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：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，过量和成瘾，突出了对新镇痛靶点的需求。 外周感觉神经系统（PSNS）是设计新治疗方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感受并且在神经元的传导中具有中心作用。 疼痛性神经病变的发展和维持。感觉神经元CaV3.2 T型钙通道 调节神经元兴奋性，是治疗疼痛的有前途的靶点，但选择性 CaV3.2抑制剂已被证明是难以捉摸的。小肽，特别是那些来自天然蛋白质， 功能抑制性肽适体（iPAs）被认为是高度有效和选择性的， 阻断特异性疼痛分子相互作用，以减少疼痛，并具有最小的脱靶效应。镇痛 在DRG中通过腺相关病毒载体（AAV）递送的PSNS中持续表达iPA是安全的， 和可行的路径慢性疼痛治疗最小的副作用和没有滥用/成瘾的责任。 分子 信号相互作用通常由缺乏确定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，我们高度定位 在CaV3.2的T型区，发现了一个新的17肽，产生持续的CaV3.2 T型 在神经病理性疼痛大鼠模型中的电流抑制和疼痛减轻，证明其治疗潜力 用于疼痛治疗拟议的两阶段研究旨在证明候选CaV3.2iPAs可递送 通过AAV进入DRG神经元具有足够的生物活性，证明进一步开发作为新型镇痛剂是合理的 approach.在第一阶段（R66）中，将使用计算和实验相结合的策略来设计 候选CaV3.2iPAs并通过基于细胞的体外研究测试它们对T型电流的抑制。里程碑 从R66阶段推进到II阶段（R33）的关键是确定符合我们要求的顶级CaV3.2iPA电极导线 选择标准（在阻断CaV3.2中更高的效力和选择性）和治疗性AAV的产生的选择标准 CaV3.2iPAs. II期旨在通过神经节内注射在体内应用AAV-CaV 3. 2 iPAs，以评估关键的 AAV-CaV 3. 2 iPAs在神经损伤诱导的大鼠疼痛模型中的特征，与对照相比。 第二阶段的结束是产生足够的体内疗效和安全性数据，这将有助于我们做出决定。 为进一步的翻译发展做出明智的决定。我们的最终目标是开发一种新的治疗方法 AAV靶向基因递送与CaV3.2的有效和选择性iPA阻断的组合在解剖学节段中 DRG治疗慢性神经性疼痛。

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.