Macrophage-mediated gene delivery of neurotrophic factors for Parkinson's disease

描述(由申请人提供)： 帕金森氏病(PD)神经保护/恢复性治疗的发展将是一个重大的治疗进展。胶质细胞源性神经营养因子(GDNF)是迄今为止检测到的最有效的营养分子，大量研究表明它具有保护和修复帕金森病患者神经元的能力。然而，GDNF在帕金森病患者的临床试验结果喜忧参半。有效的靶向疾病的特定大脑部位的传递被认为是持续成功的关键障碍。GDNF需要局部递送，因为它不会越过血脑屏障。由于人脑的靶区相对较大，而且这种分子在脑组织中的穿透性较差，因此要达到所有或大多数变性神经元的GDNF治疗水平，准确的定位是至关重要的。此外，目前的GDNF治疗存在重要的安全性问题。我们最近开发了一种新的方法-巨噬细胞介导的GDNF传递-似乎能够解决这些问题。这种独特的方法利用了众所周知的巨噬细胞特性，即定位到受损神经元附近的退化部位，利用我们强大的巨噬细胞特异性合成启动子(MSP)，并实施了造血干细胞基因治疗的最新进展。我们推测，在我们的MSP驱动下，通过慢病毒载体携带GDNF表达盒的慢病毒载体体外转导含HSC的骨髓细胞，然后将这些转导的骨髓细胞同种异体移植到巨噬细胞/小胶质细胞中，从而实现高效的CNS转导，这将极大地改善帕金森病的病理变化和神经功能缺陷。利用亚急性MPTP(1-methyl-4-phenyl-1，2，3，6-tetrahydropyridine)小鼠帕金森病神经保护模型，我们证明了基因工程骨髓细胞来源的巨噬细胞聚集在病变部位，巨噬细胞介导的胶质细胞源性神经营养因子传递显著减少黑质多巴胺能神经元及其纹状体纤维的退化，并诱导轴突再生，没有任何明显的不良反应。为了将这一新的概念和独特的方法最终应用于临床，我们建议在以帕金森病慢性渐进性变化为特征的神经毒素和遗传性小鼠模型中深入探讨这一问题。在慢性毒素模型中，我们将利用四环素可调节(Tet-On)系统，并通过多西环素诱导GDNF表达的开启和在神经修复或神经保护/修复范式中的传递来解决我们方法的有效性，模拟PD的治疗。在MitoPark条件性基因敲除模型中，我们将在小鼠达到相当于临床前、临床和晚期帕金森病的神经退行性变阶段时，对转导的骨髓细胞进行移植。我们还将通过监测短期或长期GDNF表达后的不良反应，特别是文献中描述的副作用，并检查必要时通过停用多西环素来逐渐减少或切断GDNF表达的能力，来解决安全性问题。这项转译研究将为未来临床研究这种新的帕金森病神经保护疗法的潜在益处奠定坚实的基础。

DESCRIPTION (provided by applicant): The development of a neuroprotective/restorative therapy for Parkinson's disease (PD) would be a major therapeutic advance. Glial cell line-derived neurotrophic factor (GDNF) has been the most potent trophic molecule tested so far and in numerous studies has clearly demonstrated a capacity to protect and restore neurons affected in PD. However, clinical trials of GDNF in PD patients have given mixed results. Effective targeted delivery to specific brain sites of disease is believed to be the key impediment to consistent success. GDNF requires focal delivery as it does not cross the blood- brain barrier. The precise location of the cannula for delivery is critical and it is challenging to achieve therapeutic levels of GDNF for all or most degenerating neurons due to the relatively large target area in human brain and the poor brain tissue penetration of this molecule. Additionally, current GDNF therapy entails important safety concerns. We recently developed a novel approach - macrophage- mediated GDNF delivery - that seems capable of resolving these problems. This unique approach takes advantage of the well-known macrophage property of homing to degenerating sites in proximity to damaged neurons, capitalizes on our powerful macrophage-specific synthetic promoters (MSP), and implements recent advances in hematopoietic stem cell gene therapy. We hypothesize that highly effective CNS delivery of GDNF can be achieved through its expression in macrophages / microglia by ex vivo transduction of HSC-containing bone marrow cells with lentiviral vectors carrying a cassette expressing GDNF driven by our MSP, followed by syngeneic transplantation of these transduced bone marrow cells, and this will greatly ameliorate the pathological changes and neurological defects of PD. Using a sub-acute MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) mouse model of PD in a neuroprotection paradigm, we demonstrated that genetically engineered bone marrow cell-derived macrophages accumulate in diseased sites and macrophage-mediated GDNF delivery dramatically reduces degeneration of dopaminergic neurons of the substantia nigra, as well as their fibers in the striatum, and induces axon regeneration, without any apparent adverse effects. In order to move this novel concept and unique approach eventually into clinical application, here we propose to explore it in depth in both neurotoxin and genetic mouse models featuring the chronic and progressive changes characteristic of PD. In the chronic toxin model, we will utilize a tetracycline-regulatable (Tet-on) system and address efficacy of our approach by doxycycline-induced switching on of GDNF expression and delivery in a neurorestorative or neuroprotective / restorative paradigm, simulating treatment of PD. In the MitoPark conditional gene knockout model, we will perform transplantation of the transduced bone marrow cells at ages of the mice when they reach the neurodegenerative stages equivalent to preclinical, clinical, and advanced PD. We will also address the safety issue by monitoring for adverse effects, particularly those described in the literature, after short- or long-term GDNF expression, and examining the capability of tapering or shutting off GDNF expression when necessary through withdrawal of doxycycline. This translational study will establish a solid base for future clinical investigation of the potential benefits to patients of this novel neuroprotective therapy for PD.