Fumio Takaiwa

Institute of Agrobiological Sciences, NARO, Tsukuba, Japan · Diviion of Biotechnology

Antigen-specific immunotherapy (AIT) is the only available potentially curative treatment that targets the cause of allergic or autoimmune diseases. Subcutaneous and sublingual AITs are currently achieved for 3–5 years using natural antigens, but they are sometimes accompanied by adverse effects such as anaphylaxis. Plant-based vaccines have been g...

Seed is an ideal bioreactor for the expression of high-value recombinant proteins. High production can be achieved by taking advantage of established seed-specific promoters and targeting signal. When they are produced in seeds and are deposited in the seed-specific subcellular compartments, large amounts (quantities) of expressed recombinant prote...

To exploit the rice seed-based oral vaccine against Sjögren’s syndrome, altered peptide ligand of N-terminal 1 (N1-APL7) from its M3 muscarinic acetylcholine receptor (M3R) autoantigen was expressed as fusion protein with the representative four types of rice prolamins (16 kDa, 14 kDa, 13 kDa, and 10 kDa prolamins) under the control of the individu...

We investigated the prophylactic and therapeutic effects of the oral administration of transgenic rice seeds expressing a hypoallergenic Bet v 1 derivative of allergic birch pollen conjunctivitis in mice. Transgenic rice seed depositing a chimeric molecule called TPC7 (tree pollen chimera 7) created by DNA shuffling of Bet v 1 family sequences from...

Background: We previously developed a transgenic rice that contains seven linked human predominant T-cell epitopes (7Crp) derived from Japanese cedar (JC) pollen allergens Cry j 1 and Cry j 2. Oral administration of 80 g of transgenic rice for 20 weeks suppressed allergen-specific T-cell proliferation in participants with JC pollinosis, but their c...

Fumio Takaiwa1,2 1Soul Signal Institute, Kojyohama, Shiraoi, Hokkaido, 059-0641, Japan; 2Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, 305-8602, JapanCorrespondence: Fumio TakaiwaInstitute of Agrobiological Sciences, National Agriculture and Food Research Organization, Kannondai 3-1-3,...

Human transforming growth factor-β1 (hTGF-β1) was produced in transgenic rice seeds. To boost its production yield and to extract it simply, it was expressed under the control of seed-specific promoters along with the simultaneous suppression of endogenous seed storage proteins (SSPs) through RNA interference (RNAi). When driven by the 26 kDa α-glo...

Japanese cedar (Cryptomeria japonica) pollinosis (JCP) is one of the major seasonal IgE-mediated type I allergies from February to April each year. Not only human patients but also Japanese monkeys (Macaca fuscata) are afflicted with this pollinosis in Japan, which exhibit similar clinical allergic symptoms such as allergenic rhinitis and conjuncti...

Background: A rice-based peptide vaccine containing 7 linked human predominant T-cell epitopes (7Crp) derived from Japanese cedar (JC) pollen allergens, Cry j 1 and Cry j 2, was developed. Here, we examined the efficacy and safety of this transgenic rice in JC pollinosis patients. Methods: Transgenic rice (5, 20, and 80 g) was administered orall...

Allergen-specific immunotherapy is only available curative treatment for IgE-mediated allergen diseases. Hypoallergenic allergen derivative with safety and high efficiency is required as tolerogen to induce immune tolerance to the causing allergens. In this study, to generate rice-based oral allergy vaccine for Japanese cedar (JC) pollinosis, terti...

Objective> We previously reported that Rag1-/- mice inoculated with splenocytes from M3 muscarinic acetylcholine receptor (M3R) knockout mice immunized with an M3R peptide mixture developed sialadenitis-like Sjögren’s syndrome (M3R-induced sialadenitis [MIS]). We also found that intravenous administration of altered peptide ligand (APL) of N-termin...

Transgenic rice seeds which contain genetically modified Cry j 1 and Cry j 2, the two major allergens of Cryptomeria japonica (Japanese cedar), have been developed as immunotherapeutic candidates for Japanese cedar pollinosis. Because the transgenic rice (TG‐rice) seeds express allergens containing whole amino acid sequences of Cry j 1 and Cry j 2...

