Circular RNA aptamers to regulate iron homeostasis in iron overload disorders

环状RNA适体调节铁过载疾病中的铁稳态

• 批准号: 10484377
• 负责人: Brian Frederick Pickering
• 金额: $ 25.52万
• 依托单位: CHIMERNA THERAPEUTICS INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-17 至 2024-03-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10484377
• 关键词: Acetylgalactosamine; Asialoglycoprotein Receptor; Binding; Catalytic RNA; Cell Death; Cells; Chelating Agents; Data; Disease; Disease model; Dose; Drug Kinetics; Drug Metabolic Detoxication; Excision; Exhibits; FDA approved; Ferritin; Fibrosis; Galactosamine; Genetic Transcription; Hemochromatosis; HepG2; Hepatic; Hepatocyte; Homeostasis; Human; Impairment; In Vitro; Inflammation; Iron; Iron Chelating Agents; Iron Overload; Iron-Regulatory Proteins; Legal patent; Liver; Liver diseases; Maintenance; Mediating; Messenger RNA; Modeling; Oligonucleotides; Pathway interactions; Patients; Plasma; Problem Solving; Process; Production; Proteins; RNA; RNA Binding; RNA Ligase (ATP); RNA Sequences; RNA Splicing; RNA-Binding Proteins; Reactive Oxygen Species; Resistance; Response Elements; Scientist; Small Interfering RNA; Small RNA; System; TFRC gene; Technology; Testing; Therapeutic; Time; Tornadoes; Transcript; Translations; Universities; Venous blood sampling; acquired hemochromatosis; aptamer; base; cellular targeting; circular RNA; clinical development; cohort; experimental study; ferric ammonium citrate; heme biosynthesis; hepcidin; metal transporting protein 1; novel strategies; novel therapeutics; peptide hormone; prevent; programs; response; restoration; small hairpin RNA; small molecule; therapeutic RNA; uptake

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.

摘要：铁超载障碍是由肝脏中铁的动态平衡失衡引起的。 过量的铁会产生活性氧物种，导致炎症、纤维化和铁性下垂。 铁依赖的细胞死亡形式。降低铁水平有治疗上的好处。虽然络合铁是 对于这些疾病，铁络合剂表现出较差的药代动力学，并诱导出代偿性铁。 细胞摄取反应。在这项提案中，我们提出了一种全新的除铁方法 来自肝细胞。我们的方法针对IRP1/2，这是一种控制铁稳态的蛋白质。IRP1/2结合 只有在铁含量较低的情况下，才能在某些mRNA中发现“IRE”序列。IRP1/2与mRNA结合导致 翻译引起细胞内铁离子流入的蛋白质。由于Irp1/2在铁超载时被异常激活 紊乱时，阻断IRP1/2‘S的核糖核酸结合活性可降低细胞内铁，减少铁性下垂。 由于Irp1的一个基本功能，Irp1/2不能被小分子或siRNA靶向，我们 正在提出一种新的治疗概念：我们正在使用IRE序列作为“诱饵”来阻止IRP1/2 结合信使核糖核酸，从而模拟“高铁”状态，并导致细胞降低细胞铁水平。这 这种方法通常是不可能的，因为像IRR这样的小RNA在细胞中非常不稳定。 然而，Chimerna的科学家已经开发出一种新的策略，通过合成这种RNA来稳定IRE 就像一个RNA环。我们发现，这些环状IRES诱导了一个强大的铁去除程序，并导致细胞 对铁性下垂产生抵抗力。在这一点上，主要的问题是这些循环IRES能否有效 靶向肝细胞，它们是否达到治疗浓度。为了测试这一想法， 这项建议的具体目标是：(1)优化环状ire RNA的递送和剂量 扰乱细胞铁稳态。我们将合成具有三个N-天线的圆形IRES- 乙酰半乳糖胺，允许CircRNA优先被肝脏(2)摄取，以测试 在铁超载紊乱培养模型中环状IRES减少铁性下垂的能力。在这里，我们 将使用柠檬酸铁铵处理的原代人肝细胞的铁超载紊乱模型 (FAC)。将Tri(GalNAc)修饰的环状IRES导入原代肝细胞。这些 比较将使我们能够确定循环IRES是否与或可能更有效 标准的基于螯合剂的方法。总体而言，该应用程序将(1)创建一种全新的 RNA治疗；(2)首次以细胞铁稳态为靶点；(3)提供一种全新的 治疗铁超负荷紊乱的方法。