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. 2023 Feb 24:11:1128371.
doi: 10.3389/fbioe.2023.1128371. eCollection 2023.

A universal GlycoDesign for lysosomal replacement enzymes to improve circulation time and biodistribution

Yen-Hsi Chen  1   2 , Weihua Tian  1   3 , Makiko Yasuda  4 , Zilu Ye  1   5 , Ming Song  1 , Ulla Mandel  1 , Claus Kristensen  2 , Lorenzo Povolo  1 , André R A Marques  6 , Tomislav Čaval  1   7 , Albert J R Heck  7 , Julio Lopes Sampaio  8 , Ludger Johannes  8 , Takahiro Tsukimura  4   9 , Robert Desnick  4 , Sergey Y Vakhrushev  1 , Zhang Yang  1   10 , Henrik Clausen  1
Affiliations

A universal GlycoDesign for lysosomal replacement enzymes to improve circulation time and biodistribution

Yen-Hsi Chen et al. Front Bioeng Biotechnol. .

Abstract

Currently available enzyme replacement therapies for lysosomal storage diseases are limited in their effectiveness due in part to short circulation times and suboptimal biodistribution of the therapeutic enzymes. We previously engineered Chinese hamster ovary (CHO) cells to produce α-galactosidase A (GLA) with various N-glycan structures and demonstrated that elimination of mannose-6-phosphate (M6P) and conversion to homogeneous sialylated N-glycans prolonged circulation time and improved biodistribution of the enzyme following a single-dose infusion into Fabry mice. Here, we confirmed these findings using repeated infusions of the glycoengineered GLA into Fabry mice and further tested whether this glycoengineering approach, Long-Acting-GlycoDesign (LAGD), could be implemented on other lysosomal enzymes. LAGD-engineered CHO cells stably expressing a panel of lysosomal enzymes [aspartylglucosamine (AGA), beta-glucuronidase (GUSB), cathepsin D (CTSD), tripeptidyl peptidase (TPP1), alpha-glucosidase (GAA) or iduronate 2-sulfatase (IDS)] successfully converted all M6P-containing N-glycans to complex sialylated N-glycans. The resulting homogenous glycodesigns enabled glycoprotein profiling by native mass spectrometry. Notably, LAGD extended the plasma half-life of all three enzymes tested (GLA, GUSB, AGA) in wildtype mice. LAGD may be widely applicable to lysosomal replacement enzymes to improve their circulatory stability and therapeutic efficacy.

Keywords: bioengineering; enzyme replacement therapy; glycoengineering; glycoprotein therapeutics; lysosomal storage disease.

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Conflict of interest statement

A patent application has been filed by the University of Copenhagen. GlycoDisplay ApS has license rights to the patent application. ZY, WT, CK, and HC are named co-inventors, and ZY, CK, and HC have financial interests in GlycoDisplay ApS. Y-HC is an employee of GlycoDisplay ApS. RD is a Consultant to Genzyme-Sanofi and Sangamo Therapeutics, Inc. He owns founder stock in Amicus Therapeutics and options for Sangmo Therapeutics, Inc. and receives royalities from Genzyme-Sanofi. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Depiction of the LAGD glycoengineering strategy for lysosomal enzymes. The three principle types of N-glycan structures are illustrated with the predicted outcomes indicated after KO of Gnptab in CHO cells. Further fine-tuning steps to obtain homogeneous complex biantennary N-glycans with α2-3 sialic acid capping are shown (Tian et al., 2019). Glycan symbols according to Symbol Nomenclature for Glycans format. Gal denotes galactose, Man denotes mannose, GlcNAc denotes N-Acetylglucosamine, Neu5Ac denotes N-Acetylneuraminic acid, Fuc denotes fucose, and P denotes phosphate.
FIGURE 2
FIGURE 2
GLA activities and Gb3 analysis in repeated dose study in Fabry mouse model. (A) GLA enzyme activity represented as relative fluorescence intensity in indicated organs of mice after infusion of 0.3 or 1.0 mg/kg Fabrazyme or GLA LAGD, or saline control (n = 5 in each group) every 2 weeks for 5 times. Included are data of single infusion studies at 1.0 mg/kg that were repeated to confirm findings of our previous study (Tian et al., 2019). (B) Gb3 substrate level quantified by shotgun lipidomics MS analysis of the organs of mice mentioned above. (C) Lyso Gb3 level in liver of mice mentioned above quantified by shotgun lipidomics MS analysis.
FIGURE 3
FIGURE 3
N-glycan analysis of purified recombinant lysosomal enzymes (AGA, GUSB, CTSD, TPP1, GAA, and IDS) produced in CHOWT and engineered CHO cells. (A) Summary of site-specific N-glycopeptide analysis of purified recombinant lysosomal enzymes (AGA, GUSB, CTSD and GAA) produced in CHOWT and engineered CHO cells. The most abundant glycan structure at N-glycosites of each enzyme produced in CHOWT and engineered CHO clones are displayed on the top and bottom panels, respectively, with arrows indicating genetic editing strategy. Question mark indicates that no site-specific glycan structure was obtained for the partucular site. All glycan structures at each glycosite were confirmed by tandem mass spectrometry (MS/MS) analysis. (B) Summary of released RapiFluor-labeled N-glycan profiling showing the ten most abundant predicted species (normalized to most abundant structure).
FIGURE 4
FIGURE 4
Intact MS analysis of GLA WT/glycodesign at the native conditions and after sialidase and phosphatase treatment. Most abundant glycan structures at N-glycosites (Asn108, Asn161, and Asn184) of GLA WT and GLA LAGD are illustrated next to the enzyme name. Mass deconvoluted spectra of GLA WT and GLA LAGD are shown on the left and right sides, respectively. Panel (A) shows native spectra with purple diamonds denoting the total number of sialic acid residues, as validated by sialidase treatment. Panel (B) shows deconvoluted spectra of sialidase treated enzyme, while Panel (C) shows that of sialidase and phosphatase treated enzyme.
FIGURE 5
FIGURE 5
Intact MS analysis of AGA WT/glycodesign at the native conditions. Mass deconvoluted spectra of AGA WT (top) and AGA LAGD (bottom) are shown.
FIGURE 6
FIGURE 6
PK study of (A) GLA, (B) AGA and (C) GUSB glycovariants in WT mice. Enzyme activities in plasma, represented as relative fluorescence intensity, after infusion of enzymes are shown. Blood samples were collected at 1, 20, 40, and 120 min after injection of GLA (n = 5 for WT and LAGD) and GUSB (n = 1 and 3 for WT and LAGD, respectively), and at 1, 20, 60, and 180 min after injection of AGA (n = 4 and 5 for WT and LAGD, respectively).
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Grants and funding

This work was supported by the Lundbeck Foundation, Novo Nordisk Foundation, Innovation Fund Denmark, and the Danish National Research Foundation (DNRF107). TČ and AH acknowledge support from the Netherlands Organization for Scientific Research (NWO) funding the Netherlands Proteomics Centre through the X-omics Road Map program (project 184.034.019) and further acknowledge the EU Horizon 2020 program INFRAIA project Epic-XS (Project 823839). LJ acknowledges support from Fondation pour la Recherche Médicale (EQU202103012926).