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37 Kiyono, T. et al. (1998) Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithlial cells. Nature 396, 6706 38 Esteller, M. et al. (1999) hMLH1 promoter hypermethylation is an early event in human endometrial tumorigenesis. Am. J. Pathol. 155, 1767–1772 39 Myohanen, S.K. et al. (1998) Hypermethylation can selectively silence individual p16ink4A alleles in neoplasia. Cancer Res. 58, 591–593 40 Jones, P.L. et al. (1998) Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription. Nat. Genet. 19, 187–191 41 Nan, X. et al. (1998) Transcriptional repression by the methylCpG-binding protein MeCP2 involves a histone deacetylase complex. Nature 393, 386 42 Wade, P.A. et al. (1999) Mi-2 complex couples DNA methylation to chromatin remodelling and histone deacetylation. Nat. Genet. 23, 62–66 43 Ng, H-H. et al. (1999) MBD2 is a transcriptional repressor belonging to the MeCP1 histone deacetylase complex. Nat. Genet. 23, 58–61 44 Cameron, E.E. et al. (1999) Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nat. Genet. 21, 103–107 45 Mannervik, M. et al. (1999) Transcriptional coregulators in development. Science 284, 606–609 46 Struhl, K. (1998) Histone acetylation and transcriptional regulatory mechanisms. Genes Dev. 12, 599–606 47 Toyota, M. et al. (1999) CpG island methylator phenotype in colorectal cancer. Proc. Natl. Acad. Sci. U. S. A. 96, 8681–8686 48 Melki, J.R. et al. (1999) Concurrent DNA hypermethylation of multiple genes in acute myeloid leukemia. Cancer Res. 59, 3730–3740 49 Esteller, M. et al. (1999) Hypermethylation-associated inactivation of p14ARF is independent of p16INK4A methylation and p53 mutational status. Cancer Res., 60, 129–133 50 Esteller, M. et al. (1999) Detection of aberrant promoter hypermethylation of tumor suppressor genes in serum DNA

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from non-small cell lung cancer patients. Cancer Res. 59, 67–70 Beard, C. et al. (1995) Loss of methylation activates Xist in somatic but not in embryonic cells. Genes Dev. 9, 2325–2334 Li, E. (1997) Role of DNA methylation in development in genomic imprinting. In Frontiers in Molecular Biology (Reik, W. and Surani, A., eds), pp 1–20, IRL Press Okano, M. et al. (1998) Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases. Nat. Genet. 19, 219–220 Okano, M. et al. (1999) DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99, 247–257 Lengauer, C. et al. (1997) DNA methylation and genetic instability in colorectal cancer cells. Proc. Natl. Acad. Sci. U. S. A. 94, 2545–2550 Robertson, K.D. et al. (1999) The human DNA methyltransferases (DNMTs) 1, 3a and 3b: coordinate mRNA expression in normal tissues and overexpression in tumors. Nucleic Acids Res. 27, 2291–2298 Wu, J. et al. (1993) Expression of an exogenous eukaryotic DNA methyltransferase gene induces transformation of NIH 3T3 cells. Proc. Natl. Acad. Sci. U. S. A. 90, 8891–8895 Bakin, A.V. and Curran, T. (1999) Role of DNA 5-methylcytosine transferase in cell transformation by fos. Science 283, 387–390 Laird, P.W. et al. (1995) Suppression of intestinal neoplasia by DNA hypomethylation. Cell 81, 197–205 MacLeod, A.R. and Szyf, M. (1995) Expression of antisense to DNA methyltransferase mRNA induces DNA demethylation and inhibits tumorigenesis. J. Biol. Chem. 270, 8037–8043 Vertino, P.M. et al. (1996) De Novo methylation of CpG island sequences in human fibroblasts overexpressing DNA (cytosine5-)-methyltransferase. Mol. Cell Biol. 16, 4555–4565 Rhee, I. et al. CpG methylation is maintained in cancer cells lacking DNMT. Nature (in press) Macleod, D. et al. (1998) An alternative promoter in the mouse major histocompatibility complex class II I-Abeta gene:

