WO2002095042A1

WO2002095042A1 - Recombinant adenovirus vector producing tumor-suppressor e2 protein and the use thereof

Info

Publication number: WO2002095042A1
Application number: PCT/KR2002/000962
Authority: WO
Inventors: Eun-Seong Hwang; Chan-Jae Lee
Original assignee: AMINOGEN Co Ltd
Current assignee: AMINOGEN Co Ltd
Priority date: 2001-05-21
Filing date: 2002-05-21
Publication date: 2002-11-28
Anticipated expiration: 2003-11-21
Also published as: KR20020088738A

Abstract

The present invention relates to a recombinant adenovirus vector, in which a tumor-suppressor E2 gene is inserted, and the use thereof. The E2 protein has an inhibitory effect on oncogene expression, and an effect of inducing cellular senescence. As a result, the E2 gene-inserted recombinant adenovirus vector can be used in gene therapy, capable of effectively treating patients in the terminal stage of cancer. Cell line, in which cellular senescence is induced by infection with the recombinant adenovirus vector, can be used in selecting substances inhibiting cellular senescence.

Description

RECOMBINANT ADENOVIRUS VECTOR PRODUCING TUMOR- SUPPRESSOR E2 PROTEIN AND THE USE THEREOF

Technical Field

The present invention relates to a recombinant adenovirus vector, in which a tumor- suppressor E2 gene is inserted, and the use thereof, and more particularly, to a recombinant adenovirus vector having anticancer activity, in which the E2 gene of bovine papillomavirus is inserted, and the use thereof in treatment of cancer and selection of substances inhibiting cellular senescence.

Background Art

Cervical cancer is one of the most common malignancies affecting women worldwide. It is believed that more than 90% of cervical cancers are caused by HPV (human papillomavirus). Generally, the division of cervical epithelial cells takes place in an orderly controlled manner until cellular differentiation or senescence occurs. However, if the epithelial cells are infected by HPV, E6 and E7 genes of the HPV are expressed and thus the division process gets out of control. In this state, the epithelial cells will continue to divide, a process called "immortalization".

It is known that the E6 and E7 genes interact with various other factors in cells and thus have influence on the functions of the factors (zur Hausen et al, J. Nat. Cancer Inst.,

92:690-698, 2000). Specifically, it is known that E7 protein inactivates Rb, a tumor suppressor gene, while E6 protein, together with E7 protein, induces the activity of telomerase, thus extending the lifetime of cervical epitlielial cells infected with HPV, and, consequently, contributing to the immortalization of cells. In particular, the suppression of p53 activity by the E6 protein causes inactivation of the Gl checkpoint in the cell cycle, and makes cellular chromosomes unstable. It has been reported that E7 protein induces abnormal synthesis of centrosomes by an unknown mechanism (Duensing et al, Cancer Res., 61:2356-2360, 2001). As a result, the E7 protein, together with the E6 protein, makes cellular genes unstable, and causes an accumulation of mutations in the chromosomes. That is, the infection of HPV in cells makes it possible to express E6 and E7 proteins. As a result, growth inhibition pathways in the cells are interrupted, whereby the cell cycle cannot be controlled and cellular senescence by shortening of telomeres is inhibited. Furthermore, the expressions of the E6 and E7 proteins increase instability of cellular chromosomes, whereby accumulation of mutations in the chromosomes takes place. In the end, immortalization of cells occurs and thus normal cells develop into cancer cells.

Cervical cancer caused by HPV has been treated by surgery, the administration of anticancer agents or radiotherapy, as in treatments for other cancers. However, there is a problem in that, in case of patients in the terminal stage of the cancer, the rate of complete recovery is very low. Accordingly, gene therapy studies to treat the cancer using a specific gene have been recently performed.

In gene therapy, gene related to disease is delivered into the body of patient, and is expressed in target cells, whereby the growth of the target cells is suppressed or killed. The success of gene therapy depends on strategies for delivering genes into and sufficiently expressing them in the target cells. In this regard, gene delivery vehicles such as liposomes have been used. The reason for the use of these vehicles is that nucleic acids alone pass through the target cells with a low efficiency. However, there is a problem in that most liposomes are broken down during the delivery of genes to nuclei in the target cells, thereby lowering the delivery efficiency. Many of the vectors currently in use are based on modified versions of viruses capable of infecting the human body. This is because foreign genes can be efficiently inserted into the target cells using the viral vectors.

In particular, adenoviruses have their particular mechanisms for delivering genes to nuclei in cells. Also, their gene delivery efficiencies to nuclei in cells are higher than those of other viruses such as retroviruses and adeno-associated viruses (AAVs). Accordingly, adenovirus vectors are frequently used in gene therapy.