Background Japanese cedar (JC) pollinosis is a serious type I allergic disease in Japan. Although subcutaneous immunotherapy and sublingual immunotherapy have been applied to treat JC pollinosis, high doses of allergens may cause IgE-mediated allergic reactions. The transgenic rice seeds that contain genetically modified Cry j 1 and Cry j 2, the tw...

Table S1 Relationship between the presence of T‐DNA (trigger gene) and gene silencing in progeny of CNX TGS, CNX PTGS, and OsbZIP50 TGS lines Table S2 Nucleotide sequence similarity among the coding regions (PTGS target sequences) and promoter regions (TGS target sequences) of rice glutelin genes Table S3 Gene‐specific primers used in this study...

Figure S2 TGS of the OsBiP1 gene is developmentally lethal.

Figure S1 Methylation analysis using restriction enzyme digestion in transgenic rice lines harbouring trigger genes to induce TGS of CNX, OsbZIP50, or Glb‐1.

Figure S3 TGS of the Glb‐1 gene in T1 and T2 generations with or without trigger gene.

Figure S4 Analysis of the relationship between the presence of the trigger gene and PTGS.

Key message Specific domain of the Mal d 1 was identified to be mainly involved in higher accumulation level in vegetative tissues of transgenic rice than the Bet v 1. Abstract Apple food allergen Mal d 1 and birch pollen allergen Bet v 1 belong to the same pathogen related protein 10 (PR10) family. When green fluorescent protein (GFP) fused to ei...

Pollinosis, or allergic conjunctivitis and rhinitis induced by pollen, is one of the most common diseases worldwide. In Japan, Japanese cedar (Cryptomeria japonica) pollinosis is a predominant allergic condition that affects more than one-third of all Japanese individuals. Pharmacological treatments of allergic conjunctivitis include administration...

Apple food allergen Mal d 1 and birch pollen allergen Bet v 1 belong to the same pathogen related protein 10 (PR10) family. When each of these allergens was expressed as a secretory protein by fusion with the GFP reporter in transgenic rice by ligating an N terminal signal peptide and a C terminal KDEL ER retention signal under the control of the m...

Rice-based peptide vaccine based on T cell epitopes acts as an ideal oral tolerogen for the treatment of type 1 allergic diseases. To improve production yields of oral tolerogen against Japanese cedar pollen allergy, hybrid peptide comprising seven predominant human T cell epitopes (7Crp) derived from Japanese cedar pollen allergens, Cry j 1 and Cr...

To induce transcriptional gene silencing (TGS) of endogenous genes of rice (Oryza sativa L.), we expressed double‐strand RNA of each promoter region and thus induced RNA‐directed DNA methylation (RdDM). We targeted constitutively expressed genes encoding calnexin (CNX), protein disulfide isomerase (PDIL1‐1), and luminal binding protein (BiP1); an e...

Artificial regulation of gene expression through RNA-directed DNA methylation (RdDM)-mediated epigenome editing is one the most important and attractive next-generation technologies for plant trait improvement, often called “new plant breeding techniques” (NPBTs). RdDM can induce transcriptional gene silencing (TGS) of a target gene via modificatio...

Key message: Bioactive peptide was produced by fusion to rice prolamins in transgenic rice seeds. Their accumulation levels were affected by their deposition sites and by compensatory rebalancing between prolamins within PB-Is. Peptide immunotherapy using analogue peptide ligands (APLs) is one of promising treatments against autoimmune diseases. U...

Flavonoids possess diverse health-promoting effects, including antioxidant, antiviral, anticancer, antiaging, and hepatoprotective effects. In this chapter, we introduce some studies on development of transgenic rice accumulating large amounts of flavonoids in seed by ectopic expression of the flavonoid biosynthetic enzyme genes or the transcriptio...

