Exogenous Gene Control via Modulation of RNA Self-Cleavage

描述(申请人提供)：在哺乳动物细胞中‘外源’控制基因表达的能力一直是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学过程中以及在发育和疾病状态中的作用方面发挥了重要作用。这项技术还提供了在其他一些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，由于现有系统的限制，需要开发实现基因调控的新方法，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，它涉及调节RNA的自我裂解，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基序的核酶(Rz)序列整合到哺乳动物转录单位中，可以由于有效地自切割mRNA而导致基因表达从结果转录本中消除。反过来，我们已经证明，通过使用小分子抑制剂抑制RNA的自我裂解，可以有效地诱导来自这种构型的基因表达。我们认为，与现有的基因表达控制系统相比，这种基因调控系统可能会提供明显的优势，并可能具有广泛的实验和治疗应用。这项研究计划的总体目标是进一步了解在哺乳动物细胞中控制有效RNA自切割的实验参数，并将这些知识与RZ自切割操作的新方法和标准基因转移技术的开发结合起来，开发出广泛适用和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方法，用于开发基于RZ的定制设计的基因调控系统，该系统针对任何特定的小分子或其他分子实体而量身定做。

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.