Investigation of urinary extracellular vesicles as novel and safe therapeutics for autosomal recessive polycystic kidney disease

摘要 常染色体隐性遗传性多囊肾病(ARPKD)是儿童肾功能衰竭的主要原因之一， 主要由PKHD1基因突变和Cys1突变引起。虽然ARPKD患者 终末期肾病进展在不同年龄，20%-30%的ARPKD患者有严重的疾病， 结果在出生后24-48小时内死亡。尽管有巨大的未得到满足的需求，但进展有限。 对于ARPKD治疗，没有FDA批准的疗法可用于ARPKD患者。虽然托伐普坦是一种 小分子药物于2018年被批准用于常染色体显性PKD，主要影响成年人，是 目前正在进行ARPKD的I期临床试验，托伐普坦不是针对肾脏的，会导致非靶向效应。 此外，托伐普坦的长期疗效、耐受性和对以儿科为主的 ARPKD患者群体及其提高重度ARPKD新生儿存活率的能力尚不清楚。因此， 迫切需要新的治疗策略，既能解决ARPKD的安全性，又能解决其疗效。到那时候 最后，这项建议的目的是研究尿胞外小泡(UEV)作为一种新的 ARPKD的安全治疗。EV，也被称为外体，是分泌的、膜结合的、生物的 促进细胞间通讯并含有RNA和蛋白质货物的纳米颗粒 父单元格。我们建议uEV用于ARPKD治疗有几个原因：uEV具有天然的生物相容性 这在为儿科患者开发治疗方法时是至关重要的，2)携带纤维囊藻毒素和囊藻毒素， 在ARPKD中分别突变的PKHD1和CyS1的基因产物被发现是 肾脏和转移功能蛋白以诱导肾脏疾病的治疗结果，以及4)获得 从通常被丢弃的尿液中提取大量uEV是可行的、非侵入性的和廉价的。我们假设 来自非疾病来源的uEV是一种安全的治疗方法，可以用来提供和补充 在ARPKD中有缺陷以抑制疾病进展的功能蛋白，包括纤维囊藻毒素和囊藻毒素。至 验证我们的假设，我们将首先表征uEV的纳米颗粒特性，并评估uEV纤维囊藻蛋白 以及体外肾细胞中的囊藻毒素蛋白和信使核糖核酸载量、细胞内化和治疗作用(目标1)。 接下来，我们将对进展缓慢和严重的ARPKD小鼠模型进行uEVS静脉注射。 评价其体内药代动力学性质、治疗效果和安全性(Aim 2.1)。最后，给出了大多数 人类ARPKD导致宫内死亡或新生儿出生后不久，我们将在怀孕期间在宫内交付uEV 并评估在重度ARPKD中延长胎儿分娩后存活的能力(目标2.2)。通过我们的 我们是一支多学科的纳米医学和儿科肾脏病研究团队，设备齐全 致力于成功开展ARPKD治疗中亟需的这项工作。

ABSTRACT Autosomal recessive polycystic kidney disease (ARPKD) is a leading cause of kidney failure in childhood and is caused primarily by mutations in the PKHD1 gene as well as mutations in CYS1. Although ARPKD patients progress to end stage kidney disease at varying ages, 20-30% of ARPKD patients have severe disease which results in death 24-48 hours after birth. Despite the tremendous unmet need, limited progress has been made for ARPKD treatment and no FDA-approved therapies are available for ARPKD patients. Although tolvaptan, a small molecule drug that was approved in 2018 for autosomal dominant PKD which mostly affects adults, is currently under phase I clinical trials for ARPKD, tolvaptan is not kidney-targeted and results in off-target effects. Additionally, tolvaptan’s long-term efficacy, tolerability, and developmental consequences in a primarily pediatric ARPKD patient population and its ability to enhance survival in neonates with severe ARPKD is unknown. Thus, new therapeutic strategies that can address both safety and efficacy in ARPKD are urgently needed. To that end, the purpose of this proposal is to examine the potential of urinary extracellular vesicles (uEVs) as a novel and safe therapy for ARPKD. EVs, also known as exosomes, are secreted, membrane-bound, biological nanoparticles that facilitate cell-to-cell communication and contain RNA and protein cargo characteristic to their parent cell. We propose uEVs for ARPKD therapy for several reasons: uEVs are 1) inherently biocompatible which is critically important when developing therapies for pediatric patients, 2) carry fibrocystin and cystin, the gene products of PKHD1 and CYS1 which are mutated in ARPKD, respectively, 3) have been found to home to the kidneys and transfer functional proteins to induce a therapeutic outcome in kidney disease, and 4) obtaining high quantities uEV from urine, which is normally discarded, is feasible, noninvasive, and cheap. We hypothesize that uEVs derived from non-disease sources are a safe therapy that can be used to deliver and supplement functional proteins including fibrocystin and cystin that are defective in ARPKD to inhibit disease progression. To test our hypothesis, we will first characterize the nanoparticle properties of uEVs and evaluate uEV fibrocystin and cystin protein and mRNA cargo, cell internalization, and therapeutic effects in renal cells in vitro (Aim 1). Next, we will administer uEVs intravenously in slowly progressing and severe ARPKD murine models and evaluate the pharmacokinetic properties, therapeutic efficacy, and safety in vivo (Aim 2.1). Finally, given most human ARPKD leads to death in utero or in newborns shortly after birth, we will deliver uEVs in utero in pregnant mice and evaluate the ability to extend survival in severe ARPKD upon fetal delivery (Aim 2.2). Through our multidisciplinary, investigative team of nanomedicine and pediatric nephrology, we are well-equipped and fully committed to successfully carry out this work that is much needed in ARPKD therapy.