Silencing the major apple allergen Mal d 1 by using the RNA interference approach

L.J.W.J. Gilissen, S.T.H.P. Bolhaar, C.I. Matos, G.J.A. Rouwendal, M.J. Boone, F.A. Krens, L. Zuidmeer, A. van Leeuwen, J.H. Akkerdaas, K. Hoffmann-Sommergruber, A.C. Knulst, H.J. Bosch, W.E. van de Weg, R. van Ree

    Research output: Contribution to journalArticleAcademicpeer-review

    121 Citations (Scopus)

    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
    Original languageEnglish
    Pages (from-to)364-369
    JournalJournal of Allergy and Clinical Immunology
    Volume115
    Issue number2
    DOIs
    Publication statusPublished - 2005

    Fingerprint

    Malus
    RNA Interference
    Allergens
    Gene Silencing
    Immunoglobulin E
    Immunoblotting
    Introns
    Antibodies
    Hypersensitivity
    Plant RNA
    Inverted Repeat Sequences
    Betula
    Polymerase Chain Reaction
    Pollen
    Skin Tests
    RNA
    Phenotype
    Skin
    Growth

    Keywords

    • birch pollen allergen
    • mal d 1
    • immunological relationship
    • potato
    • gene
    • ige
    • identification
    • arabidopsis
    • extracts
    • epitopes

    Cite this

    Gilissen, L.J.W.J. ; Bolhaar, S.T.H.P. ; Matos, C.I. ; Rouwendal, G.J.A. ; Boone, M.J. ; Krens, F.A. ; Zuidmeer, L. ; van Leeuwen, A. ; Akkerdaas, J.H. ; Hoffmann-Sommergruber, K. ; Knulst, A.C. ; Bosch, H.J. ; van de Weg, W.E. ; van Ree, R. / Silencing the major apple allergen Mal d 1 by using the RNA interference approach. In: Journal of Allergy and Clinical Immunology. 2005 ; Vol. 115, No. 2. pp. 364-369.
    @article{70e2d8df84194c359912ccde18d91a92,
    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",
    keywords = "birch pollen allergen, mal d 1, immunological relationship, potato, gene, ige, identification, arabidopsis, extracts, epitopes",
    author = "L.J.W.J. Gilissen and S.T.H.P. Bolhaar and C.I. Matos and G.J.A. Rouwendal and M.J. Boone and F.A. Krens and L. Zuidmeer and {van Leeuwen}, A. and J.H. Akkerdaas and K. Hoffmann-Sommergruber and A.C. Knulst and H.J. Bosch and {van de Weg}, W.E. and {van Ree}, R.",
    year = "2005",
    doi = "10.1016/j.jaci.2004.10.014",
    language = "English",
    volume = "115",
    pages = "364--369",
    journal = "Journal of Allergy and Clinical Immunology",
    issn = "0091-6749",
    publisher = "Elsevier",
    number = "2",

    }

    Gilissen, LJWJ, Bolhaar, STHP, Matos, CI, Rouwendal, GJA, Boone, MJ, Krens, FA, Zuidmeer, L, van Leeuwen, A, Akkerdaas, JH, Hoffmann-Sommergruber, K, Knulst, AC, Bosch, HJ, van de Weg, WE & van Ree, R 2005, 'Silencing the major apple allergen Mal d 1 by using the RNA interference approach', Journal of Allergy and Clinical Immunology, vol. 115, no. 2, pp. 364-369. https://doi.org/10.1016/j.jaci.2004.10.014

    Silencing the major apple allergen Mal d 1 by using the RNA interference approach. / Gilissen, L.J.W.J.; Bolhaar, S.T.H.P.; Matos, C.I.; Rouwendal, G.J.A.; Boone, M.J.; Krens, F.A.; Zuidmeer, L.; van Leeuwen, A.; Akkerdaas, J.H.; Hoffmann-Sommergruber, K.; Knulst, A.C.; Bosch, H.J.; van de Weg, W.E.; van Ree, R.

    In: Journal of Allergy and Clinical Immunology, Vol. 115, No. 2, 2005, p. 364-369.

    Research output: Contribution to journalArticleAcademicpeer-review

    TY - JOUR

    T1 - Silencing the major apple allergen Mal d 1 by using the RNA interference approach

    AU - Gilissen, L.J.W.J.

    AU - Bolhaar, S.T.H.P.

    AU - Matos, C.I.

    AU - Rouwendal, G.J.A.

    AU - Boone, M.J.

    AU - Krens, F.A.

    AU - Zuidmeer, L.

    AU - van Leeuwen, A.

    AU - Akkerdaas, J.H.

    AU - Hoffmann-Sommergruber, K.

    AU - Knulst, A.C.

    AU - Bosch, H.J.

    AU - van de Weg, W.E.

    AU - van Ree, R.

    PY - 2005

    Y1 - 2005

    N2 - 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

    AB - 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

    KW - birch pollen allergen

    KW - mal d 1

    KW - immunological relationship

    KW - potato

    KW - gene

    KW - ige

    KW - identification

    KW - arabidopsis

    KW - extracts

    KW - epitopes

    U2 - 10.1016/j.jaci.2004.10.014

    DO - 10.1016/j.jaci.2004.10.014

    M3 - Article

    VL - 115

    SP - 364

    EP - 369

    JO - Journal of Allergy and Clinical Immunology

    JF - Journal of Allergy and Clinical Immunology

    SN - 0091-6749

    IS - 2

    ER -