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implications for the origin of CpG islands. Mol. Cell Biol. 18, 4433–4443 Brandeis, M. et al. (1994) Sp1 elements protect a CpG island from de novo methylation. Nature 29, 435–538 Macleod, D. et al. (1994) Sp1 sites in the mouse aprt gene promoter are required to prevent methylation of the CpG island. Genes Dev. 8, 2282–2292. Marin, M. et al. (1997) Transcription factor Sp1 is essential for early embryonic development but dispensable for cell growth and differentiation. Cell 4, 619–628 Siegfried, Z. et al. (1999) DNA methylation represses transcription in vivo. Nat. Genet. 22, 203–206 Pollard, K.J. and Peterson, C.L. (1998) Chromatin remodeling: a marriage between two families? BioEssays 20, 771–780 Jeddeloh, J.A. et al. (1999) Maintenance of genomic methylation requires a SWI2/SNF2-like protein. Nat. Genet. 22, 94–97 Herman, J.G. et al. (1996) Methylation-specific PCR: A novel PCR assay for methylation status of CpG islands. Proc. Natl. Acad. Sci. U. S. A. 93, 9821–9826 Sadri, R. and Hornsby, P.J. (1996) Rapid analysis of DNA methylation using new restriction enzyme sites created by bisulfite modification. Nucleic Acids Res. 24, 5058–5059 Wong, I.H.N. et al. (1999) Detection of aberrant p16 methylation in the plasma and serum of liver cancer patients. Cancer Res. 59, 71–73 Momparler, R.L. et al. (1997) Pharmacological approach for optimization of the dose schedule of 5-Aza-2’-deoxycytidine (Decitabine) for the therapy of leukemia. Leukemia 11, S1–S6 Juttermann, R. et al. (1994) Toxicity of 5-aza-2’-deoxycytidine to mammalian cells is mediated primarily by covalent trapping of DNA methyltransferase rather than DNA demethylation. Proc. Natl. Acad. Sci. U. S. A. 91, 11797–11801 Ferguson, A.T. et al. (1997) Role of estrogen receptor gene demethylation and DNA methyltransferase DNA adduct formation in 5-Aza-2’-deoxycytidine-induced cytotoxicity in human breast cancer cells. J. Biol. Chem. 272, 32260–32266

Gene therapy designer promoters for tumour targeting

Dirk M. Nettelbeck [email protected] Valérie Jérôme [email protected] Rolf Müller [email protected] Institute of Molecular Biology and Tumor Research (IMT), Philipps-University Marburg, EmilMannkopff-Strasse 2, D-35033 Marburg, Germany. 174

One of the biggest challenges facing cancer therapy is to generate tumour-specific treatment strategies. Gene therapy hopes to achieve this by directing the activity of therapeutic genes specifically to the sites of disease. Of paramount importance for the success of this approach is the availability of tumour-specific delivery systems: both the transductional targeting of the vector vehicle and the restriction of transgene expression to the tumour are promising strategies towards this goal. This review will focus on the recent achievements in the field of transcriptional targeting and the different strategies to improve or design promoters with the desired specificities.

O

ne of the major problems of the conventional noninvasive cancer therapy regimen is their lack of tumour specificity, which frequently causes serious side effects and limits the therapeutic dose. Owing to this narrow therapeutic window, most metastatic and locally advanced malignancies are refractory to treatment. There is, therefore, a pressing need for the development of new approaches to cancer therapy. In this respect, the application of genetic therapy to cancer has attracted great attention1. However, gene therapy for cancer is also faced with a specificity problem: the specific targeting of transgene TIG April 2000, volume 16, No. 4

expression to the site of the tumour. Because the targeted transduction of genes still represents a major obstacle, promoters that are active in a wide range of cell types are only of limited use. This applies, for instance, to the popular cytomegalovirus (CMV) immediate-early promoter, which, as a viral sequence, has the additional problem of being frequently shut-down in vivo. Therefore, achieving high levels of transcription in defined cell populations through specific cellular promoters or regulatory elements is of considerable interest. Some of the recently described experimental gene therapy protocols do indeed make use 0168-9525/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0168-9525(99)01950-2

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of natural tissue-specific promoters, but in many instances these promoters suffer from a lack of activity, specificity or both. Many laboratories have therefore focussed on the design of improved promoters for cancer gene therapy, and this review will give a brief account of the recent successful developments in this research area, including the promising approach to restrict viral vector replication to tumour cells via the transcriptional regulation of viral genes.

Tissue-specific promoters Many tissue-specific promoters have been isolated and characterized, and some of these have been used in experimental models of cancer gene therapy (see Table 1). Obviously, tissue-specific promoters are also active in the normal tissue from which the tumour originated. Unless the loss of these normal cells is acceptable – as in the case of melanocytes [e.g. using the tyrosinase or tyrosinaserelated protein –1 (TRP-1) promoter2–7] or prostate cells [e.g. using the prostate-specific antigen (PSA) promoter8–10] – additional specificity mechanisms need consideration to make the resulting vector useful for systemic delivery. This can be achieved by targeting the vector to a defined population of cells, for instance by using a proliferation-dependent retroviral vector in conjunction with a tissue-specific promoter11. An alternative approach would be to introduce an additional level of specificity into the tissuespecific promoter, such as a specificity for proliferating cells or a selectivity for other conditions that are characteristic of tumour cells.