Adenoviruses cause acute respiratory diseases, which are common in crowded areas. They are pathogenic viruses but their symptoms are not fatal. Up until now, about 47 kinds of human adenoviruses have been isolated. Of those, adenovirus types 4 and 7 have been developed and used in particular as orally administered vaccines for prevention of acute respiratory diseases. Generally, adenovirus type 5 has been developed and used as vector for gene therapy. It has a linear structure of about 36kb. The viral origins of replication are located within both ends of the genome and are essential to the initiation of viral replication. The adenovirus type 5 also comprises four early genes, El, E2, E3 and E4, and genes encoding capsid protein. The genes, El, E2, and the portion of E4 are essential for viral replication, and the E3 gene is not essential for viral replication, but encodes proteins involved in evading host immunity. A therapeutic gene is inserted in the El site of the adenovirus genome. The El gene site can be replaced with about 4.5kb of a therapeutic gene, and the deletion of the El and E3 genes makes it possible to introduce up to about 8kb of a therapeutic gene into the deleted regions. This is called the first generation adenovirus vector. 293 cell line is human embryonic kidney cell that has been transformed with the adenovirus El gene, which is essential for the adenovirus replication. Therefore, recombinant adenoviruses that the El gene deleted can be multiplicated in the cell line. It was generally thought the El gene deleted-adenovirus vectors would not be replicated in vivo, however, the results of animal tests showed that a substitution factor by the El protein in cells can allow adenovirus replication. In this case, there are problems in that host immune response to adenovirus proteins is induced, and thus expression duration of the therapeutic gene is shortened and expression efficiency of the therapeutic gene is reduced upon second inoculation. To overcome these problems, the second or the third generation adenovirus vectors have been developed. As for tliese vectors, the site limit for a therapeutic gene to be introduced is about lOkb and the expression of the therapeutic gene lasts for a long time. Furthermore, these vectors contain no potentially harmful viral genes and little replication occurs in vivo. These adenovirus vectors are suitable for gene therapy requiring the transient expression of a therapeutic gene, because the vector genes are expressed transiently while the host cells are living, without being integrated into the host cell chromosome. Further, the virus particles of the vectors are stable and may be produced in high titers (10¹³ virus particles/ml). Still further, the size limit for a therapeutic gene to be introduced is large (10 kb) and excellent expression is accomplished, h addition to the above, a variety of cells (respiratory epithelial cell, endothelial cell, muscle cell, hepatocyte, etc.) which are actively dividing or not dividing, can be transfected with these virus vectors.

Meanwhile, it is well known that the E2 protein of bovine papillomavirus binds to gene regulatory sites of HPV and controls the expressions of the early genes E6 and E7 (Androphy et al, Nature, 325:70-73, 1987; McBride et al, J. Biol. Chem., 266:1841-1844, 1991). If the E2 protein is over-expressed, the E2 protein binds to adjacent site of P97, a promoter of HPV type 16, or PI 05, a promoter of HPV type 18 (Haugen et al, EMBO J., 6:145-152, 1987; Thierry et al, EMBO J., 6:3391-3397, 1987; Romanczuk et al, J. Virol, 64:2849-2859, 1990). As a result, factors involved in the initiation of gene transcription in cells are inhibited or formation of the transcription initiation complex is interrupted, thereby transcription being suppressed (Nishimura et al, J. Virol, 74:3752-3760, 2000). In particular, the E2 gene of bovine papillomavirus (BPV) suppresses the expressions of the E6 and E7 genes in cervical cancer cells. Therefore, the E2 gene has been effectively used to investigate the functions of the E6 and E7 proteins. Fuilhermore, it has been reported that the expression of the BPV E2 gene in cervical cancer cell line reduces the expressions of the E6 and E7 genes, and allows p53 and Rb genes, which are tumor suppressors, to activate tumor suppression pathway, thereby cell growth being suppressed (Hwang et al, J. Virol, 67:3720-3729, 1993; Dowhanick et al, J. Virol, 69:7791-7799, 1995; Hwang et al, Oncogene 12:795-803, 1996; Desaintes et al, EMBO J, 16:504-514, 1997; Sanchez-Perez etal, J. Gen. Virol, 78:3009-3018, 1997).

The present inventors have made every effort to develop a gene therapy technique capable of efficiently treating cervical cancer. And based on the fact that the E2 gene of BPV suppresses the expressions of the genes E6 and E7, known to contribute to cervical cancer, the present inventors constructed a recombinant adenovirus vector by introducing the E2 gene into an adenovirus vector. And the present inventors selected an adenovirus clone capable of producing the E2 protein after transfecting the recombinant adenovirus vector into an appropriate packaging cell line.

Disclosure of the Invention

Therefore, the present invention has been made in view of the above problems, and it is an object of the present invention to provide a recombinant adenovirus vector for treatment of cancer.

It is another object of the present invention to provide an adenovirus clone comprising the recombinant adenovirus vector for treatment of cancer. It is yet another object of the present invention to provide a cell line, in which cellular senescence is induced by the recombinant adenovirus vector.

In accordance with one aspect of the present invention, the above and other objects can be accomplished by the provision of a recombinant adenovirus vector for treatment of cancer, comprising the expression cassette consisting of a replication origin, an immediate early promoter of human cytomegalo virus, an E2 gene and a polyadenylation signal.

In accordance with another aspect of the present invention, there is provided an adenovirus clone obtained by transfecting the recombinant adenovirus vector into an appropriate packaging cell line. In accordance with yet another aspect of the present invention, there is provided a cell line, in which cellular senescence is induced by the recombinant adenovirus vector.