Cereal seed has been utilized as production platform for high-value biopharmaceutical proteins. Especially, protein bodies (PBs) in seeds are not only natural specialized storage organs of seed storage proteins (SSPs), but also suitable intracellular deposition compartment for recombinant proteins. When various recombinant proteins were produced as...

Background: We have previously shown that prophylactic oral administration of transgenic rice seeds expressing hypoallergenic modified antigens suppressed the development of allergic conjunctivitis induced by Japanese cedar pollen. We have now investigated the efficacy of oral immunotherapy with such transgenic rice for established allergic conjunc...

Objective: To investigate the effects and mechanisms of transgenic rice seeds expressing the altered peptide ligand (APL) of human glucose-6-phosphate-isomerase (hGPI325-339) in mice model of GPI induced arthritis (GIA). Methods: We generated transgenic rice expressing APL12 which was analog peptide of hGPI325-339. The transgenic rice seeds were...

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. TGF-β is one of the key players involved in the induction and maintenance of mucosal immune tolerance to dietary proteins through the indu...

Objective: To investigate the effects of transgenic rice seeds expressing the altered peptide ligand (APL) of human glucose-6-phosphate-isomerase (hGPI325-339) in mice model of GPI induced arthritis (GIA). Methods: We generated transgenic rice expressing T-cell epitope of hGPI325-339 and APL12 and contained in the seed endosperm. The transgenic...

Rice seeds are an ideal production platform for high-value recombinant proteins in terms of economy, scalability, safety, and stability. Strategies for the expression of large amounts of recombinant proteins in rice seeds have been established in the past decade and transgenic rice seeds that accumulate recombinant products such as bioactive peptid...

Transgenic rice seed can be utilized as a bioreactor to produce high-value recombinant proteins. Mouse interleukin 4 (mIL-4) and mIL-6 were specifically expressed as secretory proteins in rice endosperm by ligating the N-terminal glutelin B-1 (GluB-1) signal peptide and the C-terminal KDEL endoplasmic reticulum retention signal under control of the...

Recombinant hypoallergenic derivative is the next generation of tolerogen replacing the natural allergen extract to increase safety and efficacy. Japanese cedar pollinosis is the predominant seasonal allergy disease in Japan. A rice seed-based oral vaccine containing the recombinant hypoallergens derived from these allergens was developed. Efficacy...

In some eukaryotes, endoplasmic reticulum ( ER ) stress induces regulated inositol‐requiring enzyme 1 ( IRE 1)‐dependent decay ( RIDD ) of mRNA s. Recently, the expression levels of the mRNA s encoding some secretory proteins were reported to be downregulated by RIDD in the vegetative tissues of plants. However, the characteristics of plant RIDD ha...

Among 131 rice endosperm proteins previously identified by MS-based proteomics, most of the proteins showed low or almost no sequence similarity to known allergens in databases, whereas nine proteins did it significantly. The sequence of two proteins showed high overall identity with Hsp70-like hazel tree pollen allergen (Cor a 10) and barley α-amy...

Recombinant human interleukin-10 (hIL-10) is highly expressed in transgenic rice endosperm and forms active homodimers in the ER-derived hIL-10 body. In this study, we examined the preclinical efficacy of transgenic rice accumulating hIL-10 (hIL-10 rice) in in vivo experimental mouse models of colitis and pollen allergy. In the group of mice orally...

Plant-specialized (or secondary) metabolites represent an important source of high-value chemicals. In order to generate a new production platform for these metabolites, an attempt was made to produce flavonoids in rice seeds. Metabolome analysis of these transgenic rice seeds using liquid chromatography-photodiode array-quadrupole time-of-flight m...

Gut-associated lymphoid tissue (GALT) is the biggest lymphoid organ in the body. It plays a role in robust immune responses against invading pathogens while maintaining immune tolerance against nonpathogenic antigens such as foods. Oral vaccination can induce mucosal and systemic antigen-specific immune reactions and has several advantages includin...

