WO2001049868A1

WO2001049868A1 - Cancer cell-specific gene expression system

Info

Publication number: WO2001049868A1
Application number: PCT/KR2000/000330
Authority: WO
Inventors: Young Il Yeom; Mi Jung Lim; Jung Hee Han; Jong Seok Lim; Kwang Dong Kim; Chang Kyu Kim
Original assignee: Korea Research Institute Of Bioscience And Biotechnology; Chong Kun Dang Pharmaceutical Corporation
Priority date: 1999-12-31
Filing date: 2000-04-10
Publication date: 2001-07-12
Also published as: AU4147200A

Abstract

The present invention relates to a cancer-specific gene expression system, more particularly to a cancer-specific gene expression system characterized comprising a promoter with a binding site (E2Fbs) for E2F transcription factor expressed only in a cancerous cell and a structural gene. Said gene expression system thus can provide an effective way in conjunction with various combined structural genes to treat cancer by using the special feature of said system that specifically works on cancerous cells without affecting any normal cells.

Description

CANCER CELL-SPECIFIC GENE EXPRESSION SYSTEM

BACKGROUND OF THE INVENTION Field of the Invention π The present invention relates to an artificial gene expression system, more particularly, to an artificial cancer-specific gene expression system which comprises a promoter and a structural gene wherein said promoter contains binding sites for E2F transcription factor (E2Fbs) which is known to be specif icallv active in many types of cancers. 0

Description of the Related Art

Due to the rapid progress in molecular biology, the causes and mechanisms of various cancers have been much elucidated. In particular, mutations as well as abnormal expression of oncogenes and tumor-suppressor .^" genes have been identified as the major causes of cancers.

The retinoblastoma gene (referred to as RB hereinafter) is one of the representative tumor-suppressor genes together with the p53 gene; inactivation of RB protein or abnormalities in the structure or expression of RB gene has been identified in approximately 30% of almost all kinds of cancers [N. Engl. }. 0 Med., 323, 1457- 1462 (1990); N. Engl. ]. Med., 330, 786 ~ 787 (1994); N. Engl. ]. Med., 322, 1397-1398 (1990); N. Engl. ]. Med., 330, 757-761 (1994)].

In normal, resting-stage mammalian cells, the RB protein binds with the E2F transcription factor to form E2F-RB complex and inhibit the transcriptional activity of E2F, thus preventing cell proliferation or cell cycle progression. ,7ι When cells receive appropriate signals for cell growth, however, the E2F-RB complex becomes dissociated due to the phosphorylation of RB protein by a cell cvcle-dependent kinase and the resulting free E2F transcription factor activates the transcription of genes involved in DNA replication such as DNA polymerase α, thymidylate synthase, thymidine kinase, ribonucleotide reductase, and dihvdrofolate reductase as well as genes involved in cell cvcle progression such as cdc2, cdc6, B-myb, c-myb, c-myc, N-myc, cyclin A, cvclin D, cvclin E, cdk 2, and cdk 4, thus allowing the cell to enter the S phase of cell cvcle \Nucleic Acids Res., 24, 4139-4145 (1996); Cancer Biol, 6, 99 -108 (1995); Science, 258, 424 -429 (1992); Gene, 237, 281-302 (1999)]. Therefore, the interaction between E2F and RB plays a crucial role in regulating cell proliferation.

U.S. Pat. No. 5,885,833 discloses nuclear constructs wherein a promoter contains a nucleic acid sequence of E2F binding site and a cell cvcle gene homologv region (CHR) as essential components. However, the activities of the above nucleic acid constructs are cell cycle-dependent and thus their incessant in vivo expressions cannot be guaranteed. Also, the above U.S. patent does not provide any specific examples or preferred embodiments regarding the cancer-specificity or the intensity of activities of the nucleic acid constructs.

DNA tumor viruses can provide major carcinogenic mechanisms bv inducing the release of activated E2F from the E2F-RB complex to cause cell cvcle progression because certain viral oncoproteins can interfere with the normal interaction between E2F and RB [Nature, 334, 124 -129 (1988); Cell, 54, 275 -283 (1998)].

For example, in cells infected with human papilloma virus (HPV), the HPV E7 protein binds the RB family proteins such as pRB, pl07 and pl30, and, as the result, RB family proteins become functionally inactivated. Likewise, oncogenic viral proteins such as T antigen and E1A, which are expressed when cells are infected with SV40 or adenovirus, respectively, can bind RB protein to inhibit its activity thus destabilizing the E2F-RB complex. The excess free E2F released from this step then leads to increased expression of genes involved in cell proliferation. These properties of oncogenic proteins of DNA tumor viruses cause unregulated cell proliferation and trigger the cellular transformation.

HPV infection can result in various diseases including cervical cancer, head and neck cancer, intraepithelial neoplasias and carcinoma of larvnx, juvenile flatwarts, etc., and the severity of these disease varies greatly, ranging from an acute illness to a chronic illness [Ciur. Opm. Oncol, 3, 191 — 199 (1999); Cancer Metastasis Rev., 15, 27 - 51 (1996); hit ]. Deππatol, 27 690 - 694 (1998); Am j. Surg. Patlwl, 16, 641 - 649 (1992)].

