalization of enzyme activity and decrease in KS storage. rhGALNS treatment also modulated gene expression, increasing expression of chondrogenic genes Collagen II, Collagen X, Aggrecan and Sox9 and decreasing abnormal expression of Collagen I. Intravenous administration of rhGALNS resulted in biodistribution throughout all layers of the heart valve and the entire thickness of the growth plate in wild-type mice. We show that enzyme replacement therapy with recombinant human GALNS results in clearance of keratan sulfate accumulation, and that such treatment ameliorates aberrant gene expression in human chondrocytes in vitro. Penetration of the therapeutic enzyme throughout poorly vascularized, but clinically relevant tissues, including growth plate cartilage and heart valve, as well as macrophages and hepatocytes in wild-type mouse, further supports development of rhGALNS as enzyme replacement therapy for MPS IVA.

Human recombinant lysosomal enzymes produced in microorganisms

“…These results show for the first time the advantage of sulfatase/SUMF1 co-expression within a yeast expression system. The purified enzyme showed a specific activity of 25.3 nmol h −1 mg −1 , which is higher than the one reported for E. coli BL21(DE3) [64], but lower than that of the enzyme produced in CHO cells [61,70,71]. Nevertheless, as mentioned before the activities are not comparable between microorganisms systems and CHO cells due to substantial differences in the enzyme activity assays.…”

“…3) [64]. The purified GALNS showed a specific activity of 0.29 nmol h −1 mg −1 , which is lower than that reported for protein produced in CHO cells [60,61]. However, activity values between bacterial and CHO cells are not equivalent because they have been obtained using substantially different methods (e.g.…”

“…Recombinant GALNS enzyme for ERT has been produced in CHO cells, exhibiting after purification a specific activity of 170,000 [60] to 120,000 nmol h −1 mg −1 [61]. Both enzymes showed the highest activity at pH 5.0, contain 57, 39 and 19 kDa polypeptides, and are taken-up by cultured fibroblast and chondrocytes [48,61]. Recently ERT for Morquio A disease was approved, which showed a modest improvement in a 6-min walk test (6MWT), and a reduction of urinary KS, after a 24-weeks treatment [62].…”

Subjective and Objective Assessment of Hand Function in Mucopolysaccharidosis IVa Patients

“…ERT for Morquio patients is still undergoing clinical trials but in vitro studies and animal models are providing promising insights (Dvorak-Ewell et al 2010).…”

Innovative Therapeutic Approaches for Improving Patient Life Condition with a Neurological Lysosomal Disease

“…Thanks to these technological progresses, clinical trials were initiated by three biopharmaceutical companies: Genzyme®, BioMarin Pharmaceutical Inc, and Transkaryotic Therapies (now Shire Pharmaceuticals), for several disorders such as Fabry disease, mucopolysaccharidoses (MPS) I, II, VI, and Pompe disease, leading to the commercialization of a therapy for these diseases, improving most of the peripheric symptoms in these disorders . For several years, enzyme replacement therapy (ERT) based on intravenous injection of recombinant enzyme, whose deficiency causes the disease, has been used to treat MPS I (Kakkis et al, 2001;Wraith et al, 2004), MPS II, and MPS VI (Muenzer et al, 2002;Harmatz et al, 2006;Muenzer et al, 2006), and it appears that an analogous therapy could be also efficient for MPS IVA (clinical trial on going) (Tomatsu et al, 2008;Dvorak-Ewell et al, 2010). This therapy is effective for treating somatic symptoms of MPS I, MPS II, and MPS VI.…”