Engineering Radiation Resistance in Human Cells Through the Tardigrade-Derived DNA Repair Mechanism, the Dsup

Kurzfassung

Background Ionising radiation remains one of the most significant challenges for long-duration deep space missions due to its deleterious genomic effects, which can lead to carcinogenesis and degenerative diseases in astronauts. While physical shielding is limited by mass constraints, gene therapy has emerged as a potential mitigation strategy. A promising candidate is the Damage Suppressor (Dsup) gene from tardigrades, organisms capable of surviving radiation doses up to 5000 Gy. Methods This project, conducted at the Radiation Biology Department of the German Aerospace Center, aimed to integrate the Dsup gene into multiple mammalian cell lines (HEK293T, RPE1, and A549 with varying p53 status). The study assessed both the intrinsic toxicity of Dsup and its potential radioprotective effects on cell viability. Gene delivery was performed using chemical lipofection. Additional experiments investigated the effects of hypoxic conditions and antioxidant treatments (N-acetylcysteine [NAC] and nicotinamide [NAM]) on irradiated cells. Results Dsup was successfully cloned and expressed in HEK293T cells. However, its expression appeared to be cytotoxic in RPE1 and A549 cell lines. Overall results remain inconclusive due to low transfection efficiency using chemical lipofection. Independently, both hypoxia and antioxidant treatments demonstrated a protective “rescue effect” in cells exposed to X-ray irradiation. Discussion The limited transfection efficiency suggests that alternative delivery methods, such as lentiviral systems, are necessary to properly evaluate Dsup’s radioprotective potential. Future studies will explore combined treatments (Dsup with antioxidants and/or hypoxia) to determine whether effects are synergistic, additive, or redundant. Additionally, Dsup may be investigated for its ability to compensate for the loss of DNA damage repair proteins using CRISPR/Cas9-mediated knockouts, further elucidating its potential role in radioprotection.transfection efficiency of these cells via chemical lipofection was poor, therefore a repeat should be conducted using a lentiviral delivery system. In addition to this, this internship also conducted studies on the treatment of cells in hypoxic conditions and with NAC/NAM (seperately); both of which demonstrated a “rescue effect” when irradiated with X-rays. Future experiments are to be conducted to merge Dsup + Antioxidants, and Dsup + Hypoxia to investigate if there is a synergistic, additive or redundant effect of protectiveness against ionising radiation. Moreover, Dsup can also be tested to see if it cancompensate the activity of a DNA Damage Repair protein if it is knocked out using the CRISPR/Cas9 system.