Our laboratory has conducted two research programs at the interface of chemistry and evolution. In the first program, we developed new approaches to protein evolution and protein delivery that have dramatically increased their effectiveness. In the second program, we developed a new approach to the synthesis and discovery of bioactive small molecules that combines powerful aspects of biological evolution with synthetic organic chemistry. The resulting method of DNA-templated synthesis has enabled DNA sequences encoding synthetic molecules to undergo translation, selection, and amplification paralleling biological evolution. This proposal seeks to integrate these two research programs into a single effort under a Maximizing Investigators' Research Award (MIRA). In the first program, we developed a system that enables proteins to evolve continuously in the laboratory, requiring virtually no researcher intervention. The resulting system, phage-assisted continuous evolution (PACE), allows proteins to undergo directed evolution at a rate ~100-fold faster than conventional methods. We propose to apply these developments to continuously evolve four classes of proteins or RNAs, each with the ability to manipulate the covalent structure of genes or gene products, and each with potential relevance to the development of next-generation human therapeutics: recombinase enzymes that insert DNA of interest into safe-harbor loci in the human genome, proteases that specifically cleave disease-associated proteins, orthogonal Cas9 (CRISPR) nucleases with altered PAM specificities and enhanced activities, and smart Cas9 guide RNAs that mediate genome engineering only in those cells that are in specific disease-associated cell states. Success would establish the novel therapeutic potential of these proteins and RNAs to address a wide range of human diseases, including many human genetic disorders. In the second program, we developed the foundations of DNA-templated synthesis, generated libraries of DNA-templated small molecules, and performed in vitro selections on these libraries for affinity to an initial set of targets of biomedical interest. The results led t the discovery of novel kinase and protease inhibitors with remarkable selectivity and potency, including the first physiological inhibitor of insulin-degrading enzyme (IDE). We used this compound in mice to validate inhibition of insulin degradation as a potential therapeutic strategy for improving glucose tolerance. We propose to develop second-generation IDE inhibitors with increased therapeutic potential, to explore our recent discovery that IDE inhibition can lower blood pressure in vivo, and to expand the application of this approach by creating a new DNA-templated library of >250,000 macrocycles and to selecting this library for binding to >150 protein targets implicated in human disease. These efforts will collectively result in the evaluation of more than 30,000,000 potential small molecule-protein interactions in a manner that leverages the remarkable efficiency of in vitro selection, PCR, and modern DNA sequencing.
Our laboratory has conducted two research programs at the interface of chemistry and evolution. We developed new approaches to protein evolution and protein delivery that have dramatically increased their effectiveness, and we developed a new approach to the synthesis and discovery of bioactive small molecules that combines powerful aspects of biological evolution with synthetic organic chemistry. This proposal seeks to integrate these research programs into a single effort under a Maximizing Investigators' Research Award (MIRA).
| Hu, Johnny H; Miller, Shannon M; Geurts, Maarten H et al. (2018) Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556:57-63 |
| Komor, Alexis C; Badran, Ahmed H; Liu, David R (2018) Editing the Genome Without Double-Stranded DNA Breaks. ACS Chem Biol 13:383-388 |
| Lee, Hye Kyung; Willi, Michaela; Miller, Shannon M et al. (2018) Targeting fidelity of adenine and cytosine base editors in mouse embryos. Nat Commun 9:4804 |
| Koblan, Luke W; Doman, Jordan L; Wilson, Christopher et al. (2018) Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat Biotechnol 36:843-846 |
| Gao, Xue; Tao, Yong; Lamas, Veronica et al. (2018) Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents. Nature 553:217-221 |
| Krishnan, Yamini; Rees, Holly A; Rossitto, Christina P et al. (2018) Green fluorescent proteins engineered for cartilage-targeted drug delivery: Insights for transport into highly charged avascular tissues. Biomaterials 183:218-233 |
| Wang, Tina; Badran, Ahmed H; Huang, Tony P et al. (2018) Continuous directed evolution of proteins with improved soluble expression. Nat Chem Biol 14:972-980 |
| Yeh, Wei-Hsi; Chiang, Hao; Rees, Holly A et al. (2018) In vivo base editing of post-mitotic sensory cells. Nat Commun 9:2184 |
| Usanov, Dmitry L; Chan, Alix I; Maianti, Juan Pablo et al. (2018) Second-generation DNA-templated macrocycle libraries for the discovery of bioactive small molecules. Nat Chem 10:704-714 |
| Chen, Zhen; Lichtor, Phillip A; Berliner, Adrian P et al. (2018) Evolution of sequence-defined highly functionalized nucleic acid polymers. Nat Chem 10:420-427 |
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