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.