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

描述（由申请人提供）： 帕金森病（PD）的神经保护/恢复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）是迄今为止测试的最有效的营养分子，并且在许多研究中已经清楚地证明了保护和恢复PD中受影响的神经元的能力。然而，GDNF在PD患者中的临床试验结果喜忧参半。有效地靶向递送到疾病的特定大脑部位被认为是持续成功的关键障碍。GDNF需要局灶性递送，因为它不穿过血脑屏障。用于递送的套管的精确位置是关键的，并且由于人脑中相对大的靶区域和该分子的差的脑组织渗透，对于所有或大多数变性神经元实现GDNF的治疗水平是具有挑战性的。此外，目前的GDNF治疗带来了重要的安全性问题。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归巢到邻近受损神经元的退化位点的特性，利用了我们强大的巨噬细胞特异性合成启动子（MSP），并实现了造血干细胞基因治疗的最新进展。我们推测，GDNF的高效CNS递送可以通过其在巨噬细胞/小胶质细胞中的表达来实现，通过用携带表达GDNF的盒的慢病毒载体离体转导含有HSC的骨髓细胞，随后将这些转导的骨髓细胞同基因移植，这将极大地改善PD的病理变化和神经缺陷。使用亚急性MPTP（1-甲基-4-苯基-1，2，3，6-四氢吡啶）小鼠PD模型中，我们证明了基因工程骨髓细胞衍生的巨噬细胞在患病部位积聚，巨噬细胞介导的GDNF递送显著减少黑质多巴胺能神经元及其纹状体纤维的变性，并诱导轴突再生，没有任何明显的副作用。为了将这种新颖的概念和独特的方法最终应用于临床，我们建议在神经毒素和遗传小鼠模型中深入探讨PD的慢性和进行性变化特征。在慢性毒素模型中，我们将利用四环素可调节（Tet-on）系统，并通过在神经恢复或神经保护/恢复范例中多西环素诱导的GDNF表达和递送的开关来解决我们的方法的功效，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PD的神经退行性阶段时进行转导骨髓细胞的移植。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不良反应，并检查在必要时通过停用强力霉素来减少或关闭GDNF表达的能力，来解决安全性问题。这项转化研究将为未来的临床研究奠定坚实的基础，研究这种新型神经保护疗法对PD患者的潜在益处。

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.