Targeted integration of a DNA transposon-based nonviral vector

项目总结/摘要 Objective.基因治疗有可能永久纠正或预防单基因疾病， 囊性纤维化我们有一个利用病毒和非病毒的许多类别的证明记录 载体将基因传递到气道;然而，选择最好的基因治疗载体会导致非常 一个重大的困境。非整合载体可能不会持续存在，整合载体可能会导致 插入诱变迫切需要改进的基因递送工具来解决这个问题 难题我们的长期目标是设计一种将基因传递到细胞的载体， 有效治疗囊性纤维化。为此，这些拟议研究的目标是制定一个 整合载体具有靶向“安全港”基因组基因座的可预测整合模式。非病毒 载体系统越来越多地用作基因转移应用的工具。我们成功地使用了 piggyBac DNA转座子系统是一种有效的基因转移整合载体。 法基于我们的初步研究，piggyBac转座酶是适合修饰的。 此外，最近在工程化定制锌指蛋白的能力方面的进展使得 靶向转座的可能性有望作为一种治疗方法。总的假设是， piggyBac转座子系统可以被修饰以重新定位整合。我们将使用多个 将piggyBac转座酶导向指定基因座，增加载体递送效率， 提高了载体用于基因治疗的实用性和安全性。在这里，我们建议：1）生成和 验证“安全港”锌指蛋白/piggyBac转座酶融合蛋白的功能; 2）定位 锌指蛋白/piggyBac转座酶介导的转座子在基因组中的整合;和3）产生 杂交piggyBac/慢病毒载体系统以提高递送功效。这些研究将提供 关于对指导piggyBac整合重要的基序的重要机制信息。

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.