Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress

Giardini Naxos, Italy - 18/21 September, 2002

ISBN 88-900622-3-1

Oral Communication Abstract - S2d

AN INDUSTRIALLY-VIABLE PRODUCTION SYSTEM OF HUMAN PLACENTAL GLUCOCEREBROSIDASE FROM TRANSGENIC PLANTS

MARCHETTI S.*, REGGI S.**, PATTI T.***, BEMBI B.****, GARCIA R.*****, FOGHER C.******

*) DPVTA, Università di Udine, Via delle Scienze 208, 33100 Udine

**) Plantechno srl, Via Staffolo 60, 26040 Vicomoscano (CR)

***) TransPharma srl, AREA Science Park, Padriciano 99, 34012 Trieste

****) Laboratorio Malattie Rare, IRCCS Burlo Garofolo, Via dell’Istria 65/1, 34137 Trieste

*****) Leukocyte Biology Unit, ICGEB, AREA Science Park, Padriciano 99, 34012 Trieste

******) Istituto di Botanica e Genetica Vegetale, Università Cattolica, Via Emilia Parmense 84, 29100 Piacenza

glucocerebrosidase, Gaucher’s disease, tobacco, pharma production

Gaucher disease is the most common genetic lysosomal storage disorder. Due to the lack of functional b-glucocerebrosidase, patients accumulate glucosylceramide in tissue macrophages typically found in the liver, spleen and bone marrow. The severe effects on life quality and expectancy associated with glycosylceramide-congestion can be reversed by enzyme replacement therapy.

In the attempt to provide a safe and cost-effective way of glucocerebrosidase production, the complete coding sequence of human placental glucocerebrosidase was cloned in different plant expression vectors and inserted into tobacco (N. tabacum, cv. Xanthi) genome through Agrobacterium-mediated transformation. The expression system was found to have deep consequences on intact enzyme recovery. Furthermore, only when the glucocerebrosidase gene was put under the control of a seed-storage protein promoter was the enzyme accumulated at levels consistent with industrial-scale production. The signal peptide sequence of the soybean seed globulin 7S was effective in driving co-translationally the nascent polypeptide to the lumen of endoplasmic reticulum for glycosylation.

As far as enzyme purification is concerned, protocols were developed to approach total extraction from seeds, improve enzyme stability during processing and provide a rapid sorting of recombinant glucocerebrosidase from the bulk of soluble proteins. Even though further refinements are in progress, production levels of 250 U/Kg of seed can be anticipated. Therefore, 12 Kg of transgenic tobacco seed would provide the same enzyme amounts extractable from about 40,000 human placentae; these amounts also correspond to the total drug currently administered in Italy to a Gaucher patient of the age of 6 across one year.