Enzyme replacement therapy in newborn mucopolysaccharidosis IVA mice: early treatment rescues bone lesions?

Abstract

We treated mucopolysaccharidosis IVA (MPS IVA) mice to assess the effects of long-term enzyme replacement therapy (ERT) initiated at birth, since adult mice treated by ERT showed little improvement in bone pathology [1]. To conduct ERT in newborn mice, we used recombinant human N-acetylgalactosamine-6-sulfate sulfatase (GALNS) produced in a CHO cell line. First, to observe the tissue distribution pattern, a dose of 250units/g body weight was administered intravenously in MPS IVA mice at day 2 or 3. The infused enzyme was primarily recovered in the liver and spleen, with detectable activity in the bone and brain. Second, newborn ERT was conducted after a tissue distribution study. The first injection of newborn ERT was performed intravenously, the second to fourth weekly injections were intraperitoneal, and the remaining injections from 5th to 14th weeks were intravenous into the tail vein. MPS IVA mice treated with GALNS showed clearance of lysosomal storage in the liver and spleen, and sinus lining cells in bone marrow. The column structure of the growth plate was organized better than that in adult mice treated with ERT; however, hyaline and fibrous cartilage cells in the femur, spine, ligaments, discs, synovium, and periosteum still had storage materials to some extent. Heart valves were refractory to the treatment. Levels of serum keratan sulfate were kept normal in newborn ERT mice. In conclusion, the enzyme, which enters the cartilage before the cartilage cell layer becomes mature, prevents disorganization of column structure. Early treatment from birth leads to partial remission of bone pathology in MPS IVA mice.

Keywords: Enzyme replacement therapy; Fibrous cartilage; Hyaline cartilage; Mucopolysaccharidosis IVA; N-acetylgalactosamine-6-sulfate sulfatase.

Figure 1. Histopathology in ERT-treated mouse

Left panel shows an untreated MPS IVA mouse, middle panel shows an ERT-treated mouse, and right panel shows a wild-type control mouse. Shown are liver (A, B, C), heart valve (D, E, F), bone marrow in femur (G, H, I), growth plate in femur (hyaline cartilage) (J, K, L), articular disc (fibrous cartilage) surrounding femur (M, N, O), bone marrow in spine (P, Q, R), growth plate in spine (hyaline cartilage) (S, T, U), and intervertebral disc (fibrous cartilage) surrounding vertebrae (V, W, X). The sections were stained with toluidine blue. The arrows show the vacuolated cells. A, B, and C: Kupffer cells and sinus lining cells in an untreated mouse contain vacuoles, while no vacuole is seen in a treated or wild-type mouse. Figs. 1D and E show abundant vacuoles at the base in an untreated mouse and at the heart valve in a treated mouse. G, H, and I: Sinus lining cells in bone marrow of femur contain vacuoles in an untreated mouse, while no vacuole is seen in a treated or wild-type mouse. J, K, and L: all chondrocytes at growth plate of femur are vacuolated in an untreated mouse, while less vacuolated in a treated mouse. M, N, and O: Articular disc (meniscus) in femur contains abundant vacuolated cells in both untreated and treated mice. P, Q, and R: Sinus lining cells in bone marrow of spine in an untreated mouse, while no vacuole is seen in a treated or wild-type mouse. S, T, and U: All chondrocytes are vacuolated in an untreated mouse, while less numbers of chondrocytes are vacuolated in a treated mouse. V, W and X: Intervertebral disc in spine contains abundant vacuolated cells in untreated mice and less in treated mice. Magnifications: x100 except G, H, and I (x40). Bar in the picture indicates 100 μm. MPS = mucopolysaccharidosis; ERT = enzyme replacement therapy.

Figure 1. Histopathology in ERT-treated mouse

Left panel shows an untreated MPS IVA mouse, middle panel shows an ERT-treated mouse, and right panel shows a wild-type control mouse. Shown are liver (A, B, C), heart valve (D, E, F), bone marrow in femur (G, H, I), growth plate in femur (hyaline cartilage) (J, K, L), articular disc (fibrous cartilage) surrounding femur (M, N, O), bone marrow in spine (P, Q, R), growth plate in spine (hyaline cartilage) (S, T, U), and intervertebral disc (fibrous cartilage) surrounding vertebrae (V, W, X). The sections were stained with toluidine blue. The arrows show the vacuolated cells. A, B, and C: Kupffer cells and sinus lining cells in an untreated mouse contain vacuoles, while no vacuole is seen in a treated or wild-type mouse. Figs. 1D and E show abundant vacuoles at the base in an untreated mouse and at the heart valve in a treated mouse. G, H, and I: Sinus lining cells in bone marrow of femur contain vacuoles in an untreated mouse, while no vacuole is seen in a treated or wild-type mouse. J, K, and L: all chondrocytes at growth plate of femur are vacuolated in an untreated mouse, while less vacuolated in a treated mouse. M, N, and O: Articular disc (meniscus) in femur contains abundant vacuolated cells in both untreated and treated mice. P, Q, and R: Sinus lining cells in bone marrow of spine in an untreated mouse, while no vacuole is seen in a treated or wild-type mouse. S, T, and U: All chondrocytes are vacuolated in an untreated mouse, while less numbers of chondrocytes are vacuolated in a treated mouse. V, W and X: Intervertebral disc in spine contains abundant vacuolated cells in untreated mice and less in treated mice. Magnifications: x100 except G, H, and I (x40). Bar in the picture indicates 100 μm. MPS = mucopolysaccharidosis; ERT = enzyme replacement therapy.

Figure 2. Level of serum KS in the MPS IVA mouse

The serum was collected at 5, 7, 8, and 9 weeks old before the enzyme injection, and at 15 weeks old at necropsy. ◆ with a dash line: Wild-type control group, ■ with a solid line; Untreated MPS IVA group; ▲ with a dot line; Treated MPS IVA group. Each group comprises three mice and shows the average value with SD. Statistical analyses by Student's t-test show as follows. *: control vs. untreated p < 0.01, #: untreated vs. treated p < 0.01. MPS = mucopolysaccharidosis; SD = standard deviation