KR101464360B1 - Adenovirus Containing Ribozyme and shRNA, and Therapeutic Composition Comprising Thereof - Google Patents

Abstract

The present invention relates to adenoviruses for cancer treatment, and more particularly, to a gene construct to which a hTERT ribozyme and a therapeutic gene HSVtk (Herpes simplex virus thymidine kinase) are linked and additionally a gene construct containing an AIMP2-DX2- ≪ RTI ID = 0.0 > adenovirus < / RTI >
It is expected that the composition for treating cancer containing adenovirus of the present invention exhibits a superior tumor suppressive activity than an existing anticancer agent or an adenovirus containing conventional hTERT (human telomerase reverse transcriptase) ribozyme, .

Description

{Adenovirus Containing Ribozyme and shRNA, and Therapeutic Composition Comprising Thereof} The present invention relates to an adenovirus containing ribozyme and shRNA,

The present invention relates to adenoviruses for cancer treatment, and more particularly, to a gene construct to which a gene coding for hTERT ribozyme and a therapeutic gene HSVtk (Herpes simplex virus thymidine kinase) are linked, in addition to AIMP2-DX2 specific shRNA And a composition for treating cancer comprising the adenovirus for treating cancer.

The mortality rate from cancer is increasing year by year, both at home and abroad. Cancer is one of the leading causes of death worldwide, along with infectious and cardiovascular diseases. According to the World Health Organization (WHO) World Health Report, 12.5% of the total deaths in 2004, 7,121,000 people, died of cancer, and within the next 25 years due to changes in eating habits, Is expected to grow to about 30 million people annually, of which 20 million people will die of cancer.

Currently, the methods used to treat cancer are largely surgical surgery, radiation therapy, and drug therapy, which are used alone or in combination with two or more methods for cancer treatment, Biological therapies are booming thanks to this.

Biotherapeutics are based on the therapeutic basis of inhibiting the progression of cancer by weakening the activity of cancer cells by restoring or increasing the body's immune function. When the body's immune system functions, it can kill cancer cells effectively, but if not, cancer cells can multiply easily, or other pathogens can easily attack. It may also be used with other therapies, such as surgical surgery, radiation therapy, chemotherapy, etc., to supplement the above shortcomings of biotherapeutics. Antibiotic anticancer agents and angiogenesis inhibitors are examples of biotherapeutics that have received attention in the field of life science at present. The antisense anticancer agent induces the death of cancer cells by a method of inhibiting mRNA processing or protein expression using DNA fragments capable of complementarily binding to specific mRNA of cancer cells. As a result of the human genome project, more than 30,000 gene sequences were read and more than 100,000 mRNA sequences were identified. As a result, a large amount of information on the candidate mRNAs associated with cancer cells is obtained, and thus the antisense anticancer agent for genes related to the signal transduction system, genes involved in apoptosis and cell proliferation, and the like are screened for clinical trials.

Gene delivery technology is largely based on viral vector-based transfer methods, non-viral delivery methods using synthetic phosphatides or synthetic cationic polymers, and transient electrical stimulation on the cell membrane And physical methods such as electroporation in which a gene is introduced.

Among the above delivery techniques, a method using a viral transporter is a vector in which the replication ability of part or all of the gene having a gene replaced with a therapeutic gene is deficient. Therefore, the preferred method for gene therapy to be.

Viruses used as virus vectors or virus vectors include RNA virus vectors (retrovirus vectors, lentivirus vectors, etc.) and DNA virus vectors (adenovirus vectors, adeno-associated virus vectors, etc.) a herpes simplex viral vector, and an alpha viral vector. Among them, retroviruses and adenoviruses are particularly active in research.

Retroviruses that integrate into the genome of host cells are characterized by their ability to inhibit the function of normal cells, to infect various cells, to proliferate, , And is capable of producing replication defective viruses. However, it is difficult to infect cells after mitosis, gene transfer is difficult in vivo, and somatic tissues must be propagated in vitro at all times. In addition, retroviruses can be integrated into proto-oncogenes, which can lead to mutations and necrosis.

On the other hand, adenovirus has several advantages as a cloning vector. It can be replicated in the nucleus in a medium size, clinically non-toxic, stable even when an external gene is inserted , Can regenerate eukaryotes without rearrangement or loss of genes, and are stable and highly expressed even when integrated into the host cell chromosome. Good host cells of adenoviruses are the cells that cause human hematopoiesis, lymph, and myeloma.

