Srour Mahmoud A, Fechner Henry, Wang Xiaomin, Siemetzki Ulrike, Albert Thilo, Oldenburg Johannes, Hanfland Peter, Poller Wolfgang, Brackmann Hans-Hermann, Schwaab Rainer
Institute for Experimental Hematology and Transfusion Medicine, University Clinics Bonn, Sigmund-Freud-Str. 25, 53105 Bonn, Germany.
Thromb Haemost. 2003 Sep;90(3):398-405. doi: 10.1160/TH03-01-0010.
Following substitution therapy with human factor IX (hFIX) concentrate, therapy of haemophilia B by viral gene transfer has become an attractive alternative therapy in recent years. However, high doses of expressed hFIX, which can already be achieved in animal studies, may cause thrombosis in humans (van Hylckama Vlieg et al., 2000). Thus, it should be possible to maintain transgene expression within the therapeutic range. Therefore, we inserted elements of the tetracycline (Tet)-dependent Tet-On gene regulatory system into replication deficient adenovectors. The new system consists of two adenovectors: a response vector expressing hFIX (Ad5.TRE.hFIX), and a regulator vector expressing a second generation reverse tetracycline transactivator controlled by a CMV- (Ad5.CMV.rtTA) or human alpha1-antitrypsin-promoter (Ad5.hAAT.rtTA). Expression studies in four human cell lines showed high expression of hFIX from Ad5.TRE.hFIX in all cell lines in combination with Ad5.CMV.rtTA regulator vector, but only high specific expression in HepG2-cells in combination with Ad5.hAAT.rtTA regulator vector. Additionally, up- and down-regulation of hFIX expression could be demonstrated in vitro with the Ad5.TRE.hFIX/Ad5.CMV.rtTA combination and modulating doxycycline concentrations. When SCID-mice were infected with the Ad5.TRE.hFIX/Ad5.CMV.rtTA combination, up- and down-regulation of hFIX expression was achieved by oral doses of doxycycline for a period of at least two months. Replacement of the Ad5.CMV.rtTA vector by the Ad5.hAAT.rtTA vector showed minimal expression of hFIX in vivo. Although hFIX expression showed a slow and gradual decrease over time in vivo with the Ad5.CMV.rtTA vector, it remained within the therapeutic range. To date, regulation of hFIX has not been described in this way.
近年来，在用人凝血因子IX（hFIX）浓缩物进行替代治疗后，通过病毒基因转移治疗B型血友病已成为一种有吸引力的替代疗法。然而，在动物研究中已经能够实现的高剂量表达的hFIX可能会导致人类血栓形成（van Hylckama Vlieg等人，2000年）。因此，应该能够将转基因表达维持在治疗范围内。因此，我们将四环素（Tet）依赖性Tet-On基因调控系统的元件插入到复制缺陷型腺病毒载体中。新系统由两个腺病毒载体组成：一个表达hFIX的反应载体（Ad5.TRE.hFIX）和一个表达由CMV启动子（Ad5.CMV.rtTA）或人α1-抗胰蛋白酶启动子（Ad5.hAAT.rtTA）控制的第二代反向四环素反式激活因子的调节载体。在四种人类细胞系中的表达研究表明，Ad5.TRE.hFIX与Ad5.CMV.rtTA调节载体组合时，在所有细胞系中hFIX均有高表达，但与Ad5.hAAT.rtTA调节载体组合时，仅在HepG2细胞中有高特异性表达。此外，用Ad5.TRE.hFIX/Ad5.CMV.rtTA组合并调节强力霉素浓度，可在体外证明hFIX表达的上调和下调。当SCID小鼠感染Ad5.TRE.hFIX/Ad5.CMV.rtTA组合时，通过口服强力霉素至少两个月可实现hFIX表达的上调和下调。用Ad5.hAAT.rtTA载体替代Ad5.CMV.rtTA载体在体内显示hFIX表达极低。尽管使用Ad5.CMV.rtTA载体时hFIX表达在体内随时间缓慢逐渐下降，但仍保持在治疗范围内。迄今为止，尚未以这种方式描述hFIX的调节。
