Exososomal CNS-Delivery of Therapies for a Lysosomal Disorder

摘要 溶酶体贮积病（LSD）是由编码LSD的基因中的致病性变异引起的遗传性疾病。 溶酶体功能所必需的酶。这些先天性细胞器疾病具有累积性 发病率约为1/2000 - 3000。LSD具有广泛的临床谱，大多与进行性 神经退行性过程，导致严重残疾和死亡。LSD目前取得的进展 酶替代疗法（ERT）是治疗的示例，其由每周或每两周一次的酶替代疗法（ERT）组成。 重组酶的静脉内施用。尽管一般耐受良好，但ERT显示 在中枢神经系统（CNS）中的生物分布有限。ERT试剂是大分子， 不能通过血脑屏障（BBB）。缺乏可靠和有效的服务， BBB严重限制了ERT和其他治疗神经系统症状的疗法。因此， 用于LSD的治疗剂的递送策略是一种迫切且未满足的需求。98%的小- 分子药物和每一种生物制剂，包括蛋白质，都不能穿过血脑屏障。大量CNS输送 已经制定了一些战略来克服许多人经常说的“问题背后的问题”， 神经系统疾病最近的研究表明，外泌体提供了一种生理手段， 蛋白质或miRNA穿过BBB。外泌体，“细胞的指纹”，是细胞外的纳米囊泡， 由细胞释放到循环和体液中，显示出依赖于其 细胞起源2018年，临床批准的外泌体开始按照GMP标准生产。我们 假设外泌体是治疗性纳米囊泡，其允许穿透BBB， 治疗神经性迷幻药我们建立了一个小鼠神经细胞系， 含有特定溶酶体酶的外泌体。利用我们在全球范围内建立的独特资源， 沿着我们研究神经学LSDs和外泌体功能的工作，我们将讨论 目标：目标1。表征并进一步改进来自已建立的 来源于严重神经性LSD的Twitch（Twi）小鼠模型的鼠神经细胞系。我们 将优化来自稳定表达的神经永生化Twi鼠细胞系的外泌体的生产 人GALC（半乳糖苷酶）。使用靶向蛋白质组学和鞘脂组学，我们将表征 神经元外泌体目的2.检查神经源性外泌体作为CNS的治疗潜力- 在Twitcher小鼠模型中的递送系统。我们将研究神经- 使用Twi小鼠的神经过程中衍生的外泌体。作为细胞间通讯的介质， 作为病理状态下细胞内变化的替代物，外泌体成为递送 迷幻药的治疗这项原理验证研究将揭示外泌体是一种强大而强大的CNS， 治疗神经退行性疾病的治疗应用的“纳米囊泡递送”策略。

ABSTRACT Lysosomal storage diseases (LSDs) are genetic disorders caused by pathogenic variants in genes encoding enzymes that are essential for the lysosomal function. These inborn organelle diseases have a cumulative incidence of ~1/2,000-3,000. LSDs have a broad clinical spectrum, mostly associated with a progressive neurodegenerative course, resulting in severe disability and death. The current advances achieved in LSD treatment are exemplified by the enzyme replacement therapy (ERT), which consist of weekly or biweekly intravenous administrations of the recombinant enzymes. Although generally well-tolerated, ERT showed limited biodistribution in the central nervous system (CNS). ERT agents are large molecules, and so they are incapable of crossing the blood-brain barrier (BBB). The lack of reliable and efficacious delivery across the BBB severely limits the ERT and other therapies to treat neurological symptoms. Hence, the development of delivery strategies for therapeutic agents for LSDs is an urgent and unmet need. Unfortunately, 98% of small- molecule drugs and every biological agent, including proteins, cannot cross the BBB. Numerous CNS delivery strategies have been developed to overcome the often stated ‘problem-behind the problem’ of many neurological conditions. Recent studies have shown that exosomes provide a physiological means to deliver proteins or miRNA across BBB. Exosomes, “fingerprints of the cell,” are extracellular nanovesicles are released by cells into the circulation and bodily fluids, displaying distinct cargo profile dependable upon their cellular origin. In 2018, clinic-approved exosomes started to be produced under GMP standards. We hypothesize that exosomes are therapeutic nanovesicles that allow penetration through the BBB to treat neuropathic forms of LSDs. We established a murine neural cell line that secretes neural-derived exosomes containing specific lysosomal enzymes. Leveraging the unique resources we have built over the years, along with our work investigating neurological LSDs and function of exosomes, we will address the following aims: Aim 1. Characterize and further improve the exosomal production from an established murine neural cell line derived from a Twitcher (Twi) mouse model of a severe neurological LSD. We will optimize the production of exosomes from neural immortalized Twi murine cell line stably expressing human GALC (galactocerebrosidase). Using targeted proteomics and sphingolipidomics, we will characterize neuronal exosomes Aim 2. Examine the therapeutic potential of neural-derived exosomes as a CNS- delivery system in the Twitcher, mouse model. We will examine the biodistribution and efficacy of neural- derived exosomes in the neurological course using Twi mice. As mediators of intercellular communication and surrogates of intracellular changes in pathological states, exosomes become a reliable source to deliver therapies to LSDs. This proof-of-principle study will reveal exosomes as a powerful and robust CNS- ‘nanovesicle-delivery’ strategy of therapeutic applications to treat neurodegenerative disorders.