Ribozyme is an RNA molecule having an enzymatic function that recognizes and cleaves a specific RNA base sequence. Since it can identify single nucleotide, splice and RNA transcript differences at the target site, ribozyme can be used for gene therapy (Uhlenbeck OC, Nature, 328: 596, 1987; Woolf TM, Nat Biotech, 16: 341, 1998).

hTERT (human telomerase reverse transcriptase) RNA-targeted trans-splicing ribozyme induces hTERT-dependent expression of the reporter gene (β-galactosidase, LacZ) or the prodrug-responsive gene (herepes simplex virus thymidine kinase, HSVtk) (Kwon BS et al. , Mol. Ther. , 12: 824, 2005). In addition, adenoviral vectors containing ribozymes can be used as selectable markers for cancer cells, making cancer cells susceptible to ganciclovir in hTERT-expressing cancer cells or tumor xenografts (Hong SH, et al. , Mol. Ther 2007 ). These results suggest that trans-splicing ribozymes can be used as anticancer agents to recognize cancer-specific transcripts.

The present inventors confirmed that hTERT ribozyme recognized adenovirus-specific transcripts and reprogrammed them to inhibit tumors when adenoviruses transduced with hTERT ribozyme and HSVtk gene were prepared and injected into mice of colorectal cancer model .

Accordingly, the present inventors have made intensive efforts to develop a gene therapy agent having superior anticancer effect. As a result, the inventors have found that when a hTERT ribozyme and shRNA of AIMP2-DX2 specifically expressed in cancer cells are used, and a gene therapy agent in which HSVtk gene is further introduced into adenovirus , And it inhibits tumor growth with higher activity than adenovirus containing hTERT ribozyme, and completed the present invention.

It is an object of the present invention to provide a gene therapy adenovirus excellent in tumor suppression activity and a therapeutic composition comprising the same.

In order to achieve the above object, the present invention provides an adenovirus containing a shRNA nucleic acid molecule specific for mRNA of AIMP2-DX2 which is a protein in which the exon 2 region of hTERT (human telomerase reverse transcriptase) ribozyme and AIMP2 protein is deleted . The adenovirus may further include an HSVtk gene.

The present invention also provides a composition for treating cancer comprising the adenovirus.

The composition for treating cancer containing the adenovirus of the present invention exhibits a tumor suppressing activity higher than that of a conventional anticancer agent or an adenovirus containing a conventional hTERT (human telomerase reverse transcriptase) ribozyme, and has excellent cancer treatment efficacy.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a method for producing a recombinant plasmid for the production of adenovirus DWP 451 according to the present invention, wherein A) is a schematic representation of the production method of pE1.2-Rz-HSVtk, and B) It is a schematic representation of the production method of DX2.
FIG. 2A shows the gene structure of adenovirus DWP 451 according to the present invention, and B) shows the structure of shRNA which inhibits the expression of AIMP2-DX2 used in the present invention.
FIG. 3 shows lung cancer cell killing effect after treating adenovirus DWP 451 according to the present invention with a lung cancer cell line.
Fig. 4 shows the effect of lung cancer cell lysis upon treatment of AIMP2-DX2-specific naked siRNA with lung cancer cell lines.
FIG. 5 shows changes in tumor size when the adenovirus DWP 451 according to the present invention was treated with a mouse xenograft model for each concentration.
Figure 6 shows tumor size changes in a mouse xenograft model group treated with adenovirus DWP 451 according to the invention and DWP 455 as a positive control.
Figure 7 shows tumor size changes in a mouse xenograft model group treated with DWP418, which additionally contains adenovirus DWP 418 and AIMP2-DX2 shRNA.
Fig. 8 shows a sequence map of DWP418, an adenovirus engineered to replicate only in telomerase positive cancer cells.

In one aspect, the present invention relates to an adenovirus containing a shRNA nucleic acid molecule specific for the mRNA of AIMP2-DX2 which is a protein in which the hTERT (human telomerase reverse transcriptase) ribozyme and the exon 2 region of the AIMP2 protein are deleted.

The term "ribozyme" in the present invention is composed of an RNA molecule, or an RNA molecule and a protein, which function as an enzyme. First, ribozyme has a role of self-joining (splicing). These include the splicing RNA molecules Group I intron and the group II intron. There is also Hammerhead Ribozyme, which is contained in viroids, viruses, and satellite RNA.

