We analyze responses of three human tissue models to low-dose protracted GCR simulations, and identify and develop countermeasures using optogenetics and molecular antagonists. Human tissue models include vascular, cerebrovascular, and cardiac. These are 3D constructs generated from human pluripotent stem cells (hPSCs) and are well characterized.

Radiation exposures are in alignment with NASA guidelines. In a slight departure, we make use of a newly developed method to modify the standard GCR beam at NASA Space Radiation Laboratory (NSRL) in order to provide a GCR spectrum that better emulates one inside a spacecraft. This alleviates some of the concerns with the existing radiation sources, and provides a more direct transfer of our results to the actual spaceflight situation.

The project is organized into three specific aims. First, we determine the effect of radiation exposure on cell viability and cell cycle, tissue integrity and functionality, and the activation of oxidative stress and high mobility group box 1 (HMGB1) pathways. This will validate the usefulness of our biological models, and radiation exposures, for the subsequent investigation of countermeasures. Second, we use an integrative systems approach to identify therapeutic (countermeasure) targets to mitigate radiation damage. This is accomplished with large-scale quantitative proteomics, multi-data fusion and network analysis, and conformational inhibition tests. Ultimately, in aim three, we develop and test countermeasures based on optogenetics and protein antagonists, to activate or inhibit pathways impacted by radiation.

The results of this project will help to determine if complex human models can serve as an effective testbed for the effects of space radiation on intact humans, and will identify and assess possible countermeasures to these effects.

We analyze responses of three human tissue models to low-dose protracted GCR simulations and identify and develop countermeasures using optogenetics and molecular antagonists. Human tissue models include vascular, cerebrovascular, and cardiac. These are 3D constructs generated from hiPSCs and are well characterized.

The specific aims are: (1) Characterizing responses of 3D models of human vasculature, neurovascular, and cardiac tissues to exposure to space radiation; (2) An integrative systems-medicine approach to identifying therapeutic targets for minimizing space-radiation-induced damage; and (3) Developing and testing countermeasures using optogenetics and protein antagonist therapies to protect human tissue from radiation damage.

Preliminary findings following the first year of the project indicate that simulated GCR potentially poses a significant risk to vascular, heart, and neurovascular health. Following GCR exposure experiments, we also note the need to adjust our 3D tissue models to continue with the proposed experiments. Computational network models of drug-gene interactions that integrate across numerous data sources were created. Finessing our proteomic cell extraction protocols and workflow to fit the 3D tissue models is in progress. Countermeasure development continues with the establishment of the Opto-fibroblast growth factor receptor (Opto-FGFR) hiPSC line and engineering an antagonist to target HMGB1.

Our findings thus far do not alter our original hypothesis or the objective and aims as originally proposed. Ongoing modifications to our technologies and protocols would support the proposed experimental plan and will continue into next year. We will continue to characterize responses of the human complex model for space radiation, integrate advanced computational techniques for multi-scale modeling proteome, and begin testing countermeasures.

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Additional progress notes from PI (Ed., 10/12/22)

• While still within the preliminary stages of the research project, we have putatively identified key consequences of simulated GCR exposure on our 3D human tissues. • Improvements to our model systems are enabling us to perform in depth analyses of radiation impact. • Key modifications were made to identify protocols for cell extraction from the 3D human model systems for downstream molecular analysis. • Refinements of proteomics workflows for operating on small amounts of material were made. • Computational network models of drug-gene interactions that integrate across numerous data sources were created. • Using optogenetics we began to develop a potential countermeasure to GCR in the human iPSC line. • Ongoing countermeasure development work is designing a novel anti-inflammatory protein that promises to have utility in preventing radiation damage. • Our successful presentation of the HMGB1 box A domain on the yeast surface display platform empowers new directions in engineering immunomodulatory proteins as targeted drugs. • Monte Carlo simulations of the GCR blocker enabling us to determine the experimental exposures to more closely reflect the field that would be incurred by these cells at the depth in tissues during spaceflight.

We analyze responses of three human tissue models to low-dose protracted GCR simulations, and identify and develop countermeasures using optogenetics and molecular antagonists. Human tissue models include vascular, cerebrovascular, and cardiac. These are 3D constructs generated from human pluripotent stem cells (hPSCs) and are well characterized.

Radiation exposures are in alignment with NASA guidelines. In a slight departure, we make use of a newly developed method to modify the standard GCR beam at NASA Space Radiation Laboratory (NSRL) in order to provide a GCR spectrum that better emulates one inside a spacecraft. This alleviates some of the concerns with the existing radiation sources, and provides a more direct transfer of our results to the actual spaceflight situation.

The project is organized into three specific aims. First, we determine the effect of radiation exposure on cell viability and cell cycle, tissue integrity and functionality, and the activation of oxidative stress and high mobility group box 1 (HMGB1) pathways. This will validate the usefulness of our biological models, and radiation exposures, for the subsequent investigation of countermeasures. Second, we use an integrative systems approach to identify therapeutic (countermeasure) targets to mitigate radiation damage. This is accomplished with large- scale quantitative proteomics, multi-data fusion and network analysis, and conformational inhibition tests. Ultimately, in aim three, we develop and test countermeasures based on optogenetics and protein antagonists, to activate or inhibit pathways impacted by radiation.

The results of this project will help to determine if complex human models can serve as an effective test bed for the effects of space radiation on intact humans, and will identify and assess possible countermeasures to these effects.