Lysosomal Targeting Enhancement by Conjugation of Glycopeptides Containing Mannose-6-phosphate Glycans Derived from Glyco-engineered Yeast

Abstract

Many therapeutic enzymes for lysosomal storage diseases require a high content of mannose-6-phosphate (M6P) glycan, which is important for cellular uptake and lysosomal targeting. We constructed glyco-engineered yeast harboring a high content of mannosylphosphorylated glycans, which can be converted to M6P glycans by uncapping of the outer mannose residue. In this study, the cell wall of this yeast was employed as a natural M6P glycan source for conjugation to therapeutic enzymes. The extracted cell wall mannoproteins were digested by pronase to generate short glycopeptides, which were further elaborated by uncapping and α(1,2)-mannosidase digestion steps. The resulting glycopeptides containing M6P glycans (M6PgPs) showed proper cellular uptake and lysosome targeting. The purified M6PgPs were successfully conjugated to a recombinant acid α-glucosidase (rGAA), used for the treatment of Pompe disease, by two-step reactions using two hetero-bifunctional crosslinkers. First, rGAA and M6PgPs were modified with crosslinkers containing azide and dibenzocyclooctyne, respectively. In the second reaction using copper-free click chemistry, the azide-functionalized rGAA was conjugated with dibenzocyclooctyne-functionalized M6PgPs without the loss of enzyme activity. The M6PgP-conjugated rGAA had a 16-fold higher content of M6P glycan than rGAA, which resulted in greatly increased cellular uptake and efficient digestion of glycogen accumulated in Pompe disease patient fibroblasts.

The glycosylation design space for recombinant lysosomal replacement enzymes produced in CHO cells

“…Extended circulation is predicted to enable wider biodistribution and possibly transport across the blood–brain barrier 48 . The achieved control of N-glycosylation in CHO cells meets or surpass the glycoengineering opportunities previously presented with non-mammalian cells and postproduction modification strategies 14–19,22–24,40 . The clinical features of LSDs and the organs affected differ greatly as do the biostructural properties of the respective deficient enzymes, and the design matrix and glycoengineered CHO cells developed here will be valuable tools for production and testing of optimal designs for individual ERTs, in order to improve a class of essential drugs with high costs and poor performance.…”

“…Yeast modify human lysosomal enzymes with Man-Pi-6-Man, but the elegant introduction of an uncovering α-mannosidase enzyme results in the production of α-glucosidase rich in M6P 15 . Other strategies to increase M6P content include in vitro chemical conjugation 22,24 , or co-expression of a truncated GlcNAc-1-phosphotransferase α/β precursor 54 . These strategies do not enable fine control over the content (or site specificity) of M6P and other glycan features including SA capping, and the presented engineering of high-M6P glycoforms in CHO cells fully match these strategies (Fig.…”

“…The first recombinant GBA with Man-terminated (high-Man) N-glycans was produced in CHO cells followed by postproduction exoglycosidase treatment 19 , and similar GBA products are produced in human cells by use of mannosidase I inhibitor (kifunensine) 20 or in carrot cells 21 . Most strategies for glycoengineering of lysosomal enzymes have sought to improve targeting by MPRs and MRs by increasing the content of M6P or exposed Man residues 15,22–24 . However, these glycoforms will cause rapid and efficient uptake by especially the liver and spleen, while targeting to other organs may be limited 4 .…”

Pompe Disease: From Basic Science to Therapy

“…This glyco-engineered enzyme greatly improved muscle pathology and function even in hard-to-treat old KO mice, whereas rhGAA was inactive [107]. Another glyco-engineered rhGAA with a high content of M6P glycan has been recently successfully tested in fibroblasts from Pompe patients [108].…”

Exploiting toxin internalization receptors to enhance delivery of proteins to lysosomes for enzyme replacement therapy

“…Several strategies have been employed to increase the extent of M6P labeling on recombinantly produced lysosomal enzymes: engineering mammalian and yeast cell lines to produce more specific/uniform N-glycan modifications, 17,24,25 chemical or enzymatic modification of N-glycans post-translationally, 26 and covalent coupling of M6P 27 . Some of these strategies have made incremental improvements to uptake but do nothing to address the relatively poor affinity of M6P for its receptor which is in the low uM range.…”