In addition, ribozymes act as catalysts for the reaction of RNA molecules, DNA, and biomolecules other than themselves. hTERT (human telomerase reverse transcriptase) RNA-targeted trans splicing ribozyme induces hTERT-dependent expression of the reporter gene (β-galactosidase, LacZ) or the prodrug-responsive gene (herepes simplex virus thymidine kinase, HSVtk) (Kwon BS et al . , Mol . Ther . , 12: 824, 2005).

The present inventors have confirmed that adenoviruses containing hTERT ribozyme and HSVtk gene exhibit anticancer effects in a mouse model of colorectal cancer (Jeong, JS et al ., Cancer Theraphy: preclinical, 14: 281, 2008). Therefore, the adenovirus provided in the present invention may be further characterized by containing HSVtk (herepes simplex virus thymidine kinase) gene.

In the present invention, AIMP2 (ARS-interacting multi-functional protein 2) is one of the proteins involved in the formation of aminoacyl-tRNA synthetase (ARSs), and is called p38 / JTV-1 or p38 It has been reported that the genetic collapse of AIMP2 induces overexpression of c-myc, leading to neonatal lethality induced by the alveolar epithelial cells of the lung, -cyclo-methyl-β-glucosidase inhibits the expression of c-myc (Kim et al, Nat Genet ., 34, 330-336, 2003).

AIMP2-DX2, which is a variant of AIMP2-deficient form of AIMP2, is specifically expressed in cancer cell lines and tissues, and the sequence of AIMP2 protein (312aa version: AAC50391.1 or GI: 1215669; 320aa version: AAH13630.1 , GI: 15489023, BC013630.1) is described in the literature (312aa version: Nicolaides, NC et al., Genomics 29: 329, 1995/320 aa version: Proc. Natl. Acad. Sci. USA . It is a protein in which the region corresponding to exon 2 is deleted.

The AIMP2-DX2 protein in the present invention includes a protein having a deletion of the region of the exon 2 in the AIMP2 of the entire sequence, and the AIMP2 equivalent (substitution, deletion, insertion, or a combination of the amino acid sequences, A functional equivalent having an equivalent activity, or a functional derivative having a modification that increases or decreases physicochemical properties, but has substantially equivalent activity to AIMP2).

In the present invention, " deletion of the region of the exon 2 " in the AIMP2 protein sequence means that a mutant in which the amino acid sequence of the exon 2 region (amino acids 46 to 114) is partially or totally deleted from the AIMP2 protein forms a heterodimer with the AIMP2 protein Which interferes with the normal functioning of AIMP2. Thus, the AIMP2-DX2 protein can be obtained by deleting all of the amino acid sequences of exon 2 of the AIMP2 protein or by deleting the exon 1, exon 3, exon 4 or a portion of these regions in both of these regions, Lt; RTI ID = 0.0 > of the < / RTI > Preferably, the AIMP2-DX2 protein of the present invention is a protein in which the amino acid sequence of exon 2 of the AIMP2 protein has been deleted. It is known that AIMP2-DX2 protein is specifically expressed in cancer tissues such as lung cancer, liver cancer, breast cancer, skin cancer, kidney cancer and osteosarcoma. Therefore, anticancer effect can be expected if the expression of AIMP2-DX2 protein is inhibited.

The term "shRNA" in the present invention refers to small hairpin RNA or short hairpin RNA (shRNA), and is a small hairpin structure RNA used for silencing gene expression by RNA interference. The shRNA is introduced into the cell using a vector, and the shRNA hairpin structure is cleaved by other substances in the cell to become an siRNA. This siRNA binds to the RNA-induced silencing complex (RISC) The mRNA is attached to the mRNA with the correct sequence and the mRNA is cleaved. As a result, the mRNA is destroyed, so that the gene is not expressed and silencing of the gene occurs.

The term "siRNA" as used herein refers to short double-stranded RNA capable of inducing RNAi (RNA interference) phenomenon through cleavage of a specific mRNA. A sense RNA strand having a sequence homologous to the mRNA of the target gene And an antisense RNA strand having a complementary sequence. Since siRNA can inhibit the expression of a target gene, it is provided as an efficient gene knockdown method or a method of gene therapy.

