Targeted integration of a DNA transposon-based nonviral vector

描述（由申请人提供）：基因治疗有可能永久纠正或预防单基因疾病，如囊性纤维化。我们已经证明了利用许多类别的病毒和非病毒载体将基因递送到气道的记录;然而，选择最好的基因治疗载体导致了一个非常重大的困境。非整合载体可能不会持续存在，而整合载体可能导致插入诱变。迫切需要改进的基因递送工具来解决这个难题。我们的长期目标是设计一种将基因递送到细胞的载体，这是一种安全有效的囊性纤维化治疗方法。为此，这些研究的目标是开发具有可预测整合模式的整合载体，其靶向“安全港”基因组基因座。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piggyBac DNA转座子系统作为一个有效的整合载体的基因转移。法基于我们的初步研究，piggyBac转座酶是适合修饰的。此外，最近的进展，在工程定制的锌指蛋白的能力，使靶向转座有希望作为一种治疗方法的可能性。总的假设是piggyBac转座子系统可以被修饰以重新靶向整合。我们将使用多种方法将piggyBac转座酶定向到指定位点，提高载体递送效率，并提高载体用于基因治疗的实用性和安全性。在此，我们建议：1）产生并验证“安全港”锌指蛋白/piggyBac转座酶融合蛋白的功能; 2）绘制基因组中锌指蛋白/piggyBac转座酶介导的转座子整合;和3）创建杂合piggyBac/慢病毒载体系统以改善递送功效。这些研究将提供重要的机制信息的图案重要的指导piggyBac整合。 公共卫生相关性：这些研究的成功完成将使我们更接近纠正囊性纤维化遗传缺陷的长期目标。该项目的具体目标是创建一个靶向转座子载体系统的治疗基因转移应用。成功地将定点转基因整合到人类基因组DNA中将在基因治疗和分子遗传学领域具有令人兴奋和广泛的应用。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: The successful completion of these studies will bring us closer to the long-term goal of correcting the cystic fibrosis genetic deficiency. The specific goal of this project is to create a targetable transposon vector system for therapeutic gene transfer applications. Successful site-restricted transgene integration into human genomic DNA would have exciting and broad applications to the fields of gene therapy and molecular genetics.