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 在体内直接操作基因和基因产物具有巨大的治疗潜力，并且许多治疗方法都是有效的。 实现这些目标的策略正迅速地向临床应用发展。能够操纵DNA的蛋白质 活细胞中的RNA和蛋白质，包括基因组编辑技术， 体内致病突变，已经举例说明了这种方法对于研究和 治疗应用。虽然这些方法中的许多方法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定制，以获得所需的活性、特异性和稳定性 作为有影响力的研究工具或治疗开发的领导者。作为新型大分子治疗剂 方法继续以惊人的速度发展，在快速的时间尺度上产生蛋白质的方法 具有定制功能。理想情况下，这种方法将是通用的，可以应用于许多 生命科学中的几类问题。 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- 比使用传统的实验室进化方法快一倍，只需最少的研究人员 干预我们已经证明了PACE能够进化出许多不同种类的蛋白质， 以及改变的活性、特异性和其它期望的性质，例如在E.杆菌蛋白 使用PACE进化的药物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 已经被应用于拯救人类细胞和遗传疾病的动物模型， 能杀死农业害虫这些发展使计算机设备行动伙伴关系成为一个广泛适用和高度赋能的 用于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 用于治疗发现的技术。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和临床相关的药剂;自我递送 切割与神经变性疾病和脑癌有关的内源性蛋白质靶标的蛋白酶; 以及能够使药物诱导的靶蛋白降解的小分子结合蛋白。成功将 为创新治疗策略提供基础，以纠正导致人类遗传病的突变， 疾病，并重新编程自我递送蛋白酶作为催化药物来治疗脑部疾病。另外通过 产生药物敏感的等位基因，其允许感兴趣的蛋白质以小分子依赖性降解， 以这种方式，拟议中的研究将建立强大的新功能基因组学工具，以揭示生物学 功能和验证治疗靶点。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的进化技术 治疗学

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.