二硫代磷酸酯改性小干扰RNA（PS2-siRNA）的设计合成及其β－分泌酶基因沉默活性

Alzheimer's disease (AD) is an irreversible, progressive brain disease that slowly destroys memory and thinking skills and, eventually, the ability to carry out the simplest tasks of daily living. AD continues to be the leading cause of dementia among the aging population in the world, but no definitive treatments are currently available, and there are no drugs available that specifically and on a molecular level cure the cause of Alzheimer's disease. AD is characterized by the progressive formation of insoluble amyloid plaques and vascular deposits consisting of the amyloid β-peptide (Aβ) in the brain. Beta-Secretase (BACE1) is the major participating gene in generation of toxic Aβ. Therefore, agents that can effectively suppress the BACE1 gene are likely to be an efficient way of ameliorating AD. Since the discovery of short interfering RNA (siRNA) molecules in the late 1990s, siRNA technology has developed rapidly as a powerful tool for functional genomic analysis, target validation and therapeutic purposes. Because of its potential for highly specific targeted effects in vivo, there is significant interest in developing therapeutic agents based on siRNA. Although unmodified siRNA is able to satisfy most of the research needs in labs, therapeutic application of unmodified siRNA in vivo has proven to be much less effective mainly due to the rapid degradation of the molecules in blood and the elimination of siRNA molecules by excretion. Given these facts, finding methods for increasing the duration of siRNA activity in vivo will be important for exploiting siRNA for therapeutic applications. A wide variety of chemical modifications have been proposed to address these issues. Therefore, Developing chemically modified siRNA duplexes with improved nuclease resistance and pharmacokinetics properties is required for therapeutics applications. The objective of this application is firstly to develop novel stable ribonucleoside H-phosphonate monomers and a novel H-phosphonate solid-phase method to synthesize the PS2-siRNAs; then to design and synthesize a series of novel highly stable phosphorodithioated siRNA (PS2-siRNA) with enhanced silencing potency targeting the BACE1 gene.

阿尔茨海默病(AD)是一种不可逆的进行性大脑疾病，它缓慢破坏大脑的记忆和思考功能并最终使患者丧失最基本的日常生活能力。AD是引起老年痴呆的首要病因，但没有很有效的治疗方法。AD的特征是不断地在大脑中形成淀粉样斑块和血管内β淀粉样蛋白(Aβ)沉积物。β-分泌酶(BACE1)是参与产生Aβ的主要酶，因此抑制BACE1活性是改善AD的有效方法。自siRNA被发现以来，由于其体内的高靶向性，基于siRNA的治疗方法受到广泛重视。但由于其在血液中迅速分解使其在体内表现较低的活性，阻碍了其进一步应用。化学改性是解决此问题的有效方法。本课题拟研究H-硫代膦酸酯法固相合成高稳定的能增强BACE1沉默活性的二硫代磷酸酯改性siRNAs(PS2-siRNAs)，评价BACE1体外沉默活性，优化改性参数，筛选候选样品，为进一步评价在神经元细胞内基因沉默活性，并最终发现可用于基因治疗AD的RNAi药物奠定基础。

自小核酸（siRNA）被发现以来，由于其体内的高靶向性，基于siRNA的治疗方法受到广泛重视。但由于其在血液中迅速分解使其在体内表现较低的活性，阻碍了其进一步应用。RNA糖环的2’-OH可以和3’-磷酸酯基团形成2’,3’-环磷酸酯，使其具有较低的降解自由能，导致RNA的稳定性极差，封闭2’-羟基可改善其稳定性；同时已经证明PS2改性可以提高siRNA的稳定性和基因沉默活性，因此我们设计合成了兼具二者优点的基于淀粉样前体蛋白β位点裂解酶1(β-site amyloid precursor protein-cleaving enzyme 1, BACE1)基因的2’-MOE封闭和PS2改性的双改性siRNAs，通过分别对siRNA的正义链和反义链的单链不同位点进行双改性修饰，组装siRNA双链，基因沉默活性评价，发现了在正反义链上的化学修饰敏感区域，再将有效的修饰正反义单链组装成新的siRNA进行活性研究，筛选最佳组合。同时我们分别测定了各种修饰siRNA的理化性能譬如解链温度、圆二色光谱和血清稳定性，探索理化性能和基因沉默活性的内在联系。通过双荧光素酶报告基因检测方法分别在HELA细胞和293T细胞模型上评价所制备的2’-MOE和3’-PS2双改性siRNA体外BACE1基因沉默活性,两种细胞模型所得到的活性位点结果一致，结果表明，正义链的3’端和反义链的中间切割位点为化学改性敏感区。基于LSD1基因合成了一些PS2单改性和MOE-PS2双改性的siRNA,采用Western-Blot方法评价了对LSD1蛋白的基因沉默活性，也同样发现在正义链的3’端引入两个改性单元能明显提高siRNA的基因沉默活性，改性位点是沉默活性的决定因素，改性单元数量和活性无正相关性。