CRISPR/Cas-mediated development of an RNAi rat model system

摘要 FDA批准的每一种药物的研发成本已飙升至约26亿美元 由于缺乏有效性或安全性而失败，这表明我们目前的临床前验证过程已经 不足以预测人类的治疗潜力和毒性。动物模型是金子 在体内解剖疾病机制和评估新药靶点的标准；然而，成本和 开发它们的前期时间很长，阻碍了它们在药物发现过程中的常规使用。随着最近的 CRISPR/Cas9基因组编辑的发展，以及RNA干扰技术的进步，我们现在 有能力通过精确的基因组修改和类似人类的疾病快速开发动物模型 病理学。我们已经证明，RNAi作为基因缺失的快速替代方法，也可以用于 在基因工程小鼠模型内评估治疗潜力和预测新药物的毒性 基因靶标。这项提议的目标是将我们的能力扩展到MICE之外，并为 在短短4个月内快速、经济地生产出RNAi大鼠。尽管鼠标模型很实用，但对于 许多科学家认为，老鼠仍然是首选的啮齿动物，因为它们的体型更大，可以进行手术操作， 重复采血，他们的认知和生理特征更接近 人类比他们的老鼠同龄人。对于神经生物学、心脏生物学、免疫学和毒理学，它们是 仍然是研究中占主导地位的啮齿动物模型。我们现有客户群中有近30%的人询问过RAT 模型，注意到大多数化合物的毒理学研究仍然是在老鼠身上进行的 进入I期临床试验。我们知道，老鼠将再次成为药物领域最受欢迎的啮齿动物模型 Discovery，因为我们已经与一家大型制药公司签订了初步试点计划 这些模型。我们打算站在这个将范式转换回大鼠模型的前沿。在中使用小说 CRISPR/Cas9试剂的原位递送方法，我们现在将能够快速地操纵大鼠基因组 以经济高效的方式并系统地产生RNAi大鼠模型，允许在时间上和可逆的 模拟治疗方案的基因调控。这些RNAi大鼠模型将改变临床前验证 评估体内潜在的药物反应和耐药机制的过程，最终指导 开发更安全、更有效的药物。

Abstract The cost of drug development has skyrocketed to an estimated $2.6B for every FDA approved drug primarily due to failures from lack of efficacy or safety, suggesting that our current preclinical validation process has been insufficient in predicting therapeutic potential and toxicity in humans. Animal models are the gold standard for dissecting disease mechanisms and evaluating novel drug targets in vivo; however, the cost and long lead time to develop them has prevented their routine use in the drug discovery process. With the recent developments in CRISPR/Cas9 genome editing, and advances in RNA interference technologies, we now have the ability to rapidly develop animal models with precise genomic modifications and human-like disease pathologies. We have shown that RNAi serves as a fast alternative to gene deletion and can also be used within genetically engineered mouse models to assess the therapeutic potential and predict toxicities of novel gene targets. The goal of this proposal is to expand our capabilities beyond mice and develop a platform for rapid and cost-effective production of RNAi rats in as little as 4 months. Despite the utility of mouse models, for many scientists, the rat still remains the preferred rodent due to their larger size for surgical manipulation, repeat blood sampling, and their cognitive and physiological characteristics that more closely resemble humans than their mouse counterparts. For neurobiology, cardiobiology, immunology and toxicology, they are still the dominant rodent model in research. Nearly 30% of our current client base has inquired about rat models over the last 5 years, noting that most toxicology studies of their compounds are still done in rats prior to Phase I clinical trials. We know rats will gain popularity once again as the premier rodent model in drug discovery, as we have already been contracted a large pharmaceutical company for an initial pilot program for these models. We intend to be at the forefront of this shifting paradigm back to the rat model. Using novel in situ delivery methods of CRISPR/Cas9 reagents, we will now be able to manipulate the rat genome in a rapid and cost-efficient manner and systematically generate RNAi rat models that allow for temporal and reversible gene regulation to simulate therapeutic regimes. These RNAi rat models will transform the preclinical validation process with assessment of potential drug response and resistance mechanisms in vivo, ultimately guiding the development of safer and more effective drugs.