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

基本信息

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
项目类别：
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

项目摘要

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.

慢性疼痛是导致残疾的主要原因，影响着全世界约三分之一的成年人，其患病率比心脏病、癌症和糖尿病的总和还多。阿片类药物的误用和滥用已导致全国性的成瘾和过量危机。因此，迫切需要替代的、非成瘾性镇痛药。非选择性电压门控钠通道 (Nav) 阻滞剂是 FDA 批准的现有非成瘾药物之一有时可以缓解患者症状的药物。然而，它们的效用受到中枢神经系统和心脏副作用。人类疼痛疾病和疼痛动物模型的遗传和功能研究验证了 NaV1.7，一种优先在周围神经元中表达的电压门控钠通道，作为有吸引力的治疗目标。然而，异构体选择性 Nav 阻滞剂很难生成，而且那些已在临床试验中测试过的药物会迅速从体内清除，从而限制了其有效性。选择需要采取一些方法。我们提出了一种新颖的、非成瘾的方法来治疗慢性疼痛，通过编辑对 NaV1.7 进行编码以改变其离子选择性的消息。通过改变离子中的单个赖氨酸密码子通过对精氨酸的选择性过滤，Na 选择性通道将同时具有 Na+ 和 K+ 选择性，有效地创建会抑制兴奋性的逆流分流。定点 RNA 编辑 (SDRE) 是指在 RNA 内生成编程编辑的新机制。它依赖于内源表达的 ADAR（作用于 RNA 的腺苷脱氨酶）酶在人类细胞中，包括感觉神经元。 SDRE 系统在向导 RNA (gRNA) 的指导下精确转换选择腺苷为肌苷，肌苷是鸟苷的翻译模拟物，可以重新编码特定的氨基酸。为了作为镇痛剂，编辑 mRNA 比 DNA 更可取，因为它是短暂的，从而限制了潜在的脱靶影响，包括恶变。此外，ADAR 是内源性的，而 DNA 酶是内源性的操作（例如 Cas 蛋白）则不然，因此 SDRE 不会具有免疫原性。与小分子相比通道阻滞剂，SDRE 可以更具体，因为它依赖于 gRNA 的 Watson-Crick 碱基配对定位，其效果可能会更持久，因为只要编辑的频道存在，它们就会持续存在。表示。我们建议使用 SDRE 编辑 NaV1.7 K1395R，以使通道可渗透 Na+ 和 K+。我们将通过体外选择实验产生高效、特异的试剂，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类 DRG 神经元。对于体内测试，我们将构建专门用于测试的转基因小鼠模型 SDRE 试剂针对人类 NaV1.7，旨在改善炎症、偏头痛和神经病疼痛。