DISRUPTING VIRAL GENES WITH GROUP II INTRONS

描述：这项提案的总体目标是评估 可移动的第二组内含子干扰哺乳动物细胞中的基因，重点是HIV和 以CCR5基因为靶点。第二组内含子的移动性是由一种 核糖核蛋白(RNP)由一个切除的内含子RNA套索和一个 内含子编码的蛋白质，具有逆转录酶、DNA内切酶和RNA 剪接成熟酶活性。乳酸乳球菌II群的迁移率 内含子通过两步过程发生。首先，内含子催化 将自身反向剪接到DNA靶点，然后内含子RNA 由内含子编码的蛋白质反转录成DNA。插入位置 在DNA上被内含子RNA识别，通过大约L4碱基对，和 内含子编码的蛋白质。最近，我们证明了第二组内含子可以 被设计成专门插入HIV-1极点中的靶DNA位点 和CCR5基因，通过修改存在于内含子RNA上的DNA结合位点。 这些重定向内含子保留了插入和破坏其 瞬时导入哺乳动物细胞时的靶向DNA位点 与其内含子编码的蛋白质形成复合体。这样做的具体目的是 建议如下：1)继续探索第二组内含子 通过位点特异性DNA插入干扰HTV和CCR5基因 将重组的II组RNPs导入哺乳动物细胞，2) 评估第II组内含子剪接和哺乳动物细胞的迁移率 内含子RNA和内含子编码蛋白的细胞内表达，3) 描述乳酸乳球菌第二组内含子的序列要求 哺乳动物细胞的流动性。4)评估第二组RNPs的能力 定点插入病毒DNA灭活HIV病毒前病毒 并使细胞对艾滋病毒感染和复制具有抵抗力。 这些研究也将为第二移动集团的发展奠定基础 哺乳动物细胞中靶向基因干扰的内含子用于治疗和 研究应用。

DESCRIPTION: The overall goal of this proposal is to evaluate the ability of mobile group II introns to disrupt genes in mammalian cells focusing on HIV and CCR5 genes as targets. Group II intron mobility is mediated by a ribonucleoprotein (RNP) composed of an excised intron RNA lariat and an intron-encoded protein that has reverse transcriptase, DNA endonuclease and RNA splicing maturase activities. Mobility of the Lactococcus lactis group II intron occurs through a two-step process. First the intron catalyzes the reverse-splicing of itself into a DNA target site and then the intron RNA is reverse transcribed into DNA by the intron encoded protein. The insertion site on DNA is recognized by both the intron RNA, through about l4 base pairs, and the intron-encoded protein. Recently, we demonstrated that group II introns can be engineered to site specifically insert into targeted DNA sites in HIV- 1 pol and CCR5 genes by modifying the DNA binding sites present on the intron RNA. These retargeted introns retain the ability to insert into and disrupt their targeted DNA sites when they are transiently transfected into mammalian cells in complex with their intron-encoded protein. The specific aims of this proposal are: 1) To continue to explore the ability of group II introns to disrupt HTV and CCR5 genes via site specific DNA insertion following transfection of reconstituted group II RNPs into mammalian cells, 2) To evaluate group II intron splicing and mobility in mammalian cells following intracellular expression of the intron RNA and the intron-encoded protein, 3) To delineate the sequence requirements for Lactococcus lactis group II intron mobility in mammalian cells. 4) To evaluate the ability of group II RNPs to inactivate HIV proviruses by site-specifically inserting into viral DNA in infected cells and to render cells resistant to HIV infection and replication. These studies will also lay the foundation for development of mobile group II introns for targeted gene disruption in mammalian cells for therapeutic arid research applications.