According to recent reports, the oncogenic properties of the Epstein-Barr virus (EBV) is also attributable to the functional inactivation of the RB protein; In particular, BRLFl, an immediate-earlv lytic gene product, can bind RB and inhibit its activity. EBV is a kind of a herpes virus that can infect all human races and also known to induce diseases such as Burkitt's lvmphoma, lvmphoproliferatrve syndrome, Hodgkin's disease and nasopharyngeal carcinoma {Leukemia, 12, 1796 -1805 (1998); Am }. Pathol, 155, 619 - 625 (1999); /. Virol, 73, 1630 - 1636 (1999); Indian /. Cancer, 35, 47 ~ 56 (1998)]. Above examples well demonstrate that a number of human cancers have oncogenic viral proteins as the causative agents. A major challenge in treating these malignant diseases by a gene therapeutic approach has been to direct the therapeutic action specifically to tumor cells so that the disease could be effectively coped with without affecting normal cells. . >

SUMMARY OF THE INVENTION

To solve the above-mentioned problem, the inventors of the present invention devised an artificial gene expression system comprising a therapeutic gene, inserted as a part of the structural gene and located downstream of the

1 ' ) E2F binding site which specifically directs expression of the downstream therapeutic gene to cancer cells. Said artificial gene expression system was transfected in a variety of cells and its ability to induce cancer-cell-specific gene expression was confirmed accordingly. Therefore, the objective of the present invention is to provide an artificial gene expression system whereby the i .^'"> expression of therapeutic genes can be selectively targeted to such cancer cells that have abnormally elevated activity of the E2F transcription factor due to inactivation of the RB function, which was, in turn, caused either by mutations of genes involved in the RB pathway or by infections with DNA tumor viruses.

O Brief Description of the Drawings

Fig. 1 shows a diagram of the plasmid (E2Fbs) /tk-LacZ constructed according to the example 1 of the present invention.

Fig. 2 shows a diagram of the plasmid (E2Fbs)6/Spl/tk-LacZ constructed according to the example 2 of the present mvention. J^") Fig. 3 shows a diagram of the plasmid (E2Fbs)₆/Spl/tk-CD constructed according to the example 3 of the present invention.

Fig. 4 is a graph showing the IL-2 production profile in COS-7 cells bv plasmids (E2Fbs)β/Spl/tk-IL-2 and Spl/tk-IL-2 constructed according to the example 4 of the present invention.

Fig. 5 is a graph showing the TNF production profile in COS-7 cells bv plasmids (E2Fbs)₆/Spl/tk-TNFα and Spl/tk-TNF constructed according to the example 5 of the present invention.

Detailed Description of the Invention

This invention relates to a cancer-specific gene expression system characterized by having a promoter with E2F binding sites (referred to as E2Fbs hereinafter) and structural genes.

This invention is explained in more detail as set forth hereunder.

This invention relates to an artificial cancer-specific gene expression system comprising binding sites for E2F and a structural gene, wherein said gene expression system is designed to utilize the unique characteristics of E2F proteins that are released in transcriptionally active forms from E2F-RB complexes to increase the expression of genes involved in cell proliferation in cancer cells where RB family proteins are inactivated; ultimately, said gene expression system intends to change the target genes of the E2F-mediated transcription from the normal endogenous ones to therapeutic genes. That is, bv recombining therapeutic genes with E2Fbs the therapeutic genes can now be expressed in E2F-dependent manner specifically in cancer cells that show abnormal proliferation due to over-activation of E2F, and thus the system provides an efficient molecular tool whereby cancer cells can be eradicated within a relatively short period of time. In this schema, E2F-RB will be disintegrated by the phosphorylation of RB protein by cell cvcle-dependent kinase in cancer cells, and the activated E2F, which would normally transactivate the genes involved in cell proliferation, is then able to promote the expression of therapeutic genes thus eliminating the cancer cells.

Therapeutic genes that can be utilized in this system include suicide genes, cvtokine genes, immune-regulatorv genes, antisense genes, tumor- suppressor genes and other therapeutically effective anti-cancer genes. For example, one or more of such genes encoding cytosine deaminase, thvmidine kinase, tumor necrosis factor (TNF) α, interleukin-2, interleukin-12, interleukin- 18, granulocvte/macrophage-colony stimulating factor (GM-CSF), and the tumor suppressor gene p53 are expected to exhibit excellent anti-cancer activity when inserted into said expression system according to the present invention.

The E2Fbs, the binding sites for the E2F transcription factor, can be either a monomer or a multimer, and mav consist of 1-24 subunits. Further, it is also possible to insert other transcription factor binding sites together with the E2Fbs in the present invention. The expression system where more than one transcription factor binding site is inserted along with E2Fbs can exhibit an increased transcriptional activity in transfected cancer cells. The increased transcriptional activity of the expression system having additional transcription factor binding sites together with E2Fbs is rnainly ascribed to the fact that the additional transcription factor can exert a synergistic effect in combination with E2F. Examples of transcription factors whose binding sites can be inserted along with the E2Fbs are Spl, API, NFl(ATF), C/EBP and other transcription factors known to be involved in gene expression.

Said cancer-specific gene expression system can not only provide a veπ^r high level expression of foreign genes but also show a sustained expression property for a reasonably long period of time, so it should be useful for therapeutic intervention of all kinds of cancers wherein E2F is specifically activated. Examples of such cancers include those ones wherein RB gene is genetically impaired as in retinoblastoma or functionally inactivated due to DNA tumor virus infection as in cervical cancer, head and neck cancer, intraepithelial neoplasias and carcinoma of larynx, juvenile flat warts, Burkitt's lvmphoma, lymphoproliferative syndrome, Hodgkin's disease and nasopharvngeal carcinoma.

