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Chimeric transcriptional control units for improved liver-specific transgene expression
Gene 322 (2003) 137 – 143 www.elsevier.com/locate/gene
Chimeric transcriptional control units for improved liver-specific transgene expression Stephan Gehrke, Vale´rie Je´roˆme, Rolf Mu¨ller * Institute of Molecular Biology and Tumor Research (IMT), Philipps-University, Emil-Mannkopff-Strasse 2, 35033 Marburg, Germany Received 2 July 2003; received in revised form 5 August 2003; accepted 18 August 2003 Received by M. Schartl
Abstract Hepatocyte-directed delivery of therapeutic genes is a major field of gene therapy. An important issue in this context is the availability of promoters units providing for maximum transcriptional activity and specificity. Although a number of liver-specific promoters and transcriptional control elements have been identified and used for gene delivery, no systematic study has been performed to identify the best suitable combination of known liver-specific promoter and enhancer elements. We now report the results of a comparative investigation addressing this issue. We tested a total of 25 synthetic transcriptional control units consisting of either of the four core promoters from liverspecific genes linked in various combinations and configurations to hepatocyte-specific enhancer elements. These constructs were analyzed for transcriptional activity in different cell types in cell culture and in mouse liver in vivo. The data lead to the clear conclusion that a combination of the alcohol dehydrogenase 6 (ADH6) basal promoter linked to two tandem copies of an apoplipoprotein E enhancer element is the transcriptional control unit of choice for the liver-specific expression of transgenes. D 2003 Elsevier B.V. All rights reserved. Keywords: Liver-specific genes; ADH promoter; apoE promoter; Alpha1-microglobulin/bikunin promoter; Alpha-antitrypsin promoter
1. Introduction The liver represents a target of major interest for gene therapy. Hepatocytes fulfill a great variety of essential functions ranging from metabolism and homeostasis to immune responses that are frequently perturbed due to monogenetic dysfunctions. Moreover, the liver is a potentially well suitable site for the ectopic expression of therapeutic proteins with, for instance, immune modulatory, tumoricidal or anti-angiogenic properties. Indeed, a large number of studies, including clinical trials in humans, have addressed the possibility to employ gene therapeutic strategy for the treatment of diverse diseases, such as defect in lipid metabolism or blood clotting, liver cancer or metastasis, hepatitis and liver cirrhosis (FujiAbbreviations: ABP, alpha1-microglobulin/bikunin; ADH6, alcohol dehydrogenase 6; CMV, cytomegalo virus; CYP, cholesterol 7alphahydroxylase; FBS, fetal bovine serum; FIX, clotting factor IX; apoE, apolipoprotein E/C-I; hAAT, human alpha-antitrypsin; HCR-1, hepatic control region-1; SV40, simian virus 40. * Corresponding author. Tel.: +49-6421-286236; fax: +49-6421288923. E-mail address: [email protected] (R. Mu¨ller). 0378-1119/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2003.08.010
moto, 2000; Ghosh et al., 2000; Mitry et al., 2000; Oka et al., 2000; Schmitz et al., 2002; Prieto et al., 2003). In most cases, gene transfer into the liver has been attempted by the administration via different routes of DNA or viral vectors based on retroviruses (Hafenrichter et al., 1994), adenoviruses (Okuyama et al., 1998), adeno-associated viruses (Xiao et al., 1998) or lentiviruses (Park et al., 2000). Major limitations of these transduction systems include an inefficient or unstable transduction and a lack of tissue-specificity (Ghosh et al., 2000). These problems may be in part interrelated, as non-specific transgene expression can cause immune responses against the transduced cells which in turn severely limits the stability of transduction. Tissue-specific transgene expression is obviously highly desirable for gene therapy and can in principle be achieved through transcriptional targeting, i.e. the use of transcriptional control units that restrict transgene expression to the cell type of interest (Nettelbeck et al., 2000). Transcriptional targeting can be used to compensate for the lack of specificity of the available vector systems, thereby allowing for the efficient and stable expression of transgenes, including genes coding for proteins whose proper function is depen-
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dent on their synthesis in the correct cell type due to, for instance, the requirement for defined post-translational modifications. An example in this context is the synthesis of blood clotting factors in the liver (Manno, 2002). Even though many tissue-specific promoters have been described their applicability to gene therapy is frequently limited due to low activity and/or large size which can pose a serious obstacle for the construction of viral vectors. This also applies to hepatocyte-directed gene therapy, where a number of tissue-specific promoters have been used, but systematic studies pertaining at the design of an optimal transcriptional control unit have not been published. Ideally, such a control unit should be as small as possible while maintaining a high transcriptional activity and cell type specificity. In the present study, we have addressed this problem by constructing a total of 25 synthetic transcriptional control units consisting of core promoters from liverspecific genes linked in various combinations and configurations to hepatocyte-specifc enhancer elements, and analyzing these constructs for transcriptional activity in different cell types in cell culture and in mouse liver in vivo. The transcription regulatory elements used in this study were derived from the human genes coding for alcohol dehydrogenase 6 (ADH6) (Yasunami et al., 1991; Zhi et al., 2000), human alpha-antitrypsin (hAAT) (Shen et al., 1989; Hafenrichter et al., 1994), cholesterol 7alpha-hydroxylase (CYP) (Molowa et al., 1992), clotting factor IX (FIX) (Boccia et al., 1996; Hoag et al., 1999), apolipoprotein E/C-I (apoE) (Simonet et al., 1993; Allan et al., 1997) and alpha1-microglobulin/bikunin (ABP) (Rouet et al., 1992). These studies led to the clear conclusion that the ADH6 basal promoter linked to two tandem copies of an apoE enhancer element allows for the highest level of liver-specific transcription.
