Somatic Gene Therapy for Mosquitoes

项目总结/摘要 疟疾仍然是一个全球性的健康问题，特别是在撒哈拉以南非洲， 冈比亚按蚊是原生动物疟原虫的主要传播媒介 恶性疟原虫。媒介能力的一个关键组成部分是蚊子的免疫系统。到 确定蚊子免疫的分子机制，载体生物学家目前使用RNAi作为 一种反向遗传学工具来敲除目标基因。虽然高效且易于使用， RNAi的缺点是缺乏组织特异性。绕过这一障碍的一种方法是 使用转基因蚊子。不幸的是，转基因品系制备起来很麻烦， 维持，并且良好表征的组织特异性启动子的数量有限。由于 大量有吸引力的基因在多种组织中表达，这是一个可塑性系统， 组织特异性基因敲低将对载体生物界非常有益。我们 建议通过结合两种成分来驱动蚊子中的组织特异性RNAi，（1）泛- 组织嗜性、非致病性病毒和（2）组织特异性miRNA。病毒会感染 递送短发夹RNA分子以诱导RNAi。为了限制输送系统， 内源性miRNA将用于阻断病毒复制或转基因表达; 最终导致组织特异性基因敲除。长期目标是破译基因 在蚊子体内以组织特异性方式发挥作用。对于这项提议，组织特异性RNAi 原理验证实验将集中在卵巢上。具体目标是（1）驱动RNAi， 每个组织，但卵巢，（2）驱动RNAi特异性卵巢，并获得更深的 了解蚊子体内miRNA的表达，（3）确定蚊子体内miRNA的转录组， 多个冈比亚按蚊组织。这项建议的健康相关性是知识 从了解疟疾病媒组织水平的基因功能中获得的信息对于 今后制定病媒控制战略。一旦优化，所提出的体细胞基因 蚊子的治疗方法将为媒介生物学提供一种独特的有价值的遗传工具 整个社区。

Project Summary/Abstract Malaria continues to be a global health problem, especially in sub-Saharan Africa where the mosquito Anopheles gambiae s.s. serves as the major vector for the protozoan Plasmodium falciparum. A critical component of vector competency is the mosquito immune system. To determine molecular mechanisms of mosquito immunity, vector biologists currently use RNAi as a reverse genetics tool to knockdown genes of interest. While efficient and easily employed, a weakness of RNAi is its lack of tissue specificity. One approach to get around this barrier is the use of transgenic mosquitoes. Unfortunately, transgenic lines are cumbersome to make and maintain, and the number of well characterized tissue-specific promoters is limited. Due to the vast number of attractive genes expressed in multiple tissues, a malleable system for producing tissue-specific gene knockdown would be highly beneficial to the vector biology community. We propose to drive tissue-specific RNAi in the mosquito by combining two components, (1) a pan- tissue tropic, non-pathogenic virus and (2) tissue-specific miRNAs. The virus will infect and deliver short-hairpin RNA molecules to induce RNAi. To restrict the delivery system, endogenous miRNAs will be used to block either virus replication or transgene expression; ultimately resulting in tissue specific gene knockdown. The long-term goal is to decipher gene function in a tissue-specific manner in the mosquito. For this proposal, tissue-specific RNAi proof of principle experiments will focus on the ovary. The specific aims are (1) drive RNAi to every tissue but the ovary, (2) drive RNAi specifically to the ovary, and to obtain a deeper understanding of miRNA expression in mosquitoes, (3) determine miRNA transcriptome for multiple Anopheles gambiae tissues. The health relevance of this proposal is the knowledge gained from understanding gene function at the tissue level in the malaria vector is important for future development of vector control strategies. Once optimized, the proposed somatic gene therapy method for mosquitoes will provide a unique genetic tool of value to the vector biology community as a whole.