Brief Description of the Drawings

The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description talcen in conjunction with the accompanying drawings, in which:

Fig. 1 is a schematic genetic map of the recombinant adenovirus vector of the present invention; Fig. 2 is a PCR result showing degree of suppression of expression of HPV E6 gene by BPV E2 gene;

- : No expression of E2 gene in the absence of infection by AvBPVE2 +: Expression of E2 gene with infection by AvBPVE2

Fig. 3 is a Western blot result showing degree of suppression of HPV E7 gene expression by BPV E2 gene; MOCK: treatment with PBS buffer instead of AvBPVE2

Fig. 4 is an nmunoblot result showing the expressions of p53, p21 and Rb genes induced by BPV E2 gene in cells;

Fig. 5 is a photograph showing cellular senescence induced by BPV E2 transfection using β -galactosidase staining;

Fig. 6 is an electrophoresis gel showing the relative activity of telomerase, after the introduction of BPV E2 gene into cervical cancer cell line; - : No expression of E2 gene in the absence of infection by AvBPVE2

+: Expression of E2 gene with infection by AvBPVE2

Fig. 7 is an Immunoblot result showing the expressions of hTERT and c-myc, after the introduction of BPV E2 gene into cervical cancer cell line;

- : No expression of E2 gene in the absence of infection by AvBPVE2 +: Expression of E2 gene with infection by AvBPVE2; and

Fig. 8 is an Immunoblot result showing the expressions of E6, E7, p53/p21, c-myc and hTERT at various times, after the introduction of BPV E2 gene into cervical cancer cell line;

- : No expression of E2 gene in the absence of infection by AvBPVE2 +: Expression of E2 gene with infection by AvBPVE2.

Best Mode for Carrying Out the Invention

Hereinafter, the present invention will be described in detail. Adenoviruses are DNA viruses and have a genome of 36 kb consisting of the El gene essential for viral replication, genes essential for virus packaging, and the like. In order to use adenoviruses for gene therapy, essential genes for viral replication must be inactivated, so as to prevent any other diseases capable of being caused by the self-replication of the virus in the human body. Therefore, the deletion of the El gene essential for viral replication among adenovirus genome, makes it possible to prevent self-replication of the virus in normal cells, thereby achieving a replication-deficient adenovirus capable of being used in gene therapy.

In order to prepare a recombinant adenovirus vector for anticancer therapy in the present invention, the present inventors replaced the El gene of adenovirus with an expression cassette consisting of an immediate early promoter of human cytomegalovirus or CMV, an E2 gene, and a polyadenylation signal. The E2 gene can be selected from the group consisting of bovine papillomavirus E2 gene, human papillomavirus E2 gene, or deer papillomavirus E2 gene, and the like. Preferably, the E2 gene of bovine papillomavirus can be used. The polyadenylation signal can be derived from SV40 (simian virus 40), human growth hormone gene, and the like. Preferably, the polyadenylation signal of the SV40 can be used. h accordance with one embodiment of the present invention, the present inventors constructed a recombinant adenovirus vector, pΔACMV-BPVE2 for anticancer therapy, comprising the expression cassette consisting of a replication origin, an immediate early promoter of human CMV, an E2 gene of bovine papillomavirus and a polyadenylation signal of SV40. The schematic genetic map of the pΔ ACMV-BPVE2 is shown in Fig.1. In order to obtain adenovirus clone capable of producing the E2 protein, the recombinant adenovirus vector was transfected into an appropriate packaging cell line. Specifically, the 293 cell line was used as a packaging cell line. 293 cells are transformed with the adenovirus El gene and thus can produce the El protein.

Adenoviruses have a large genome of 36 kb and thus their gene manipulation is not easy. To overcome this problem, part of the adenovirus genome, not the entire of adenovirus genome is inserted into the adenovirus vector, and the genes and proteins necessary for the multiplication of adenovirus are provided from an adenovirus helper vector. In the present invention, the pJM17 helper vector was used for this purpose.

If the recombinant adenovirus vector of the present invention and the adenovirus helper vector are co-transfected into the packaging cell line, the recombinant adenovirus vectors containing the E2 gene are replicated in large quantities. At the same time, genes necessary for the packaging of adenoviruses are expressed, thereby complete adenoviruses being prepared. Generally, about ten thousand adenovirus particles are produced in about one 293 cell. Viruses so accumulated in the cells can be purified by centrifugation following lysing of the cells.

In order to prepare adenovirus clone useful for gene therapy, it is important to exclude the possibility of the unwanted formation of RCVs (replication competent recombinant viruses), in the above process. In the recombinant adenovirus vector of the present invention, pΔ ACMV-BPVE2, the El gene of the original adenovirus genome is replaced with the expression cassette containing the E2 gene of BPV. Viruses produced from the recombinant adenovirus vector cannot self-replicate in normal cells, except for the 293 cell line. However, RCVs can be produced in high numbers in the 293 cell line. This is because homologous recombination occurs between the recombinant adenovirus vector and the adenovirus constructs present in the 293 cell line. Specifically, the expression cassette containing the BPV E2 gene of the recombinant adenovirus vector is replaced with adenovirus El gene in the 293 cell line by homologous recombination in the 293 cell line, which is transfected with the recombinant adenovirus vector of the present invention. As a result, a new viral DNA containing the El gene can be constructed and can be replicated in normal cells with no El protein. RCVs so produced may give rise to host infection and thus cannot be clinically used. Only viruses with negative results in RCV tests can be used clinically.

In accordance with another embodiment of the present invention, the present inventors investigated whether the adenovirus clone expresses BPV E2 protein after being infected in host cells and is contaminated with RCVs. As a result of the investigation, the present inventors recognized that the adenovirus clone containing the recombinant adenovirus vector of the present invention suppresses cancer cell growth and does not give rise to virus infections in the host cells, thereby capable of being used for gene therapy for treatment of cancers.

