Assessing and improving the safety profile of Sleeping Beauty transposon-mediated gene transfer in human cells

评估和改善睡美人转座子介导的人体细胞基因转移的安全性

• 批准号: 22667427
• 负责人: Professor Dr. Zoltan Ivics
• 金额: --
• 依托单位: Max-Delbrück-Centrum für Molekulare Medizin (MDC) in der Helmholtz-Gemeinschaft
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2006
• 资助国家: 德国
• 起止时间: 2005-12-31 至 2013-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/22667427?language=en
• 关键词: Assessing; improving; safety; profile; Sleeping

Transposons are natural gene delivery vehicles. The Sleeping Beauty (SB) transposon shows efficient transposition and long-term expression of transgenes in cells of a wide range of vertebrates, including humans. SB transposition, during which the element ¿jumps¿ from one DNA molecule to another, occurs into human chromosomal DNA in a fairly random manner. Thus, SB has a mutagenic potential, because it can insertionally activate or inactivate genes. This is useful for gene discovery in vertebrate models, but is clearly undesired for human gene therapeutic applications. In this project, we will determine the effects of transposase expression and transposon integration on cellular processes. We propose to investigate the molecular mechanisms involved in SB¿s target site selection, by mutagenizing the region of the SB transposase responsible for target DNA capture. Mutants will be analyzed for altered function on the levels of primary DNA binding, transposon excision and transposon integration in human cells. We further propose to manipulate SB¿s target site selection in order to achieve targeted transposition into predetermined loci or chromosomal regions. We consider experimental strategies based on targeting fusion proteins composed of a specific DNAbinding protein domain, responsible for binding to chromosomal DNA, and either the transposase polypeptide or another protein that interacts with the transposase or with the transposon DNA. Insights into the molecular mechanisms involved in target site selection of transposable elements can lead to powerful methods for safe transgene integration in human applications, and for target-selected gene knock-outs in vertebrate models.

转座子是天然的基因传递载体。睡美人（SB）转座子在包括人类在内的多种脊椎动物细胞中显示出高效的转座子和转基因的长期表达。在SB转位过程中，元素从一个DNA分子“跳跃”到另一个DNA分子，以相当随机的方式发生在人类染色体DNA中。因此，SB具有诱变潜力，因为它可以插入激活或灭活基因。这对于脊椎动物模型中的基因发现是有用的，但对于人类基因治疗应用显然是不希望的。在这个项目中，我们将确定转座酶表达和转座子整合对细胞过程的影响。我们提议通过诱变SB转座酶负责靶DNA捕获的区域来研究SB¿s靶位点选择的分子机制。将分析突变体在人类细胞中原代DNA结合、转座子切除和转座子整合水平上的功能改变。我们进一步建议操纵SB¿s的目标位点选择，以实现靶向转位到预定的位点或染色体区域。我们考虑了基于靶向融合蛋白的实验策略，这些融合蛋白由特定的DNA结合蛋白结构域组成，负责与染色体DNA结合，以及转座酶多肽或与转座酶或转座子DNA相互作用的另一种蛋白质。深入了解转座因子靶位选择的分子机制，可以为人类应用中的安全转基因整合以及脊椎动物模型中的靶位选择基因敲除提供强有力的方法。