Cell-based Platform for Gene Delivery to the Brain

摘要 帕金森氏症(PD)在美国影响着100多万人，新增病例高达6万人 每年都会确诊。目前，还没有治疗方法可以阻止或逆转帕金森病的进程；只有姑息治疗 治疗方法已经存在，例如对缺失的神经递质的替代策略。帕金森病的病理生物学是 与多巴胺(DA)能神经元的丧失有关。因此，神经营养因子的成功输送，在 尤其是胶质细胞源性神经营养因子(GDNF)，它能促进神经元存活并逆转 帕金森病的进展是非常重要的。令人遗憾的是，血脑屏障(BBB)似乎仍然是一种 常规使用系统管理的大分子--包括GDNF1--的不可逾越的障碍。 为了绕过这个问题，我们建议使用转基因外周血单核细胞(PBM)来 全身性基因输送到大脑。免疫系统的特化细胞，包括 单核细胞、巨噬细胞和T细胞可以很容易地穿透血脑屏障，并迅速迁移到大脑的各个部位 炎症和变性2，3。我们的研究先前证明，巨噬细胞转染EX 体内携带治疗性蛋白编码DNA质粒(PDNA)，可携带治疗性基因治疗中毒小鼠 急性脑炎，在转基因小鼠中，Parkin-Q311X(A)。机械论研究表明， 转基因巨噬细胞释放胞外囊泡，外体，充满编码蛋白质 遗传物质、pDNA和信使核糖核酸，以及参与编码基因表达的转录因子。 重要的是，在帕金森病小鼠模型中观察到了多种治疗效果的证据，包括 减少脑部炎症，显著的神经保护，并改善运动功能。 计划中的研究包括评估工程化PBM和GDNF基因转移的脑生物利用度 PARKIN-Q311X(A)小鼠模型(SA1)。为了执行新提法的结果，PBM将有所区别 到特定的具有再生功能的“交替激活”(M2)巨噬细胞亚群。这一机制 巨噬细胞介导的基因转移，包括参与细胞与细胞之间的相互作用和/或由 转GDNF的PBM将被阐明(SA2)。然后我们将评估这本小说的治疗潜力 通过测量其抗炎和神经保护作用，最后通过广泛的行为 Parkin-Q311X(A)小鼠(SA3)的分析。为了提供翻译联系，人类诱导的多能干细胞 具有几乎无限扩展能力的(IPSCs)将进行测试。此外，为了获得一个通用的蜂窝载体，iPSCS 对于敲除的MHC第二类(MHC-II)受体将被利用。 我们的研究将为PBM如何与脑细胞相互作用和促进 神经变性时的水平基因转移，可能打开其他基于细胞的基因传递 系统连接到CNS和更远的地方。

ABSTRACT Parkinson’s disease (PD) affects more than a million individuals in the U.S. with up to 60,000 new cases diagnosed each year. Currently, there are no treatments that can halt or reverse the course of PD; only palliative therapies, such as replacement strategies for missing neurotransmitters, exist. The pathobiology of PD is associated with the loss of dopaminergic (DA) neurons. Thus, the successful delivery of neurotrophic factors, in particular, glial cell line-derived neurotrophic factor (GDNF), that promote neuronal survival and reverse the progression of PD is of great importance. Regrettably, the blood brain barrier (BBB) remains a seemingly insurmountable obstacle to the routine use of systemically administered macromolecules - including GDNF 1. To circumvent this problem, we propose using genetically modified peripheral blood monocytes (PBM) for systemic gene delivery to the brain. It is well established that specialized cells of the immune system, including monocytes, macrophages, and T cells, can easily penetrate the BBB and migrate rapidly to sites of brain inflammation and degeneration 2,3. Our research has previously demonstrated that macrophages transfected ex vivo with therapeutic protein-encoding DNA plasmid (pDNA), can deliver therapeutic gene in intoxicated mice with acute brain inflammation, and in the transgenic mice, Parkin-Q311X(A). Mechanistic studies revealed that genetically modified macrophages release extracellular vesicles, exosomes, packed with protein-encoding genetic material, pDNA and mRNA, as well as a transcription factor involved in the encoded gene expression. Importantly, multiple lines of evidence for therapeutic efficacy were observed in PD mouse models, including decreased brain inflammation, significant neuroprotection, and improved locomotor functions. Planned studies include the evaluation of brain bioavailability for engineered PBM and GDNF gene transfer in Parkin-Q311X(A) mouse model (SA1). To enforce outcomes of the new formulation, PBM will be differentiated to a specific subset of “alternatively activated” (M2) macrophages with regenerative functions. The mechanism of macrophage-mediated gene transfer including involvement cell-cell interactions and/or exosomes secreted by GDNF-transfected PBM will be elucidated (SA2). We will then assess the therapeutic potential of this novel product by measuring its anti-inflammatory and neuroprotective effects and, lastly, by extensive behavioral analysis in Parkin-Q311X(A) mice (SA3). To provide translational link, human induced pluripotent stem cells (iPSCs) with almost unlimited expandability will be tested. Furthermore, to obtain a universal cell-carrier, iPSCs with knockout MHC class II (MHC-II) receptor will be utilized. Our studies will provide fundamental insights into how PBM interact with brain cells and facilitate horizontal gene transfer upon neurodegeneration, potentially opening up other cell-based gene delivery systems to the CNS and beyond.