Targeted integration of a DNA transposon-based nonviral vector

项目摘要/摘要 目标。基因疗法有可能永久纠正或预防这样的单基因疾病 为囊性纤维症。我们在使用多种病毒型和非病毒型病毒方面有良好的记录 载体将基因输送到呼吸道；然而，选择最好的基因治疗载体会导致非常 严重的进退两难。非积分向量可能不会持久存在，积分向量可能会导致 插入突变。迫切需要改进的基因传递工具来解决这一问题 难解之谜。我们的长期目标是设计一种向细胞运送基因的工具，它是一种安全和 有效治疗囊性纤维化。为此，这些拟议研究的目标是制定一项 将载体与可预测的整合模式整合在一起，以“安全港”为目标的基因组座位。非病毒型 载体系统越来越多地被用作基因转移应用的工具。我们成功地使用了 猪Bac DNA转座子系统是一种有效的基因转移整合载体。 方法。根据我们的初步研究，iggyBac转座酶是可以修饰的。 此外，最近在定制锌指蛋白工程能力方面的进展使 靶向转位有望成为一种治疗方法。总体假设是 可以修改iggyBac转座子系统以重定向整合。我们将使用多个 将PiggyBac转座酶定向到指定位置的方法，提高载体传递效率， 提高了基因治疗载体的实用性和安全性。在这里，我们建议：1)生成和 验证安全港锌指蛋白/猪转座酶融合蛋白的功能；2)图谱 锌指蛋白/猪Bac转座酶介导的转座子在基因组中的整合；以及3)创建 一种混合的猪Bac/慢病毒载体系统，以提高递送效率。这些研究将提供 关于基序的重要机械信息对指导iggyBac整合非常重要。

PROJECT SUMMARY/ABSTRACT Objective. Gene therapy has the potential to permanently correct or prevent monogenic disorders such as cystic fibrosis. We have a demonstrated track record of utilizing many categories of viral and non-viral vectors to deliver genes to the airways; however, choosing the best gene therapy vector leads to a very significant dilemma. Non-integrating vectors may not persist and integrating vectors may cause insertional mutagenesis. There is a critical need for improved gene delivery tools that address this conundrum. Our long-term goal is to engineer a vehicle for gene delivery to cells that is a safe and effective therapeutic for cystic fibrosis. To this end, the goal for these proposed studies is to develop an integrating vector with a predictable integration pattern that targets 'safe harbor' genomic loci. Nonviral vector systems are used increasingly as tools for gene transfer applications. We successfully used the piggyBac DNA transposon system as an effective integrating vector for gene transfer. Method. Based on our preliminary studies, piggyBac transposase is amenable to modification. Furthermore, recent advances in the ability to engineer customized zinc finger proteins make the possibility of targeted transposition promising as a therapeutic approach. The overall hypothesis is that the piggyBac transposon system may be modified to retarget integration. We will use multiple approaches to direct the piggyBac transposase to designated loci, increase vector delivery efficiency, and improve the utility and safety of the vector for gene therapy. Here we propose to: 1) generate and validate the function of 'safe harbor' zinc finger protein/piggyBac transposase fusion proteins; 2) map zinc finger protein/piggyBac transposase mediated transposon integrations in the genome; and 3) create a hybrid piggyBac/lentivirus vector system to improve delivery efficacy. These studies will provide important mechanistic information regarding the motifs important for directing piggyBac integrations.