Toward Engineering the Mannose 6-Phosphate Elaboration Pathway in Plants for Enzyme Replacement Therapy of Lysosomal Storage Disorders

Abstract

Mucopolysaccharidosis (MPS) I is a severe lysosomal storage disease caused by α-L-iduronidase (IDUA) deficiency, which results in accumulation of non-degraded glycosaminoglycans in lysosomes. Costly enzyme replacement therapy (ERT) is the conventional treatment for MPS I. Toward producing a more cost-effective and safe alternative to the commercial mammalian cell-based production systems, we have produced recombinant human IDUA in seeds of an Arabidopsis mutant to generate the enzyme in a biologically active and non-immunogenic form containing predominantly high mannose N-linked glycans. Recombinant enzyme in ERT is generally thought to require a mannose 6-phosphate (M6P) targeting signal for endocytosis into patient cells and for intracellular delivery to the lysosome. Toward effecting in planta phosphorylation, the human M6P elaboration machinery was successfully co-expressed along with the recombinant human IDUA using a single multi-gene construct. Uptake studies using purified putative M6P-IDUA generated in planta on cultured MPS I primary fibroblasts indicated that the endocytosed recombinant lysosomal enzyme led to substantial reduction of glycosaminoglycans. However, the efficiency of the putative M6P-IDUA in reducing glycosaminoglycan storage was comparable with the efficiency of the purified plant mannose-terminated IDUA, suggesting a poor in planta M6P-elaboration by the expressed machinery. Although the in planta M6P-tagging process efficiency would need to be improved, an exciting outcome of our work was that the plant-derived mannose-terminated IDUA yielded results comparable to those obtained with the commercial IDUA (Aldurazyme ® (Sanofi, Paris, France)), and a significant amount of the plant-IDUA is trafficked by a M6P receptor-independent pathway. Thus, a plant-based platform for generating lysosomal hydrolases may represent an alternative and cost-effective strategy to the conventional ERT, without the requirement for additional processing to create the M6P motif.Toward producing a more cost-effective and safe alternative to the commercial mammalian cell-based production systems, we have established an efficient plant-based recombinant protein production platform resulting in high-level synthesis of active human IDUA in seeds of the complex-glycan-deficient (cgl) mutant of Arabidopsis thaliana. The seed-based platform accumulates human IDUA at~5.7% total soluble protein and the enzyme is generated in a non-immunogenic form containing predominantly high mannose N-linked glycans (~94%) [3][4][5][6]. Purified cgl-recombinant IDUA was used to determine the 3-D structure of the human protein along with the essential details of its catalytic mechanism [7].The seed-derived IDUA possesses biological activities and kinetic properties that are very similar to the commercial IDUA (Aldurazyme ® (Sanofi, Paris, France)) used for ERT and derived from cultured Chinese hamster ovary (CHO) cells [6]. Our high-yielding Arabidopsis cgl seed line is thus a viable system for generating IDUA that is potentially suita...

Mucopolysaccharidosis: An overview and new treatment modalities

“…2 Alpha -L -Iduronidase is present in precursor form, which undergoes intracellular modification to a shorter form by losing 10 amino acids and contains Mannose -6-phosphate marker for targeting lysosomes. 3 Beta -glucuronidase detaches glucuronic acid in dermatan, chondroitin, and heparan sulfate. A deficiency of this enzyme is seen in Mucopolysaccharidosis VII (MPS VII).…”

A homozygous hypomorphicBNIP1variant causes an increase in autophagosomes and reduced autophagic flux and results in a spondylo‐epiphyseal dysplasia

“…The enzymatic activities of the lysosomal enzymes arylsulfatase B, β-glucuronidase, β-hexosaminidase, and α-iduronidase in protein extracts of cultured fibroblasts were assayed by estimation of 4-nitrophenol or 4-methylumbelliferone liberated from the enzymespecific substrate as described previously (Di Lorenzo et al, 2018;Zeng et al, 2019). Cells were harvested and whole-cell lysates were immunoblotted with anti-LC3B and anti-GAPDH antibodies.…”

Chemoenzymatic glycan-selective remodeling of a therapeutic lysosomal enzyme with high-affinity M6P-glycan ligands. Enzyme substrate specificity is the name of the game

“…[6][7][8][9] Thus, enhancement of the CI-MPR-mediated endocytosis represents a major strategy to improve the overall efficiency of the ERT-based treatments. [10][11][12] Toward this end, several approaches for increasing M6P modication of lysosomal enzymes have been attempted, including chemical conjugation of synthetic M6P-containing glycans, [13][14][15][16][17][18][19][20][21][22] construction of non-mammalian based platforms to improve the M6P content, [23][24][25][26] gene engineering of the glycosylation pathways, 27,28 and a chemoenzymatic remodeling approach to introduce synthetic phosphorylated N-glycans. 29,30 There are nice examples that the resulting modied enzymes show increased binding to CI-MPR and enhanced uptake by cells compared with the unmodied enzymes.…”