2. Materials and methods 2.1. Cell culture HepG2 (ATCC HB 8065; hepatoma), MeWo (Bean et al., 1975), MCF7 (ATCC HTB-22; breast cancer) and A431 (ATCC CRL-1555; epidermoid carcinoma) were cultured in DMEM with L-glutamine (Boehringer Ingelheim BioProducts, Heidelberg, Germany) and 10% fetal bovine serum (FBS; Life Technologies, Eggenstein, Germany). H322 (Falzon et al., 1986) and PC3 (ATCC CRL 1435; prostate carcinoma) were grown in RPMI (Boehringer Ingelheim BioProducts) with 10% FBS. All cells were kept at 37 jC in a humidified atmosphere of 5% CO2. 2.2. Construction of plasmids All core promoters were synthesized by PCR from HepG2 genomic DNA using pfu DNA-polymerase and cloned as BglII and HindIII-fragments into the pGL3-basic luciferase vector (Promega, Mannheim, Germany). Sequence and ori-
entation were verified by DNA sequencing. The promoters contained the following sequences: ADH6, 342 to + 57 (Yasunami et al., 1991; Zhi et al., 2000); hAAT, 347 to + 56 (Shen et al., 1989; Hafenrichter et al., 1994; Bowling et al., 1996; Miao et al., 2000); CYP, 370 to + 63 (Molowa et al., 1992); and FIX, 219 to + 21 (Boccia et al., 1996). The core promoters were linked to the following enhancer sequences in the pGL3-basic luciferase vector by standard ligation techniques: a 319-bp apoE enhancer containing the hepatic control region (HCR-1) (Simonet et al., 1993; Dang et al., 1995; Allan et al., 1997); a 190-bp ABP enhancer region spanning nucleotides 2824 to 2634 (Rouet et al., 1992); a quadruplet of the site 5 enhancer element of the FIX gene spanning nucleotides 219 to 190 (Hoag et al., 1999). The simian virus 40 (SV40) promoter/enhancer luciferase vector pGL3 was obtained from Promega. 2.3. Transfection of plasmids Transfection was performed with DOTAP following the supplier’s protocol (Roche diagnostics, Mannheim, Germany). For each assay 105 cells were transfected with 5 Ag of plasmid DNA. Luciferase assays were performed as described (Nettelbeck et al., 1998). The SV40 promoter luciferase vector was used for standardization. Relative promoter activities were calculated using the following formula: standardized relative RLUs=(RLUs sample/background RLUs)/(RLUs pGL3-background). The standardized relative RLUs from three independent experiments were combined to calculate averages and standard deviations. 2.4. In vivo studies The hydrodynamics-based transfection technique described previously (Liu et al., 1999; Zhang et al., 1999) was followed. Briefly, NMRI nu/nu mice were rapidly injected into the tail vein with plasmid DNA (1 Ag/g body weight) in Ringer’s solution (147 mM NaCl, 4 mM KCl, 1.13 mM CaCl2). For standardization, a cytomegalo virus (CMV) promoter/enhancer-renilla luciferase plasmid was co-injected at a ratio of 1:50 (renilla luciferase plasmid: firefly luciferase plasmid). The mice were killed 24 h after injection, the livers were removed and lysed in Passive lysis buffer (Promega) and renilla and firefly luciferase activities were determined using the dual-luciferase reporter assay system as previously described (Je´roˆme and Mu¨ller, 2001).
3. Results and discussion 3.1. Comparison of core promoters derived from different liver-specific genes Our strategy for constructing an optimal liver-specific expression cassette involved four steps: (i) identification of a core promoter enabling efficient basal transcription in cul-
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tured hepatoma cells; (ii) identification of an enhancer module providing for a strong and liver-specific stimulation of transcription and (iii) verification of the results in vivo. To identify a suitable basal promoter we assessed the strength of different core promoters of liver-specific genes in transient luciferase assays in human hepatoma cells. Promoter sequences were chosen for analyses on the basis that their function as basal promoters had been defined in previous studies. These included the promoters of the alcohol dehydrogenase 6 (ADH6) (Yasunami et al., 1991; Zhi et al., 2000), the human alpha-antitrypsin (hAAT) (Shen et al., 1989; Hafenrichter et al., 1994), the cholesterol 7alpha-hydroxylase (CYP) (Molowa et al., 1992) and the clotting factor IX (FIX) (Boccia et al., 1996; Hoag et al., 1999) genes. Transient transfection experiments using the human hepatoma cell line HepG2 (Fig. 1) showed that both the ADH6 and the hAAT promoter gave rise to high levels of basal transcription (9.6- and 6.3-fold, respectively, relative to the SV40 promoter), while both the CYP and FIX promoters were considerably weaker (16% and 12%, respectively, of the SV40 promoter). 3.2. Effect of different liver-specific enhancers on transcriptional activity We next addressed the question which enhancer elements would work best in conjunction with the basal promoter of the ADH6 gene. For this purpose, we constructed enhancer modules containing elements from three different liverspecific genes: the HCR-1 region of the apoE gene (Allan et al., 1997), a far upstream enhancer region of the ABP gene (Rouet et al., 1992) and the site 5 enhancer domain of the FIX gene (Hoag et al., 1999). In total 19 transcriptional control units containing these enhancer elements in different configurations were designed, i.e. single copies, two copies of the same element in the same or opposite orientation and combinations of different enhancer elements in different orientations and orders (see Fig. 2). The results corroborated
Fig. 1. Transcriptional activities of core promoters derived from liverspecific genes. Promoter – luciferase constructs were transiently transfected into HepG2 cells and luciferase activities were determined 48 h later. Values are given as multiples of basal SV40 promoter activity and represent average values of three independent experiments F standard deviations. ADH6: human alcohol dehydrogenase 6; hAAT: human alpha-antitrypsin; CYP: human cholesterol 7alpha-hydroxylase; FIX: human clotting factor IX.
