I-Corps: A C. elegans model using human genes for high-throughput screening

这个I-Corps项目的更广泛的影响/商业潜力在于改进药物发现。制药公司在选择一种药物进行人体临床试验之前，要筛选数百万种化合物。潜在治疗剂的初步筛选在细胞培养测定中进行，其允许低成本评价，但在确定治疗相关性方面受到限制。通过在多种哺乳动物模型中进行额外筛选，可以克服这一限制，其中进一步评估潜在化合物的治疗效果、毒理学等。然而，与哺乳动物模型测试相关的费用和时间意味着只有一小部分通过细胞培养筛选的化合物能够进入这一阶段，实际上忽略了数千种可能具有有效治疗作用的化合物。这个项目？的技术旨在绕过这一瓶颈。这是劳动友好和成本效益，但同时将使公司能够测试化合物的疗效在动物模型，而不是细胞培养，从而允许进一步知情决策方面的发展，他们的化合物。这个I-Corps项目提供了一个创新的方法来筛选化合物针对肌动蛋白细胞骨架及其相关基因。C.将线虫改造成用其人同源物（ARHGEF 10）替换细胞骨架基因OSG-1，并将缺乏OSG-1的蠕虫（OSG-1 KO）保持在96孔板中，每个板含有不同的化合物。使蠕虫经受4小时热休克并对存活进行评分。由于OSG-1 KO蠕虫的死亡率显著高于ARHGEF 10，因此与ARHGEF 10蠕虫相比，只有靶向肌动蛋白细胞骨架途径的化合物才能差异性地改变KO的死亡率。有效但以相同程度影响KO和ARHGEF 10蠕虫死亡率的化合物不影响肌动蛋白细胞骨架途径（它们代表第二次切割，因为它们可能作用于在人类中不保守的途径和机制）。蠕虫被设计成在身体的大部分区域表达GFP。只有活的蠕虫表达GFP，而死的蠕虫不表达。存活评估仅需要对荧光蠕虫进行评分。由于成本仅来自琼脂和平板成本，因此可以以传统早期药物发现方法的一小部分成本筛选早期化合物的效价。

The broader impact/commercial potential of this I-Corps project is in the improvement of drug discovery. Pharmaceutical companies screen millions of compounds before a single agent is selected to undergo human clinical trials. Initial screening of potential therapeutics is conducted in cell culture assays that allow for low-cost evaluation, but are limited in determining therapeutic relevance. This limitation is overcome by additional screening in multiple mammalian models where potential compounds are further assessed for their therapeutic effects, toxicology etc. Yet, the expenses and time associated with mammalian model testing means that only a small percentage of compounds that pass cellculture screening will make it to this stage, effectively overlooking thousands of compounds that may be potent therapeutics. This project?s technology aims at bypassing this bottleneck. It is labor friendly and cost-effective but at the same time will enable companies to test compounds efficacy on an animal model rather than cells in culture thus allowing for further informed decision making in regards to the development of their compounds.This I-Corps project offers an innovative method to screen compounds targeting the actin cytoskeleton and its associated genes. C. elegans worms are engineered to replace a cytoskeletal gene, OSG-1, with its human homolog, (ARHGEF10), and worms lacking OSG-1, (OSG-1 KO), are maintained in 96-well plates each containing a different compound. Worms are subjected to a 4-hr heat shock and scored for survival. Because the mortality of OSG-1 KO worms is significantly higher than ARHGEF10, only compounds that target the actin cytoskeleton pathway can differentially change the mortality rates of the KO compared to the ARHGEF10 worm. Compounds that are efficacious but affect the mortality rates of the KO and the ARHGEF10 worm to the same extent do not impinge on the actin cytoskeleton pathway (they represent a second cut because they might act on pathways and mechanisms that are not conserved in humans). Worms are engineered to express GFP through most of the body. Only living worms express GFP whereas dead ones do not. Survival assessment only requires scoring fluorescent worms. With costs only accruing from agar and plate costs, early compounds can be screened for potency at a fraction of the cost of traditional early phase drug discovery methods.