Production of recombinant human granulocyte macrophage‐colony stimulating factor in rice cell suspension culture with a human‐like N‐glycan structure

Abstract

SummaryThe rice a-amylase 3D promoter system, which is activated under sucrose-starved conditions, has emerged as a useful system for producing recombinant proteins. However, using rice as the production system for therapeutic proteins requires modifications of the N-glycosylation pattern because of the potential immunogenicity of plant-specific sugar residues. In this study, glyco-engineered rice were generated as a production host for therapeutic glycoproteins, using RNA interference (RNAi) technology to down-regulate the endogenous a-1,3-fucosyltransferase (a-1,3-FucT) and b-1,2-xylosyltransferase (b-1,2-XylT) genes. N-linked glycans from the RNAi lines were identified, and their structures were compared with those isolated from a wild-type cell suspension. The inverted-repeat chimeric RNA silencing construct of a-1,3-fucosyltransferase and b-1,2-xylosyltransferase (D3FT ⁄ XT)-9 glyco-engineered line with significantly reduced core a-1,3-fucosylated and ⁄ or b-1,2-xylosylated glycan structures was established. Moreover, levels of plant-specific a-1,3-fucose and ⁄ or b-1,2-xylose residues incorporated into recombinant human granulocyte ⁄ macrophage colony-stimulating factor (hGM-CSF) produced from the N44 + D3FT ⁄ XT-4 glyco-engineered line co-expressing ihpRNA of D3FT ⁄ XT and hGM-CSF were significantly decreased compared with those in the previously reported N44-08 transgenic line expressing hGM-CSF. None of the glyco-engineered lines differed from the wild type with respect to cell division, proliferation or ability to secrete proteins into the culture medium.

Biological significance of complex N-glycans in plants and their impact on plant physiology

“…Developments during the last 10 years have shown that plants are amenable to glyco-engineering and capable of producing valuable recombinant glycoproteins with defined human-like structures (Castilho and Steinkellner, 2012; Nagels et al, 2012; Bosch et al, 2013). The absence of any growth phenotype in Arabidopsis cgl1 laid the foundation for N -glycan engineering of other species like Nicotiana benthamiana and Lemna minor as well as of rice suspension cells (Cox et al, 2006; Strasser et al, 2008; Shin et al, 2011). In these studies, gene silencing of XYLT and FUT11/12 was used to eliminate the non-human and potentially immunogenic β1,2-xylose and core α1,3-fucose residues from complex N -glycans of recombinant proteins.…”

Improving Pharmaceutical Protein Production in Oryza sativa

“…The first report of human GM-CSF (hGM-CSF) production in rice was in 2003 through suspension cell cultures, and the maximum yield obtained was 129 mg/L [30]. Since then, improvements have been made in rice suspension cell systems producing hGM-CSF by using methods such as humanizing N -Glycan structure and increasing yield by 2–3 folds through reduction of endogenous α-amylase expression, co-expression of proteinase inhibitor, and suppression of cellular cysteine proteinase [8,58–61]. The protein is also produced in other expression systems such as rice seeds and tobacco seeds using rice glutelin promoters Gt1 and Gt3 .…”

Engineering of human-type O-glycosylation inNicotiana benthamianaplants

“…These two glycosyltransferases are not present in mammals and the administration of glycoprotein therapeutics with xylosylated and fucosylated N-glycans might therefore lead to adverse immunogenic reactions in mammals 16 . As a consequence the N-glycosylation pathway of several plant species has been engineered in a way to eliminate the expression of the corresponding non-mammalian glycosyltransferases 17 - 21 . The glyco-engineered N. benthamiana plants known as ΔXF plants produce predominately GlcNAc 2 Man 3 GlcNAc 2 structures on recombinant proteins 20 .…”