Generation of a transgenic rice seed-based edible vaccine against house dust mite allergy

As an alternative approach to conventional allergen-specific immunotherapy, transgenic rice seed expressing a major house dust mite (HDM) allergen, Der p 1, was developed as an edible vaccine. The C-terminal KDEL-tagged Der p 1 allergen specifically accumulated in seed endosperm tissue under the control of the endosperm-specific GluB1 promoter. Der p 1 reached a maximum concentration of 58 μg/grain and was deposited in the endoplasmic reticulum (ER)-derived protein body I (PB-I). Plant-derived Der p 1 was posttranslationally modified with high-mannose-type glycan structures. Glycosylated Der p 1 displayed reduced IgE binding capacity in comparison with its unglycosylated counterpart in vitro. Our results indicate that transgenic Der p 1 rice seeds are a safe, potential oral delivery vaccine for the treatment of HDM allergy.     

Prevention of allergic asthma by vaccination with transgenic rice seed expressing mite allergen: induction of allergen-specific oral tolerance without bystander suppression

This study tested the feasibility of oral immunotherapy for bronchial asthma using a newly developed subunit vaccine in which a fragment (p45-145) of mite allergen (Der p 1) containing immunodominant human and mouse T cell epitopes was encapsulated in endoplasmic reticulum-derived protein bodies of transgenic (Tg) rice seed. Allergen-specific serum immunoglobulin responses, T cell proliferation, Th1/Th2 cytokine production, airway inflammatory cell infiltration, bronchial hyper-responsiveness (BHR) and lung histology were investigated in allergen-immunized and -challenged mice. Prophylactic oral vaccination with the Tg rice seeds clearly reduced the serum levels of allergen-specific IgE and IgG. Allergen-induced CD4(+) T cell proliferation and production of Th2 cytokines in vitro, infiltration of eosinophils, neutrophils and mononuclear cells into the airways and BHR were also inhibited by oral vaccination. The effects of the vaccine were antigen-specific immune response because the levels of specific IgE and IgG in mice immunized with Der f 2 or ovalbumin were not significantly suppressed by oral vaccination with the Der p 1 expressing Tg rice. Thus, the vaccine does not induce nonspecific bystander suppression, which has been a problem with many oral tolerance regimens. These results suggest that our novel vaccine strategy is a promising approach for allergen-specific oral immunotherapy against allergic diseases including bronchial asthma.     

Formation mechanism of the internal structure of type I protein bodies in rice endosperm: relationship between the localization of prolamin species and the expression of individual genes

Rice prolamins, a group of seed storage proteins, are synthesized on the rough endoplasmic reticulum (ER) and form type I protein bodies (PB-Is) in endosperm cells. Rice prolamins are encoded by a multigene family. In this study, the spatial accumulation patterns of various prolamin species in rice endosperm cells were investigated to determine the mechanism of formation of the internal structure of PB-Is. Immunofluorescence microscopic analysis of mature endosperm cells showed that the 10 kDa prolamin is mainly localized in the core of the PB-Is, the 13b prolamin is localized in the inner layer surrounding the core and the outermost layer, and the 13a and 16 kDa prolamins are localized in the middle layer. Real-time RT-PCR analysis showed that expression of the mRNA for 10 kDa prolamin precedes expression of 13a, 13b-1 and 16 kDa prolamin in the developing stages. mRNA expression for 13b-2 prolamin occurred after that of the other prolamin species. Immunoelectron microscopy of developing seeds showed that the 10 kDa prolamin polypeptide initially accumulates in the ER, and then 13b, 13a, 16 kDa and 13b prolamins are stacked in layers within the ER. Studies with transgenic rice seeds expressing prolamin-GFP fusion proteins under the control of native and constitutive promoters indicated that the temporal expression pattern of prolamin genes influenced the localization of prolamin proteins within the PB-Is. These findings indicate that the control of gene expression of prolamin species contributes to the internal structure of PB-Is.     

