Primary sensory neuron-targeted block of Cav3.2 for treatment of chronic neuropathic pain

选择性阻断初级感觉神经元CaV3.2治疗慢性神经病理性疼痛 摘要/摘要：慢性疼痛是一个毁灭性的问题，而阿片类药物治疗慢性疼痛 许多风险，包括滥用、过量用药和成瘾，突显了需要新的止痛药靶点。 周围感觉神经系统(PSNS)是设计新疗法的一个特别容易接近的部位， 其中背根神经节(DRG)的感觉神经元启动伤害性感觉并在 痛性神经病的发展和维持。感觉神经元CaV3.2 T型钙通道 调节神经元兴奋性是治疗疼痛的有希望的靶点，但选择性 事实证明，CaV3.2抑制剂难以捉摸。小肽小肽，尤指从天然蛋白质中提取的小肽 功能抑制肽适配子(IPA)，被认为是高效和选择性的，允许 阻断特定的疼痛分子相互作用，以最大限度地减少偏离目标的影响，减少疼痛。止痛药 在DRG中腺相关病毒载体(AAV)递送的PSN中IPA持续表达是安全的 以及治疗慢性疼痛的可行途径，副作用最小，没有滥用/成瘾倾向。 分子 信号相互作用通常由缺乏明确的三级结构的蛋白质区域介导，称为 蛋白质固有无序区(IDR)。使用已建立的IDR预测算法，我们高度本地化 CaV3.2中的无序区，并鉴定了一种新的能产生持续的CaV3.2 T型的17聚体多肽 神经病理性疼痛大鼠模型的电流抑制和疼痛减弱，显示其治疗潜力 用来治疗疼痛。拟议的两个阶段的研究是为了发展候选CaV3.2 iPA提供的证据 通过AAV进入DRG神经元具有足够的生物活性，因此有理由进一步开发为一种新型止痛药 接近。在第一阶段(R66)，将使用计算和实验相结合的策略来设计 候选CaV3.2iPA，并通过体外细胞实验测试其对T-型电流的抑制作用。里程碑 对于从R66阶段到第二阶段(R33)的进展，是确定符合我们的 筛选标准(在CaV3.2阻断中更高的效价和选择性)和治疗性AAV的生产 CaV3.2iPas。第二阶段旨在通过神经节内注射在体内应用AAV-CaV3.2iPAs来评估危重患者 AAV-CaV3.2iPAs在神经损伤诱导的大鼠疼痛模型中的特点，与对照组相比具有里程碑意义 第二阶段的结束是产生足够的体内有效性和安全性的数据，这将帮助我们进行/不进行- 为进一步的翻译开发做出明智的决定。我们的最终目标是开发一种新的疗法 AAV靶向基因联合高效选择性IPA阻断CaV3.2的解剖节段性研究 DRG用于治疗慢性神经病理性疼痛。

Selective block of CaV3.2 in primary sensory neurons for treatment of chronic neuropathic pain Summary/Abstract: Chronic pain is a devastating problem, while opioid treatment of chronic pain has numerous risks, including misuse, overdose, and addiction, highlighting the need for new analgesic targets. The peripheral sensory nervous system (PSNS) is a particularly accessible site for devising new treatments, where the sensory neurons of the dorsal root ganglia (DRG) initiate nociception and have a central role in the development and maintenance of painful neuropathy. Sensory neuronal CaV3.2 T-type calcium channels regulate neuronal excitability and are a promising target for treatment of pain, but the development of selective CaV3.2 inhibitors has proved elusive. Small peptides, especially those derived from the natural proteins as functionally inhibitory peptide aptamers (iPAs), are recognized as being highly effective and selective, allowing blockade of specific pain molecular interactomes to reduce pain with minimal off-target effects. The analgesic iPA-sustained expression in the PSNS delivered by adeno-associated viral vectors (AAV) in the DRG is a safe and feasible path to chronic pain treatment with minimal side effects and no abuse/addiction liability. Molecular signaling interactions are often mediated by regions of proteins lacking a defined tertiary structure, known as protein intrinsically disordered regions (IDRs). Using established IDR prediction algorithms, we localized highly disordered regions in CaV3.2, and identified a novel 17mer peptide that produces sustained CaV3.2 T-type current inhibition and pain attenuation in a neuropathic pain rat model, demonstrating its therapeutic potential for pain treatment. The proposed Two-Phase study is to develop evidence that candidate CaV3.2iPAs delivered by AAV into DRG neurons have sufficient biological activity to justify further development as a novel analgesic approach. In Phase I (R66), a combined computational and experimental strategy will be used to design candidate CaV3.2iPAs and test their inhibition of T-type current by in vitro of cell-based studies. The milestone for advancement from R66 Phase to Phase II (R33) is identification of the top CaV3.2iPA leads that meet our selection criteria (higher potency and selectivity in block of CaV3.2) and of production of therapeutic AAV- CaV3.2iPAs. Phase II aims to apply AAV-CaV3.2iPAs in vivo by intraganglionic injection to evaluate critical features of AAV-CaV3.2iPAs in nerve injury-induced rat pain model, compared to controls The milestone for the end of Phase II is generation of sufficient data of in vivo efficacy and safety which will help us to make a go/no- go informed decision for further translational development. Our ultimate goal is to develop a novel therapeutics combined AAV-targeted gene delivery with potent and selective iPA block of CaV3.2 in anatomically segmental DRG to treat chronic neuropathic pain.