The preferred embodiments of the present invention are as follows.

First, the cancer cells used in the present invention are characterized as follows.

COS-7 is a transformed mutant obtained from kidney cells of the African green monkey bv infecting said kidney cells with SV40 and constitutively expresses T antigen [Cell, 23, 175-182 (1981)]. HeLa is a cell line derived from undifferentiated cervical cancer and constitutively expresses E7 [Cancer Res., 12, 264 (1952)]. 293 is a cell line derived from renal cells of a human fetus transformed by adenovirus and constitutively expresses E1A and E1B [/. Gen. Virol, 36, 59 - 72 (1977)]. Saos-2 is a cell line derived from osteogenic sarcoma wherein p53 and RB are not produced but pi 07 or pi 0 is normally expressed [/. Natl. Cancer hist. (Bethesda), 58, 209-214 (1997)]. CaSki is a human cancer cell line derived from cervical epidermoid carcinoma, and expresses E6 and E7 from the HPV typelό genome inserted into the host genome [Science, 196, 1456 — 1458 (1997)]. C3 is a mouse cell line transformed with human papilloma virus type 16, and expresses E6 and E7 proteins [Em: /. Immunol, 23, 2242 -2249 (1993)]. NIH/3T3 is a mouse fibroblast cell line which is used as a normal control [/. Virol. 4, 549 — 553 (1969)].

The procedure for estimating the activity of gene expression system in the present invention using above-mentioned cells is as follows.

First, β-galactosidase (LacZ) gene is selected as a reporter gene to estimate the activity of a given expression system and then plasmid tk-LacZ is constructed so that LacZ gene can be expressed under the control of minimal promoter of thymidine kinase (tk) of herpes simplex virus. Next, E2Fbs is manufactured by PCR (polymerase chain reaction) and inserted into the plasmid pBS KS to clone (E2Fbs)_n, where n is 2, 4 or 6. The (E2Fbs)_n is then inserted into the plasmid tk-LacZ to construct plasmids (E2Fbs)_n/tk-LacZ. Said plasmids (E2Fbs)_n/tk-LacZ were then transfected into the normal control group (NIH/3T3) or cancer cell lines such as COS-7, HeLa, 293, Saos-2, CaSki and C3 by means of calcium phosphate co-precipitation and then cultured for 48 hr in 5% CO2 incubator. The cultured cells were then lvsed by three freeze- thaw cycles using liquid nitrogen and a 37 ^°C bath, and then measured for the activities of β-galactosidase and luciferase to estimate the expression level of each gene expression system. Here, the luciferase was used as an internal control to normalize the experimental results for variations in the transfection efficiency⁷. As the results of these series of experiments said cancer-specific gene expression system of the present invention, (E2Fbs)_n/tk-LacZ, showed high level of β-galactosidase activities in transfected cancer cells where it could expedite the expression of LacZ gene in E2F-dependent manner.

This invention is explained in greater detail based on the following examples but they should not be construed as limiting the scope of this invention.

Example 1: Construction of p(E2Fbs)η/tk-LacZ

(1) Construction of tk-LacZ

A plasmid pBlueLacZ (kindly provided by Dr. Hans Scholer from EMBL) was serially treated with the restriction enzyme Bamlil, Klenow enzyme, and another restriction enzyme Xhol to isolate a 3.8kb DNA fragment containing LacZ gene. Meanwhile, another plasmid tk-CAT which (also kindly provided by Dr. Hans Scholer from EMBL) was double-digested with restriction enzvmes Xhol and Smal to get rid of CAT gene fragment and obtain a plasmid fragment containing the promoter region of the tk gene. Then, the above separated LacZ gene of 3.8kb was ligated with the plasmid containing tk promoter, and the resulting plasmid was partially digested with EcoRI. The insert of the partially digested plasmid was then ligated into pBluescript also digested with EcoRI, and, as the result, the plasmid tk-LacZ was finally constructed that contains one Spl binding site and TATA box.

(2) Construction of pBS-(E2Fbs)_n

30 cycles of PCR were performed under the conditions of 1 min at 94°C for denaturation, 1 min at 63°C for annealing, and 45 sec at 72°C for extension by using 20 ng of plasmid DNA containing the promoter of adenovirus E2A gene [kindly provided by Dr. Dong-Soo Im from KRIBB (Korea Research Institute of Bioscience and Biotechnology)], and 50 pmol of E2-3 primer of SEQ. ID. NO. 1 and E2-4 primer of SEQ. ID. NO. 2, respectively. The resulting 93 bp DNA fragment was then digested with Xhol and Sail and then ligated to form a dimer ((E2Fbs)₂), which was then inserted into Xhol/ Sail restriction site of pBS KS (Stratagene Co., Ltd., U.S.A.) to construct pBS-(E2Fbs)₂. The above plasmid pBS-(E2Fbs)₂ was digested with X/;σI and Sail, and then inserted with (E2Fbs)₂ fragment which were also digested with Xhol and Sail to construct the plasmid i o pBS-(E2Fbs)₄. The above steps were repeated to obtain pBS-(E2Fbs)β. (3) Construction of (E2Fbs)_n/tk-LacZ

Each of the plasmids pBS-(E2Fbs)₂, pBS-(E2Fbs)₄ and pBS-(E2Fbs)_h was digested with EcoRI, and inserted with a 4.2 kb EcoRI fragment of tk-Lac-Z vector to construct plasmids (E2Fbs)₂/tk-LacZ, (E2Fbs)₄/ tk-LacZ and

I .^") (E2Fbs)(-,/tk-LacZ, respectively. E. coli transformed with thus obtained (E2Fbs)_h/tk-LacZ was deposited to KCTC (Korea Collection for Type Cultures) in KRIBB on November 22, 1999 and was assigned the depositary number KCTC 0698 BP.

