Efficient Uptake of Recombinant α-Galactosidase A Produced with a Gene-Manipulated Yeast by Fabry Mice Kidneys

Abstract

To economically produce recombinant human α-galactosidase A (GLA) with a cell culture system that does not require bovine serum, we chose methylotrophic yeast cells with the OCH1 gene, which encodes α-1,6-mannosyltransferase, deleted and overexpressing the Mnn4p (MNN4) gene, which encodes a positive regulator of mannosylphosphate transferase, as a host cell line. The enzyme (yr-hGLA) produced with the gene-manipulated yeast cells has almost the same enzymological parameters as those of the recombinant human GLA produced with cultured human fibroblasts (agalsidase alfa), which is currently used for enzyme replacement therapy for Fabry disease. However, the basic structures of their sugar chains are quite different. yr-hGLA has a high content of phosphorylated N-glycans and is well incorporated into the kidneys, the main target organ in Fabry disease, where it cleaves the accumulated glycosphingolipids. A glycoprotein production system involving this gene-manipulated yeast cell line will be useful for the development of a new enzyme replacement therapy for Fabry disease.

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

“…In yeast, mannose phosphorylation occurs, however, the phosphorylated sugars are capped by terminal mannose residues (Ballou 1990 ) that block the binding of M6PR to its ligands. Efforts have been applied to add M6P tag to plant-made enzymes (He et al 2012 ), or increase M6P content or expose the covered M6P in yeast-made enzymes (Chiba et al 2002 ; Tsukimura et al 2012 ). Although the role of MR in the therapeutic outcome of ERT can be α-gal A-specific, our study proposed a possibility that mannose-terminated enzymes produced from these non-mammalian cell systems, without M6P manipulations, may be sufficiently effective for some LSDs in which non-macrophage cells are affected.…”

Human recombinant lysosomal enzymes produced in microorganisms

“…Purified recombinant GLA (yr-hGLA) showed a mammalian-like phosphorylated N-glycans, with a specific activity of 2.0 × 10 6 nmol h −1 mg −1 , similar to that of agalsidase alfa (2.1 × 10 6 nmol h − 1 mg − 1 ). Although the basic structure of N-glycans was different to those of agalsidase alfa, administration to Fabry mice showed that yr-hGLA was distributed to the kidney and liver, reducing the levels of glycosphingolipids in these tissues [94].…”

“…A combination of yeast cultivation optimization, growth medium supplementation, and glyco-engineering allowed the production of a recombinant GLA with a high mannose-6-phosphate content, and expression levels comparable to those observed in mammalian cells [93]. Finally, recombinant GLA was produced in the yeast O. minuta, glyco-engineered through the disruption of OCH1 gene and the overexpression of the S. cerevisiae MNN4 gene [94]. Purified recombinant GLA (yr-hGLA) showed a mammalian-like phosphorylated N-glycans, with a specific activity of 2.0 × 10 6 nmol h −1 mg −1 , similar to that of agalsidase alfa (2.1 × 10 6 nmol h − 1 mg − 1 ).…”

PER1, GUP1 and CWH43 of methylotrophic yeast Ogataea minuta are involved in cell wall integrity

“…The methylotrophic yeast Ogataea minuta , which can utilize methanol as a sole carbon source, is a suitable host for producing exogenous glycoproteins with human‐compatible sugar chains at large scale and low cost (Kuroda, Kobayashi, Tsumura, et al, ; Kuroda, Kitagawa, Kobayashi, et al, ; Kuroda, Kobayashi, Kitagawa, et al, ; Tsukimura, Kawashima, Togawa, et al, ). To date, several recombinant enzymes with potential for clinical use in enzyme replacement therapies have been successfully expressed in O. minuta cells (Akeboshi, Chiba, Kasahara, et al, ; Akeboshi, Kasahara, Tsuji, et al, ; Tsukimura et al, ). Moreover, the endo‐ β ‐ N ‐acetylglucosaminidase (ENGase) of O. minuta has been identified and characterized, and this enzyme represents the first known yeast ENGase (Murakami, Takaoka, Ashida, et al, ).…”