Retrotransposons as site-specific gene delivery vectors

描述(申请人提供)：L1反转录转座子是可移动的DNA 通过复制和粘贴机制移动且没有强烈插入的元素 站点首选项。初步研究表明，LIS可以将附件 报告基因进入培养的人类细胞的DNA中，这些基因可以 被表达出来。这些研究表明，L1可能被用作一种基因 转移载体与其他基因治疗载体相比有几个优点 目前正在进行临床前和临床试验。 本提案旨在探讨修改L1的策略 反转录转座子产生靶向特定靶点的基因传递载体 人类基因组中的序列。这可以通过更换L1 具有来自特定位点的核酸内切酶结构域的核酸内切酶结构域 其他物种的反转录转座子，包括昆虫的R1元件和 Tx1L元件，或通过将L1内切酶结构域连接到 特定部位的结合基序(例如GAL4结合结构域、锌指结构域)。我 还将测试野生型R1、R2和Tx1L元件的能力 逆转座子在人类细胞中的应用，并将其直接应用于 基因传递载体。此外，我还将在细胞培养中检测其效率 L1的外源DNA的整合长度增加。

DESCRIPTION (provided by applicant): L1 retrotransposons are mobile DNA elements which move by a copy and paste mechanism and show no strong insertion site preference. Preliminary studies have shown that Lis can deliver attached reporter genes into the DNA of human cells in culture and that these genes can be expressed. Such studies suggest that the L1 might be utilized as a gene transfer vector having several advantages over other gene therapy vectors currently in preclinical and clinical trials. This proposal is designed to explore strategies for modifying the L1 retrotransposon to produce a gene delivery vector which can target to specific sequences in the human genome. This may be accomplished by replacing the L1 endonuclease domain with endonuclease domains from site-specific retrotransposons of other species, including the R1 element of insects and the Tx1L element from Xenopus, or by linking the L1 endonuclease domain to site-specific binding motifs (eg. GAL4 binding domain, zinc finger domains). I also will test the ability of wild-type R1, R2, and Tx1L elements to retrotranspose in human cells and will consider their direct application as gene delivery vectors. In addition, I will assay in cell culture the efficiency of the L1 at integrating increasing lengths of exogenous DNA.