O Example 2: Construction of (E2Fbs)β/Spl/tk-LacZ

Plasmid pBS-(E2Fbs)β was digested with Sail and inserted with a 0.2 kb Sail fragment to construct pBS-(E2Fbs)6/SPl. Thus obtained plasmid was digested with EcoRI and inserted with a 4.2 kb EcoRI fragment of tk-Lac-Z vector in example 1 to construct p(E2Fbs)ή/SPl/tk-LacZ. E. coli transformed with thus obtained (E2Fbs)6/Spl/tk-LacZ was deposited to KCTC in KRIBB on March 16, 2000 and was assigned the depositary number KCTC 0756 BP.

Example 3: Construction of (E2Fbs)f/Spl/tk-CD

Plasmid (E2Fbs)6/Spl/tk-CD was constructed in order to study the therapeutic characteristics and the applicability of the gene expression system in example 1 by combining E2Fbs and a therapeutic gene, cytosine deaminase (referred to as CD hereinafter). First, plasmid pCD2 (kindly provided bv Dr. Dong-Soo Im from KRIBB) was double-digested with EcoRI and BamHl, treated with Klcnow enzyme and a 1.5 DNA fragment was isolated. To increase the transcription efficiency and mediate the expedition of protein translation, plasmid pGLN/SP73/bGH that contains rabbit β-globin(bGLN) intron and 3'- flanking sequence of bovine growth hormone was double-digested with EroRI and BamHl, treated with Klenow enzyme and ligated with the above 1.5 kb DNA fragment containing CD gene. This plasmid was then digested with Notl, treated with Klenow enzyme, and inserted with a 0.8 kb DNA fragment of (E2Fbs)ή/Spl/tk, which was separated from pBS-(E2Fbs)e/Spl by digestion with Xhol and Klenow enzyme, and the plasmid p(E2Fbs)_< /Spl/tk-CD was constructed.

Experimental Example 1: The Estimate of Transcriptional Activity of an E2Fbs-containing Plasmid in Normal Cells

10 μg each of the plasmids (E2Fbs)₂/tk-LacZ, (E2Fbs)₄/tk-LacZ and (E2Fbs)₆/ tk-LacZ constructed in example 1 and 1 μg of plasmid pRSV-luciterase were added with 250 μL of solution consisting of 2x BBS (50mM N,N-bis(2- hydroxyethvl)-22-minoethanesulfonic acid), 280mM NaCl and 1.5mM Na₂HP0₄ (pH 6.95) and 250 μL of 250mM CaCl₂ in this order to form a co-precipitation solution. Plasmid HβA-LacZ was used as a positive control while plasmid pBS was used as a negative control. 3x IO⁵ cells of NIH/3T3 cultured in DMEM containing 10% fetal bovine serum and 0.45% glucose were plated in a > dish of 6 cm diameter, subcultured in a 5% C0₂ incubator for 16 hr and replaced with 3 mL of fresh medium. After 4 hr, the co-precipitation solution was added and cultured further for 16 hr. After removing the medium the cells were washed with phosphate buffered saline solution, added with 4m L of fresh medium and continued to be cultured for another 16 hr. After washin.tr the cultured cells twice with phosphate buffered saline solution, 700 μL of a phosphate buffer solution was added and the cells were collected bv using rubber policeman into a 1.5 mL centrifuge tube, and centrifuged at 4,000 rpm for 4 min at 4°C. After centrifugation, supernatant was discarded and cells were resuspended in 50μL of a dipersion solution (ImM DTT, pH 7.8 in 250mM ) Tris). This cell suspension was then frozen in liquid nitrogen and thawed at 37°C incubator and this freeze-thaw process was repeated 3 times to lyse cells. The lvsate was centrifuged at 12,000 rpm for 15 min at 4°C to obtain the cell extract.

The relative activity of β-galactosidase expressed in NIH/3T3 cells transfected with each plasmid was measured to estimate the transcriptional activities conferred by E2Fbs. Further, the activity of luciferase in the cell extract was also measured to normalize the variation in the transfection efficiency of every sample.

The methods of estimating the activities of β-galactosidase and

U luciferase are as follows.

1) The method of estimating the activity of β-galactosidase

On ice 40μL of the above cell extract was added to 800 μL of solution 1 (60mM Na₂HP0₄, 40mM NaH₂P0₄, lOrnM KC1, ImM MgCb, and 50mM β- mercaptoethanol) followed by addition of 200 μL of solution II (60mM Na₂HP0₄, 40mM NaH P0₄ and ONPG 2mg/mL) at a 30 sec interval between consecutive samples, mixed vigorous!}⁷ and incubated at 37°C. When there was a chromophoric response after the reaction, 500 μL of ImM a2CO-, was added every 30 sec to terminate the reaction and the absorbance was measured at 420 ran.

2) The method of estimating the activity of luciferase

1 OμL of the above cell extract was added to 350 μL of solution A (2mM ATP, OmM MgS0₄ and 22.5mM Gly-glycine at pH 7.8) and the luminescence was measured by using a luminometer (Lumat, EG & G Berthold) which adds 100 μL of solution B (0.3 mg/mL of D-luciferin and 20mM Gly-glycine at pH 7.8), and the results are shown in the following table 1.

