Developing tools for genetic manipulation of hookworms

描述（由申请人提供）：钩虫感染仍然是全球最大的公共卫生威胁之一，估计有 8 亿人受到感染。严重的钩虫感染是热带地区贫血的主要原因，由于喂养肠道中的成虫而失血，导致虚弱甚至有时致命的缺铁性贫血。儿童、孕妇和老年人特别容易受到钩虫感染的影响。控制策略仅限于对感染个体进行定期驱虫，这受到快速再感染率和耐药蠕虫种群发展的限制。疫苗工作因缺乏有效的靶抗原而受到影响。新药物靶点的开发和改进的疫苗抗原将需要更好地了解钩虫生物学，特别是感染过程。然而，直到最近，对宿主的必然要求以及无法在体外培养完整生命周期的能力阻碍了钩虫研究的强大遗传工具的开发。我们建议利用其他寄生线虫来适应和利用最新进展来开发钩虫转染系统。我们将采取两种并行但独立的方法来开发钩虫的瞬时转染方法，为未来遗传转化技术的发展奠定基础。在目标 1 中，将使用粒子轰击将报告基因引入胚胎和幼虫阶段，并分析转化体的存活、生长和报告基因表达。我们还将使用最近描述的抗生素选择技术从非转化体中选择转化体。在目标 2 中，我们将使用基于化学和脂质的技术将报告基因引入蜕皮幼虫中，该技术最近在寄生线虫马来丝虫中取得了成功。在这两个目标中，我们将使用基于 Piggybac 逆转录转座子的整合载体将报告转基因引入钩虫染色体。体外转染的发展将代表钩虫研究的重大进步，并将首次在同源遗传背景下确定钩虫基因表达和功能。此外，转基因将使我们能够利用即将发布的几种已测序的钩虫基因组和转录组的数据来开发新的钩虫控制策略。

DESCRIPTION (provided by applicant): Hookworm infection remains one of the greatest public health threats worldwide, with an estimated 800 million people infected. Heavy hookworm infection is the leading cause of anemia in the tropics, resulting in debilitating and sometimes fatal iron-deficiency anemia caused by blood loss to feeding adult worms in the intestine. Children, pregnant women, and the elderly are particularly susceptible to morbidity from hookworm infection. Control strategies are restricted to periodic de-worming of infected individuals, which is limited by rapid re-infection rates and the development of drug resistant worm populations. Vaccine efforts suffer for the lack of effective target antigens. Development of new drug targets and improved vaccine antigens will require a better understanding of hookworm biology, particularly the infective process. However, the obligate requirement for a host and the inability to grow the complete life cycle in vitro precluded development of powerful genetic tools for hookworm research until recently. We propose to adapt and build upon recent advances using other parasitic nematodes to develop a transfection system for hookworms. We will take two parallel but independent approaches to develop transient transfection methods for hookworms that will provide a foundation for the future development of heritable transformation technology. In Aim 1, particle bombardment will be used to introduce reporter genes into embryos and larval stages, and transformants will be assayed for survival, growth and reporter gene expression. We will also use a recently described antibiotic selection technique to select transformants from non-transformants. In Aim 2, we will use chemical and lipid-based technology to introduce reporter genes into molting larvae, a technique that was recently successful in the parasitic nematode Brugia malayi. In both aims we will use a piggybac retrotransposon-based integrating vector to introduce the reporter transgenes into hookworm chromosomes. Development of in vitro transfection would represent a significant advance for hookworm research, and will allow determination of hookworm gene expression and function in a homologous genetic context for the first time. Furthermore, transgenesis would enable us to exploit the data from the imminent release of several sequenced hookworm genomes and transcriptomes for the development of novel hookworm control strategies.