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

项目摘要 临床上使用的大多数阿片类镇痛药，包括吗啡和芬太尼，主要通过μ阿片类药物起作用 受体。然而，阿片类镇痛和副作用，如便秘，瘙痒，镇静和成瘾， 在患者和动物模型中的应用非常广泛。这些观察结果表明存在多个mu 阿片受体，一个最初基于药理学研究提出的概念， 通过大量的μ阿片受体变体的分子克隆得到加强，这些变体通过广泛的 从单拷贝μ阿片受体（OPRM 1）基因的选择性前mRNA剪接。越来越多的证据 表明OPRM 1剪接变异体在介导多核苷酸的复杂和可变作用中是重要的。 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以下基因的表达： E1相关或E11相关剪接变体。E1相关变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E11相关变体 编码截短的6-TM结构域受体。基因靶向小鼠模型显示，E1相关 全长7-TM C-末端变体介导吗啡和美沙酮的作用，而E11相关的 截短的6-TM变体介导μ阿片类药物的子集，包括芬太尼、M6 G和海洛因，以及IBNtxA （3-碘苯甲酰基-6 β-纳洛酮酰胺），一种新型镇痛药，没有许多传统的阿片类药物副作用。而 小鼠模型是有价值的，大鼠在行为建模和体内操作方面都具有许多优势。 几十年来，大鼠已被广泛用于研究阿片类药物的作用，其中许多尚未被广泛应用。 在小鼠中进行。在许多人类条件下，大鼠被认为是比小鼠更好的模型。具有 大鼠模型将为研究界提供重要的优势和资源，并将扩大 我们有能力用在小鼠中不可行的方法和技术探索μ阿片药理学。剪接 大鼠OPRM 1基因与小鼠和人OPRM 1基因相似，两者都有广泛的排列 E1相关和E11相关变体。然而，缺乏合适的大鼠基因靶向模型限制了进一步的研究。 探索其功能。我们建议产生三种大鼠OPRM 1敲除/敲入（KO/KI）模型，E1- KO/KI、E11-KO/KI和E1/E11-KO/KI，使用最近开发的CRISPR/Cas9（定期更新） 间隔短回文重复序列/Cas9核酸酶）技术。我们将表征E1-KO/KI和E11-KO/KI。 KO/KI模型，通过检查OPRM 1剪接变体的表达，选择的镇痛作用， 阿片类药物，以及内源性E1和E11启动子活性。这些突变的老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学功能， 大鼠OPRM 1基因的转录调控。这些研究将使我们更好地了解 各种阿片类药物的复杂作用，并设计新颖的，有效的阿片类镇痛药，缺乏传统的 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.