CHIMERNA THERAPEUTICS, INC. — Department of Health and Human Services SBIR Phase I: NINDS

Description

SUMMARY: Iron overload is found in nearly all patients with Parkinsonandapos;s disease and can lead to ferroptosis, an iron-dependent form of cell death. The importance of iron in Parkinsonandapos;s disease pathology is supported by the beneficial effects of iron chelators in culture and animal models of Parkinsonandapos;s disease. However, iron chelators have poor pharmacokinetics, poor blood-brain barrier permeability, and they activate a compensatory cellular response that involves activating iron uptake into cells. Thus, the cellandapos;s compensatory response to chelators could eventually counteract any beneficial iron-removal effect of the chelator. Therefore, it is important to develop alternative iron removal therapies that may be more effective than conventional chelators. In this proposal, we are proposing a completely novel approach for removing iron from neurons. Rather than using a chelator, we are activating the cellandapos;s endogenous iron removal and detoxification programs. Iron homeostasis in the cell relies on two iron-sensing proteins, IRP1 and IRP2 (iron-response protein 1 and 2). IRP1 and 2 are RNA-binding proteins that bind to mRNAs that contain an IRE (iron-response element) hairpin sequence. Inhibiting IRP1 and 2 would cause the cell to activate pathways that reduce intracellular iron levels. We are creating a new type of RNA therapy in which we use the IRE RNA hairpin as a “decoy” to block IRP1 and 2 from binding its target mRNAs. Although small RNAs are unstable in cells, Chimerna has developed a novel technology that allows small RNAs to be rapidly circularized, either in vitro, or when expressed in cells. Our studies in HEK293 cells show that circular IREs induce a robust iron removal program and confer resistance to ferroptosis. At this point, the major question is whether circular IREs can block ferroptosis in models of Parkinsonandapos;s disease. In order to test this idea, the specific aims of this proposal are: (1) To optimize transfection of circular IRE RNA and circular IRE- expressing plasmids for iron depletion in mesencephalic neurons. In this aim, we will optimize two distinct delivery modes for circular IRE RNAs: (A) direct transfection of circular IRE RNAs; and (B) plasmid- based expression of circular IREs. We will test the efficiency of total iron reduction, transferrin receptor, ferritin and ferroportin levels in cultured neurons. Overall, these experiments will optimize two different approaches for achieving circular IRE RNA in neurons. (2) To compare deferoxamine and circular IRE as inhibitors of neurodegeneration in a cultured Parkinsonandapos;s disease model neurons. Here, we will use two Parkinsonandapos;s disease models: MPTP toxicity and alpha-synuclein toxicity. We will compare circular IREs to desferoxamine, and iron chelator, to determine whether circular IREs are as, or potentially more effective, than standard chelator-based approaches. If these approaches are successful, it would suggest that IRP1/2 is a therapeutic target, and that circular RNAs represented new modality distinct from small molecule chelators for Parkinsonandapos;s disease.PROJECT NARRATIVE Although Parkinsonandapos;s disease is associated with iron overload and exhibits iron-mediated neurodegeneration, strategies to reverse this pathology with iron chelators have shown limited efficacy due to their poor pharmacokinetics and inability to quantitatively deplete iron from the brain. Here we describe a new approach to restore iron homeostasis in neurons using an RNA therapeutic that selectively binds and blocks the two iron-regulatory proteins, resulting in decreased iron-uptake proteins and increased iron- storage proteins. We will determine the feasibility and rationale for preclinical development of this RNA- based inhibitor by testing its ability to reduce iron levels and preventing iron-mediated neurodegeneration in cultured neurons from a murine model of Parkinsonandapos;s disease.