Oral immunotherapy against a pollen allergy using a seed-based peptide vaccine

Peptide immunotherapy using dominant T-cell epitopes is safer and more effective than conventional immunotherapy for the treatment of immunoglobulin E (IgE)-mediated allergic diseases. When allergenic T-cell epitope peptides are expressed in the edible part of transgenic plants, successful mucosal immune tolerance to these allergens may be attainable by the consumption of these plants. In this study, we generated transgenic rice seed that accumulated high concentrations (about 60 microg per grain) of polypeptide consisting of seven dominant human T-cell epitopes derived from the Japanese cedar pollen allergens, Cry j 1 and Cry j 2, in the endosperm. Oral administration of these transgenic rice seeds to B10.S mice before or after they were immunized with Cry j 1 holoprotein reduced not only their T-cell proliferative response to Cry j 1, but also their serum IgE levels, proving the efficacy of oral immunotherapy for the treatment of pollinosis.     

Secretory type of recombinant thioredoxin h induces ER stress in endosperm cells of transgenic rice

Thioredoxin h (TRX h) functions as a reducing protein and is present in all organisms. As a new approach for inducing the endoplasmic reticulum (ER) stress, TRX h (OsTRX23) was expressed as a secretory protein using the endosperm-specific glutelin GluB-1 promoter and a signal peptide. In transgenic rice seeds, the majority of the recombinant TRX h accumulated in the ER but some was also localized to the protein body IIs (PB-IIs). The rice grain quality was dependent on the TRX h accumulation level. Increased TRX h expression resulted in aberrant phenotypes, such as chalky and shriveled features, lower seed weight and lower seed protein content. Furthermore, the accumulation of some seed storage proteins (SSPs) was significantly suppressed and the morphology of the protein bodies (PB-Is and PB-IIs) changed according to the level of TRX h. SSPs, such as 13 kDa prolamin and GluA, were specifically modified via the reducing action of TRX h. These changes led to the activation of the ER stress response, which was accompanied by the expression of several chaperone proteins. Specifically, the ER stress markers BiP4 and BiP5 were significantly up-regulated by an increase in the level of TRX h. These results suggest that changes in the conformation of certain SSPs via the action of recombinant TRX h lead to an induced ER stress response in transgenic rice seeds.     

Expression of house dust mite allergens Der f 1 and Der f 2 in leaves of Nicotiana benthamiana

In test systems for diagnostics of type I allergy, recombinant allergens in native conformation are used, because immunoglobulins E (IgE), responsible for the development of this type of allergy, recognize conformational epitopes of protein allergens. To obtain recombinant allergens of the Dermatophagoides farinae house dust mites (HDM), Der f 1 and Der f 2, two systems of heterological expression were used: Escherichia coli and Nicotiana benthamiana plants. IgE from sera of children allergic to HDM were shown to recognize the recombinant Der f 2 protein synthesized in both E. coli and N. benthamiana plants, as well as the recombinant Der f 1 protein produced in N. benthamiana plants, while mature form of Der f 1 produced in E. coli did not interact with IgE.     

Oral administration of recombinant human granulocyte-macrophage colony stimulating factor expressed in rice endosperm can increase leukocytes in mice

Human granulocyte-macrophage colony stimulating factor (hGM-CSF) is used clinically to treat leucopenia typically caused by cancer chemotherapy or radiotherapy. This study used multiple strategies to obtain very high expression levels of OsrhGM-CSF (14 mug/seed) in rice endosperm. Electron micrographs of immunogold-labeled transgenic endosperm showed that rhGM-CSF was not only localized in protein bodies but was also distributed in the apoplast. A biological activity assay indicated that OsrhGM-CSF stimulated the growth of TF-1 cells in vitro. In addition, the transgene was used to effectively treat leucopenia by oral administration of the unprocessed transgenic grains. In cyclophosphamide-induced leucopenic mice, transgenic seeds produced a 27% (t = 0.021) gain in leukocytes after 14 days feeding. Even in non-leucopenic mice, leukocyte gain was 37% (t = 0.002) more than that of mice fed non-transgenic seeds. This study provides a novel approach to the use of oral unprocessed transgenic OsrhGM-CSF seeds to treat leucopenia.     