O

ι :i Table 1.

As shown in the above table 1, there was no noticeable increase in LacZ gene expression due to E2Fbs in NIH/3T3, which are normal cells, but there was an increase in LacZ gene expression by the presence of binding sites for Spl, API and NF1. These results indicate that in normal cells RB protein inhibits the transcriptional activity of E2F by binding to E2F.

Experimental Example 2: The Estimate of Transcriptional Activity of E2Fbs in the Presence of the Oncoprotein El A in Normal Cells

0.4 μg of ElA-expressing plasmid (kindly supplied by Dr. Dong-Soo Im from KRIBB) was transfected into NIH/3T3 cells along with the plasmid pRSV- luciferase and the E2Fbs-containing plasmids in the same way as in the experimental example 1, and the relative activities of β-galactosidase was measured in each sample. The results are shown in table 2.

Table 2.

As shown above, the level of LacZ gene expression was increased due to the expression of El A, an oncogenic protein. The level of LacZ gene expression from (E2Fbs)₄/tk-LacZ was about 30 times higher than that from tk- LacZ (without E2Fbs), while (E2Fbs)e/ Spl/tk-LacZ showed about 9 times higher LacZ gene expression as compared to that of Spl/tk-LacZ. In plasmids (E2Fbs)6/APl/tk-LacZ and (E2Fbs)_ft/NFl/tk-LacZ, the level of LacZ gene expression was also about 9 times higher than that of APl/tk-LacZ and NFl/tk-LacZ, respectively. The results imply that binding between El A and RB resulted in activation of E2F resultine_. in the induction of the LacZ ene expression. Experimental Example 3: The Estimate of Transcriptional Activity of E2Fbs in the Presence of the Oncoprotein E7 in Normal Cells

0.2 μg of E7-expressing plasmid (kindly provided by Dr. Soo-Jong Uhm from The Sejong University of Korea) was transfected into NIH/3T3 cells along with the plasmid pRSV-luciferase and the E2Fbs-containing plasmids in the same way as in experimental example 1, and the relative activities of β- galactosidase was measured in each sample. The results are shown in table 3.

Table 3.

As shown above, the level of LacZ gene expression was increased due to the expression of E7, an oncogenic protein of human papilloma virus. The level of LacZ gene expression in (E2Fbs)₄/tk-LacZ was about 10 times higher than that of tk-LacZ(without E2Fbs), whereas (E2Fbs)₆/ Spl/tk-LacZ doubled the LacZ gene expression as compared to that of Spl/tk-LacZ. In plasmids (E2Fbs)f,/APl/tk-LacZ and (E2Fbs)₆/NFl/tk-LacZ, the level of LacZ gene expression was also about 2-3 times greater than that of APl/tk-LacZ and NFl/tk-LacZ, respectively. These results show that the inactivation of RB protein bv E7 activated the E2F thus inducing the increase in LacZ gene expression.

Experimental Example 4: The Estimate of Transcriptional Activity of an E2Fbs containing Plasmid in Cells where SV40T gene is expressed

Experiment was performed in the same way as in the experimental example 1 with the exception that the cell line used was COS-7, a transformed mutant wherein T antigen of SV40 is expressed and that the culture medium was replaced by DMEM-L containing 10% fetal bovine serum. The results are shown in table 4.

Table 4.

As shown above, the level of LacZ gene expression in (E2Fbs)6/tk-LacZ was about 51 times higher than that of tk-LacZ(without E2Fbs). Also, the level of LacZ gene expression from (E2Fbs)e/ Spl/tk-LacZ was 123 times greater than that of Spl/tk-LacZ. In plasmids (E2Fbs)₆/APl/tk-LacZ and

(E2Fbs)6/NFl/tk-LacZ, the level of LacZ gene expression was also about 100 and 124 times greater than APl/tk-LacZ and NFl/tk-LacZ, respectively.

Experimental Example 5: The Estimate of Transcriptional Activity of E2Fbs containing Plasmids in Cells that contain low copy number of HPV (human papilloma virus) genome

Experiment was performed in the same way as in the experimental example 1 with the exception that the cell line used was HeLa, a transformed mutant wherein E7 gene is expressed and that the culture medium was replaced by DMEM-L containing 10% fetal bovine serum. The results are shown in table 5.

Table 5.

As shown above, the level of LacZ gene expression in (E2Fbs)r-,/ tk-LacZ was about 12 times higher than that of tk-LacZ(without E2Fbs). Also, the level of LacZ gene expression in (E2Fbs)_h/ Spl/tk-LacZ was 10 times greater than that of Spl/tk-LacZ. In plasmids (E2Fbs)e/APl/tk-LacZ and

(E2Fbs)₍-,/NFl/tk-LacZ, the level of LacZ gene expressiori was also about 10 -11 times greater than APl/tk-LacZ and NFl/tk-LacZ, respectively.

l ' Experimental Example 6: The Estimate of Transcriptional Activity of E2Fbs- containing Plasmids in Cells where adenovirus E1A gene is expressed

Experiment was performed in the same way as in the experimental example 1 with the exception that the cell line used was 293, a transformed mutant wherein El A gene is expressed and that the culture medium was replaced by MEM (minimal essential medium) containing 10% fetal bovine serum. The results are shown in table 6.

