A Novel TDP-43-Targeting Circular RNA to Treat Amyotrophic Lateral Sclerosis (ALS)

一种新型 TDP-43 靶向环状 RNA 治疗肌萎缩侧索硬化症 (ALS)

• 批准号: 10547146
• 负责人: Jacob Litke
• 金额: $ 25.62万
• 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: 2022-09-08 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10547146
• 关键词: ALS patients; Affect; Affinity; Amyotrophic Lateral Sclerosis; Binding; Binding Proteins; Biological Assay; Catalytic RNA; Cell Death; Cell Nucleus; Cells; Clinical Trials; Cytoplasm; Cytoplasmic Protein; Cytosol; DNA Sequence Alteration; Data; Directed Molecular Evolution; Disease; Dose; Evolution; Exhibits; Exonuclease; FDA approved; Genetic Transcription; Goals; Human; Inclusion Bodies; Kerckring' s valve; Legal patent; Link; Mediating; Messenger RNA; Modeling; Motor Neurons; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Oligonucleotides; Oxidative Stress; Pathway interactions; Patients; Phase; Predisposition; Prevention; Problem Solving; Process; RNA; RNA Binding; RNA Instability; RNA Ligase (ATP); RNA Recognition Motif; RNA Sequences; RNA Splicing; RNA-Binding Proteins; Resistance; Scientist; Small RNA; Stress; Structure; System; TDP-43 aggregation; Technology; Testing; Therapeutic; Time; Tissues; Tornadoes; Toxic effect; Transcription Repressor; Universities; Viral; appropriate dose; aptamer; base; circular RNA; clinical development; experimental study; induced pluripotent stem cell; inhibitor; innovation; lipid nanoparticle; nanoparticle delivery; neuron loss; novel; novel strategies; novel therapeutics; optogenetics; preclinical development; prevent; protein TDP-43; protein aggregation; recruit; stem cell model; stress granule; therapeutic RNA

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.

概括 肌萎缩性外侧硬化症（ALS）是一种致命的神经退行性疾病，其特征是包容物体 运动神经元的细胞质中包含TDP-43蛋白。这些包容体是由TDP-引起的 43聚集是由导致TDP-43定位于细胞质的途径或突变引起的。 TDP-43是一种RNA结合蛋白，其结合mRNA的能力是该过程中最终的关键步骤 导致TDP-43聚集体。当TDP-43结合细胞RNA时，它可以“分开”并变为 富含应力颗粒，其中TDP-43慢慢形成不溶性骨料。广泛接受的疗法 目标是防止TDP-43将相位分为压力颗粒，因为这是先决条件 用于TDP-43聚合。为了阻止TDP-43相分离，我们使用的是绑定TDP-43的RNA适体 RNA结合结构域并因此充当阻断TDP-43与细胞mRNA结合的“诱饵”。然而， RNA适体在细胞质中迅速降解。 Chimerna科学家开发了一种创新的 允许RNA适体迅速转化为细胞中的“圆形” RNA的表达系统，从而使 它们对细胞外切核酸酶有抗性。这大大提高了RNA APATMER稳定性，导致 表达水平至少比任何以前的表达系统高100倍。使用此系统， 我们发现，圆形TDP-43结合适体可以使用光遗传学阻断TDP-43的相位分离 TDP-43相分离测定法。由于圆形RNA是胞质的，因此它们有选择地阻断胞质 TDP-43的形式不影响核中TDP-43的正常功能。关键问题是是否 TDP-43结合适体将防止疾病中的TDP-43聚集，毒性和神经变性 相关模型。为此，该提案的具体目的是：（1）进化TDP-43适体 较高的亲和力和最低的IC50用于阻断相分离。我们将使用定向进化来进化 高亲和力TDP-43适体。我们将使用我们的光遗传平台进行诱导的TDP-43相分离 细胞计算顶部进化的适体的IC50值。这些实验将揭示适体 具有破坏TDP-43相分离的最大潜力。 （2）测试圆形TDP-43的能力 可以阻止人IPS神经元中TDP-43的相分离，聚集和毒性的适体。这里， 我们将使用IPS衍生的ALS运动神经元暴露于TDP-43骨料，骨料毒性的标记和 增加对氧化应激介导的细胞死亡的敏感性。我们将确定TDP-43适体是否可以 阻止TDP-43总毒性的这些关键参数，从而提供有关我们的有效性的关键数据 使用与疾病相关的模型的APATMER方法。总体而言，这些实验将测试一个新概念 使用高度稳定的圆形TDP-43适体阻止TDP-43聚集 光遗传相分离测定法和ALS IPS衍生的运动神经元。