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

转座子是一种天然的基因传递工具。睡美人(SB)转座子显示了转基因在包括人类在内的多种脊椎动物细胞中的有效转座和长期表达。Sb转座，在此过程中，元素从一个DNA分子跳到另一个分子，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失活基因。这对于脊椎动物模型中的基因发现是有用的，但对于人类的基因治疗应用显然是不受欢迎的。在这个项目中，我们将确定转座酶的表达和转座子整合对细胞过程的影响。我们建议通过突变负责靶基因捕获的SB转座酶区域来研究Sb？S靶点选择的分子机制。将分析突变体在人类细胞中初级DNA结合、转座子切除和转座子整合水平上的功能变化。我们进一步建议操纵SB‘S的靶点选择，以实现靶向转座到预定的基因座或染色体区域。我们考虑基于靶向融合蛋白的实验策略，该融合蛋白由负责与染色体DNA结合的特定DNA结合蛋白结构域以及转座酶多肽或另一种与转座酶或转座子DNA相互作用的蛋白质组成。深入了解转座元件的靶点选择所涉及的分子机制，可以为人类应用中安全的转基因整合和脊椎动物模型中的靶向选择基因敲除提供强有力的方法。

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.