We propose substantial improvements to a powerful targeted cell ablation system we developed to create novel animal models for regenerative medicine research, and to provide a corresponding set of versatile new toolsets. This system uniquely facilitates: 1) Studies of cell-specific regeneration for nearly any cellular subtype; 2) Inducible degenerative disease modeling; and 3) Whole organism HTS discovery of regenerative therapeutics. Importantly, this approach can be deployed in any transgenic animal model system, and is capable of modeling any disease linked to the loss of specific cell or tissue types. The system employs a prodrug converting nitroreductase (NTR) enzyme, which was first developed to [eradicate human cancer cells via gene therapy.] To create novel cell-specific regeneration paradigms, a NTR enzyme and a fluorescent reporter gene are placed under the control of a cell-type specific promoter in a transgenic organism [(i.e., only expressed in the targeted cells/tissues)]. NTR expression selectively sensitizes those cells to prodrugs that produce cytotoxic derivatives [(e.g., metronidazole), enabling precise] targeted cell ablation, on [demand, without harming surrounding cells]. The reporter allows subsequent regenerative processes to be characterized in detail. This versatile NTR system has been adapted] to multiple species, promoting a wide array of cell-specific regeneration paradigms and degenerative disease models. However, inadequate prodrug activation compromises the full potential of this approach; this limits the types of cells that can be ablated, slows cell loss kinetics, and necessitates semi-toxic prodrug regimens that can confound key data. We propose to develop superior iterations of NTR/prodrug systems that will: 1) Provide additional dimensions of cell targeting; 2) Accelerate cell ablation kinetics, to facilitate better resolutionof regeneration kinetics; and 3) Alleviate systemic semi-toxicity associated with current prodrug treatment regimens.
The specific aims of this study are: 1) Identify NTR variants with improved cell-specific ablation efficacy; 2) Define selective NTR prodrug pairs to facilitate independent ablation of multiple cell types; and 3) Develop pan-species transgenic [vectors to create improved] NTR resources. To achieve these aims we have assembled a library of NTR variants from 24 bacterial species, used directed evolution (random mutagenesis at a single-gene level, followed by selection for improved enzyme variants) to tailor desirable NTR activities, identified new prodrug substrates, and developed high-throughput methods to quantify ablation efficacy in comparative assays. We will apply these resources to develop NTR [variants with improved enzymatic activity, and novel NTR/prodrug pairs with enhanced specificity. Our improved NTRs will: 1) Facilitate the creation of improved animal models to yield insights] into cell-specific regeneration; 2) Enable identification of factors that regulate the regenerative potential of discrete stem cell niches; and 3) Aid development of curative therapies for the many degenerative conditions linked to the loss of specific cells.
The fact that humans cannot replace certain cell types makes us susceptible to numerous diseases. To discover drugs that protect us from these conditions, [and that may even promote] cell replacement, new animal models of degenerative disease are needed-specifically, [models that are uniquely] tailored to large- scale chemical testing. Here, we [propose to substantially improve] an elegant and flexible animal model system that we developed to facilitate better understanding of regenerative processes, and which is designed specifically to enable the discovery of drugs that promote the survival, sustained function, and regeneration of cell types lost to human disease.
|Vergara, M Natalia; Flores-Bellver, Miguel; Aparicio-Domingo, Silvia et al. (2017) Three-dimensional automated reporter quantification (3D-ARQ) technology enables quantitative screening in retinal organoids. Development 144:3698-3705|