• i Table 6.

! Plasmid Activity of β-galactosidase tk-LacZ 1

! (E2Fbs)₂/tk-LacZ 10.05

(E2Fbs)₄/tk-LacZ 16.42

(E2Fbs)₆/tk-LacZ 18.21

Spl/tk-LacZ 7.86

APl/tk-LacZ 4.96

NFl/tk-LacZ 4.39

' (E2Fbs)₆/ Spl/tk-LacZ 165.07

(E2Fbs)₆/ APl/tk-LacZ 114.08

(E2Fbs)β/ NFl/tk-LacZ 105.60

Hβ A-LacZ 105.05 pBS 0.50

As shown above, the level of LacZ gene expression in (E2Fbs)_h/tk-LacZ was about 18 times higher than that of tk-LacZ(without E2Fbs). Also, the level

JO of LacZ gene expression in (E2Fbs)β/ Spl/tk-LacZ was 21 times greater than that of Spl/tk-LacZ. In plasmids of (E2Fbs)₆/APl/tk-LacZ and

(E2Fbs)ή/NFl/tk-LacZ, the level of LacZ gene expression was also about 23-24 times greater than those of APl/tk-LacZ and NFl/tk-LacZ, respectively.

Experimental Example 7: The Estimate of Transcriptional Activity of E2Fbs- containing Plasmids in Cells where the expression of RB and p53 is impaired

Experiment was performed in the same way as in the experimental example 1 with the exception that the cell line used was Saos-2 wherein RB and p53 proteins are not expressed but pl07 a d pl30, the RB family members, are normally expressed. The results are shown in the following table 7.

Table 7.

As shown above, the level of LacZ gene expression in p(E2Fbs)_h/tk- LacZ was threefold higher than that of tk-LacZ(without E2Fbs); however, the rate of increase was markedly lower as compared to those in the previous

l experimental examples. These results suggest that pl07 or pl30, which belong to RB family and expressed normally in Saos-2 cells, can bind to E2F and thus inhibit the increase of LacZ gene expression.

Experimental Example 8: The Estimate of Transcriptional Activity of E2Fbs containing Plasmids in Cells that contain high copy number of HPV (human papilloma virus) genome

Experiment was performed in the same way as in the experimental example 1 with the exception that the cell line used was CaSki wherein E6 and E7 proteins are expressed due to the presence of HPV typelό genome and the culture medium used was RPMI 1640 containing 10% fetal bovine serum. The results are shown in the table 8.

Table 8.

As shown above, the

of LacZ gene expression in (E2Fbs)₆/tk-LacZ was about 475 times higher than that of tk-LacZ (without E2Fbs). Also, the

JJ level of LacZ gene expression in (E2Fbs)_b/ Spl/tk-LacZ was 35 times greater than that of Spl/tk-LacZ.

Experimental Example 9: The Estimate of Transcriptional Activity of E2Fbs- containing Plasmids in Cells that contain artificially introduced HPV(human papilloma virus) genome

Experiment was performed in the same way as in the experimental example 1 with the exception that the cell line used was C3, a transformed cell line wherein E6 and E7 proteins are constitutively expressed due to the presence of HPV typelό genome, and that the culture medium was replaced by IMDM containing 8% fetal bovine serum and lx β-mercaptoethanol. The results are shown in the table 9.

Table 9.

As shown above, the level of LacZ gene expression in (E2Fbs)6/tk-LacZ was about 4.8 times higher than that of tk-LacZ (without E2Fbs). Also, the

9 ' ! level of LacZ gene expression in (E2Fbs)₆/ Spl/tk-LacZ was 11 times greater than that of pSpl/tk-LacZ.

Experimental Example 10: The Estimate of Cytotoxic Activity in Cancer cells of an E2Fbs-containing Plasmid encoding a therapeutic gene CD

Experiment was performed in the same way as in the experimental example 1 with the exception that three different plasmids (E2Fbs)r->/ Spl/ tk-CD, Spl/tk-CD and pBS were used to transfect C3 cells wherein RB protein was inactivated by the expression of E7. Then, the culture medium of transfected cells were replaced with fresh one containing 0, 0.5, 1, 5, 0, 50, 100, 500, 1 ,000, 5,000, and 10,000 μM of 5-FC (5-fluorocvtosine), and the cells were cultured for additional 3 days. The cultured cells were then washed with phosphate- buffered saline, added with 1 mL of MTT (5mg/mL) and incubated in a 5% CO2 incubator for 4 hr. After discarding the supernatant bv aspiration, IrnL of DMSO was added, mixed thoroughly, and the number of live cells -were estimated by measuring the absorbance at 590 ran. The IC50 [the amount of 5- FC which gives 50% growth inhibition; calculated from log (volume-response) curve] of each plasmid was calculated based on the above MTT assay results, and is shown in the table 10.

Table 10.

J4 As shown above, the IC50 value of (E2Fbs)6/tk-LacZ decreased about 23 folds compared to that of tk-LacZ (without E2Fbs). This result suggests that expression of E7 protein abolished the function of RB protein; as the result, the activity of tk promoter became increased by the E2F transcription factor to produce high level of CD thus increasing the sensitivity of transfected C3 cells to 5-FC

Experimental Example 11: The Safety Evaluation of a Vector containing E2Fbs and a Spl binding site using Mouse Embryonic Fibroblast cells

The plasmid vector constructed according to the present invention that contains both E2Fbs and an Spl binding site may be toxic to normal cells when the inserted therapeutic genes in said plasmid vector are expressed depending on the activity of Spl, a transcription factor which is active in most cell types. Therefore, the inventors of the present invention used mouse embryonic fibroblast cells as the normal control to measure the activity level of said plasmid vector in normal cells.