Different anti-apoptotic effects of normal and asthmatic serum on normal eosinophil apoptosis depending on house dust mite-specific IgE

We investigated the effect of asthmatic serum on constitutive eosinophil apoptosis in normal subjects. Eosinophil apoptosis in normal subjects was inhibited by asthmatic serum but not normal serum. In a detailed analysis based on the presence of house dust mite (HDM) IgE, HDM IgE-positive (+) asthmatic serum was more effective for eosinophil apoptosis than that of HDM IgE-negative (?) asthmatic serum. HDM IgE+ asthmatic serum inhibited both HDM IgE? and HDM IgE+ normal eosinophil apoptosis, and HDM IgE? asthmatic serum suppressed eosinophil apoptosis of HDM IgE+ normal. HDM IgE? normal serum did not inhibit either HDM IgE? or HDM IgE+ normal eosinophil apoptosis, and HDM IgE+ normal serum inhibited HDM IgE+ normal eosinophil apoptosis. The kind of HDM IgE (Dermatophagoides pteronissinus-specific IgE and Dermatophagoides farinae-specific IgE) was not related to the effect of asthmatic serum on eosinophil apoptosis. Extracts of DP and DF, Der p1, and Der p2, were not effective for eosinophil apoptosis. HDM IgE+ asthmatic serum inhibited cleavage of procaspase 9 and procaspase 3. Asthmatic serum induced Akt and ERK phosphorylation, and ERK activation was suppressed by AKTi. Taken together, asthmatic serum inhibited normal eosinophil apoptosis via PI3K/Akt/ERK cascade. The novel approach taken in this study provided better insight into HDM-associated anti-apoptotic mechanism of eosinophils in patients with asthma.     

Accumulation of rice prolamin–GFP fusion proteins induces ER-derived protein bodies in transgenic rice calli

Key message

We showed that rice prolamin polypeptides formed ER-derived PBs in transgenic rice calli, and that this heterologous transgene expression system is suitable for studying the mechanism of rice PB-I formation.

Abstract

Rice prolamins, alcohol-soluble seed storage proteins, accumulate directly within the rough endoplasmic reticulum (ER) lumen, leading to the formation of ER-derived type I protein bodies (PB-Is) in rice seed. Because rice prolamins do not possess a well-known ER retention signal such as K(H)DEL, or a unique sequence for retention in the ER such as a tandem repeat domain of maize and wheat prolamins, the mechanisms of prolamin accumulation in the ER and PB-I formation are poorly understood. In this study, we examined the formation mechanisms of PBs by expressing four types of rice prolamin species fused to green fluorescent protein (GFP) in transgenic rice calli. Each prolamin–GFP fusion protein was stably accumulated in rice calli and formed ER-derived PBs. In contrast, GFP fused with the signal peptide of prolamin was secreted into the intercellular space in rice calli. In addition, each of the four types of prolamin–GFP fusion proteins was co-localized with the ER chaperone binding protein. These results suggest that the mature polypeptide of prolamin is capable of being retained in the ER and induce the formation of PBs in non-seed tissue, and that the rice callus heterologous transgene expression system is useful for studying the mechanisms of rice PB-I formation.     

Development of transgenic rice seed accumulating a major Japanese cedar pollen allergen (Cry j 1) structurally disrupted for oral immunotherapy

Rice seed-based edible vaccines expressing T-cell epitope peptides derived from Japanese cedar major pollen allergens have been used to successfully suppress allergen-specific Th2-mediated immunoglobulin E (IgE) responses in mouse experiments. In order to further expand the application of seed-based allergen-specific immunotherapy for controlling Japanese cedar pollinosis, we generated transgenic rice plants that specifically express recombinant Cry j 1 allergens in seeds. Cry j 1 allergens give low specific IgE-binding activity but contain all of the T-cell epitopes. The allergens were expressed directly or as a protein fusion with the major rice storage protein glutelin. Fusion proteins expressed under the control of the strong rice endosperm-specific GluB-1 promoter accumulated in rice endosperm tissue up to 15% of total seed protein. The fusion proteins aggregated with cysteine-rich prolamin and were deposited in endoplasmic reticulum-derived protein body I. The production of transgenic rice expressing structurally disrupted Cry j 1 peptides with low IgE binding activity but spanning the entire Cry j1 region can be used as a universal, safe and effective tolerogen for rice seed-based oral immunotherapy for cedar pollen allergy in humans and other mammals.     

Control of foreign polypeptide localization in specific layers of protein body type I in rice seed

Key message

Prolamin–GFP fusion proteins, expressed under the control of native prolamin promoters, were localized in specific layers of PB-Is. Prolamin–GFP fusion proteins were gradually digested from outside by pepsin digestion.