Large amounts of seed storage proteins (SSPs) are produced in the maturing endosperm of rice seeds. Rice SSPs are synthesized as secretory proteins on the rough endoplasmic reticulum (ER), and are transported and deposited into protein complexes called protein bodies (PB-I and PB-II). Due to the high production of SSPs, unfolded SSPs may be generat...

Background/aims: To determine whether oral immunotherapy with transgenic rice seeds expressing hypoallergenic modified antigens suppresses cedar pollen–induced allergic conjunctivitis by eliciting immune tolerance in mice. Methods: BALB/c mice were fed once a day for 20 days with 220 mg of transgenic rice expressing modified Japanese cedar pollen a...

The endoplasmic reticulum-derived type-I protein body (PB-I) from rice endosperm cells is an ideal candidate formulation for the oral delivery of bioencapsulated peptides as tolerogens for allergen-specific immunotherapy. In the present study, PBs containing the deconstructed Japanese cedar pollen allergens Cryptomeria japonica 1 (Cry j 1) and Cry...

According to the amyloid hypothesis, amyloid β accumulates in brains with Alzheimer's disease (AD) and triggers cell death and memory deficit. Previously, we developed a rice Aβ vaccine expressing Aβ, which reduced brain Aβ levels in the Tg2576 mouse model of familial AD. We used senescence-accelerated SAMP8 mice as a model of sporadic AD and inves...

Rheumatoid arthritis is an autoimmune disease associated with the recognition of self proteins secluded in arthritic joints. We generated transgenic rice seeds expressing three types of altered peptide ligands (APL) and the T cell epitope of type II collagen (CII256-271). When these transgenic rice and non-transgenic rice seeds were orally administ...

Rice seed proteins contain antigens that provoke allergic responses in some individuals with food allergy, particularly in those with cereal allergy, and these antigens can elicit clinical symptoms such as eczema and dermatitis. We previously generated transgenic rice with reduced accumulation of the three major allergens, which dramatically reduce...

Tree pollen chimera 7 (TPC7), a hypoallergenic Bet v 1 tolerogen against birch pollen allergy, induces the formation of novel, huge protein bodies (referred to as TPC7 bodies) in rice endosperm, and is accumulated in high level. In the present study, we found that native Bet v 1 and TPC9, analog proteins of TPC7, were also deposited into novel prot...

Rheumatoid arthritis (RA) is an autoimmune disease associated with the recognition of self proteins secluded in arthritic joints. We previously reported that altered peptide ligands (APLs) of type II collagen (CII256-271) suppress the development of collagen-induced arthritis (CIA). In this study, we generated transgenic rice expressing CII256-271...

Extraction and purification of recombinant proteins rendered difficult to extract from transgenic plants by using an extraction solution containing reducing agents and surfactants or an extraction solution containing reducing agents and organic solvents.

The endoplasmic reticulum (ER) stress response is widely known to function in eukaryotes to maintain the homeostasis of the ER when unfolded or misfolded proteins are overloaded in the ER. To understand the molecular mechanisms of the ER stress response in rice (Oryza sativa L.), we previously analyzed the expression profile of stably transformed r...

Soybean 7S globulin, known as β-conglycinin, has been shown to regulate human plasma cholesterol and triglyceride levels. Furthermore, the α' subunit of β-conglycinin has specifically been shown to possess low-density lipoprotein (LDL)-cholesterol-lowering activity. Therefore, accumulation of the α' subunit of β-conglycinin in rice seeds could lead...

Peptide immunotherapy using dominant T-cell epitopes is a safe treatment alternative to conventional subcutaneous injection of natural crude allergen extract, which is sometimes accompanied by anaphylactic shock. For Japanese cedar pollinosis (JCP), hybrid peptides composed of six to seven major T-cell epitopes (7Crp peptide) from the causative all...

J-proteins are co-chaperone components of the HSP70 system. J-proteins stimulate Hsp70ATPase activity, which is responsible for stabilizing the interaction of Hsp70 with client proteins. J-proteins are localized in various intracellular compartments including the cytoplasm, mitochondria and endoplasmic reticulum (ER). Five types of ER resident J-pr...

