Neuron-specific block of T-type calcium channels

T 型钙通道的神经元特异性阻断

• 批准号: 8235787
• 负责人: EDWARD PEREZ-REYES
• 金额: $ 24.61万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-15 至 2013-08-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8235787
• 关键词: Address; Adverse effects; Affect; Afferent Neurons; American; Analgesics; Animal Model; Animals; Antisense Oligonucleotides; Biological Assay; Calcium Channel; Capsid; Capsid Proteins; Cell Line; Cell Nucleus; Chronic; Clinical; Clinical Trials; DNA delivery; Development; Diabetic Neuropathies; Direct Costs; Doxycycline; Elements; Facilities and Administrative Costs; Funding; Future; Gene Transfer; Genes; Goals; Grant; Health; Human; Hyperalgesia; In Vitro; Intrathecal Injections; Ion Channel; Knockout Mice; Magic; Measures; Mechanics; Mediating; Medical; Messenger RNA; Modeling; Mus; Neurons; Nociception; Nociceptors; Pain; Patients; Pharmaceutical Preparations; Physiology; Population; Production; Productivity; Quality of life; Rattus; Recombinant adeno-associated virus (rAAV); Regulation; Research; Reverse Transcriptase Polymerase Chain Reaction; Rodent; Role; Serotyping; Signal Transduction; Slice; Small Interfering RNA; Sodium Channel; Spinal Ganglia; Staining method; Stains; T-Type Calcium Channels; Testing; Tetanus Helper Peptide; Tetracyclines; Thioctic Acid; United States; Validation; Viral; allodynia; base; chronic pain; clinically relevant; cost; design; diabetic rat; drug development; ganglion cell; gene therapy; human ZNF45 protein; human tissue; in vivo; innovation; nerve injury; novel; pain inhibition; painful neuropathy; particle; promoter; selective expression; small hairpin RNA; tool; transmission process; vector; voltage

DESCRIPTION (provided by applicant): Pain is a leading health problem in the United States with 1 in 10 Americans suffering from moderate to severe pain. Yet, the treatment of chronic pain remains a clinical challenge, with only half of patients receiving adequate pain relief. Therefore, the development of novel analgesics without abuse potential and side effects would have significant impact on the treatment of pain. The hypothesis guiding these exploratory studies is that chronic pain can be treated by inhibiting ion channels in nociceptors such as the Cav3.2 T-type voltage-gated calcium channel. T-currents are upregulated in animal models of chronic constrictive nerve injury and diabetic neuropathy, and Cav3.2 directed antisense oligonucleotides reverse the mechanical hyperalgesia and allodynia observed in these animals. Importantly, Cav3.2 is important for pain signaling in both rodents and humans, since the clinically relevant analgesic, lipoic acid, blocks human Cav3.2 currents in vitro yet is ineffective in Cav3.2 knockout mice in vivo. These studies will develop short hairpin RNAs (shRNA) that inhibit the expression of mouse and human Cav3.2 channels. In contrast to chemically synthesized siRNA, delivery of DNA-based shRNA can be specifically targeted to neuronal subtypes using gene promoters. The present study will explore the use of the sodium channel promoter Scn10a, whose expression is largely limited to nociceptors. An important feature for clinical use is the ability to regulate gene therapy. This will be accomplished using a newly developed version of the tetracycline repressor, which requires drug for activation (doxycycline-ON). Recombinant adeno-associated virus (rAAV) has emerged as the top choice for human gene therapy. AAV particles can be produced with protein coats that effectively infect sensory neurons and are retrogradely transported to the nucleus (e.g. serotype 8). The goal of these studies is to develop rAAV targeting vectors that direct shRNA- mediated knockdown of Cav3.2, use this to prepare viral particles, and then test for nociceptor- specific expression in rats with neuropathic pain. The research team includes Dr. Edward Perez- Reyes, a T-channel expert, Dr. Guanping Gao, an AAV expert, and Dr. Hui-Lin Pan, a neuropathic pain expert.

描述（由申请人提供）：疼痛是美国的一个主要健康问题，十分之一的美国人患有中度至重度疼痛。然而，慢性疼痛的治疗仍然是一个临床挑战，只有一半的患者得到足够的疼痛缓解。因此，开发无滥用潜力和副作用的新型镇痛药将对疼痛的治疗产生重大影响。指导这些探索性研究的假设是，可以通过抑制伤害感受器中的离子通道（例如 Cav3.2 T 型电压门控钙通道）来治疗慢性疼痛。 在慢性缩窄性神经损伤和糖尿病神经病变的动物模型中，T 电流上调，Cav3.2 定向反义寡核苷酸逆转了在这些动物中观察到的机械痛觉过敏和异常性疼痛。 重要的是，Cav3.2 对于啮齿动物和人类的疼痛信号传导都很重要，因为临床相关的镇痛剂硫辛酸在体外阻断人类 Cav3.2 电流，但在体内对 Cav3.2 敲除小鼠无效。这些研究将开发抑制小鼠和人类 Cav3.2 通道表达的短发夹 RNA (shRNA)。与化学合成的 siRNA 相比，基于 DNA 的 shRNA 的递送可以使用基因启动子特异性靶向神经元亚型。本研究将探索钠通道启动子 Scn10a 的使用，其表达很大程度上限于伤害感受器。临床应用的一个重要特征是调节基因治疗的能力。这将使用新开发的四环素阻遏物来实现，该阻遏物需要药物（多西环素-ON）来激活。重组腺相关病毒（rAAV）已成为人类基因治疗的首选。 AAV 颗粒可以产生具有有效感染感觉神经元的蛋白质外壳并逆行转运至细胞核（例如血清型 8）。这些研究的目标是开发 rAAV 靶向载体，指导 shRNA 介导的 Cav3.2 敲低，用其制备病毒颗粒，然后测试患有神经性疼痛的大鼠中伤害感受器特异性表达。研究团队包括T通道专家Edward Perez-Reyes博士、AAV专家高冠平博士和神经病理性疼痛专家潘慧琳博士。