Targeted integration of a DNA transposon-based nonviral vector

基于 DNA 转座子的非病毒载体的靶向整合

• 批准号: 8237293
• 负责人: PATRICK L SINN
• 金额: $ 48.21万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8237293
• 关键词: Address; Animal Model; Binding; Bioinformatics; Categories; Cells; Chimeric Proteins; Collaborations; Cystic Fibrosis; DNA; DNA Transposable Elements; DNA Transposons; Data; Disease; Engineering; Excision; Feline Immunodeficiency Virus; Future; Gene Delivery; Gene Transduction Agent; Gene Transfer; Genes; Genetic; Genome; Genomics; Goals; Human; Hybrids; In Vitro; Insertional Mutagenesis; Integrase; Lactones; Lentivirus Vector; Maps; Mediating; Methods; Modification; Molecular Genetics; Non-Viral Vector; Oxidases; Pattern; Plasmids; Proteins; Pseudogenes; Safety; Sequence Analysis; Site; Somatic Cell; Subfamily lentivirinae; System; Therapeutic; Transgenes; Transposase; Viral Vector; Zinc Fingers; airway epithelium; base; cellular transduction; design; efficacy testing; gene therapy; gene transfer vector; improved; in vivo; mutant; novel; prevent; success; therapeutic gene; tool; vector

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.

描述（由申请人提供）：基因疗法有可能永久纠正或预防囊性纤维化等单基因疾病。我们在利用多种病毒和非病毒载体将基因输送到呼吸道方面拥有良好的记录；然而，选择最佳的基因治疗载体会导致一个非常严重的困境。非整合载体可能不会持续存在，而整合载体可能会导致插入突变。迫切需要改进基因传递工具来解决这一难题。我们的长期目标是设计一种将基因传递到细胞的载体，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整合模式的整合载体，以“安全港”基因组位点为目标。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转座子系统作为基因转移的有效整合载体。方法。根据我们的初步研究，piggyBac 转座酶易于修饰。此外，最近在设计定制锌指蛋白的能力方面取得的进展使得靶向转座成为一种有望成为治疗方法的可能性。总体假设是piggyBac 转座子系统可能被修改以重新定位整合。我们将采用多种方法将piggyBac转座酶引导至指定位点，提高载体递送效率，提高基因治疗载体的实用性和安全性。在这里，我们建议：1）生成并验证“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的图谱； 3) 创建混合piggyBac/慢病毒载体系统以提高递送效率。这些研究将提供有关对指导piggyBac整合很重要的基序的重要机制信息。 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用于治疗性基因转移应用的靶向转座子载体系统。成功的位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。