ADENOVIRAL GENE TRANSFER OF APOE IN APOE DEFICIENT MICE

APOE 缺陷小鼠中 APOE 的腺病毒基因转移

• 批准号: 3757644
• 负责人: S SANTAMARINA-FOJO
• 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/3757644
• 关键词: Adenoviridae; apolipoproteins; atherosclerosis; blood lipoprotein metabolism; disease /disorder model; gene expression; gene therapy; genetic models; genetic transduction; high performance liquid chromatography; human genetic material tag; hypercholesterolemia; hyperlipoproteinemia; laboratory mouse; nonhuman therapy evaluation; protein purification; recombinant DNA; transfection /expression vector; western blottings

Apolipoprotein E (apoE) is a 299 amino acid protein present in VLDL, IDL and HDL that plays a major role in the metabolism of plasma lipoproteins. ApoE is a major ligand for the LDL and remnant receptors and thus, necessary for the normal clearance of remnant particles from the circulation. Patients with a functional deficiency of apoE can develop Type III hyperlipoproteinemia and premature atherosclerosis. Recently, apoE deficient mice have been generated by homologous recombination. These animals develop marked hypercholesterolemia and spontaneous vascular lessions and are a useful model for evaluating the potential for gene therapy. We have generated a recombinant adenoviral vector containing human apoE cDNA (rAdV) for injection of apoE def mice (n=8) with pre-tx lipids:TC=609plus/minus108mg/dl,TG=101plus/minus50mg/dl and chol-rich VLDL/IDL present on FPLC. After IV infusion of either ten to the seventh or ninth pfu, apoE-def mice had peak (day 6) plasma h-apoE levels of 2.3 mg/dl and 648 mg/dl, respectively. Western blot analysis demonstrated the expression of a normal sized h-apoE. Expression of these two different levels of apoE in plasma resulted in markedly different lipoprotein changes. Mice achieving physiologic apoE levels (2.3 mg/dl) normalized their lipids (TC=109plus/minus19mg/dl, TG=56plus/minus29mg/dl) at days 4-8; FPLC was normalized with loss of all VLDL/IDL and generation of HDL. Animals with 200X increase in apoE had a biphasic lipid response with initial decrease in TC (230mg/dl) but increase in TG (652 mg/dl); FPLC shifted from chol-rich VLDL/IDL to Tg-rich LDL remnants. By 8-12d apoE (less than 10mg/dl) and TG (66plus/minus40mg/dl) decreased and FPLC revealed a normal HDL profile. ApoE expression and normal plasma lipids were maintained for a period of 4 weeks after virus injection. Our studies indicate successful physiologic replacement (2.3mg/dl) as well as marked overexpression (more than500mg/dl) of h-apoE in apoE def mice using recombinant adenovirus as vehicle.Physiologic levels of h-apoE normalized plasma lipids whereas apoE overexpression resulted in transient formation of TG-rich remnants possibly due to high apoE levels blocking receptor mediated remnant clearance. Successful replacement of apoE in apoE-def mice demonstrates the feasibility of gene therapy in human apolipoprotein deficiencies.

载脂蛋白E（apoE）是存在于VLDL、IDL中的299个氨基酸的蛋白质， 和在血浆脂蛋白代谢中起主要作用的HDL。 ApoE是LDL和残余受体的主要配体，因此， 必要的残留颗粒的正常清除 流通apoE功能缺陷的患者可以发展为 III型高脂蛋白血症与过早动脉粥样硬化。 最近，apoE缺陷小鼠已经通过同源重组产生。 重组这些动物出现明显的高胆固醇血症， 自发性血管病变，是一个有用的模型，用于评估 基因治疗的潜力。我们制造了一种重组腺病毒 用于apoE def小鼠注射的含有人apoE cDNA（rAdV）的载体 （n=8）治疗前血脂：TC= 609 ± 108 mg/dl，TG= 101 ± 50 mg/dl 和富胆VLDL/IDL存在于FPLC上。静脉输注10种 至第7或第9 pfu，apoE-def小鼠的血浆h-apoE峰值（第6天 水平分别为2.3 mg/dl和648 mg/dl。蛋白质印迹分析 显示正常大小的h-apoE的表达。表达这些 血浆中两种不同水平的apoE导致显著不同的 脂蛋白变化。达到生理apoE水平（2.3 mg/dl）的小鼠 血脂正常（TC= 109 ± 19 mg/dl，TG= 56 ± 29 mg/dl） 在第4-8天; FPLC标准化，所有VLDL/IDL丢失，并产生 的HDL。apoE增加200倍的动物具有双相脂质反应， TC开始下降（230 mg/dl），TG开始升高（652 mg/dl）; FPLC从富含胆固醇的VLDL/IDL转变为富含TG的LDL残余物。8- 12天 apoE（<10 mg/dl）和TG（66 ± 40 mg/dl）降低，FPLC 显示高密度脂蛋白含量正常ApoE表达与正常血脂 在病毒注射后维持4周。 我们的研究表明，成功的生理替代（2.3mg/dl）， 在apoE缺陷型中，h-apoE明显过表达（> 500 mg/dl）， 小鼠使用重组腺病毒作为载体。h-apoE的生理水平 正常化的血脂，而apoE过表达导致 可能由于高apoE水平而短暂形成富含TG的残留物 阻断受体介导的残留清除。成功替代 apoE-def小鼠中的apoE证实了基因治疗在 人载脂蛋白缺乏症。