Gene Silencing in adult hookworms using RNA interference

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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.