Manipulation of microRNA for CNS delivery: implication to treatment of neurological LSD

摘要 溶酶体储存障碍(LSD)是一组以功能障碍为特征的遗传性代谢性疾病 在溶酶体中，累积频率为每7000名活产儿中就有一名(尽管个别情况很少)。超过2/3的LSD 患者表现为中枢神经系统(CNS)受累，严重程度广泛(NLSD)， 这使得LSD成为儿童神经退行性疾病的最常见原因。异基因造血剂 干细胞移植(HSCT)或酶替代疗法(ERT) 酶是治疗nLSD的主要选择。然而，他们在逆转神经学方面基本上是不成功的。 由于酶不能穿过血脑屏障(BBB)进入中枢神经系统而引起的并发症 治疗nLSD的主要障碍。阳离子非依赖性甘露糖-6-磷酸受体(M6PR，又称 IGF2R)在溶酶体酶的运输和大多数溶酶体酶的细胞间转移中起关键作用， 这对于治疗LSD的代谢交叉校正是必不可少的。血脑屏障中M6PR的发育衰退 在小鼠和人类出生后的早期阶段，已有文献记载，这可归因于缺乏 中枢神经系统酶递送。使用双荧光素酶报告系统和点突变，我们最近已经 发现microRNA-143(MiR143)调节形成血脑屏障的脑毛细血管上M6PR蛋白水平 通过靶向M6PR基因3‘端非翻译区诱导内皮细胞(BrMV)表达。使用赫勒的鼠标模型 α-L-艾杜糖苷酶缺乏所致的综合征(重症粘多糖病I型) (IDUA)，我们进一步展示了M6PR介导的IDUA在双侧脑内转移的功能挽救。 具有长期中枢神经系统治疗益处的MPS/miR-143KO基因敲除小鼠，以及在人类血管中的作用 通过用miR-143-海绵序列下调miR-143对内皮细胞的影响。这些数据提供了强有力的 开发一种新的中枢神经系统靶向治疗方法的科学前提 许多神经学LSD涉及M6PR通路，或在提供能够适应M6PR-1的脑部疗法方面。 介导的跨细胞途径。在这项建议中，我们的目标是开发一种新型的腺相关病毒载体(AAV)- 基于可翻译平台在成熟血脑屏障上恢复M6PR通路，以实现治疗的高级传递 具有3个目标的酶进入中枢神经系统，包括开发优化的人工miR143抑制剂(143in)和表达 有效和靶向减少BrMV上miR143的盒(S)(目标1)，生物分布和 AAV-143在小鼠体内的非靶点表达和作用(AIM 2)，以及临床前评估 BBB靶向AAV/miR143in对MPS-I小鼠中枢神经系统异常的作用 转基因红系/巨核系(目标3)。这项研究的影响是由未被满足的 有效治疗遗传性nLSD的医疗需要以及跨血脑屏障给药的主要限制。

Abstract Lysosomal storage disorders (LSDs) are a group of inherited metabolic diseases characterized by a dysfunction in lysosomes, with cumulative frequency of 1 in 7000 live births (although individually rare). Over 2/3 of LSD patients present an involvement of the central nerve system (CNS) with a broad spectrum of severity (nLSD), which makes LSDs the most common cause of pediatric neurodegenerative disease. Allogeneic hematopoietic stem cell transplantation (HSCT) or enzyme replacement therapy (ERT) by periodical injection of recombinant enzyme are main treatment options for nLSD. However, they are largely unsuccessful in reversing neurological complications due to the poor penetration of the enzymes across the blood-brain-barrier (BBB) to the CNS, a major obstacle in treating nLSD. The cation-independent mannose-6-phosphate receptor (M6PR, also called IGF2R) plays a critical role in lysosomal enzyme trafficking and intercellular transfer of most lysosomal enzymes, which is essential for metabolic cross-correction in treating LSDs. Developmental decline of M6PR on the BBB during early postnatal period in mouse and human has been documented, which is attributable to the lack of CNS enzyme delivery. Using a dual luciferase reporter system with site-mutagenesis, we have recently discovered that microRNA-143 (miR143) modulate M6PR protein levels on BBB-forming brain capillary endothelial cells (BrMV) by targeting to 3' untranslated region of M6PR mRNA. Using a mouse model of Hurler syndrome (severe mucopolysaccharidosis type I, MPS I), which is caused by the deficiency of α-L-iduronidase (IDUA), we further demonstrated functional rescue of M6PR-mediated IDUA transfer in the brain of double- knockout (MPS/miR-143KO) mice with long-term CNS therapeutic benefits, as well as in human vascular endothelial cells by down-regulation of miR-143 with miR-143-sponge sequences. The data provide strong scientific premise for the development of a novel CNS-targeted approach that would be applicable in treating many neurologic LSDs involving M6PR pathway, or in delivering brain therapeutics that can adapting M6PR- mediated transcytosis pathway. In this proposal, we aim to develop a novel adeno-associated viral vector (AAV)- based translatable platform to “restore” M6PR pathway on mature BBB for advanced delivery of therapeutic enzymes into the CNS with 3 aims, including developing optimal artificial miR143 inhibitor (143in) and expression cassette(s) for efficient and targeted reduction of miR143 on BrMV (aim 1), examination of biodistribution and “off-target” expression and effects in mice with AAV-143in delivery (aim 2), as well as preclinical evaluation of BBB-targeted AAV/miR143in in correcting CNS abnormalities in MPS I mice by enzyme therapy derived from genetically modified erythroid/ megakaryocytic lineages (aim 3). The impact of the study is driven by the unmet medical need for efficient treatment of inherited nLSDs AND the major limitation of drug-delivery across the BBB.