To investigate if RCVs exist among the adenovirus clone, general methods known in the pertinent art can be used. Specifically, based on the fact that RCVs contain the El gene, which is not present in recombinant adenovirus vector, the present inventors determined the presence or absence of the El gene using PCR (polymerase chain reaction), after the isolation of genes from the adenovirus clone. Further, to search for RCVs, which may be present in small numbers in a large population of non-replicable viruses, a modified method of Zhang et al., in which HeLa cells were used to subculture viruses was used (Zhang etal, Human Gene Therapy, 6:155-164, 1995).

As a result, the present inventors selected RCV-nonproducing adenovirus clone, which may produce active BPV E2 proteins in host cells. It was named AvBPVE2. The AvBPVE2 clone was deposited in the Korean Culture Center of Microorganisms (KCCM) under Deposit No. KCCM 10264 on April 30, 2001.

In accordance with still another embodiment of the present invention, the present inventors investigated degrees of expressions of the E2, E6 and E7, after infection of AvBPVE2 into cervical cancer cell line in order to confirm that the expression of the BPV E2 protein can suppress the expression of the oncogenes E6 and E7. Caski, HKcHPVl 6.dl and CXI 6.11 may be used as the cervical cancer cell lines. The Caski is a cervical cancer cell line and has about 600 copies of HPV type 16 DNA. The HKcHPV16.dl and CX16.11 are derived from the epidermal keratin cells and the cervical epidermal cells of the uterus, and are immortalized cell lines transformed with two copies of HPV type 16 DNA. As shown in Fig.2, recognizable amounts of the E2 genes were expressed in the Caski cell line and the two immortalized cell lines, which were infected with AvBPVE2. The number of transcripts of the E6 genes dropped to nearly undetectable levels in all the cell lines infected with AvBPVE2. Furthermore, the degree of suppression of HPV E7 gene expression by BPV E2 gene was investigated. As shown in Fig.3, in contrast to the Caski cells infected with Avβ-Gal, an adenovirus expressing bacterial β-galactosidase, the amount of the E7 protein was dramatically reduced in Caski cells infected with AvBPVE2. From the above results, it can be recognized that the transcription of the E6 and E7, early expression genes of HPV, can be suppressed by the BPV E2 protein, regardless of whether the transcriptions of the E6 and E7 are carried out in immortalized cell lines such as HKcHPVl 6.dl and CXI 6.11, or in a transformed cancer cell line such as Caski, and how many copies of HPV DNA exist in the cell lines.

In accordance with the still another embodiment of the present invention, the present inventors investigated the expressions of the p53, p21WAFl and Rb genes in cell lines infected with the E2 genes, in order to determine how the expression of the E2 gene affects the expression of each of the tumor suppressor genes. The p53, p21WAFl and Rb genes are representative genes known as suppressors of cancer cells proliferation. As shown in Fig.4, the expressions of above three proteins were increased in cell lines, in case that the E2 genes were introduced. That is, it could be seen that the expression levels of the tumor suppressor genes increased in the presence of E2 genes. This fact suggests the possibility of anticancer gene therapy by the recombinant adenovirus of the present invention. Accordingly, the present invention provides a gene therapy method and means capable of treating cancers, by infecting the adenovirus clone AvBPVE2 comprising BPV E2 gene-inserted recombinant adenovirus vector into the affected cells. Examples of cancers which may be treated by the adenovirus clone, AvBPVE2, include, but are not limited to, cervical cancer or viral laryngeal cancer. hi accordance with the yet another embodiment of the present invention, it was confirmed that the expression of BPV E2 gene induces senescence of cell lines. A variety of methods known in the pertinent art can be used to confirm senescence of cell lines. The measurement of the activity of SA-β-galactosidase (senescence-associated-β-galactosidase, refer to 'SA-β-GaP, hereinafter), the measurement of the activity of telomerase, and the measurement of the expression of each of hTERT (human telomerase reverse transcriptase) and c-myc were used in the present invention.

Because the activity of the SA-β-Gal generally increases in senescent cells, the measurement of the activity of SA-β-Gal is mainly used as a method measuring degree of cellular senescence. The present inventors observed the appearance of Caski cells under microscope after the infection of AvBPVE2 into the Caski and staining of the Casld with SA- β-Gal stain. As shown in Fig.5, the Caski cells, in which the E2 gene was expressed, had a large and flat form, which is common in senescent cells.

Meanwhile, hTERT, a catalytic subunit of telomerase complex, plays an important role to the activity of telomerase, and is expressed at a high level in most cancer cells. However, as shown in Fig.7, in case BPV E2 gene was introduced into cells, the expression of hTERT dropped. That is, it could be seen that the reduction of the expression of the hTERT in the cells capable of expressing BPV E2 proteins lowered the activity of telomerase. The c-myc protein binds the E-box site located in the promoter of the hTERT gene and controls the expression of hTERT gene. Accordingly, the expression of the c-myc gene is reduced in senescent cells. In contrast, the increased expression of p53 suppresses the expression of hTERT, thereby the activity of telomerase being reduced. The inventors observed that the level of the c-myc protein dropped and the activity of the p53 increased in the BPV E2 expressing cells.

In Fig.8, the activity of p53 was expressed as that of p21WAFl. The activity of p53 appeared 24 hours after the infection of the AvBPVE2 of the present invention. At 30 hours after the infection, the activity of p53 reached maximum and remained constant, while, the expression of hTERT began falling 30 hours after infection with AvBPVE2. At 36 hours after an infection, the expression of hTERT was remarkably reduced.

