# Patent

## Title
REIC-expressing adenovirus vector

## Abstract

An objective of the present invention is to provide an adenovirus vector expressing a REIC/Dkk-3 protein at a high level and containing a DNA construct for expression of REIC/Dkk-3 DNA, wherein the DNA construct is prepared by ligating, from the 5′ terminal side,

    
    
        (i) a CMV promoter,
        (ii) REIC/Dkk-3 DNA,
        (iii) a polyA addition sequence, and
        (iv) enhancers prepared by linking an hTERT (Telomerase Reverse Transcriptase) enhancer, an SV40 enhancer, and a CMV enhancer in this order.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a national phase of international application PCT/JP2012/064250, filed May 25, 2012, which was published on Nov. 29, 2012, as WO 2012/161352, which claims the benefit of Japanese application No. 2011-117321, filed May 25, 2011. Each of these applications is incorporated herein by reference in its entirety.
TECHNICAL FIELD
The present invention relates to an adenovirus vector containing a promoter, an enhancer, and the like and expressing the REIC/Dkk-3 protein at a high level.
BACKGROUND ART
Various gene expression promoters such as CMV and CAG promoters have been developed to increase gene expression efficiency (Patent Documents 1-4). However, in the field of biotechnology, even when these conventional techniques are used, problems regularly occur, such that almost no gene expression takes place or the amount of the thus expressed protein is extremely low, depending on cell types or gene types. These problems cause big barriers to the development of medical care using gene expression for diagnosis or treatment.
Meanwhile, the REIC/Dkk-3 gene is known to be a gene relating to cell immortalization. It has been reported that the expression of this gene is suppressed in cancer cells. It has also been reported that the REIC/Dkk-3 gene has been used for cancer therapy (Patent Document 5).
CITATION LIST
Patent Documents
Patent Document 1 JP Patent Publication No. 2814433
Patent Document 2 JP Patent Publication No. 2814434
Patent Document 3 U.S. Pat. No. 5,168,062 Description
Patent Document 4 U.S. Pat. No. 5,385,839 Description
Patent Document 5 International Patent Publication WO01/038523 pamphlet
SUMMARY OF THE INVENTION
An objective of the present invention is to provide an adenovirus vector expressing high levels of the REIC/Dkk-3 protein.
The present inventors have examined the use of the REIC/Dkk-3 gene for gene therapy against cancer and have found that the incorporation of the REIC/Dkk-3 gene into an expression vector that is administered to a living body exhibits an effect in cancer therapy. The present inventors have intensively examined a method to cause the expression of the REIC/Dkk-3 gene at a higher level in vivo, considering the possibility that in vivo expression of the REIC/Dkk-3 gene at such a high level could increase therapeutic effects against cancer.
The present inventors have attempted the development of a new gene expression system using a promoter, which enables gene expression with higher efficiency. Specifically, promoter activity has been compared and examined using combinations of various gene promoters and enhancers.
As a result, they have discovered that the REIC/Dkk-3 gene is expressed at a significantly high level in vivo and exhibits significantly improved therapeutic effects against cancer when a CMV (cytomegalovirus) promoter was used as a prompter and a construct prepared by ligating REIC/Dkk-3 DNA to a site downstream of a CMV (cytomegalovirus) promoter, ligating a polyA sequence to a site downstream of the DNA, and ligating enhancers prepared by linking an hTERT (Telomerase Reverse Transcriptase) enhancer, an SV40 enhancer, and a CMV enhancer in this order to a site downstream of the polyA sequence is inserted into an adenovirus vector, which is then administered to a living body. They thus have completed the present invention. Specifically, the present invention is as described below.

