Development and Validation of Animal Models and/or Outcome Measures

动物模型和/或结果测量的开发和验证

基本信息

批准号：
10398390
负责人：
JOSHUA J.C. ROSENTHAL
金额：
$ 85.73万
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/10398390
关键词：
Action Potentials Acute Adenosine Adult Affect Afferent Neurons Amino Acids Analgesics Animal Model Animals Arginine Base Pairing Base Sequence Behavior Capsaicin Capsid Cardiac Cells Chemicals Closure by clamp Codon Nucleotides Complement DNA Data Development Diabetes Mellitus Disease Disease model Effectiveness Electrophysiology (science)Ensure Enzymes Evaluation Exons FDA approved Face Female Gene-Modified Genes Genetic Goals Guanosine Guide RNA Headache Heart Diseases Human Hypersensitivity IL6 gene Injury Inosine Interleukin-1 Ions Knock-in Laboratories Letters Lysine Malignant Neoplasms Measures Mechanics Messenger RNA Migraine Modeling Mouse Strains Multiple Trauma Mus Muscle Neurons Nociceptors Nucleotides Operative Surgical Procedures Outcome Measure Overdose Pain Pain Disorder Pain management Patients Peripheral Permeability Pharmaceutical Preparations Plasmids Prevalence Property Protein Isoforms Proteins RNA Binding RNA Editing Reagent Research Project Grants Sensory Thresholds Shunt Device Site Skin Sodium Channel Stimulus Surgical incisions System Testing Transcript Validation Work addiction base behavior test chronic pain density design disability dsRNA adenosine deaminase efficacy testing experimental study immunogenic in vivo in vivo evaluation indium arsenide induced pluripotent stem cell male multi-electrode arrays neuronal excitability novel opioid abuse pain model patch clamp prevent response side effect small molecule targeted treatment transcriptome sequencing voltage voltage clamp

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 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 tested are rapidly cleared from the body, limiting their effectiveness. We propose a novel, non-addictive approach to treat pain by editing mRNA for 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) 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, and ADARs are endogenous thus SDRE will not be as immunogenic. Compared to small molecule NaV blockers, SDRE can be more specific, because it relies on base-pairing of gRNAs for targeting, and its effects are likely to be longer lasting 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+. Work in RC2 will generate the Scn9ahSF mouse producing mNaV1.7hSF so that human-specific gRNA/NDD combinations could be tested in vivo. We will characterize acute sensory thresholds of WT and Scn9ahSF mice and will test their behaviors in the SNI, post-surgical, and headache models used throughout RC5. Recordings of NaV1.7 currents will be conducted on DRG/TG neurons from Scn9ahSF mice compared to WT mice to determine whether replacement of nucleotides influences mNaV1.7hSF gating properties and current density. Experiments will also test the ability of SDRE reagents developed in RC3 to modify the mNaV1.7hSF ion selectivity and excitability of fully mature DRG neurons where the full complement of NaV channels is present. Since these mice will be used in RC5 to test efficacy of the NaV1.7 SDRE reagents, these electrophysiology studies will provide the initial evaluation of whether the human-specific editing reagents can effectively edit mRNA for the humanized mNaV1.7hSF channels.

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，其患病率超过心脏病、癌症和糖尿病的总和。因此，迫切需要非成瘾性的替代品。非选择性电压门控钠通道 (NaV) 阻滞剂属于现有的非成瘾药。 FDA 批准了哪些药物有时可以缓解患者的症状，但它们的效用却很有限。受到人类疼痛疾病和动物的中枢神经系统和心脏副作用的遗传和功能研究的限制。疼痛模型已验证 NaV1.7，这是一种电压门控 Na 通道，优先在然而，外周神经元作为同种型选择性 Nav 阻滞剂的一个有吸引力的治疗靶标，却很难实现。产生的物质和经过测试的物质会迅速从体内清除，限制了它们的有效性。提出了一种新的、非成瘾的方法来治疗疼痛，通过编辑 NaV1.7 的 mRNA 来改变其通过将离子选择性过滤器中的单个赖氨酸密码子改变为精氨酸，通道将从 Na+ 选择性变为 Na+ 和 K+ 选择性，从而有效地产生逆流抑制兴奋性的分流依赖于 ADAR（腺苷）。作用于 RNA 的脱氨酶）酶，这些酶在人体细胞中内源表达，包括感觉细胞 SDRE 系统在引导 RNA (gRNA) 的指导下，将精确选择的腺苷转化为肌苷。鸟苷的翻译模拟物，可以重新编码特定氨基酸，用作镇痛剂、编辑剂。 mRNA 优于 DNA，因为它是瞬时的，因此限制了潜在的脱靶效应，而 ADAR 是与小分子 NaV 阻断剂相比，SDRE 是内源性的，因此 SDRE 不会具有免疫原性。更具体，因为它依赖于 gRNA 的碱基配对来进行靶向，并且其效果可能会更长只要编辑的通道被表达，我们建议使用 SDRE 将 NaV1.7 K1395R 编辑为使通道对 Na+ 和 K+ 都可渗透。在 RC2 中工作将生成 Scn9ahSF 小鼠。 mNaV1.7hSF，以便可以在体内测试人类特异性 gRNA/NDD 组合。 WT 和 Scn9ahSF 小鼠的急性感觉阈值，并将测试它们在 SNI、手术后和整个 RC5 中使用的头痛模型将在 DRG/TG 神经元上进行 NaV1.7 电流的记录。将 Scn9ahSF 小鼠与 WT 小鼠进行比较，以确定核苷酸替换是否会影响 mNaV1.7hSF 门控特性和电流密度实验还将测试 SDRE 试剂的能力。在 RC3 中开发，用于修改完全成熟的 DRG 神经元的 mNaV1.7hSF 离子选择性和兴奋性，其中由于这些小鼠将用于 RC5 来测试 NaV 通道的功效，因此存在完整的 NaV 通道。 NaV1.7 SDRE 试剂，这些电生理学研究将提供是否人类特异性编辑试剂可以有效编辑人源化 mNaV1.7hSF 通道的 mRNA。