Extracellular Vesicles for CNS Delivery of Therapeutic Enzymes to Treat Lysosomal Storage Disorders

神经元蜡样质脂褐质沉积症或Batten病（BD）是一类严重的神经退行性疾病， 这些疾病被称为溶酶体贮积症（LSD）。BD的特征在于细胞内 神经组织中储存物质的积累以及神经功能和视力的进行性丧失， 主要影响儿童。CLN 2是一种特定类型的BD，其由TPP 1基因突变引起， 可溶性溶酶体酶三肽基肽酶-1（TPP 1）不足或完全缺乏。去年首届 FDA批准的治疗，脑室内输注重组形式的人TPP 1，显示出提供 儿科患者的症状改善。不幸的是，这种侵入性手术具有很高的风险， 不良影响侵入性较小的全身给药不提供治疗益处，因为血液 脑屏障（BBB）严重限制了大分子（包括TPP 1）向脑的转运。规避 针对这一问题，我们提出使用巨噬细胞释放的细胞外囊泡（EV）作为生物相容性的 用于全身递送TPP 1的纳米载体。由具有多种粘附蛋白的细胞膜组成 在它们的表面上，已知EV专门进行细胞间通信，促进蛋白质的运输， 遗传物质的靶细胞。 我们的研究先前已经证明，治疗性蛋白质，包括TPP 1，可以有效地 在不丧失其生物活性的情况下并入EV。特别地，使用两个微处理器将TPPl装载到EV中。 方法：（i）用编码TPP 1的质粒DNA（pDNA）转染亲代EV产生巨噬细胞，或 (ii)将治疗性蛋白质TPP 1装载到初始空EV中。所得到的EV载体集合被示出为 在全身给药后， 局重要的是，在LINCL小鼠中观察到治疗功效的多条证据， 包括显著的神经保护和延长的寿命。值得注意的是，我们开发了不同的方法， 足够数量的EV分离、纯化、表征和储存以用于治疗应用。 在当前的提案中，我们将利用两个基于EV的TPP 1公式来证明概念验证 使用BD小鼠模型，LINCL小鼠。计划的研究包括：（SA 1）阐明选择性 巨噬细胞衍生的EV的指纹，以及EV相互作用的机制和TPP 1促进了 神经血管单位的细胞;（SA 2）用MRI和光学成像评价EV-TPP 1的脑生物利用度， 体内，和（SA 3）验证这种新型药物递送系统的治疗潜力，通过测量其 在LINCL小鼠中的神经保护和抗炎作用。拟议的研究解决了一个关键问题， 本发明解决了将治疗性蛋白质有效递送至中枢神经系统（CNS）的问题，并将提供 关于电动汽车如何与目标脑细胞通信并选择性地运送货物的基本见解。

The neuronal ceroid lipofuscinoses or Batten disease (BD) is one of a broad class of severe neurodegenerative diseases that are known as lysosomal storage disorders (LSDs). BD is characterized by the intracellular accumulation of storage material in neural tissues and progressive loss of neurological functions and vision that primarily affect children. CLN2 is a specific type of BD that results from mutations in the TPP1 gene causing an insufficiency or complete lack of a soluble lysosomal enzyme tripeptidyl peptidase-1 (TPP1). Last year, the first FDA-approved treatment, intraventricular infusions of a recombinant form of human TPP1, was shown to provide symptomatic improvements in pediatric patients. Unfortunately, this invasive procedure carries a high risk of adverse effects. The less invasive systemic administration provides no therapeutic benefits, because the blood brain barrier (BBB) severely restricts transport of macromolecules - including TPP1- to the brain. To circumvent this problem, we propose using extracellular vesicles (EVs) released by macrophages as biocompatible nanocarriers for systemic delivery of TPP1. Comprised of cellular membranes with multiple adhesive proteins on their surface, EVs are known to specialize in cell-cell communications facilitating transport of proteins and genetic material to target cells. Our research has previously demonstrated that therapeutic proteins, including TPP1, can be efficiently incorporated into EVs without losing their biological activity. In particular, TPP1 was loaded into EVs using two methods: (i) transfection of parental EV-producing macrophages with TPP1-encoding plasmid DNA (pDNA), or (ii) loading therapeutic protein TPP1 into naive empty EVs. The resulting EVs carrier ensemble was shown to readily migrate into the brains of late-infantile neuronal ceroid lipofuscinosis (LINCL) mice upon systemic administration. Importantly, multiple lines of evidence for therapeutic efficacy were observed in LINCL mice, including significant neuroprotection and improved life span. Noteworthy, we developed different methods for EVs isolation, purification, characterization, and storage in sufficient quantities for therapeutic application. In the current proposal, we will utilize two EV-based formulations of TPP1 to demonstrate the proof of concept using a BD mouse model, LINCL mice. Planned studies include: (SA1) elucidation the nature of selective fingerprinting of macrophage-derived EVs, and mechanism of EVs interactions and TPP1 facilitated uptake in cells of neurovascular unit; (SA2) evaluation of brain bioavailability for EV-TPP1 with MRI and optical imaging in vivo, and (SA3) validation the therapeutic potential of this novel drug delivery system by measuring its neuroprotective and anti-inflammatory effects in LINCL mice. The proposed research addresses a critical problem in the effective delivery of therapeutic proteins to the central nervous system (CNS), and will provide fundamental insights into, how EVs communicate with target brain cells, and selectively deliver their cargo.