Conditional Gene Silencing in Ischemia/Stroke Research

描述（由申请人提供）：缺血/中风是世界范围内的主要健康问题。由于脑缺血是一种严重的代谢应激形式，干扰了所有主要的生化和分子生物学途径，因此很难确定直接参与最终导致神经元细胞死亡的病理过程的干扰。遗传方法提供了有希望的新工具来验证被认为在这种病理过程中发挥作用的蛋白质是否确实是关键的贡献者。小干扰rna （sirna）的基因沉默可以用来确定消除相应的基因产物是否会加剧或阻断导致神经元细胞死亡的病理过程。为了避免可能与永久表达的sirna相关的副作用，已经建立了条件基因沉默方法，其中沉默RNA序列的转录被外部药物（如四环素）修饰。由于这些系统依赖于可能产生各种副作用的药物，这些副作用可能会显著改变细胞应激反应，从而使结果的解释复杂化，因此迫切需要不需要外部化学物质激活的新基因沉默系统。拟议的项目重点关注两个新的条件基因沉默系统，使用内源性转录因子激活的启动子或神经元细胞特异性聚合酶II启动子作为诱导剂。我们的具体目标是基于微RNA （miRNA）方法开发新的条件基因沉默系统，该系统将特异性靶向神经元或通过在正在研究的病理过程中激活的启动子控制所设计的miRNA序列的表达。为了保证神经元特异性沉默效应，我们将设计miRNA序列的表达由神经元特异性启动子控制的结构。为了保证应激诱导性，我们将使用热休克启动子或缺氧诱导启动子在缺血或其他已知与热休克因子和/或缺氧诱导因子激活相关的严重应激形式后诱导设计的miRNA序列的表达。使用这些结构，我们将能够以有条件的方式沉默凋亡蛋白编码基因的表达。细胞凋亡是一个活跃的过程，需要表达编码细胞凋亡诱导蛋白的基因。这种基于条件miRNA的新方法可以有效地抑制这些基因在缺血后的表达。我们将用我们最有效的构建体制造慢病毒，感染原代神经细胞，并将培养物暴露于短暂的氧气/葡萄糖剥夺或缺氧中，以测试所开发系统的功效。新的条件基因沉默系统将是阐明缺血性细胞死亡机制的宝贵工具，也可用于包括癌症研究在内的基础和应用研究的各个领域。公共卫生相关性：迫切需要更好地了解由缺血/中风引发并最终导致神经元细胞死亡的病理过程。条件基因表达或基因沉默是研究缺血性细胞死亡机制的最新工具。这一建议的重点是开发两种新的系统条件基因沉默使用应激诱导或神经元细胞特异性的Pol II启动子。新的条件基因沉默系统将是阐明缺血性细胞死亡机制的宝贵工具，也可以在包括癌症研究在内的各个领域的基础和应用研究中得到利用。

DESCRIPTION (provided by applicant): Ischemia/stroke presents a major health problem worldwide. Since cerebral ischemia is a severe form of metabolic stress that interferes with all major biochemical and molecular biological pathways, it is difficult to identify the disturbances that are directly involved in the pathological process culminating in neuronal cell death. Genetic approaches provide promising new tools to verify whether proteins believed to play a role in this pathological process are indeed key contributors. Gene silencing by small interfering RNAs (siRNAs) can be used to establish whether eliminating the respective gene product aggravates or blocks the pathological process resulting in neuronal cell death. To avoid possible side effects that could be associated with permanently expressed siRNAs, conditional gene silencing approaches have been established where transcription of the silencing RNA sequence is modified by external drugs such as tetracycline. Since these systems rely on drugs potentially producing various side effects that could significantly modify the cellular stress response and thus complicate the interpretation of result, new systems of gene silencing that do not require activation by external chemicals are urgently required. The proposed project focuses on two new systems of conditional gene silencing using promoters activated by endogenous transcription factors or neuronal cell-specific polymerase II promoters as inducers. Our specific aims are to develop new systems of conditional gene silencing, based on the micro RNA (miRNA) approach that will target neurons specifically or control expression of the designed miRNA sequence by promoters that are activated in the course of the pathological process under investigation. To guarantee a neuron-specific silencing effect, we will de- sign constructs where expression of the miRNA sequence is controlled by a neuron-specific promoter. To guarantee stress inducibility, we will use a heat shock promoter or hypoxia-inducible promoter to induce expression of the designed miRNA sequence after ischemia or other severe forms of stress known to be associated with activation of heat shock factors and/or the hypoxia-inducible factor. Using these constructs, we will be able to silence expression of genes coding for apoptotic proteins in a conditional manner. Apoptosis is an active process requiring expression of genes coding for apoptosis-inducing proteins. Post-ischemic expression of these genes could be suppressed efficiently using the new conditional miRNA- based approach. We will produce lentivirus with our most potent constructs, infect primary neuronal cells and expose cultures to transient oxygen/glucose deprivation or hypoxia to test the efficacy of the systems developed. The new conditional gene silencing systems will be invaluable tools for the elucidation of mechanisms of ischemic cell death and could also be exploited in various areas of basic and applied research, including cancer research. PUBLIC HEALTH RELEVANCE: There is urgent need to better understand the pathological process triggered by ischemia/stroke and culminating in neuronal cell death. Conditional gene expression or gene silencing are state-of-the-art tools for investigating mechanisms of ischemic cell death. This proposal focuses on developing two new systems of conditional gene silencing using stress-inducible or neuronal cell specific Pol II promoters. The new conditional gene silencing systems will be invaluable tools for the elucidation of mechanisms of ischemic cell death and could also be exploited in various areas of basic and applied research, including cancer re- search.