Neuron-specific block of T-type calcium channels

描述(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到重度疼痛。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了足够的疼痛缓解。因此，开发无滥用潜力和副作用的新型止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，慢性疼痛可以通过抑制伤害性感受器上的离子通道来治疗，例如Cav3.2T型电压门控钙通道。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，而Cav3.2定向的反义寡核苷酸逆转了在这些动物中观察到的机械性痛觉过敏和痛觉异常。重要的是，Cav3.2对于啮齿动物和人类的疼痛信号都很重要，因为临床上相关的止痛剂硫辛酸在体外阻断了人类Cav3.2电流，但在体内对Cav3.2基因敲除的小鼠无效。这些研究将开发抑制小鼠和人类Cav3.2通道表达的短发夹状RNA(ShRNA)。与化学合成的siRNA不同，基于DNA的shRNA的传递可以使用基因启动子针对神经元亚型。本研究将探索钠通道启动子Scn10a的用途，其表达在很大程度上限于伤害性感受器。临床使用的一个重要特征是能够规范基因治疗。这将使用一种新开发的四环素抑制物来实现，这需要药物来激活(强力环素-ON)。重组腺相关病毒(RAAV)已成为人类基因治疗的首选。AAV颗粒可以与有效感染感觉神经元的蛋白质外壳一起产生，并被逆行运输到细胞核(例如，8型血清)。这些研究的目标是开发rAAV靶向载体，指导shRNA介导的Cav3.2基因敲除，使用它来制备病毒颗粒，然后在神经病理性疼痛的大鼠中测试伤害性感受器的特异性表达。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。

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.