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 #
5R01EB022376-11
Application #
9211342
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
2017-02-01
Budget End
2018-01-31
Support Year
11
Fiscal Year
2017
Total Cost
$406,339
Indirect Cost
$162,790
Name
Harvard University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
082359691
City
Cambridge
State
MA
Country
United States
Zip Code
02138
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Tang, Weixin; Liu, David R (2018) Rewritable multi-event analog recording in bacterial and mammalian cells. Science 360:

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