Development of Gene Therapy for Familial Hypercholesterolemia-in vivo gene transfer to hepatocytes by HVJ-liposome-retrovirus method-

为了研究基因转移的效率，我们用具有细胞融合能力的仙台病毒（HVJ）和核蛋白（hmg - 1）构建了一个脂质体，使DNA有效地转移到细胞核中。然后将报告基因（β -半乳糖苷酶）引入脂质体中，通过门静脉或直接注射到大鼠和小鼠肝脏中，使报告基因在5% ~ 10%的肝细胞中表达1 ~ 2周。然后，将人低密度脂蛋白受体基因导入脂质体，并通过该方法转移到大鼠体内肝脏。结果发现，人LDL受体mRNA在肝细胞中表达，体内血清总胆固醇水平降低约20%。然而，免疫组化检查显示，低密度脂蛋白受体蛋白在肝细胞中表达不足。因此，我们将pMy3的表达载体换成了比pMy3更小（7.5kb）且含有鸡β -肌动蛋白启动子的pCL1，观察到基因转移效率得到了提高。将人LDL受体基因移植到渡边遗传性高脂血症（Watanabe遗传性高脂血症，WHHL）家兔体内，发现人LDL受体基因在肝细胞中表达，血清胆固醇水平降低约20%。然而，仅转染hvj -脂质体的家兔血清胆固醇水平也出现了类似的下降，因此，我们将通过Northern blotting、RNase保护实验和免疫组织化学研究来探讨这些家兔LDL受体基因的表达机制，并探讨hvj -脂质体降低血清胆固醇水平的机制。我们还在开发逆转录病毒的有效转移，这种方法在美国已被用于体外基因治疗，使用的是hvj脂质体方法。为了将来将基因治疗应用于人类，我们将研究将人类LDL受体基因转移到敲除LDL受体的小鼠和WHHL兔。少

To investigate the efficiency of gene transfer, we constructed a liposome with the Sendai-virus(HVJ), which has the ability for cell fusion, and the nuclear protein (HMG-l) to make DNA to transfer efficiently into the nucleus. Then the reporter gene (BETA-galaczosidase) was introduced to the liposome, and the construct was injected to the livers of rats and mice via portal vein or directly, resulting in the expression of the reporter gene in 5 to 10% of the hepatocytes for I or 2 weeks. Next, human low density lipoprotein (LDL) receptor gene was introduced to the liposome and transferred to rat liver in vivo using this method. It was found that the human LDL receptor mRNA was expressed in the hepatocytes, and approximately 20% reduction of serum total cholesterol level was observed in vivo. However, immunohistochemical examinations revealed that the expression of the LDL receptor protein was not adequate in the hepatocytes. Thus we exchanged the expression vector from pMy3 to pCL1, whi … More ch is smaller in size (7.5kb) than pMy3 and has chicken BETA-actin promoter, and it was observed that the efficiency of gene transfer was improved. Then we transferred the human LDL receptor gene to Watanabe heritable hyperlipidemic (WHHL) rabbits using this procedure, and found that the human LDL receptor gene was expressed in the hepatocytes and that serum cholesterol levels were decreased about 20% before the gene transfer. However, control rabbits to which only the HVJ-liposome was transferred also showed the similar reduction of serum cholesterol levels, thus we are investigating the mechanism of the expression of the LDL receptor gene in these rabbits by Northern blotting, RNase protection assay and immunohistochemical studies, and examining the mechanism of the reduction of serum cholesterol level by the HVJ-Iiposome. We are also developing the efficient transfer of retrovirus, which has been used for ex vivo gene therapy in the United States, using the HVJ-liposome method. For the future application of the gene therapy to the human, we are going to investigate the transfer of the human LDL receptor gene to the LDL receptor knock out mice and WHHL rabbits. Less