KR100450450B1 - Adenovirus vector producing antisense telomerase and the methods for anti-cancer treatments - Google Patents

Abstract

The present invention relates to adenoviruses which produce antisense capable of inhibiting telomerase enzyme activity associated with tumor development and the use thereof for anticancer therapy. Specifically, the present invention relates to an adenovirus vector capable of inhibiting the activity of telomerase whose activity is reported in 90% or more tumors and its application method, wherein the adenovirus clone Ad-OA of the present invention is lung cancer, prostate cancer, or the like. It can be usefully used for treating various tumors.

Description

Adenovirus vector producing antisense telomerase and the methods for anti-cancer treatments

The present invention relates to adenoviruses which produce antisense capable of inhibiting telomerase activity reported to be associated with tumor development and the use thereof for anticancer therapy. Specifically, the present invention relates to an adenovirus vector capable of inhibiting telomerase activity whose activity is reported in 90% or more tumors and its application method, wherein the adenovirus clone Ad-OA of the present invention is a lung cancer, prostate cancer, or the like. It can be usefully used for treating various tumors.

Telomere is a specific structure at the end of eukaryotic chromosomes that repeats short DNA fragments (TTAGGG in vertebrates) over 100 kb. These have been reported to exhibit a variety of functions in the form of protein binding. Telomerase 1) protects the chromosome by preventing recombination, interchromosomal fusion, and disruption between chromosomal ends, 2) assists in chromosomal migration during somatic or germ cell division, and 3) ends that occur naturally in straight eukaryotic chromosomes. It helps prevent end-replication problems. In other words, the DNA primer cannot be synthesized by the RNA primer, which is removed at the end of replication by lagging strand DNA synthesis, and the site disappears permanently. . In germ cells, there is an enzyme called telomerase, which continuously synthesizes the TTAGGG repeat at the end of the chromosome, whereas somatic cells lack this telomerase activity, resulting in shortening of telomeres in each cell cycle and eventually death (David et al., 1997). ). 4) Telomerase is a riboprotein, which is a template of the RNA region of telomerase itself, and serves to connect telomeres to the 3 'end of the existing telomeres. That is, telomerase is thought to play an important role in the replication and protection of chromosomes, in particular, it is thought to play an important role in the immortalization of cells and telomerase activity is observed in approximately 80-95% of cancer cells (Feng et al. al., 1997) Therefore, there is an active research worldwide to use the inhibition of telomerase activity in anti-cancer treatment. In other words, according to Bisoffi (Bisoffi et al., 1998), when malignant glioma (MG) cells are infected with an antisense vector against human telomoerase RNA component (hTR), a large part of MG cells dies or differentiates. And reacted more sensitively to anticancer drugs such as cisplatin. In addition, treatment of retrovirus reverse transcriptase inhibitors to cancer cells resulted in inhibition of telomerase activity and apoptosis, and the same effect in the treatment of various peptide nucleic acids and antisense oligomers against hTR (Bhaswati et al., 1998). , AIGlukhov et al., 1998). The relationship between telomerase activity and other tumor suppressor genes or oncogenes in the cell has not been elucidated yet, but recent papers have suggested that telomerase activity is inhibited by inhibiting c-myc expression. It has been found that normal p53 reduces telomere length and induces cell death (kohtaro et al., 1997).

The theory of anticancer therapy with complementary antisense is very simple. In other words, the target gene mRNA to be removed is prevented from working with complementary antisense. Methods using such antisense molecules include methods using synthetic antisense oligomers and methods using expressed antisense sequences. However, synthetic antisense oligomers have many problems such as 1) manufacturing difficulties, 2) degradation by nucleases, and 3) the absence of efficient intracellular delivery methods (Scanlon et al., 1995). Therefore, much attention is paid to methods using expression antisense sequences (Lee et al., 1996; Steiner et al., 1998). The principle of this method is to suppress the expression of a specific gene by cloning a cDNA part of the target mRNA in the eukaryotic gene expression vector in the antisense direction to express complementary RNA sequences.

