Development of Therapy for Refractory Hyperlipidemia with LDL Receptor Gene Introduction by Lipofection Method

本研究试图开发一种基因治疗低密度脂蛋白（LDL）受体基因异常引起的家族性高胆固醇血症（FH）。首先，我们开发了一种高效的体内基因导入系统，HVJ-脂质体法。第二，利用该方法将人LDL受体基因导入成年大鼠肝脏。将含有人LDL受体基因的HVJ-脂质体颗粒通过门静脉或尾静脉导入肝脏，用RT-PCR方法证实人LDL受体mRNA在肝脏中的表达。此外，与高胆固醇饮食的对照大鼠相比，静脉转染大鼠的血清胆固醇降低了约20%。但HVJ-脂质体法的转染效率较低，不能满足FH基因治疗的需要，因此我们尝试对表达载体进行改进。我们首先使用的载体（pMy 3）较长，其启动子（转铁蛋白启动子）在肝脏中可被调控，因此我们开发了一种新的载体，该载体包含鸡β肌动蛋白启动子和人LDL受体cDNA，并在体外比较了这两种载体的效率。北方印迹分析表明，新载体（pCL 1）比pMy 3更有效。因此，我们使用该载体进行体内转染，但与pMy 3相比，血清胆固醇没有显著降低。目前，我们正在尝试开发另一种新的表达载体，利用EB病毒或逆转录病毒载体，并通过改变脂质体的脂质组成来改进HVJ-脂质体法。

In this report, we tried to develop a gene therapy for famillial hypercholesterolemia (FH) which is caused by the abnormality in the low density lipoprotein (LDL) receptor gene. First, we developed a highly efficient in vivo gene introduction system, HVJ-liposome method. Second, we introduced human LDL receptor gene into adult rat liver with this method. HVJ-liposome particles containing human LDL receptor gene were introduced to the liver through portal vein or tail vein and the expression of human LDL receptor mRNA was confirmed in the liver with RT-PCR method. Furthermore, serum cholesterol was decreased about 20% in the intravenously transfected rats compared with control rats which were loaded with a high-cholesterol diet. However, the efficiency of transfection with HVJ-liposome method was a little lower than expected and was considered to be insufficient for the gene therapy for FH.So, we tried to improve the expression vector. The vector we used first (pMy3) was long and its promoter (transferrin promoter) could be regulated in the liver, so we developed a new vector which contains chicken beta actin promoter and included human LDL receptor cDNA and compared in vitro the efficiency of these two vectors. Northern blot analysis demonstrated that the new vector (pCL1) was more efficient than pMy3. So we used this vector for transfection in vivo, but serum cholesterol was not reduced markedly compared with pMy3. Now, we are trying to develop another new expression vector, using Epstein-Barr virus or retrovirus vector and improve HVJ-liposome method by changing the lipid composition of liposomes.