Targeted integration of a DNA transposon-based nonviral vector

描述(申请人提供)：基因疗法有可能永久纠正或预防单基因疾病，如囊性纤维化。我们在利用多种类型的病毒和非病毒载体将基因输送到呼吸道方面有着良好的记录；然而，选择最好的基因治疗载体会导致一个非常重要的两难境地。非整合载体可能不会持续存在，整合载体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个难题。我们的长期目标是设计一种将基因输送到细胞的载体，这是一种安全有效的囊性纤维化治疗方法。为此，这些拟议研究的目标是开发一种具有可预测的整合模式的整合载体，该整合模式以“安全港”基因组座位为靶标。非病毒载体系统越来越多地被用作基因转移应用的工具。我们成功地利用了猪Bac DNA转座子系统作为基因转移的有效整合载体。方法。根据我们的初步研究，iggyBac转座酶是可以修饰的。此外，最近在设计定制锌指蛋白的能力方面的进展使靶向转座有望成为一种治疗方法。总体假设是，iggyBac转座子系统可能会被修改以重定向整合。我们将通过多种途径将猪转座酶定向到指定的位点，提高载体的传递效率，提高载体用于基因治疗的实用性和安全性。在这里，我们建议：1)构建并验证“安全港”锌指蛋白/猪转座酶融合蛋白的功能；2)定位锌指蛋白/猪转座酶介导的转座子在基因组中的整合；3)建立一个杂交的猪Bac/慢病毒载体系统，以提高递送效率。这些研究将提供重要的机制信息，关于指导iggyBac整合的重要基序。 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种靶向转座子载体系统，用于治疗性基因转移应用。成功地将定点限制性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.