Sleeping beauty transposon for gene therapy

用于基因治疗的睡美人转座子

• 批准号: 6861185
• 负责人: PERRY B. HACKETT
• 金额: $ 17.83万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-01 至 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6861185
• 关键词: disease /disorder model; gene delivery system; gene therapy; genetically modified animals; human tissue; laboratory mouse; liver cells; mucopolysaccharidosis; tissue /cell culture; transfection /expression vector; transposon /insertion element

New non-viral gene transfer procedures are needed for human gene therapy in order to achieve long-term maintenance and expression of newly introduced genes. The purpose of this project is to evaluate the Sleeping Beauty (SB) transposon system for its efficacy to catalyze integration of several transgenes into selected tissues in mice and rats. The SB system is binary, consisting of a transposon containing a transgene and a source of transposase enzyme. The SB transposase is able to mediate enhanced integration of marker/reporter genes in several mammalian culture cell lines in vitro as well as in fertilized embryos of zebrafish and Xenopus. Here, we propose to extend these studies in order to determine the efficacy of the SB transposon system as a gene transfer vector. Preliminary results indicate that the transposon can be delivered to livers of adult mice for long-term expression using hydrodynamic pressure. This procedure may not be possible to use in humans. Accordingly, we will evaluate the efficiencies of alternative delivery of the SB system to livers of whole animals affected by single-gene disorders that mimic conditions of human disease. Furthermore, as an additional precaution to curtail potential long-term effects of SB transposase, we will examine two strategies to curtail transposase activity. We will concentrate on gene therapy for mucopolysaccharidosis. The Specific Aims of this project are to: 1) Construct safer transposon vectors with either insulator elements, immediately inside both ends of the transposon vector to keep the enhancers driving a transgene from activating neighboring genes or that contain a "suicide gene". 2) Determine the efficiency of SB for gene transfer and expression in the livers of mice; SB will be tested as a vector system for gene transfer into the liver using a transposon engineered to express genes whose activities are deficient in certain human diseases. 3) Determine the efficiency of SB for gene transfer and expression in the livers of mice using purified SB transposase rather than its gene; to assure limitation of transposase activity, the efficacy of using purified SB transposase accompanying transposons will be tested in liver cells. The experiments in this project will provide an assessment of the capacity of SB as a gene transfer vector targeting therapeutically important organs in an in vivo gene therapy protocols. The results will lay the groundwork for optimization of this vector system and its future application to human gene therapy.

人类基因治疗需要新的非病毒基因转移方法，以实现新引入基因的长期维持和表达。本项目的目的是评估睡美人（SB）转座子系统催化几种转基因整合到小鼠和大鼠的选定组织中的功效。SB系统是二元的，由含有转基因的转座子和转座酶的来源组成。SB转座酶能够介导标记/报告基因在几种哺乳动物体外培养细胞系以及斑马鱼和非洲爪蟾的受精胚胎中的整合增强。在这里，我们建议扩展这些研究，以确定SB转座子系统作为基因转移载体的功效。初步结果表明，转座子可以交付到成年小鼠的肝脏长期表达使用流体动力学压力。这种方法可能不适用于人类。因此，我们将评估SB系统替代递送至受模拟人类疾病条件的单基因疾病影响的整个动物的肝脏的效率。此外，作为减少SB转座酶潜在长期效应的额外预防措施，我们将研究减少转座酶活性的两种策略。我们将专注于粘多糖样沉积症的基因治疗。本项目的具体目标是：1）构建更安全的转座子载体，其中任一绝缘子元件都位于转座子载体的两端， 防止驱动转基因的增强子激活邻近基因或含有“自杀基因”的基因。2)确定SB在小鼠肝脏中基因转移和表达的效率;将SB作为载体系统进行试验，以使用转座子将基因转移到肝脏中，转座子经工程改造以表达在某些人类疾病中缺乏活性的基因。3)使用纯化的SB转座酶而不是其基因确定SB在小鼠肝脏中的基因转移和表达效率;为了确保转座酶活性的限制， 将在肝细胞中测试使用纯化的SB转座酶伴随转座子的效力。本项目的实验将评估SB作为体内基因治疗方案中靶向治疗重要器官的基因转移载体的能力。本研究结果将为该载体系统的优化及其在人类基因治疗中的应用奠定基础。