Advancing CNS drug delivery via epigenetic modulation

摘要 溶酶体贮积症（LSD）是一组遗传性疾病，其特征在于： 溶酶体，累积频率为1/7000活产。超过三分之二的LSD患者表现为 中枢神经系统（CNS）具有广泛的严重性（nLSD），这使得LSD是最 小儿神经病的常见病因异基因造血干细胞移植 或酶替代疗法（ERT）是LSD的主要治疗选择。然而，它们在很大程度上 由于酶对CNS的渗透性差，在逆转神经并发症方面不成功， 这是治疗非迷幻药的主要障碍拟议研究的影响是由未满足的医疗需求驱动的， 遗传性nLSD的有效治疗和酶递送到CNS的主要限制。 非阳离子依赖性甘露糖-6-磷酸受体（M6 PR）在溶酶体酶中起关键作用 运输和细胞间转移的大多数溶酶体酶，这是必不可少的代谢交叉， 治疗LSD的方法生后早期血脑屏障M6 PR表达的发育性下降 在小鼠和人中，这一时期的延迟可归因于缺乏CNS酶递送到成年脑中。使用双 荧光素酶报告系统与位点诱变，我们最近确定microRNA-143（miR 143）作为一个 表观遗传调节剂，以降低脑微血管（BrMV）上的M6 PR蛋白水平。使用小鼠模型， Hurler综合征（I型严重粘多糖沉积症，MPS I），由α-L- 艾杜糖醛酸酶（IDUA），我们证明了M6 PR介导的IDUA转移在脑中的功能拯救， 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-海绵序列隔离miR-143来抑制内皮细胞的增殖。数据显示， 这是开发一种新方法的科学前提，该方法将选择性地“打开”BBB， 任何当前治疗选择或未来酶/基因/细胞疗法提供的酶，用于协同CNS 许多nlsd的好处。在本提案中，我们的目标是开发一种基于腺相关病毒载体（腺相关病毒）的 用于治疗酶的高级递送的在成熟BBB上“恢复”M6 PR途径的可翻译平台 研究miR 143抑制剂（143 in）和miR 143在中枢神经系统的表达 用于稳健和靶向减少脑内皮细胞上的miR 143的试剂盒（目的1）， 用AAV/143 in递送的小鼠中的“中靶”和“脱靶”表达和作用（Aim 2），以及临床前 BrMV靶向的AAV/143 in通过酶疗法校正MPS I小鼠中CNS异常的评价 衍生自遗传修饰的红细胞/巨核细胞谱系（Aim 3）。这些研究将提供一个证据- 一种新的体内miRNA抑制剂介导的脑靶向方法的概念， 涉及M6 PR通路的许多其他nLSD和受益于高级CNS递送的神经系统疾病 通过用M6 P残基或IGF 2-标签修饰来适应M6 PR介导的转运途径。

Abstract Lysosomal storage disorders (LSDs) are a group of inherited diseases characterized by dysfunctions in lysosomes, with cumulative frequency of 1 in 7000 live births. 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 neuronopathic diseases. Allogeneic hematopoietic stem cell transplantation (HSCT) or enzyme replacement therapy (ERT) are main treatment options for LSDs. However, they are largely unsuccessful in reversing neurological complications due to the poor penetration of the enzymes into the CNS, a major obstacle in treating nLSD. The impact of the proposed study is driven by the unmet medical need for efficient treatment of inherited nLSDs AND the major limitation of enzyme-delivery into the CNS. The cation-independent mannose-6-phosphate receptor (M6PR) plays a critical role in lysosomal enzyme trafficking and intercellular transfer for the majority of lysosomal enzymes, which is essential for metabolic cross- correction in treating LSDs. Developmental decline of M6PR on blood-brain-barrier (BBB) during early postnatal period in mouse and human is attributable to the lack of CNS enzyme delivery into adult brain. Using a dual luciferase reporter system with site-mutagenesis, we have recently identified microRNA-143 (miR143) as an epigenetic modulator to reduce M6PR protein levels on brain microvessels (BrMV). 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 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 sequestration of miR-143 with miR-143-sponge sequences. The data provide strong scientific premise for the development of a novel approach that would selectively “open” BBB to systemic enzymes provided by any current treatment options or future enzyme/gene/cell therapies for synergistic CNS benefits in many nLSDs. In this proposal, we aim to develop an 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 robust and targeted reduction of miR143 on brain endothelia cells (Aim 1), in vivo examination of “on-target” and “off-target” expression and effects in mice with AAV/143in delivery (Aim 2), as well as preclinical evaluation of BrMV-targeted AAV/143in in correcting CNS abnormalities in MPS I mice by enzyme therapy derived from genetically modified erythroid/megakaryocytic lineages (Aim 3). The studies will provide a proof- of concept for a new in vivo miRNA-inhibitor mediated, brain-targeted approach that could be applicable for many other nLSDs involving M6PR pathway AND neurological diseases benefiting from advanced CNS delivery of therapeutics via adapting M6PR-mediated transport pathway by modification with M6P residues or IGF2-tag.