    [1] A DNA construct for the expression of REIC/Dkk-3 DNA, which is prepared by ligating, from the 5′ terminal side:
    
        (i) a CMV promoter;
        (ii) the following REIC/Dkk-3 DNA:
        
            (a) DNA comprising the nucleotide sequence shown in SEQ ID NO: 1,
            (b) DNA hybridizing under stringent conditions to DNA comprising a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1 and encoding a protein having cancer cell death-inducing activity and/or tumor cell growth-suppressing activity,
            (c) a polynucleotide comprising a nucleotide sequence ranging from the 1st nucleotide to any single nucleotide from the 117th nucleotide to the 234th nucleotide in the nucleotide sequence shown in SEQ ID NO: 1, or
            (d) a polynucleotide hybridizing under stringent conditions to a polynucleotide that comprises a nucleotide sequence complementary to a nucleotide sequence ranging from the 1st nucleotide to any single nucleotide from the 117th nucleotide to the 234th nucleotide in the nucleotide sequence shown in SEQ ID NO: 1, and encoding a polypeptide having cancer cell death-inducing activity and/or tumor cell growth suppressing activity;
        
        
        (iii) a polyA addition sequence; and
        (iv) enhancers prepared by linking an hTERT (Telomerase Reverse Transcriptase) enhancer, an SV40 enhancer, and a CMV enhancer in this order.
    
    
    [2] The DNA construct of [1], wherein the polyA addition sequence is a polyA addition sequence (BGA polyA) derived from a bovine growth hormone gene.
    [3] The DNA construct of [1] or [2], containing the nucleotide sequence shown in SEQ ID NO: 6, wherein (ii) REIC/Dkk-3 DNA, (iii) the polyA addition sequence, and (iv) enhancers prepared by linking the hTERT (Telomerase Reverse Transcriptase) enhancer, the SV40 enhancer, and the CMV enhancer in this order, are ligated.
    [4] An adenovirus vector, containing the DNA construct of [1] or [2].
    [5] A cancer cell death-inducing agent, containing the adenovirus vector of [4].
    [6] A tumor cell growth-suppressing agent, containing the adenovirus vector of [4].
    [7] A cancer therapeutic drug, containing the adenovirus vector of [4].


The adenovirus vector contains a DNA construct for expression of REIC/Dkk-3 DNA, wherein the DNA construct is prepared by ligating, from the 5′ terminal side, (i) a CMV promoter; (ii) REIC/Dkk-3 DNA, (iii) a polyA addition sequence, and (iv) enhancers prepared by linking an hTERT (Telomerase Reverse Transcriptase) enhancer, an SV40 enhancer, and a CMV enhancer in this order. The adenovirus vector can: express REIC/Dkk-3 DNA at a high level in vivo; exhibit a higher gene expression level than an adenovirus vector containing a DNA construct that is prepared by ligating REIC/Dkk-3 DNA to a site downstream of a CMV promoter or a CAG promoter, but lacks the above enhancers; induce cancer-selective cell death; and suppress tumor growth. Therefore, the adenovirus vector of the present invention can be favorably used for gene therapy against cancer using REIC/Dkk-3 DNA.
This description includes part or all of the contents as disclosed in the description and/or drawings of Japanese Patent Application No. 2011-117321, which is a priority document of the present application.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the structure of the REIC/Dkk-3 DNA expression construct of the present invention.
FIG. 2 shows the nucleotide sequence of the REIC/Dkk-3 DNA expression construct of the present invention.
FIG. 3 shows the results of confirming inserts in the constructed adenovirus vector containing the construct of the present invention.
FIG. 4 shows the results of confirming the structure of the constructed adenovirus vector containing the construct of the present invention.
FIG. 5 shows the results of confirming the structure of the purified adenovirus vector containing the construct of the present invention.
FIG. 6 shows the results of confirmation (check) regarding the RCA (Replication Competent Adenovirus) for the constructed adenovirus vector containing the construct of the present invention.
FIG. 7 shows the intensities of REIC-Dkk-3 gene expression when various vectors were used.
FIG. 8 shows the degrees of cell death induction when various vectors were used.
FIG. 9 shows the effects of tumor growth suppression when various vectors were used (No. 1).
FIG. 10 shows tumors after treatment, indicating tumor growth-suppressing effects when various vectors were used.
FIG. 11 shows the effects of suppressing tumor growth when various vectors were used (No. 2).



