Prevention of Celiac Disease by Transgenic Removal of Prolamin T-cell Epitopes

通过转基因去除谷醇溶蛋白 T 细胞表位来预防乳糜泻

• 批准号: 7600499
• 负责人: DITER H VON WETTSTEIN
• 金额: $ 20.22万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-02 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7600499
• 关键词: Abstinence; Address; Autoimmune Process; Autoimmune Responses; Barley; Biological; C hordein; Celiac Disease; Cells; Cereals; Characteristics; Chronic; Code; Codon Nucleotides; CpG Islands; Cysteine; Cysteine Protease; D hordein; Development; Digestion; Disease; Drug Metabolic Detoxication; Elastin; Enzymes; Epithelium; Epitopes; Escherichia coli; Excision; Exposure to; Food; Generations; Genes; Genetic Transcription; Genome; Gliadin; Glutamine; Gluten; Goals; HLA Antigens; Hand; Human; Hypermethylation; In Vitro; Individual; Inflammatory; Ingestion; Injury; Intestinal Mucosa; Intestines; Knock-out; Knowledge; Lead; Left; Life; Methylation; Modeling; Molecular; Molecular Conformation; Molecular Weight; Monoclonal Antibodies; Morbidity - disease rate; Mucous Membrane; Mutation; Orthologous Gene; Pain; Pancreas; Patients; Peptide Hydrolases; Peptides; Perfusion; Plants; Population; Prevalence; Prevention; Prolamine; Proline; Proteins; Proteolysis; Rattus; Reaction; Recombinants; Research; Resistance; Rye cereal; Seeds; Site; Small Intestines; Sprue; Stomach; Structural Genes; Structure; Symptoms; System; T-Lymphocyte; T-Lymphocyte Epitopes; Testing; Tissues; Transcriptional Regulation; Transgenic Organisms; Vacuole; Wheat; brush border membrane; cellular microvillus; early childhood; effective therapy; glutenin; in vivo; interest; microbial; mortality; mutant; plant prolamin protein; prevent; prolyl oligopeptidase; promoter; research study; resilin

DESCRIPTION (provided by applicant): Celiac disease is the most common food-sensitive enteropathological condition in humans and caused by an autoimmune reaction against wheat, barley and rye prolamins. In human leukocyte antigen (HLA) DQ2 (or DQ8)-positive individuals exposure to gluten can lead to a painful chronic erasure of the microvilli of the epithelium in the intestine and to a permanent intolerance of dietary gluten. It is commonly detected in early childhood in cases with severe symptoms. In many patients symptoms arise only later in life and, if untreated, it is associated with increased morbidity and mortality. Despite its prevalence in most populations (up to 1 in 200), the only effective therapy is strict dietary abstinence from these food grains. Recent research has established that the autoimmune response results from the resistance to digestion of certain prolamine/glutamine rich peptides of the prolamins by gastric, pancreatic and brush-border membrane proteases. These peptides are taken up through the intestinal mucosa and initiate the autoimmune response. In this proposal we wish to address the following testable hypotheses for removal of the prolamin T-cell stimulating epitopes in barley and wheat. 1) The fully viable barley mutation lys3a prevents transcription of all prolamin genes requiring de-methylation of their promoters in the developing endosperm except of the single gene with a CpG island encoding the High Molecular (HMW) D-hordein. This prolamin is highly homologous to the wheat HMW prolamins that are responsible for baking quality. We ask the question if we can exploit this mutation to obtain barley grain that provides celiac safe food products. 2) The close evolutionary relationship of barley and wheat makes it likely that the two cereals employ the same transcriptional control system for the synthesis of the storage proteins in the grain. We want to test this by selecting ortholog mutations of lys3a in wheat and ask the question if it prevents the transcription of the gliadin genes but not the 6 HMW genes present in hexaploid wheat. 3) Proline/glutamine rich peptides containing T-cell stimulating epitopes can be detoxified by prolyl endopeptidases and the barley cysteine endoprotease EP-B in vitro and in vivo with rat intestinal perfusion experiments. We want to test if it is possible to synthesize these enzymes in transgenic barley and wheat during endosperm development, target the enzymes into the storage vacuoles and detoxify the prolamins during grain development.Celiac disease or sprue is the most common food-sensitive enteropathy in humans resulting from chronic inflammatory injury to the mucosa of the small intestine after ingestion of proline- and glutamine-rich prolamins present in wheat gluten as well as in barley and rye grain storage proteins. To date the only effective treatment of this debilitating disease is life-long strict abstinence from the staple food grains. The goal of this research is to eliminate the celiac causing epitopes of prolamins by exploiting the cell biological features of prolamin synthesis in the endosperm of barley and wheat.

描述（由申请人提供）：乳糜泻是人类最常见的食物敏感的肠道病理状况，由对小麦、大麦和黑麦蛋白的自身免疫反应引起。在人白细胞抗原（HLA） DQ2（或DQ8）阳性的个体中，暴露于谷蛋白可导致肠上皮微绒毛的慢性清除和对饮食谷蛋白的永久不耐受。通常在儿童早期发现有严重症状的病例。在许多患者中，症状仅在生命后期才出现，如果不加以治疗，则与发病率和死亡率增加有关。尽管它在大多数人群中普遍存在（高达1 / 200），但唯一有效的治疗方法是严格禁食这些食物。最近的研究表明，自身免疫反应是由于胃、胰腺和刷缘膜蛋白酶对某些富含脯胺/谷氨酰胺的脯胺肽的消化产生抵抗。这些肽通过肠黏膜吸收并启动自身免疫反应。在这一建议中，我们希望解决以下可测试的假设，以去除大麦和小麦中的蛋白蛋白t细胞刺激表位。1)在发育中的胚乳中，完全活的大麦突变lys3a阻止了所有需要启动子去甲基化的prolamin基因的转录，除了编码HMW - D-hordein的CpG岛基因。这种蛋白与小麦HMW蛋白高度同源，后者负责烘焙质量。我们的问题是，我们是否可以利用这种突变来获得大麦谷物，提供乳糜泻安全的食品。2)大麦和小麦的密切进化关系使得这两种谷物可能采用相同的转录控制系统来合成谷物中的贮藏蛋白。我们想通过选择小麦中lys3a的同源突变来验证这一点，并询问它是否阻止麦胶蛋白基因的转录，而不是六倍体小麦中存在的6个HMW基因的转录。3)在体外和体内大鼠肠道灌注实验中，含有t细胞刺激表位的富含脯氨酸/谷氨酰胺的肽可被脯氨酸内肽酶和大麦半胱氨酸内蛋白酶EP-B解毒。我们想测试是否有可能在转基因大麦和小麦胚乳发育期间合成这些酶，并在籽粒发育期间将这些酶靶向到储存液泡中并解毒蛋白。乳糜泻是人类最常见的食物敏感性肠病，是由于摄入了小麦麸质以及大麦和黑麦谷物储存蛋白中富含脯氨酸和谷氨酰胺的脯氨酸后，小肠黏膜受到慢性炎症损伤而引起的。迄今为止，治疗这种使人衰弱的疾病的唯一有效方法是终生严格禁食主食谷物。本研究的目的是利用大麦和小麦胚乳中蛋白合成的细胞生物学特性，消除引起乳糜泻的蛋白表位。