Molecular mechanism of NAT1.7-modulated negative regulation of SCN9A expression

疼痛是一个主要的临床问题，影响到的人比糖尿病、心脏病和癌症加起来还要多。在任何时候，大约20%的人患有慢性疼痛，大约6%的人疼痛严重，导致严重的个人和经济后果。疼痛是许多医疗条件的辅助因素，但止痛药通常只有部分有效，而且随着人口老龄化，这个问题正在增加。新镇痛药开发进展缓慢的一个主要原因是由于疼痛的复杂性，我们对疼痛作为一种疾病的理解不完整。通过了解控制疼痛感觉的细胞和分子过程，可以开发出有效的靶向治疗方法来减轻痛苦。一种方法是分析患者疼痛敏感程度改变的遗传条件，特别是疼痛敏感性和感知能力下降的情况。识别负责这种表型及其功能产物的人类基因，使我们能够确定提供痛觉的分子事件链中的关键角色。通过这种方式，SCN9A已被确定为关键的疼痛基因之一。该基因编码一种叫做Nav1.7的蛋白质，这是一种钠跨膜通道，由膜的电荷调节。在此过程中，这种蛋白质将疼痛信号从其主要表达的背根神经节神经元传递到大脑。该基因的缺失和/或使该蛋白失活的突变导致痛觉丧失，因此SCN9A是制药行业的重要靶点，目前正在开发几种选择性药物阻断剂。另一种针对Nav1.7的治疗策略是通过关注SCN9A基因本身，并利用任何下调基因表达的自然机制，从而潜在地降低疼痛敏感性。我们最近发现并鉴定了一个位于SCN9A旁边的新调控基因，该基因编码在人类和小鼠中保守的天然反义转录物（NAT1.7）。本质上，NAT1.7是由与主SCN9A基因直接相反的第二条互补DNA链编码的，两个基因部分重叠。在最近发表的研究结果中，我们证明了这两个基因的表达水平呈现出明显的不一致模式，即表达高水平SCN9A转录物的组织（如DRG）具有低水平的NAT1.7，而高水平NAT1.7的组织（如脊髓和脑）具有低水平或不表达SCN9A。此外，当我们生成具有高水平NAT1.7的细胞时，它导致Nav1.7表达水平显著降低，重要的是其活性降低，这表明NAT1.7是SCN9A的负调节因子。我们现在提出了一个实验计划，旨在阐明NAT1.7作为SCN9A负调节因子的分子机制。为了揭示这一机制，我们的目标是回答三个主要问题：NAT1.7如何，在何处以及何时下调SCN9A表达？通过在新开发的小鼠模型中详细分析NAT1.7在分子和小鼠行为水平上的功能，本提案旨在全面了解NAT1.7在调控SCN9A表达中的作用。我们还将关注NAT1.7调节的任何其他基因，因为它将为我们提供关于使用NAT1.7作为镇痛剂可能产生的任何潜在副作用的重要信息。此外，通过控制升高的NAT1.7水平，我们的目标是确定这种新基因治疗疼痛的翻译潜力。我们相信，了解NAT1.7下调SCN9A的机制，并在小鼠行为模型中评估其镇痛潜力，将为进一步研究这一保守基因的转化潜力和开发针对疼痛疾病的新疗法提供坚实的平台。

Pain is a major clinical problem and affects more people than diabetes, heart disease and cancer combined. At any one time about 20% of the population is in chronic pain and in approximately 6% the pain is severe, resulting in serious personal and economic consequences. Pain is a cofactor in many medical conditions yet pain medicines are often only partially effective, and the problem is increasing with an aging population. A major reason for the poor progress in the development of new analgesics is our incomplete understanding of pain as a disease due to its complex nature. By understanding the cellular and molecular processes that control the sensation of pain, effective targeted therapies can be developed to alleviate suffering. One way of doing so is analysing genetic conditions in which patients have altered levels of pain sensitivity, in particular the cases where pain sensitivity and perception are diminished. Identifying human genes which are responsible for such a phenotype and their functional products, allows us to pinpoint the key players in the chain of molecular events providing for pain sensation. In this way, SCN9A has been identified as one of the key pain genes. The gene codes for a protein called Nav1.7, a sodium transmembrane channel which is regulated by the membrane's charge. In doing so the protein transmits pain signalling from the dorsal root ganglia neurons where it is expressed predominantly, towards the brain. Loss of this gene and/or mutations deactivating the protein cause loss of pain sensation, and thus SCN9A is an important target for the pharmaceutical industry with several selective drug blockers of its activity currently in development. A different strategy targeting Nav1.7 therapeutically is by focusing on the SCN9A gene itself and exploit any natural mechanism that downregulates the gene expression and therefore potentially reduce pain sensitivity. We have recently discovered and characterised a new regulatory gene located next to SCN9A that encodes a natural antisense transcript that is conserved in humans and mice (NAT1.7). Essentially NAT1.7 is coded by the second complementary strand of DNA that is directly opposite to the main SCN9A gene, with the two genes partially overlapping.In recently published results we demonstrated that the expression levels of these two genes show a clear discordant pattern, that is tissues expressing high levels of SCN9A transcript have low levels of NAT1.7 (such as DRG) and tissues with high levels of NAT1.7 have low or no expression of SCN9A (such as spinal cord and brain). Moreover, when we generated cells with high levels of NAT1.7 it led to a significant reduction in Nav1.7 expression levels, and importantly its activity, pointing out that NAT1.7 is a negative regulator of SCN9A. We now propose an experimental plan aimed to elucidate the molecular mechanism(s) by which NAT1.7 acts as a negative regulator of SCN9A. In order to uncover this mechanism we aim to answer three main questions: how, where and when does NAT1.7 downregulate SCN9A expression? Through the detailed analysis of NAT1.7 function at the molecular and mouse behavioural levels in a newly developed mouse model, this proposal aims to provide a comprehensive understanding of the NAT1.7 role in the regulatory control of SCN9A expression. We will also pay attention to any other genes being modulated by NAT1.7, as it will give us important information on any potential side effects arising if NAT1.7 is used as an analgesic agent. In addition, by manipulating elevated NAT1.7 levels we aim to determine the translational potential of this novel gene for treating pain. We believe that understanding of the mechanism by which NAT1.7 downregulates SCN9A and assessment of its analgesic potential in our mouse behavioural models will form a solid platform for further research into the translational potential of this conserved gene and the development of novel therapies targeting pain conditions.