An object of the present invention is to obtain a tumor specific anticancer effect by inhibiting telomerase activity using an expression antisense sequence. Telomerase consists largely of the hTERT site with the activity of the RNA template site (hTR) and reverse transcriptase. To date, research has been reported to have the greatest effect when inhibiting the RNA template region (hTR) of telomerase. Also, according to Kondo et al., The analysis of telomerase mRNA using the MFOLD program showed the effect of killing cancer cells when the most open sites (94-76 residues) were inhibited with synthetic oligomers (Kondo et al. , 1998). Accordingly, the present invention provides a viral vector that inserts antisense DNA for a telomerase open hTR site into an adenovirus vector, thereby inhibiting telomerase activity by transcribed mRNA.

The present invention provides an antisense DNA synthesis method for producing the adenovirus clone that can be used for anticancer gene therapy. Specifically, the present invention synthesized two primers (SEQ ID NO: 1, SEQ ID NO: 2) to make the antisense adenovirus expression vector, and the two primers at the end of the primers to facilitate cloning.

The adenoviruses provided herein provide adenovirus expression vectors capable of producing antisense telomerase in cells without including the E1 gene region involved in virus propagation. Specifically, the present invention can produce an antisense telomerase using an expression cassette consisting of the latest promoter, polycloning site and post adenine multiplex signal of Simian virtus 40 (SV 40) of Cytomegalovirus. Adenovirus expression vector pΔACMV-OA (see FIG. 1). It is also an object of the present invention to provide an adenovirus clone that can be used for anticancer gene therapy by producing antisense telomerase in cells. Specifically, the present invention provides an adenovirus clone that does not contain proliferative recombinant mutant virus (RCV) by propagating the adenovirus expression vector in packaging cells (293 cells). At this time, adenovirus vector pJM17 was used as an adenovirus parent vector containing a gene necessary for propagation of adenovirus, and 293 cells were used as packaging cells. The adenovirus clones selected in the present invention were named Ad-OA and deposited on August 29, 2001 at the Korea Microbial Conservation Center (Accession No .: KCCM 10312).

It is also an object of the present invention to provide a use of the adenovirus clone for the treatment of cancer. The adenovirus clone of the present invention can be particularly useful for lung cancer, prostate cancer and the like. Specifically, the present invention may provide an anticancer gene therapy for treating cancer by inhibiting cancer progression or killing cancer cells by infecting the adenovirus clone to a site where cancer occurs in the human body.

1 is a genetic map of the adenovirus expression vector pΔACMV-OA of the present invention

2 is a dot-blot analysis of antisense expression in 293 cell lines infected with adenovirus Ad-OA obtained using the adenovirus expression vector of the present invention.

Mock: DNA of 293 cell lines without pretreatment

Ad-CMV: Viral DNA from Ad-CMV null infected cells

Ad-OA: Viral DNA from Ad-OA Treated Cells

Figure 3 is confirmed by the polymerase chain reaction (PCR) using 239 DNA and adenovirus DNA whether the presence of proliferative recombinant mutant virus (RCV) in the adenovirus clones of the present invention

Lane 1-3: Amplification of Ad-OA viral DNA using primers, E1A and E1B primers that do not correspond to the adenovirus E1 gene region

M: 100 base pair marker

Lanes 4-6: 293 DNA amplification using primers that do not correspond to the adenovirus E1 gene region, E1A, E1B primers

4 is confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) whether the adenovirus clone of the present invention selectively removes the target hTR site

M: 100 base pair markers.

Lanes 1, 4, 7: Mock Infection

Lanes 2, 5 and 8: Ad-CMV null infection

Lanes 3, 6 and 9: Ad-OA infection

Figure 5 is a photograph showing the cytotoxicity of NCI-H358 by Ad-OA

A, B, C: Ad-CMV null infected NCl-H358. D, E, F: Ad-OA infected NCl-H358

6 is a result of measuring the growth inhibition of NCI-H358 and Du-145 cells by Ad-OA by trypan blue staining

A: IMR-90

B: Du-145

C: NCI-H358

7 is a result of measuring the effect of inhibiting the telomerase activity in NCI-H358 cells by Ad-OA by the telomere repeat amplification protocol (TRAP)

FIG. 8 shows that Ad-OA infected NCI-H358, Du-145 cells were apoptotic as a result of the TUNEL assay using the ApopTag Apoptosis Detection Kit.

