Efficacy and PK/PD Studies

慢性疼痛是致残的主要原因，全世界约三分之一的成年人受到影响，患病率 比心脏病、癌症和糖尿病的总和还要大。阿片类药物的滥用和滥用导致了 全国范围内的吸毒和吸毒过量危机。因此，迫切需要替代的、不会上瘾的止痛药。 非选择性电压门控钠通道(NAV)阻滞剂是FDA批准的现有非成瘾性药物之一 有时可以缓解患者症状的药物。然而，它们的应用受到中枢神经系统和 心脏副作用。人类疼痛障碍的遗传和功能研究以及疼痛的动物模型 有效的Nav1.7，一个电压门控的钠通道，优先在外周神经元中表达，作为一种 有吸引力的治疗目标。然而，选择异构体的NAV阻滞剂很难产生，而那些 已经产生的毒素迅速从体内清除，限制了它们的效力。另一种方法是 需要的。我们提出了一种新的、非上瘾的方法来治疗慢性疼痛，方法是编辑编码的消息 Nav1.7，以改变其电生理特性。通过将单个赖氨酸密码子改变为精氨酸 离子选择性过滤器，通道将从对Na+的选择性转变为对Na+和K+的选择性，有效地创造 一种会抑制兴奋性的逆流分流。 Site-Directed RNA Editing(SDRE)是指在RNA中生成程序化编辑的新机制。它 依赖于ADAR(作用于RNA的腺苷脱氨酶)酶，这些酶在体内表达 人类细胞，包括感觉神经元。在引导RNA(GRNA)的指导下，SDRE系统精确地转换 精选的腺苷为肌苷，这是鸟苷的翻译模拟物，可以重新编码特定的氨基酸。 作为止痛药，编辑mRNA比编辑DNA更可取，因为它是瞬时的，从而限制了潜在的脱氧核糖核酸。 靶效应，包括恶性转化和ADAR是内源性的，而酶对DNA是内源性的 操纵(例如，CaS蛋白)不具有免疫原性，因此SDRE不具有免疫原性。与小分子相比 通道阻滞剂，SDRE可以更具体，因为它依赖于Watson-Crick碱基配对 目标，它的影响可能会持续更长时间，因为只要编辑的频道 表达。我们建议使用SDRE编辑Nav1.7 K1395R，以使通道对Na+和 K+。RC5的目的是测试促进Nav1.7的4组候选人SDRE试剂的有效性 使用体内小鼠行为疼痛模型编辑备用神经损伤(SNI)、手术后疼痛和 偏头痛。RC5还将在高峰期从小鼠的感觉神经元中测试Nav1.7的功能编辑 行为效能感的时间点。作为通向下一个发展阶段的最后桥梁，人类SDRE试剂将 在来自组织捐赠者的培养的人背根神经节神经元中进行测试。

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 RC5 is to test efficacy of 4 candidate sets of human-SDRE reagents promoting NaV1.7 editing using in vivo mouse behavioral pain models of spared-nerve injury (SNI), post-surgical pain, and migraine. RC5 will also test for functional editing of NaV1.7 in sensory neurons taken from mice at the peak time point of behavioral efficacy. As a final bridge to the next development stage, human-SDRE reagents will be tested in cultured human DRG neurons from tissue donors.