Tumour endothelium-directed promoters Targeting the tumour vasculature by gene therapy has several potential advantages compared with direct tumourcell targeting. The tumour blood vessels are more readily accessible to vectors, endothelial cells (ECs) are not known to undergo mutations or to gain resistance to treatment, and the endothelium represents a target that is largely independent of tumour type. The tumour endothelium differs from the normal vasculature not only in the expression of membrane-associated receptors, adhesion molecules and other proteins, but also in the high proportion of proliferating cells12,13. Exploiting these differences might represent a powerful strategy in the designing of vectors that selectively target the tumour vasculature through transductional or transcriptional targeting. The latter could be achieved by using synthetic promoters combining multiple specificities (e.g. cell-cycle regulation and EC specificity) or natural promoters that are preferentially active in tumour-associated ECs. Promoters that are upregulated in tumour ECs are exemplified by those of the genes encoding vascular endothelial growth factor receptor kinase insert domain receptor (KDR; mouse homologue Flk1)14, E-selectin14,15 and the transforming growth factor-b binding protein endoglin/CD105 (Ref. 16; see also Table 1).

Tumour-selective promoters A set of genes has been identified that show little or no activity in the cells of an adult organism under non-pathological conditions, but that are turned on or upregulated in certain types of tumours (see Table 1). Exploiting these tumour-specific abnormalities might represent a powerful way to improve the targeting achieved by tumour-directed vectors. This applies, for instance, to the promoter of the gene encoding oncofetal a-fetoprotein (AFP)17–21, which, under non-pathological conditions, is active specifically in

the foetal liver, but becomes reactivated in hepatoma cells. Another example is the promoter of the gene encoding carcinoembryonal antigen (CEA)22–27, which is reactivated in different types of adenocarcinoma. Another interesting group of promoters is induced by disease-specific conditions (see Table 1). Disease-specific conditions with respect to malignant tumours are caused, for instance, by their highly pathological blood vessels. The resulting inadequate blood supply leads to hypoxic conditions in most areas of the tumour. Based on these observations, a promoter responding to hypoxia through activation by the hypoxia-inducible factor-1 (HIF-1) has been successfully used for experimental gene therapy for cancer28. Disease-specific conditions in tumour cells can also directly result from genetic alterations or from altered signaling pathways. The latter is presumably the reason for the frequent upregulation of the erbB-2 promoter in a range of adenocarcinomas, a feature that has also been exploited in experimental therapeutic models29–32 (see also Table 1). Another promoter often found upregulated in human adenocarcinomas is the mucin-1 gene (muc-1) promoter, although the underlying mechanism of overexpression in tumour cells remains unknown. Nevertheless, the muc-1 promoter has been used successfully in experimental gene therapy for cancer31,33,34. Tumour-specific genomic rearrangements have also been exploited to construct tumour-specific artificial promoters. A translocation found in some rhabdomyosarcomas leads to the expression of a protein that comprises the DNA-binding and the transactivation domains of two different transcription factors, the paired box gene 3 (PAX3) and forkhead in rhabdomyosarcoma (FKHR). This novel transcriptional activator is tumour-cell specific because PAX3 is normally expressed specifically during prenatal development. Thus, an artificial promoter, consisting of multiple PAX3-binding sites in front of a minimal adenoviral E1B promoter, could be used to drive the expression of the desired transgene in a rhabdomyosarcoma-specific fashion (Fig. 1). Because the PAX3-binding sites repress transcription in the absence of PAX3–FKHR, a remarkable specificity (.100-fold compared with normal cells) was achieved35. Translocations generating transcription factors with novel properties are frequently found in human cancers suggesting that the strategy described for the PAX3–FKHR rearrangement might also prove useful for other malignancies. Finally, the expression of viral genes is a hallmark of certain tumours, such as Burkitt lymphoma. To direct gene expression selectively to Burkitt lymphoma cells promoter elements have been used that are responsive to the Epstein–Barr Virus (EBV)-encoded transcriptional activators EBV nuclear antigen 1 (EBNA1) or EBNA2 (Refs 36–38). In addition, as described above for the PAX3 elements, the EBNA1-binding sites act as repressors of transcription in the absence of the viral proteins, so that upon activation an extremely high degree of specificity is achieved (up to 1000-fold relative to non-infected cells). Because viruses have been associated with several human cancers, in particular cervical carcinoma, this kind of approach might be applicable to a variety of human tumours.

