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巴顿病 (BD) 是一大类严重神经退行性疾病之一 称为溶酶体贮积症（LSD）的疾病。 BD的特点是细胞内 神经组织中储存物质的积累以及神经功能和视力的逐渐丧失 主要影响儿童。 CLN2 是一种特殊类型的 BD，由 TPP1 基因突变导致 可溶性溶酶体酶三肽基肽酶-1 (TPP1) 不足或完全缺乏。去年，第一 FDA 批准的治疗方法，即脑室内注射重组形式的人 TPP1，被证明可以提供 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没有提供治疗益处，因为血液 脑屏障 (BBB) 严重限制大分子（包括 TPP1）向大脑的运输。规避 针对这个问题，我们建议使用巨噬细胞释放的细胞外囊泡（EV）作为生物相容性 用于全身递送 TPP1 的纳米载体。由具有多种粘附蛋白的细胞膜组成 在其表面，电动汽车专门从事细胞通信，促进蛋白质和 将遗传物质传递给靶细胞。 我们之前的研究表明，包括 TPP1 在内的治疗性蛋白质可以有效地 并入电动汽车而不失去其生物活性。特别是，TPP1 使用两个 方法：(i) 用 TPP1 编码质粒 DNA (pDNA) 转染亲代产生 EV 的巨噬细胞，或 (ii) 将治疗蛋白 TPP1 加载到初始空 EV 中。由此产生的电动汽车载体群被证明 在全身性治疗后很容易迁移到婴儿晚期神经元蜡样脂褐素沉积症（LINCL）小鼠的大脑中 行政管理。重要的是，在 LINCL 小鼠中观察到了多种治疗效果的证据， 包括显着的神经保护和延长寿命。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、表征和存储以用于治疗应用。 在当前的提案中，我们将利用两种基于 EV 的 TPP1 公式来演示概念验证 使用BD小鼠模型，LINCL小鼠。计划的研究包括： (SA1) 阐明选择性的本质 巨噬细胞来源的 EV 的指纹图谱，以及 EV 相互作用和 TPP1 促进摄取的机制 神经血管单元细胞； (SA2) 使用 MRI 和光学成像评估 EV-TPP1 的脑生物利用度 体内，并（SA3）通过测量这种新型药物递送系统的治疗潜力来验证其治疗潜力 LINCL 小鼠的神经保护和抗炎作用。拟议的研究解决了一个关键问题 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.