title: Silencing the major apple allergen Mal d 1 by using the RNA interference approach.
abstract: BACKGROUND
  Apple allergy is dominated by IgE antibodies against Mal d 1 in areas where birch pollen is endemic. Apples with significantly decreased levels of Mal d 1 would allow most patients in these areas to eat apples without allergic reactions.
  
  
  OBJECTIVE
  The aim of this study was to inhibit the expression of Mal d 1 in apple plants by RNA interference.
  
  
  METHODS
  In vitro -grown apple plantlets were transformed with a construct coding for an intron-spliced hairpin RNA containing a Mal d 1-specific inverted repeat sequence separated by a Mal d 1-specific intron sequence. The presence of the construct in transformants was checked by PCR. Expression of Mal d 1 in leaves was monitored by prick-to-prick skin testing in 3 patients allergic to apples and by immunoblotting with a Mal d 1-reactive mAb and with IgE antibodies against Mal d 1.
  
  
  RESULTS
  After transformation, plantlets were selected on the basis of having a normal phenotype and growth rate. With PCR, in 6 of 9 selected plantlets, the presence of the gene-silencing construct was demonstrated. By skin prick test it was shown that a wild-type plantlet had significantly ( P < .05) higher allergenicity than 5 of the transformants. Reduction of expression of Mal d 1 was confirmed by immunoblotting. In wild-type and unsuccessful transformants, a strong band was detected with Mal d 1-reactive mAb 5H8 at the expected apparent M r of 17 kDa. This band was virtually absent in the transformants that carried the gene-silencing construct. With human IgE antibodies, the same observations were made.
  
  
  CONCLUSIONS
  Mal d 1 expression was successfully reduced by RNA interference. This translated into significantly reduced in vivo allergenicity. These observations support the feasibility of the production by gene silencing of apples hypoallergenic for Mal d 1.
authors.affiliation: Allergy Consortium Wageningen, Wageningen, The Nertherlands. luud.gilissen@wur.nl
authors.forename: Luud J W J
authors.initials: LJ
authors.lastname: Gilissen
authors.forename: Suzanne T H P
authors.initials: ST
authors.lastname: Bolhaar
authors.forename: Catarina I
authors.initials: CI
authors.lastname: Matos
authors.forename: Gerard J A
authors.initials: GJ
authors.lastname: Rouwendal
authors.forename: Marjan J
authors.initials: MJ
authors.lastname: Boone
authors.forename: Frans A
authors.initials: FA
authors.lastname: Krens
authors.forename: Laurian
authors.initials: L
authors.lastname: Zuidmeer
authors.forename: Astrid
authors.initials: A
authors.lastname: Van Leeuwen
authors.forename: Jaap
authors.initials: J
authors.lastname: Akkerdaas
authors.forename: Karin
authors.initials: K
authors.lastname: Hoffmann-Sommergruber
authors.forename: André C
authors.initials: AC
authors.lastname: Knulst
authors.forename: Dirk
authors.initials: D
authors.lastname: Bosch
authors.forename: W Eric
authors.initials: WE
authors.lastname: Van de Weg
authors.forename: Ronald
authors.initials: R
authors.lastname: Van Ree
mesh_terms: D000328:Adult; D000485:Allergens / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D052179:Antigens, Plant; D005638:Fruit / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D010940:Plant Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D034622:RNA Interference; D012882:Skin Tests
chemical_list: D000485:Allergens; D052179:Antigens, Plant; C095551:MALD1 protein, Malus domestica; D010940:Plant Proteins