eukaryotic expression vectors resistant to transgene silencing

抗转基因沉默的真核表达载体

• 批准号: 7264338
• 负责人: James Williams
• 金额: $ 14.55万
• 依托单位: NATURE TECHNOLOGY CORPORATION
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-10 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7264338
• 关键词: Accounting; Alzheimer' s Disease; Antibiotic Resistance; Autoimmune Diseases; Binding Sites; CMV promoter; Cell Line; Cells; Communicable Diseases; Cultured Cells; DNA Polymerase II; DNA Vaccines; Development; Disease; Engineering; Eukaryotic Cell; Evaluation; Exonuclease; Feasibility Studies; Follicle Stimulating Hormone; Gene Transduction Agent; Genes; Genetic Transcription; Glycoproteins; Goals; Heterochromatin; Hypersensitivity; In Vitro; Licensing; Malignant Neoplasms; Mammalian Cell; Matrix Attachment Regions; Mediating; Methodology; Methods; Non-Viral Vector; Performance; Pharmaceutical Preparations; Phase; Plasmid Cloning Vector; Plasmids; Production; Protein Binding; Proteins; Recombinant Proteins; Recruitment Activity; Replication Origin; Replicon; Repressor Proteins; Resistance; Runaway; Safety; Site; Structure; System; Technology; Technology Transfer; Temperature; Testing; Time; Titrations; Transfection; Transgenes; Vaccination; Vertebral column; Viral; base; cost; design; expression vector; gene replacement; gene therapy; improved; in vivo; next generation; novel; prevent; programs; promoter; protein misfolding; size; transgene expression; vector; vector vaccine

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.

描述（由申请人提供）：真核表达载体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）。这种持续转基因表达的缺乏对重组蛋白的细胞培养生产的成本产生负面影响，并且限制了非病毒载体系统在短期应用中的应用。在这里，我们提出创建使能技术，以改善从非病毒载体在哺乳动物细胞中的表达的持续时间，通过开发新的真核表达载体抗转基因沉默。这些研究将利用在NTC开发的真核表达载体骨架，其含有嵌合SV 40-CMV启动子，其比替代CMV启动子载体提高表达水平10倍。我们建议通过评估两种方法来进一步改进这些载体，以防止转基因沉默。首先，我们假设原核区域的转录将破坏异染色质的形成，并提高附加型或整合型细胞系的表达。我们将使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。第二，我们假设复制起点内的一个或多个结构化区域形成不寻常的结构，该结构招募异染色质并解释沉默。将构建消除这些推定的异染色质诱导区域的高产最小来源构建体。将在整合和瞬时转染的细胞系中检测载体，以提高性能。该可行性研究的总体目标是确定任一策略是否代表相对于防止转基因沉默的替代方法（例如小环或基质附着区（MAR）载体）的显著优势。该技术与优化的NTC表达载体相结合，应使NTC能够创建下一代表达系统，用于低成本生产细胞培养衍生的重组蛋白。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，用于生产糖蛋白，如FSH和生物仿制药。