Allergen-specific immunotherapy (SIT) is the only clinically available treatment that targets the cause of allergic disease and leads to long-lasting relief from allergic symptoms. Conventional treatment involves repeated subcutaneous injections with increasing doses of a crude allergen extract for at least 3 years, sometimes accompanied by adverse...

Japanese cedar pollen and house dust mite allergens are major causes of immunoglobulin (Ig)E-mediated type I allergy. For these allergies allergen-specific immunotherapy using systemic immunization with crude allergen extracts has been achieved as an only curative treatment. Here, we introduce a novel allergen-specific immunotherapy using rice seed...

A protein disulfide isomerase (PDI) family oxidoreductase, PDIL2-3, is involved in endoplasmic reticulum (ER) stress responses in rice. We identified a critical cis-element required for induction of the ER stress response. The activation of PDIL2-3 in response to ER stress strongly depends on the IRE1-OsbZIP50 signaling pathway.

The heat shock protein 70 (Hsp70) chaperone system participates in protein folding and quality control of unfolded proteins. To examine the roles of co-chaperones in the rice Hsp70 chaperone system in the endoplasmic reticulum (ER), the functions of six ER-resident J-proteins (OsP58A, OsP58B, OsERdj2, OsERdj3A, OsERdj3B, and OsERdj7) in rice were i...

The present inventors identified a novel rice glutelin gene, GluD-1, which is expressed specifically in seeds. The promoter of the GluD-1 gene was confirmed to induce seed-specific gene expression, and to induce expression of downstream genes specifically in the endosperm during the early stage of seed maturation process. More specifically, the Glu...

Rice (Oryza sativa L.) is the major staple food consumed by half of the world's population. Rice seeds have gained recent attention as bioreactors for the production of human pharmaceuticals such as therapeutic proteins or peptides. Rice seed production platforms have many advantages over animal cell or microbe systems in terms of cost-effectivenes...

Genetic modification (GM) by Agrobacterium-mediated transformation is a robust and widely employed method to confer new traits to crops. In this process, a transfer DNA is delivered into the host genome, but it is still unclear how the host genome is altered by this event at single-base resolution. To decipher genomic discrepancy between GM crops a...

IRE1 is an endoplasmic reticulum (ER) stress sensor protein in eukaryotes. In this study, we generated transgenic rice plants overexpressing three types of OsIRE1, including wild-type OsIRE1 (IRE1-OE) and two disrupted-IRE1s deficient in either kinase activity (K519A-OE) or RNase activity (K833A-OE), under the control of a constitutive promoter. Ov...

Pollen proteins from several grass species have been identified and characterized as causative allergens in grass pollinosis. By contrast, allergenic potential of pollen proteins from rice, which belongs to the same Poaceae family, has not well been investigated, despite that a few clinical cases have been reported on rice pollen allergy. In this s...

The membrane transport system is built on the proper functioning of the endoplasmic reticulum (ER). The accumulation of unfolded proteins in the ER lumen (ER stress) disrupts ER homeostasis and disturbs the transport system. In response to ER stress, eukaryotic cells activate intracellular signaling (named the unfolded protein response, UPR), which...

Seeds must maintain a constant level of nitrogen in order to germinate. When recombinant proteins are produced while endogenous seed protein expression is suppressed, the production levels of the foreign proteins increase to compensate for the decreased synthesis of endogenous proteins. Thus, exchanging the production of endogenous seed proteins fo...

Flavonoids possess diverse health-promoting benefits but are nearly absent from rice, because most of the genes encoding enzymes for flavonoid biosynthesis are not expressed in rice seeds. In the present study, a transgenic rice plant producing several classes of flavonoids in seeds was developed by introducing multiple genes encoding enzymes invol...

A versatile hypoallergenic allergen derivative against multiple allergens is an ideal tolerogen for allergen-specific immunotherapy. Such a tolerogen should exhibit high efficacy, without side effects, when administered at high doses and should be applicable to several allergens. Tree Pollen Chimera 7 (TPC7), a hypoallergenic Bet v 1 tolerogen agai...