Experiment was performed in the same way as in the experimental example 1 with the exception that the cell used as the normal cell type was a primary culture of the mouse embryonic fibroblast.

Table 11.

As shown above, there was no noticeable increase in transcriptional activitλ⁷ of (E2Fbs)6/tk-LacZ due to the presence of E2Fbs in normal cells; furthermore, the E2Fbs even repressed the activity of Spl thus avoiding unnecessary gene expressions in normal cells and also further specifying the cancer-specific gene expression.

Example 4: The Construction of a Plasmid that contains IL-2 gene and E2Fbs and the Estimate of its IL-2 production capacity in Cancer cells

Plasmids (E2Fbs)f-,/Spl/tk-IL-2 and Spl/tk-IL-2 were constructed by using the same method in the example 3 with the exception that cDNA of human IL-2 gene was inserted as the therapeutic gene instead of CD gene. Thus obtained plasmids were transfected into COS-7 cells wherein RB protein was inactivated by the expression of T antigen of SV40, using the same method in the experimental example 1. Starting from 24 hr after the transfection, 300 μL of the culture medium was collected everyday for 7 days while the culture medium was replenished with equal amount of medium.

MTT analysis using IL-2-dependent cell line CTLL-2 was employed to measure the biological activity of IL-2 in the collected culture medium. Briefly, CTLL-2 cells were cultured for 2 days in a medium wherein a serial dilution of

JO the above collected culture medium was added, washed with phosphate- buffered saline, added with 1 mL of MTT (5mg/mL) and cultured in a 5% CO2 incubator for 4 hr. Then, the supernatant was discarded, lmL of DMSO λvas added, mixed thoroughly, and the number of live cells were estimated bv measuring the absorbance at 590 nm. The amount of IL-2 produced in COS-7 cells was calculated from the absorbance data, and the result is shown in Fitr. 4. As shown in Fig. 4, COS-7 cells, wherein the E2Fbs-containing expression vector was transfected, showed a continuous increase in IL-2 production for up to 3 days; the maximum amount of expression was 112,200 unit/mL and the amount gradually decreased after the fourth day of transfection. The level of IL-2 production was shown to stay relatively high even after day 7, reaching 24,980 unit/mL; this result suggests that production of therapeuticallv effective level of IL-2 can last for a fairly long period of time.

In contrast, the amount of IL-2 production from COS-7 cells transfected with Spl/tk-IL-2 vector without E2Fbs was negligible, and there was almost 180 folds difference in the maximum level of IL-2 production between Spl/tk- IL-2 and (E2Fbs)6/ Spl/tk-IL-2. This result proves that T antigen of SV40 in COS-7 cell induces dissociation of RB-E2F complex by binding to RB protein, and the transcriptional activity of the released E2F expedites the cancer-specific gene expression from the vector (E2Fbs)ή/ Spl/tk-IL-2. Accordingly, a novel therapeutic gene expression system was developed whereby high level of IL-2 can be selectively produced in cancer cells with impaired RB function. This novel gene expression system should prove highly useful in inducing IL-2- mediated anti-cancer therapeutic activities such as the anti-cancer reaction bv T Ivmphocλ'tes, increased NK cell activity, and induction of various other

J7 cvtokine production. The gene expression vector proposed in the present invention shows a very high level of foreign gene expression which is specific to cancer cells and lasting a reasonably long period of time, and thus is expected to make a very useful molecular tool for the gene therapy of cancers associated .1 with DNA tumor virus infections such as the cervical cancer.

Example 5: The Construction of a Plasmid that contains TNF α gene and E2Fbs and the Estimate of its TNF α production capacity in Cancer cells

Plasmids (E2Fbs)₆/Spl/tk-TNF and Spl/tk- TNF were

J ) constructed by using the same method as in the example 3 with the exception that cDNA of human TNF α gene was inserted as the therapeutic gene instead of CD gene. Thus obtained plasmids were transfected into COS-7 cells using the same method in the experimental example 1. Starting from 24 hr after the transfection, 300 μL of the culture medium was collected everyday for 7 davs i r> while the culture medium was replenished with equal amount of medium.

MTT analysis using Wehi 164 cells that show TNF α-dependent apoptosis was employed to measure the biological activity of TNF α in the collected culture medium. Briefly, Wehi 164 cells were cultured for 2 davs in a medium wherein a serial dilution of the above collected culture medium was

^0 added, washed with phosphate-buffered saline, added with 1 mL of MTT(5mg/mL) and cultured in a 5% C0₂ incubator for 4 hr. Then, the supernatant was discarded, lmL of DMSO was added, mixed thoroughly, and the number of live cells were estimated by measuring the absorbance at 590 ran. The amount of TNF α produced in COS-7 cells was calculated from the

.0 absorbance data, and the result is shown in Fig. 5. As shown in Fig. 5, COS-7 cells, wherein the E2Fbs-containing expression vector was transfected, showed a continuous increase in TNF α production for up to 5 days; the maximum amount of expression was 16.15 μg/mL and the amount gradually decreased after the sixth day of transfection. However, the amount of TNF α production ^" was drastically decreased after day 7 due to massive death of COS-7 cells themselves by TNF α. It is speculated that TNF α production could have lasted for a fairly long period of time as in the case of IL-2 in example 4 if there were no TNF α-mediated toxicities against the COS-7 cells.

