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

Verona, Italy - 24/27 September, 2003

ISBN 88-900622-4-X

Poster Abstract - 3.26

BOOSTING HUMAN GAD EXPRESSION IN TRANSGENIC PLANTS

L. AVESANI*, A. FALORNI**, E. DALLA POZZA*, G.B. TORNIELLI*, F. MORANDINI*, L. BORTESI*, M. PEZZOTTI*

*) Dip.Scientifico e Tecnologico, Università degli Studi di Verona

**) Dip. di Medicina Interna, Sezione di Medicina Interna e Scienze Endocrine e Metaboliche, Università degli Studi di Perugia

autoimmunity, GAD65, oral tolerance, transgenic plants

The smaller isoform of the enzyme glutamic acid decarboxylase (GAD65) is a major islet autoantigen in autoimmune type 1 diabetes mellitus (T1DM). Transgenic plants expressing human GAD65 (hGAD65) are a potential means of direct oral administration of the islet autoantigen in order to induce tolerance and prevent clinical onset of disease. We have previously reported the successful generation of transgenic tobacco and carrot that express immunoreactive, full-length hGAD65. Successively, we tested the hypothesis that the expression levels of recombinant hGAD65 in transgenic plants can be increased by targeting the enzyme to the plant cell cytosol and by the mediating expression through the potato virus X (PVX) vector. By substituting the NH2-terminal region of hGAD65 with a homologous region of rat GAD67, a chimeric GAD67/GAD65 molecule was expressed via-Agrobacterium in transgenic tobacco plants. Immunolocalisation analysis confirms cytosolic and a radio-immunoassay analysis demonstrates that expression levels of hGAD65 were increased five times compared with that obtained in the previous stable transformation (0.19% of the total soluble protein, compared to only 0.04% of wild-type hGAD65). Transient expression of wild-type, full-length hGAD65 in N. benthamiana mediated by PVX infection was associated with expression levels of immunoreactive protein as high as 2.2% of total soluble protein.

In the present study we report new strategies aimed to boost hGAD65 expression in tobacco plants in order to plan oral tolerance studies on animal models. To increase stable expression of hGAD65 in plants Agrobacterium-mediated gene transfer expression vectors were developed. By using such vectors we tested the hypothesis that the expression levels of hGAD65 could be enhanced by targeting the human protein to the ER and by using an enzymatic inactive form of the recombinant protein.

Moreover, to express hGAD65 in edible plant organs, transgenic tomato plants were also generated; therefore we constructed an Agro-based vector in which hGAD65 expression was driven by a tomato fruit-specific promoter.