The present invention provides cellular senescence-induced cell lines, resulting from Hie provision of the BPV E2 gene-introduced recombinant adenovirus vector. The cell lines to be used in inducing cellular senescence tests comprise Caski, HeLa, HT-3, HKcHPVl 6.dl and CXI 6.11. Preferably, the Caski cells can be used. The senescent cell lines by the expression of BPV E2 gene, were large and flat in form (see Fig.5), and had a low activity of telomerase (see Fig.6). The cellular senescence-induced cell line by the expression of BPV E2 gene can be used in the selection of substances inhibiting cellular senescence.

Hereinafter, the present invention will be illustrated by way of examples. It is, however, to be borne in mind that the present invention is by no means limited to or by them.

Example 1 Cell line culture

Cervical cancer cell lines, HeLa, HT-3 and Caski, were obtained from ATCC. The cell lines were cultures in 10% FBS (fetal bovine serum) containing DMEM (Dulbeco's Modified Eagle's medium, Gibco BRL). HKcHPV16.dl and CX16.11, which are cervical epidermal cell lines immortalized by HPV type 16 DNA, were obtained from Lucia Pirisi (University of South Carolina, SC, USA). The above cell lines were cultures in Keratinocyte- SFM (Gibco BRL, Bethesda, MD, USA) media containing EGF (epithelial growth factor) and pituitary extracts. The 293 cell line, which is a kidney embryo cell line, was cultured in 10% FBS containing DMEM.

Example 2

Construction of recombinant adenovirus vector containing BPV E2 gene The recombinant adenovirus vector was constructed to express the E2 gene of BVP at ahigh level in the Casld, HKcHPVlό.dl or CX16.11 cell lines cultured in the example 1.

The El gene of adenovirus type 5 DNA (0.95-9.8 map unit) was replaced with the expression cassette consisting of a replication origin, an immediate early promoter of CMV,

1,506 bp (2405-3910) E2 gene of BPV DNA, and a polyadenylation signal of SV40 (simian virus 40), so as to prepare the recombinant adenovirus vector (see Fig.l). The recombinant adenovirus vector was named pΔ ACMV-BPVE2.

Example 3

Preparation of adenovirus clone producing BPV E2 protein To prepare adenovirus clone capable of producing BPV E2 protein, the recombinant adenovirus vector, pΔACMV-BPVE2, constructed in the example 2, and adenovirus helper vector pJM17 (Doctor Graham, McMaster university, Ontario, Canada) were co-transfected into the 293 cell line, a packaging cell line, using a calcium phosphate method. The co- transfection was carried out in a 24-well plate. As a result, the formation of plaques was observed in two wells (number 1 and 2, each). To corifirm that adenovirus clones produce the E2 protein and can be used in anticancer therapy, the inventors investigated whether the above two types of adenovirus clones express the active E2 protein upon infection into host cells and are contaminated with RCVs.

First, 2 x 10⁵ HeLa cells were placed in 60 mm dishes and incubated for 48 hours. 20 μi (at a multiplicity of infection (M.O.I.) of 25) of each of the two types of the adenovirus clones was diluted with 1 ml of FBS-free media and infected into the HeLa cells. At 1 hour after the infection, the FBS-free media was replaced with FBS-containing media and the HeLa cells were incubated for 48 hours. Upon investigating the levels of BPV E2 proteins, it was confirmed that the E2 proteins were expressed in both of the two types of the adenovirus clones.

Meanwhile, the adenovirus clone with no RCV was isolated from the two types of the adenovirus clones obtained previously, according to the RCV determination method described in example 4 below. It was named AvBPVE2, and was deposited in the Korean Culture Center of Microorganisms (KCCM) under Deposit No. KCCM 10264 on April 30, 2001. The entire nucleotide sequence of BPV E2 gene present in the adenovims clone,

AvBPVE2, was determined, confhπiing that the E2 gene contained in the adenovirus clone was wild type. The titer of the adenovirus clone was determined from the measurement of the number of plaques in the 293 cell line.

Example 4

Analysis of replication competent recombinant viruses

The presence of RCVs, which can be present in the adenovirus clones (number 1 and 2) obtained in the example 3, was determined as the following manner.

First, to confirm that the El A gene and E1B gene of adenovirus type 5 are present in DNAs isolated from the adenovirus clones, the DNAs were subjected to PCR using a pair of El A primers of SEQ ID NO. 1 and SEQ ID NO. 2, and a pair of E1B primers of SEQ ID NO. 3 and SEQ ID NO. 4. The adenovirus DNAs were treated with proteinase K (2 mg/ml) in the presence of 0.5% SDS. Then, the treated DNAs were isolated using the conventional phenol extraction and ethanol precipitation. According to PCR using the El A primers and E1B primers, the El gene present in adenovirus DNA corresponds to 752 bp and 1818 bp DNA bands on agarose gel. In case of using adenovirus primers having nucleotide sequences of SEQ ID NO. 5 and SEQ ID NO. 6 as controls, an 861 bp DNA band was detected by PCR.

