Wilson J M, Johnston D E, Jefferson D M, Mulligan R C
Whitehead Institute for Biomedical Research, Cambridge, MA.
Proc Natl Acad Sci U S A. 1988 Jun;85(12):4421-5. doi: 10.1073/pnas.85.12.4421.
Familial hypercholesterolemia is an inherited disease in humans that is caused by a defect in the receptor for low density lipoproteins (LDLR). The existence of an animal model for this disease, the Watanabe heritable hyperlipidemic (WHHL) rabbit, makes it an attractive candidate for developing new therapies that involve gene transfer into liver. As a first step toward the development of these therapies, we report the use of retrovirus-mediated gene transfer to correct the genetic defect in hepatocytes isolated from WHHL rabbits. A series of retroviral vectors that express the gene for human LDLR were constructed, each differing in the transcriptional elements used to drive LDLR expression. Helper-free amphotropic virus stocks representing each construct were then used to infect primary cultures of hepatocytes that were isolated from newborn WHHL rabbits. The efficiency of transduction, as measured by Southern analysis of integrated proviral sequences, ranged from 20% to 100%. Expression of human LDLR was analyzed by blot hybridization analysis of total cellular RNA and by biochemical and in situ analyses of transduced cultures for receptor function. The vector in which the expression of LDLR was driven by the viral long terminal repeat sequence produced the greatest quantity of LDLR RNA and protein in WHHL hepatocytes; LDLR activity approached normal levels in these cultures.
家族性高胆固醇血症是人类的一种遗传性疾病，由低密度脂蛋白受体（LDLR）缺陷引起。这种疾病的动物模型——渡边遗传性高脂血症（WHHL）兔的存在，使其成为开发涉及基因导入肝脏的新疗法的有吸引力的候选对象。作为开发这些疗法的第一步，我们报告了使用逆转录病毒介导的基因转移来纠正从WHHL兔分离的肝细胞中的遗传缺陷。构建了一系列表达人LDLR基因的逆转录病毒载体，每个载体在用于驱动LDLR表达的转录元件上有所不同。然后使用代表每个构建体的无辅助嗜异性病毒株感染从新生WHHL兔分离的肝细胞原代培养物。通过对整合的前病毒序列进行Southern分析来测量转导效率，范围为20%至100%。通过对总细胞RNA进行印迹杂交分析以及对转导培养物的受体功能进行生化和原位分析来分析人LDLR的表达。由病毒长末端重复序列驱动LDLR表达的载体在WHHL肝细胞中产生了最大量的LDLR RNA和蛋白质；这些培养物中的LDLR活性接近正常水平。