A: Ad-CMV null infection

B: Ad-OA Infection

Figure 9 shows the apoptosis of NCI-H358 cells by flow cytometry analysis after Ad-OA infection

A: Ad-CMV null infection

B: Ad-OA Infection

The present invention provides adenovirus vectors expressing antisense telomerase. In order to prepare the adenovirus of the present invention, an expression vector capable of producing antisense telomerase by removing the E1 gene region from the genomic DNA of the adenovirus and inserting an expression cassette containing antisense telomerase DNA in its place. Manufacture.

Specifically, the present invention includes the remaining genes from which the E1 gene region is removed in adenovirus type 5, and is an early early promoter, multiple cloning site, and simian virus 40 of human cytomegalovirus. An adenovirus expression vector pΔACMVp (A) containing an expression cassette composed of a late polyadenylation signal of (simian virus, SV40) is used. Antisense telomerase synthesizes two primers (SEQ ID NOs: 1, 2) and complements them, inserts them into the Bam HI and Hind III restriction sites of the polyclonal region of the expression vector pΔACMVp (A) and expresses antisense telomerase. An expression vector pΔACMV-OA can be prepared (see FIG. 1).

In order to investigate the antisense telomerase expression, the adenovirus clone was isolated and subjected to dot blot using SEQ ID NO: 1 to confirm that antisense telomerase is expressed in 293 cells (see FIG. 2).

In addition, to analyze the proliferative recombinant mutant virus (RCV) present in the adenovirus clone, the E1A gene region of adenovirus type 5 of the present invention based on the fact that RCV contains an E1 gene region which is not present in the adenovirus expression vector. And the presence of the E1B gene region is confirmed by performing a polymerase chain reaction (PCR) (see FIG. 3). At this time, a pair of E1A primers having the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4 and a pair of E1B primers having the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 6 are used (SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, see SEQ ID NO: 6). As a result, it was confirmed that 752 bp and 1818 bp sized DNA fragments were not present in the viral DNA used as the template. As a result, adenovirus clones that can produce antisense telomerase in 293 cells and do not produce RCV were selected and named Ad-OA. The adenovirus clone of the present invention was deposited in Korea microbial conservation center on August 29, 2001 (Accession No .: KCCM 10312). The virus produces antisense sequences and confirms by reverse transcriptase-polymerase chain reaction that selectively removes the desired site, hTR (see FIG. 4).

In order to investigate whether the adenovirus clone of the present invention specifically works on tumor cells, the adenovirus clone Ad-OA is infected with lung cancer, prostate cancer cell lines and normal fibroblasts to inhibit specific killing of tumor cells and cell growth. Confirm the effect (see FIGS. 5 and 6). Specifically, the present invention uses an NCI-H358 cell line derived from lung cancer as a cancer cell line, Du-145, etc. derived from prostate cancer. In addition, IMR-90, a normal fibroblast, is used as a control to examine the effect on normal cell lines.

The effect of the adenovirus clone Ad-OA of the present invention on cell growth is determined by trypan blue staining (cell exclusion assay), where cells are infected with adenovirus Ad-CMV null that does not express any genes as a control. . As a result, the cell number decrease was observed in NCI-H358 and Du-145 cells, but it was confirmed that normal fibroblast cells were hardly affected (see FIG. 6). In addition, to determine whether the adenovirus inhibits telomerase activity, the telomerase activity inhibitory effect is confirmed by measuring by a telomere repeat amplification protocol (TRAP) (see FIG. 7). The breakdown of nucleosome DNA among apoptosis induced in cells infected with Ad-OA is analyzed by cell histochemical analysis using ApopTag Apoptosis Detection Kit (Intergen) (see FIG. 8). Specifically, the free 3′-OH group generated by internucleosomal fragmentation between the nucleosomes, which are cellular markers of apoptosis, by treating adenovirus Ad-OA or Ad-CMV null in cells, the terminal deoxynucleotide transferase Labeling and analysis using deoxynucleotide transferase and dioxyxigenin-labeled nucleotides. As a result, the phenomenon in which apoptosis was induced in NCI-H358 and Du-145 cell lines was observed by optical microscopy of browned nucleus and membrane blebbing cells (see FIG. 8). Flow cytometry analysis using Annexin V-FITC kit (Clontech) confirms the apoptosis of Ad-OA infected NCI-H358 cells (FIG. 9).