To increase sensitivity of searching for RCVs which can be present in small numbers in a large population of non-replicable viruses, the inventors used a modified method of Zhang et al. (Zhang et al, Human Gene Therapy, 6:155-164, 1995). To amplify a few RCVs in a large population of non-replicable viruses, the above number 1 and 2 viral clones were subcultured in HeLa cells three times. The three subcultures were carried out as follows: HeLa cells were infected with the viral clones. After 48 hours, the HeLa cells were lysed by freeze-thawing process to obtain the supernatants of the lysate. The supernatants were infected into fresh HeLa cells to repeat the previous procedure. To obtain viral DNAs for PCR, clear lysates were obtained from the final subcultured cells, treated with proteinase K, extracted from phenol and precipitated with ethanol. The obtained DNA pellets were dissolved in distilled water, and PCR was performed using the El A primer.

As for the adenovirus clone AvBPVE2 (number 1) selected in the present invention, when it was subcultured in HeLa cell line, DNA fractions corresponding to 752 bp and 1818 bp DNA bands were not discovered. While, judging from the fact that number 2 adenovirus clone was multiplicated in HeLa cell line, it can be seen that RCVs were mixed in the number 2 adenovirus clone.

Example 5 Inhibition of the expressions of the E6 and E7 genes by the E2 protein 5-1) Identification of inhibition of expression of the E6 gene by PCR To confirm whether the E2 protein suppresses the expressions of the oncogenes E6 and E7, AvBPVE2 (at a M.O.I. of 50) obtained in the example 3 were infected into each of the cervical cancer cell lines, HeLa, HT-3, Caski, HKcHPVl 6.dl and CXI 6.11.

At 48 hours after the infection, RNAs were isolated from each of the cell lines using

Trizol reagent (Gibco BRL). From 5 mg of the isolated RNAs, cDNAs were synthesized using Superscript II reverse transcriptase (Gibco BRL). 1/40 of the synthesized cDNAs were used in PCR. The PCR was carried out at 95 ^°C for 30 seconds, at 58 ^°C for 30 seconds, and at 72 ^°C for 1 minute, for 30 cycles.

E2 genes were expressed in all the cell lines infected with AvBPVE2. The results were confirmed by PCR using two primers of SEQ ID NO.7 and SEQ ID NO. 8, 2 days after the viral infection (see Fig.2). The effect on transcription of HPV early expression genes, e.g. HPV E6 gene, by the expression of BPV E2, was confirmed by PCR using two primers of SEQ ID NO. 9 and SEQ ID NO. 10. According to the PCR result, the amount of the E6 DNA fell to undetectable levels in all the cell lines infected with AvBPVE2.

5-2) Identification of E7 protein using Western blotting

At 48 hours after the infection of Caski cells with each of AvBPVE2 and Avβ-gal (at M.O.I, of 50), proteins were isolated from the Caski cells. Proteins were isolated using RIPA buffer and 50 μg of the protein was used in electrophoresis. The proteins were subjected to 12% gel electrophoresis and then transferred to nitrocellulose membrane (Hybond ECL, Amersham) using trnasfer buffer (12.5 mM Tris, 0.1M glycine, 0.05%) SDS, 20% methanol). The nitrocellulose membrane was blocked with 5% skin milk powder in buffer (25 mM Tris- HC1 pH 8.0, 125 mM NaCl, 0.1% Tween-20) and then 3 ml of anti-E7 antibody from VD6 cell line culture, which produces and secretes monoclonal antibody against the E7 protein of HPV type 16, was added thereto. The nitrocellulose membrane was washed with TBST buffer and incubated with horseradish peroxidase conjugated anti-mouse antibody. The protein bands were developed using LumiLightplus Western Blotting Substrate (Roche, Germany). The Avβ -gal viruses were provided by Jung et al. (Urology Research Laboratory, University of Virginia Health Science Center, Charlottesville, Virginia, U.S.A.).

Example 6

Measurements of expressions of tumor suppressor genes induced by BPV E 2 gene In order to measure degrees of expressions of p53, p21WAFl and Rb proteins in cell lines transfected with adenovirus clone producing BPV E2 protein, immunoblotting was performed.

Similar to the example 5, at 48 hours after infection of HeLa, Casl , HKcHPVl 6.dl and CXI 6.11 cell lines with AvBPVE2 (at M.O.I. of 50), proteins were isolated from the cell lines. Proteins were isolated using RIPA buffer and 15 mg of the protein was used in electrophoresis. The Rb protein was subjected to 7.5% gel electrophoresis, while p53 and p21WAFl proteins were subjected to 12% gel electrophoresis was used. Then, the proteins were transferred to nitrocellulose membrane (Hybond ECL, Amersham) using transfer buffer (Rb protein: 12.5 mM Tris/O.IM glycine, p53 and p21WAFl proteins: 12.5 mM Tris/O.IM glycine + 0.05% SDS, 20% methanol). The Rb protein-transferred nitrocellulose membrane was blocked with 5% sldn millc powder in TNET buffer (10 mM Tris-HCl (pH 7.4), 2.5 mM EDTA, 50 Mm NaCl, 0.2% Tween-20) and incubated with anti-Rb antibody. The p53 and p21WAFl -transferred nitrocellulose membranes were blocked with 5% sldn millc powder in TBST buffer (25 mM Tris-HCl (pH 8.0), 125 mM NaCl, 0.1% Tween-20) and incubated with anti-p53 antibody (DO-1; Calbiochem) and anti-p21WAFl antibody (EA10; Calbiochem), respectively. The nitrocellulose membranes were washed with TNET or TBST buffer and incubated with horseradish peroxidase conjugated anti-mouse antibody. The protein bands were developed using LumiLightplus Western Blotting Substrate (Roche, Germany).