Treatment-responsive promoters Another interesting scenario is the use of promoters that are inducible by certain conventional cancer therapy TIG April 2000, volume 16, No. 4

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TABLE 1. Natural promoters used in experimental cancer gene therapy Promoter

Target

Tissue-specific Tyrosinase, TRP-1

Melanocytes

PSA

Prostate cancer

Albumin

Liver

MCK

Muscle

MBP

Oligodendrocytes glial cells

GFAP

Glial cells

NSE

Neurons

Tumour endothelium-directed KDR E-selectin Endoglin Tumour-selective AFP

Liver tumour

in vivo application

Reporter/ effector gene

Vector

Therapeutic effect

In vitro

In animal model

i.t. i.t., transduced cells i.v. i.t. i.v. Transduced cells i.m. and other tissues i.m. (producer cells) Injection into brain Injection into brain Injection into brain i.v. i.v.

LacZ tk tk Luc, PNP IL-2 Luc LacZ, antisense tk, PNP VZV-tk hAAT F VIII LacZ Luc, LacZ LacZ LacZ, tk LacZ GFP LacZ, tk LacZ tk FasL FasL

nv, re nv re nv re nv nv ad re re ad re ad re re ad AAV re HSV-1 nv ad ad

2 1 2 1 2 2 1 2 1 2 2 2 2 2 1 2 2 1 2 1 1 1

2 1 1 2 2 2 2 1 2 2 2 2 2 2 2 2 2 2 2 2 1* 1*

-

LacZ, TNF LacZ, TNF Luc Luc

re re ad nv

2 2 2 2

2 2 2 2

i.t Transduced cells i.p. i.t., i.v. Transduced cells, i.p. i.t., i.p. i.v., i.t. i.t. i.v. Transduced cells Transduced cells -

tk, VZV-tk tk, LacZ, CD CAT, tk LacZ, tk LacZ, CD tk CD CD tk GRPr LacZ, tk, Bax GRPr LacZ, tk tk LacZ, tk tk CD2, CD Neo, tk LacZ CAT, LacZ, tk

re ad nv ad ad ad re re ad, re, nv ad ad ad ad ad ad nv nv re ad nv, ad

1 1 1 1 2 1 1 1 1 2 + 2 1 1 1 1 1 1 2 1

2 1 1 2 1* 1* 1** 2 2 2 + 2 1 1 1 1 2 1 2 2

TNFa TNFa LacZ Luc 1 tk TNFa LacZ, CD-tk

nv ad, nv ad nv re ad

2 1 2 1 1 1

1 1 2 1*** 2 2

LacZ, tk Luc

ad nv

1 2

1 2

CEA

Many adenocarcinomas (breast, lung, colorectal carcinoma)

erbB2

Breast and pancreatic cancer

muc-1 (DF3)

Breast cancer

ALA, BLG Osteocalcin

Breast cancer Osteosarcoma, prostate cancer

SLPI HRE Grp78 (BIP) L-plastin hexokinase II

Ovarial, cervical carcinoma Solid tumours Solid tumours Cancer cells Cancer cells

Treatment-responsive egr-1, t-PA

Radiation induced

mdr-1 hsp70

Chemotherapy induced Heat-induced

Transduced cells i.t., i.m. i.t. i.t. -

Cell cycle-regulated E2F-1 cyclin A, cdc25C

Proliferating/malignant cells Proliferating cells

i.t. i.t.

-: not determined; * reduced liver toxicity in vivo compared to the same construct with a constitutive promoter; ** no bone marrow suppression (which is observed with the same construct including a constitutive promoter); *** egr-1 promoter activity upregulated in hepatocellular carcinoma compared with normal liver cells. Abbreviations: (vectors) AAV, adenoassociated virus; ad, adenoviral vector; HSV-1, herpes simplex virus-1; nv, non-viral vector; re, retroviral vector; (cDNAs), CAT, chloramphenicol acetyl-transferase; CD, Escherichia coli cytosine deaminase; CD2, cluster of differentiation antigen 2; aF VIII, blood coagulation factor VIII; GFP, green fluorescent protein; GRPr, gastrin releasing peptide receptor; tk, HSV thymidine kinase; neo, neomycine resistance; LacZ, b-galactosidase; Luc, luciferase; PNP, E.coli purine nucleoside phosphorylase; TNFa, tumor necrosis factor a; VZV-tk, varicella-zoster virus thymidine kinase; [genes (59-regulatory regions)], AFP, a-fetoprotein; ALA, a-lactalbumin; BLG, b-lactoglobin; CEA, carcinoembryonic antigen; egr-1, early growth response-1 gene; GFAP, glial fibrillary acidic protein; grp78 (BIP), glucose regulated protein 78; hsp70, heat shock protein 70; HRE, hypoxia response element; KDR, kinase insert domain containing receptor (human homologue of flk-1); MBP, myelin basic protein; MCK, muscle creatinine kinase; mdr-1, multiple drug resistance gene 1; muc-1, mucin-1; PSA, prostate-specific antigen; SLPI, secretory leukoprotease inhibitor; t-PA, tissue plasminogen activator; TRP-1, tyrosinase related protein-1; i.v., intravenous; i.p., intraperitoneal; i.t., intrautumoural. A full table including all references can be requested from the authors.

