Gene Silencing in adult hookworms using RNA interference

描述（由申请人提供）：钩虫性贫血是一个重大的全球健康问题，影响全世界多达10亿人。本R21提案（响应PAS-02-160：探索性/开发技术在niaid资助研究中的应用）的目标是利用RNA干扰（RNAi）技术开发一种可靠的方法来沉默成虫的基因。使用双链RNA （dsRNA）沉默特定基因表达的方法已经成功地应用于自由生活的秀丽隐杆线虫，但迄今为止还没有适用于任何人类寄生线虫。该提案与NIH拨款1 R01 AI47929有关，题为“钩虫贫血的分子发病机制”。本亲本基金的目的是在钩虫感染的动物模型中，利用疫苗方法对钩虫中主要的抗凝血剂和血小板抑制剂进行分子表征。目前的建议旨在应用RNAi分子技术更清楚地定义钩虫分泌蛋白，包括抗凝血剂（AceAP1）和血小板抑制剂，在寄生虫生物学和贫血发病机制中的作用。利用先前描述的方法将dsRNA引入秀丽隐杆线虫；将开发一种针对人类钩虫的RNAi技术。dsRNA介导的基因沉默对钩虫生存能力的影响将使用最近开发的离体培养系统进行评估。目的基因的表达将采用单虫RT-PCR法进行评估，相应的蛋白表达水平将采用生化和免疫学检测方法进行表征。一旦优化了RNAi技术的效率，就可以确定沉默钩虫抗凝基因表达对疾病发病机制的影响。暴露于四种候选基因靶点（钩虫抗凝血剂、肠丝氨酸蛋白酶抑制剂AceKI、β -微管蛋白和钩虫血小板抑制剂）对应的dsRNA的成虫将被转移到允许的田鼠身上，并对这些动物进行随访，以观察其贫血、生长迟缓和粪卵排泄的情况。最终，钩虫RNAi的发展代表了一种创新策略，用于表征特定基因产物在疾病发病机制中的作用，以及确定潜在的人类疫苗靶点。

DESCRIPTION (provided by the applicant): Hookworm anemia is a significant global health problem, affecting up to one billion people worldwide. The goal of this R21 proposal (in response to PAS-02-160: Application of exploratory/developmental technologies to NIAID-funded research) is to develop a reliable method of gene silencing in adult Ancylostoma ceylanicum hookworms utilizing the technique of RNA interference (RNAi). The use of double stranded RNA (dsRNA) to silence specific gene expression has been successfully developed for the free-living nematode Caenorhabditis elegans, but to date has not been adapted to any human parasitic nematode. This proposal is linked to NIH grant 1 R01 AI47929, entitled "Molecular pathogenesis of hookworm anemia." The goals of the parent grant involve the molecular characterization of the major anticoagulant and platelet inhibitor from the hookworm A. ceylanicum using a vaccine approach in an animal model of hookworm infection. The current proposal seeks to apply the molecular technique of RNAi to more clearly define the role of hookworm secretory proteins, including the anticoagulant (AceAP1) and platelet inhibitor, in the biology of the parasite and pathogenesis of anemia. Utilizing previously described methods of introducing dsRNA into C. elegans; a technique for RNAi will be developed for the human hookworm A. ceylanicum. The effect of dsRNA mediated gene silencing on hookworm viability will be assessed using a recently developed ex vivo cultivation system. Expression of the targeted genes will be assessed using a single worm RT-PCR assay, and the corresponding levels of protein expression will be characterized using biochemical and immunologic detection methods. Once the efficiency of the RNAi technique has been optimized, the effect of silencing of hookworm anticoagulant gene expression on disease pathogenesis will be determined. Adult worms that have been exposed to dsRNA corresponding to four candidate gene targets (the hookworm anticoagulant, the intestinal serine protease inhibitor AceKI, beta tubulin, and the hookworm platelet inhibitor), will be transferred to permissive hamsters, and the animals will be followed for evidence of anemia, growth delay, and fecal egg excretion. Ultimately, the development of RNAi for hookworm represents an innovative strategy for characterizing the role of specific gene products in disease pathogenesis, as well as defining potential human vaccine targets.