Continuous Evolution of Proteins with Novel Therapeutic Potential

项目摘要：具有新治疗潜力的蛋白质的持续进化 体内基因和基因产物的直接操作具有巨大的治疗潜力，并且许多 实现这些目标的策略正在迅速推进临床应用。可以操纵DNA的蛋白质， 活细胞中的 RNA 和蛋白质，包括能够精确校正基因组编辑技术 体内引起疾病的突变，已经证明了这种方法对于研究和应用的前景。 治疗应用。虽然其中许多方法在初步研究中显示出前景， 蛋白质通常需要广泛的开发和定制才能获得所需的活性、特异性和稳定性 充当有影响力的研究工具或治疗开发的先导。作为新型大分子治疗药物 模式、快速生成蛋白质的方法继续以惊人的速度发展 需要具有定制功能。理想情况下，此类方法具有通用性，可以应用于许多领域 生命科学中的问题类别。 我们的实验室开发了噬菌体辅助连续进化（PACE），这是一种进化生物分子≥100-的技术 折叠速度比使用传统的实验室进化方法更快，所需的研究人员最少 干预。我们已经证明了 PACE 能够利用新的技术进化出许多不同类别的蛋白质。 并改变活性、特异性和其他所需特性，例如大肠杆菌中的可溶性表达。蛋白质 使用 PACE 进化而来的技术在多种非细菌环境中显示出广泛的实用性，包括基因组编辑剂 已应用于挽救遗传性疾病的人类细胞和动物模型以及杀虫蛋白 杀死农业害虫。这些发展使 PACE 成为一种广泛适用且高度赋能的 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型蛋白质，或者使新的蛋白质成为可能 治疗发现技术。这些蛋白质包括下一代精确基因组编辑 可以更容易地在体内递送或更有效且具有临床相关性的药物；自我交付 裂解与神经退行性疾病和脑癌有关的内源性蛋白质靶点的蛋白酶； 以及能够实现药物诱导的靶蛋白降解的小分子结合蛋白。成功将会 为纠正导致人类基因突变的创新治疗策略奠定基础 疾病，并将自传递蛋白酶重新编程为治疗脑部疾病的催化药物。此外，通过 创建药物敏感的等位基因，使感兴趣的蛋白质以小分子依赖性方式降解 以这种方式，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 功能并验证治疗目标。总的来说，拟议的研究整合了强大的蛋白质 利用酶精确操纵基因组和蛋白质组以推进进化的技术 治疗学科学。

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.