Abstract

In rice seed endosperm, protein body type I (PB-I) has a layered structure consisting of prolamin species and is the resistant to digestive juices in the intestinal tract. We propose the utilization of PB-Is as an oral vaccine carrier to induce mucosal immune response effectively. If vaccine antigens are localized in a specific layer within PB-Is, they could be protected from gastric juice and be delivered intact to the small intestine. We observed the localization of GFP fluorescence in transgenic rice endosperm expressing prolamin–GFP fusion proteins with native prolamin promoters, and we confirmed that the foreign proteins were located in specific layers of PB-Is artificially. Each prolamin–GFP fusion protein was localized in specific layers of PB-Is, such as the outer-most layer, middle layer, and core region. Furthermore, to investigate the resistance of prolamin–GFP fusion proteins against pepsin digestion, we performed in vitro pepsin treatment. Prolamin–GFP fusion proteins were gradually digested from the peripheral region and the contours of PB-Is were made rough by in vitro pepsin treatment. These findings suggested that prolamin–GFP fusion proteins accumulating specific layers of PB-Is were gradually digested and exposed from the outside by pepsin digestion.

     

Expression and localization of human lysozyme in the endosperm of transgenic rice

In order to understand the characteristics of recombinant protein expression and sublocalization in rice ( Oryza sativa L.) endosperm, we examined the expression level of human lysozyme protein and its subcellular location in transgenic rice seeds driven by rice glutelin and globulin promoters and signal peptides. A time course of human lysozyme expression during endosperm development was analyzed. The results showed that the expression profile of recombinant protein accumulation in endosperm paralleled that of the two storage proteins. Immunofluorescence microscopy revealed that human lysozyme and storage proteins co-localized to type-II protein bodies. Both promoter-signal peptide parings targeted recombinant protein to the protein bodies. In addition, a transgenic line with a higher lysozyme expression level exhibited morphologically different protein bodies with an unbalanced composition of lysozyme and native storage proteins. The high-level expression of recombinant protein distorted the trafficking and sorting of native storage proteins in rice endosperm and affected the expression of native storage protein.     

Characterization of an immunomodulatory Der p 2-FIP-fve fusion protein produced in transformed rice suspension cell culture

Der p 2, a major allergen of Dermatophagoides pteronyssinus mites, is one of the most clinically relevant allergens to allergic patients worldwide. FIP-fve protein (Fve) from the golden needle mushroom (Flammulina velutipes) is an immunomodulatory protein with potential Th1-skewed adjuvant properties. Here, we produced and immunologically evaluated a Der p 2-Fve fusion protein as a potential immunotherapeutic for allergic diseases. Using an inducible expression system in cultured rice suspension cells, the recombinant Der p 2-Fve fusion protein (designated as OsDp2Fve) was expressed in rice cells under the control of an α-amylase gene (αAmy8) promoter and secreted under sucrose starvation. OsDp2Fve was partially purified from the cultured medium. The conformation of Der p 2 in OsDp2Fve remains intact as reflected by its unaltered allergenicity, as assessed by human IgE ELISA and histamine release assays, compared to non-fusion Der p 2 protein. Furthermore, the Fve protein expressed in OsDp2Fve retains its in vitro lymphoproliferative activity but loses its hemagglutination and lymphoagglutination effects compared to the native protein. Notably, in vivo evaluation showed that mice administered with OsDp2Fve possessed an enhanced production of Der p 2-specific IgG antibodies without potentiating the production of Der p 2-specific IgE and Th2 effector cytokines in comparison with mice co-administered with native Fve and Der p 2 proteins. These results suggest that the recombinant Der p 2-Fve fusion protein produced in rice suspension cell cultures has a great potential for allergy immunotherapy.     

Oral immunotherapy with transgenic rice seed containing destructed Japanese cedar pollen allergens,Cry j 1 and Cry j 2, against Japanese cedar pollinosis

Transgenic rice accumulating the modified major Japanese cedar pollen allergens, Cryptomeria japonica 1 (Cry j 1) and Cryptomeria japonica 2 (Cry j 2), which were deconstructed by fragmentation and shuffling, respectively, in the edible part of the seed was generated by transformation of a good‐tasting rice variety, ‘Koshihikari’. These modified cedar pollen antigens were deposited in ER‐derived protein bodies (PB‐I), which are suitable for delivery to the mucosal immune system in gut‐associated lymphoid tissue when orally administered because antigens bioencapsulated in PB‐I are resistant against hydrolysis by intestinal enzymes and harsh environments. Mice fed transgenic seeds daily for three weeks and then challenged with crude cedar pollen allergen showed marked suppression of allergen‐specific CD4⁺ T‐cell proliferation, IgE and IgG levels compared with mice fed nontransgenic rice seeds. As clinical symptoms of pollinosis, sneezing frequency and infiltration of inflammatory cells such as eosinophils and neutrophils were also significantly reduced in the nasal tissue. These results imply that oral administration of transgenic rice seeds containing the structurally disrupted Cry j 1 and Cry j 2 antigens, serving as universal antigens, is a promising approach for specific immunoprophylaxis against Japanese cedar pollinosis.     

