Rho GTPase siRNA as a Therapeutic Strategy in Experimental Autoimmune Neuritis

描述（由申请人提供）： 获得性炎症性神经病是一个相当大的社会和经济负担，我们的退伍军人和退伍军人健康管理局。炎症性神经病变包括已知的感染性或可能的感染性病因，构成神经系统疾病的最大和最不了解的光谱之一。这些疾病包括急性炎性脱髓鞘性多神经根病（AIDP），这是一种高度致残的外周神经系统炎性自身免疫性疾病，其特征为急性/亚急性对称性轻瘫，伴有进展为神经肌肉麻痹的反射消失。尽管其压倒性的患病率和社会经济影响，治疗退伍军人炎症性周围神经病变，包括AIDP，仍然姑息。细胞因子介导的自身反应性白细胞的募集和运输穿过血-神经屏障并进入外周神经是炎症性周围神经病（包括AIDP）的公认早期病理学标志。外周神经血管内皮细胞对促炎细胞因子的局部GTP酶依赖性激活是一种起始性病理损伤。来自我们实验室的同行评审和发表的初步研究强烈支持在AIDP期间自身反应性白细胞向外周神经的运输可能通过涉及Cdc 42 GT3依赖性CCL 2分泌的机制进行。迫切需要新的转化研究来开发治疗和康复策略，用于因获得性炎性神经病而虚弱的退伍军人的高级护理。在这项为期两年的SPiRE研究中，我们将确定siRNA介导的GT3基因敲低是否能在治疗上防止炎性神经病变的发生和进展。假设：治疗性施用的针对关键单体GTP酶的siRNA将通过抑制内皮细胞CCL 2表达来减弱实验性自身免疫性神经炎的发展和进展。将使用已建立的AIDP（实验性自身免疫性神经炎，EAN）的临床上可转化的大鼠模型，通过以下两个具体目的在体内测试该假设。具体目的1将确定预防性或治疗性施用的靶向关键单体GTP酶的siRNA是否防止EAN的发展和进展。我们将使用诱发反应电生理学确定靶向Cdc 42或RalA GTP酶的siRNA对（a）EAN的临床严重性和病程以及对（B）EAN诱导的外周神经功能变化的剂量依赖性作用。具体目标2将确定预防性或治疗性施用靶向关键单体GTP酶的siRNA是否减弱EAN期间自身反应性白细胞向外周神经的运输。我们将（a）与乱序siRNA处理的EAN对照相比，验证siRNA处理的EAN大鼠坐骨神经内关键单体GTP酶（Cdc 42或RalA）的siRNA介导的敲低，以及（B）与乱序siRNA处理的EAN对照相比，用免疫组织化学定量siRNA处理的大鼠坐骨神经内CCL 2、CCR 2和免疫浸润物（巨噬细胞和白细胞）的含量和分布。本研究的目的是建立siRNA介导的Rho GT3基因敲低作为一种可行的治疗策略，用于炎症性周围神经病变的管理。我们认为，这项研究的成功完成将加快siRNA技术的翻译成主流管理的退伍军人获得性炎症性神经病变，包括AIDP。

DESCRIPTION (provided by applicant): Acquired Inflammatory neuropathies are a considerable social and economic burden to our Veterans and to the Veterans Health Administration. Encompassing both known infectious or possibly infectious etiologies, inflammatory neuropathies constitute one of the largest and least understood spectrums of neurologic disorders. Inclusive among these disorders is acute inflammatory demyelinating polyradiculopathy (AIDP), a highly disabling inflammatory autoimmune disease of the peripheral nervous system that is characterized by acute/subacute symmetrical paresis with areflexia progressing to neuromuscular paralysis. Despite its overwhelming prevalence and socioeconomic impact, the treatment of Veterans with inflammatory peripheral neuropathies, including AIDP, remains palliative. Cytokine-mediated recruitment and trafficking of autoreactive leukocytes across the blood-nerve barrier and into peripheral nerves is a well-established early pathological hallmark of inflammatory peripheral neuropathies, including AIDP. Localized GTPase-dependent activation of the peripheral nerve vascular endothelium in response to proinflammatory cytokines represents an initiating pathological insult. Peer- reviewed and published preliminary studies from our laboratory strongly support that trafficking of autoreactive leukocytes into peripheral nerves during AIDP may proceed by a mechanism that involves Cdc42 GTPase- dependent secretion of CCL2. Novel translational studies are critically needed to develop therapeutic and rehabilitative strategies for the advanced care of Veterans debilitated by acquired inflammatory neuropathies. In this two-year SPiRE study, we will determine whether siRNA-mediated GTPase knockdown therapeutically protects against the development and progression of inflammatory neuropathy. Hypothesis: Therapeutically administered siRNA directed against key monomeric GTPases will attenuate the development and progression of experimental autoimmune neuritis by inhibiting endothelial cell CCL2 expression. This hypothesis will be tested in vivo with the following two Specific Objectives using an established clinically- translatable rat model of AIDP (experimental autoimmune neuritis, EAN). Specific Objective 1 will determine whether prophylactic or therapeutically administered siRNAs targeting key monomeric GTPases protects against the development and progression of EAN. We will determine the dose- dependent effect of siRNAs targeting Cdc42 or RalA GTPases on (a) the clinical severity and course of EAN and on (b) EAN-induced changes in peripheral nerve function, using evoked-response electrophysiology. Specific Objective 2 will determine whether prophylactic or therapeutically administered siRNAs targeting key monomeric GTPases attenuates trafficking of autoreactive leukocytes into peripheral nerves during EAN. We will (a) validate siRNA-mediated knockdown of key monomeric GTPases (Cdc42 or RalA) within sciatic nerves of siRNA-treated EAN rats, compared with scrambled siRNA-treated EAN controls, and (b) quantify the content and distribution of CCL2, CCR2, and immune infiltrates (macrophages and leukocytes) within sciatic nerves of siRNA-treated rats, compared with scrambled siRNA-treated EAN controls, with immunohistochemistry. The goal of this study is to establish siRNA-mediated Rho GTPase knockdown as a viable therapeutic strategy for the management of inflammatory peripheral neuropathies. We argue that successful completion of this study will expedite the translation of siRNA technology into mainstream management of Veterans with acquired inflammatory neuropathies, including AIDP.