凝血因子VIII在稳定转导了包含最小因子VIII启动子的第三代慢病毒载体的肝细胞系和非肝细胞系中的重组表达。

Recombinant expression of coagulation factor VIII in hepatic and non-hepatic cell lines stably transduced with third generation lentiviral vectors comprising the minimal factor VIII promoter.

作者信息

Picanço V, Heinz S, Bott D, Behrmann M, Covas D T, Seifried E, Tonn T

机构信息

Institute for Transfusion Medicine and Immunohematology, Red Cross Blood Donor Service Baden-Wuerttemberg-Hesse, Johann Wolfgang Goethe University Clinics, Frankfurt/Main, Germany.

出版信息

Cytotherapy. 2007;9(8):785-94. doi: 10.1080/14653240701656053. Epub 2007 Oct 4.

DOI:10.1080/14653240701656053

PMID:17917890

Abstract

BACKGROUND

Lentiviral vectors have the capacity to transduce stably non-dividing, differentiated and undifferentiated cells of various tissues, including liver. To obtain high-level expression of transgenes, vectors often rely on viral promoters. However, recent data suggest that the supraphysiologic expression from ubiquitous viral promoters may not be beneficial and harbor the risk of oncogene activation. Therefore this study explored the lentiviral-mediated expression of human coagulation factor VIII (FVIII) driven by the physiologic FVIII gene promoter (FVIII-p), the liver-specific human alpha-1-antitrypsin gene promoter (hAAT-p), the ubiquitous but non-viral EF1alpha promoter (EF1alpha-p) and the viral CMV promoter.

METHODS

Hepatic and non-hepatic cell lines were stably transduced with lentiviral vectors encoding FVIIIdelB and EGFP. To compare the different promoters, lentiviral vectors were cloned to drive FVIII expression from FVIII-p, EF1alpha-p, hAAT-p and CMV-p.

RESULTS

As expected, the strong viral CMV-p and the ubiquitous EF1alpha-p resulted in the highest FVIII expression in all cell lines tested (CMV-p 1.85 IU/mL/10(6) cells for 293T, 3.15 for HepG2, 5.03 for SK-Hep, 0.91 for Hepa1-6; EF1-alpha promoter 0.30 IU/mL/10(6) cells for 293T, 0.04 for HepG2, 2.75 for SK-Hep, 0.46 for Hepa1-6). While the hAAT-p resulted in low FVIII levels (0.10 IU/mL/10(6)cells in HepG2 and 0.04 in Hepa1-6), the FVIII promoter gave reasonable expression levels in hepatic cells (0.47 IU/mL/10(6)cells in Hepa1-6 and 0.44 in SK-Hep).

DISCUSSION

These results indicate the potential usefulness of the FVIII-p for hemophilia A gene therapy.

摘要

背景

慢病毒载体能够稳定转导包括肝脏在内的各种组织中的非分裂、分化和未分化细胞。为了获得转基因的高水平表达，载体通常依赖病毒启动子。然而，最近的数据表明，来自普遍存在的病毒启动子的超生理表达可能并无益处，且存在激活致癌基因的风险。因此，本研究探索了由生理凝血因子VIII基因启动子（FVIII-p）、肝脏特异性人α-1-抗胰蛋白酶基因启动子（hAAT-p）、普遍存在但非病毒的EF1α启动子（EF1α-p）和病毒CMV启动子驱动的慢病毒介导的人凝血因子VIII（FVIII）表达。

方法

用编码FVIIIdelB和EGFP的慢病毒载体稳定转导肝细胞系和非肝细胞系。为了比较不同的启动子，克隆慢病毒载体以驱动FVIII从FVIII-p、EF1α-p、hAAT-p和CMV-p表达。

结果

正如预期的那样，强病毒CMV-p和普遍存在的EF1α-p在所有测试的细胞系中导致最高的FVIII表达（293T细胞中CMV-p为1.85 IU/mL/10(6)细胞，HepG2为3.15，SK-Hep为5.03，Hepa1-6为0.91；EF1-α启动子在293T细胞中为0.30 IU/mL/10(6)细胞，HepG2为0.04，SK-Hep为2.75，Hepa1-6为0.46）。虽然hAAT-p导致FVIII水平较低（HepG2中为0.10 IU/mL/10(6)细胞，Hepa1-6中为0.04），但FVIII启动子在肝细胞中给出了合理的表达水平（Hepa1-6中为0.47 IU/mL/10(6)细胞，SK-Hep中为0.44）。