Project Grant R43NS129441

A NOVEL TDP-43-TARGETING CIRCULAR RNA TO TREAT AMYOTROPHIC LATERAL SCLEROSIS (ALS) - SUMMARY AMYOTROPHIC LATERAL SCLEROSIS (ALS) IS A FATAL NEURODEGENERATIVE DISEASE CHARACTERIZED BY INCLUSION BODIES CONTAINING THE TDP-43 PROTEIN IN THE CYTOPLASM OF MOTOR NEURONS. THESE INCLUSION BODIES ARE CAUSED BY TDP- 43 AGGREGATION, WHICH IS CAUSED BY PATHWAYS OR MUTATIONS THAT CAUSE TDP-43 TO LOCALIZE TO THE CYTOPLASM. TDP-43 IS AN RNA-BINDING PROTEIN AND ITS ABILITY TO BIND MRNA IS A CRITICAL STEP IN THE PROCESS THAT ULTIMATELY LEADS TO TDP-43 AGGREGATES. WHEN TDP-43 BINDS CELLULAR RNAS, IT CAN "PHASE SEPARATE" AND BECOME ENRICHED IN STRESS GRANULES, WHERE TDP-43 SLOWLY FORMS INSOLUBLE AGGREGATES. A WIDELY ACCEPTED THERAPEUTIC GOAL IS TO PREVENT TDP-43 FROM UNDERGOING PHASE SEPARATION INTO STRESS GRANULES, SINCE THIS IS A PREREQUISITE FOR TDP-43 AGGREGATION. TO BLOCK TDP-43 PHASE SEPARATION, WE ARE USING RNA APTAMERS THAT BIND TDP-43'S RNA-BINDING DOMAIN AND THUS ACT AS A "DECOY" TO BLOCK TDP-43 FROM BINDING TO CELLULAR MRNA. HOWEVER, RNA APTAMERS ARE RAPIDLY DEGRADED IN THE CYTOSOL. CHIMERNA SCIENTISTS HAVE DEVELOPED AN INNOVATIVE EXPRESSION SYSTEM THAT ALLOWS RNA APTAMERS TO RAPIDLY BE CONVERTED INTO "CIRCULAR" RNAS IN CELLS, THUS MAKING THEM RESISTANT TO CELLULAR EXONUCLEASES. THIS DRAMATICALLY INCREASES RNA APTAMER STABILITY, LEADING TO EXPRESSION LEVELS THAT ARE AT LEAST 100 TIMES HIGHER THAN ANY PREVIOUS EXPRESSION SYSTEM. USING THIS SYSTEM, WE FOUND THAT CIRCULAR TDP-43-BINDING APTAMERS CAN BLOCK PHASE SEPARATION OF TDP-43 USING AN OPTOGENETIC TDP-43 PHASE-SEPARATION ASSAY. SINCE THE CIRCULAR RNAS ARE CYTOSOLIC, THEY SELECTIVELY BLOCK THE CYTOSOLIC FORM OF TDP-43 WITHOUT AFFECTING THE NORMAL FUNCTION OF TDP-43 IN THE NUCLEUS. THE CRITICAL QUESTION IS WHETHER TDP-43-BINDING APTAMERS WILL PREVENT TDP-43 AGGREGATION, TOXICITY, AND NEURODEGENERATION IN DISEASE- RELEVANT MODELS. TO TEST THIS, THE SPECIFIC AIMS OF THIS PROPOSAL ARE: (1) TO EVOLVE TDP-43 APTAMERS FOR HIGHER AFFINITY AND LOWEST IC50 FOR BLOCKING PHASE SEPARATION. WE WILL USE DIRECTED EVOLUTION TO EVOLVE HIGH-AFFINITY TDP-43 APTAMERS. WE WILL USE OUR OPTOGENETIC PLATFORM FOR INDUCING TDP-43 PHASE SEPARATION IN CELLS TO CALCULATE IC50 VALUES FOR THE TOP EVOLVED APTAMERS. THESE EXPERIMENTS WILL REVEAL THE APTAMERS THAT HAVE THE GREATEST POTENTIAL TO DISRUPT TDP-43 PHASE SEPARATION. (2) TO TEST THE ABILITY OF CIRCULAR TDP-43 APTAMERS TO BLOCK PHASE SEPARATION, AGGREGATION, AND TOXICITY OF TDP-43 IN HUMAN IPS NEURONS. HERE, WE WILL USE IPS-DERIVED ALS MOTOR NEURONS THAT EXHIBIT TDP-43 AGGREGATES, MARKERS OF AGGREGATE TOXICITY, AND INCREASED SUSCEPTIBILITY TO OXIDATIVE STRESS-MEDIATED CELL DEATH. WE WILL DETERMINE IF TDP-43 APTAMERS CAN BLOCK THESE KEY PARAMETERS OF TDP-43 AGGREGATE TOXICITY, THUS PROVIDING CRITICAL DATA ABOUT THE VALIDITY OF OUR APTAMER APPROACH USING DISEASE-RELEVANT MODEL. OVERALL, THESE EXPERIMENTS WILL TEST A NEW CONCEPT FOR BLOCKING TDP-43 AGGREGATION USING HIGHLY STABLE CIRCULAR TDP-43 APTAMERS, THAT WILL BE TESTED USING AN OPTOGENETIC PHASE SEPARATION ASSAY AND ALS IPS-DERIVED MOTOR NEURONS.