Abstract
Pompe disease is an autosomal recessive lysosomal storage disorder (LSD) caused by deficiency of lysosomal acid alpha-glucosidase (GAA). The result of the GAA deficiency is a ubiquitous lysosomal and non-lysosomal accumulation of glycogen. The most affected tissues are heart, skeletal muscle, liver, and the nervous system. Replacement therapy with the currently approved enzyme relies on M6P-mediated endocytosis. However, therapeutic outcomes still leave room for improvement, especially with regard to skeletal muscles. We tested the uptake, activity, and effect on glucose metabolism of a non-phosphorylated recombinant human GAA produced in moss (moss-GAA). Three variants of moss-GAA differing in glycosylation pattern have been analyzed: two with terminal mannose residues in a paucimannosidic (Man3) or high-mannose (Man 5) configuration and one with terminal N-acetylglucosamine residues (GnGn). Compared to alglucosidase alfa the moss-GAA GnGn variant showed increased uptake in differentiated myotubes. Moreover, incubation of immortalized muscle cells of Gaa−/− mice with moss-GAA GnGn led to similarly efficient clearance of accumulated glycogen as with alglucosidase alfa. These initial data suggest that M6P-residues might not always be necessary for the cellular uptake in enzyme replacement therapy (ERT) and indicate the potential of moss-GAA GnGn as novel alternative drug for targeting skeletal muscle in Pompe patients.
Highlights
Pompe disease is caused by compound heterozygous or homozygous mutations of the GAA gene, which encodes the acid alpha-1,4-glucosidase (GAA) [1,2]
Like in the case of some other lysosomal storage disorders (LSDs), alglucosidase alfa is being produced in Chinese hamster ovary (CHO) cells to ensure a human-like glycosylation profile, including terminal mannose 6-phosphate (M6P)-rests
Comparable specific activity could be shown for all three variants and the marketed alglucosidase alfa (Figure 1C)
Summary
Pompe disease ( termed glycogen storage disease type II; OMIM #232300) is caused by compound heterozygous or homozygous mutations of the GAA gene, which encodes the acid alpha-1,4-glucosidase (GAA) [1,2]. Since 2006, a licensed enzyme replacement therapy (ERT) is available for all Pompe patients: alglucosidase alfa, a recombinant human GAA (rhGAA) carrying mannose 6-phosphate (M6P) glycans. Like in the case of some other lysosomal storage disorders (LSDs), alglucosidase alfa is being produced in Chinese hamster ovary (CHO) cells to ensure a human-like glycosylation profile, including terminal M6P-rests. Several different modifications of the alglucosidase alfa, aimed to improve enzyme uptake have been proposed e.g.,: crafting of M6P analogues [7,8], increasing the number of M6P residues on the enzyme [9], co-administration with a small molecule pharmacological chaperone [10], or using a glycosylation-independent lysosomal targeting tag–insulin-like growth factor [11]. Several lysosomal enzymes for therapeutic use in ERT have been successfully produced using plant-based expression platforms [12,13,14,15] (see [16] for review). In this study we tested the uptake and activity of moss-produced recombinant human GAA (moss-GAA) in murine (Pompe model mouse) as well as human (originating from Pompe patients) myoblasts and differentiated myotubes, in comparison to alglucosidase alfa
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