Development of hypoallergenic GMO-foods: friend or foe?
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1 Development of hypoallergenic GMO-foods: friend or foe?
2 Food allergy no cure so far symptom suppression with antiallergic medication not reliable avoidance measures=only causal treatment social burden/retreat and isolation severe allergic reactions (life threatening) Longo G et al.. Lancet 2013 Jul 8 (Epub ahead of print)
3 causes of life threatening allergic reactions (adults) From: German anaphylaxis registry
4 causes of life threatening allergic reactions (children) Notfälle From: German anaphylaxis registry
5 Thresholds (objective symptoms) single dose cumulative dose carrot (mg) Dau c Dau c Dau c 4 (µg) carrot (mg) Dau c Dau c Dau c 4 (µg) Foetisch K et al.. J Allergy Clin Immunol 2013; 131: e2
6 Allergen content (storage effects) Foetisch K, Scheurer S, Vieths S, Hanschmann KM, Lidholm J, Mahler V. Identification of allergen-resolved threshold doses of carrot (Daucus carota) by means of oral challenge and ELISA. J Allergy Clin Immunol 2013; 131: e2
7 If you can t change the individual, change the environment. Ronald van Ree, EAACI congress 2009, Warsaw
8 Therapeutic target structures in planta? Silencing of allergens? RNAinterference (RNAi) natural mechanism of gene silencing
9 Allergen reduction in plant-foods: Arachis hypogaea Ara h 2 Dodo HW et al.. Alleviating peanut allergy using genetic engineering: the silencing of the immunodominant allergen Ara h 2 leads to its significant reduction and a decrease in peanut allergenicity. Plant Biotechnol J 2008;6:135-45
10 Allergen reduction in plant-foods: Malus domestica Mal d 1 Gilissen LJ, Bolhaar ST, Matos CI, Rouwendal GJ, Boone MJ, Krens FA, Zuidmeer L, Van Leeuwen A, Akkerdaas J, Hoffmann-Sommergruber K, Knulst AC, Bosch D, Van de Weg WE, Van Ree R. Silencing the major apple allergen Mal d 1 by using the RNA interference approach. J Allergy Clin Immunol 2005;115:364-9.
11 Allergen reduction in plant-foods: Daucus carota Dau c 1 Peters S, Imani J, Mahler V, Foetisch K, Kaul S, Paulus KE, Scheurer S, Vieths S, Kogel KH. Dau c 1.01 and Dau c 1.02-silenced transgenic carrot plants show reduced allergenicity to patients with carrot allergy. Transgenic Res 2011; 20:547-56
12 Allergen reduction in plant-foods: Lycopersicon esculentum Lyc e 1 Le LQ, Mahler V, Lorenz Y, Scheurer S, Biemelt S, Vieths S, Sonnewald U. Reduced allergenicity of tomato fruits harvested from Lyc e 1-silenced transgenic tomato plants. J Allergy Clin Immunol 2006; 118:
13 Allergen reduction in plant-foods: Lycopersicon esculentum Lyc e 2 Paulus KE, Mahler V, Pabst M, Kogel KH, Altmann F, Sonnewald U. Silencing β1,2-xylosyltransferase in Transgenic Tomato Fruits Reveals xylose as Constitutive Component of Ige-Binding Epitopes. Front Plant Sci 2011; 2:42.
14 Allergen reduction in plant-foods: Lycopersicon esculentum Lyc e 3 Le LQ, Lorenz Y, Scheurer S, Fotisch K, Enrique E, Bartra J, Biemelt S, Vieths S, Sonnewald S. Design of tomato fruits with reduced allergenicity by dsrnai-mediate inhibition of ns-ltp (Lyc e 3) expression Plant Biotechnol J 2006; 4:231-42
