Assay Development, Screening and Early Optimization

检测方法开发、筛选和早期优化

• 批准号: 10398391
• 负责人: JOSHUA J.C. ROSENTHAL
• 金额: $ 160.36万
• 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/10398391
• 关键词: Adenosine; Adult; Affect; Afferent Neurons; Amino Acids; Analgesics; Animal Model; Arginine; BCAR1 gene; Base Pairing; Behavioral Assay; Biological Assay; Capsid; Cardiac; Catalysis; Cells; Codon Nucleotides; Complex; Cultured Cells; Cytomegalovirus; DNA; DRADA2b protein; Diabetes Mellitus; Disease model; Effectiveness; Electrophysiology (science); Elements; Enzymes; Event; FDA approved; Generations; Genetic; Goals; Guanosine; Guide RNA; Heart Diseases; Human; In Vitro; Inosine; Ions; Link; Lysine; Malignant - descriptor; Malignant Neoplasms; Messenger RNA; Monitor; Mus; Mutation; Natural regeneration; Neurons; Nuclear Localization Signal; Outcome; Overdose; Pain Disorder; Patients; Peptides; Peripheral; Permeability; Pharmaceutical Preparations; Plasmids; Positioning Attribute; Prevalence; Procedures; Property; Protein Isoforms; RNA; RNA Editing; RNA Polymerase II; RNA Sequences; Randomized; Reaction; Reagent; Recombinants; Research; Resources; Shunt Device; Site; Sodium Channel; Sorting - Cell Movement; Specificity; System; Testing; Viral; Virus; addiction; assay development; base; channel blockers; chronic pain; design; disability; dsRNA adenosine deaminase; flexibility; immunogenic; improved; in vivo; innovation; mutant; novel; opioid abuse; pain model; pain reduction; pain sensitivity; promoter; screening; side effect; small molecule; stoichiometry; targeted treatment; transcriptome; 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. 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 RC3 is to optimize SDRE components so that they efficiently and selectively drive K1395R editing in cellula and in vivo. Top gRNAs and RNA editing enzymes will be selected and then tested in cultured cells. Components will be rigorously screened for on-target and off-target editing and the best pairs will be combined in AAV capsids and then provided to the other RCs for testing in neurons and mice.

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 Na 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 产生的物质会迅速从体内清除，从而限制了其有效性。替代方法是 需要。我们提出了一种新颖的、非成瘾的方法，通过编辑编码的信息来治疗慢性疼痛 NaV1.7 以改变其电生理特性。通过将单个赖氨酸密码子更改为精氨酸 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 ADAR（作用于 RNA 的腺苷脱氨酶）酶，该酶内源性表达于 人类细胞，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 包括恶性转化和 ADAR 在内的效应是内源性的，而用于 DNA 操作的酶 （例如 Cas 蛋白）则不然，因此 SDRE 不会具有免疫原性。与小分子通道相比 阻滞剂，SDRE 可以更具体，因为它依赖于 gRNA 的 Watson-Crick 碱基配对来进行靶向， 而且它的影响可能会更持久，因为只要编辑的频道被表达，它们就会一直存在。 我们建议使用 SDRE 编辑 NaV1.7 K1395R，以使通道可渗透 Na+ 和 K+。这 RC3 的目的是优化 SDRE 组件，以便它们高效且有选择性地驱动 K1395R 编辑 细胞和体内。将选择顶级 gRNA 和 RNA 编辑酶，然后在培养细胞中进行测试。 组件将经过严格筛选，以进行目标编辑和脱目标编辑，并将最好的组合组合起来 在 AAV 衣壳中，然后提供给其他 RC 在神经元和小鼠中进行测试。