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples are illustrative of the present invention, and the content of the present invention is not limited by the examples.

Example 1 Preparation of Adenovirus Expression Vectors Containing Antisense Telomerase

The present invention removes the E1 gene site from adenovirus type 5 and thus produces an early early promoter, polycloning site and simeon of human cytomegalovirus to prepare an adenovirus expression vector comprising antisense telomerase. An expression vector pΔACMVp (A) (manufactured by Chinghai Cao) containing an expression cassette consisting of a post-adenine polyadenylation signal of virus 40 was used.

In order to prepare an adenovirus expression vector capable of producing the antisense telomerase of the present invention in cells, a pair of primers having restriction enzymes BamHI and HindIII sites at each end were prepared (SEQ ID NO: 1, SEQ ID NO: 2) It was complementarily bound to 10 mM Tris-HCl (pH 8.0), 1 mM EDTA buffer at 90 ° C. for 10 minutes and 55 ° C. for 4 hours, and the Bam HI / was present at the polycloning site of the expression vector pΔACMVp (A). Inserted into the HindIII restriction enzyme site. As a result, an adenovirus expression vector pΔACMV-OA was prepared (see FIG. 1).

Example 2 Preparation of Adenovirus Clones capable of Intracellular Production of Antisense Telomerase

In order to prepare adenovirus clones capable of infecting cells and producing antisense telomerase, the adenovirus expression vector pΔACMV-OA of the present invention was packaged together with the adenovirus parent vector pJM17 (manufactured by Microbix, Canada). 293 cells were transformed using liposomes (FuGENE6, manufactured by Rosh). At this time, transfection was performed using 24-well plates, and as a result, it was confirmed that virus plaques were generated in four wells.

Example 3 Analysis of Proliferative Recombinant Mutant Virus

In order to analyze the replication competent recombinant virus (RCV) that may be present in the adenovirus clone obtained in Example 2, RCV is characterized by the presence of the overlapping site of adenovirus genes and adenovirus expression vectors present in 293 cells. Based on the fact that the E1A gene sequence was inserted into the adenovirus expression vector by recombination, the following investigation was carried out.

Specifically, a pair of E1A primers having the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4 to determine whether the E1A gene region and E1B gene region of adenovirus type 5 are present in the DNA isolated from the adenovirus clone (SEQ ID NO: 3 and SEQ ID NO: 4) and a polymerase chain reaction (PCR) using a pair of E1B primers (see SEQ ID NO: 5 and SEQ ID NO: 6) having the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 6 Was performed. At this time, adenovirus DNA was isolated by treating proteinase K (proteinase K, 2mg / ml) in the presence of 0.5% SDS, followed by phenol extraction and ethanol precipitation. The polymerase chain reaction using the E1A primers and the E1B primers confirmed that there was no E1 gene region (752bp and 1818bp sizes) on the agarose gel in the viral DNA used as a template (see FIG. 3). 861bp irrelevant to the presence or absence of the E1 gene when the polymerase chain reaction was carried out with an adenovirus primer (see SEQ ID NO: 7 and SEQ ID NO: 8) having the nucleotide sequences of SEQ ID NO: 7 and SEQ ID NO: 8, which does not correspond to the E1 gene region as a control. DNA fragments of size can be identified.

Example 4 Culture of Various Cell Lines

In order to investigate whether the adenovirus clone of the present invention acts on cancer cells and exhibits anticancer effects, 10% of the lung cancer cell line NCI-H358, the prostate cancer cell line Du-145 and the control human normal skin fibroblast cell IMR90, etc. It was cultured in a medium RPMI1640 containing fetal bovine serum. Cell lines were all purchased from ATCC.

