In vivo efficacy of a kinase inhibitor, roscovitine, in HD mouse model

项目摘要（Abstract）： 亨廷顿病（HD）是由亨廷顿基因（HTT）突变引起的。扩增CAG三核苷酸 外显子1中的重复编码突变亨廷顿蛋白（mHTT）中的多聚谷氨酰胺，导致优先 纹状体、皮质和其他区域的神经变性。目前尚无改善疾病的治疗方法。突变体 HTT降低方法是非常有前途的;然而，递送剂大多是大分子，具有有限的 脑部穿透需要侵入性手术小分子RNA导向疗法也正在出现，但 它们通常是非等位基因特异性的，并且可能对其它mRNA具有脱靶效应。 亨廷顿蛋白（HTT）具有许多翻译后修饰位点（PTM）。作为长期以来 为了研究HTT的PTM，我们已经确定了少量PTM位点可以改变mHTT的毒性， 特别是那些具有丝氨酸和通过磷酸化靶向的位点。使用体外激酶筛选，我们发现 这些丝氨酸之一，S1181，被CDK（包括CDK 1、CDK 2和CDK 5等）主动磷酸化。 CDK抑制剂roscovitine（也称为seliciclib，CYC 202）对mHTT毒性具有高度神经保护作用 在我们的HD细胞模型中。作为这一策略的原理证明，我们现在建议确定roscovitine是否 可以在HD小鼠模型体内治疗有效。 Roscovitine是一种众所周知的CDKs抑制剂，已进入多项人体II期临床试验。一个先前 在HD小鼠模型中研究显示roscovitine可以改善HD小鼠的抑郁样行为。 然而，这项研究使用脑内输注将roscovitine短时间内输送到大脑中。 其他研究发现，单次口服roscovitine可以渗透大脑。我们假设 Roscovitine的慢性外周给药可以达到足够的脑浓度， 抑制过度活化的CDK 5，其参与HD发病机制。因此，它将在HD中产生有益的效果 小鼠模型 我们已经证实单次注射roscovitine可以穿透大脑。每天注射三周就好了 在zQ 175 HD小鼠中耐受，zQ 175 HD小鼠是在小鼠体内表达全长人外显子1 HTT敲入小鼠 亨廷顿蛋白基因具有大约175个CAG重复。我们将注射roscovitine在表现zQ 175 HD小鼠 长期（6个月）。体重、行为任务和MRI扫描将用于比较治疗的HD小鼠 不管有没有Roscovitine将采集脑组织样本并进行脑病理、分子生物学改变 将在HD小鼠中进行检查。我们期待roscovitine保护HD小鼠，并可进一步转化为HD 诊所

Project Summary (Abstract): Huntington’s disease (HD) is caused by a mutation in the huntingtin gene (HTT). Expanded CAG trinucleotide repeats in exon 1 encode polyglutamine in the mutant huntingtin protein (mHTT), causing preferential neurodegeneration in striatum, cortex and other regions. No disease-modifying treatment is available. Mutant HTT lowing approaches are very promising; However, the delivery agents are mostly large molecules with limited brain penetration, requiring invasive procedures. Small molecule RNA-directed therapies are also emerging, but they are generally non-allele specific and may have off target effects on other mRNAs. Huntingtin protein (HTT) has many sites of post-translational modification (PTM). As part of a long-standing program to study PTMs of HTT, we have identified a small number of PTM sites can modify mHTT toxicity, especially those sites with serine and targeting by phosphorylation. Using in vitro kinase screens, we have found that one of those serines, S1181, is actively phosphorylated by CDKs, including CDK1, CDK2 and CDK5, etc. A CDK inhibitor, roscovitine (also known as seliciclib, CYC202), is highly neuroprotective against mHTT toxicity in our HD cell model. As a proof of principle for this strategy, we now propose to determine whether roscovitine can be therapeutically efficacious in an HD mouse model in vivo. Roscovitine is a well-known CDKs inhibitor and has been in several human phase II clinical trials. One previous study in an HD mouse model showed that roscovitine can ameliorate depressive-like behavior in HD mice. However, this study used intracerebral infusion to deliver roscovitine into the brain for a short period of time. Other studies have found that a single oral administration of roscovitine can penetrate brain. We hypothesize that chronic peripheral administration of roscovitine can achieve adequate brain concentrations to inhibit overactivated CDK5, which involves in HD pathogenesis. Thus, it will have beneficial effect in HD mouse model. We have confirmed that single injection of roscovitine can penetrate brain. Three weeks of daily injection is well tolerable in zQ175 HD mice, which is a knock-in mouse express full length human exon1 HTT under mouse huntingtin gene with approximate of 175 CAG repeats. We will inject roscovitine in manifested zQ175 HD mice for long term (6 months). Body weight, behavioral task and MRI scan will be used to compare HD mice treated with or without roscovitine. Brain samples will be collected and brain pathology, molecular biological changes will be examined in HD mice. We expect that roscovitine protect HD mice and can be further translate into HD clinic.