Site-directed RNA editing of Nav1.7 as a novel analgesic

Nav1.7 的定点 RNA 编辑作为新型镇痛药

• 批准号: 10398386
• 负责人: GREGORY O DUSSOR
• 金额: $ 678.99万
• 依托单位: MARINE BIOLOGICAL LABORATORY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-23 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10398386
• 关键词: Acute Pain; Adenosine; Adult; Adverse effects; Affect; Afferent Neurons; Amino Acid Substitution; Amino Acids; Analgesics; Animal Model; Animals; Arginine; Attenuated; Axon; BCAR1 gene; Base Pairing; Behavioral; Biological; Biological Assay; Capsaicin; Capsid; Cardiac; Cells; Characteristics; Clinical; Clinical Trials; Codon Nucleotides; DNA; DRADA2b protein; Data; Deaminase; Dependovirus; Development; Diabetes Mellitus; Disease model; Effectiveness; Enzymes; FDA approved; Gene Transfer; Genetic; Goals; Guanosine; Guide RNA; Heart Diseases; Human; Impairment; In Vitro; Inflammatory; Inosine; Ions; Lead; Lysine; Malignant - descriptor; Malignant Neoplasms; Measures; Mediating; Messenger RNA; Migraine; Modeling; Mus; Mutate; Nervous system structure; Neurons; Nociceptors; Opioid; Overdose; Pain; Pain Disorder; Patients; Peripheral; Permeability; Pharmaceutical Preparations; Plasmids; Positioning Attribute; Postoperative Pain; Preparation; Prevalence; Protein Isoforms; RNA; RNA Binding; RNA Editing; Reagent; Research; Role; Shunt Device; Signal Transduction; Site; Sodium Channel; Spinal Ganglia; Structure; Synapses; System; Testing; Time; Tissue Donors; Transgenic Mice; Validation; addiction; assay development; channel blockers; chronic pain; design; disability; dorsal horn; dsRNA adenosine deaminase; efficacy testing; efficacy validation; immunogenic; improved; in silico; in vivo; in vivo evaluation; induced pluripotent stem cell; mouse model; nerve injury; neuronal cell body; novel; novel therapeutic intervention; opioid abuse; pain model; pain relief; painful neuropathy; pharmacodynamic biomarker; pharmacokinetics and pharmacodynamics; screening; side effect; small molecule; spared nerve; stable cell line; targeted treatment; tool; transmission process; voltage

Chronic pain is a leading cause of disability, affecting about one-third of adults worldwide, with a prevalence greater than heart disease, cancer, and diabetes combined. Misuse and abuse of opiates have led to a nationwide addiction and overdose crisis. Thus, there is an urgent need for alternative, non-addictive analgesics. Non-selective voltage-gated sodium channel (Nav) blockers are among existing non-addictive FDA-approved drugs which can sometimes provide symptomatic relief for patients. However, their utility is limited by CNS and cardiac side effects. Genetic and functional studies of human pain disorders and animal models of pain have validated NaV1.7, a voltage-gated sodium channel that is preferentially expressed in peripheral neurons, as an attractive target for therapy. Isoform-selective Nav blockers, however, are difficult to generate and those that have been tested in clinical trials are rapidly cleared from the body, limiting their effectiveness. Alternative approaches are needed. We propose a novel, non-addictive approach to treat chronic pain by editing the messages that encode NaV1.7 in order to alter its ion selectivity. By changing a single lysine codon in the ion selectivity filter to arginine, the Na selective channel will become both Na+ and K+ selective, effectively creating a counter-current shunt that will dampen excitability. Site-Directed RNA Editing (SDRE) refers to novel mechanisms to generate programmed edits within RNAs. It relies on the ADAR (Adenosine Deaminase that Acts on RNA) enzymes, which are endogenously expressed in human cells, including sensory neurons. Directed by a guide RNA (gRNA), SDRE systems convert precisely selected adenosines to inosine, a translational mimic for guanosine, which can recode specific amino acids. For use as an analgesic, editing mRNA is preferable to DNA because it is transient, thus limiting potential off-target effects, including malignant transformations. In addition, ADARs are endogenous while enzymes for DNA manipulation (e.g., Cas proteins) are not, thus SDRE will not be as immunogenic. Compared to small molecule channel blockers, SDRE can be more specific, because it relies on Watson-Crick base-pairing of gRNAs for targeting, and its effects are likely longer lasting because they will remain as long as the edited channels are expressed. We propose to use SDRE to edit NaV1.7 K1395R to render the channel permeable to both Na+ and K+. We will generate efficient and specific reagents through an in vitro selection assay, and then test their efficacy in cells, human sensory neurons induced from pluripotent stem cells, and cultured mouse and human DRG neurons. For in vivo testing, we will construct a transgenic mouse model that is specifically designed to test SDRE reagents targeting human NaV1.7 with the goal of ameliorating inflammatory, migraine and neuropathic pain.

慢性疼痛是残疾的主要原因，影响了全球约三分之一的成年人，患病率很高 比心脏病，癌症和糖尿病大。滥用和滥用阿片类药物已导致 全国成瘾和过量危机。因此，迫切需要替代性，非添加性镇痛药。 非选择性电压门控钠通道（NAV）阻滞剂是现有的非添加性FDA批准 有时可以为患者提供症状的药物。但是，它们的效用受到CNS的限制， 心脏副作用。人类疼痛障碍和疼痛动物模型的遗传和功能研究已有 经过验证的NAV1.7，一种在周围神经元中优先表达的电压门控钠通道 有吸引力的治疗靶标。但是，同工型选择的NAV阻滞剂很难生成 已经在临床试验中进行了测试，从人体中迅速清除，限制了它们的有效性。选择 需要方法。我们提出了一种新颖的，非依恋的方法来通过编辑来治疗慢性疼痛 编码NAV1.7的消息以更改其离子选择性。通过更改离子中的单个赖氨酸密码子 选择性过滤到精氨酸，NA选择性通道将同时成为Na+和K+选择性，有效地创建 一个反流的分流，会降低兴奋性。 位置定向的RNA编辑（SDRE）是指在RNA内生成编程编辑的新型机制。 它依赖于adar（作用于RNA的腺苷脱氨酶），该酶是内源表达的 在人类细胞中，包括感觉神经元。 SDRE系统由导向RNA（GRNA）指导，精确转换 选定的腺苷对肌苷，这是一种转化的鸟嘌呤模仿，可以重新构建特定的氨基酸。为了 用作镇痛作用，编辑mRNA比DNA更可取 效果，包括恶性转化。另外，ADAR是内源性的，而DNA的酶 操纵（例如CAS蛋白）不是，因此SDRE不会那么免疫原性。与小分子相比 渠道阻滞剂，SDRE可以更具体，因为它依赖于grnas的watson-Crick碱基对 定位及其影响可能更长，因为只要编辑渠道是 表达。我们建议使用SDRE编辑NAV1.7 K1395R，以使通道可渗透到Na+和 K+。我们将通过体外选择测定产生高效而特定的试剂，然后测试其功效 在细胞中，由多能干细胞诱导的人类感觉神经元以及培养的小鼠和人DRG 神经元。对于体内测试，我们将构建一个专门设计用于测试的转基因鼠标模型 针对人NAV1.7的SDRE试剂的目标是改善炎症，偏头痛和神经性 疼痛。