Example 5 Antisense Expression and Selective Specific Removal of Target Site hTR

Recombinant virus produces antisense, extracts viral DNA from Ad-OA infected 293 cells, labels SEQ ID NO: 1 using DIG-DNA labeling and detection kit (Boehringer Mannheim) and attaches viral DNA to the company's nylon membrane After that, a dot blot was performed using the kit (FIG. 2). In addition, after extracting whole cell RNA from Ad-OA-infected NCI-H358 with TRIZol reaction solution (Gibco Co., Ltd.), after cDNA synthesis using oligo (dT) 15 (Promega) and Superscript II reverse transcriptase (Gibco Co.). Expression of antisense was confirmed by polymerization with r-Taq (product of Takara) using an expression antisense specific primer (see SEQ ID NOS: 9, 10) (see Figure 4) and hTR specific primer (see SEQ ID NOs: 9, 11). The polymerization reaction was performed to confirm the selective specific removal effect of hTR, the target site by Ad-OA (see FIG. 4). At this time, the human glyceraldehyde-3 phosphate dehydrogenase (GAPDH) primer having the nucleotide sequences of SEQ ID NO: 12 and SEQ ID NO: 13 is used as a control.

Example 6 Effect of Inhibiting Telomerase Activity

To investigate the effect of inhibiting telomerase activity in cells infected with adenovirus Ad-OA, cells were cultured in 6-well and treated with adenovirus Ad-OA or Ad-CMV null at 100 pfu / cell concentration, followed by Kim ( The telomerase activity was measured in 2000 cells by the telomeric repeat amplification protocol (TRAP) by Kim et al., 1994). As a result, telomerase activity of Ad-OA infected cells was reduced by about 40% compared to Ad-CMV null infected cells (FIG. 7). Relative activity was analyzed using a density analysis program (Total Lab.).

Example 7 Anticancer Effect of Adenovirus Ad-OA

In order to confirm that the adenovirus clone of the present invention can be effectively used for anticancer gene therapy, the cancer cell lines listed in Example 4 were treated with adenovirus clone Ad-OA to investigate the anticancer effect therefrom.

The effect of the adenovirus Ad-OA infection of the present invention on cell growth was measured by trypan blue dye exclusion assay. Approximately 5 × 10 ⁴ cells were planted in a 60-mm dish and treated with adenovirus Ad-OA or Ad-CMV null at a concentration of 100 pfu / cell after one day incubation, and then cell numbers were measured for 6 days at two-day intervals. As a result, the cell number was reduced in NCI-H358 and Du-145, but it was confirmed that normal fibroblast IMR-90 cells were hardly affected (see FIGS. 5 and 6).

Example 8 Cell Apoptosis Assay

To investigate apoptosis induced in cells infected with adenovirus Ad-OA, cells were cultured in 4-chamber slides and treated with adenovirus Ad-OA or Ad-CMV null at 100 pfu / cell concentration and then with 4% formalin. Fixed. The breakdown of nucleosome DNA caused by apoptosis was analyzed histochemically according to the manufacturer's method using an Apop Tag Apoptosis Detection Kit (available from Intergen). Specifically, free 3'-OH groups formed by internucleosomal fragmentation between nucleosomes, which are cellular markers of apoptosis, by treating cells with adenovirus Ad-OA or Ad-CMV null for 6 days, are terminal deoxynucleotide transfers. Labeling was done using an enzyme (terminal deoxynucleotide transferase) and dioxyxigenin-labeled nucleotides. As a result, it was confirmed that the NCI-H358 cell line and Du-145 cell line responded positively to the staining that DNA fragmentation was caused by infection of adenovirus Ad-OA to induce apoptosis (FIG. 8). In addition, flow cytometry analysis using Annexin V-FITC kit (Clontech) confirmed the apoptosis of Ad-OA infected NCI-H358 cells. Specifically, the cells were cultured in 6-well and treated with adenovirus Ad-OA or Ad-CMV null at 100 pfu / cell for 6 days, and then stained with anacsin V (AnnexinV-FITC) and propidium ioda. Analysis was performed using propidium iodide (PI). As a result, it was confirmed that about 55.91% of NCI-H358 cells were apoptotic (FIG. 9).

The adenovirus expression vector of the present invention and the viral clone Ad-OA obtained therefrom can be confirmed to have an excellent effect on killing cancer cells or inhibiting growth. In addition, the adenovirus clone is not expected to produce a proliferative mutant virus at all as a result of performing various polymerase chain reactions, so it is easy to use directly in the human body is expected to be anti-cancer gene therapy directly used in the future cancer patients.

