Investigate stability of RNA interference in human lymphocytes

描述(申请人提供)：我们的总体假设是，可以开发出RNA干扰(RNAi)来长期减少人类淋巴细胞中特定基因的表达，而不会产生毒性影响。使用siRNA抑制特定基因表达的RNAi技术在基础研究和治疗干预方面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，这项技术在哺乳动物细胞中具有意想不到的非特异性细胞毒作用，这引发了人们对RNAi在人类治疗应用中的长期使用的担忧。细胞毒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表达有关。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，并且对这些水平的优化可以产生既有效又不具有或最低细胞毒性的shRNA。我们还将探讨shRNA的细胞定位和/或其加工途径是否会影响细胞内的细胞毒性程度。通过我们提出的研究，我们将了解shRNA在体外成熟淋巴样细胞中表达的影响，以及在体内环境下对淋巴细胞群分化的影响。我们将最大限度地减少RNAi对原代T淋巴细胞的细胞毒作用，最大限度地发挥其稳定有效的功能。这项研究与公共卫生的相关性。RNA干扰是一种强大而有前景的技术，可以抑制特定基因的表达，无论是在基础研究还是治疗干预方面。对特定基因表达的抑制需要是有效的，没有意外的副作用。这项建议建议研究RNAi在人T淋巴细胞中的作用，以最大限度地发挥RNAi效应，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.