Project Grant R43DK133031

CIRCULAR RNA APTAMERS TO REGULATE IRON HOMEOSTASIS IN IRON OVERLOAD DISORDERS - SUMMARY: IRON OVERLOAD DISORDERS ARE CAUSED BY HOMEOSTATIC IMBALANCE OF IRON IN THE LIVER. EXCESS IRON PRODUCES REACTIVE OXYGEN SPECIES THAT INDUCE INFLAMMATION, FIBROSIS, AND CAUSE FERROPTOSIS, AN IRON-DEPENDENT FORM OF CELL DEATH. REDUCING IRON LEVELS HAS A THERAPEUTIC BENEFIT. ALTHOUGH CHELATING IRON IS APPEALING FOR THESE DISORDERS, IRON CHELATORS EXHIBIT POOR PHARMACOKINETICS AND INDUCE A COMPENSATORY IRON UPTAKE RESPONSE IN CELLS. IN THIS PROPOSAL, WE ARE PROPOSING A COMPLETELY NOVEL APPROACH FOR REMOVING IRON FROM HEPATOCYTES. OUR APPROACH TARGETS IRP1/2, THE PROTEINS THE CONTROL IRON HOMEOSTASIS. IRP1/2 BINDS "IRE" SEQUENCES FOUND IN CERTAIN MRNAS ONLY WHEN IRON LEVELS ARE LOW. IRP1/2 BINDING TO MRNA LEADS TO TRANSLATION OF PROTEINS THAT CAUSE IRON INFLUX IN CELLS. SINCE IRP1/2 IS ABNORMALLY ACTIVATED IN IRON OVERLOAD DISORDERS, BLOCKING THE IRP1/2'S RNA-BINDING ACTIVITY COULD REDUCE INTRACELLULAR IRON AND REDUCE FERROPTOSIS. SINCE IRP1/2 CANNOT BE TARGETED WITH SMALL MOLECULES OR SIRNA DUE TO AN ESSENTIAL FUNCTION OF IRP1, WE ARE PROPOSING A NEW THERAPEUTIC CONCEPT: WE ARE USING IRE SEQUENCES AS "DECOYS" TO BLOCK IRP1/2 FROM BINDING MRNA, THUS SIMULATING A "HIGH IRON" STATE, AND CAUSING THE CELL TO REDUCE CELLULAR IRON LEVELS. THIS APPROACH WOULD NORMALLY BE IMPOSSIBLE SINCE SMALL RNAS, LIKE THE IRE, ARE HIGHLY UNSTABLE IN CELLS. HOWEVER, CHIMERNA SCIENTISTS HAVE DEVELOPED A NEW STRATEGY TO STABILIZE THE IRE BY SYNTHESIZED THIS RNA AS AN RNA CIRCLE. WE FIND THAT THESE CIRCULAR IRES INDUCE A ROBUST IRON REMOVAL PROGRAM AND CAUSE CELLS TO BECOME RESISTANT TO FERROPTOSIS. AT THIS POINT, THE MAJOR QUESTION IS CAN THESE CIRCULAR IRES BE EFFECTIVELY TARGETED TO HEPATOCYTES AND DO THEY REACH A THERAPEUTIC CONCENTRATION. IN ORDER TO TEST THIS IDEA, THE SPECIFIC AIMS OF THIS PROPOSAL ARE: (1) TO OPTIMIZE THE DELIVERY AND DOSE OF CIRCULAR IRE RNA FOR PERTURBING CELLULAR IRON HOMEOSTASIS. WE WILL SYNTHESIZE CIRCULAR IRES WITH A TRIANTENNARY N- ACETYLGALACTOSAMINE THAT ALLOWS THE CIRCRNAS TO BE PREFERENTIALLY TAKEN UP BY THE LIVER (2) TO TEST THE ABILITY OF CIRCULAR IRES TO REDUCE FERROPTOSIS IN CULTURE MODELS OF IRON OVERLOAD DISORDERS. HERE, WE WILL USE AN IRON OVERLOAD DISORDER MODEL OF PRIMARY HUMAN HEPATOCYTES TREATED WITH FERRIC AMMONIUM CITRATE (FAC). PRIMARY HEPATOCYTES WILL BE TRANSFECTED WITH TRI(GALNAC)-MODIFIED CIRCULAR IRES. THESE COMPARISONS WILL ALLOW US TO DETERMINE WHETHER CIRCULAR IRES ARE AS, OR POTENTIALLY MORE EFFECTIVE, THAN STANDARD CHELATOR-BASED APPROACHES. OVERALL, THIS APPLICATION WILL (1) CREATE A FUNDAMENTALLY NEW TYPE OF RNA THERAPEUTIC; (2) TARGET CELLULAR IRON HOMEOSTASIS FOR THE FIRST TIME; AND (3) PROVIDE A FUNDAMENTALLY NEW APPROACH TO TREAT IRON OVERLOAD DISORDERS.