As shown in Fig.4, the levels of the p53 and p21WAFl proteins increased markedly in all the cell lines expressing BPV E2. The Rb proteins also increased in terms of total amounts, and were transfonried into active dephosphorylated forms.

Accordingly, it was confirmed that E2 protein induces the expressions of tumor suppressor genes, resulting in effectively inhibiting cancers.

Example 7

Measurement of inhibition of cell proliferation by BPV E2 protein In order to confirm that inhibition of expressions of E6 and E7 proteins by BPV E2 protein makes it possible to inhibit cell proliferation, DNA synthesis was monitored. At 48 hours after infection of HeLa, Caski, HKcHPV16.dl and CX16.11 cell lines with AvBPVE2, the cells were labeled with ³H-thymidine (1.5 mCi/ml, Amersham, England) for 4 hours. The cells infected with Avβ -Gal were used as controls. Then, the amount of ³H-thymidine incorporated into chromosomal DNAs of each of the cells was measured and compared with controls. The results are presented as DNA synthesis inhibition (%) in Table 1.

Table 1

As can be seen from the Table 1, in HKcHPV16.dl and CX16.11 cell lines, the expression of BPV E2 markedly inhibited DNA synthesis (89.5% and 87%, respectively).

The expression of BPV E2 also inhibited DNA synthesis to a large degree in Caski cell line.

Accordingly, it can be seen that the inhibition of expressions of E6 and E7 proteins by BPV E2 reduces the proliferation of the cells expressing HPV E6 and E7 proteins.

Example 8

Measurements of activities of SA-β-Gal in cells

HeLa cells, when the growth which is interrupted by BPV E2, have a flat cellular form, enlarged cytoplasm, high levels of SA-β-Gal activity, and self-fluorescence by accumulation of lipofuscin. These phenomena are indicators of cellular senescence.

Meanwhile, it was investigated whether the treatment of Caski, HKcHPVl 6.dl and CXI 6.11 cell lines with AvBPVE2 increases the activity of SA-β-Gal.

At 96 hours after infection of the cell lines with AvBPVE2, the cell lines were washed with phosphate buffered saline (PBS, pH 7.4) and then fixed using 3% formaldehyde for 5 minutes. MOCK treated with only PBS, and the cell lines infected with Av-β-Gal, were used as controls. The cells were washed with 1 mM MgCl₂ containing PBS (pH 7.4) and then with 1 mM MgCl₂ containing PBS (pH 6.0). Then, the resultant cells were incubated with SA-β- Gal stain (lmg/ml 5-bromo-4-chloro-3-indol β -D-galactopyranoside (X-gal), 5 mM K₃Fe(CN)₆, 5 mM K₄Fe[CN]₆, and 2 mM MgCl₂ in PBS (pH 6.0)) at 37^°C overnight. Then, the activities of SA-β-Gal in each of the cell lines were measured as the number of SA-β-Gal positive cells (%), and the cellular form of the cells was observed under microscope.

At 5 days after infection of the above viruses, the cells infected with AvBPVE2 showed higher activities of SA-β-Gal than controls (see Table 2). Furthermore, as shown in Fig.5, the Caski cells infected with AvBPVE2 had flat cellular form and enlarged cytoplasm. Accordingly, it can be seen that Caski cell line and immortalized cell lines proceed to cellular senescence, due to the inhibition of expressions of E6 and E7 proteins.

Table 2

Example 9

Effect of BPV E2 on telomerase activity

Telomerase activity was measured to confirm whether BPV E2 protein induces cellular senescence.

The measurement of telomerase activity was carried out according to telomerase repeat amplification protocol (TRAP). Caski, HKcHPVl 6.dl and CXI 6.11 cells were dissolved with dissolving buffer (10 mM Tris-HCl (pH 7.5), 1 mM MgCl₂, ImM EGTA, 0.1 mM phenylmethylsulfonyl fluoride, 5 mM β -mercaptoethanol, 0.5% CHAPS, and 10% glycerol in buffer), and then TRAP buffer (20 mM Tris-HCl(pH 8.3), 1.5 mM MgCl₂, 63 mM KC1, 0.005% Tween 20, ImM EGTA, 50 mM dNTP, 0.1 mg/ml bovine serum albumin) and 0.1 μg of TS primer of SEQ ID NO. 19 were added to Hie dissolved cells, which were let stand at room temperature for 30 minutes. 0.1 g of CX primer (5'-CCCTAA-3')₃ 2 units of Taq DNA polymerase, and ³²P labeled dGTP were added to the resultant and then PCR was performed. PCR was performed for 27 cycles under the reaction conditions as follows: at 94 ^°C for 30 seconds, at 50 ^°C for 30 seconds, and at 72 ^°C for 1 minute. The products so obtained were subjected to 15% polyacrylamide gel electrophoresis and the electrophoresis photograph was obtained to visualize the results.

As can be seen from Fig.6, Caski, HKcHPVl 6.dl and CXI 6.11 cells had high levels of telomerase activities in the absence of AvBPVE2. However, after infection of the cells with AvBPVE2, the telomerase activities were remarkably reduced. The above result shows that the inhibition of expressions of HPV early genes by BPV E2 makes it possible to reduce the activity of telomerase, inducing cellular senescence.

