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

摘要 溶酶体贮积症（LSD）是一组以功能障碍为特征的遗传性代谢疾病 在溶酶体中，累积频率为七千分之一的活产（尽管个别罕见）。超过 2/3 的 LSD 患者出现中枢神经系统（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。异体造血 干细胞移植（HSCT）或酶替代疗法（ERT）通过定期注射重组 酶是 nLSD 的主要治疗选择。然而，他们在逆转神经系统疾病方面基本上没有成功。 由于酶穿过血脑屏障（BBB）到达中枢神经系统的渗透性差而引起的并发症， 治疗 nLSD 的主要障碍。不依赖于阳离子的 6-磷酸甘露糖受体 (M6PR，也称为 IGF2R）在溶酶体酶运输和大多数溶酶体酶的细胞间转移中起着关键作用， 这对于治疗 LSD 的代谢交叉校正至关重要。 BBB 上 M6PR 发育衰退 在小鼠和人类的产后早期已有记录，这是由于缺乏 中枢神经系统酶递送。使用具有位点诱变的双荧光素酶报告系统，我们最近 发现 microRNA-143 (miR143) 调节 BBB 形成脑毛细血管中的 M6PR 蛋白水平 通过靶向 M6PR mRNA 的 3' 非翻译区来抑制内皮细胞 (BrMV)。使用 Hurler 的鼠标模型 由 α-L-艾杜糖醛酸酶缺乏引起的综合征（I 型严重粘多糖贮积症，MPS I） (IDUA)，我们进一步证明了 M6PR 介导的 IDUA 转移在双脑大脑中的功能性拯救 敲除 (MPS/miR-143KO) 小鼠具有长期中枢神经系统治疗效果以及人类血管 通过 miR-143-sponge 序列下调 miR-143 来抑制内皮细胞。数据提供了强有力的 开发适用于治疗的新型中枢神经系统靶向方法的科学前提 许多涉及 M6PR 通路的神经LSD，或提供可以适应 M6PR 的大脑治疗药物 介导的转胞吞途径。在本提案中，我们的目标是开发一种新型腺相关病毒载体（AAV）- 基于可翻译平台来“恢复”成熟 BBB 上的 M6PR 通路，以实现治疗的高级传递 将酶引入中枢神经系统有 3 个目的，包括开发最佳的人工 miR143 抑制剂 (143in) 和表达 用于有效和有针对性地减少 BrMV 上的 miR143（目标 1）、检查生物分布和 AAV-143 递送小鼠中的“脱靶”表达和影响（目标 2），以及临床前评估 BBB靶向AAV/miR143in通过酶疗法纠正MPS I小鼠的中枢神经系统异常 转基因红细胞/巨核细胞谱系（目标 3）。该研究的影响是由未满足的问题驱动的 有效治疗遗传性 nLSD 的医疗需求以及跨血脑屏障药物输送的主要限制。

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.