The vast majority of current therapeutic agents function by binding to disease-associated macromolecules and modulating their activity. Recent developments, however, have made increasingly realistic the possibility of developing next-generation therapeutics that do not simply bind targets implicated in disease, but instead alter the covalent structure of genes and gene products in ways that can more effectively treat-or even cure-many diseases. While the possibility of precisely manipulating genes and proteins in mammalian cells and, eventually, in humans, has enormous potential, several major challenges must be overcome to fully realize this vision. Perhaps the most significant of these challenges is the efficient creation of the macromolecules that are needed to alter genomes or proteomes with a high degree of selectivity and potency. To realize a vision in which arbitrary genes or proteins can be manipulated in mammalian cells to treat disease thus requires new approaches to rapidly generating macromolecules with precise, tailor-made properties. During the last granting period, 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. In the first applications of PACE, we rapidly evolved RNA polymerases with dramatically different DNA promoter specificities. We also identified the vulnerabilities of drug candidates to the evolution of drug resistance by using PACE to evolve proteases that are resistant to HCV protease inhibitors currently used in human clinical trials. In addition, we developed important PACE capabilities beyond basic positive selection, including small- molecule modulation of selection stringency and negative selection against undesired activities. These initial studies established PACE as a robust and general platform to evolve proteins with tailor-made properties at an unprecedented speed. In the next granting period, 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.

Public Health Relevance

We recently developed a method to evolve proteins continuously in the laboratory for the first time, enabling evolution at a rate 100-fold faster tha that of traditional laboratory evolution methods. We propose to use this method to generate highly evolved proteins that precisely modify the structure of genes and gene products implicated in human disease. The resulting proteins have the potential to serve as next-generation protein therapeutics to treat human diseases including genetic disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Research Project (R01)
Project #
5R01EB022376-13
Application #
9419171
Study Section
Therapeutic Approaches to Genetic Diseases Study Section (TAG)
Program Officer
Rampulla, David
Project Start
2002-04-01
Project End
2020-01-31
Budget Start
2018-02-01
Budget End
2019-01-31
Support Year
13
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Broad Institute, Inc.
Department
Type
DUNS #
623544785
City
Cambridge
State
MA
Country
United States
Zip Code
02142
Rees, Holly A; Komor, Alexis C; Yeh, Wei-Hsi et al. (2017) Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery. Nat Commun 8:15790
Packer, Michael S; Rees, Holly A; Liu, David R (2017) Phage-assisted continuous evolution of proteases with altered substrate specificity. Nat Commun 8:956
Tang, Weixin; Hu, Johnny H; Liu, David R (2017) Aptazyme-embedded guide RNAs enable ligand-responsive genome editing and transcriptional activation. Nat Commun 8:15939
Kim, Y Bill; Komor, Alexis C; Levy, Jonathan M et al. (2017) Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions. Nat Biotechnol 35:371-376
Bryson, David I; Fan, Chenguang; Guo, Li-Tao et al. (2017) Continuous directed evolution of aminoacyl-tRNA synthetases. Nat Chem Biol 13:1253-1260
Gaudelli, Nicole M; Komor, Alexis C; Rees, Holly A et al. (2017) Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551:464-471
Chan, Alix I; McGregor, Lynn M; Jain, Tara et al. (2017) Discovery of a Covalent Kinase Inhibitor from a DNA-Encoded Small-Molecule Library × Protein Library Selection. J Am Chem Soc 139:10192-10195
Komor, Alexis C; Zhao, Kevin T; Packer, Michael S et al. (2017) Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity. Sci Adv 3:eaao4774
Komor, Alexis C; Badran, Ahmed H; Liu, David R (2017) CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes. Cell 168:20-36
Suzuki, Tateki; Miller, Corwin; Guo, Li-Tao et al. (2017) Crystal structures reveal an elusive functional domain of pyrrolysyl-tRNA synthetase. Nat Chem Biol 13:1261-1266

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