EMBODIMENTS FOR CARRYING OUT THE INVENTION
Hereafter, the present invention is described in detail.
The present invention relates to a DNA construct containing REIC/Dkk-3 DNA, which can be used for REIC protein expression, and further relates to a recombinant adenovirus vector containing the DNA construct. The structure of the DNA construct of the present invention is shown in FIG. 1.
The nucleotide sequence of REIC/Dkk-3 DNA is shown in SEQ ID NO: 1. Furthermore, the amino acid sequence of the REIC/Dkk-3 protein that is encoded by REIC/Dkk-3 DNA is shown in SEQ ID NO: 2. In the present invention, REIC/Dkk-3 may also be referred to as “REIC.”
Moreover, the DNA of REIC/Dkk-3 contained in the DNA construct of the present invention is:

    DNA hybridizing under stringent conditions to DNA having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1;
    DNA having at least 85%, preferably at least 90%, further preferably at least 95%, and particularly preferably at least 97% sequence identity with the nucleotide sequence shown in SEQ ID NO: 1, when calculated using BLAST (Basic Local Alignment Search Tool) at the National Center for Biological Information (NCBI) or the like (with the use of, for example, default (i.e., initial) parameters); or
    DNA encoding a protein that comprises an amino acid sequence having a substitution, a deletion and/or an addition of one or a plurality of or several (1 to 10, preferably 1 to 5, and further preferably 1 or 2) amino acids with respect to the amino acid sequence of the protein encoded by the above DNA; which
    encodes a protein having cancer cell death-inducing activity and/or tumor cell growth-suppressing activity.
    Under “stringent conditions” referred to herein, for example, hybridization is carried out with about 1×SSC, 0.1% SDS, and 37° C. Under more stringent conditions, it is carried out with about 0.5×SSC, 0.1% SDS, and 42° C. Under further stringent conditions, it is carried out with about 0.2×SSC, 0.1% SDS, and 65° C. With the higher stringency of hybridization conditions, the isolation of DNA having higher homology with a probe sequence can be expected. However, the above combinations of SSC, SDS and temperature conditions are given only for illustrative purposes. Through appropriate combination of probe concentration, probe length, hybridization reaction time, and the like, required stringency can be realized. Persons skilled in the art can adequately determine such “stringent conditions” as conditions where DNA with high sequence identity can hybridize. Furthermore, the DNA of REIC/Dkk-3 contained in the DNA construct of the present invention is DNA encoding the protein shown in SEQ ID NO: 2.


