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 肌萎缩侧索硬化症（ALS）是一种致命的神经退行性疾病，其特征是包涵体 运动神经元细胞质中含有 TDP-43 蛋白，这些包涵体是由 TDP- 引起的。 43 聚集，这是由导致 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 的能力是最终实现这一过程的关键步骤。 当 TDP-43 结合细胞 RNA 时，它会导致 TDP-43 聚集，并发生“相分离”。 富含应激颗粒，其中 TDP-43 缓慢形成不溶性聚集体，是一种广泛接受的治疗方法。 目标是防止 TDP-43 相分离成应力颗粒，因为这是先决条件 为了阻止 TDP-43 相分离，我们使用结合 TDP-43 的 RNA 适体。 RNA 结合结构域，因此充当“诱饵”来阻止 TDP-43 与细胞 mRNA 结合。 Chimerna 科学家开发了一种创新的 RNA 适体在细胞质中快速降解。 表达系统允许RNA适体在细胞内快速转化为“环状”RNA，从而使 它们对细胞核酸外切酶具有抵抗力，这极大地提高了 RNA 适体的稳定性，从而导致 表达水平比使用该系统的任何以前的表达系统至少高100倍， 我们发现圆形 TDP-43 结合适体可以使用光遗传学阻断 TDP-43 的相分离 TDP-43 相分离测定由于环状 RNA 是胞质的，因此它们选择性地阻断胞质。 TDP-43的形式而不影响TDP-43在细胞核中的正常功能。 TDP-43 结合适体将防止疾病中的 TDP-43 聚集、毒性和神经退行性变 为了测试这一点，该提案的具体目标是：（1）进化TDP-43适体。 更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。 我们将使用我们的光遗传学平台来诱导 TDP-43 相分离。 细胞计算顶级进化适体的IC50值这些实验将揭示适体 (2) 测试圆形TDP-43的能力 适配体可阻断人 iPS 神经元中 TDP-43 的相分离、聚集和毒性。 我们将使用 iPS 衍生的 ALS 运动神经元，这些神经元表现出 TDP-43 聚集体、聚集毒性标记物，以及 我们将确定 TDP-43 适体是否可以增加对氧化应激介导的细胞死亡的敏感性。 阻断 TDP-43 聚集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 总体而言，这些实验将测试使用疾病相关模型的适体方法。 使用高度稳定的圆形 TDP-43 适体阻断 TDP-43 聚集，该适体将使用 光遗传学相分离测定和 ALS iPS 衍生的运动神经元。