Anti-inflammatory and hMSC combination therapy for traumatic brain injury

我们研究计划的长期目标是为创伤性脑损伤开发安全有效的治疗方法 创伤性脑损伤（TBI），这是我们的军事人员在战斗中的主要健康问题。TBI的发病率 据估计，仅在美国就有约250万至370万例。目前，没有FDA- 治疗创伤性脑损伤的药物虽然TBI疾病的范围从轻度到重度，中度TBI是 临床上最相关，如果不立即治疗，这些患者的继发性损伤可能会扩散 并可能导致包括认知和记忆丧失在内的长期后果。充质 干细胞（MSC）移植已经成为治疗TBI的主要方法之一。然而，在这方面， 单一MSC治疗的可行性正在争论，因为在 炎症状态因此，存在发现和开发新的治疗药物的真正未满足的需求。 用于所有形式的TBI，尤其是中度TBI的药剂。 据报道，在MSC治疗前加入抗炎剂增加了MSC的免疫反应。 后者的功效。在上一个资助期间，我们建立了一个实验小鼠模型， 能够表征闭合性颅脑损伤中反复TBI（rTBI）的后果， 与认知缺陷相关的广泛神经病理学变化，与人类TBI一致。我们 发现趋化因子（C-C基序）配体20（CCL 20）（MIP 3 α）的表达 是TBI炎症的关键，暗示CCL 20是潜在的TBI药物靶点。我们有 证明了CCL 20抑制剂吡格列酮（PG）在减少星形胶质细胞增生中起关键作用， TBI中的小胶质细胞活化和炎性小体活化。PG和hMSC治疗的组合 不仅减少了炎症，而且通过增加表达， 脑源性神经营养因子（BDNF）因为PG不是特异性的CCL 20抑制剂， 具有脱靶效应，我们已经建立并验证了短发夹RNA的纳米制剂， 使用树枝状聚合物的编码CCL 20及其受体CCR 6的shRNA质粒。的组合治疗 涉及shCCL 20/shCCR 6（shCombo）和人MSC（hMSC）的细胞凋亡显著降低。 在rTBI小鼠模型中，炎症、BDNF和神经发生的伴随增加。 基于我们的初步数据，我们假设shCombo树枝状复合物（DPX）或/和 hMSC组合方案将通过以下方式减弱急性脑炎症和全身炎症两者： 减少脑中神经胶质细胞和星形胶质细胞的活化，并分泌营养因子超过 这反过来又促进受损大脑中的神经元重塑， 持续缓解TBI引起的病理。提出了三个具体目标来检验这一假设。在 目标#1，我们将确定pshCombo-DPX的最佳剂量和治疗窗 rTBI小鼠。在目标#2中，我们将检查shCombo-DPX或/和 hMSC治疗对rTBI小鼠中病理生理学和行为缺陷的影响。在目标#3中，我们将确定 CCL 20在rTBI诱导的发病机制和行为缺陷中的作用机制。 这项高度创新的提案旨在研究shCombo-DPX作为单一药物或 与hMSC组合将减少急性脑和全身炎症和行为缺陷， 并计划根据诱导的基因来解决作用机制和疗效的替代标志物， 在急性炎症期间。建议的翻译研究基于合理设计的更好的 抗炎治疗有望增加我们对长期结果的理解。 治疗轻中度TBI，并为未来的临床转化铺平道路。

The long-term goal of our research program is to develop safe and effective therapies for traumatic brain injury (TBI), which is a major health issue with our military personnel in combat. The prevalence of TBI from all causes is estimated at ~2.5 to 3.7 million cases in the USA alone. Currently, there are no FDA- approved drugs available to treat TBI. While TBI illness ranges from mild to severe, moderate TBI is clinically most relevant, and If not treated immediately the secondary injury in these patients could spread quickly and may lead to long-term consequences including cognitive and memory loss. Mesenchymal stem cells (MSCs) transplantation has emerged as one of the major approaches to treat TBI. However, the viability of sole MSC therapy is being debated due to the uncertainty of variable engraftment in inflammatory state. Hence, there is a genuine unmet need to discover and develop new therapeutic agents for all forms of TBI, especially moderate TBI. It has been reported that adding an anti-inflammatory agent prior to MSC treatment increased the latter’s efficacy. During the last funding period, we have established an experimental mouse model to enable characterization of the consequences of repeated TBI (rTBI) in closed head injury, which shows extensive neuropathologic changes associated with the cognitive deficit, consistent with human TBI. We made the striking discovery that the expression of chemokine (C-C motif) ligand 20 (CCL20) (MIP3α) is pivotal to inflammation in TBI, implicating CCL20 as a potential TBI drug target. We have demonstrated that CCL20 inhibitor, pioglitazone (PG), played a key role in reducing astrogliosis, microglial activation and inflammasome activation in TBI. A combination of PG and hMSC treatment not only reduced inflammation but also improved behavioral outcomes by increasing the expression of brain-derived neurotrophic factor (BDNF). Because PG is not a specific CCL20 inhibitor and may have off-target effects, we have established and validated a nanoformulation of short hairpin RNA (shRNA) plasmids encoding CCL20 and its receptor CCR6 using dendrimer. A combination treatment involving shCCL20/shCCR6 (shCombo) and human MSC (hMSC) showed a significant reduction in inflammation, a concomitant increase in BDNF and neurogenesis in rTBI mouse model. Based on our preliminary data, we hypothesize that the shCombo dendriplex (DPX) or/and hMSC combination regimen will attenuate both acute brain- and systemic-inflammation by decreasing activation of glial cells and astrocytes in the brain, and secreting trophic factors over a prolonged period, which in turn promotes neuronal remodeling in the damaged brain and provides sustained relief from TBI-induced pathology. Three specific aims are proposed to test this hypothesis. In Aim #1, we will determine the optimal dose of pshCombo-DPX and the therapeutic window of treatment in rTBI mice. In Aim #2 we will examine the short-term and long-term effects of shCombo-DPX or/and hMSC therapy on pathophysiology and behavioral deficits in rTBI mice. In Aim #3, we will determine the mechanism of action of CCL20 in rTBI-induced pathogenesis and behavioral deficits. This highly innovative proposal aims to investigate whether shCombo-DPX as a single agent or in combination with hMSC will reduce acute brain- and systemic-inflammation and behavioral deficits, and plans to address the mechanism of action and surrogate markers of efficacy based on genes induced during acute inflammation. The proposed translational research based on a rationally designed better anti-inflammatory therapy is expected to increase our understanding of the long-term outcome of the therapy for mild to moderate TBI and pave the way for future clinical translation.