TARGETABLE RETROVIRAL VECTORS--IN VIVO LIVER GENE TRANSFER

靶向逆转录病毒载体——体内肝脏基因转移

• 批准号: 3843316
• 负责人: L BALTRUCKI
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3843316
• 关键词: Retroviridae; biotechnology; chimeric proteins; clone cells; disease /disorder model; gene expression; gene therapy; genetic disorder; glycoproteins; human tissue; isozymes; liver cells; liver disorder; mixed tissue /cell culture; molecular cloning; receptor binding; transfection /expression vector

Our work is based on the premise that the ability to target retroviral vectors to specific cell surface receptors will increase the efficiency of gene transfer, and that this along with other advances will lead to significant therapeutic benefits to patients. To target vectors specifically to the hepatocytes' asialo-glycoprotein receptor, (ASG-R), we have constructed chimeric envelope glycoproteins, in which the native receptor binding domain of gp70 (ecotropic), has been replaced by peptide sequences derived from alpha-1-acid glycoprotein (AGP). Once we can target HepG2 cells in vitro these vectors will be tested in vivo. Our ultimate goal is to transfer genes of therapeutic value to the liver. The c-DNAs encoding two isoenzymes with (human) bilirubin-UDP-glucuronyl transferase activity, have been subcloned into retroviral vectors, and their expression has been tested in vitro. The Gunn rat animal model, for Crigler-Najjar syndrome type I, will be our test system. Optimizing gene transfer efficiency, by comparing alternate routes of virus delivery, and increasing the titer and stability of vector formulations, will represent major ongoing efforts in this system. Protein C deficiency represents another hereditary liver disease, which for homozygotes is invariably fatal in the neonatal period. Our near-term application for vectors producing this key anticoagulant is to optimize its production in vitro. Another possible goal is to test its ability to prevent intra-vascuar thrombosis, particularly in conjunction with tPA. Finally, new retroviral packaging cell lines based on hepatocytes, bone marrow stromal and thymic epithelial cells, are under development. These may have certain advantages over the current cell lines which are derived from 3T3 cells.

我们的工作是基于这样一个前提： 将载体与特定的细胞表面受体结合将提高 基因转移，这沿着其他进展将导致 对患者有显著的治疗益处。 以载体为目标 我们专门针对肝细胞的脱唾液酸糖蛋白受体（ASG-R）， 已经构建了嵌合包膜糖蛋白，其中天然的 gp 70的受体结合结构域（亲嗜性）已被肽取代 衍生自α-1-酸性糖蛋白（AGP）的序列。 一旦我们能够 体外靶向HepG 2细胞这些载体将在体内进行测试。 我们的最终目标是将具有治疗价值的基因转移到肝脏。 编码两种（人）葡萄糖醛酸-UDP同工酶的c-DNA 转移酶活性，已经亚克隆到逆转录病毒载体中， 它们的表达已经在体外进行了测试。 古恩大鼠动物模型， Crigler-Najjar综合征I型，将是我们的测试系统。 优化基因 转移效率，通过比较病毒传递的替代途径， 增加载体制剂的滴度和稳定性，将代表 在这一系统中不断努力。 蛋白C缺乏症代表另一种遗传性肝病， 在新生儿期是致命的。 我们的近期 生产这种关键抗凝剂的载体的应用是优化 其在体外的生产。另一个可能的目标是测试它的能力， 预防血管内血栓形成，特别是与tPA联合使用。 最后，新的逆转录病毒包装细胞系基于肝细胞，骨 骨髓基质细胞和胸腺上皮细胞正在开发中。 这些 可能比目前的细胞系具有某些优势， 3 T3细胞