SITE-SPECIFIC GENE EXPRESSION IN VIVO

尽管最近在真核细胞基因调控的研究中取得了进展， 人类疾病治疗管理的障碍仍然是 基因在体内的位点特异性表达。在原则上， 基因工程内皮细胞可用于提供 心血管和其他疾病的位点特异性基因治疗。 内皮细胞在血管内皮细胞损伤的发病机制中起着重要的作用。 心血管疾病和全身性动脉粥样硬化。这些细胞 影响动脉壁的完整性，调节血栓溶解， 调节血管平滑肌细胞张力。因为血栓形成与 动脉粥样硬化斑块通常是局部的， 局部动脉节段，期望将治疗引导至 具体的受伤部位。转基因内皮或其他 血管细胞可用于递送抗凝剂，血管舒张剂， 血管生成因子或生长因子。因为它们靠近 循环，他们也有可能治疗全身先天性遗传 疾病这种方法还可以提供一种解决基本问题的手段 血管生物学。 在这个建议中，我们开发了一种新的方法来传递蛋白质 使用内皮或其他血管技术在体内分离动脉段 经过基因改造的细胞。尤卡坦迷你猪 选择作为动物模型，用于位点特异性基因的开发 疗法来自近交系尤卡坦小型猪的内皮细胞将被感染 与表达重组β- 半乳糖苷酶。这些受感染的细胞将通过导管导入 同系动物裸露的髂股动脉。动脉段 术后2周至12个月将进行检查。我们最近 表明表达重组β-半乳糖苷酶的内皮细胞具有 使用这种方法成功植入血管壁。在这 建议，这项技术将得到优化，并用于引入一个 血管生成蛋白碱性成纤维细胞生长因子进入动脉壁 并测定其原位生物学效应。此外，初步 实验表明逆转录病毒载体可用于引入 通过直接感染将重组基因转移到脉管系统中的特定位点。 这些技术提供了内皮细胞的转移， 重组基因在体内脉管系统中的表达，可以允许 引入具有治疗价值的蛋白质， 心血管、全身和其他疾病。

Despite recent advances in the study of eukaroytic gene regulation, a major obstacle to the therapeutic management of human disease remains the site-specific expression of genes in vivo. In principle, genetically-engineered endothelial cells can be used to provide site-specific gene therapy for cardiovascular and other diseases. Endothelial cells play an important role in the pathogenesis of cardiovascular disease and in systemic atherosclerosis. These cells influence the integrity of the arterial wall, regulate thrombolysis, and modulate vascular smooth muscle cell tone. Because thromboses associated with atherosclerotic plaques are usually focal and represent injury to localized arterial segments, it is desirable to direct treatment to specific sites of injury. Genetically-modified endothelial or other vascular cells could be used to deliver anticoagulant, vasodilatory, angiogenic or growth factors in vivo. Because of their proximity to the circulation, they also have potential to treat systemic inborn inherited diseases. This approach may also provide a means to address basic questions of vascular biology. In this proposal, we develop a novel approach to the delivery of proteins to discrete arterial segments in vivo using endothelial or other vascular cells which have been genetically-modified. The Yucatan minipig has been chosen as an animal model for the development of site-specific gene therapy. Endothelial cells from the inbred Yucatan minipig will be infected with a replication-defective retroviral vector expressing recombinant beta- galactosidase. These infected cells will be introduced with a catheter into denuded illofemoral arteries of syngeneic animals. Arterial segments explanted 2 weeks to 12 months later will be examined. We have recently shown that endothelial cells expressing recombinant beta-galactosidase have successfully implanted on the vessel wall using this method. In this proposal, this technique will be optimized and used to introduce an angiogenic protein, basic fibroblast growth factor, into the arterial wall and to determine its biologic effect in situ. In addition, preliminary experiments indicate that retroviral vectors can be used to introduce recombinant genes to specific sites in the vasculature by direct infection. These techniques, providing for the transfer of endothelial cells and the expression of recombinant genes in the vasculature in vivo, may allow the introduction of proteins with therapeutic value in the management of cardiovascular, systemic, and other diseases.