Moreover, REIC/Dkk-3 DNA contained in the DNA construct of the present invention is a fragmental nucleotide comprising a partial nucleotide sequence of the nucleotide sequence of the DNA, including nucleotides which encode a peptide having cancer cell death-inducing activity and/or tumor cell growth-suppressing activity. Such a fragmental nucleotide can be easily obtained by cleaving the full-length REIC/Dkk-3 DNA at appropriate sites and then determining whether the resultant has cancer cell death-inducing activity and/or tumor cell growth-suppressing activity. Examples of such a fragmental nucleotide include: a polynucleotide comprising a nucleotide sequence ranging from the 1st nucleotide to any single nucleotide from the 117th to the 234th nucleotides in the nucleotide sequence of REIC/Dkk-3 DNA shown in SEQ ID NO: 1; and a polynucleotide hybridizing under stringent conditions to a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence ranging from the 1st nucleotide to any single nucleotide from the 117th to the 234th nucleotides in the nucleotide sequence of REIC/Dkk-3 DNA shown in SEQ ID NO: 1; each of which encodes a polypeptide having cancer cell death-inducing activity and/or tumor cell growth-suppressing activity. Examples of such a polynucleotide comprising a nucleotide sequence ranging from the 1st nucleotide to any single nucleotide from the 117th to the 234th nucleotides in the nucleotide sequence of REIC/Dkk-3 DNA shown in SEQ ID NO: 1 include the polynucleotide (SEQ ID NO: 3) ranging from the 1st to the 117th nucleotides and the polynucleotide (SEQ ID NO: 4) ranging from the 1st to the 234th nucleotides. Polypeptides encoded by these polynucleotides having cancer cell death-inducing activity and/or tumor cell growth-suppressing activity are described in U.S. patent application publication No. 2011-0269824, which corresponds to JP Patent Publication (Kokai) No. 2009-114103 A, for example.
REIC/Dkk-3 DNA can be obtained from human cells, human tissue, and the like based on the sequence information of SEQ ID NO: 1. REIC/Dkk-3 DNA can also be obtained according to WO01/038523.
The DNA construct is prepared by ligating a CMV (cytomegarovirus) promoter to a site upstream of REIC/Dkk-3 DNA, and a polyA addition sequence (polyadenylation sequence, polyA) to a site downstream of REIC/Dkk-3 DNA. Examples of the origin of the polyA addition sequence (polyadenylation sequence, polyA) include, but are not limited to, a growth hormone gene-derived polyA addition sequence (e.g., a bovine growth hormone gene-derived polyA addition sequence (BGA polyA)) (contained in the nucleotide sequence shown in SEQ ID NO: 5 (the sequence of the 13th nucleotide and nucleotides following thereto)), a human growth hormone gene-derived polyA addition sequence, an SV40 virus-derived polyA addition sequence, and a human or rabbit globin gene-derived polyA addition sequence. Transcriptional efficiency is increased by causing the DNA construct to contain such a polyA addition sequence. Moreover, enhancers (3×enh) prepared by linking an hTERT (Telomerase Reverse Transcriptase) enhancer, an SV40 enhancer, and a CMV (cytomegarovirus) enhancer in this order are ligated to a site downstream of the polyA addition sequence (in FIG. 2, the “3×enh” portion denotes the relevant sequence; and the sequence in FIG. 2 is shown in SEQ ID NO: 6). Specifically, the DNA construct is prepared by ligating, from the 5′ terminal side, (i) a CMV promoter, (ii) REIC/Dkk-3 DNA, (iii) a polyA addition sequence, and (iv) enhancers prepared by linking the hTER (Telomerase Reverse Transcriptase) enhancer, the SV40 enhancer, and the CMV enhancer in this order.
The above elements should be functionally linked (ligated) to each other. The expression used herein, “functionally linked (ligated) to each other” means that elements are linked or ligated to each other so that each element can exhibit its functions so as to enhance the expression of a gene to be expressed.
The above expression cassette can be obtained by inserting REIC/Dkk-3 DNA into a pShuttle vector (Clonetech) containing a foreign gene insertion site downstream of a commercial CMV promoter, and a BGA polyA sequence downstream of the insertion site, and then ligating an hTERT (Telomerase Reverse Transcriptase) enhancer, an SV40 enhancer, and a CMV enhancer in this order to a site downstream of the BGA polyA sequence.
The structure of a portion of the DNA construct containing REIC/Dkk-3 DNA of the present invention, which lacks the CMV promoter, is shown in FIG. 2, and the sequence thereof is shown in SEQ ID NO: 6. In FIG. 2, a BGA polyA sequence is contained between REIC/Dkk-3 DNA and 3×enh. The DNA construct containing REIC/Dkk-3 DNA of the present invention has a CMV promoter upstream (5′ side) of the sequence shown in SEQ ID NO: 4.