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modalities (see Table 1). Such promoters are interesting for the temporally and spatially restricted expression of genes whose products are able to sensitize the tumour cells to the actual treatment. These proteins – like tumour necrosis factor-a (TNF-a) – are often highly toxic, which precludes their systemic application and prevents the accumulation of therapeutically desirably levels at the site(s) of the tumour. In this context, promoters that are induced by therapeutic doses of ionizing radiation, such as the early growth response 1 (egr-1) promoter39–45, or by chemotherapeutic agents, such as the promoter of the P-glycoprotein/multidrug resistance-1 gene (mdr-1)46, have attracted some attention (see Table 1). Other promoters that might fulfil a similar purpose are those activated through the transcription factor nuclear factor kB (NFkB) in response to ionizing radiation and certain chemotherapeutic agents.

Cell cycle-regulated promoters Unrestrained cell proliferation is a hallmark of cancer. This is exemplified by the G1 checkpoint which is governed by the retinoblastoma protein (Rb) pathway. This pathway is lost in nearly all tumour cells47 owing to genetic alterations affecting Rb itself or its upstream regulators, cyclin D, CDK4 and p16INK4a. As a consequence, the transcription factor E2F loses its G0/G1-specific repressor function and is constitutively active. In an attempt to make use of these observations for gene therapy, the E2Fregulated promoter of the E2F-1-encoding gene has been incorporated into an adenoviral vector and shown to confer tumour-cell specific transgene expression in a setting of experimental gene therapy for glioma48. As hyperproliferation is characteristic of most tumour cells and tumour ECs, the promoters of cell-cycle genes that are regulated by other mechanisms, such as cyclin A or cdc25C, also represent potentially interesting tools for gene therapy in the treatment of cancer49,50 (see also Table 1).

Enhancing the activity of tissue-specific or other selective promoters As pointed out above, selectively active or inducible promoters play an important role in the development of siteselective vectors for gene therapy. Some of these promoters appear to be ideally suited for gene therapy because they combine strong transcriptional activity with a high degree of specificity. This is true, for instance, for the melanocyte-specific tyrosinase promoter2–7. Frequently, however, tissue-specific or other selective promoters are inefficient activators of transcription, which severely limits their applicability. A typical example is the von Willebrand factor (vWF) promoter, which exhibits a particularly high degree of EC specificity compared with other EC promoters, but which is a poor activator of transcription51. Several strategies for improving promoter strength, while maintaining specificity, have been described. Frequently, these approaches have also led to the design of promoters that are substantially smaller than the corresponding wild-type sequences, which can be a great advantage in view of the size limitations of many vectors currently used in gene therapy. The first and simplest approach is to eliminate from a natural promoter all the regions that do not contribute to its transcriptional strength or specificity and to multimerize the positive regulatory promoter elements or enhancer domains. This strategy has been used in several instances, for example: the carcinoembryonic antigen (CEA)

FIGURE 1. A synthetic promoter that binds a tumour-specific transcription factor Transcription start site

FKHR TAD

CAT DT-A

PAX3 DBD Rhabdomyosarcoma-specific SV40polyA chimaeric transcription factor (due to translocation)

PAX3 bs TATA (E1B promoter) trends in Genetics

A genomic translocation in alveolar rhabdomyosarcoma (ARMS) results in the expression of a fusion protein that consists of the paired box gene 3 (PAX3) DNA-binding domain (DBD) and the potent transactivation domain (TAD) of the unrelated transcription factor forkhead in rhabdomyosarcoma (FKHR). An artificial promoter consisting of six PAX3 binding sites (bs) and the adenovirus E1B basal promoter (including a TATA box) shows ARMS-specific activity. The PAX3–FKHR chimaeric transcription factor binds to the PAX3-binding sites and activates transcription from the basal E1B promoter. As PAX3 is not expressed in adults, the artificial promoter is silent in normal cells. Abbreviations: DT-A, cDNA encoding diphteria toxin A chain; CAT, chloramphenicol acetyl-transferase.

