Espressione in planta di autoantigeni associati al diabete mellito di tipo I

Type 1 insulin-dependent diabetes mellitus (T1DM), afflicting 0.3-0.4% of human individuals, is the most frequent chronic disease of childhood. T1DM is a syndrome characterized by inappropriate hyperglycaemia due to a deficiency of insulin secretion caused by the autoimmune destruction of insulin-secreting pancreatic b cells. The young age of the affected patients, the need of life-long insulin therapy and the high prevalence of late-onset complications make T1DM a major health problem. No effective immunotherapy for the prevention of human T1DM, in either at-risk subjects (secondary prevention) or in the general population (primary prevention) is today available. Several immunoprevention strategies have been tested in human T1DM; however, based on the fact that at least 90% of T1DM subjects have no affected relatives, primary prevention is the ultimate goal. Actually there are no therapies available combining all the characteristics required for primary prevention: efficacy, safety, specificity of action, low cost and applicability to the general population. Studies in animal models of spontaneous autoimmune diabetes have shown that GAD65 plays a critical role in the destruction of pancreatic islets (Yoon et al., 1999). In fact, parenteral and nasal administration of GAD65 or GAD65- peptides can either prevent or delay the onset of disease (Kaufman et al., 1993; Tish et al., 1993; Peterson et al., 1994; Tian et al., 1996). Moreover, in rodents, immunological tolerance can also be induced by oral administration of diseaseassociated autoantigens (Zhang et al., 1991; Arakawa et al., 1998), suggesting that the clinical onset of T1DM might be prevented by oral prophylaxis with GAD65. Induction of oral tolerance would satisfy all the requirements for primary prevention with the exception of the present high cost for producing large quantities of recombinant human autoantigens. A major issue concerning the induction of oral tolerance is the dose of autoantigens to be fed. Studies on animal models of autoimmune diseases indicate that high doses could induce deletion or anergy of specific T cell clones while low to intermediated doses could activate regulatory T-cells in the gut, with subsequent active suppression (Chen et al, 1996). Induction of oral tolerance requires the prolonged administration of autoantigens in the range of milligrams/week/mouse (Zhang et al., 1991; Arakawa et al., 1998). Poor GAD protein solubility in bacteria and inadequate production from eukaryotic cells have so far precluded the use of this approach for the large-scale production of GAD65 in long-term oral tolerance studies. Transgenic plants expressing high levels of recombinant proteins would allow large quantities of autoantigens to be produced at relatively low costs. Moreover plants are highly amenable to oral administration and could provide local deliver of the drug directly to the gut associated lymphoid tissue (GALT), becoming effective delivery vehicles without the need for extensive protein purification. The production and characterization of transgenic plants expressing full-length recombinant human GAD65 has been previously reported (Porceddu et al., 1999; Avesani et al., 2003). Radioimmunoassay results indicate that hGAD65 expressed in transgenic plants was specifically immunoreactive with T1DM associated autoantibodies but the expression levels obtained in transgenic plants were not high enough to plan immunoprevention studies in animal models.