Retrotransposons as site-specific gene delivery vectors

描述（由申请人提供）：L1反转录转座子是移动的DNA 通过复制和粘贴机制移动且没有显示强插入的元素 网站偏好初步研究表明，Lis可以将附着在 报告基因进入培养的人类细胞的DNA，这些基因可以 表达出来。这些研究表明，L1可能被用作基因， 与其它基因治疗载体相比具有几个优点的转移载体 目前处于临床前和临床试验阶段。 本研究旨在探讨母语修饰策略 逆转录转座子，以产生基因递送载体，该载体可以靶向特定的 人类基因组中的序列这可以通过更换L1来实现 核酸内切酶结构域与来自位点特异性 其他物种的反转录转座子，包括昆虫的R1元件和 Tx1L元件，或者通过将L1内切核酸酶结构域连接到 位点特异性结合基序（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 逆转录转座在人类细胞中，并将考虑其直接应用， 基因传递载体。此外，我将在细胞培养中测定 L1在整合长度增加的外源DNA方面的作用。

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.