Fig. 2. Schematic representation of enhancer modules analyzed in the present study. apoE: apolipoprotein E/C-I; ABP: alpha1-microglobulin/ bikunin; site5: enhancer box of the clotting factor IX.
in Fig. 3 lead to the following conclusions: (i) apoE inserted in ‘‘sense’’ orientation is the strongest enhancer when used as a single copy (50-fold relative to SV40 promoter); (ii) a strong enhancement of transcription was seen when two copies of the apoE or ABP elements were present in the expression cassette. In case of apoE, the orientation was critical (‘‘sense’’ only; 117-fold relative to SV40 promoter), whereas for ABP both ‘‘sense’’ and ‘‘antisense’’ orientations functioned equally (84-fold relative to SV40 promoter); (iii) of all combinations of enhancer elements, only the combination of site5 and ABP showed a high transcriptional activity (44-fold relative to SV40 promoter). These data clearly indicate that the highest level of transcription was achieved by the 2apoEs module. To confirm and extend these results we next generated constructs where the 2apoEs and 2ABPas enhancer modules were tested in the context of the four core promoters already used in Fig. 1. As shown in Fig. 4 and in keeping with the results described above, the combination of the ADH6 core promoter and the 2apoEs enhancer module proved to drive transcription in HepG2 cells most efficiently (117-fold relative to SV40 promoter), followed by the 2apoEs + hAAT, 2ABPas + ADH6 and 2ABPas + hAAT constructs, which worked equally well (84-fold relative to SV40 promoter). In addition, in agreement with the data in Fig. 1, the ADH6 and hAAT core promoters were clearly superior to the basal promoters of the CYP and FIX genes with respect to transcriptional activity in HepG2 cells.
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Fig. 3. Transcriptional activities in HepG2 cells of synthetic expression cassettes combining the ADH6 core promoter and different enhancer modules derived from liver-specific genes (see Fig. 2) after transient transfection into HepG2 cells. pGL3-control: SV40 promoter with early region enhancer; core: ADH6 core promoter. Values are given as multiples of basal SV40 promoter activity and represent average values of three independent experiments F standard deviations.
3.3. Cell type specificity of the chimeric transcriptional control units The next important issue to address was cell type specificity of the transcriptional control units. We therefore transfected in addition to HepG2 several other cell lines originating from different tissues with the 2apoEs + ADH6, 2ABPas + ADH6, 2apoEs + hAAT, and 2ABPas + hAAT enhancer –promoter units (and for comparison with the ADH6 and hAAT core promoter constructs). The transfected cell lines included H322 lung carcinoma, PC-3 prostate carcinoma, A431 vulva carcinoma, MCF-7 mammary carcinoma and MeWo melanoma. The data in Fig. 5 support the following conclusions: (i) all constructs showed the highest level of transcriptional activity in HepG2 cells; (ii) cell type
specificity was already evident with the core promoter constructs, but the degree of specificity was relatively low when compared to PC-3 cells (7- to 18-fold); this specificity could be greatly enhanced by addition of either the 2apoE or the ABP enhancer modules. With all four enhancer –promoter constructs the highest degree of non-specific expression was seen with H322 cells, but specificity was very high even in this case (76- to 117-fold). Specificity was greatest with the constructs containing the ADH6 basal promoter, as confirmed by Student’s t-test. This is the same enhancer – promoter combination that we had identified above as the transcriptional control unit giving rise to the highest level of transcription (Figs. 3 and 4), and thus appears to be the combination of choice for liver-specific expression of transgenes.
Fig. 4. Transcriptional activities in HepG2 cells of different basal promoters linked to apoE- and ABP-derived enhancer modules. Values are given as multiples of basal SV40 promoter activity and represent average values of three independent experiments F standard deviations.
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Fig. 5. Cell type specificity of expression cassettes consisting of different basal promoters and enhancer modules. Luciferase – promoter constructs were transiently transfected into different cell lines and luciferase activities were determined 48 h later. Values are given as multiples of basal SV40 promoter activity and represent average values of three independent experiments F standard deviations. HepG2: hepatoma; H322: lung carcinoma; PC-3: prostate carcinoma; A431: vulva carcinoma; MCF-7; mammary carcinoma; MeWo: melanoma.
3.4. Transcriptional activity in vivo
3.5. Conclusions
To test the optimal promoter –enhancer combinations identified above in an in vivo setting, we determined the transcriptional activity of four apoEs-based enhancer – promoter constructs in mouse liver relative to the SV40 promoter. Plasmid DNA was delivered i.v. and the firefly luciferase activity in liver was determined 24 h later. For standardization we co-injected a plasmid containing a renilla luciferase gene driven by the CMV-promoter/early-enhancer. As can be seen in Fig. 6, the two plasmids with two copies of the apoEs enhancer module gave rise to a f 3.5-fold higher level of gene luciferase activity compared to the constructs with single apoEs modules, irrespective of the basal promoter used (ADH6 or hAAT). The transcriptional activity achieved with both 2apoEs plasmids was very high and reached 40- to 45-fold the activity of the SV40 promoter activity. In contrast to the liver, other organs (spleen, kidney, heart, lung and muscle) showed only very little luciferase activity that was too low to allow for a precise determination of tissue-specificity (data not shown). Taken together, the in vivo data fully confirm the observations made with cultured HepG2 cells and support the conclusion that the 2apoEs + ADH6 combination is a particularly well suitable liver-specific transcriptional unit.
Successful liver-directed gene therapy will critically depend on the availability of small, efficient and specific transcriptional control units. In the present study, we pursued this goal by constructing chimeric regulatory units
Fig. 6. Transcriptional activities of the ADH6 and hAAT basal promoters linked to apoE-derived enhancer modules in mouse liver. Promoter constructs were injected i.v. along with a reporter plasmid used for standardization (CMV promoter/enhancer linked to renilla luciferase) and both luciferase activities were determined 24 h later.
