Production of α-L-iduronidase in maize for the potential treatment of a human lysosomal storage disease

Abstract

Lysosomal storage diseases are a class of over 70 rare genetic diseases that are amenable to enzyme replacement therapy. Towards developing a plant-based enzyme replacement therapeutic for the lysosomal storage disease mucopolysaccharidosis I, here we expressed α-l-iduronidase in the endosperm of maize seeds by a previously uncharacterized mRnAtargeting-based mechanism. Immunolocalization, cellular fractionation and in situ RT-PCR demonstrate that the α-l-iduronidase protein and mRnA are targeted to endoplasmic reticulum (ER)-derived protein bodies and to protein body-ER regions, respectively, using regulatory (5′-and 3′-uTR) and signal-peptide coding sequences from the γ-zein gene. The maize α-l-iduronidase exhibits high activity, contains high-mannose n-glycans and is amenable to in vitro phosphorylation. This mRnA-based strategy is of widespread importance as plant n-glycan maturation is controlled and the therapeutic protein is generated in a native form. For our target enzyme, the n-glycan structures are appropriate for downstream processing, a prerequisite for its potential as a therapeutic protein.

Lysosomal enzyme replacement therapies: Historical development, clinical outcomes, and future perspectives

“…Hence, strategies to reduce plant-specific maturation have been attempted [199]. One approach tried to retain proteins in the ER, preventing their transit to Golgi compartments, attempted for production of α-L-iduronidase in maize seeds [201]. Another approach deals with trafficking proteins from the ER directly to vacuolar compartments bypassing the Golgi, as for glucocerebrosidase produced from carrot cells [202, 203].…”

Brassica rapa hairy root based expression system leads to the production of highly homogenous and reproducible profiles of recombinant human alpha‐L‐iduronidase

“…Indeed, the site specific glycosylation pattern that characterize Aldurazyme is: Asn‐110, complex type glycans; Asn‐190, complex type glycans; Asn‐336, bisphosphorylated oligomannosidic glycan (P2Man7GlcNAc2); Asn‐372, high mannose type glycans (mainly Man9GlcNAc2, some of which are monoglucosylated); Asn‐415, mixed oligomannosidic and complex type glycans; Asn‐451, bisphosphorylated oligomannosidic glycan (P2Man7GlcNAc2; Zhao et al ., ); which contrasts with the single paucimmanose profile found in all N‐glycosylation sites of rIDUA_RLT. Similarly, the recombinant IDUA proteins produced in other plant‐based expression systems than the hairy roots are characterized by a heterogeneity of the N‐glycosylation profiles with nevertheless a predominance of high mannose profiles (He et al ., , ). The absence of such a large heterogeneity when analysing the N‐ glycosylation profiles of the rIDUA_RLT produced using the B. rapa hairy root system is of particular interest at a regulatory level to increase the reproducibility of the batches that may be used in clinical trials.…”

“…Regarding the treatment of other lysosomal disorders, the addition of mannose‐6‐phosphate (M6P) residues would be ideally required in vivo as the plants are not naturally able to phosphorylate the mannose residue s . Several strategies are described in the literature allowing the addition of such residues on plant‐based recombinant proteins (He et al ., , ). The existence of alternate M6P‐independent pathways for lysosomal enzyme sorting has also been largely described (Markmann et al ., ).…”

“…IDUA is a secreted protein presenting a signal peptide ( 1 M‐ 23 A) which is released in its final secreted form and six potential N ‐glycosylation sites as well as hydroxylation. Such protein have already being produced in plants (Acosta et al ., ; He et al ., , ; Pierce et al ., ) and in CHO cells (Aldurazyme ® , Sanofi, Paris, France; Kakkis et al ., ). This gave the opportunity to compare the characteristics of the rIDUA_RLT protein produced in hairy root clones with its benchmark produced by using a mammalian‐based production system or other production systems.…”

In situ Recovery of hCTLA4Ig from Suspension Cell Cultures of Oryza sativa

“…결과적으로 목적생 산물의 in situ 회수를 통해 하류 공정의 단계도 줄이면서 부 피당 생산성까지 증가시키는 효과를 얻을 수 있다. 형질전환 식물세포를 이용한 재조합 단백질 생산은 세포의 내부에 단백질을 축적해 생산하거나, 세포 외부로 단백질을 분비하여 생산한다 [9,10]. 세포 외부로의 재조합 단백질의 분비는 세포를 파쇄하지 않고 배지 내 단백질의 분석과 정제 를 할 수 있기 때문에 생물공정에 있어 유리하다 [11].…”