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

长期抑制 BACE1 的 RNAi 策略：非编码 RNA 的作用

• 批准号: 7470593
• 负责人: Claes Robert Wahlestedt
• 金额: $ 18.02万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7470593
• 关键词: Active Sites; Alzheimer' s Disease; Alzheimer' s disease model; Amyloid; Amyloid beta-Protein Precursor; Behavioral; Bioinformatics; Brain; Brain region; Cultured Cells; Data; Development; Endopeptidases; Grant; Human; Laboratories; Messenger RNA; Modeling; Modification; Molecular; Mus; Mutate; Non-Viral Vector; Nucleotides; Outcome; Pathology; Peptide Hydrolases; Peptides; Physiological; Play; Protein Overexpression; Proteins; RNA Interference; Research Proposals; Resistance; Role; Site; Small Interfering RNA; Specificity; System; Testing; Thinking; Transcript; Transgenic Mice; Untranslated RNA; Validation; Viral; Viral Vector; base; beta-site APP cleaving enzyme 1; cognitive function; concept; day; drug discovery; heuristics; in vivo; inhibitor/antagonist; innovation; insight; locked nucleic acid; mouse model; novel; novel therapeutics; nuclease; protein degradation; research study; secretase; small molecule; tool; transcriptomics

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.

描述（由申请人提供）：这是一份重新提交的阿尔茨海默病药物发现R21资助申请（PAS-06-261）。淀粉样蛋白（A）肽在脑中的积累被认为在阿尔茨海默病（AD）的病理学中起关键作用。A肽是淀粉样前体蛋白（APP）被β-分泌酶-1（BACE 1）降解的副产物。尽管进行了大量的药物发现工作，但很少有针对BACE 1的小分子抑制剂被开发出来，这可能是因为BACE 1的活性位点比许多其他蛋白酶更“开放”且疏水性更低。因此，抑制BACE 1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。该提议的第一个目的是开发一种基于非病毒/非载体的、无毒的经修饰的siRNA，其适用于体内BACE 1活性的延长敲低。为了实现这一目标，我们将通过掺入锁核酸（LNA）核苷酸来修饰针对BACE 1 mRNA的常规siRNA。LNA掺入siRNA增加稳定性、对核酸酶的抗性，并增加siRNA效力和链特异性。因此，可以预测，LNA修饰的针对BACE 1的siRNA将比常规siRNA更有效地降低APP过表达小鼠脑中BACE 1和A？的表达。最近的证据表明，除了正义mRNA转录本，人类和小鼠也合成许多天然的非编码反义转录本。我们的实验室最近证明，干扰反义转录本可以深刻地改变其正义mRNA对应物的表达。有趣的是，我们实验室的生物信息学和转录组学分析现在已经确定了一个高度保守的BACE 1非编码反义转录本，提高了BACE 1表达水平可能不仅受到针对BACE 1 mRNA的siRNA的调节，而且还受到非编码BACE 1反义转录本的敲低的可能性。这一假设得到了令人信服的细胞培养数据和本提案中的初步体内数据的支持。该提案的第二个目的将测试以下假设：BACE 1反义转录物的敲低将诱导APP过表达小鼠脑中BACE 1本身的一致减少，可能揭示BACE 1表达可能被调节的全新机制。总的来说，我们试图定义一种优化的非病毒体内RNAi方法，专注于BACE 1，一种主要的阿尔茨海默病靶标。

项目成果

Natural antisense transcripts as therapeutic targets.

• DOI: 10.1016/j.ddstr.2013.03.001
• 发表时间: 2013
• 期刊: Drug discovery today. Therapeutic strategies

Regulation of chromatin structure by long noncoding RNAs: focus on natural antisense transcripts.

• DOI: 10.1016/j.tig.2012.03.013
• 发表时间: 2012-08
• 期刊: TRENDS IN GENETICS
• 影响因子: 11.4
• 作者: Magistri, Marco;Faghihi, Mohammad Ali;St Laurent, Georges, III;Wahlestedt, Claes
• 通讯作者: Wahlestedt, Claes