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）：乳糜泻是人类中最常见的食物敏感肠病理学状况，是由针对小麦，大麦和黑麦酸性菜单的自身免疫反应引起的。在人白细胞（HLA）DQ2（或DQ8）阳性个体中，暴露于面筋的个体会导致肠道上上皮细胞的慢性慢性擦除，并导致饮食中的饮食面筋的永久性不耐症。在患有严重症状的情况下，通常在幼儿期检测到它。在许多患者中，症状仅在生命的晚期出现，如果未经治疗，它与发病率和死亡率的增加有关。尽管在大多数人群中均患病率（最多200分之一），但唯一有效的疗法是严格的饮食戒酒。最近的研究表明，自身免疫反应是由于胃，胰腺和刷子膜膜蛋白酶的抗药性对催化蛋白的某些衍生物/谷氨酰胺富含肽的抗药性而产生的。这些肽是通过肠粘膜吸收的，并启动自身免疫反应。在此提案中，我们希望解决以下可检验的假设，以取消大麦和小麦中的刺激性T细胞刺激表位。 1）完全可行的大麦突变Lys3a防止了所有催化剂基因的转录，这些基因需要在发育中的胚乳中脱甲基化的启动子，除了具有编码高分子（HMW）D荷链的CPG岛的单个基因。这种脯氨酰蛋白与负责烘烤质量的小麦HMW溶素高度同源。我们问一个问题，我们是否可以利用这种突变以获取提供乳糜泻的食品的大麦谷物。 2）大麦和小麦的密切进化关系使两种谷物可能采用相同的转录控制系统来合成谷物中的储存蛋白。我们想通过选择小麦中的Lys3a的直系同源突变来测试这一点，并询问该问题是否阻止了麦醇溶蛋白基因的转录，而不是六磷酸小麦中存在的6个HMW基因。 3）可以通过大鼠肠道灌注实验在体外和体内对含有T细胞刺激表位的脯氨酸/富含T细胞刺激表位的蛋白肽进行解毒。我们要测试是否有可能在胚乳发育过程中在转基因大麦和小麦中合成这些酶，将酶靶向储存液泡中的酶，并在谷物发育过程中排毒蛋白酶蛋白酶蛋白质或春季是由慢性炎性造成的肥胖型和刺激性的摄入型肠胃疾病的最常见的肠肠疾病 - 刺激性刺激性的肠胃nig虫，并累及了小小的刺激性。出现在小麦面筋以及大麦和黑麦谷物储存蛋白中。迄今为止，这种使人衰弱的疾病的唯一有效治疗方法是终身严格戒酒。这项研究的目的是通过利用大麦和小麦胚乳中的丙书蛋白合成的细胞生物学特征来消除腹腔。