Development of Novel RNA Sensor-Based Treatment for Ewing’s Sarcoma

Chromosomal translocations creating novel fusion molecules occur frequently in sarcomas. These not only contribute to oncogenesis, but also serve as unique molecular tags distinguishing cancerous from non-cancerous cells. The rationale for this proposal is that these unique mRNA tags can be used to selectively target cells for death using cutting-edge programmable gene editing technologies. Reprogrammable ADAR Sensors (RADARS), is a newly developed system that recognizes a specific RNA transcript, and if present in the cell, activates the expression of a payload protein. The system works by expressing a “sensor” mRNA that includes a translational start site, a short initial open reading frame containing a UAG stop codon, followed in-frame by the sequence encoding a “payload” protein. The sequence flanking the UAG is designed to be complementary to the “detected” RNA and centered on a CCA codon in the “detected RNA”. If the “detected” RNA is not present in the cells, the payload protein is not produced. But, if the “detected” RNA is present in the cells, it forms a dsRNA substrate near the UAG, including an A-C mismatch bubble at the center of the UAG codon. This recruits the RNA editing enzyme ADAR, which edits the UAG to UIG, allowing read-through and translation of the downstream payload protein. We hypothesize that ADARS can be adapted to selectively kill sarcoma cells harboring oncogenic fusion mRNAs. The specific aim of this proposal is to develop RADARs to recognize EWS-FLI1 fusion mRNAs in Ewing’s sarcoma and test their ability to activate the HSV-TK ganciclovir-inducible suicide gene. The approach will involve generating and optimizing RADARS constructs to detect EWS-FLI1 using a GFP payload. We will then replace GFP with HSV-TK and test their ability to kill cells harboring EWS-FLI fusion mRNA transcripts. Specificity and sensitivity of the system will then be established. Positive results from this proof-of-principle study could then be extended to other fusion transcripts associated with other sarcomas in the future. RADARS is an mRNA-based method and is compatible with new synthetic mRNA/lipid nanoparticle delivery systems. Therefore, positive results from this study would be able to harness this new therapeutic platform, which of high current commercial/pharmaceutical interest. This will facilitate future clinical translation of this approach. In addition, the HSV-TK suicide gene system is currently in clinical use with CAR T-cell immunotherapy and other applications, further facilitating clinical translation of the work from this project.