Advancing CNS drug delivery via epigenetic modulation

摘要 溶酶体储存障碍(LSD)是一组遗传性疾病，其特征是 溶酶体，累积频率为每7000名活产儿中有1名。超过2/3的LSD患者存在受累 具有广谱严重程度(NLSD)的中枢神经系统(CNS)，这使得LSD成为最 儿科神经性疾病的常见原因。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，他们很大程度上 由于酶对中枢神经系统的渗透不良而导致的神经系统并发症逆转不成功， 治疗nLSD的一个主要障碍。拟议研究的影响是由未得到满足的医疗需求推动的 遗传性nLSD的有效治疗和酶进入中枢神经系统的主要限制。 阳离子非依赖性甘露糖-6-磷酸受体(M6PR)在溶酶体酶中起关键作用 大多数溶酶体酶的运输和细胞间转移，这是代谢交叉必不可少的。 更正了LSD的治疗。M6PR在血脑屏障(BBB)上的发育衰退 小鼠和人的经期可归因于中枢神经系统酶缺乏对成人大脑的输送。使用DUAL 荧光素酶报告系统与点突变，我们最近发现microRNA-143(MiR143)是一种 表观遗传调节剂，降低脑微血管(BrMV)上M6PR蛋白水平。使用鼠标模型 α-L缺乏所致的Hirler综合征(重症粘多糖病I型，MPS I) IDUA，我们展示了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miR-143与miR-143-海绵序列隔离内皮细胞。这些数据提供了强有力的 开发一种新方法的科学前提，该方法将选择性地向全身性血脑屏障开放 任何目前的治疗选择或未来的酶/基因/细胞疗法为协同中枢神经系统提供的酶 在许多nLSD中受益。在这项建议中，我们的目标是开发一种基于腺相关病毒载体(AAV)的 可翻译平台在成熟的血脑屏障上“恢复”M6PR通路，用于高级递送治疗酶 以3个目标进入中枢神经系统，包括开发优化的人工miR143抑制剂(143 In)和表达 强健和靶向降低miR143对脑血管内皮细胞作用的盒(目标1)，体内检测 携带AAV/143的小鼠体内“靶上”和“非靶向”表达及其影响(AIM 2) BrMV靶向AAV/143In对MPS-I小鼠中枢神经系统异常的酶治疗作用 来自转基因红系/巨核系(目标3)。这些研究将提供证据-- 一种新的体内miRNA抑制物介导的脑靶向方法的概念，该方法可能适用于 许多其他涉及M6PR通路的nLSD和受益于高级中枢神经系统递送的神经系统疾病 通过用M6P残基或IGF2-Tag修饰M6PR介导的转运途径来改变治疗方法。

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.