Example 10

Expressions of hTERT and c-myc by BPV E2 in cells To confirm how the activities of telomerase were controlled in cells expressing E6 and E7 proteins at low levels, the inventors investigated the expression levels of factors involved in the control of telomerase activities.

First, the inventors carried out PCR using two primers having nucleotide sequences of

SEQ ID NO. 9 and SEQ ID NO. 10, and two primers having nucleotide sequences of SEQ ID NO. 11 and SEQ ID NO. 12, respectively, to confirm the effects of E2 gene expression on expressions of E6 and E7 genes. As can be seen from Fig.8, the expression of E2 gene inhibited the expressions of E6 and E7.

Meanwhile, hTERT, a catalytic subunit of telomerase complex, plays an important role to the activity of telomerase, and is expressed at a high level in most cancer cells. However, the result of PCR performed with two primers of SEQ ID NO. 13 and SEQ ID NO. 14 shows that the expression of hTERT was extremely reduced in Caski, HKcHPVl 6.dl and CXI 6.11 cells infected with AvBPVE2. Furthermore, the result of PCR performed with two primers of SEQ ID NO. 15 and SEQ ID NO. 16, shows that the expression of c- myc was reduced in the cells (see Fig.7). The inventors investigated the expression levels of p53, c-myc and hTERT in HeLa cells infected with AvBPVE2 at various times, to confirm that the expression of hTERT depends on the expressions of p53 and c-myc. As shown in Fig.8, the activity of p53 was expressed as that of p21 WAF1. The result of PCR with two primers of SEQ ID NO. 17 and SEQ ID NO. 18, shows that p53 activity appeared 24 hours after infection with AvBPVE2. At 30 hours after infection, the activity of p53 reached maximum and remained constant, while the expression of hTERT began falling 30 hours after infection with AvBPVE2. At 36 hours after the infection, the expression of hTERT was remarkably reduced.

Industrial Applicability

As apparent from the above description, according to the present invention, bovine papillomavirus E2 gene-inserted recombinant adenovirus vector is constructed and adenovirus clone, containing the recombinant adenovirus vector nucleic acids and producing the E2 protein, also are prepared. The E2 protein has an inhibitory effect on oncogene expression. Adenovirus clone containing the E2 gene-inserted recombinant adenovirus vector can be used in gene therapy, thus being capable of treating patients in the terminal stage of cancer. Also, cell line, in which cellular senescence is induced by infection with the recombinant adenovirus vector producing the E2 protein, can be used in selecting substances inhibiting cellular senescence.

Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims. Applicant's or agent's International application No. file reference : OP02-1009

INDICATIONS RELATING TO DEPOSITED MICROOGANISM

OR OTHER BIOLOGICAL MATERIAL

(PCT Rule 13 to)

A. The indications made below relate to tlie deposited microorganism or other biological material referred to in the description on page 10. 15 , line 20. 14

B. IDENTIFICAΗON OF DEPOSIT Further deposits are identified on an additional sheeϋ

Name of depositary institution

Korean Culture Center of Microorganisms

Address of depositary institution (including postal code and country)

Korean Culture Center of Microorganisrns(KCCM)

361-221, Yurim B D, Hongje-1-dong, Seodaemun-gu, Seoul 120-091, Republic of Korea

Date of deposit Accession Number

2001. 04. 30. KCCM-10264

C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheeO

D. DESIGNATED STATES FOR WHICH INDICAΗONS ARE MADE (if the indications are not for all designated States)

E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)

The indications listed below will be submitted to the International Bureau later (specify the general nature of the indications e.g., "Accession Number ofDeposit")

For International Bureau use only

□ This sheet was received by tlie International Bureau on:

lAuthorized officer

FormPCT/RO/134(July 1998)

Claims

What is claimed is:

1. A recombinant adenovirus vector for treatment of cancer, comprising the expression cassette consisting of: a replication origin; an immediate early promoter of human cytomegalovirus; an E2 gene; and a polyadenylation signal.

2. The recombinant adenovirus vector for treatment of cancer according to claim 1 , wherein the E2 gene is selected from the group consisting of bovine papillomavirus E2 gene, human papillomavirus E2 gene and deer papillomavirus E2 gene.

3. The recombinant adenovirus vector for treatment of cancer according to claim 1, wherein the polyadenylation signal is derived from SV40 or human growth hormone gene.

4. The recombinant adenovirus vector for treatment of cancer according to claim 1, which is pACMV-BPVE2 comprising the expression cassette consisting of: a replication origin; an immediate early promoter of human cytomegalovirus; an E2 gene of bovine papillomavirus; and a polyadenylation signal of SV40.

5. A pharmaceutical composition for treatment of cancer, comprising the recombinant adenovirus vector according to any one of claims 1 to 4 as an active component.

6. The pharmaceutical composition for treatment of cancer according to claim 5, which is used for the treatment of cervical cancer.

7. An adenovirus clone, obtained by transfecting a packaging cell line with the recombinant adenovirus vector according to claim 4 (Deposit No: KCCM 10264).

8. A cell line, in which cellular senescence is induced by infection with the recombinant adenovirus vector according to claim 4.

9. The cell line according to claim 8, wherein the cell line is selected from the group consisting of Caski, HeLa, HT-3, HKcHPV16.dl and CX16.11.

10. The cell line according to claim 8 or claim 9, wherein the cell line is used in selecting substances inhibiting cellular senescence.