The siRNA is not limited to a complete pair of double-stranded RNA portions that are paired with each other, but is paired by a mismatch (the corresponding base is not complementary), a bulge (no base corresponding to one chain) May be included. The total length is 10 to 100 bases, preferably 15 to 80 bases, more preferably 20 to 70 bases. The siRNA terminal structure is capable of blunt or cohesive termini as long as it can inhibit the expression of the target gene by the RNAi effect. The sticky end structure can have both a 3-terminal protruding structure and a 5-terminal protruding structure. The number of protruding bases is not limited.

For example, the base water may be from 1 to 8 bases, preferably from 2 to 6 bases. In the present specification, the total length of the siRNA is represented by the sum of the length of the central double-stranded portion and the length of the single-stranded protrusion at both ends. In addition, the siRNA may be added to a protruding portion at one end in a range that can maintain the effect of suppressing the expression of the target gene, for example, a small RNA (for example, natural RNA molecules such as tRNA, rRNA, Molecules). The siRNA terminal structure does not need to have a cleavage structure on both sides, and a stem loop structure in which one terminal region of double-stranded RNA is connected by linker RNA may be used. The length of the linker is not particularly limited as long as it does not interfere with the pairing of the stem portions.

The term " specific " or " specific " in the present invention means an ability to suppress expression of a target gene without affecting other genes in the cell. In the present invention, "AIMP2- Lt; RTI ID = 0.0 > DX2 < / RTI > The shRNA and siRNA of the present invention are characterized in that a sense RNA strand comprising a sequence homologous to the mRNA corresponding to the border between exon 1 and exon 3 of AIMP2-DX2 so as to function specifically for AIMP2-DX2 and an antisense It has RNA strands.

The term " inhibition of gene expression " as used herein means that the level of mRNA and / or protein produced in a target gene is removed or decreased, and this means that RNA interference (RNAi) caused by cleavage of mRNA : RNA interference).

Methods for preparing siRNA include direct synthesis of siRNA in a test tube, transfection into a cell, and introduction of siRNA expression vector or PCR-derived siRNA expression cassette into a cell Transformation, or infection. The determination of how to prepare siRNA and introduce it into a cell or animal may depend on the purpose of the experiment and the cellular biological function of the target gene product.

In one embodiment of the present invention, adenovirus DWP 451 containing a shRNA nucleic acid molecule specific for mRNA of hTERT ribozyme and AIMP2 - DX2 was treated to a lung cancer cell line A459 by concentration, and as the concentration of virus particles increased, The survival rate was decreased. In the present invention, a preferred shRNA is characterized by having the nucleotide sequence of SEQ ID NO: 1.

In another aspect of the invention, hTERT ribozyme and AIMP2-mouse tumor DX2 shDNA deletion virus in DWP455 to 10 ⁶ virus particles concentration - from adenovirus DWP451 and DWP451 containing specific shRNA nucleic acid molecule to the mRNA of DX2 AIMP2 As a result of the treatment with DWP 455, DWP 451 treatment significantly reduced tumor size.

In another embodiment of the present invention, adenovirus further comprising AIMP2-DX2 shRNA is administered in a mouse tumor model in which adenovirus containing AIMP2-DX2 shRNA is incorporated into DWP418 which is not an adenovirus containing hTERT ribozyme The tumor size of mice in the group administered with adenovirus not containing AIMP2-DX2 shRNA was further reduced.

Therefore, even if the same shRNA is used, it can be seen that there is a difference in anticancer efficacy depending on the type of adenovirus used as a transporter.

In another aspect, the present invention relates to a composition for treating cancer comprising the adenovirus.

The composition for treating cancer of the present invention may be administered together with a pharmaceutically acceptable carrier. In oral administration, a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, A buffer, a preservative, an anhydrous agent, a solubilizer, an isotonic agent, a stabilizer and the like may be mixed. In case of topical administration, a base, an excipient, a lubricant, a preservative and the like may be used . Formulations of the pharmaceutical compositions of the present invention may be prepared in a variety of ways by mixing with pharmaceutically acceptable carriers as described above. For example, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers and the like in the case of oral administration, and in the case of injections, unit dosage ampoules or a plurality of dosage forms.

