eukaryotic expression vectors resistant to transgene silencing

抗转基因沉默的真核表达载体

基本信息

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
项目类别：
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&apos 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

项目摘要

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，包括蛋白质产生，基因治疗和基因疫苗。一个关键的障碍是表达向量会随着时间的流逝而经历启动子灭活（沉默）。这种缺乏持续的转基因表达对重组蛋白的细胞培养成本产生负面影响，并限制了非病毒载体系统在短期应用中的应用。在这里，我们建议创建能够通过开发新型的真核表达载体来改善哺乳动物细胞中非病毒载体的表达持续时间的能力，以提高技术的表达持续。这些研究将利用在NTC开发的真核表达载体主链，其中包含嵌合SV40-CMV启动子，该促进剂在替代CMV启动子向量上提高了表达水平10倍。我们建议通过评估两种方法来进一步改善这些向量，以防止转基因沉默。首先，我们假设原核生物区域的转录会破坏异染色质形成，并改善偶发或整合细胞系表达。我们将使用集成到矢量主链中的启动子来促进质粒形成的质粒区域。其次，我们假设复制起源中的一个或多个结构化区域形成了异常结构，这些结构募集了异染色质并解释了沉默。将构建消除这些推定的异染色质诱导区域的高产量最小起源构建体。向量将在集成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任何一种策略是否代表了防止转基因沉默的替代方法的重要优势，例如微米或基质附着区域（MAR）向量。这项技术与优化的NTC表达向量相结合，应使NTC能够创建下一代表达系统，以低成本生产细胞培养的重组蛋白。在第二阶段，NTC将使矢量可用于许可，并将应用该技术开发基于细胞培养的方法来生产糖蛋白（例如FSH和生物基因药物）。