Exogenous Gene Control via Modulation of RNA Self-Cleavage

描述（由申请人提供）：“外源性”控制哺乳动物细胞中基因表达的能力已经成为生物医学研究的有力工具。特别是，基因调控技术在了解特定基因产物在基本生物过程以及发育和疾病状态中的作用方面发挥了重要作用。该技术还提供了在许多其他研究领域产生重大影响的机会，甚至可能形成新的治疗范例的重要基础。然而，由于现有系统的局限性，需要开发新的方法来实现基因调控，以实现该技术的全部潜力。这项拨款提案的重点是开发一种新的基因调控技术平台，该平台涉及RNA自切割的调节，而不是转录，作为控制基因表达的一种手段。提出的研究计划的基础是我们实验室最近的研究，这些研究表明，将编码自切割RNA基序的序列，核酶（rz）结合到哺乳动物转录单元中，由于mRNA的有效自切割，可以导致从所产生的转录物中消除基因表达。反过来，我们已经证明，通过使用小分子抑制剂抑制RNA自裂，可以有效地“诱导”这种结构的基因表达。我们相信这样的基因调控系统可能比现有的控制基因表达的系统有明显的优势，并且可以有广泛的实验和治疗应用。这里描述的研究计划的总体目标是进一步了解控制哺乳动物细胞中有效RNA自裂的实验参数，并将这些知识应用于rz自裂操作和标准基因转移技术的新方法的开发，以开发广泛适用且“用户友好”的基因调控技术平台。该研究计划的一个重要目标将是建立一种通用的方法，用于开发“定制设计”的基于rz的基因调控系统，以响应任何特定的小分子或其他分子实体。

DESCRIPTION (provided by applicant): The ability to 'exogenously' control the expression of genes in mammalian cells has been a powerful tool of biomedical research. In particular, gene regulation technology has played a major role in efforts to understand the role of specific gene products in fundamental biological processes and in development and disease states. The technology also offers the opportunity to have a major impact in a number of other areas of research and could even form an important basis for new therapeutic paradigms. However, due to limitations of existing systems, there is a need for the development of new methods for achieving gene regulation in order to realize the full potential of the technology. The focus of this grant proposal is the development of a new technology platform for gene regulation which involves the modulation of RNA self-cleavage, rather than transcription, as a means of controlling gene expression. The basis for the proposed research program are recent studies in our laboratory which demonstrate that incorporation of sequences encoding self-cleaving RNA motifs, ribozymes (rz), into a mammalian transcription unit can lead to the elimination of gene expression from the resulting transcript, due to efficient self-cleavage of the mRNA. In turn, we have shown that gene expression from such a configuration can be effectively 'induced' via inhibition of RNA self-cleavage using small molecule inhibitors. We believe that such a system for gene regulation may offer distinct advantages over existing systems for controlling gene expression and could have broad experimental and therapeutic application. The overall objective of the research program described here is to further understand the experimental parameters which govern efficient RNA self-cleavage in mammalian cells, and to apply that knowledge, in conjunction with development of new methodologies for the manipulation of rz self-cleavage and standard gene transfer technology, to the development of a widely applicable and 'user-friendly' technology platform for gene regulation. An important goal of the research program will be to establish a general methodology for the development of 'custom-designed' rz-based gene regulation systems tailored to respond to any specific small molecule or other molecular entity.