15 Why tomato as model allergen source?
16 Tomato as model allergen source Prevalence of tomato allergy 16% in food allergic populations Nature of tomato allergens are largely known well characterized plant species established transformation protocols non-glycosylated (Lyc e 1) and glycosylated (Lyc e 2) allergens can be studied
17 Lyc e 1 (Profilin): crossreactive model allergen
18 RNAi-construct for tomato profilin (constitutive silencing) XbaI MspI FokI XbaI EcoRI FokI MspI HindIII p35s attr1 PFN attr2 INTRON attr2 PFN attr1 T35S IgG-Immunoblot: Strong reduction of profilin accumulation green fruit extracts WT WT WT WT WT WT red fruit extracts
19 Profilin (Lyc e 1)-deficient tomato plants 15 cm WT PFN_RNAi 11 PFN_RNAi 32 PFN_RNAi 39 reduction of plant growth and fruit set Le LQ et al. J Allergy Clin Immunol 2006; 118:
20 Yeast profilin complementation of profilin deficient transgenic tomato fruits?
21 Comparison of YPFN and Lyc e 1 folding structure Lyc e 1 YPNF
22 AA sequence alignment of YPFN and Lyc e 1 Cross-reactivity expected, if 35% aa-id
23 Purification and circular dichroism spectroscopy of rypfn and rlyc e 1 Lyc e 1 YPFN
24 dsrnai inhibition of tomato profilin and over-expression of yeast profilin in transgenic tomato plants (A) Tandem orientation of dsrnai and OX genes p35s PFN I PFN T p35s Yeast PFN T (B) Immunoblot analysis of transgenic tomato leaves
25 Complementation of profilin deficiency by overexpression of yeast profilin WT YPFN/PFN_RNAi PFN_RNAi Correlation of green biomass and residual Lyc e 1: WT phenotype: >5% residual Lyc 1 or simulataneous YPFN complementation Le LQ et al.. FASEB J 2010; 24:
26 Dose dependent IgE-Inhibition to rlyc e 1 (ELISA) Inhibition [%] microtom wild type Lyce1_RNAi/YPFN (71) Lyce1_RNAi/YPFN (28) Lyce1_RNAi/YPFN (53) rypfn rlyc e ,0E-03 1,0E-02 1,0E-01 1,0E+00 1,0E+01 1,0E+02 1,0E+03 Inhibitor [µg/ml]
27 Reduced in vivo allergenic reactivity of YPFN/Lyc e1_rnai crops Patient 1 Patient 2 Hist. Hist. 0.9 % sodium chloride WT WT Lyc e 1_RNAi Lyc e 1_RNAi YPFN/PFN_RNAi YPFN/PFN_RNAi
28 Conclusion allergenic potential of plant-derived food can be reduced by RNAi yeast profilin supplementation enables regular plant growth and fruits set minor allergens contribute to the overall IgE-binding multiple silencing strategies are required oral food challenge (read out system) popularity of GMO-food? EFSA Panel on Genetically Modified Organisms (GMO Panel). Scientific Opinion on the assessment of allergenicity of GM plants and microorganisms and derived food and feed. EFSA Journal 2010: 8:1700
29 Outlook Multi-target silencing of multiple allergens at once (in planta) clinical validation of hypoallergenic RNAi silenced and supplemented crops by oral challenge application of new breeding techniques for hypoallergenic non-gmo variants (e.g. TILLING=Targeting Induced Local Lesions in Genomes, Transcription activator-like effector nucleases (TALENs)) identification of naturally occuring low allergenic variants Paulus KE, Schmid B, Zajic D, Schäfer A, Mahler V, Sonnewald U. Hypoallergenic profilin: a new way to identify allergenic determinants. FEBS J 2012:279: JRC Scientific and Technical Report: New plant breeding techniques
30 Coworkers and cooperations Coworkers Andrea Kerpes Antje Golka Maria Portisch Andreas Maronna Cooperation partners Lehrstuhl für Biochemie, FAU Erlangen Prof. U. Sonnewald Dr. S. Sonnewald K. Paulus Alice Schäfer Paul-Ehrlich Institut, Langen Prof. S. Vieths Dr. S. Scheurer Dr. K Fötisch Sponsors DFG Adolf-Rohrschneider- Stiftung Inst. für Phytopathologie und angewandte Zoologie, Giessen Prof. K. Kogel Dr. J. Imani Susanna Peters
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Accepted: 7 April 2009
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