promoter52; (Fig. 2a), the PSA promoter53 and the tyrosinase promoter54,55. A drawback of this approach is that it might only be applicable successfully to a subset of promoters and that the precise strategy has to be explored for each promoter. In another scenario, promoters with activating point mutations have been used, but such promoters have been found only in a few specific cases, such as that encoding AFP (Ref. 56) or the multidrug resistance 1 promoter (mdr-1; Ref. 57). The third strategy involves the construction of chimaeric promoters by combining the transcription regulatory elements from different promoters that are specific for the same tissue. A particularly interesting strategy that might also be applicable to many other promoters is the random combination of multiple tissue-specific elements followed by the identification of the optimal construct. In one example58, 5–20 DNA elements involved in muscle-specific transcriptional activation (Fig. 2) were assembled in random order in such a way that they were exposed on the same side of the double-helix. These synthetic cassettes were linked to a minimal chicken a-actin promoter. One of these combinations (see Fig. 2c) was investigated in detail and shown to be sixfold more active than the CMV immediate early promoter/enhancer in differentiating muscle cells in culture. Of particular interest is that this promoter construct gave levels of transgene expression in mice that were higher than those obtained with the CMV immediate early promoter/enhancer. In another approach, a liver-specific promoter was constructed by combining regions from the albumin promoter and the AFP enhancer59,60, although in this case it is not clear what degree of promoter enhancement could be achieved. The fourth approach makes use of recombinant transcriptional activators (RTAs) and appears to be the most generally applicable system at present. The construction of RTAs is based on the modular structure of transcription factors, which allows the combination of DNA-binding and transactivation domains derived from different proteins. For example, RTAs have been used to establish a positive feedback loop initiated by transcription from a weak celltype specific promoter (Fig. 3a)51. Such a promoter drives the simultaneous expression of the desired effector/ reporter gene product and a strong artificial transcriptional TIG April 2000, volume 16, No. 4

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FIGURE 2. Improving promoter activity and specificity (a)

hCEA

—13.6

—10.7

Transcription start site

—6.1 —4.0

—89—40+69

Luc

(b)

Tyr Human Murine

—2014 —1811 —12.3 —12.1

—209 —815

+51 —46

LacZ Human Murine

(c)

Chicken skeletal Luc α-actin promoter LacZ hGHRH (144 bp) TATA Sp1 site

SRE (skeletal α-actin) MEF-1 site (E box)

MEF-2 site (myosin LC 2A)

TEF-1 site (CAT motif) trends in Genetics

Reducing promoter size by joining 59-regulatory elements that confer promoter selectivity. (a) Carcinoembryonic antigen (CEA) promoter: multimerized proximal promoter elements (shown in green) and distal enhancer elements (orange and red) are cloned immediately upstream of the core promoter (blue) and the reporter gene that encodes luciferase (Luc). (b) Tyrosinase promoter: a duplicate short enhancer (red) is cloned upstream of the proximal promoter (blue) and the LacZ reporter gene. (c) Synthetic promoter combining tissue-specific elements of different genes: randomly assembled muscle-specific elements [serum response element (SRE), MADS box transcription enhancer factor (MEF) -1 and -2 sites, and thyrotropic embryonic factor (TEF)-1 site] were capped by Sp1 elements and cloned upstream of a 144 bp chicken a-actin promoter. Shown is the most potent construct obtained after screening hundreds of individual clones for transcriptional activity in vitro and in vivo. Arrows indicate orientation of elements (if changed in the synthetic promoter). Numbers indicate nucleotide positions relative to the transcription start site.

activator, comprising the HSV-derived VP16 transactivation domain and the bacterial LexA DNA-binding domain, which subsequently stimulates transcription through appropriate LexA-binding sites in the promoter. This approach leads to an enhancement of up to .100-fold while maintaining a 30- to .1000-fold cell type specificity, with the EC-specific vWF promoter and with the gastro-intestinal cell–specific sucrase isomaltase promoter. A related approach has also been described by Segawa et al. using the PSA promoter as a model (Fig. 3b)61. In this instance, the prostate-specific PSA promoter was used to drive the expression of a Gal4–VP16 fusion protein. This chimaeric transcription factor then activates the therapeutic gene or reporter gene through Gal4-binding sites placed upstream of a minimal tk promoter. 178

