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

• 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
• 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

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、非成瘾的 止痛药非选择性电压门控钠通道（NaV）阻断剂是现有的非成瘾性药物之一。 FDA批准的药物，有时可以为患者提供症状缓解。然而，它们的效用是 受中枢神经系统和心脏副作用的限制。人类和动物疼痛障碍的遗传和功能研究 疼痛模型已经验证了NaV1.7，这是一种电压门控Na通道，优先表达于 外周神经元，作为一个有吸引力的治疗目标。然而，异构体选择性Nav阻断剂难以 产生和那些已经测试过的被迅速从体内清除，限制了它们的有效性。我们 提出了一种新的，非成瘾性的方法来治疗疼痛，通过编辑NaV1.7的mRNA来改变其表达。 电生理特性通过在离子选择性过滤器中将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，有效地产生逆流 会抑制兴奋性分流。定点RNA编辑（SDRE）依赖于阿达尔（ADAR） 作用于RNA的脱氨酶）酶，其在人类细胞中内源性表达，包括感觉神经细胞， 神经元在向导RNA（gRNA）的指导下，SDRE系统将精确选择的腺苷转化为肌苷， 鸟苷的翻译模拟物，其可以重新编码特定的氨基酸。用作止痛剂，编辑 mRNA优于DNA，因为它是瞬时的，从而限制了潜在的脱靶效应，并且ADAR是 因此，SDRE不会是内源性的。与小分子NaV阻断剂相比，SDRE可以是 更具体，因为它依赖于gRNA的碱基配对进行靶向，并且其效果可能更长 只要被编辑的频道被表达就持续。我们建议使用SDRE编辑NaV1.7 K1395R， 使通道对Na+和K+都是可渗透的。RC2中的工作将产生Scn9ahSF小鼠，其产生 mNaV1.7hSF，使得可以在体内测试人特异性gRNA/cDNANDD组合。我们将描述 WT和Scn9ahSF小鼠的急性感觉阈值，并将测试它们在SNI中、手术后和术后的行为。 RC5中使用的头痛模型。将在DRG/TG神经元上记录NaV1.7电流 比较来自Scn9ahSF小鼠与WT小鼠的核苷酸，以确定核苷酸的替换是否影响 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，以改变完全成熟DRG神经元的mNaV1.7hSF离子选择性和兴奋性，其中 存在NaV通道的全部补充。由于这些小鼠将用于RC 5中以测试本发明的组合物的功效， NaV1.7 SDRE试剂，这些电生理学研究将提供是否 人特异性编辑试剂可以有效地编辑人源化mNaV1.7hSF通道的mRNA。