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consisting of basal promoter and enhancer elements from different liver-specific genes. The analysis of a total of 19 such constructs in transient transfection assays with hepatoma cells showed that the isolated core promoters of CYP and FIX genes were clearly inferior to the core promoter regions of the hAAT and ADH6 genes, the latter giving rise to the highest level of basal transcription. Of the different enhancer configurations tested, the most efficient construction turned out to be a head-to-tail tandem apoE sequence which was superior to similar configurations made up of the ABP enhancer domain, single copies of the apoE, ABP or site 5 enhancers or the combinations of heterologous enhancer modules. Importantly, the ability of the tandem apoE construct to drive transcription in a particularly efficient way was associated with a very high degree of cell type specificity, which was in the range of two orders of magnitude when compared to tumor cell lines derived from other tissues of origin, including lung, breast, prostate, epidermis and melanocytes. The functionality of the 2apoEs + ADH6 transcriptional control unit for liver-directed gene delivery could be confirmed in vivo by measuring reporter gene activity in mouse tissues after i.v. delivery of a plasmid vector. These results clearly demonstrate that 2apoEs + ADH6 is a novel regulatory module that allows for a highly efficient and specific expression of transgene in liver cells. Of particular importance is the small size of this module (1068 bp), which makes it possible to accommodate this transcriptional control unit in any type of viral or non-viral vector, even in conjunction with large effector genes. The 2apoEs + ADH6 transcriptional control unit therefore represents an invaluable new tool to explore the feasibility of liver-directed gene therapy in further detail. Acknowledgements We thank Claudia Cybon for excellent technical assistance with the animal experiments. This work was supported by a grant from the Dr. Mildred Scheel Stiftung fu¨r Krebsforschung. References Allan, C.M., Taylor, S., Taylor, J.M., 1997. Two hepatic enhancers, HCR.1 and HCR.2, coordinate the liver expression of the entire human apolipoprotein E/C-I/C-IV/C-II gene cluster. J. Biol. Chem. 272, 29113 – 29119. Bean, M.A., Bloom, B.R., Herberman, R.B., Old, L.J., Oettgen, H.F., Klein, G., Terry, W.D., 1975. Cell-mediated cytotoxicity for bladder carcinoma: evaluation of a workshop. Cancer Res. 35, 2902 – 2913. Boccia, L.M., Lillicrap, D., Newcombe, K., Mueller, C.R., 1996. Binding of the Ets factor GA-binding protein to an upstream site in the factor IX promoter is a critical event in transactivation. Mol. Cell. Biol. 16, 1929 – 1935. Bowling, W.M., Kennedy, S.C., Cai, S.R., Duncan, J.R., Gao, C., Flye, M.W., Ponder, K.P., 1996. Portal branch occlusion safely facilitates in vivo retroviral vector transduction of rat liver. Hum. Gene Ther. 7, 2113 – 2121.
Dang, Q., Walker, D., Taylor, S., Allan, C., Chin, P., Fan, J., Taylor, J., 1995. Structure of the hepatic control region of the human apolipoprotein E/C-I gene locus. J. Biol. Chem. 270, 22577 – 22585. Falzon, M., McMahon, J.B., Gazdar, A.F., Schuller, H.M., 1986. Preferential metabolism of N-nitrosodiethylamine by two cell lines derived from human pulmonary adenocarcinomas. Carcinogenesis 7, 17 – 22. Fujimoto, J., 2000. Gene therapy for liver cirrhosis. J. Gastroenterol. Hepatol. 15, D33 – D36 (Suppl.). Ghosh, S.S., Takahashi, M., Thummala, N.R., Parashar, B., Chowdhury, N.R., Chowdhury, J.R., 2000. Liver-directed gene therapy: promises, problems and prospects at the turn of the century. J. Hepatol. 32, 238 – 252. Hafenrichter, D.G., Wu, X., Rettinger, S.D., Kennedy, S.C., Flye, M.W., Ponder, K.P., 1994. Quantitative evaluation of liver-specific promoters from retroviral vectors after in vivo transduction of hepatocytes. Blood 84, 3394 – 3404. Hoag, H., Gore, J., Barry, D., Mueller, C., 1999. Gene therapy expression vectors based on the clotting Factor IX promoter. Gene Ther. 6, 1584 – 1589. Je´roˆme, V., Mu¨ller, R., 2001. A synthetic leucine zipper-based dimerization system for combining multiple promoter specificities. Gene Ther. 8, 725 – 729. Liu, F., Song, Y., Liu, D., 1999. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Ther. 6, 1258 – 1266. Manno, C.S., 2002. Gene therapy for bleeding disorders. Curr. Opin. Hematol. 9, 511 – 515. Miao, C.H., Ohashi, K., Patijn, G.A., Meuse, L., Ye, X., Thompson, A.R., Kay, M.A., 2000. Inclusion of the hepatic locus control region, an intron, and untranslated region increases and stabilizes hepatic factor IX gene expression in vivo but not in vitro. Mol. Ther. 1, 522 – 532. Mitry, R.R., Mansour, M.R., Havlik, R., Habib, N.A., 2000. Gene therapy for liver tumours. Adv. Exp. Med. Biol. 465, 193 – 205. Molowa, D.T., Chen, W.S., Cimis, G.M., Tan, C.P., 1992. Transcriptional regulation of the human cholesterol 7 alpha-hydroxylase gene. Biochemistry 31, 2539 – 2544. Nettelbeck, D.M., Je´roˆme, V., Mu¨ller, R., 1998. A strategy for enhancing the transcriptional activity of weak cell type-specific promoters. Gene Ther. 5, 1656 – 1664. Nettelbeck, D.M., Je´roˆme, V., Mu¨ller, R., 2000. Gene therapy: designer promoters for tumour targeting. Trends Genet. 16, 174 – 181. Oka, K., Davis, A.R., Chan, L., 2000. Recent advances in liver-directed gene therapy: implications for the treatment of dyslipidemia. Curr. Opin. Lipidol. 