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Dual-specificity promoters combining tissuespecificity and cell-cycle regulation A frequently employed strategy in cancer gene therapy makes use of effector systems – the efficacy of which is dependent on cell division – such as the thymidine kinase/ganciclovir system. A limiting factor of this approach is the fact that non-proliferating cells are not affected. Therefore, it would be advantageous to achieve cell-cycle dependence through the vector rather than the therapeutic gene. One way to accomplish this goal would be to combine cell-type-specific and cell-cycle-regulated gene expression in the same promoter. Two different strategies that seem to be generally applicable for the construction of such dual specificity promoters have been described. In the first approach, the gene of interest is driven by an artificial heterodimeric transcription factor, the DNAbinding subunit of which is expressed from a tissue-specific promoter, whereas the transactivating subunit is transcribed from a cell-cycle-regulated promoter (Fig. 4a)62. As a result, gene expression occurs preferentially in the proliferating cells of a specific type of tissue. The selectivity of this strategy has been demonstrated for the expression of a transgene in proliferating melanoma cells, using cyclin A and the tyrosinase promoter elements. The second approach is based on the recently discovered mechanism of cell-cycle-regulated transcriptional repression mediated by a repressor protein termed cell-cycledependent factor 1 (CDF1)63. This repressor functions by blocking transcriptional activation in resting cells by specific factors binding to the upstream activating sequence, most notably the CCAAT-box binding factor (CBF), also termed nuclear factor-Y (NF-Y)64. Based on this work, a dual specificity promoter system was developed that combines celltype specificity with cell-cycle regulation (Fig. 4b)65. A chimaeric transcription factor (Gal4–NF-Y), consisting of the transactivation domain of NF-Y (A subunit, NF-YA) and the DNA-binding domain of Gal4, is expressed from a tissuespecific promoter. Gal4–NF-Y can bind to a second promoter, consisting of the cyclin A CDF-1 binding site downstream of multiple Gal4-binding sites. As a result, the stimulatory activity of Gal4–NF-Y is restrained in resting cells by the recruitment of the CDF-1 repressor to the promoter. The functionality of this system has been demonstrated for the specific transcriptional targeting of proliferating melanoma cells, where cell-cycle regulation was .20-fold and cell-type specificity was .50-fold.

Transcriptional targeting of viral replication To circumvent the problem of inefficient in vivo transduction, conditionally replication-competent viruses have recently been introduced as a new concept. With this approach, it is obviously necessary to prevent viral replication in cells outside the tumour. One strategy to achieve this restriction has been the deletion of viral genes, such as the adenoviral E1B/55 kD-encoding gene, which is essential for viral replication but is dispensable in p53 (TP53)-deficient tumour cells66. An alternative approach is the transcriptional targeting of viral replication – that is, the expression of an essential viral gene under the control of a promoter that is preferentially or specifically active in tumour cells. This strategy has been used successfully to construct replication-competent prostate carcinoma-specific adenoviruses. This was achieved by placing the E1A gene under the control of either the PSA or the kallikrein-2 promoter, or

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FIGURE 3. Enhancing promoter activity by using recombinant transcriptional activators (RTAs) (a)

Binding and tissuespecific transcriptional activation

Tissue-specific expression

nVP16LexA fusion NLS VP16TA 2nd tissuedomain specific promoter (vWF or SI)

Luc

LexADB domain

LexA bs

(b) Tissue-specific expression

Tissue-specific promoter (vWF or SI)

Binding and transcriptional activation +

Gal4 —VP16 fusion

Tissue-specific promoter (PSA)

Gal4DB domain

VP16TA domain

Luc or 79 CAG repeats ( Q79)

4 x Gal4 bs Minimal tk promoter

RTA Amplification system

Transactivation domain

Specific elements

DNA-binding domain trends in Genetics

Recombinant transcriptional activators (RTAs) are synthetic proteins that contain a potent transactivation (TA) domain (here of the Herpes Simplex virus VP16 protein) fused to a DNA-binding (DB) domain of a transcription factor that recognizes synthetic, non-human DNA elements [here of yeast Gal4 or bacterial LexA proteins and corresponding binding sites (bs)]. The RTA is expressed from a cell-type-specfic promoter. (a) The endothelial-specific von Willebrand factor (vWF) promoter or the enterocyte-specific sucrase isomaltase (SI) promoter. (b) The prostate-specific PSA promoter. The RTA binds to synthetic binding sites upstream of a second promoter leading to its activation and expression of the transgene. A second level of specificity might be incorporated if the second promoter is also tissue-specific and the activation of this promoter is repressed in non-target cells (a).

by driving the expression of both E1A and E1B by the prostate-specific promoters of the PSA and kallikrein-2encoding genes, respectively67,68. Replication-competent hepatoma-specific adenoviruses have been generated by placing the E1A gene under the control of the AFP promoter69,70. A similar approach has been used to generate replication-competent hepatoma-directed herpes viruses, where the viral gene IE-3 encoding ICP4 was placed under the control of the human albumin gene promoter/ enhancer71. In cell culture, these replication-competent adenoviruses and herpes viruses have been shown to be highly specific (up to 10 000-fold, relative to non-target cells). In addition, animal models have demonstrated their safety and efficacy. Based on these observations, it can be concluded that tumour-specific replication-competent viruses generated through the transcriptional targeting of viral replication hold some promise as a novel efficacious tool for cancer gene therapy.

