Cichon Guenter, Willnow Thomas, Herwig Susanne, Uckert Wolfgang, Löser Peter, Schmidt Hartmut H, Benhidjeb Tahar, Schlag Peter M, Schnieders Frank, Niedzielska Dagmara, Heeren Joerg
Department of Molecular Cell Biology, Humboldt-University Berlin at the Max Delbrueck Center for Molecular Medicine, Robert-Roessle-Str. 10, 13125 Berlin, Germany.
J Gene Med. 2004 Feb;6(2):166-75. doi: 10.1002/jgm.473.
Gene therapy of familial hypercholesterolemia (FH) requires successful transfer and lifelong expression of a functional low density lipoprotein receptor (LDLr) gene in the liver. Most of the vector systems currently employed for gene therapy use promoter elements which do not modulate transgene expression in a physiological manner.
To study the in vivo effects of constitutive LDLr gene expression in the absence of interfering immunological reactions we established a new mouse model which combines homozygous LDLr deficiency and severe combined immune deficiency (SCID).
Adenovirus-mediated transfer and expression of the LDLr gene under the control of a commonly used virus-derived promoter (minimal CMV promoter) leads to prolonged reduction of serum cholesterol levels in LDLr-deficient SCID mice. During the first 10 days after gene therapy serum cholesterol drops to about 10% of pretherapeutic values. Serum cholesterol persists on this level for 2 weeks and then slowly starts to rise again. Four months after vector application serum levels have reached about 40% of pretherapeutic values. However, as early as 5 days after gene transfer, the histological analysis of liver sections revealed the formation of crystalline lipid/cholesterol deposits in the cytosol of hepatocytes. During the following 8 weeks the amount of crystals increased in size and density. The intracellular storage of lipid and cholesterol reduced cell viability and induced an accelerated loss of therapeutic DNA from mice livers as was shown in a comparative expression study employing a transgene with a different metabolic function (human alpha 1-antitrypsin).
The non-physiological constitutive overexpression of an LDL receptor gene induces an imbalance between the speed of LDL uptake and metabolism which leads to pathological accumulation of lipids and cholesterol in hepatocytes. To protect cells from negative effects of LDLr overexpression, future vector design should consider the use of physiologically controlled expression elements.
家族性高胆固醇血症（FH）的基因治疗需要在肝脏中成功转移并长期表达功能性低密度脂蛋白受体（LDLr）基因。目前用于基因治疗的大多数载体系统使用的启动子元件不能以生理方式调节转基因表达。
为了研究在无干扰免疫反应情况下组成型LDLr基因表达的体内效应，我们建立了一种新的小鼠模型，该模型结合了纯合LDLr缺乏和严重联合免疫缺陷（SCID）。
腺病毒介导的在常用病毒衍生启动子（最小CMV启动子）控制下的LDLr基因转移和表达导致LDLr缺陷型SCID小鼠血清胆固醇水平长期降低。基因治疗后的前10天，血清胆固醇降至治疗前值的约10%。血清胆固醇在该水平持续2周，然后又开始缓慢上升。载体应用4个月后，血清水平达到治疗前值的约40%。然而，早在基因转移后5天，肝切片的组织学分析就显示肝细胞胞质中形成了结晶性脂质/胆固醇沉积物。在接下来的8周内，晶体的数量、大小和密度都增加了。脂质和胆固醇的细胞内储存降低了细胞活力，并导致治疗性DNA从小鼠肝脏中加速丢失，这在一项使用具有不同代谢功能的转基因（人α1-抗胰蛋白酶）的比较表达研究中得到了证实。
LDL受体基因的非生理性组成型过表达导致LDL摄取和代谢速度之间的失衡，从而导致脂质和胆固醇在肝细胞中病理性蓄积。为了保护细胞免受LDLr过表达的负面影响，未来的载体设计应考虑使用生理控制的表达元件。
