Therapeutic Potential of Intracerebroventricular Replacement of Modified Human β-Hexosaminidase B for GM2 Gangliosidosis

Abstract

To develop a novel enzyme replacement therapy for neurodegenerative Tay-Sachs disease (TSD) and Sandhoff disease (SD), which are caused by deficiency of β-hexosaminidase (Hex) A, we designed a genetically engineered HEXB encoding the chimeric human β-subunit containing partial amino acid sequence of the α-subunit by structure-based homology modeling. We succeeded in producing the modified HexB by a Chinese hamster ovary (CHO) cell line stably expressing the chimeric HEXB, which can degrade artificial anionic substrates and GM2 ganglioside in vitro, and also retain the wild-type (WT) HexB-like thermostability in the presence of plasma. The modified HexB was efficiently incorporated via cation-independent mannose 6-phosphate receptor into fibroblasts derived from Tay-Sachs patients, and reduced the GM2 ganglioside accumulated in the cultured cells. Furthermore, intracerebroventricular administration of the modified HexB to Sandhoff mode mice restored the Hex activity in the brains, and reduced the GM2 ganglioside storage in the parenchyma. These results suggest that the intracerebroventricular enzyme replacement therapy involving the modified HexB should be more effective for Tay-Sachs and Sandhoff than that utilizing the HexA, especially as a low-antigenic enzyme replacement therapy for Tay-Sachs patients who have endogenous WT HexB.

Protease-resistant modified human β-hexosaminidase B ameliorates symptoms in GM2 gangliosidosis model

“…We herein demonstrated that repeated administration by i.c.v.ERT using mod2B had remarkable therapeutic effects, including improvement of the motor dysfunction and life extension of SD model mice. This was caused by the incorporation of the therapeutic molecule via neural cell surface CI-M6PR, followed by delivery to lysosomes and a reduction of the accumulated substrates, as supported by our previous studies (4,5,26) in addition to the in vivo stability due to the protease resistance in the brain parenchyma. The GSEPLDS loop structure of modB may be cleaved by lysosomal endoproteases, possibly cathepsins L and B, after being incorporated and delivered to lysosomes because the proteolytic cleavage was inhibited by pretreatment of the microglial cells derived from SD mouse brain with leupeptin, a thiol protease inhibitor for cathepsins L and B (data not shown), although the enhancement of intracellular GM2 degradation by the modB replacement should be proved under the conditions in which the endoprotease activities are inhibited.…”

“…The fibroblasts were fixed with 4% paraformaldehyde. After the samples were washed with PBS, the intracellular GM2 and Hex were detected by Ab against GM2 and human HexA (5). The specimens were viewed with the LSM700.…”

“…Then β-Hex activities toward 4-methylumbelliferyl-6-sulfo-N-acetyl-β-D-glucosaminide (MUGS; Merck) were measured in 0.1 M sodium citrate buffer (pH 4.2) (57). The kinetic parameters and in vitro thermostability was analyzed (5,26). K m and V max values for 4-MUGS were determined by the method of Lineweaver-Burk plot at various substrate concentrations (0.125-4 mM).…”

“…The underlying mechanism of the neuropathogenesis is still unclear, although it has been reported that lysosomal accumulation of their substrates, including glycosphingolipids, oligosaccharides, and glycosaminoglycans, leading to inflammatory responses (microglial activation and astrogliosis) and neuronal injury are the hallmarks during the course of these disorders (1,2). There are currently no effective treatments for GM2 gangliosidoses, although several therapeutic approaches have been developed, including gene therapy (GT) (2,3), enzyme replacement therapy (ERT) (4,5), substrate reduction therapy (SRT) (6,7), and pharmacological chaperone therapy (PCT) (8). An SD model mouse was generated by targeted disruption of the Hexb gene allele (9,10) and was utilized for investigating the neuropathogenic mechanism and evaluating the therapeutic approaches as an authentic infantile GM2 gangliosidosis model, which develops the pathological features -including spasticity, muscle weakness, rigidity, tremor, and ataxia -that closely resemble those observed in the acute form of human disorders.…”

“…The modB is composed of the homodimeric modified β subunits (β 1 ') secreted by a CHO cell line, which retained thermostability similar to that of human HexB in vitro and could reduce the GM2 and GA2 accumulation in the brains of SD mice following i.c.v. administration (5). In this study, we elucidated the x-ray crystal structure of purified modB (newly registered PDB ID: 5BRO), which retained a local conformation of the active pocket of β 1 ' subunits very similar to that of the α subunits and the introduced GSEP loops ( Figure 1A and Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI85300DS1).…”