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对退伍军人和退伍军人健康管理局来说是一个相当大的社会和经济负担。炎症性神经病包括已知的感染性或可能的感染性病因，是神经系统疾病中规模最大、最不为人所知的疾病之一。这些疾病包括急性炎症性脱髓鞘多神经根病(AIDP)，这是一种高度致残性的周围神经系统炎症性自身免疫性疾病，其特征是急性/亚急性对称性瘫痪，反射障碍进展为神经肌肉瘫痪。尽管它的压倒性流行和社会经济影响，退伍军人炎症性周围神经病的治疗，包括AIDP，仍然是姑息性的。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进入周围神经是包括AIDP在内的炎症性周围神经病的一个公认的早期病理特征。外周神经血管内皮细胞对促炎症细胞因子的反应依赖于GTPase的局部激活是一种起始的病理损害。我们实验室的同行评审和发表的初步研究有力地支持了AIDP期间自身反应性白细胞向外周神经的运输可能是通过一种涉及CDC42 GTP酶依赖的CCL2分泌的机制进行的。迫切需要新的翻译研究来开发治疗和康复策略，为因获得性炎症性神经病而虚弱的退伍军人提供高级护理。在这项为期两年的SPIRE研究中，我们将确定siRNA介导的GTP酶基因敲除是否在治疗上对炎症性神经病的发生和进展具有保护作用。假设：针对关键单体GTP酶的治疗给予siRNA将通过抑制内皮细胞CCL2的表达来减缓实验性自身免疫性神经炎的发展和进展。这一假说将利用已建立的临床可翻译的AIDP(实验性自身免疫性神经炎，EAN)大鼠模型，通过以下两个特定目标在体内进行验证。具体目标1将确定针对关键单体GTP酶的预防性或治疗性给药siRNAs是否可以预防EAN的发生和发展。我们将利用诱发反应电生理学，确定针对CDC42或Rala GTP酶的siRNAs对(A)EAN的临床严重程度和病程以及(B)EAN引起的周围神经功能变化的剂量依赖性影响。具体目标2将确定针对关键单体GTP酶的预防性或治疗性给予的siRNAs是否能减少EAN期间自身反应性白细胞向周围神经的运输。我们将(A)验证siRNA介导的关键单体GTP酶(CDC42或Rala)在siRNA处理的EAN大鼠和杂乱的siRNA处理的EAN对照大鼠的坐骨神经中的敲除，以及(B)用免疫组织化学方法定量比较siRNA处理的EAN大鼠的坐骨神经中CCL2、CCR2和免疫浸润物(巨噬细胞和白细胞)的含量和分布。这项研究的目的是建立siRNA介导的Rho GTP酶基因敲除作为治疗炎症性周围神经病的一种可行的治疗策略。我们认为，这项研究的成功完成将加快siRNA技术在包括AIDP在内的获得性炎症性神经病退伍军人的主流管理中的转换。

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.