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.

Project Narrative: Continuous Evolution of Proteins with Novel Therapeutic Potential We have successfully evolved many different classes of proteins with new and altered activities using a technology that we developed to evolve biomolecules ≥100-fold faster than conventional laboratory evolution approaches. We propose to use this technology to evolve novel proteins with therapeutic potential, or that serve as new tools for therapeutics discovery. Success would enable the development of next-generation therapeutic agents to treat human diseases including genetic and neurodegenerative disorders.