TILLING wheat for reduced celiac disease causing proteins

描述（由申请人提供）：乳糜泻是一种自身免疫性消化系统疾病，在遗传易感个体中，肠道病变是摄入特定谷物种子储存蛋白的反应。唯一的治疗方法就是严格遵守不含有害谷物的饮食。因此，大量的研究一直致力于确定小麦种子储存蛋白中引起乳糜泻患者免疫反应的特定氨基酸序列，这项研究激发了人们的希望，即可以开发出减少或消除这些蛋白质的小麦和其他谷物品系。在NIH STTR第一阶段的研究过程中，华盛顿州立大学的Diter von Wettstein博士和阿卡迪亚生物科学公司合作进行了研究，我们已经克隆并开始系统分析以前未表征的？麦胶蛋白基因家族位于小麦六倍体同源染色体6D上。在这个麦胶蛋白基因家族中，存在一个或多个麦胶蛋白亚型基因，该基因在乳糜泻中具有免疫优势，含有一种高度耐消化的33-mer肽（Molberg等人，Gastroenterology 128:393-401,2005； Shan等人，Science 297:2275-2279,2002）。鉴定编码这种麦胶蛋白亚型的基因是使用TILLING将其从小麦籽粒中清除的先决条件。TILLING （Targeting Induced Local lesion in Genomes）是一种新的非转基因生物技术，由阿卡迪亚生物科学公司的一位科学家发明，是一种靶向选择变异的突变育种。在STTR I期研究中，我们还确定了DNA 5-甲基胞嘧啶脱糖基酶对小麦和大麦麸质基因表达的重要性，并通过TILLING和转基因手段将小麦同源物灭活。最后，我们开发了大量含有候选转录因子错义和无义突变的新型小麦品系，这些候选转录因子对于积累对乳糜泻患者有毒的种子储存蛋白很重要。STTR II期研究将继续朝着开发乳糜泻患者可接受的小麦的方向发展：1。利用TILLING和转基因方法培育胚乳DNA 5-甲基胞嘧啶去糖基化活性下调的小麦植株，并对其种子贮藏成分进行表征。2. 在候选转录因子中含有优先错义和无义等位基因的植物之间进行杂交，并表征后代的表型。这项NIH STTR II期研究的结果将是突变和转基因小麦品系，其创造是小麦品种商业化的必要前提，为乳糜泻患者所接受。公共卫生相关性：拟议的研究采用一种创新的方法，通过开发缺乏引起乳糜泻的主要麸质成分的小麦品种，来改善乳糜泻的病因。乳糜泻是人类中最常见的食物敏感性肠道病理疾病，患病率为0.5-1%，目前对患者的唯一治疗方法是从饮食中完全排除谷物蛋白。因此，开发乳糜泻患者可接受的小麦解决了一个迫切的、未满足的需求。

DESCRIPTION (provided by applicant): Celiac disease is an autoimmune digestive disorder in which intestinal lesions develop in genetically susceptible individuals in response to the ingestion of specific cereal seed storage proteins. The only treatment is strict adherence to a diet free from the offending grains. Therefore, considerable research has been devoted to identifying the specific amino acid sequences in wheat seed storage proteins responsible for eliciting the immune reaction in celiac patients, and this research has motivated the hope that strains of wheat and other grains could be developed in which these proteins have been reduced or eliminated. During the course of NIH STTR phase I research conducted in collaboration between Dr. Diter von Wettstein of Washington State University and Arcadia Biosciences, we have cloned and begun a systematic analysis of the composition of the previously uncharacterized ? gliadin gene family located on homoeologous chromosome 6D of hexaploid wheat. Among this gliadin gene family there exist one or more genes for the gliadin subtype immunodominant in celiac disease containing a 33-mer peptide highly resistant to digestion (Molberg et al., Gastroenterology 128:393-401,2005; Shan et al., Science 297:2275-2279,2002). Identifying the gene(s) encoding this gliadin subtype is a prerequisite for using TILLING to attempt to eliminate it from the wheat grain. TILLING (Targeting Induced Local Lesions in Genomes) is a novel, non-GMO (genetically modified organism) technique invented by an Arcadia Biosciences scientist that is a target-selected variation of mutation breeding. During STTR phase I research we have also determined the importance of a DNA 5- methylcytosine deglycosylase for gluten gene expression in wheat and barley and are targeting the wheat homologues for inactivation via TILLING and transgenic means. Finally, we have developed a large number of novel wheat lines containing missense and nonsense mutations in candidate transcription factors known to be important for the accumulation of seed storage proteins toxic to celiac patients. STTR phase II research will continue progress towards the development of wheat acceptable for celiac patients by: 1. Using TILLING and transgenic approaches to develop wheat plants down-regulated for endosperm DNA 5- methylcytosine deglycosylation activity and characterizing their seed storage composition. 2. Making crosses between plants harboring priority missense and nonsense alleles in the candidate transcription factors and characterizing the phenotypes of the progeny. The outcome of this NIH STTR Phase II research will be mutant and transgenic wheat lines whose creation are a necessary prerequisite for the commercialization of wheat cultivars acceptable for sufferers of celiac disease. PUBLIC HEALTH RELEVANCE: The proposed research takes an innovative approach to ameliorating the cause of celiac disease by developing wheat varieties that lack the major gluten components that elicit the disease. Celiac disease is the most common food-sensitive enteropathological condition in humans with a prevalence of 0.5-1%, and currently the only treatment for patients is the complete exclusion of cereal proteins from the diet. Developing wheat that is acceptable for celiac patients thus addresses an urgent, unmet need.