The DNA construct of the present invention is used after introduction thereof into an adenovirus vector. The present invention also encompasses an adenovirus vector containing the DNA construct for expression of REIC/Dkk-3 DNA. A vector system containing the DNA construct of the present invention is referred as an SGE (Super Gene Expression) system. For example, an adenovirus vector containing a DNA construct that contains REIC/Dkk-3 DNA and a CMV promoter is referred to as Ad-SGE-CMV-REIC. The above adenovirus vector containing the DNA construct is obtained by preparing a recombinant adenovirus through introduction of the DNA construct into an adenovirus vector. Introduction of the DNA construct into an adenovirus can be performed by introducing the DNA construct in a pShuttle vector containing the DNA construct of the present invention into an adenovirus, for example.
An adenovirus vector is characterized in that: (1) it enables gene transfer into many types of cells; (2) it enables efficient gene transfer into even cells at the stationary phase; (3) it can be concentrated by centrifugation, and thus a high-titer virus (10-11 PFU/ml or more) can be obtained; (4) and it is suitable for direct gene transfer into in vivo tissue cells.
As adenoviruses for gene therapy, the first generation adenovirus vector prepared by deleting the E1/E3 region (Miyake, S., et al., Proc. Natl. Acad. Sci. U.S.A., 93, 1320, 1996), the second generation adenovirus vector prepared by deleting, in addition to the E1/E3 region, the E2 or E4 region (Lieber, A., et al., J. Virol., 70, 8944, 1996; Mizuguchi, H. & Kay, M. A., Hum. Gene Ther., 10, 2013, 1999), and the third generation adenovirus vector prepared by almost completely deleting the adenovirus genome (GUTLESS) (Steinwaerder, D. S., et al., J. Virol., 73, 9303, 1999) have been developed. Any of these adenovirus vectors can be used without particular limitation for the gene transfer according to the present invention.
A recombinant adenovirus vector containing the DNA construct that contains REIC/Dkk-3 DNA of the present invention is administered to a human subject or a subject that is another mammal, so that a gene for cancer therapy is delivered to cancer cells of the subject, the gene is expressed in cancer cells, cell death is induced selectively for cancer cells, and/or tumor cell growth is suppressed so that therapeutic effects are exhibited against cancer. The present invention encompasses a viral preparation for cancer therapy containing such an adenovirus vector. Examples of cancer to be treated herein include, but are not limited to, brain/nerve tumor, skin cancer, gastric cancer, lung cancer, hepatic cancer, lymphoma/leukemia, colon cancer, pancreatic cancer, anal/rectal cancer, esophageal cancer, uterine cancer, breast cancer, adrenal cancer, kidney cancer, renal pelvic and ureteral cancer, bladder cancer, prostate cancer, urethral cancer, penile cancer, testicular cancer, osteoma/osteosarcoma, leiomyoma, rhabdomyoma, and mesoepithelioma. The adenovirus vector of the present invention can also be used for treatment of primary cancer and metastatic cancer. Furthermore, the present invention further encompasses a method for treating cancer by administering the above adenovirus vector to a subject.
The adenovirus vector of the present invention can be administered by methods that can be used in the field of gene therapy, such as via intravascular administration (e.g., intravenous administration and intraarterial administration), peroral administration, intraperitoneal administration, intratracheal administration, intrabronchial administration, subcutaneous administration, or transdermal administration. In particular, the adenovirus vector of the present invention has strong directivity toward a specific tissue or cells, and thus is capable of efficiently delivering a target gene to a specific tissue or cells. Therefore, efficient diagnosis and treatment can be performed even through intravascular administration of the adenovirus vector.