11, 179 – 186. Okuyama, T., Fujino, M., Li, X.K., Funeshima, N., Kosuga, M., Saito, I., Suzuki, S., Yamada, M., 1998. Efficient Fas-ligand gene expression in rodent liver after intravenous injection of a recombinant adenovirus by the use of a Cre-mediated switching system. Gene Ther. 5, 1047 – 1053. Park, F., Ohashi, K., Kay, M.A., 2000. Therapeutic levels of human factor VIII and IX using HIV-1-based lentiviral vectors in mouse liver. Blood 96, 1173 – 1176. Prieto, J., Herraiz, M., Sangro, B., Qian, C., Mazzolini, G., Melero, I., Ruiz, J., 2003. The promise of gene therapy in gastrointestinal and liver diseases. Gut 52 (Suppl. 2), ii49 – ii54. Rouet, P., Raguenez, G., Tronche, F., Yaniv, M., N’Guyen, C., Salier, J.P., 1992. A potent enhancer made of clustered liver-specific elements in the transcription control sequences of human alpha 1-microglobulin/bikunin gene. J. Biol. Chem. 267, 20765 – 20773. Schmitz, V., Qian, C., Ruiz, J., Sangro, B., Melero, I., Mazzolini, G., Narvaiza, I., Prieto, J., 2002. Gene therapy for liver diseases: recent strategies for treatment of viral hepatitis and liver malignancies. Gut 50, 130 – 135. Shen, R.F., Clift, S.M., DeMayo, J.L., Sifers, R.N., Finegold, M.J., Woo, S.L., 1989. Tissue-specific regulation of human alpha 1-antitrypsin gene expression in transgenic mice. DNA 8, 101 – 108. Simonet, W.S., Bucay, N., Lauer, S.J., Taylor, J.M., 1993. A far-downstream hepatocyte-specific control region directs expression of the
S. Gehrke et al. / Gene 322 (2003) 137–143 linked human apolipoprotein E and C-I genes in transgenic mice. J. Biol. Chem. 268, 8221 – 8229. Xiao, W., Berta, S.C., Lu, M.M., Moscioni, A.D., Tazelaar, J., Wilson, J.M., 1998. Adeno-associated virus as a vector for liver-directed gene therapy. J. Virol. 72, 10222 – 10226. Yasunami, M., Chen, C.S., Yoshida, A., 1991. A human alcohol dehydrogenase gene (ADH6) encoding an additional class of isozyme. Proc. Natl. Acad. Sci. U. S. A. 88, 7610 – 7614.
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Chimeric transcriptional control units for improved liver-specific transgene expression Stephan Gehrke, Vale´rie Je´roˆme, Rolf Mu¨ller * Institute of Molecular Biology and Tumor Research (IMT), Philipps-University, Emil-Mannkopff-Strasse 2, 35033 Marburg, Germany Received 2 July 2003; received in revised form 5 August 2003; accepted 18 August 2003 Received by M. Schartl
Abstract Hepatocyte-directed delivery of therapeutic genes is a major field of gene therapy. An important issue in this context is the availability of promoters units providing for maximum transcriptional activity and specificity. Although a number of liver-specific promoters and transcriptional control elements have been identified and used for gene delivery, no systematic study has been performed to identify the best suitable combination of known liver-specific promoter and enhancer elements. We now report the results of a comparative investigation addressing this issue. We tested a total of 25 synthetic transcriptional control units consisting of either of the four core promoters from liverspecific genes linked in various combinations and configurations to hepatocyte-specific enhancer elements. These constructs were analyzed for transcriptional activity in different cell types in cell culture and in mouse liver in vivo. The data lead to the clear conclusion that a combination of the alcohol dehydrogenase 6 (ADH6) basal promoter linked to two tandem copies of an apoplipoprotein E enhancer element is the transcriptional control unit of choice for the liver-specific expression of transgenes. D 2003 Elsevier B.V. All rights reserved. Keywords: Liver-specific genes; ADH promoter; apoE promoter; Alpha1-microglobulin/bikunin promoter; Alpha-antitrypsin promoter
1. Introduction The liver represents a target of major interest for gene therapy. Hepatocytes fulfill a great variety of essential functions ranging from metabolism and homeostasis to immune responses that are frequently perturbed due to monogenetic dysfunctions. Moreover, the liver is a potentially well suitable site for the ectopic expression of therapeutic proteins with, for instance, immune modulatory, tumoricidal or anti-angiogenic properties. Indeed, a large number of studies, including clinical trials in humans, have addressed the possibility to employ gene therapeutic strategy for the treatment of diverse diseases, such as defect in lipid metabolism or blood clotting, liver cancer or metastasis, hepatitis and liver cirrhosis (FujiAbbreviations: ABP, alpha1-microglobulin/bikunin; ADH6, alcohol dehydrogenase 6; CMV, cytomegalo virus; CYP, cholesterol 7alphahydroxylase; FBS, fetal bovine serum; FIX, clotting factor IX; apoE, apolipoprotein E/C-I; hAAT, human alpha-antitrypsin; HCR-1, hepatic control region-1; SV40, simian virus 40. * Corresponding author. Tel.: +49-6421-286236; fax: +49-6421288923. E-mail address: [email protected] (R. Mu¨ller). 0378-1119/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2003.08.010
moto, 2000; Ghosh et al., 2000; Mitry et al., 2000; Oka et al., 2000; Schmitz et al., 2002; Prieto et al., 2003). In most cases, gene transfer into the liver has been attempted by the administration via different routes of DNA or viral vectors based on retroviruses (Hafenrichter et al., 1994), adenoviruses (Okuyama et al., 1998), adeno-associated viruses (Xiao et al., 1998) or lentiviruses (Park et al., 2000). Major limitations of these transduction systems include an inefficient or unstable transduction and a lack of tissue-specificity (Ghosh et al., 2000). These problems may be in part interrelated, as non-specific transgene expression can cause immune responses against the transduced cells which in turn severely limits the stability of transduction. Tissue-specific transgene expression is obviously highly desirable for gene therapy and can in principle be achieved through transcriptional targeting, i.e. the use of transcriptional control units that restrict transgene expression to the cell type of interest (Nettelbeck et al., 2000). Transcriptional targeting can be used to compensate for the lack of specificity of the available vector systems, thereby allowing for the efficient and stable expression of transgenes, including genes coding for proteins whose proper function is depen-
