Resource Core

资源核心

• 批准号: 10398389
• 负责人: Eli Eisenberg
• 金额: $ 73.73万
• 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/10398389
• 关键词: Adenosine; Adult; Affect; Afferent Neurons; Amino Acids; Analgesics; Animal Model; Arginine; BCAR1 gene; Base Pairing; Benchmarking; Bioinformatics; Cardiac; Cell model; Cells; Code; Codon Nucleotides; Complementary DNA; Computer Analysis; Correlation Studies; DNA; DNA sequencing; Data; Data Analyses; Data Set; Detection; Diabetes Mellitus; Dideoxy Chain Termination DNA Sequencing; Disease model; Effectiveness; Electrophysiology (science); Ensure; Enzymes; Experimental Designs; FDA approved; Future; Genetic; Goals; Guanosine; Guide RNA; Heart Diseases; Human; Individual; Infrastructure; Inosine; Institution; Ions; Lysine; Malignant - descriptor; Malignant Neoplasms; Messenger RNA; Methods; Monitor; Mus; Neurons; Outcome; Overdose; Pain Disorder; Patients; Peripheral; Permeability; Pharmaceutical Preparations; Population; Prevalence; Process; Property; Protein Isoforms; Proteins; RNA; RNA Editing; Reagent; Research; Research Personnel; Resources; Reverse Transcriptase Polymerase Chain Reaction; Reverse Transcription; Role; Sample Size; Sampling; Shunt Device; Signal Transduction; Site; Sodium Channel; Specificity; Statistical Data Interpretation; System; Testing; Therapeutic; Variant; Work; addiction; base; bioinformatics pipeline; channel blockers; chronic pain; de novo mutation; design; disability; dsRNA adenosine deaminase; exome sequencing; experimental analysis; immunogenic; member; novel; opioid abuse; pain model; process optimization; reference genome; side effect; small molecule; targeted treatment; tool; transcriptome; transcriptome sequencing; 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 Na 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 generated 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 electrophysiological properties. By changing a single lysine codon to arginine in the ion selectivity filter, the channel will go from being Na+ selective to 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. Off- target RNA editing is a significant consideration for SDRE that must be taken into account as part of reagent design. For use as an analgesic, editing mRNA is preferable to DNA because it is transient, thus limiting potential off-target effects, including malignant transformations and 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+. The purpose of the Resource Core is to supply all investigators involved in this project with the required RNA editing quantification and statistical analyses. Primarily, the Resource Core will quantify on-target and off-target RNA editing activity for the various experimental systems to be used by the different research components, from cells to mice. We will introduce off-target analysis as an integral part of the reagent optimization process. In addition, the Resource Core will provide the statistical guidance required for experimental design and analysis.

慢性疼痛是导致残疾的主要原因，影响全球约三分之一的成年人， 比心脏病、癌症和糖尿病加起来还要严重。滥用和滥用阿片类药物导致了 全国性的吸毒过量危机因此，迫切需要替代的非成瘾性镇痛剂。 非选择性电压门控钠通道（Nav）阻滞剂是FDA批准的现有非成瘾性药物之一。 有时可以缓解患者症状的药物。然而，它们的实用性受到CNS的限制， 心脏副作用对人类疼痛障碍和疼痛动物模型的遗传和功能研究， 经验证的NaV1.7是一种优先在外周神经元中表达的电压门控Na通道， 有吸引力的治疗目标。然而，亚型选择性Nav阻断剂难以产生， 产生的毒素迅速从体内清除，限制了它们的有效性。替代方法有 needed.我们提出了一种新的，非成瘾性的方法来治疗慢性疼痛，通过编辑编码的信息， NaV1.7，以改变其电生理特性。通过改变一个单一的赖氨酸密码子精氨酸在 离子选择性过滤器，通道将从Na+选择性变为Na+和K+选择性，有效地产生 一个能抑制兴奋性的逆流分流器 定点RNA编辑（SDRE）是指在RNA内产生程序化编辑的新机制。 它依赖于内源性表达的阿达尔（腺苷脱氨酶，作用于RNA）酶 包括感觉神经元。在向导RNA（gRNA）的指导下，SDRE系统精确地转化 选择的腺苷转化为肌苷，肌苷是鸟苷的翻译模拟物，可以重新编码特定的氨基酸。关- 靶向RNA编辑是SDRE重要考虑因素，其必须作为试剂的一部分考虑 设计对于用作止痛剂，编辑mRNA比DNA更可取，因为它是短暂的，因此限制了潜力。 脱靶效应，包括恶性转化和ADAR是内源性的，而DNA酶 操作（例如Cas蛋白）的SDRE不会是免疫原性的，因此SDRE不会是免疫原性的。与小分子相比 通道阻断剂，SDRE可以更特异，因为它依赖于gRNA的沃森-克里克碱基配对， 目标定位，其影响可能会持续更长时间，因为只要编辑过的频道 表达。我们建议使用SDRE编辑NaV1.7 K1395 R，以使通道对Na+和Na+都是可渗透的。 K+。资源核心的目的是为参与本项目的所有研究者提供所需的RNA 编辑量化和统计分析。首先，资源核心将量化达标和脱靶 RNA编辑活动的各种实验系统所使用的不同的研究组成部分，从 细胞对小鼠。我们将介绍脱靶分析作为试剂优化过程的一个组成部分。在 此外，资源核心将提供实验设计和分析所需的统计指导。