Mannose receptor-mediated delivery of moss-made α-galactosidase A efficiently corrects enzyme deficiency in Fabry mice

Abstract

Enzyme replacement therapy (ERT) is an effective treatment for several lysosomal storage disorders (LSDs). Intravenously infused enzymes are taken up by tissues through either the mannose 6-phosphate receptor (M6PR) or the mannose receptor (MR). It is generally believed that M6PR-mediated endocytosis is a key mechanism for ERT in treating LSDs that affect the non-macrophage cells of visceral organs. However, the therapeutic efficacy of MR-mediated delivery of mannose-terminated enzymes in these diseases has not been fully evaluated. We tested the effectiveness of a non-phosphorylated α-galactosidase A produced from moss (referred to as moss-aGal) in vitro and in a mouse model of Fabry disease. Endocytosis of moss-aGal was MR-dependent. Compared to agalsidase alfa, a phosphorylated form of α-galactosidase A, moss-aGal was more preferentially targeted to the kidney. Cellular localization of moss-aGal and agalsidase alfa in the heart and kidney was essentially identical. A single injection of moss-aGal led to clearance of accumulated substrate in the heart and kidney to an extent comparable to that achieved by agalsidase alfa. This study suggested that mannose-terminated enzymes may be sufficiently effective for some LSDs in which non-macrophage cells are affected, and that M6P residues may not always be a prerequisite for ERT as previously considered.

Figures

Fig. 1

In vitro characterization and uptake studies a Enzyme preparations separated in SDS-PAGE and stained with Coomassie blue. Lanes 1 and 2 are moss-aGal and agalsidase alfa respectively. Lanes 3 and 4 are moss-aGal and agalsidase alfa digested with PNGase F. Arrow, α-gal A enzymes after digestion; arrowhead, PNGase F (36 KDa). Protein standard and molecular weights are shown on left. b Moss-aGal and agalsidase alfa (1 ng each) detected by western blot using a polyclonal antibody specific to human α-gal A. Representative data from three independent experiments was shown. c Specific α-gal A activities of enzyme preparations determined using artificial substrate 4-MU-α-D-galactopyranoside. d Plots of reaction velocities of moss-aGal and agalsidase alfa assessed with artificial substrate 4-nitrophenyl α-D-galactopyranoside (pNP-Gal). e Stability of the enzymes diluted in buffered human plasma and heated at 37 °C (data are means of triplicates). f Intracellular α-gal A activities of Fabry patient fibroblasts after overnight incubation with different enzymes in the presence or absence of 5 mM M6P or 2 mg/ml yeast mannan. g Gb3 immunofluorescence staining shows massive lysosomal accumulation of Gb3 in untreated Fabry patient fibroblasts (upper) and significantly decreased Gb3 in the cells that were treated with moss-aGal (lower). h and i MR expression in Fabry patient fibroblasts and microvascular endothelial cells IMFE1. IMFE1 cells were MR-positive determined by both western blot (h) and immunofluorescence staining (i), while the fibroblasts were MR-negative. j Intracellular α-gal A activities of IMFE1 cells after overnight incubation with different enzymes in the presence or absence of M6P or mannan. k Uptake rates of different enzymes in IMFE1 cells. Cells were harvested at indicated time points and intracellular activities were measured. ***P < 0.001, moss-aGal vs. high-mann moss-aGal or agalsidase alfa. l Western blot analysis of internalized α-gal A in IMFE1 cells after 3 h incubation with different enzyme preparations. m Binding of different enzymes (10 μg/ml) to IMFE1 cells. After 3 h incubation at 4 °C, cell surface-bound enzymes were determined by enzyme assay. The dotted line indicates activity level of mock-treated IMFE1 cells in this assay (i.e., background level). *P < 0.05, ***P < 0.001. All the data in graphs are presented as mean ± SEM (n = 3-4). High-mann: high-mann moss-aGal; Agal-alfa: agalsidase alfa; Agal-beta: agalsidase beta

Fig. 2

Tissue and cellular distribution of infused enzymes a-c Enzyme preparations were injected into Fabry mice, and α-gal A activities in the kidney, heart, spleen, and liver were measured 2 h post-injection. a Specific activities in organs. Data are presented as mean ± SEM (n = 5). *P < 0.05, **P < 0.01. b Activities in whole organs were calculated and data are presented as % of total activity recovered from four organs. c α-gal A protein in kidney homogenates detected by western blot. Arrow, specific α-gal A band in moss-aGal-injected mice. No detectable specific band was seen in agalsidase alfa-injected mice. Arrowhead, approximate position where agalsidase alfa band may migrate (based on findings shown in Fig. 1b). d Cellular distribution of infused enzymes in the heart and kidney was determined by immunohistochemistry (n = 2). Heart: asterisks indicate the blood vessels with immunostaining positive cells (most likely endothelial cells), and arrows indicate positive perivascular cells (presumably macrophages). Kidney: arrows indicate immunostaining positive tubular epithelial cells. Scale bar: 25 μm. Original magnification: 400×. Agal-alfa: agalsidase alfa

Fig. 3

Efficacy of moss-aGal in clearing accumulated Gb3 in tissues. Gb3 contents in kidney (a), heart (b), and liver (c) were analyzed 7 days after a single infusion of either moss-aGal or agalsidase alfa at various doses. Data are presented as mean ± SEM (n = 4-5). *P < 0.05, ***P < 0.001. Statistical significance shown on top of each agalsidase alfa-injected group indicates difference between agalsidase alfa and the same dose of moss-aGal. Agal-alfa: agalsidase alfa