Continuous Evolution of Proteins with Novel Therapeutic Potential

项目总结：具有新治疗潜力的蛋白质的持续进化 在体内直接操纵基因和基因产品具有巨大的治疗潜力，而且有许多 实现这些目标的策略正在迅速走向临床应用。可以操纵DNA的蛋白质， RNA和活细胞中的蛋白质，包括基因组编辑技术，使精确纠正 体内致病突变，已经证明了这种方法在研究和 治疗应用。虽然这些方法中的许多在最初的研究中显示出了希望， 蛋白质通常需要广泛的开发和调整才能获得所需的活性、特异性和稳定性。 作为有影响力的研究工具或治疗发展的先导。作为新的大分子疗法 模式继续以惊人的速度发展，在快速时间尺度上产生蛋白质的方法 需要量身定做的功能。理想情况下，这样的方法将是多才多艺的，可以应用于许多 生命科学中的问题类别。 我们的实验室开发了噬菌体辅助持续进化(PACE)，这是一种进化生物分子≥100- 与使用传统的实验室进化方法相比，速度更快，需要的研究人员最少 干预。我们已经证明了PACE的能力，可以进化出许多不同类别的蛋白质 以及改变的活性、特异性和其他所需的性质，例如在大肠杆菌中的可溶表达。蛋白质 使用PACE的进化已经在包括基因组编辑试剂在内的多种非细菌环境中显示出广泛的用途 已经被应用于拯救人类细胞和动物的遗传病模型，以及杀虫蛋白 杀死农业害虫的物质。这些发展确立了PACE作为一种广泛适用和高度赋能的 产生治疗和生物技术相关蛋白质的技术。 我们建议应用PACE来进化具有治疗潜力的新蛋白质，或者使新的 治疗学发现的技术。这些蛋白质包括下一代精确基因组编辑 体内更容易给药或更有效和临床相关的药物；自我给药 切割与神经退行性疾病和脑癌有关的内源性蛋白靶点的蛋白酶； 和小分子结合蛋白，使药物诱导的目标蛋白降解。成功将会 为纠正导致人类基因突变的创新治疗策略提供基础 并将自我递送的蛋白酶重新编程为治疗脑部疾病的催化药物。此外，由 创造药物敏感的等位基因，使感兴趣的蛋白质在小分子依赖的情况下被降解 通过这种方式，拟议的研究将建立强大的新功能基因组学工具来揭示生物学 功能和验证治疗目标。总的来说，拟议的研究整合了强大的蛋白质 利用精确操纵基因组和蛋白质组的酶来推进进化技术 治疗科学。

Project Summary: Continuous Evolution of Proteins with Novel Therapeutic Potential The direct manipulation of genes and gene products in vivo has enormous therapeutic potential, and many strategies to achieve these goals are swiftly advancing toward clinical use. Proteins that can manipulate DNA, RNA, and proteins in living cells, including genome editing technologies that enable the precise correction of disease-causing mutations in vivo, have exemplified the promise of such approaches both for research and therapeutic applications. While many of these approaches have shown promise in initial research studies, proteins often require extensive development and tailoring to acquire the activity, specificity, and stability needed to serve as impactful research tools or leads for therapeutic development. As new macromolecular therapeutic modalities continue to be developed at a remarkable rate, methods to generate proteins on a rapid time scale with tailor-made functions are needed. Ideally such methods will be versatile and can be applied to many classes of problems in the life sciences. Our lab developed phage-assisted continuous evolution (PACE), a technology to evolve biomolecules ≥100- fold faster than using conventional laboratory evolution approaches, with minimal required researcher intervention. We have demonstrated the ability of PACE to evolve many different classes of proteins with new and altered activities, specificities, and other desirable properties such as soluble expression in E. coli. Proteins evolved using PACE have shown broad utility in multiple non-bacterial settings, including genome editing agents that have been applied to rescue human cell and animal models of genetic diseases, and insecticidal proteins that kill agricultural pests. These developments establish PACE as a broadly applicable and highly enabling technology for generating therapeutically and biotechnologically relevant proteins. We propose to apply PACE to evolve novel proteins with therapeutic potential, or that enable new technologies for therapeutics discovery. These proteins include next-generation precision genome editing agents that can be more easily delivered in vivo or are more efficient and clinically relevant; self-delivering proteases that cleave endogenous protein targets implicated in neurodegenerative disorders and brain cancer; and small molecule-binding proteins that enable drug-induced target protein degradation. Success would provide a foundation for innovative therapeutic strategies to correct mutations that cause human genetic diseases, and to reprogram self-delivering proteases as catalytic drugs to treat brain diseases. In addition, by creating drug-sensitive alleles that allow a protein of interest to be degraded in a small molecule-dependent manner, the proposed research would establish powerful new functional genomics tools to reveal biological functions and validate therapeutics targets. Collectively, the proposed research integrates powerful protein evolution technologies with enzymes that precisely manipulate genomes and proteomes to advance therapeutics science.