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dent on their synthesis in the correct cell type due to, for instance, the requirement for defined post-translational modifications. An example in this context is the synthesis of blood clotting factors in the liver (Manno, 2002). Even though many tissue-specific promoters have been described their applicability to gene therapy is frequently limited due to low activity and/or large size which can pose a serious obstacle for the construction of viral vectors. This also applies to hepatocyte-directed gene therapy, where a number of tissue-specific promoters have been used, but systematic studies pertaining at the design of an optimal transcriptional control unit have not been published. Ideally, such a control unit should be as small as possible while maintaining a high transcriptional activity and cell type specificity. In the present study, we have addressed this problem by constructing a total of 25 synthetic transcriptional control units consisting of core promoters from liverspecific genes linked in various combinations and configurations to hepatocyte-specifc enhancer elements, and analyzing these constructs for transcriptional activity in different cell types in cell culture and in mouse liver in vivo. The transcription regulatory elements used in this study were derived from the human genes coding for alcohol dehydrogenase 6 (ADH6) (Yasunami et al., 1991; Zhi et al., 2000), human alpha-antitrypsin (hAAT) (Shen et al., 1989; Hafenrichter et al., 1994), cholesterol 7alpha-hydroxylase (CYP) (Molowa et al., 1992), clotting factor IX (FIX) (Boccia et al., 1996; Hoag et al., 1999), apolipoprotein E/C-I (apoE) (Simonet et al., 1993; Allan et al., 1997) and alpha1-microglobulin/bikunin (ABP) (Rouet et al., 1992). These studies led to the clear conclusion that the ADH6 basal promoter linked to two tandem copies of an apoE enhancer element allows for the highest level of liver-specific transcription.
2. Materials and methods 2.1. Cell culture HepG2 (ATCC HB 8065; hepatoma), MeWo (Bean et al., 1975), MCF7 (ATCC HTB-22; breast cancer) and A431 (ATCC CRL-1555; epidermoid carcinoma) were cultured in DMEM with L-glutamine (Boehringer Ingelheim BioProducts, Heidelberg, Germany) and 10% fetal bovine serum (FBS; Life Technologies, Eggenstein, Germany). H322 (Falzon et al., 1986) and PC3 (ATCC CRL 1435; prostate carcinoma) were grown in RPMI (Boehringer Ingelheim BioProducts) with 10% FBS. All cells were kept at 37 jC in a humidified atmosphere of 5% CO2. 2.2. Construction of plasmids All core promoters were synthesized by PCR from HepG2 genomic DNA using pfu DNA-polymerase and cloned as BglII and HindIII-fragments into the pGL3-basic luciferase vector (Promega, Mannheim, Germany). Sequence and ori-
entation were verified by DNA sequencing. The promoters contained the following sequences: ADH6, 342 to + 57 (Yasunami et al., 1991; Zhi et al., 2000); hAAT, 347 to + 56 (Shen et al., 1989; Hafenrichter et al., 1994; Bowling et al., 1996; Miao et al., 2000); CYP, 370 to + 63 (Molowa et al., 1992); and FIX, 219 to + 21 (Boccia et al., 1996). The core promoters were linked to the following enhancer sequences in the pGL3-basic luciferase vector by standard ligation techniques: a 319-bp apoE enhancer containing the hepatic control region (HCR-1) (Simonet et al., 1993; Dang et al., 1995; Allan et al., 1997); a 190-bp ABP enhancer region spanning nucleotides 2824 to 2634 (Rouet et al., 1992); a quadruplet of the site 5 enhancer element of the FIX gene spanning nucleotides 219 to 190 (Hoag et al., 1999). The simian virus 40 (SV40) promoter/enhancer luciferase vector pGL3 was obtained from Promega. 2.3. Transfection of plasmids Transfection was performed with DOTAP following the supplier’s protocol (Roche diagnostics, Mannheim, Germany). For each assay 105 cells were transfected with 5 Ag of plasmid DNA. Luciferase assays were performed as described (Nettelbeck et al., 1998). The SV40 promoter luciferase vector was used for standardization. Relative promoter activities were calculated using the following formula: standardized relative RLUs=(RLUs sample/background RLUs)/(RLUs pGL3-background). The standardized relative RLUs from three independent experiments were combined to calculate averages and standard deviations. 2.4. In vivo studies The hydrodynamics-based transfection technique described previously (Liu et al., 1999; Zhang et al., 1999) was followed. Briefly, NMRI nu/nu mice were rapidly injected into the tail vein with plasmid DNA (1 Ag/g body weight) in Ringer’s solution (147 mM NaCl, 4 mM KCl, 1.13 mM CaCl2). For standardization, a cytomegalo virus (CMV) promoter/enhancer-renilla luciferase plasmid was co-injected at a ratio of 1:50 (renilla luciferase plasmid: firefly luciferase plasmid). The mice were killed 24 h after injection, the livers were removed and lysed in Passive lysis buffer (Promega) and renilla and firefly luciferase activities were determined using the dual-luciferase reporter assay system as previously described (Je´roˆme and Mu¨ller, 2001).
