TILLING wheat for reduced celiac disease causing proteins

描述(申请人提供)：乳糜泻是一种自身免疫性消化疾病，遗传易感个体因摄入特定的谷类种子储存蛋白而出现肠道损伤。唯一的治疗方法是严格遵守不含有害谷物的饮食。因此，大量的研究致力于鉴定小麦种子贮藏蛋白中的特定氨基酸序列，这些氨基酸序列负责在乳糜泻患者中引发免疫反应，这项研究激发了人们的希望，即可以培育出减少或消除这些蛋白质的小麦和其他谷物品系。在华盛顿州立大学的Diter von Wettstein博士和阿卡迪亚生物科学公司合作进行的NIH STTR第一阶段研究过程中，我们已经克隆并开始了对以前未知的？醇溶蛋白基因家族位于六倍体小麦的同源染色体6D上。在这个醇溶蛋白基因家族中，存在一个或多个以免疫为主的醇溶蛋白亚型基因，该基因包含一个高度耐消化的33肽(Molberg等人，胃肠病128：393-401,2005；单等人，Science 297：2275-2279,2002)。确定编码这种醇溶蛋白亚型的基因(S)是利用分蘖技术试图将其从小麦籽粒中消除的先决条件。TILLING(靶向诱导的基因组局部损伤)是阿卡迪亚生物科学公司的一位科学家发明的一种新的非转基因生物技术，是突变育种的目标选择变体。在STTRI阶段的研究中，我们还确定了DNA 5-甲基胞嘧啶脱糖基酶对小麦和大麦面筋基因表达的重要性，并通过耕作和转基因手段针对小麦同源物进行失活。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小麦品系，这些转录因子已知对对乳糜泻患者有毒的种子储存蛋白的积累具有重要作用。STTR第二阶段的研究将通过以下几个方面继续取得进展：1.利用分蘖和转基因方法培育胚乳DNA 5-甲基胞嘧啶脱糖基活性下调的小麦植株，并对其种子贮藏成分进行表征。2.对候选转录因子中含有优先错义和无义等位基因的植物进行杂交，并对后代的表型进行鉴定。NIH STTR第二阶段研究的结果将是产生突变和转基因小麦品系，这些品系的创造是乳糜泻患者可接受的小麦品种商业化的必要先决条件。与公共卫生相关：拟议的研究采取了一种创新的方法来改善乳糜泻的原因，方法是开发缺乏导致乳糜泻的主要面筋成分的小麦品种。乳糜泻是人类最常见的食物敏感型肠道病理疾病，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类蛋白。因此，开发可被乳糜泻患者接受的小麦解决了一个紧急的、未得到满足的需求。

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.