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）是儿童肾衰竭的主要原因， 主要由PKHD 1基因突变以及CYS 1突变引起。虽然ARPKD患者 在不同的年龄发展为终末期肾病，20-30%的ARPKD患者患有严重疾病， 导致出生后24-48小时死亡。尽管有巨大的未满足需求，但取得的进展有限 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， 2018年批准用于常染色体显性PKD的小分子药物，主要影响成人， 目前处于ARPKD的I期临床试验中，托伐普坦不是肾靶向的，并导致脱靶效应。 此外，托伐普坦的长期疗效，耐受性和发育后果，主要是儿科 ARPKD患者人群及其提高重度ARPKD新生儿生存率的能力尚不清楚。因此，在本发明中， 迫切需要能够解决ARPKD的安全性和有效性的新的治疗策略。与 最后，本建议的目的是检查尿细胞外囊泡（uEVs）作为一种新的 和安全的治疗方法EV，也称为外泌体，是分泌的、膜结合的、生物相容的。 促进细胞间通讯并含有RNA和蛋白质货物特征的纳米颗粒， 父细胞。我们建议将uEV用于ARPKD治疗有几个原因：1）uEV具有固有的生物相容性 这在为儿科患者开发治疗方法时至关重要，2）携带纤维囊蛋白和囊蛋白， 分别在ARPKD中突变的PKHD 1和CYS 1的基因产物，3）已经发现归巢于 肾和转移功能蛋白以诱导肾病的治疗结果，和4）获得 从通常被丢弃的尿液中提取大量的uEV是可行的、非侵入性的和便宜的。我们假设 来自非疾病来源的uEV是一种安全的治疗方法，可用于提供和补充 在ARPKD中有缺陷的功能性蛋白质，包括纤维囊蛋白和囊蛋白，以抑制疾病进展。到 为了验证我们的假设，我们将首先描述uEV的纳米颗粒特性，并评估uEV纤维囊蛋白 以及cystin蛋白和mRNA货物、细胞内化和体外肾细胞中的治疗效果（目的1）。 接下来，我们将在缓慢进展和严重的ARPKD鼠模型中静脉内施用uEV， 评价体内药代动力学特性、疗效和安全性（目标2.1）。最后，鉴于大多数 人ARPKD导致子宫内或出生后不久的新生儿死亡，我们将在妊娠期子宫内递送uEV。 小鼠，并评估在胎儿分娩时延长严重ARPKD存活的能力（目的2.2）。通过我们 多学科，纳米医学和儿科肾病学的研究团队，我们装备精良， 致力于成功开展ARPKD治疗中急需的这项工作。

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.