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1/7000活产（尽管个别罕见）。超过2/3的LSD 患者表现出中枢神经系统（CNS）受累，具有广谱严重性（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 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，它们在逆转神经系统疾病方面基本上是不成功的。 由于酶穿过血脑屏障（BBB）到达CNS的渗透性差而引起的并发症， 治疗NLSD的主要障碍。阳离子非依赖性甘露糖-6-磷酸受体（M6 PR，也称为 IGF 2 R）在溶酶体酶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。BBB上M6 PR的发育衰退 在小鼠和人的出生后早期，已经有记录，这是由于缺乏 CNS酶递送。使用双荧光素酶报告系统与位点突变，我们最近 发现microRNA-143（miR 143）调节BBB形成脑毛细血管上的M6 PR蛋白水平 通过靶向M6 PR mRNA的3'非翻译区，对内皮细胞（BrMV）进行转染。使用Hurler的小鼠模型 α-L-艾杜糖醛酸酶缺乏引起的严重粘多糖样沉积症（I型，MPS I） （IDUA），我们进一步证明了M6 PR介导的IDUA转移在脑中的功能性拯救。 具有长期CNS治疗益处的MPS/miR-143 KO敲除小鼠以及人血管 通过用miR-143-sponge序列下调miR-143来抑制内皮细胞。数据显示， 开发一种新的CNS靶向方法的科学前提， 许多涉及M6 PR通路的神经学LSD，或在递送可适应M6 PR的脑治疗剂中， 介导的转胞吞途径。在这项提案中，我们的目标是开发一种新的腺相关病毒载体（AAV）- 在成熟BBB上“恢复”M6 PR通路，用于治疗性药物的高级递送 研究miR 143抑制剂（143 in）的合成和miR 143的表达 用于有效和靶向减少BrMV上miR 143的试剂盒（目的1），检查生物分布， AAV-143在小鼠中的“脱靶”表达和作用（目的2），以及临床前评价 靶向BBB的AAV/miR 143 in通过来源于 遗传修饰的红细胞/巨核细胞谱系（AIM 3）。这项研究的影响是由未满足的 有效治疗遗传性nLSD的医学需求和药物递送穿过BBB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.