Circular mimics of Iron-response elements to inhibit ferroptosis

铁反应元件的圆形模拟物抑制铁死亡

• 批准号: 10258929
• 负责人: Brian Frederick Pickering
• 金额: $ 43.34万
• 依托单位: CHIMERNA THERAPEUTICS INC.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10258929
• 关键词: Animal Model; Binding; Binding Proteins; Brain; Catalytic RNA; Cell Death; Cells; Chelating Agents; Cleaved cell; Data; Deferoxamine; Disease; Disease model; Drug Kinetics; Drug Metabolic Detoxication; Excision; Exhibits; FDA approved; Ferritin; Genetic Transcription; Homeostasis; In Vitro; Iron; Iron Chelating Agents; Iron Overload; Iron-Regulatory Proteins; Lead; Legal patent; Link; MPTP Poisoning; MPTP model; Mediating; Messenger RNA; Modality; Modeling; Nerve Degeneration; Neurologic; Neurons; Oligonucleotides; Parkinson Disease; Pathology; Pathway interactions; Patients; Phase; Plasmids; Problem Solving; Process; Protein Export Pathway; Proteins; RNA; RNA Binding; RNA Ligase (ATP); RNA Sequences; RNA Splicing; RNA-Binding Proteins; Resistance; Response Elements; Scientist; Small Interfering RNA; Small RNA; System; TFRC gene; Technology; Testing; Therapeutic; Time; Tornadoes; Toxic effect; Transcript; Transfection; Translations; Universities; alpha synuclein; base; blood-brain barrier permeabilization; circular RNA; clinical development; cohort; experimental study; heme biosynthesis; inhibitor/antagonist; metal transporting protein 1; mouse model; new technology; novel strategies; novel therapeutics; preclinical development; prevent; programs; response; restoration; small hairpin RNA; small molecule; therapeutic RNA; therapeutic target; uptake

SUMMARY: Iron overload is found in nearly all patients with Parkinson's disease and can lead to ferroptosis, an iron-dependent form of cell death. The importance of iron in Parkinson's disease pathology is supported by the beneficial effects of iron chelators in culture and animal models of Parkinson'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 cell'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 cell'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 Parkinson'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 Parkinson's disease model neurons. Here, we will use two Parkinson'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 Parkinson's disease.

摘要：铁超载几乎存在于所有帕金森病患者中，并可导致 铁凋亡，一种依赖铁的细胞死亡形式。铁在帕金森病病理学中的重要性 铁螯合剂在帕金森病的培养和动物模型中的有益作用支持了这一观点。 然而，铁螯合剂具有差的药代动力学、差的血脑屏障渗透性，并且它们 激活代偿性细胞反应，包括激活铁吸收进入细胞。因此，细胞的 对螯合剂的代偿反应最终可能抵消铁的任何有益的去除效果。 螯合剂。因此，开发可能更有效的替代性除铁疗法非常重要 比传统的螯合剂。在这项提案中，我们提出了一种全新的方法， 神经元中的铁我们不使用螯合剂，而是激活细胞的内源性铁去除， 戒毒项目细胞内的铁稳态依赖于两种铁敏感蛋白，IRP 1和IRP 2 （铁反应蛋白1和2）。IRP 1和IRP 2是RNA结合蛋白，其结合含有RNA的mRNA。 IRE（铁反应元件）发夹序列。抑制IRP 1和IRP 2会导致细胞激活 降低细胞内铁水平的途径。我们正在创造一种新型的RNA疗法， IRE RNA发夹作为“诱饵”来阻断IRP 1和2结合其靶mRNA。虽然小RNA 在细胞中不稳定，Chimerna开发了一种新技术，可以快速地将小RNA 在体外或当在细胞中表达时环化。我们在HEK 293细胞中的研究表明， 诱导强有力铁去除程序并赋予对铁凋亡的抗性。在这一点上，主要的问题是 在帕金森病模型中，环形IRE是否可以阻断铁下垂。为了验证这个想法， 本发明的具体目的是：（1）优化环状IRE RNA和环状IRE- 表达质粒用于中脑神经元中的铁耗竭。为此，我们将优化两个 环状IRE RNA的不同递送模式：（A）环状IRE RNA的直接转染;和（B）质粒- 基于圆形IRE的表达。我们将测试总铁还原效率，转铁蛋白受体， 培养的神经元中的铁蛋白和膜铁转运蛋白水平。总的来说，这些实验将优化两种不同的 在神经元中获得环状IRE RNA的方法。(2)比较去铁胺和圆形IRE， 在培养的帕金森病模型神经元中的神经变性抑制剂。在这里，我们将使用 两种帕金森病模型：MPTP毒性和α-突触核蛋白毒性。我们将比较圆形IRE 去铁胺和铁螯合剂，以确定环状IRE是否与，或可能更有效， 比标准的基于螯合剂的方法。如果这些方法取得成功，就意味着， IRP 1/2是一个治疗靶点，环状RNA代表了不同于小分子RNA的新模式， 帕金森氏症的分子螯合剂