Site-directed RNA editing of Nav1.7 as a novel analgesic

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， 验证了NaV1.7，一种优先在外周神经元中表达的电压门控钠通道，作为一种 有吸引力的治疗目标。然而，亚型选择性Nav阻断剂难以产生， 已经在临床试验中测试过的药物很快就从体内清除，限制了它们的有效性。替代 需要采取一些办法。我们提出了一种新的，非成瘾性的方法来治疗慢性疼痛， 编码NaV1.7以改变其离子选择性的消息。通过改变离子中的一个赖氨酸密码子 选择性过滤精氨酸，钠选择性通道将成为Na+和K+选择性，有效地创造 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。为 作为止痛药，编辑mRNA优于DNA，因为它是瞬时的，因此限制了潜在的脱靶 影响，包括恶性转化。此外，ADAR是内源性的，而DNA酶 操纵（例如，Cas蛋白）不是，因此SDRE不会具有免疫原性。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+。我们将通过体外筛选试验产生高效和特异的试剂，然后测试其功效 在细胞中，从多能干细胞诱导的人感觉神经元，以及培养的小鼠和人DRG 神经元对于体内测试，我们将构建一个专门设计用于测试的转基因小鼠模型， 靶向人NaV1.7的SDRE试剂，目的是改善炎症、偏头痛和神经性疾病 痛苦

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 sodium 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 in clinical trials 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 ion selectivity. By changing a single lysine codon in the ion selectivity filter to arginine, the Na selective channel will become 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. In addition, 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+. We will generate efficient and specific reagents through an in vitro selection assay, and then test their efficacy in cells, human sensory neurons induced from pluripotent stem cells, and cultured mouse and human DRG neurons. For in vivo testing, we will construct a transgenic mouse model that is specifically designed to test SDRE reagents targeting human NaV1.7 with the goal of ameliorating inflammatory, migraine and neuropathic pain.