Exploring function of mu opioid receptor splice variants in rat by gene targeting

项目摘要 大多数临床上使用的阿片类镇痛剂，包括吗啡和芬太尼，主要通过u阿片类药物起作用。 感受器。然而，阿片类止痛和副作用，如便秘、瘙痒、镇静和成瘾，各有不同。 在患者和动物模型中非常常见。这些观察结果表明多个单位的存在。 阿片受体，这一概念最初是基于药理学研究提出的，并已 通过大量的Mu阿片受体变异体的分子克隆，通过广泛的 单拷贝u阿片受体(OPRM1)基因的选择性前-mR NA剪接。越来越多的证据 表明OPRM1剪接变异体在介导Mu的复杂和多变的作用中起着重要作用 阿片类药物。与外显子1(E1)或外显子11(E11)相关的两个不同的启动子控制着 E1相关或E11相关的剪接变异体。E1相关变异体主要编码全长羧基(C- )末端，7-跨膜(7-TM)结构域受体，而大多数与E11相关的变体 编码截短的6-TM结构域受体。基因靶向小鼠模型显示，与E1相关的 全长7-TM C末端突变体介导吗啡和美沙酮的作用，而与E11相关的 截短的6-TM变体介导MU阿片类药物的子集，包括芬太尼、M6G和海洛因，以及IBNtxA (3-碘苯甲酰基-6-β-纳曲胺)，一种新型的止痛药，没有许多传统的阿片类药物副作用。而当 小鼠模型是有价值的，大鼠在行为建模和活体操作方面都有许多优势。 几十年来，大鼠一直被广泛用于研究阿片类药物的作用，其中许多还没有被 在小鼠身上进行。在许多人类条件下，大鼠被认为是比小鼠更好的模型。拥有 大鼠模型将为研究界提供显著的优势和资源，并将扩大 我们有能力用在小鼠身上不可行的方法和技术来探索Mu阿片类药物。拼接 大鼠的OPRM1基因与小鼠和人类的OPRM1基因相似，具有广泛的两者 E1相关和E11相关的变异体。然而，缺乏合适的大鼠基因打靶模型进一步限制了 探索它们的功能。我们建议建立三种大鼠OPRM1基因敲除/敲除(KO/KI)模型， KO/KI、E11-KO/KI和E1/E11-KO/KI，使用最近开发的CRISPR/CAS9(定期集群 间隔短回文重复/Cas9核酸酶)技术。我们将表征E1KO/KI和E11- 通过检测KO/KI模型中OPRM1剪接变异体的表达，筛选其镇痛作用 阿片类药物，以及内源性的E1和E11启动子活性。这些突变的大鼠模型将提供独特的 和有价值的工具，为研究界进一步探索药理作用和 大鼠OPRM1基因的转录调控。这些研究将使我们能够更好地了解 各种阿片类药物的复杂作用，并设计缺乏传统阿片类药物的新型、有效的阿片类镇痛剂 阿片类药物的副作用和滥用潜力。

Project Summary Most opioid analgesics used clinically, including morphine and fentanyl, act primarily through the mu opioid receptors. However, opioid analgesia and side-effects, such as constipation, itch, sedation and addiction, vary immensely among patients and animal models. These observations suggest the existence of multiple mu opioid receptors, a concept that was initially proposed based on pharmacological studies and has been reinforced by molecular cloning of a vast array of mu opioid receptor variants, generated through extensive alternative pre-mRNA splicing from the single-copy mu opioid receptor (OPRM1) gene. Increasing evidence indicates that the OPRM1 splice variants are important in mediating the complex and variable actions of mu opioids. Two distinct promoters, associated with either exon 1 (E1) or exon 11 (E11), control the expression of E1-associated or E11-associated splice variants. E1-associated variants mainly encode full-length carboxyl (C- ) terminal, 7-transmembrane (7-TM) domain receptors, whereas the majority of E11-associated variants encode truncated, 6-TM domain receptors. Gene targeted mouse models have revealed that E1-associated full-length 7-TM C-terminal variants mediate the actions of morphine and methadone, whereas E11-associated truncated 6-TM variants mediate a subset of mu opioids, including fentanyl, M6G and heroin, as well as IBNtxA (3-iodobenzoyl-6β-naltrexamide), a novel analgesic that lacks many traditional opioid side-effects. While the mouse models are valuable, rats have many advantages both in behavioral modeling and in vivo manipulation. Rats have been used extensively to study opioid actions for many decades, many of which have not been conducted in mice. Rats are considered as better models than mice in a number of human conditions. Having rat models would provide significant advantages and resources for the research community, and would expand our ability explore mu opioid pharmacology with approaches and techniques not feasible in mice. Splicing in the rat OPRM1 gene is similar to that in the mouse and human OPRM1 genes, with an extensive array of both E1-associated and E11-associated variants. Yet, the lack of appropriate rat gene targeting models limits further exploration of their functions. We propose generating three rat OPRM1 knockout/knockin (KO/KI) models, E1- KO/KI, E11-KO/KI and E1/E11-KO/KI, using recently developed CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 nuclease) technology. We will characterize E1-KO/KI and E11- KO/KI models by examining the expression of OPRM1 splice variants, the analgesic actions of selected opioids, as well as the endogenous E1 and E11 promoter activity. These mutant rat models will provide unique and valuable tools for the research community to further explore the pharmacological functions and transcriptional regulation of the rat OPRM1 gene. These studies will allow us to obtain a better understanding of the complex actions of various opioids, and to design novel, potent opioid analgesics that lack traditional opioid side-effects and abuse potential.