Investigate stability of RNA interference in human lymphocytes

描述（由申请人提供）：我们的总体假设是，可以开发RNA干扰（RNAi）来长期降低特定基因表达，而不会对人淋巴细胞产生毒性作用。利用siRNA抑制特异性基因表达的RNAi技术是一种强有力的基础研究和治疗干预技术。然而，我们的结果和最近的几项研究表明，在哺乳动物细胞中与该技术相关的非特异性细胞毒性作用引起了对RNAi在人类治疗应用中长期使用的担忧。细胞毒性作用至少部分归因于细胞凋亡，尽管其他机制也是可能的。细胞毒性与较高水平的shRNA表达相关。基于这些结果，我们假设shRNA的水平对于确定给定的shRNA是否具有细胞毒性是重要的，并且这些水平的优化可以产生既有效又无细胞毒性或具有最小细胞毒性的shRNA。我们还将讨论shRNA的细胞定位和/或其加工途径是否会影响细胞内细胞毒性的程度。通过我们提出的研究，我们将了解shRNA表达在体外成熟淋巴细胞中的作用，以及在体内环境中对淋巴细胞群体分化的作用。我们将最大限度地减少细胞毒性效应，并最大限度地提高RNAi在原代T淋巴细胞中的稳定和有效功能。这项研究与公共卫生的相关性。RNA干扰是一种有效的、有前途的抑制特异性基因表达的技术，可用于基础研究和治疗干预。特定基因表达的抑制需要是有效的，没有意外的副作用。该提案建议研究RNAi在人类T淋巴细胞中的作用，以最大限度地提高RNAi效果并最大限度地减少副作用。

DESCRIPTION (provided by applicant): Our overall hypothesis is that RNA interference (RNAi) can be developed to reduce specific gene expression over long-term without toxic effects in human lymphocytes. RNAi using siRNA to inhibit specific gene expression is a powerful and promising technology for both basic research and therapeutic intervention. However, our results and several recent studies have shown unintended nonspecific cytotoxic effects associated with this technology in mammalian cells raising a concern for the long term use of the RNAi in therapeutic applications in humans. The cytotoxic effects were attributed at least in part to apoptosis, although other mechanisms are also possible. The cytotoxicity was associated with higher levels of shRNA expression. Based upon these results, we hypothesized that that the levels of shRNA are important for determining whether a given shRNA will be cytotoxic or not and that optimization of those levels can yield shRNAs that are both effective and not or minimally cytotoxic. We will also address whether the cell localization of the shRNA and/or its pathway for processing can affect the extent of cytotoxicity within cells. Through our proposed studies we will understand the effects of shRNA expression in mature lymphoid cells in vitro as well as the effects on the differentiation of the lymphocyte populations in an in vivo setting. We will minimize the cytotoxic effect and maximize stable and effective function of RNAi in primary T-lymphocytes. The relevance of this research to public health. RNA interference is a powerful and promising technology to inhibit specific gene expression for both basic research and therapeutic intervention. The inhibition of specific gene expression needs to be effective without unintended side effects. This proposal propose to study the effects of RNAi in human T lymphocytes to maximize the RNAi effect and minimize side effects.