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

基本信息

批准号：
8403682
负责人：
PATRICK L SINN
金额：
$ 45.9万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
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/8403682
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 deep sequencing design efficacy testing gene therapy gene transfer vector improved in vivo mutant novel prevent success therapeutic gene tool vector

项目摘要

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.

项目概要/摘要客观的。基因疗法有可能永久纠正或预防单基因疾病，例如如囊性纤维化。我们在利用许多类别的病毒和非病毒方面拥有良好的记录将基因传递到气道的载体；然而，选择最好的基因治疗载体会导致非常重大的困境。非整合向量可能不会持续存在，而整合向量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个问题难题。我们的长期目标是设计一种安全可靠的将基因传递到细胞的载体囊性纤维化的有效治疗方法。为此，这些拟议研究的目标是开发一种将载体与针对“安全港”基因组位点的可预测整合模式进行整合。非病毒性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，设计定制锌指蛋白的能力的最新进展使得靶向转座的可能性有望成为一种治疗方法。总体假设是可以修改piggyBac转座子系统以重新定位整合。我们将使用多个将piggyBac转座酶引导至指定位点、提高载体递送效率的方法，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）地图锌指蛋白/piggyBac 转座酶介导的基因组转座子整合； 3）创建混合的piggyBac/慢病毒载体系统可提高递送效率。这些研究将提供关于对指导piggyBac整合很重要的主题的重要机制信息。