Establishment of a tobacco BY2 cell line devoid of plant-specific xylose and fucose as a platform for the production of biotherapeutic proteins.

Uri Hanania,Dina Oz,Mara Weiss,Maor Sheva,Albina Turbovski,Yoram Tekoah,Yehuda Gubbay,Tami Ariel,Liat Fux,Yaniv Azulay,Yehava Forster,Yoseph Shaaltiel

doi:10.1111/pbi.12702

Abstract

SummaryPlant‐produced glycoproteins contain N‐linked glycans with plant‐specific residues of β(1,2)‐xylose and core α(1,3)‐fucose, which do not exist in mammalian‐derived proteins. Although our experience with two enzymes that are used for enzyme replacement therapy does not indicate that the plant sugar residues have deleterious effects, we made a conscious decision to eliminate these moieties from plant‐expressed proteins. We knocked out the β(1,2)‐xylosyltranferase (XylT) and the α(1,3)‐fucosyltransferase (FucT) genes, using CRISPR/Cas9 genome editing, in Nicotiana tabacum L. cv Bright Yellow 2 (BY2) cell suspension. In total, we knocked out 14 loci. The knocked‐out lines were stable, viable and exhibited a typical BY2 growing rate. Glycan analysis of the endogenous proteins of these lines exhibited N‐linked glycans lacking β(1,2)‐xylose and/or α(1,3)‐fucose. The knocked‐out lines were further transformed successfully with recombinant DNaseI. The expression level and the activity of the recombinant protein were similar to that of the protein produced in the wild‐type BY2 cells. The recombinant DNaseI was shown to be totally free from any xylose and/or fucose residues. The glyco‐engineered BY2 lines provide a valuable platform for producing potent biopharmaceutical products. Furthermore, these results demonstrate the power of the CRISPR/Cas9 technology for multiplex gene editing in BY2 cells.

Highlights

The use of plant cell suspension culture as a host for the production of recombinant proteins is gaining more and more popularity (Santos et al, 2016) and the Nicotiana tabacum (N. tabacum) cv
While there is no clinical evidence indicating differences in immunogenic response to plant-specific glycans versus mammalian-derived glycans (Santos et al, 2016; Tekoah and Shaaltiel, 2016), extensive research efforts have been undertaken in recent years to modulate the plant-specific N-glycosylation machinery aiming at the production of recombinant proteins with mammalian-like modifications
The first 2161-bp product was identical to a fragment of the published Ntab-XylT-A sequence and was labelled Bright yellow 2 (BY2)-XylT-A (Figure S1)

Summary

Introduction

The use of plant cell suspension culture as a host for the production of recombinant proteins is gaining more and more popularity (Santos et al, 2016) and the Nicotiana tabacum (N. tabacum) cv. The transfer and the attachment of plant-specific sugar moieties to the developing glycan structure are carried out by the two Golgi resident enzymes a(1,3)-fucosyltransferase (FucT) and b(1,2)-xylosyltransferase (XylT) (Strasser et al, 2004). RNAi methodology was used to modulate the GMD gene, encoding the Guanosine 50-diphosphate (GDP)-Dmannose 4,6-dehydratase enzyme, which is associated with GDPL-fucose biosynthesis in Nicotiana benthamiana (N. benthamiana) plants. This resulted in N-glycans with decreased a(1,3)-fucose residues (Matsumura, 2010)

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