Investigate stability RNA interference 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）来长期减少特定基因的表达，而不会对人类淋巴细胞产生毒性作用。利用siRNA抑制特定基因表达的RNAi技术在基础研究和治疗干预中都是一项强大而有前途的技术。然而，我们的结果和最近的几项研究表明，这种技术在哺乳动物细胞中产生了意想不到的非特异性细胞毒性作用，这引起了人们对RNAi在人类治疗应用中的长期使用的关注。细胞毒性作用至少部分归因于细胞凋亡，尽管其他机制也可能存在。细胞毒性与shRNA表达水平升高有关。基于这些结果，我们假设shRNA的水平对于确定给定shRNA是否具有细胞毒性非常重要，并且这些水平的优化可以产生既有效又无或最低细胞毒性的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，我们将了解体外成熟淋巴样细胞中shRNA表达的影响，以及体内淋巴细胞群体分化的影响。我们将最大限度地减少RNAi在原代t淋巴细胞中的细胞毒性作用，最大限度地提高RNAi在原代t淋巴细胞中的稳定有效功能。这项研究与公众健康的相关性。RNA干扰是一种强大而有前途的技术，可以抑制特定基因的表达，用于基础研究和治疗干预。对特定基因表达的抑制必须是有效的，而不会产生意想不到的副作用。本课题拟对RNAi在人T淋巴细胞中的作用进行研究，使RNAi的作用最大化，副作用最小化。

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.