Outlook The rapid progress in the field of transcriptional regulation in the past years has strongly stimulated translational

research, especially in the application to gene therapy for cancer. This includes the discovery and application of novel cell-type-specific, cell-cycle-regulated or tumourselective promoters, as well as promoters that respond to radiation, chemotherapy, tumour-specific environmental conditions, infection by tumour viruses or specific genetic alterations affecting the structure, expression or activity of transcription factors. Research has also involved the recombination of promoter elements or transcription factors to create regulatory units that have an enhanced transcriptional activity and/or an improved specificity. Another recent strategy aims at restricting the replication of viral vectors to tumour cells by placing the transcription of an essential viral gene under the control of a specific promoter. Clearly, all this work has advanced the field considerably, but it is also obvious that additional layers of selectivity are necessary to construct highly efficient and specific vectors. In this context, the combination of transcriptional and transductional targeting is of particular interest, because it is unlikely that the systemic delivery of a non-targeted vector vehicle will lead to a sufficiently high concentration of the therapeutic gene at TIG April 2000, volume 16, No. 4

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FIGURE 4. Dual-specificity promoter systems: combining tissue specificity and cell-cycle regulation Tissue-specific module

Cell cycle-regulated module

Reporter/therapeutic module

(a) Gal4/LCK fusion protein

VP16/CD4 fusion protein

Tissue-specific expression

RTA (heterodimer)

Cell cycle-regulated expression Transactivation

Tissue-specific promoter

Cell cycleregulated promoter

Gal p56LCK DNA-binding domain domain (aa2—71)

PolyA

PolyA

VP16 CD4 transactivation domain domain (aa397—435)

PolyA

PolyA

+ Luc/ TNFα

PolyA

Gal4bs

SV40

(b) Transactivation

RTA (Gal4/NF-YA fusion protein)

PolyA

Tissue-specific expression Tissue-specific promoter

Luc/ TNFα

Gal4bs CDE/CHR Proliferating cell

Gal NF-YA DNA-binding transactivation domain domain

PolyA

+

Transactivation repressed

Repressor (CDF-1)

PolyA

Non-proliferating cell trends in Genetics

(a) A DNA-binding subunit (shown in light blue) expressed from a tissue-specific promoter (dark blue) and a transactivating subunit (light green) expressed from a cell-cycle-regulated promoter (dark green) interact to form a heterodimeric recombinant transcriptional activator (RTA) via the p56Lck and CD4 dimerization domains. The expression of a functional (i.e. heterodimeric) RTA will, therefore, be restricted to proliferating cells of a certain tissue type. The RTA binds to Gal4-binding sites (bs) of a synthetic reporter/effector construct and, as a consequence, activates the downstream minimal SV40 promoter. (b) An RTA consisting of the Gal4 DNA-binding domain fused to the NF-YA transactivation domain (light blue) is expressed from a tissue-specific promoter (dark blue) and binds constitutively to Gal4-binding sites of a synthetic reporter/effector construct. A repressor named CDF-1 (dark green) binds to the downstream cell cycle dependent/cell cycle homology region (CDE/CHR) element specifically in the G0/G1 phases of the cell cycle and inhibits transactivation by NF-YA. As a consequence expression of the transgene is restricted to proliferating cells of a specific type.

the tumour site(s). The retargeting of vector vehicles to tumour cells or ECs has been achieved in several cases, as exemplified by the fibre-modified retargeted adenoviruses72. Another area that deserves particular attention are the pharmacologically regulatable promoters that are inducible by analogues of tetracycline, steroid hormone or rapamycin. Such promoter systems make it possible to turn the expression of the transgene off and on at will, which not only increases the safety of such vectors but, in some cases, provides the basis for new therapeutic strategies73. This primarily applies to those approaches that do not aim at killing the cancer cells but require the longterm expression of the transgene, as for instance in the control of graft-versus-host disease after allogeneic bone marrow transplantation.

References 1 Culver, K.W. and Blaese, R.M. (1994) Gene therapy for cancer. Trends Genet. 10, 174–178

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Several selective promoters have now been tested in preclinical models (see Table 1). To date, two major conclusions can be drawn from this work. First, selective transcriptional activity could be demonstrated in vivo, and promoter activity was sufficient to achieve therapeutic effects. Second, it has become clear that selective promoters lead to reduced side effects compared with nonspecific promoters, owing to the targeted expression of the transgene. As these studies mostly used rather simple promoters, further improvement can be expected in the near future when the more specific designer promoters will be exploited for in vivo use.

Acknowledgement We are grateful to F.C. Lucibello for critical reading of the manuscript.

2 Vile, R.G. and Hart, I.R. (1993) In vitro and in vivo targeting of gene expression to melanoma cells. Cancer Res. 53, 962–967 3 Vile, R.G. and Hart, I.R. (1993) Use of tissue-specific

expression of the herpes simplex virus thymidine kinase gene to inhibit growth of established murine melanomas following direct intratumoral injection of DNA. Cancer Res. 53, 3860–3864

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