Abstract
BackgroundThe deficiency of human acid beta-glucosidase (hGCase) causes Gaucher disease, a rare genetically-inherited disorder currently treated by enzyme replacement therapy using recombinant CHO-derived GCase. In an attempt to provide an alternative and more efficient production system, a chimeric cDNA coding for hGCase operatively linked to the signal peptide of rice glutelin 4 (GluB4) was put under the control of the GluB4 endosperm-specific promoter and inserted into the genome of a waxy rice.ResultsMolecular, immunological and biochemical analyses showed that recombinant hGCase, targeted to the protein storage vacuoles of rice endosperm cells, is equivalent to the native protein and has a glycosylation pattern compatible with direct therapeutic use. Compared to a previous study carried out on transgenic tobacco seeds, enzyme contents per unit of biomass were drastically increased; in addition, differently from what observed in tobacco, rice seed viability was unaffected by hGCase even at the highest production level. Transgenic seed polishing combined with a pretreatment of seed flour greatly facilitated hGCase extraction and purification with an industrially-scalable procedure.ConclusionsThis study opens up the possibility to efficiently produce in the rice seed pharmaceutical compounds which are available in limited amounts or completely excluded from clinical practice due to the inadequacy of their production systems.Electronic supplementary materialThe online version of this article (doi:10.1186/1939-8433-5-34) contains supplementary material, which is available to authorized users.
Highlights
The deficiency of human acid beta-glucosidase causes Gaucher disease, a rare genetically-inherited disorder currently treated by enzyme replacement therapy using recombinant CHO-derived GCase
Rice transformation and molecular analyses on primary transformants Compared to other japonica varieties, the waxy rice CR W3 is less responsive in vitro, preliminary experiments were done to select the best conditions for embryogenic callus formation and plant regeneration
The endogenous reference was the sucrose phosphate synthase (SPS) gene, which is present in a single copy in the haploid rice genome
Summary
The deficiency of human acid beta-glucosidase (hGCase) causes Gaucher disease, a rare genetically-inherited disorder currently treated by enzyme replacement therapy using recombinant CHO-derived GCase. Human acid beta-glucosidase (hGCase; EC 3.2.1.45) catalyzes the hydrolytic cleavage of glucose from glucosylceramide in lysosomes (Brady et al 1965). It is a homomeric glycoprotein of 497 amino acids with a molecular mass ranging from 60 to 68 kDa, containing five putative N-glycosylation sites, four of which are normally occupied (Berg-Fussman et al 1993; Brumshtein et al 2006). GD is currently treated by enzyme replacement therapy (ERT) using mainly a recombinant processed form of human acid beta-glucosidase (commercial imiglucerase) obtained from cultured CHO cells. The current production system based on CHO is economically very expensive and limits the drug amounts present on the market, seriously hindering the maintenance and the diffusion of therapeutic regimes
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