Mucopolysaccharidosis II (MPS II) is an X-linked recessive lysosomal storage disease caused by mutations in the iduronate-2-sulfatase (IDS) gene. Since IDS catalyzes the degradation of glycosaminoglycans (GAGs), deficiency in this enzyme leads to accumulation of GAGs in most cells in all tissues and organs, resulting in severe somatic and neurological disorders. Although enzyme replacement therapy with human IDS (hIDS) has been used for the treatment of MPS II, this therapy is not effective for defects in the CNS mainly because the enzyme cannot cross the blood-brain barrier (BBB). Here, we developed a BBB-penetrating fusion protein, JR-141, which consists of an anti-human transferrin receptor (hTfR) antibody and intact hIDS. The TfR-mediated incorporation of JR-141 was confirmed by using human fibroblasts in vitro. When administrated intravenously to hTfR knockin mice or monkeys, JR-141, but not naked hIDS, was detected in the brain. In addition, the intravenous administration of JR-141 reduced the accumulation of GAGs both in the peripheral tissues and in the brain of hTfR knockin mice lacking Ids, an animal model of MPS II. These data provide a proof of concept for the translation of JR-141 to clinical study for the treatment of patients with MPS II with CNS disorders.

Mucopolysaccharidosis II (MPS II), a lysosomal storage disease caused by mutations in iduronate-2-sulfatase (IDS), is characterized by a wide variety of somatic and neurologic symptoms. The currently approved intravenous enzyme replacement therapy with recombinant IDS (idursulfase) is ineffective for CNS manifestations due to its inability to cross the blood-brain barrier (BBB). Here, we demonstrate that the clearance of heparan sulfate (HS) deposited in the brain by a BBB-penetrable antibody-enzyme fusion protein prevents neurodegeneration and neurocognitive dysfunctions in MPS II mice. The fusion protein pabinafusp alfa was chronically administered intravenously to MPS II mice. The drug reduced HS and attenuated histopathological changes in the brain, as well as in peripheral tissues. The loss of spatial learning abilities was completely suppressed by pabinafusp alfa, but not by idursulfase, indicating an association between HS deposition in the brain, neurodegeneration, and CNS manifestations in these mice. Furthermore, HS concentrations in the brain and reduction thereof by pabinafusp alpha correlated with those in the cerebrospinal fluid (CSF). Thus, repeated intravenous administration of pabinafusp alfa to MPS II mice decreased HS deposition in the brain, leading to prevention of neurodegeneration and maintenance of neurocognitive function, which may be predicted from HS concentrations in CSF.

Mucopolysaccharidosis type II (MPS II or Hunter syndrome) is a lysosomal storage disorder caused by a deficiency of iduronate-2-sulfatase (IDS), an enzyme that catabolizes glycosaminoglycans (GAGs) including heparan sulfate (HS) and dermatan sulfate (DS). GAG accumulation leads to severe neurological and somatic impairments. At present, the most common treatment for MPS II is intravenous enzyme replacement therapy; however, the inability of recombinant IDS to cross the blood-brain barrier (BBB) restricts therapeutic efficacy for neurological manifestations. We recently developed a BBB-penetrating IDS fusion protein, JR-141, and demonstrated its ability to reduce GAG accumulation in the brain of human transferrin receptor knock-in and Ids knock-out mice (TFRC-KI/Ids-KO), an animal model of MPS II, following intravenous administration. Given the impossibility of measuring GAG accumulation in the brains of human patients with MPS II, we hypothesized that GAG content in the cerebrospinal fluid (CSF) might serve as an indicator of brain GAG burden. To test this hypothesis, we optimized a high-sensitivity method for quantifying HS and DS in low-volume samples by combining acidic methanolysis and liquid chromatography-tandem mass spectrometry (LC/MS/MS). We employed this method to quantify HS and DS in samples from TFRC-KI/Ids-KO mice and revealed that HS but not DS accumulated in the central nerve system (CNS). Moreover, concentrations of HS in CSF correlated with those in brain. Finally, intravenous treatment with JR-141 reduced levels of HS in the CSF and brain in TFRC-KI/Ids-KO mice. These results suggest that CSF HS content may be a useful biomarker for evaluating the brain GAG accumulation and the therapeutic efficacy of drugs in patients with MPS II.

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The hydrozoan is the simplest organism whose movements are governed by the neuromuscular system, and its de novo morphogenesis can be easily induced by the removal of body parts. These features make the hydrozoan an excellent model for studying the regeneration of tissues in vivo, especially in the nervous system. Although glycosaminoglycans (GAGs) and proteoglycans (PGs) have been implicated in the signaling functions of various growth factors and play critical roles in the development of the central nervous system, the isolation and characterization of GAGs from hydrozoans have never been reported. Here, we characterized GAGs of Hydra magnipapillata. Immunostaining using anti-GAG antibodies showed chondroitin or chondroitin sulfate (CS) in the developing nematocyst, which is a sting organelle specific to cnidarians. The CS-PGs might furnish an environment for assembling nematocyst components, and might themselves be components of nematocysts. Therefore, GAGs were isolated from Hydra and their structural features were examined. A considerable amount of CS, three orders of magnitude less heparan sulfate (HS), but no hyaluronan were found, as in Caenorhabditis elegans. Analysis of the disaccharide composition of HS revealed glucosamine 2-N-sulfation, glucosamine 6-O-sulfation, and uronate 2-O-sulfation. CS contains not only nonsulfated and 4-O-sulfated N-acetylgalactosamine (GalNAc) but also 6-O-sulfated GalNAc. The average molecular size of CS and HS was 110 and 10 kDa, respectively. It has also been established here that CS chains are synthesized on the core protein through the ubiquitous linkage region tetrasaccharide, suggesting that indispensable functions of the linkage region in the synthesis of GAGs have been conserved during evolution.