Identification of target-structures for gene suppression and post-translational changes in plant foods and their impact upon alteration of the allergenicity and immunogenicity in man

• Applicant: Professorin Dr. Vera Mahler
• Subject Area: Dermatology
• Term: from 2007 to 2011
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 30717786

Based on the findings of the previous funding period the translational approach is further strengthened in this joint application.The major aims of this subproject for the requested funding period are(i) to characterize and express further relevant allergens from tomato(ii) to investigate the allergenicity of mutagenized profilin variants in vivo(iii) provide clinical evidence that RNAi multi-targeted silenced tomatoes and carrots are hypoallergenic.To achieve the first aim, the characterization and purification of the ubiquitous glycoproteins polygalacturonase (PG) and pectinesterase (PE), cDNA-cloning of the suspected target proteins will be pursued on the basis of the published genomic sequence by PCR.Subsequently, recombinant proteins will be overexpressed in prokaryotic and plant expression systems according to established methods. The recombinant proteins and transgenic plant material carrying a chimeric gene encoding a hairpin RNA directed against PG and PE will complement each other in the investigations on allergenicity.For the second aim selected profilin variants -identified by a yeast-based screening system which allows selection for functional variants- will be tested for their IgE-inducing capacity in vivo in a mouse model.Furthermore, to test for the clinical relevance of the mutations within the Lyc e 1 coding region for man, besides of IgE-Immunoblot examination of a larger group of patients, the respective recombinant Lyc e 1 variants will be produced and subsequently be used in skinprick- tests (SPT) by the applicant to analyse their allergenic potential in allergic volunteers. Moreover, T cell reactivity to specific hypoallergenic Lyc e 1 variants will be tested in T cell lines and clones obtained from profilin allergic patients.Our third aim, proof of principle of a sufficient allergen silencing in multi-target knock-down plants and its benefit for tomato and carrot allergic patients, we will first approach with IgEimmunoblots and SPTs with multiple-target knock-down fruits. Since these are surrogate markers for allergenicity, subsequently, a titrated food challenge as gold standard with the modified tomato fruits (without kernels) compared to the unmodified wild type is intended. Positive statements for these investigations have been obtained from the Bundesamt für Verbraucherschutz und Lebensmittelsicherheit and the Bayerische Landesamt für Gesundheit und Lebensmittelsicherheit, as well as consent of index patients.Additionally, we will identify threshold allergen levels of no observed adverse effect levels (NOAEL) and lowest observed adverse effect level (LOAEL) in Double Blind Placebo Controlled Food Challenges (DBPCFC) in parallel with specific measurement of individual allergens contained in WT carrot samples.Furthermore, we will maintain and expand our cohort of patients allergic to the model allergen plants to provide well characterized volunteers for all subprojects within this research consortium.

• DFG Programme: Research Grants