title: First successful reduction of clinical allergenicity of food by genetic modification: Mal d 1-silenced apples cause fewer allergy symptoms than the wild-type cultivar.
abstract: BACKGROUND
  Genetic modification of allergenic foods such as apple has the potential to reduce their clinical allergenicity, but this has never been studied by oral challenges in allergic individuals.
  
  
  METHODS
  We performed oral food challenges in 21 apple-allergic individuals with Elstar apples which had undergone gene silencing of the major allergen of apple, Mal d 1, by RNA interference. Downregulation of Mal d 1 gene expression in the apples was verified by qRT-PCR. Clinical responses to the genetically modified apples were compared to those seen with the wild-type Elstar using a visual analogue scale (VAS).
  
  
  RESULTS
  Gene silencing produced two genetically modified apple lines expressing Mal d 1.02 and other Mal d 1 gene mRNA levels which were extensively downregulated, that is only 0.1-16.4% (e-DR1) and 0.2-9.9% (e-DR2) of those of the wild-type Elstar, respectively. Challenges with these downregulated apple lines produced significantly less intense maximal symptoms to the first dose (Vmax1) than with Elstar (Vmax1 Elstar 3.0 mm vs 0.0 mm for e-DR1, P = 0.017 and 0.0 mm for e-DR2, P = 0.043), as well as significantly less intense mean symptoms per dose (meanV/d) than with Elstar (meanV/d Elstar 2.2 mm vs 0.2 mm for e-DR1, P = 0.017 and 0.0 mm for e-DR2, P = 0.043). Only one subject (5%) remained symptom-free when challenged with the Elstar apple, whereas 43% did so with e-DR1 and 63% with e-DR2.
  
  
  CONCLUSIONS
  These data show that mRNA silencing of Mal d 1 results in a marked reduction of Mal d 1 gene expression in the fruit and reduction of symptoms when these apples are ingested by allergic subjects. Approximately half of the subjects developed no symptoms whatsoever, and virtually all subjects wished to consume the apple again in the future.
authors.affiliation: Department of Pediatric Pulmonology and Pediatric Allergy, and GRIAC Research Institute, Beatrix Children's Hospital, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
authors.forename: A E J
authors.initials: AE
authors.lastname: Dubois
authors.affiliation: Wageningen UR Plant Breeding, Wageningen University and Research Centre, Wageningen, The Netherlands.
authors.forename: G
authors.initials: G
authors.lastname: Pagliarani
authors.affiliation: Department of Pediatric Pulmonology and Pediatric Allergy, and GRIAC Research Institute, Beatrix Children's Hospital, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
authors.forename: R M
authors.initials: RM
authors.lastname: Brouwer
authors.affiliation: Department of General Practice, and GRIAC Research Institute, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
authors.forename: B J
authors.initials: BJ
authors.lastname: Kollen
authors.affiliation: Department of Nutrition, Exercise and Sports, University of Copenhagen, Frederiksberg, Denmark.
authors.forename: L O
authors.initials: LO
authors.lastname: Dragsted
authors.affiliation: Technical University of Denmark, National Food Institute, Toxicology and Risk Assessment, Søborg, Denmark.
authors.forename: F D
authors.initials: FD
authors.lastname: Eriksen
authors.affiliation: Department of Horticulture, Faculty of Agricultural Sciences, University of Aarhus, Årslev, Denmark.
authors.forename: O
authors.initials: O
authors.lastname: Callesen
authors.affiliation: Bioscience, Wageningen University and Research Centre, Wageningen, The Netherlands.|Allergy Consortium Wageningen, Wageningen University and Research Centre, Wageningen, The Netherlands.
authors.forename: L J W J
authors.initials: LJ
authors.lastname: Gilissen
authors.affiliation: Wageningen UR Plant Breeding, Wageningen University and Research Centre, Wageningen, The Netherlands.
authors.forename: F A
authors.initials: FA
authors.lastname: Krens
authors.affiliation: Allergy Consortium Wageningen, Wageningen University and Research Centre, Wageningen, The Netherlands.
authors.forename: R G F
authors.initials: RG
authors.lastname: Visser
authors.affiliation: Wageningen UR Plant Breeding, Wageningen University and Research Centre, Wageningen, The Netherlands.|Allergy Consortium Wageningen, Wageningen University and Research Centre, Wageningen, The Netherlands.
authors.forename: M J M
authors.initials: MJ
authors.lastname: Smulders
authors.affiliation: Department of Pediatric Pulmonology and Pediatric Allergy, and GRIAC Research Institute, Beatrix Children's Hospital, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
authors.forename: B J
authors.initials: BJ
authors.lastname: Vlieg-Boerstra
authors.affiliation: Department of General Practice, and GRIAC Research Institute, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
authors.forename: B J
authors.initials: BJ
authors.lastname: Flokstra-de Blok
authors.affiliation: Wageningen UR Plant Breeding, Wageningen University and Research Centre, Wageningen, The Netherlands.|Allergy Consortium Wageningen, Wageningen University and Research Centre, Wageningen, The Netherlands.
authors.forename: W E
authors.initials: WE
authors.lastname: van de Weg
mesh_terms: D000328:Adult; D052179:Antigens, Plant / Q000235:genetics* / Q000276:immunology*; D015536:Down-Regulation; D005260:Female; D005512:Food Hypersensitivity / Q000175:diagnosis / Q000276:immunology* / Q000517:prevention & control; D015870:Gene Expression; D020868:Gene Silencing; D006801:Humans; D008297:Male; D027845:Malus / Q000009:adverse effects* / Q000235:genetics*; D010940:Plant Proteins / Q000235:genetics* / Q000276:immunology*; D030821:Plants, Genetically Modified; D055815:Young Adult
chemical_list: D052179:Antigens, Plant; C095551:MALD1 protein, Malus domestica; D010940:Plant Proteins
keywords: RNAi; apple allergy; food allergy; food challenge; genetically modified food