Induction of Allergic Responses to Peanut Allergen in Sheep

Peanut allergy is the leading cause of deaths due to food-induced anaphylaxis but despite continued research, there are currently no specific treatments available. Challenge testing is limited in patients due to the high risk of adverse reactions, emphasising the need for an appropriate animal model. In the present study we examine the induction of allergic responses in a sheep model for peanut allergy. Sheep were sensitised with peanut (PN) extract and in separate injections with ovalbumin (OVA) or house dust mite (HDM) extract. Serum PN-specific IgE responses were detected in 40–50% of immunised sheep, while only 10% (1 of 10 sheep) showed detectable OVA-specific IgE. All PN-allergic sheep tested showed an Ara h 1-specific IgE response, while four out of five allergic sheep showed an Ara h 2-specific IgE response. Animals with high serum IgE levels to HDM were also PN IgE-positive. Of the PN-sensitised animals with high PN-specific IgE, 80% also showed an immediate hypersensitivity reaction following an intradermal PN injection. This new large animal model of peanut allergy may provide a useful tool for future investigations of allergen-associated immune mechanisms and specific immunotherapy.     

High-level production of lactostatin, a hypocholesterolemic peptide, in transgenic rice using soybean A1aB1b as carrier

Hypercholesterolemia, a form of cardiovascular disease, is one of the leading causes of deaths worldwide. Lactostatin (Ile-Ile-Ala-Glu-Lys), derived from β-lactoglobulin in cow’s milk, is a bioactive peptide with hypocholesterolemic activity higher than sitosterol, a known anti-hypercholesterolemic drug. Here, we successfully developed a transgenic rice accumulating a much higher level of lactostatin by inserting 29 IIAEK sequences into the structurally flexible (nonconserved) regions of soybean seed storage protein, A1aB1b, and introducing it into LGC-1 (low glutelin content mutant 1) as host variety. A1aB1b containing 29 lactostatins was expressed in the endosperm of rice seed cells by using seed specific promoters and sorted into novel compartments distinct from normal PB-I (ER-derived protein body) and PB-II (protein storage vacuoles). Transgenic rice seeds accumulated approximately 2 mg of lactostatins/g of dry seeds, which is relatively high compared with previous reports. Our findings suggest that the introduction of a high copy number of bioactive peptide into seed storage proteins as carrier is one of the effective means in producing higher amounts of bioactive peptides in rice.     

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

Peanut allergy is one of the most life-threatening food allergies and one of the serious challenges facing the peanut and food industries. Current proposed solutions focus primarily on ways to alter the immune system of patients allergic to peanut. However, with the advent of genetic engineering novel strategies can be proposed to solve the problem of peanut allergy from the source. The objectives of this study were to eliminate the immunodominant Ara h 2 protein from transgenic peanut using RNA interference (RNAi), and to evaluate the allergenicity of resulting transgenic peanut seeds. A 265-bp-long PCR product was generated from the coding region of Ara h 2 genomic DNA, and cloned as inverted repeats in pHANNIBAL, an RNAi-inducing plant transformation vector. The Ara h 2-specific RNAi transformation cassette was subcloned into a binary pART27 vector to construct plasmid pDK28. Transgenic peanuts were produced by infecting peanut hypocotyl explants with Agrobacterium tumefaciens EHA 105 harbouring the pDK28 construct. A total of 59 kanamycin-resistant peanut plants were regenerated with phenotype and growth rates comparable to wild type. PCR and Southern analyses revealed that 44% of plants stably integrated the transgene. Sandwich ELISA performed using Ara h 2-mAbs revealed a significant ( P <  0.05) reduction in Ara h 2 content in several transgenic seeds. Western immunobloting performed with Ara h 2-mAb corroborated the results obtained with ELISA and showed absence of the Ara h 2 protein from crude extracts of several transgenic seeds of the T₀ plants. The allergenicity of transgenic peanut seeds expressed as IgE binding capacity was evaluated by ELISA using sera of patients allergic to peanut. The data showed a significant decrease in the IgE binding capacity of selected transgenic seeds compared to wild type, hence, demonstrating the feasibility of alleviating peanut allergy using the RNAi technology.     

