RNAi strategy for prolonged inhibition of BACE1: Role for noncoding RNA

描述(由申请人提供)：这是一份针对阿尔茨海默病药物发现的R21拨款申请的重新提交(PAS-06-261)。大脑中淀粉样多肽的积累被认为在阿尔茨海默病(AD)的病理中起着关键作用。A肽是分泌酶-1(BACE1)降解淀粉样前体蛋白(APP)的副产物。尽管进行了大量的药物发现工作，但很少有针对BACE1的小分子抑制剂被开发出来，这可能是因为BACE1的活性部位比许多其他蛋白酶更开放，疏水性更小。因此，抑制BACE1的替代方法已经被证明是合理的，例如基于小干扰RNA(SiRNA)的策略。这项建议的第一个目的是开发一种基于非病毒/非载体、无毒的修饰siRNA，适用于体内BACE1活性的长期敲除。为了实现这一目标，我们将通过掺入锁定核酸(LNA)核苷酸来修饰针对BACE1 mRNA的传统siRNAs。在siRNAs中掺入LNA可提高稳定性、对核酸酶的抵抗力，并增加siRNA的效力和链的特异性。因此，可以预测，针对BACE1的LNA修饰的siRNA将比传统的siRNA更有效地降低APP过度表达小鼠脑内BACE1和A？的表达。最近的证据表明，除了正义mRNA转录本外，人类和小鼠还合成了许多天然的非编码反义转录本。我们的实验室最近证明，反义转录的干扰可以深刻地改变其正义mRNA的表达。有趣的是，我们实验室的生物信息学和转录学分析现在已经确定了一个高度保守的BACE1非编码反义转录本，这增加了BACE1表达水平不仅可能受到针对BACE1 mRNA的siRNA的调控，还可能受到非编码BACE1反义转录本的下调。这一假设得到了令人信服的细胞培养数据和初步体内数据的支持。这项提议的第二个目的将检验这样的假设，即敲除BACE1反义转录本将导致APP过度表达小鼠大脑中BACE1本身的一致性下降，可能揭示一种调控BACE1表达的全新机制。总体而言，我们寻求定义一种优化的非病毒体内RNAi方法，重点放在BACE1，阿尔茨海默病的主要靶点。

DESCRIPTION (provided by applicant): This is a resubmission of an application for an R21 Grant for Alzheimer's Disease Drug Discovery (PAS-06-261). Accumulation of amyloid-¿ (A¿) peptides in the brain is thought to play a key role in the pathology of Alzheimer's disease (AD). A¿ peptides are generated as byproducts of the amyloid precursor protein (APP) degradation by ¿-secretase-1 (BACE1). Despite intense drug discovery efforts, few small molecule inhibitors have been developed for BACE1, likely because the active site of BACE1 is more 'open' and less hydrophobic than in many other proteases. Thus, alternative approaches to inhibiting BACE1 have been justified, such as small interfering RNA (siRNA) based strategies. The first aim of this proposal is to develop a non-viral/non- vector based, non-toxic modified siRNA suitable for prolonged knockdown of BACE1 activity in vivo. To accomplish this aim, we will modify conventional siRNAs directed against BACE1 mRNA by incorporation of locked nucleic acid (LNA) nucleotides. LNA incorporation into siRNAs increases stability, resistance to nucleases, and increases siRNA potency and strand specificity. Thus, it is predicted that LNA-modified siRNA directed against BACE1 will decrease BACE1 and A¿ expression in the brains of APP overexpressing mice more potently that conventional siRNA. Recent evidence suggests that, in addition to sense mRNA transcripts, humans and mice also synthesize many natural noncoding antisense transcripts to their sense counterparts. Our laboratory has recently demonstrated that perturbation of antisense transcripts can profoundly alter the expression of their sense mRNA counterparts. Intriguingly, bioinformatic and transcriptomic analyses in our laboratory have now identified a highly conserved BACE1 noncoding antisense transcript, raising the possibility that BACE1 expression levels may be modulated not just by siRNA directed against BACE1 mRNA, but also by knockdown of noncoding BACE1 antisense transcript. This hypothesis is supported by compelling cell culture data and preliminary in vivo data in this proposal. The second aim of this proposal will test the hypothesis that knockdown of BACE1 antisense transcripts will induce a concordant decrease in BACE1 itself in the brains of APP overexpressing mice, potentially revealing a fundamentally new mechanism by which BACE1 expression may be modulated. Overall, we seek to define an optimized non-viral in vivo RNAi approach focusing on BACE1, a prime Alzheimer's disease target.