The cancer or carcinoma which can be treated with the composition according to the present invention is not particularly limited, and includes all kinds of solid cancer and blood cancer. Cancer, cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, pituitary cancer, kidney cancer, breast cancer, ovarian cancer, hepatoma, stomach cancer, bronchial cancer, nasopharyngeal cancer, pancreatic cancer, pancreatic cancer, colon cancer, pancreatic cancer, But are not limited to, cancer, esophageal cancer, biliary cancer, testicular cancer, rectal cancer, head and neck cancer, cervical cancer, ureteral cancer, osteosarcoma, neuronal subpopulation, melanoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma or glioma Lung cancer, liver cancer, breast cancer, kidney cancer, skin cancer, osteosarcoma, colon cancer, ovarian cancer, stomach cancer, pancreatic cancer and the like.

The effective dose range of the adenovirus contained in the therapeutic composition of the present invention may vary depending on various factors such as sex, severity, age, administration method, target cell, expression level, and the like, Can be determined.

The pharmaceutical composition according to the present invention is orally or parenterally administered to humans and animals such as intravenously, subcutaneously, intranasally or intraperitoneally.

Parenteral administration includes injection and drip, such as subcutaneous, intramuscular, and intravenous. Other pharmaceutical compositions of the invention may be prepared according to techniques commonly used in the form of various formulations.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the present invention is not construed as being limited by these embodiments.

Example 1: Preparation of adenoviruses for cancer treatment according to the present invention

AdenoQuick 13.1 Kit (O.D.260, Inc., USA) was used to prepare adenovirus DWP451 for cancer treatment. First, to insert the Rz-HSVtk gene into the adenovirus cosmid vector pE1.2, PCR was performed using DWP455 as a template DNA using primers of SEQ ID NOS: 2 to 3

 Sense primer: 5'-TTT TCT AGA CCT GGC ATT ATG CCC AGT AC-3 '(SEQ ID NO: 2)

antisense prime: 5'-AAG GGA TCC TCA GTT AGC CTC CCC CAT C-3 '(SEQ ID NO: 3)

The resulting PCR product was digested with XbaI / BamHI restriction enzyme and cloned into the XbaI / BamHI site of pE1.2 to construct pE1.2-Rz-HSVtk (FIG. 1A).

Two cosmids of pE3.1-DX2 and pE1.2-Rz-HSVtk were digested with DraIII restriction enzyme and inserted into Sfi-digested pAD-13.1. The adenovirus pAd-Rz-HSVtk-DX2, in which the Rz-HSVtk gene was inserted into the adenovirus E1 region and the DX2 was inserted into the E3 region, was constructed. Plasmid pAd-Rz-HSVtk-DX2 prepared by homologous recombination was digested with Pac I and transformed into 293 cell lines to prepare DWP451 recombinant adenovirus (Fig. 1B).

Example 2: Effect of adenovirus on lung cancer cell killing effect

A549 cells (ATCC, American Type Culture Collection, Manassas, Va., USA) were dispensed in 96 well plates at 4000 cells / well / 1% FBS F12 kaighn's strain medium (Life technologies, USA) , DWP451 virus prepared in Example 1 at various concentrations (0.5, 2.5, 5, 50, 500, 5000 virus particles / cell) was treated in each well and Ganciclovir (Roche, USA) Well / 1% FBS, and cultured for 3 days.

MTS (tetrazolium compound [3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- ) Were treated at a concentration of 20 μL / well, cultured at 37 ° C., and then read until the control value reached 1.5-2.

As a result, as shown in FIG. 3, when DWP 541 was treated at various concentrations, it was confirmed that lung cancer killing effect was dependent on DWP 541 concentration.

Example  3: Naked  s iRNA  Evaluation of efficacy using

When the DX2 specific siRNA used in the present invention was treated with lung cancer cell line H1703 using an iMAX carrier, survival rate of cancer cells was confirmed.

The cells were cultured at 37 ° C for 1 day, and then cultured in a variety of media (100 μl / well) in a 96 well plate at 4000 cells / well / 1% FBS RPMI 1640 medium (Life technologies, USA) (2.812, 5.625, 11.25, 22.5, 45 nM) siRNA were mixed with iMAX and then treated in each well. After culturing for 3 days, CTG (CellTiter-Glo ™, Promega, USA) was treated at a concentration of 100 μL / well in each well and luminescence was read.

As a result, as shown in FIG. 4, when Naked siRNA against AIMP2-DX2 was treated, it was confirmed that lung cancer cell kill effect was proportional to the concentration in lung cancer cell line H1703.

Example 4: xenograft experiment using a mouse model

Nude mice were subcutaneously injected into lung cancer cell lines to form tumors, and adenovirus DWP 451 of the present invention and DWP455 (adenovirus without AIMP2-DX2 shRNA in DWP451) as a control group were injected to confirm tumor inhibiting ability.

