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

9612334 Lauffenburger尽管它们具有高效的基因转移能力，但目前的病毒载体还不能满足人类体内基因治疗的理想载体。其中最严重的问题是基因随机插入宿主染色体，天然病毒蛋白引起的免疫反应，以及细胞在转移过程中的低选择性和低效率。此外，腺病毒载体和逆转录病毒载体都不能整合大小超过7或8kb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来影响DNA转移到细胞中。因此，结合基因传递提供了更好的传递潜力，但还没有得到足够详细的研究来证明其可行性。用于基因转移。这一建议寻求获得关于受体介导的DNA/配体共轭转运从细胞表面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.确定内体分选途径中的何处共轭化合物逃逸到胞浆中；c.预测和测试所需的配基性质以影响转移。该计划将研究将表皮生长因子(EGF)基因转移到含有先前转基因的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。P.I.S实验室对表皮生长因子系统有着丰富的工作经验，并且已经证明了将表皮生长因子转移到小鼠成纤维细胞上是相当成功的。

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. ***