eukaryotic expression vectors resistant to transgene silencing

描述(申请人提供)：真核表达载体被用于各种生物医学应用，包括蛋白质生产、基因治疗和基因疫苗接种。一个关键的障碍是表达载体随着时间的推移经历启动子失活(沉默)。缺乏持续的转基因表达对细胞培养生产重组蛋白的成本产生了负面影响，并限制了非病毒载体系统在短期应用中的应用。在这里，我们建议通过开发新型的抗转基因沉默的真核表达载体来创造能够延长非病毒载体在哺乳动物细胞中表达的持续时间的技术。这些研究将利用NTC开发的包含SV40-CMV嵌合启动子的真核表达载体骨架，其表达水平比替代CMV启动子载体提高10倍。我们建议通过评估两种防止转基因沉默的方法来进一步改进这些载体。首先，我们假设原核区的转录将扰乱异染色质的形成，并改善表型或整合细胞系的表达。我们将利用整合到载体骨架中的启动子转录促进异染色质形成的质粒区域。其次，我们假设复制起点内的一个或多个结构化区域形成了招募异染色质的不寻常结构，并解释了沉默的原因。将构建高产量的最小起源构建物，消除这些假定的异染色质诱导区。这些载体将在整合和瞬时转染的细胞系中进行测试，以提高性能。这项可行性研究的总体目标是确定这两种策略是否代表了防止转基因沉默的替代方法的显著优势，例如微环或基质附着区(MAR)载体。这项技术与优化的NTC表达载体相结合，将使NTC能够创建下一代表达系统，用于低成本生产细胞培养来源的重组蛋白。在第二阶段，NTC将使载体可用于许可，并将应用该技术开发基于细胞培养的方法，用于生产FSH和生物仿制药等糖蛋白。

DESCRIPTION (provided by applicant): Eukaryotic expression vectors are utilized for various biomedical applications including protein production, gene therapy and gene vaccination. A key barrier is that expression vectors undergo promoter inactivation (silencing) over time. This lack of sustained transgene expression negatively impacts the cost of cell culture production of recombinant proteins, and has limited the application of non-viral vector systems to short term applications. Here we propose to create enabling technology to improve the duration of expression from non-viral vectors in mammalian cells by developing novel eukaryotic expression vectors resistant to transgene silencing. These studies will utilize a eukaryotic expression vector backbone developed at NTC, containing a chimeric SV40-CMV promoter, which improves expression levels 10 fold over alternative CMV promoter vectors. We propose to further improve these vectors through evaluation of two methodologies to prevent transgene silencing. First, we hypothesize that transcription of the prokaryotic region will disrupt heterochromatin formation, and improve episomal or integrated cell line expression. We will transcribe the region of the plasmid that promotes heterochromatin formation using promoters integrated into the vector backbone. Second, we hypothesize that one or more structured regions within the replication origin form unusual structures that recruits heterochromatin and accounts for the silencing. High yield minimal origin constructs, that eliminate these putative heterochromatin inducing regions, will be constructed. The vectors will be tested in integrated and transiently transfected cell lines for improved performance. The overall goal of this feasibility study is to determine whether either strategy represents a significant advantage over alternate approaches to prevent transgene silencing, such as minicircle or matrix attached region (MAR) vectors. This technology, combined with the optimized NTC expression vectors, should enable NTC to create next generation expression systems for low cost production of cell culture derived recombinant proteins. In Phase II, NTC will make the vectors available for licensing, and will apply the technology to develop cell culture based methods for manufacture of glycoproteins such as FSH and biogeneric drugs.