In contrast, the amount of TNF α production from COS-7 cells

■ 0 transfected with Spl/tk- TNF α vector without E2Fbs was negligible, and there was almost 163,838 folds difference in the maximum level of IL-2 production between Spl/tk- TNF α and (E2Fbs)β/ Spl/tk- TNF α. This result proves that T antigen of SV40 in COS-7 cell induces dissociation of RB-E2F complex bv binding to RB protein, and the transcriptional activity of the released E2F

.^" expedites the cancer-specific gene expression from the vector (E2Fbs)β/Spl /tk- TNF α. Accordingly, a novel therapeutic gene expression system was developed whereby high level of TNF α can be selectively produced in cancer cells with impaired RB function. This novel gene expression system should prove highly useful in inducing TNF α-mediated anti-cancer therapeutic

10 activities. The gene expression vector proposed in the present invention shows a very high level of foreign gene expression which is specific for cancer cells arid lasting a reasonably long time period, and thus is expected to make a very useful molecular tool for the gene therapy of cancers associated with DNA tumor virus infections such as the cervical cancer.

jy As shown above, the present invention relates to a cancer-specific gene expression system which comprises a promoter and a structural gene wherein said promoter contains binding sites for E2F transcription factor (E2Fbs) which is specifically active in many types of cancers. According to the present . invention, oncogenic proteins can increase the transcriptional activity of E2F bλ⁷ inhibiting the E2F-RB interaction specifically in cancer cells. Further, bv replacing the target genes regulated by E2F from those involved in cell proliferation to therapeutic genes, the therapeutic genes can now be expressed only in cancer cells where E2F is abnormally activated, thus resulting in very o high anti-cancer effects. For example, when cytosine deaminase, a therapeutic gene with a suicidal type of anti-cancer activity, was transfected, it was specifically expressed in cancer cells at high concentrations, resulting in elimination of the cancer cells. Experiments with the plasmids wherein LacZ gene is ligated to (E2Fbs)₆ showed that there was approximately 3.2-475 fold

0 increase in the LacZ expression depending on the kind of cancers. Also, experiments with plasmids inserted with other anti-cancer therapeutic genes such as interleukin-2 and tumor necrosis factor α (TNF α) showed that vectors containing (E2Fbs)ή resulted in approximately 180-163,838 folds higher level of therapeutic gene expression as compared to that without E2Fbs. Still further,

: > when a vector containing, in addition to E2Fbs, other transcription factor binding sites such as Spl is transfected into cancer cells, it showed 2-11.5 folds higher gene expression rates as compared to vectors inserted with E2Fbs only. However, in normal cells the vector inserted with both SP1 binding site and E2Fbs did not show any E2F activity, and even repressed the gene expression

^" capacity that would be normally expected by the presence of the Spl binding

0.0 site. Thus, by combining the Spl binding site with the E2Fbs, we can now more efficiently direct the cancer cell specificity of gene expression. In contrast, the vector inserted with Spl binding site only without E2Fbs showed a considerable amount of gene expression in both normal and cancer cells. In other words, vectors inserted with other transcription factor binding sites in addition to E2Fbs can be utilized as an excellent expression system showing a high level gene expression in a cancer-cell-specific manner. Consequenth⁷, the present invention provides a highly efficient gene expression system which is specifically active in cancer cells without affecting normal cells. Bv combining with appropriate structural genes the present invention should provide a highly useful and safe gene therapeutic tool for the treatment of many types of cancers.

81

Claims

CLAIMSWhat is claimed is:

1. A cancer-specific gene expression system characterized by having a promoter and a structural gene wherein said promoter contains E2Fbs, a binding site for the transcription factor E2F.

2. A cancer-specific gene expression system according to claim 1, wherein said structural gene is selected from the group consisting of suicide genes, cvtokine genes, immune-regulatorv genes, antisense genes, and tumor- suppressor genes.

3. A cancer-specific gene expression system according to claim 1, wherein said structural gene is selected from the group consisting of genes encoding cytosine deaminase, thymidine kinase, tumor necrosis factor (TNF) α, interleukin-2, interleukin-12, interleukin-18, granulocyte/macrophage- colony stimulating factor (GM-CSF), and the tumor suppressor gene p53.

4. A cancer-specific gene expression system according to claim 1, wherein said binding site E2Fbs for said transcription factor E2F is either a monomer or a multimer.

5. A cancer-specific gene expression system according to claims 1 and 4, wherein said binding site E2Fbs for said transcription factor E2F consists of 1-24 subunits.

6. A cancer-specific gene expression system according to claim 1, wherein said promoter additionally contains one or more of the binding sites for transcription factors selected from the group consisting of Spl, API, NF1 (ATF) and C/EBP.

7. A cancer-specific gene expression system according to claims 1 - 6, wherein said cancer is a kind where RB gene is genetically or functionally impaired or a kind that is infected with DNA tumor viruses.

8. A cancer-specific gene expression system according to claim 7, wherein said cancer is the one selected from the group consisting of retinoblastoma, cervical cancer, head and neck cancer, intraepithelial neoplasias and carcinoma of larynx, juvenile flat warts, Burkitt's lymphoma, lymphoproliferative syndrome, Hodgkin's disease and nasopharvngeal carcinoma.