EGF Receptor-Mediated DNA Uptake and Expression: A Model System for Engineering Selective Gene Delivery

EGF 受体介导的 DNA 摄取和表达：工程选择性基因传递的模型系统

• 批准号: 9612334
• 负责人: Douglas Lauffenburger
• 金额: $ 5万
• 依托单位: Massachusetts Institute of Technology
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9612334&HistoricalAwards=false
• 关键词: EGF; Receptor; Mediated; DNA; Uptake

9612334 Lauffenburger In spite of their efficient gene transfer capabilities, current viral vectors fall short of ideal vector for human in-vivo gene therapy. Among the most serious problems are the random gene insertion into the host chromosome, immune responses caused by the natural viral proteins, and low cell selectivity and efficiency in the transfer. Also, neither adenoviral nor retrovial vectors can incorporate genes over 7 or 8 kb in size. There is a growing body of work demonstrating that certain ligands have the ability to bind DNA and effect its transfer into cells through very selective surface receptor binding. Thus conjugate gene delivery offers and improved delivery potential but has not been studied in enough detail to demonstrate feasibility. For gene transfer use. This proposal seeks to gain information on the rate- limiting steps of the receptor-mediated DNA/ligand- conjugate transport from the cell surface to the nucleus. Specific aims include: A. Determine whether cellular internalization is a limiting factor; B. Determine where in the endosomal sorting pathways conjugates escape to the cytosol; C. Predict and test desirable ligand properties to effect the transfer. The program will study the transfer of the epidermal growth factor (EGF) gene into mouse fibroblast cells that contain previously transfected EGF receptors. This robust system will allow variation of all the key factors. The P.I.'s lab has extensive experience working with the EGF system and has already demonstrated reasonable success transferring EGF to the mouse fibroblasts. ***

9612334 Lauffenburger尽管它们具有有效的基因转移能力，但目前的病毒载体达不到用于人体内基因治疗的理想载体。 其中最严重的问题是随机基因插入宿主染色体，天然病毒蛋白引起的免疫反应，以及低细胞选择性和转移效率。 此外，无论是腺病毒载体还是逆转录病毒载体都不能掺入大小超过7或8 kb的基因。 有越来越多的工作表明，某些配体具有结合DNA的能力，并通过非常选择性的表面受体结合将其转移到细胞中。 因此，偶联基因递送提供了改进的递送潜力，但尚未进行足够详细的研究以证明可行性。 用于基因转移。 该提议试图获得关于受体介导的DNA/配体缀合物从细胞表面转运到细胞核的限速步骤的信息。 具体目标包括：A.确定细胞内化是否是限制因素; B. 确定结合物在内体分选途径中逃逸到胞质溶胶的位置; 预测和测试所需的配体特性以实现转移。 该计划将研究表皮生长因子（EGF）基因转移到含有先前转染的EGF受体的小鼠成纤维细胞中。 这个强大的系统将允许所有关键因素的变化。 私家侦探实验室有丰富的经验与EGF系统的工作，并已证明合理的成功转移EGF到小鼠成纤维细胞。 ***