OPIOID RECEPTOR GENE TRANSFER-PAIN AND TOLERANCE

这项研究的长期目标是开发慢性疼痛的基因疗法。阿片类药物仍然是治疗严重疼痛患者的首选药物。阿片类药物的副作用，包括呼吸抑制、耐受性和依赖性，往往限制了它们的使用。这项资助的重点是设计病毒载体，使野生型或突变的mu阿片受体（muOR）基因能够有效和稳定地传递到感觉神经元。我们假设，在背根神经节（DRG）神经元中适当的muOR基因传递和表达将降低镇痛所需的mu-阿片类药物的剂量并降低耐受性，从而减少阿片类药物的副作用。为了验证这一假设，我们将(1)使用腺相关病毒（AAV）载体将野生型或突变型的mu or基因传递到感觉神经元中，(2)在注射重组AAV介导的muOR转基因（rAAV-muOR）的炎症大鼠中评估mu-阿片类药物对疼痛行为的影响。(3)评估在接受rAAV-muOR注射的耐受大鼠中mu阿片的作用；(4)评估mu阿片对非耐受和耐受大鼠rAAV-muOR转导的DRG神经元膜电导和受体运输的影响。实验将在接受完全弗氏佐剂（CFA）治疗的大鼠和从这些大鼠分离的DRG神经元上进行。疼痛行为将通过足爪退缩潜伏期来监测，膜电流将通过穿孔贴片电极来测量，受体运输将通过免疫荧光染色和受体结合试验来检测。这些实验将确立用基因方法治疗疼痛的可行性。如果成功，该研究将为临床医生提供治疗严重疼痛患者的新工具，并为研究阿片受体调节在阿片耐受性发展中的作用提供模型。

The long-term goal of this research is to develop gene therapy for chronic pain. Opiates have remained to be the drugs of choice for treating patients with severe pain. The side effects of opiates, including respiratory depression, tolerance and dependence, have often limited their use. The focus of this grant is to design viral vectors that allow for efficient and stable delivery of wild type or mutant mu opioid receptor (muOR) genes to sensory neurons. We hypothesize that appropriate muOR gene delivery and expression in dorsal root ganglion (DRG) neurons will lower the doses of mu-opioids required for analgesia and reduce tolerance, thus decreasing the side effects of opioids. To test this hypothesis, we will (1) deliver the wild type or mutant mu OR gene into sensory neurons using an adeno- associated virus (AAV) vector, (2) evaluate the effects of mu- opioids on pain behaviors in inflamed rats injected with recombinant AAV-mediated muOR transgene (rAAV-muOR), (3) evaluate the effects of mu-opioid in tolerant rats that receive injections of rAAV-muOR and (4) evaluate the effects of mu-opioids on membrane conductance and receptor trafficking in rAAV-muOR- transduced DRG neurons isolated from non tolerant and tolerant rats. The experiments will be performed on rats treated with complete Freund's adjuvant (CFA) and on DRG neurons isolated from those rats. Pain behaviors will be monitored by paw withdrawal latencies, membrane currents will be measured with perforated patch electrodes and receptor trafficking will be examined with immunofluorescence staining and receptor binding assays. These experiments will establish the feasibility of a genetic approach to pain treatment. If successful, the study will provide clinicians with a new tool to treat patients with severe pain and provide researchers a model for studying the role of opioid receptor regulation in the development of opioid tolerance.