First, the A549 cells, 10% FBS, 2mM L-glutamine , 100U / ml penicillin and 100μg / ml streptomycin was added the RPMA-1640 (Gibco, BRL, USA) inoculated in culture medium in 37 ℃, 5% CO ₂ conditions Lt; / RTI >

5 × 10 ⁷ of the cultured A549 cells were suspended in 0.1 mL HBSS and subcutaneously injected into the right lower abdomen of BALB / c nude mice (18-22 g) at 5-8 weeks of age and the size of tumor was 100-150 mm ³ The ramen group was isolated and adenovirus was administered.

The administration groups are shown in Table 1.

Mouse model experiment group group Marie Drug treatment group Route of administration Interval of administration One 10 1x PBS IT (intra-tumor) 1 time / 2 weeks 2 10 Cisplatin (3 mg / kg) IP (abdominal cavity) 2 times / 1 week 3 10 DWP455 (1 x 10 ⁶ )
GCV 50 mg / kg IT
IP 1 time / 2 weeks
2 times / day 4 10 DWP451 (1x10 ⁹ )
GCV 50 mg / kg IT
IP 1 time / 2 weeks
2 times / day 5 10 DWP451 (1 x ^{10 8} )
GCV 50 mg / kg IT
IP 1 time / 2 weeks
2 times / day 6 10 DWP451 (1 x 10 ⁷ )
GCV 50 mg / kg IT
IP 1 time / 2 weeks
2 times / day 7 10 DWP451 (1 x 10 < ⁶ >)
GCV 50 mg / kg IT
IP 1 time / 2 weeks
2 times / day

As a result, as shown in FIG. 5, it was confirmed that the size of the abdominal tumors of the mice treated with the adenovirus DWP451 of the present invention decreased in a virus concentration-dependent manner, and the anti-cancer effect was superior to that of the positive control substance Cisplatin .

In addition, as shown in Fig. 6, in the group treated with 10 < ⁶ > virus particles, the tumor size in the group of DWP451-administered mice of the present invention was much larger than that of the adenovirus-containing DWP 455 group containing hTERT ribozyme I can confirm that it has decreased a lot. Therefore, it can be confirmed that when the AIMP2-DX2 shRNA and the hTERT ribozyme are administered together, they show a remarkable synergistic effect.

Comparative Example: Mouse xenograft analysis of DWP 418 containing AIMP2-DX2 shRNA

Using the adenovirus containing AIMP2-DX2 shRNA in DWP418 (adenovirus engineered to be replicated only in telomerase positive cancer cells, not the adenovirus containing hTERT ribozyme, see Fig. 8), the tumor Inhibition experiments were carried out and treated with 10 ⁹ virus particles, respectively.

As a result, as shown in Fig. 7, the tumor size of the mouse of the group administered adenovirus not containing AIMP2-DX2 shRNA was decreased more than that of the group administered with adenovirus further containing AIMP2-DX2 shRNA Respectively.

Therefore, even if the same shRNA was used, it was confirmed that the anticancer efficacy was different depending on the type of the adenovirus used as the transporter.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments, It will be obvious. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Deawoong. Co., LTD <120> Adenovirus Containing Ribozyme and shRNA, and therapeutic          < <130> P12-B208 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 65 <212> RNA <213> Artificial Sequence <220> <223> shRNA <400> 1 ggatccgctg gccacgtgca ggattattca agagataatc ctgcacgtgg ccagtttttt 60 ggaaa 65 <210> 2 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 2 ttttctagac ctggcattat gcccagtac 29 <210> 3 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 aagggatcct cagttagcct cccccatc 28

Claims (5)

A shRNA nucleic acid molecule having a nucleotide sequence of SEQ ID NO: 1 specific to the mRNA of AIMP2-DX2 which is a protein in which the exon 2 region of the AIMP2 (ARS-interacting multi-functional protein 2) protein has been deleted and hTERT (human telomerase reverse transcriptase) Adenovirus. &Lt; / RTI &gt;
delete The adenovirus according to claim 1, further comprising a HSV tk (herepes simplex virus thymidine kinase) gene.
A composition for treating cancer comprising adenovirus according to any one of claims 1 to 3, selected from the group consisting of lung cancer, liver cancer, breast cancer, kidney cancer, skin cancer and osteosarcoma.
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