Neuron-specific block of T-type calcium channels

描述（由申请人提供）：疼痛是美国的主要健康问题，每10个美国人中就有1个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，只有一半的患者接受足够的疼痛缓解。因此，开发无滥用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，慢性疼痛可以通过抑制伤害感受器中的离子通道，如Cav3.2 t型电压门控钙通道来治疗。在慢性收缩性神经损伤和糖尿病性神经病变的动物模型中，t电流上调，Cav3.2导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异常性疼痛。重要的是，Cav3.2对啮齿动物和人类的疼痛信号都很重要，因为临床相关的镇痛药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，其表达主要局限于伤害感受器。临床应用的一个重要特点是调控基因治疗的能力。这将使用一种新开发的四环素抑制因子来完成，它需要药物激活（多西环素- on）。重组腺相关病毒（rAAV）已成为人类基因治疗的首选。AAV颗粒可以产生具有有效感染感觉神经元的蛋白质外壳，并逆行运输到细胞核（例如血清型8）。这些研究的目的是开发直接shRNA介导的Cav3.2敲低的rAAV靶向载体，并利用该载体制备病毒颗粒，然后在神经性疼痛大鼠中检测伤害感受器特异性表达。研究团队包括T-channel专家Edward Perez- Reyes博士、AAV专家高冠平博士、神经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.