Rice seed provides an ideal production platform for pharmaceuticals in terms of high productivity and stability, as well as the scalability, safety and economy that are expected in plant production systems. Furthermore, these therapeutic products are bioencapsulated in protein bodies, which are seed-specific storage organelles that provide protecti...

Rice seed has been used as a production platform for high value recombinant proteins. When mature human interleukin 7 (hIL-7) was expressed as a secretory protein in rice endosperm by ligating the N terminal glutelin signal peptide and the C terminal KDEL endoplasmic reticulum (ER) retention signal to the hIL-7 cytokine to improve production yield,...

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) lumen leads to ER stress. Intracellular signalling pathways are activated to alleviate the stress. The ER stress sensor IRE1 induces the active form of key transcription factors, such as XBP1 in mammals and bZIP50 in rice, by mediating unconventional splicing of their mRNAs. Al...

The endoplasmic reticulum (ER) stress sensor, IRE1, contains a kinase domain and a ribonuclease domain. Ribonuclease mediates the unconventional splicing of mRNA encoding the transcription factor AtbZIP60 in Arabidopsis, or OsbZIP50 in rice, and thereby transduces signals from stressed ER. Here, we demonstrate the additional roles of plant IRE1 usi...

Cholera toxin B subunit (CTB) is widely used as a carrier molecule and mucosal adjuvant and for the expression of fusion proteins of interest. CTB-fusion proteins are also expressed in plants, but the N-glycan structures of CTB have not been clarified. To gain insights into the N-glycosylation and N-glycans of CTB expressed in plants, we expressed...

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...

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 ce...

Conditions within the endoplasmic reticulum (ER) influence most secretory proteins that pass through the ER. Therefore, eukaryotic cells must strike a balance between the ER stress response, which changes the conditions in the ER, and other considerations associated with protein secretion. Here, an interaction between the ER stress and defence resp...

Supplementary information

Human IL-10 (hIL-10) is a therapeutic treatment candidate for inflammatory allergy and autoimmune diseases. Rice seed-produced IL-10 can be effectively delivered directly to gut-associated lymphoreticular tissue (GALT) via bio-encapsulation. Previously, the codon-optimized hIL-10 gene was expressed in transgenic rice with the signal peptide and end...

A method for Agrobacterium-mediated co-transformation of rice (Oryza sativa L.) was developed using rice-derived selection markers. Two T-DNAs were efficiently introduced into separate loci using selectable marker gene cassettes consisting of the mutated acetolactate synthase gene (mALS) under the control of the callus-specific promoter (CSP) (CSP:...

Posttranscriptional gene silencing (PTGS) is a sequence-specific mRNA degradation caused by small RNA, such as microRNA (miRNA) and small interfering RNA (siRNA). miRNAs are generated from MIRNA loci, whereas siRNAs originate from various sources of double-stranded RNA. In this study, an artificial RNA silencing inducible sequence (RSIS) was identi...

Binding protein (BiP) is a chaperone protein involved in the folding of secretory proteins in the ER lumen. OsBiP1 is constitutively expressed in various tissues, whereas the expression of OsBiP4 and OsBiP5 (OsBiP4&5) is not detected in any tissue under normal conditions. However, expression of OsBiP4&5 was highly and specifically activated under E...

Rab family proteins are small GTP-binding proteins involved in intracellular trafficking. They play critical roles in several plant development processes. Different expression patterns of 46 Rabs in the rice genome were examined in various rice tissues and in leaves treated with plant growth regulators and under senescence conditions. One of the Os...

Oral immunotherapy is potentially useful for the treatment of allergic diseases. We previously demonstrated that allergen-induced airway inflammation and immunoglobulin E (IgE) production in mice were suppressed by oral administration of high-dose transgenic (Tg) rice seeds (approximately 50 g/kg/day) expressing a T cell epitope of Dermatophagoides...