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.