Virus nanoparticles as autophagy activators

描述（由申请人提供）：自噬通过溶酶体介导细胞质物质的降解，在这一重要的体内平衡过程中效率低下导致毁灭性疾病。例如，神经退行性溶酶体贮积症的特征在于溶酶体降解和自噬功能障碍的缺陷。低效的自噬还导致聚集蛋白的积累和蛋白质错误折叠疾病如帕金森病中的神经变性。因此，激活自噬的转化研究策略可能会极大地影响大范围神经退行性疾病的治疗方法的发展。有趣的是，最近出现了纳米材料诱导自噬的证据。不幸的是，目前大多数合成纳米颗粒系统无法穿过血脑屏障（BBB），从而无法用于治疗中枢神经系统。此外，大多数纳米材料破坏溶酶体功能，最终导致自噬通量受阻和清除受损。为了克服这些限制，我们提出开发一种病毒纳米颗粒（VNP）平台，可以诱导自噬和溶酶体生物合成的协调激活。我们的VNP技术是基于腺相关病毒（AAV），并将特别侧重于AAV血清型9，已被证明可以穿过BBB。最近发现了自噬和溶酶体生物发生的主要调节因子转录因子EB（TFEB），证明了溶酶体和自噬体的整合和共调节作用。这项工作的长期目标是开发基于VNP的平台技术，以激活TFEB并增强溶酶体对自噬货物的清除。该提案的目的是确定用于产生可以激活TFEB并增强体外清除的VNPs的设计规则。基于我们的试验数据，本研究的中心假设是VNP的TFEB激活特性取决于颗粒的细胞摄取，而不是衣壳的感染性，因为不能有效感染细胞的缺陷VNP仍然可以激活TFEB。如果成功，拟议的研究将为治疗以自噬底物积累为特征的神经退行性疾病提供一个可行的治疗平台。具体地，我们提出定义用于构建TFEB的基于VNP的激活剂的设计规则（目的1），并且我们将通过评估i）来源于患有神经元蜡样脂褐质沉积症的患者的成纤维细胞中的脂褐质和ii）神经胶质瘤细胞中的聚集的突触核蛋白的清除来测试VNP的体外性能（目的2）。这项研究意义重大，因为它将产生一个治疗平台，能够促进溶酶体自噬系统的共调节激活，以有效治疗神经退行性疾病。这种方法具有创新性，因为它有望克服目前可用的合成纳米材料无法穿过血脑屏障的局限性。这项研究的结果也将大大提高我们对AAV生物学的认识，因为目前还没有关于AAV对自噬途径影响的科学信息。

DESCRIPTION (provided by applicant): Autophagy mediates degradation of cytoplasmic material through the lysosomes, and inefficiencies in this vital homeostatic process result in devastating diseases. For example, neurodegenerative lysosomal storage disorders are characterized by deficiencies in lysosomal degradation and autophagy dysfunction. Inefficient autophagy also leads to accumulation of aggregated proteins and neurodegeneration in protein misfolding diseases such as Parkinson's disease. Thus, translational research strategies to activate autophagy could dramatically impact the development of therapeutics for a large range of neurodegenerative diseases. Interestingly, evidence of autophagy induction by nanomaterials has recently emerged. Unfortunately, most current synthetic nanoparticle systems are unable to cross the blood-brain barrier (BBB), precluding their use to treat the central nervous system. In addition, most nanomaterials disrupt lysosomal function, ultimately leading to block of autophagy flux and impaired clearance. To overcome these limitations, we propose to develop a platform of virus nanoparticles (VNP) that can induce the coordinated activation of autophagy and lysosomal biogenesis. Our VNP technology is based on the adeno-associated virus (AAV), and will particularly focus on AAV serotype 9 that was shown to cross the BBB. Evidence of the integrated and co-regulated roles of lysosomes and autophagosomes emerged from the recent discovery of a master regulator of autophagy and lysosome biogenesis, the transcription factor EB (TFEB). The long-term goal of this work is the development of a VNP-based platform technology to activate TFEB and enhance clearance of autophagic cargo by lysosomes. The objective of this proposal is to identify the design rules for generating VNPs that can activate TFEB and enhance clearance in vitro. The central hypothesis of this study, based on our pilot data, is that the TFEB-activating properties of VNPs depend on cellular uptake of the particle and not on the infectivity of the capsid since a defective VNP unable to productively infect cells can still activate TFEB. If successful, the proposed research will produce an enabling therapeutic platform for the treatment of neurodegenerative diseases characterized by accumulation of autophagic substrates. Specifically, we propose to define the design rules for building VNP-based activators of TFEB (aim 1), and we will test the performance of VNPs in vitro by evaluating clearance of i) lipofuscin in fibroblasts derived from patients with Neuronal Ceroid Lipofuscinosis and ii) aggregated ¿-synuclein in neuroglioma cells (aim 2). The proposed research is significant because it will generate a therapeutic platform able to promote co-regulated activation of the lysosome-autophagy system for effective treatment of neurodegenerative diseases. This approach is innovative because it promises to overcome limitations of currently available synthetic nanomaterials that cannot cross the BBB. Results from this study will also significantly advance our knowledge of AAV biology, as no scientific information is currently available concerning the impact of AAV on the autophagy pathway.