Project Grant R43AG085837

CELL-BASED RNA DEGRADATION ASSAY FOR C9ALS/FTD DRUG DISCOVERY - PROJECT SUMMARY TO OVERCOME THE "UNDRUGGABLE" PROTEIN PROBLEM, RESEARCHERS HAVE TURN TO DRUGGING MRNAS THAT TRANSLATE INTO THESE UNDRUGGABLE PROTEINS OR NON-CODING RNAS THAT REGULATE THE BIOGENESIS OF THESE PROTEINS. AVENUES OF CONTROLLING RNA DEGRADATION INCLUDE INDUCED PROXIMITY DEGRADATION OR SPLICING MODULATED NONSENSE-MEDIATED DECAY (NMD). INDUCED PROXIMITY DEGRADATION IS A STRATEGY WHERE A BIFUNCTIONAL DRUG BINDS A TARGET RNA VIA A SMALL MOLECULE (SM) ENTITY ON ONE ARM AND INDUCES SELECTIVE RNA DEGRADATION VIA A RIBONUCLEASE ON THE OTHER ARM. THERAPEUTIC MODULATION OF NMD VIA RNA SPLICING IS ANOTHER STRATEGY TO SELECTIVELY CONTROL RNA DEGRADATION. EARLY-STAGE RNA DEGRADATION DRUG DISCOVERY HAS BEEN HINDERED BY CURRENT HIGH-THROUGHPUT SCREENING (HTS) ASSAY TECHNOLOGIES DESIGNED FOR PROTEIN TARGETS. SPECIFICALLY, CELL-BASED DRUG SCREENS USE MINIGENE REPORTERS THAT RELY ON LUCIFERASE OR FLUORESCENT PROTEIN SIGNALS FOR ASSAY READ-OUT. MINIGENE REPORTER DESIGN IS STRAIGHTFORWARD BUT MINIGENE REPORTER REQUIRES MRNA EXPORT TO THE CYTOSOL AND PROTEIN TRANSLATION. THIS SIGNIFICANTLY INCREASES THE RATE OF FALSE HITS PER SCREEN SINCE COMPOUNDS THAT INHIBIT GLOBAL PROTEIN TRANSLATION MACHINERY OR RNA EXPORT WILL ALSO IMPACT REPORTER READ-OUT. FURTHER, PROTEIN-BASED REPORTERS CANNOT BE USED TO STUDY NUCLEAR RNA OR NON- CODING RNA TURNOVER, AND IN DISEASES WITH NUCLEAR EXPORT DEFECTS. THUS, THERE IS AN UNMET NEED FOR A CELL-BASED HTS ASSAY PLATFORM THAT MONITORS SELECTED RNA TURNOVER DIRECTLY, REFLECTS REAL- TIME RNA DYNAMICS, AND COMPATIBLE FOR DIFFERENT TYPES OF RNA. A PRIME EXAMPLE WHERE A PREVIOUSLY INTRACTABLE DISEASE CAN BE UNLOCKED BY RNA DEGRADING DRUGS IS THE G4C2 HEXANUCLEOTIDE REPEAT EXPANSION IN THE C9ORF72 GENE - THE MOST FREQUENT GENETIC CAUSE OF AMYOTROPHIC LATERAL SCLEROSIS (ALS) AND FRONTOTEMPORAL DEMENTIA (FTD). IT IS NOW KNOWN THAT THE TOXIC GAIN-OF-FUNCTION FROM THE EXPANDED G4C2 REPEATS INDUCES ALS/FTD PATHOLOGY. ANTISENSE OLIGONUCLEOTIDES (ASOS) DESIGNED TO BIND EITHER THE G4C2 SEQUENCES OR INTRONIC REGIONS DOWNSTREAM OF THE REPEAT SEQUENCES SHOWED REDUCTION IN DISEASE PHENOTYPES IN C9ORF72 MODELS. HOWEVER, ASOS CAN TRIGGER IMMUNE RESPONSES AND SUFFER FROM TISSUE DELIVERY ISSUES. A CELL- BASED ASSAY THAT CAN REPORT EXPANDED TRANSCRIPT TURNOVER TO IDENTIFY EFFECTIVE C9ORF72 RNA GAIN- OF-FUNCTION SM INHIBITORS WILL HAVE HIGH COMMERCIAL POTENTIAL. THE GOALS OF THIS PHASE I SBIR APPLICATION WILL BE CELL-BASED RNA DEGRADATION REPORTERS FOR VALIDATED RNA TARGETS IN NEURODEGENERATIVE DISEASES. THE RESULTS WILL FORM THE FOUNDATION OF AN HTS ASSAY PLATFORM FOR EARLY-STAGE DISCOVERY SMS THAT MODULATE RNA STABILITY.