3. Results and discussion 3.1. Comparison of core promoters derived from different liver-specific genes Our strategy for constructing an optimal liver-specific expression cassette involved four steps: (i) identification of a core promoter enabling efficient basal transcription in cul-
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tured hepatoma cells; (ii) identification of an enhancer module providing for a strong and liver-specific stimulation of transcription and (iii) verification of the results in vivo. To identify a suitable basal promoter we assessed the strength of different core promoters of liver-specific genes in transient luciferase assays in human hepatoma cells. Promoter sequences were chosen for analyses on the basis that their function as basal promoters had been defined in previous studies. These included the promoters of the alcohol dehydrogenase 6 (ADH6) (Yasunami et al., 1991; Zhi et al., 2000), the human alpha-antitrypsin (hAAT) (Shen et al., 1989; Hafenrichter et al., 1994), the cholesterol 7alpha-hydroxylase (CYP) (Molowa et al., 1992) and the clotting factor IX (FIX) (Boccia et al., 1996; Hoag et al., 1999) genes. Transient transfection experiments using the human hepatoma cell line HepG2 (Fig. 1) showed that both the ADH6 and the hAAT promoter gave rise to high levels of basal transcription (9.6- and 6.3-fold, respectively, relative to the SV40 promoter), while both the CYP and FIX promoters were considerably weaker (16% and 12%, respectively, of the SV40 promoter). 3.2. Effect of different liver-specific enhancers on transcriptional activity We next addressed the question which enhancer elements would work best in conjunction with the basal promoter of the ADH6 gene. For this purpose, we constructed enhancer modules containing elements from three different liverspecific genes: the HCR-1 region of the apoE gene (Allan et al., 1997), a far upstream enhancer region of the ABP gene (Rouet et al., 1992) and the site 5 enhancer domain of the FIX gene (Hoag et al., 1999). In total 19 transcriptional control units containing these enhancer elements in different configurations were designed, i.e. single copies, two copies of the same element in the same or opposite orientation and combinations of different enhancer elements in different orientations and orders (see Fig. 2). The results corroborated
Fig. 1. Transcriptional activities of core promoters derived from liverspecific genes. Promoter – luciferase constructs were transiently transfected into HepG2 cells and luciferase activities were determined 48 h later. Values are given as multiples of basal SV40 promoter activity and represent average values of three independent experiments F standard deviations. ADH6: human alcohol dehydrogenase 6; hAAT: human alpha-antitrypsin; CYP: human cholesterol 7alpha-hydroxylase; FIX: human clotting factor IX.
Fig. 2. Schematic representation of enhancer modules analyzed in the present study. apoE: apolipoprotein E/C-I; ABP: alpha1-microglobulin/ bikunin; site5: enhancer box of the clotting factor IX.
in Fig. 3 lead to the following conclusions: (i) apoE inserted in ‘‘sense’’ orientation is the strongest enhancer when used as a single copy (50-fold relative to SV40 promoter); (ii) a strong enhancement of transcription was seen when two copies of the apoE or ABP elements were present in the expression cassette. In case of apoE, the orientation was critical (‘‘sense’’ only; 117-fold relative to SV40 promoter), whereas for ABP both ‘‘sense’’ and ‘‘antisense’’ orientations functioned equally (84-fold relative to SV40 promoter); (iii) of all combinations of enhancer elements, only the combination of site5 and ABP showed a high transcriptional activity (44-fold relative to SV40 promoter). These data clearly indicate that the highest level of transcription was achieved by the 2apoEs module. To confirm and extend these results we next generated constructs where the 2apoEs and 2ABPas enhancer modules were tested in the context of the four core promoters already used in Fig. 1. As shown in Fig. 4 and in keeping with the results described above, the combination of the ADH6 core promoter and the 2apoEs enhancer module proved to drive transcription in HepG2 cells most efficiently (117-fold relative to SV40 promoter), followed by the 2apoEs + hAAT, 2ABPas + ADH6 and 2ABPas + hAAT constructs, which worked equally well (84-fold relative to SV40 promoter). In addition, in agreement with the data in Fig. 1, the ADH6 and hAAT core promoters were clearly superior to the basal promoters of the CYP and FIX genes with respect to transcriptional activity in HepG2 cells.
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Fig. 3. Transcriptional activities in HepG2 cells of synthetic expression cassettes combining the ADH6 core promoter and different enhancer modules derived from liver-specific genes (see Fig. 2) after transient transfection into HepG2 cells. pGL3-control: SV40 promoter with early region enhancer; core: ADH6 core promoter. Values are given as multiples of basal SV40 promoter activity and represent average values of three independent experiments F standard deviations.
3.3. Cell type specificity of the chimeric transcriptional control units The next important issue to address was cell type specificity of the transcriptional control units. We therefore transfected in addition to HepG2 several other cell lines originating from different tissues with the 2apoEs + ADH6, 2ABPas + ADH6, 2apoEs + hAAT, and 2ABPas + hAAT enhancer –promoter units (and for comparison with the ADH6 and hAAT core promoter constructs). The transfected cell lines included H322 lung carcinoma, PC-3 prostate carcinoma, A431 vulva carcinoma, MCF-7 mammary carcinoma and MeWo melanoma. The data in Fig. 5 support the following conclusions: (i) all constructs showed the highest level of transcriptional activity in HepG2 cells; (ii) cell type
specificity was already evident with the core promoter constructs, but the degree of specificity was relatively low when compared to PC-3 cells (7- to 18-fold); this specificity could be greatly enhanced by addition of either the 2apoE or the ABP enhancer modules. With all four enhancer –promoter constructs the highest degree of non-specific expression was seen with H322 cells, but specificity was very high even in this case (76- to 117-fold). Specificity was greatest with the constructs containing the ADH6 basal promoter, as confirmed by Student’s t-test. This is the same enhancer – promoter combination that we had identified above as the transcriptional control unit giving rise to the highest level of transcription (Figs. 3 and 4), and thus appears to be the combination of choice for liver-specific expression of transgenes.
