Targeted Therapies for Rett Syndrome

项目摘要 大脑中的免疫失调和谷氨酸神经传递的破坏，都是由胶质细胞介导的， 与Rett综合征（RTT）的发病机制和症状恶化有关， 与癫痫发作、智力残疾、运动和自主神经相关的发育障碍 功能障碍和无目的的手部运动。小胶质细胞和星形胶质细胞似乎介导免疫 反应、氧化损伤和谷氨酸毒性。针对这些关键机制的治疗， 调节神经胶质反应可以通过阻止损伤和促进修复来影响RTT， 再生在Mecp 2 null和Het小鼠中使用树枝状聚合物的阳性初步结果的基础上 共轭抗氧化剂N-乙酰半胱氨酸（D-NAC），我们提出了一种创新的纳米方法， 减轻/阻止RTT中的损伤。我们的总体假设是，靶向递送抗- 炎性/抗氧化剂，沿着对小胶质细胞和星形胶质细胞有效的β-氨基转移酶抑制剂， Mecp 2缺失和Mecp 2杂合（HET）小鼠将导致氧化损伤和谷氨酸毒性降低 导致Mecp 2-null和HET小鼠中改善的长期神经行为结果， 在Mecp 2缺失小鼠中的存活率。我们在RTT中的初步结果表明：（1）全身给药 树枝状聚合物纳米颗粒特异性地定位于RTT小鼠脑中的小胶质细胞中，但不在野生型小鼠脑中。 型小鼠;（2）D-NAC单一疗法每周一次全身性施用给有症状的Mecp 2缺失小鼠， 结果在6-7周龄时神经行为评分显著改善，而游离药物则没有 （3）D-NAC能有效改善大鼠行为表型和海马谷胱甘肽水平， HET小鼠;和（4）谷氨酸盐合成酶-谷氨酸脱氢酶在MeCP 2中上调 缺乏的小胶质细胞，并通过全身施用的树枝状聚合物-转氨酶抑制剂特异性抑制 共轭在R21的支持下，我们在Mecp 2-null和HET小鼠中完成了D-NAC单药治疗， 鉴定谷氨酰胺拮抗剂DON（6-重氮-5-氧代-L-正亮氨酸）为有效的谷氨酰胺酶。唐 由于严重的毒性而导致临床试验失败。我们建议确定是否1）D-DON全身治疗 导致小胶质细胞转氨酶的特异性抑制，从而改善神经学结局， 谷氨酸毒性，同时消除游离DON的严重外周毒性，2）与D- NAC+D-DON在改善Mecp 2-null和Mecp 2-null患者的生存率和长期神经功能结局方面更有效， HET小鼠，和3）用D-NAC+D-DON的全身治疗导致免疫应答的改善， 从RTT小鼠脑中分离的Mecp 2-null小胶质细胞的氧化损伤和功能。体内影响 将评价存活率（Mecp 2缺失小鼠）、行为、呼吸、睡眠（HET）。如果成功，这些初始 概念验证研究将为未来的临床翻译工作奠定基础。

PROJECT SUMMARY Immune dysregulation in the brain and disruption of glutamate neurotransmission, both mediated by glia, have been implicated in the pathogenesis and worsening of symptoms in Rett syndrome (RTT), a debilitating, developmental disorder that is associated with seizures, intellectual disability, motor and autonomic dysfunction, and non-purposeful hand movements. Microglia and astrocytes appear to mediate the immune response, oxidative injury and glutamate toxicity in RTT. Therapies targeting these key mechanisms by modulating the glial responses could have an impact in RTT by arresting the injury and promoting repair and regeneration. Building on positive preliminary results in the Mecp2 null and Het mice with dendrimer conjugated antioxidant N-acetyl cysteine (D-NAC), we propose an innovative nanotherapeutic approach to attenuate/arrest the injury in RTT. Our overall hypothesis is that targeted delivery of a combination of an anti- inflammatory/anti-oxidant agent, along with a potent glutaminase inhibitor to microglia and astrocytes in Mecp2-null and Mecp2-heterozygous (HET) mice will lead to decreased oxidative injury and glutamate toxicity resulting in improved long term neurobehavioral outcomes in Mecp2-null and HET mice and symptom free survival in Mecp2-null mice. Our preliminary results in RTT demonstrates that (1) systemically administered dendrimer nanoparticles localize specifically in microglia in the RTT mouse brain but not in the brain of wild type mice; (2) D-NAC monotherapy administered systemically once a week to symptomatic Mecp2-null mice, results in significant improvement in neurobehavioral scores at 6-7 weeks of age, while the free drug is not effective; (3) D-NAC is effective in improving the behavioral phenotype and hippocampal glutathione levels in HET mice; and (4) glutaminase, the enzyme responsible for glutamate synthesis, is upregulated in MeCP2 deficient microglia and is specifically inhibited by systemically administered dendrimer-glutaminase inhibitor conjugate. Supported by an R21, we completed D-NAC monotherapy in Mecp2-null and HET mice, and identified the glutamine antagonist DON (6-Diazo-5-Oxo-L-Norleucine) as a potent glutaminase. DON has failed clinical trials due to severe toxicity profile. We propose to determine if 1) Systemic treatment with D-DON results in specific inhibition of microglial glutaminase resulting in improved neurologic outcomes and decreased glutamate toxicity while eliminating the severe peripheral toxicities of free DON, 2) combination therapy with D- NAC+D-DON is more effective in improving survival and long-term neurologic outcomes in Mecp2-null and HET mice, and 3) systemic treatment with D-NAC+D-DON results in improvement in immune response, oxidative injury and function of Mecp2-null microglia isolated from RTT mouse brains. In vivo effects on survival (Mecp2-null mice), behavior, respiration, sleep (HET) will be evaluated. If successful, these initial proof-of-concept studies will lay the groundwork for future work crucial for clinical translation.