While the adenovirus Ad-OA of the present invention is less toxic or side effects than conventional antisense oligomers, the adenovirus Ad-OA is considered to have almost the same effect as the existing antisense oligomers and thus exhibits very good safety when used in anticancer gene therapy.

[Sequence list]

SEQ ID NO: 1

Sequence length: 27

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acids (oligonucleotides)

[SEQ ID NO: 1]

5 '-GATCCCGCGCGGGGAGCAAAAGCACGA-3'

[Sequence list]

SEQ ID NO: 2

Sequence length: 27

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acids (oligonucleotides)

[SEQ ID NO: 2]

5'- AGCTTCGTGCTTTTGCTCCCCGCGCGG-3 '

[Sequence list]

SEQ ID NO: 3

Length of sequence: 20

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acids (oligonucleotides)

[SEQ ID NO: 3]

5'-AGCTGATCGAAGAGGTACTG-3 '

[Sequence list]

SEQ ID NO: 4

Sequence length: 19

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acids (oligonucleotides)

[SEQ ID NO: 4]

5'-GAGTCACAGCTATCCGTAC-3 '

[Sequence list]

SEQ ID NO: 5

Sequence length: 22

Type of sequence: nucleic acid

Number of chains: 1 chain

Type: Straight

Type of sequence: Other nucleic acids (oligonucleotides)

[SEQ ID NO: 5]

5'-GGTTACATCTGACCTCATGGAG-3 '

[Sequence list]

SEQ ID NO: 6

Sequence length: 22

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acids (oligonucleotides)

[SEQ ID NO: 6]

5'-CAGTACCTCAATCTGTATCTTC-3 '

[Sequence list]

SEQ ID NO: 7

Length of sequence: 20

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acids (oligonucleotides)

[SEQ ID NO: 7]

5'-TCGTTCTCAGCAGCTGTTG-3 '

[Sequence list]

SEQ ID NO: 8

Length of sequence: 20

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acids (oligonucleotides)

[SEQ ID NO: 8]

5'-CATCTGAACTCAAAGCGTGG-3 '

[Sequence list]

SEQ ID NO: 9

Sequence length: 24

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acids (oligonucleotides)

[SEQ ID NO: 9]

5'-TTTGTCTAACCCTAACTGAGAAGG-3 '

[Sequence list]

SEQ ID NO: 10

Sequence length: 24

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Types of Sequences Other Nucleic Acids (oligonucleotides)

[SEQ ID NO: 10]

5'-TGTGAGCCGAGTCCTGGGTGCACG-3 '

[Sequence list]

SEQ ID NO: 11

Sequence length: 19

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acids (oligonucleotides)

[SEQ ID NO: 11]

5'-CCACAACTAGAATGCAGTG-3 '

[Sequence list]

SEQ ID NO: 12

Length of sequence: 20

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acids (oligonucleotides)

[SEQ ID NO 12]

5'-TTCGTCATGGGTGTGAACCA-3 '

[Sequence list]

SEQ ID NO: 13

Length of sequence: 20

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acids (oligonucleotides)

[SEQ ID NO: 13]

5'-TGGCAGGTTTTTCTAGACGG -3 '

Claims (6)

The antisense telomerase DNA having the 6-26th sequence of SEQ ID NO: 1, which is involved in the propagation of the virus, is removed and the post-adenylation of the last promoter of cytomegalovirus (CMV) and Simeon virus (SV) 40 An adenovirus expression vector capable of being inserted at the polycloning site between signals to produce antisense telomerase in cells. The adenovirus expression vector according to claim 1, which has a cleavage map and a nucleotide sequence of pΔACMV-OA of FIG. 1. An adenovirus clone Ad-OA which does not contain a proliferative mutant virus obtained by propagating the adenovirus expression vector of claim 1 in a packaging cell and can be used for anticancer gene therapy. 4. Adenovirus clone Ad-OA according to claim 3, deposited with accession number KCCM 10312. The adenovirus clone Ad-OA of claim 3, which is used to treat cancer. The adenovirus clone Ad-OA of claim 5, wherein the cancer is prostate cancer or lung cancer.