Fig. 4. Transcriptional activities in HepG2 cells of different basal promoters linked to apoE- and ABP-derived enhancer modules. Values are given as multiples of basal SV40 promoter activity and represent average values of three independent experiments F standard deviations.
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Fig. 5. Cell type specificity of expression cassettes consisting of different basal promoters and enhancer modules. Luciferase – promoter constructs were transiently transfected into different cell lines and luciferase activities were determined 48 h later. Values are given as multiples of basal SV40 promoter activity and represent average values of three independent experiments F standard deviations. HepG2: hepatoma; H322: lung carcinoma; PC-3: prostate carcinoma; A431: vulva carcinoma; MCF-7; mammary carcinoma; MeWo: melanoma.
3.4. Transcriptional activity in vivo
3.5. Conclusions
To test the optimal promoter –enhancer combinations identified above in an in vivo setting, we determined the transcriptional activity of four apoEs-based enhancer – promoter constructs in mouse liver relative to the SV40 promoter. Plasmid DNA was delivered i.v. and the firefly luciferase activity in liver was determined 24 h later. For standardization we co-injected a plasmid containing a renilla luciferase gene driven by the CMV-promoter/early-enhancer. As can be seen in Fig. 6, the two plasmids with two copies of the apoEs enhancer module gave rise to a f 3.5-fold higher level of gene luciferase activity compared to the constructs with single apoEs modules, irrespective of the basal promoter used (ADH6 or hAAT). The transcriptional activity achieved with both 2apoEs plasmids was very high and reached 40- to 45-fold the activity of the SV40 promoter activity. In contrast to the liver, other organs (spleen, kidney, heart, lung and muscle) showed only very little luciferase activity that was too low to allow for a precise determination of tissue-specificity (data not shown). Taken together, the in vivo data fully confirm the observations made with cultured HepG2 cells and support the conclusion that the 2apoEs + ADH6 combination is a particularly well suitable liver-specific transcriptional unit.
Successful liver-directed gene therapy will critically depend on the availability of small, efficient and specific transcriptional control units. In the present study, we pursued this goal by constructing chimeric regulatory units
Fig. 6. Transcriptional activities of the ADH6 and hAAT basal promoters linked to apoE-derived enhancer modules in mouse liver. Promoter constructs were injected i.v. along with a reporter plasmid used for standardization (CMV promoter/enhancer linked to renilla luciferase) and both luciferase activities were determined 24 h later.
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consisting of basal promoter and enhancer elements from different liver-specific genes. The analysis of a total of 19 such constructs in transient transfection assays with hepatoma cells showed that the isolated core promoters of CYP and FIX genes were clearly inferior to the core promoter regions of the hAAT and ADH6 genes, the latter giving rise to the highest level of basal transcription. Of the different enhancer configurations tested, the most efficient construction turned out to be a head-to-tail tandem apoE sequence which was superior to similar configurations made up of the ABP enhancer domain, single copies of the apoE, ABP or site 5 enhancers or the combinations of heterologous enhancer modules. Importantly, the ability of the tandem apoE construct to drive transcription in a particularly efficient way was associated with a very high degree of cell type specificity, which was in the range of two orders of magnitude when compared to tumor cell lines derived from other tissues of origin, including lung, breast, prostate, epidermis and melanocytes. The functionality of the 2apoEs + ADH6 transcriptional control unit for liver-directed gene delivery could be confirmed in vivo by measuring reporter gene activity in mouse tissues after i.v. delivery of a plasmid vector. These results clearly demonstrate that 2apoEs + ADH6 is a novel regulatory module that allows for a highly efficient and specific expression of transgene in liver cells. Of particular importance is the small size of this module (1068 bp), which makes it possible to accommodate this transcriptional control unit in any type of viral or non-viral vector, even in conjunction with large effector genes. The 2apoEs + ADH6 transcriptional control unit therefore represents an invaluable new tool to explore the feasibility of liver-directed gene therapy in further detail. Acknowledgements We thank Claudia Cybon for excellent technical assistance with the animal experiments. This work was supported by a grant from the Dr. Mildred Scheel Stiftung fu¨r Krebsforschung. References Allan, C.M., Taylor, S., Taylor, J.M., 1997. Two hepatic enhancers, HCR.1 and HCR.2, coordinate the liver expression of the entire human apolipoprotein E/C-I/C-IV/C-II gene cluster. J. Biol. Chem. 272, 29113 – 29119. Bean, M.A., Bloom, B.R., Herberman, R.B., Old, L.J., Oettgen, H.F., Klein, G., Terry, W.D., 1975. Cell-mediated cytotoxicity for bladder carcinoma: evaluation of a workshop. Cancer Res. 35, 2902 – 2913. Boccia, L.M., Lillicrap, D., Newcombe, K., Mueller, C.R., 1996. Binding of the Ets factor GA-binding protein to an upstream site in the factor IX promoter is a critical event in transactivation. Mol. Cell. Biol. 16, 1929 – 1935. Bowling, W.M., Kennedy, S.C., Cai, S.R., Duncan, J.R., Gao, C., Flye, M.W., Ponder, K.P., 1996. Portal branch occlusion safely facilitates in vivo retroviral vector transduction of rat liver. Hum. Gene Ther. 7, 2113 – 2121.
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