Anti-inflammatory and hMSC combination therapy for traumatic brain injury

我们研究计划的长期目标是开发安全有效的脑创伤治疗方法。 受伤(TBI)，这是我们的军事人员在战斗中的一个主要健康问题。颅脑损伤的患病率 据估计，仅在美国就有约250万至370万病例死于各种原因。目前，没有FDA- 可用于治疗脑损伤的批准药物。虽然脑损伤的病情从轻微到严重不等，但中度的脑损伤是 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蔓延 而且可能会导致包括认知和记忆丧失在内的长期后果。间充质 干细胞移植已成为治疗颅脑损伤的主要方法之一。然而， 由于可变植入的不确定性，单一MSC疗法的可行性正在争论中。 炎症状态。因此，发现和开发新的治疗方法是一种真正未得到满足的需求。 适用于所有形式的创伤性脑损伤，尤其是中度脑损伤。 据报道，在MSC治疗之前加入抗炎剂会增加 后者的功效。在上一次资助期间，我们建立了一个实验性的小鼠模型，以 能够描述反复颅脑损伤(RTBI)在闭合性脑损伤中的后果，这表明 与认知缺陷相关的广泛神经病理改变，与人类脑外伤相一致。我们 发现趋化因子(C-C基序)配体20(CCL20)(MIP3α)的表达 是脑损伤炎症的关键，暗示CCL20是潜在的脑损伤药物靶点。我们有 证明CCL20抑制剂吡格列酮(PG)在减少星形胶质细胞增生方面发挥关键作用， 脑外伤后小胶质细胞激活和炎性小体激活。PG与hMSC联合治疗的实验研究 不仅可以减少炎症，还可以通过增加表达改善行为结果 脑源性神经营养因子(BDNF)。因为PG不是特定的CCL20抑制剂，并且可能 我们已经建立并验证了一种短发夹状RNA的纳米配方 (ShRNA)利用树状大分子编码CCL20及其受体CCR6。一种综合治疗 涉及shCCL20/shCCR6(ShCombo)和人类MSC(HMSC)显示显著减少 炎症、伴随的脑源性神经营养因子增加和RTBI小鼠模型中的神经再生。 根据我们的初步数据，我们假设shCombo Dendriplex(DPX)或/和 HMSC联合方案将通过以下方式减轻急性脑和全身炎症 减少大脑中胶质细胞和星形胶质细胞的激活，并在长时间内分泌营养因子 延长，这反过来又促进受损大脑中的神经元重塑，并提供 持续缓解脑损伤所致的病理改变。为了检验这一假说，本文提出了三个具体目标。在……里面 目的#1，我们将确定pshCombo-DPX的最佳剂量和治疗窗口 在RTBI小鼠身上。在目标2中，我们将研究shCombo-DPX或/和 HMSC治疗对RTBI小鼠病理生理和行为障碍的影响。在目标3中，我们将确定 CCL20在RTBI致病机制和行为障碍中的作用机制 这项高度创新的提案旨在调查ShCombo-DPX作为单一代理还是 与hMSC联合使用将减少急性脑部和全身炎症以及行为缺陷， 并计划解决作用机制和基于基因诱导的疗效替代标记 在急性炎症期间。建议在合理设计的基础上更好地进行翻译研究 抗炎治疗有望增加我们对慢性阻塞性肺疾病长期结局的了解 治疗轻中度颅脑损伤，为未来的临床翻译铺平道路。

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.