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，全世界有多达10亿人受到影响。这项R21提案(响应PAS-02-160：探索/开发技术在NIAID资助的研究中的应用)的目标是利用RNA干扰(RNAi)技术开发一种可靠的方法来抑制成虫的基因沉默。利用双链RNA(DsRNA)沉默特定基因的表达已被成功地用于自由生活的线虫，但到目前为止还没有适应任何人类寄生线虫。这项建议与美国国立卫生研究院第1号拨款R01 AI47929有关，题为“钩虫贫血的分子发病机制”。父母资助的目标包括在钩虫感染的动物模型中使用疫苗方法对钩虫的主要抗凝血剂和血小板抑制物进行分子表征。目前的提议试图应用RNAi的分子技术来更清楚地确定钩虫分泌蛋白，包括抗凝剂(AceAP1)和血小板抑制物，在寄生虫的生物学和贫血的发病机制中的作用。利用前面描述的将dsRNA导入线虫的方法；将为人类钩虫A.ceylanicum开发一种RNAi技术。DsRNA介导的基因沉默对钩虫存活的影响将使用最近开发的体外培养系统进行评估。目标基因的表达将使用单虫RT-PCR分析进行评估，相应的蛋白质表达水平将使用生化和免疫学检测方法进行表征。一旦RNAi技术的效率得到优化，将确定沉默钩虫抗凝基因表达在疾病发病机制中的作用。暴露于四个候选基因靶点(钩虫抗凝剂、肠道丝氨酸蛋白酶抑制剂AceKI、β-微管蛋白和钩虫血小板抑制剂)的dsRNA的成虫将被转移到允许的仓鼠身上，并将对这些动物进行跟踪，以寻找贫血、生长迟缓和粪卵排泄的证据。最终，针对钩虫的RNAi的开发代表了一种创新的策略，用于表征特定基因产物在疾病发病机制中的作用，以及定义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.