1. NTR 2.0: a rationally engineered prodrug-converting enzyme with substantially enhanced efficacy for targeted cell ablation
- Author
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Abigail V. Sharrock, Timothy S. Mulligan, Kelsi R. Hall, Elsie M. Williams, David T. White, Liyun Zhang, Kevin Emmerich, Frazer Matthews, Saumya Nimmagadda, Selena Washington, Katherine D. Le, Danielle Meir-Levi, Olivia L. Cox, Meera T. Saxena, Anne L. Calof, Martha E. Lopez-Burks, Arthur D. Lander, Ding Ding, Hongkai Ji, David F. Ackerley, and Jeff S. Mumm
- Subjects
Technology ,Green Fluorescent Proteins ,Genetically Modified ,CHO Cells ,Protein Engineering ,Regenerative Medicine ,Biochemistry ,Medical and Health Sciences ,Retina ,Animals, Genetically Modified ,Cricetulus ,Metronidazole ,Animals ,Humans ,Prodrugs ,Molecular Biology ,Zebrafish ,Vibrio ,5.2 Cellular and gene therapies ,Cell Biology ,Nitroreductases ,Biological Sciences ,Stem Cell Research ,Recombinant Proteins ,HEK293 Cells ,Stem Cell Research - Nonembryonic - Non-Human ,Development of treatments and therapeutic interventions ,Biotechnology ,Developmental Biology - Abstract
Transgenic expression of bacterial nitroreductase (NTR) enzymes sensitizes eukaryotic cells to prodrugs such as metronidazole (MTZ), enabling selective cell-ablation paradigms that have expanded studies of cell function and regeneration in vertebrates. However, first-generation NTRs required confoundingly toxic prodrug treatments to achieve effective cell ablation, and some cell types have proven resistant. Here we used rational engineering and cross-species screening to develop an NTR variant, NTR 2.0, which exhibits ~100-fold improvement in MTZ-mediated cell-specific ablation efficacy, eliminating the need for near-toxic prodrug treatment regimens. NTR 2.0 therefore enables sustained cell-loss paradigms and ablation of previously resistant cell types. These properties permit enhanced interrogations of cell function, extended challenges to the regenerative capacities of discrete stem cell niches, and novel modeling of chronic degenerative diseases. Accordingly, we have created a series of bipartite transgenic reporter/effector resources to facilitate dissemination of NTR 2.0 to the research community.
- Published
- 2022