Deposition mode of transforming growth factor-β expressed in transgenic rice seed

Key message

Mouse TGF-β highly accumulated by expressing as a secretory homodimeric protein in transgenic rice endosperm. It was tightly deposited in ER-derived PBs by interaction with cysteine-rich prolamins.

Abstract

TGF-β is one of the key players involved in the induction and maintenance of mucosal immune tolerance to dietary proteins through the induction of regulatory T cells. In order to utilize rice-based TGF-β as a tool to promote oral immune tolerance induction, high production of TGF-β is essentially required. When the codon-optimized mTGF-β was expressed as a secretory protein by ligating an N-terminal signal peptide and C-terminal KDEL ER retention signal under the control of the endosperm-specific rice storage protein glutelin GluB-1 promoter, accumulation level was low in stable transgenic rice seeds. Then, to increase the accumulation level of mTGF-β, it was expressed as fusion proteins by inserting into the C terminus of acidic subunit of glutelin GluA and the variable region of 26 kDa globulin. When fused with the glutelin, it could accumulate well as visible bands by CBB staining gel, but not for the 26 kDa globulin. Unexpectedly, expression of homodimeric mTGF-β linked by a 6×Gly1×Ser linker as secretory protein resulted in higher level of accumulation. This expression level was further enhanced by reduction of some endogenous prolamins by RNA interference. The monomeric and dimeric mTGF-βs were deposited in ER-derived PBs containing prolamins. When highly produced in rice seed, it is notable that most of ER-derived PBs were distorted and granulated. Step-wise extraction of storage proteins from rice seeds suggested that the mTGF-β strongly interacted with cysteine-rich prolamins via disulfide bonds. This result was also supported by the finding that reducing agent was absolutely required for mTGF-β extraction.

     

Native and Artificial Reticuloplasmins Co-Accumulate in Distinct Domains of the Endoplasmic Reticulum and in Post-Endoplasmic Reticulum Compartments

We compared the subcellular distribution of native and artificial reticuloplasmins in endosperm, callus, and leaf tissues of transgenic rice (Oryza sativa) to determine the distribution of these proteins among endoplasmic reticulum (ER) and post-ER compartments. The native reticuloplasmin was calreticulin. The artificial reticuloplasmin was a recombinant single-chain antibody (scFv), expressed with an N-terminal signal peptide and the C-terminal KDEL sequence for retrieval to the ER (scFvT84.66-KDEL). We found that both molecules were distributed in the same manner. In endosperm, each accumulated in ER-derived prolamine protein bodies, but also in glutelin protein storage vacuoles, even though glutelins are known to pass through the Golgi apparatus en route to these organelles. This finding may suggest that similar mechanisms are involved in the sorting of reticuloplasmins and rice seed storage proteins. However, the presence of reticuloplasmins in protein storage vacuoles could also be due to simple dispersal into these compartments during protein storage vacuole biogenesis, before glutelin deposition. In callus and leaf mesophyll cells, both reticuloplasmins accumulated in ribosome-coated vesicles probably derived directly from the rough ER.     

Rice Allergenic Protein and Molecular-genetic Approach for Hypoallergenic Rice

Allergenic proteins with a molecular mass of about 14 to 16 kDa were isolated from a rice salt-soluble fraction based on the reactivity with IgE antibodies from patients allergic to rice. cDNA clones encoding these allergenic proteins were isolated from a cDNA library of maturing rice seeds, and the deduced amino acid sequences showed considerable similarity to wheat and barley α-amylase/trypsin inhibitors, which have recently been identified as major allergens associated with baker’s asthma. An antisense RNA strategy was applied to repress the allergen gene expression in maturing rice seeds. Immunoblotting and ELISA analyses of the seeds using a monoclonal antibody to a 16-kDa allergen showed that allergen content of seeds from several transgenic rice plants was markedly lower than that of the seeds from parental wild type rice.