The adenovirus vector may be administered at a therapeutically effective dose, which can be easily determined by persons skilled in the field of gene therapy. Furthermore, the dose can be adequately varied depending on the severity of the pathological condition, gender, age, body weight, lifestyle, and the like of the subject. For example, the adenovirus vector may be administered in doses ranging from 0.5×1011 to 2.0×1012 viral genome/kg body weight, preferably ranging from 1.0×1011 to 1.0×1012 viral genome/kg body weight, and further preferably ranging from 1.0×1011 to 5.0×1011 viral genome/kg body weight. The term “viral genome” represents the number of molecules of the genome of an adenovirus (viral particle count), and is also referred as “particle(s).” The adenovirus vector contains a carrier, a diluent, and an excipient which are generally used in the field of formulation. For example, lactose, magnesium stearate, and the like are used as carriers or excipients for tablets. An aqueous solution is used for injection, such as physiological saline or an isotonic solution containing dextrose or another adjuvant, and this can be used in combination with an appropriate solubilizing agent (e.g., alcohol, polyalcohol such as propylene glycol, and nonionic surfactant). As an oily fluid, sesame oil, soybean oil, or the like is used. As a solubilizing agent, benzyl benzoate, benzyl alcohol, or the like can also be used in combination therewith.
The present invention is hereafter described in detail with reference to the following examples, although the present invention is not limited thereto.
Example 1
Construction of a pShuttle-REIC-TSC Plasmid
The DNA construct of the present invention containing a human REIC/Dkk-3 gene as a foreign gene (i.e., a gene to be inserted) was prepared. The DNA construct (FIG. 1) used herein was constructed by inserting DNA encoding the REIC/Dkk-3 protein into the insertion site of Xba 1-Kpn 1 of a commercial pShuttle plasmid vector (Clontech), and further inserting three enhancers (an hTERT enhancer, an SV40 enhancer, and a CMV enhancer; such enhancers are hereinafter referred to as “TSC”) into the insertion site of Kpn 1-EcoR 1 downstream of the DNA encoding the REIC/Dkk-3 protein to form BGH poly A+3 enhancers. FIG. 2 shows the nucleotide sequence [SEQ ID NO: 6] of a construct in which DNA encoding REIC was inserted (excluding the CMV promoter sequence). SEQ ID NO: 7 shows the nucleotide sequence of the region containing BGH poly A and three enhancers (contained in the above construct). In FIG. 2, portions (1) and (2) enclosed by frames in the nucleotide sequence indicate DNA encoding the REIC/Dkk-3 protein and the three enhancers (3×enh), respectively. The pShuttle plasmid vector containing the above construct is referred to as a “pShuttle-REIC-TSC plasmid.”
Example 2
Preparation of Ad-SGE-CMV-REIC
A recombinant adenovirus vector containing the above construct was further constructed.
For construction, an Adeno-X (trademark) Expression System 1 (TaKaRa Code.Z1513N) and a Plasmid Midi kit (QIAGEN Code.12143) were used.
The recombinant adenovirus vector containing the construct shown in FIG. 1 is referred to as “Ad-SGE-CMV-REIC.”
1. Construction of Adeno-X Plasmid
The pShuttle-REIC-TSC plasmid was digested with restriction enzymes, PI-Sce I, I-Ceu I, and thus an expression cassette specific to the target gene was obtained. The expression cassette was ligated to Adeno-X Viral DNA, and then the ligation product was digested with Swa I. Electro competent cells, DH10B, were transformed with the ligation product, and then the cells were plated on an LB agar medium supplemented with ampicillin. The thus obtained colonies were picked up and then amplified with an Adeno-X System PCR Screening Primer Set. Amplification bands were confirmed by electrophoresis. A 287-bp amplification band was obtained for the Adeno-X plasmid DNA containing the target gene expression cassette, and thus the selected colonies #1, 2, 4, 5, and 7 were confirmed to be target plasmids (FIG. 3). Sequencing was performed for the selected colony #4 using primers constructed upstream and downstream of the cloning site Swa I. The analytical results confirmed that the target gene had been inserted forward in the recombinant plasmid, and that the ligation had caused no excessive insertion or deletion of nucleotides.
Escherichia coli was transformed with the selected clone #4, and then cultured with shaking in an LB medium containing ampicillin. On the next day, plasmid DNA was purified from the cultured cells using a DNA purification kit. The resultant was partially digested with restriction enzymes Xho I, Pac I, and then electrophoretic bands indicating expected fragment sizes were detected by agarose gel electrophoresis (digested with Xho I: 0.6 kbp, 1.4 kbp, 2.6 kbp, 8 kbp, 9 kbp, and 14.5 kbp; and digested with Pac I: 2.9 kbp and 33.3 kbp in FIG. 4)
It was thus confirmed that clones obtained by scale-up preparation were target recombinant plasmids.
2. Preparation of Recombinant Adenovirus
(i) Preparation of Primary Virus
293 cells were cultured in DMEM containing 10% FBS, 2 mML (final concentration)-glutamine and a 1% penicillin-streptomycin solution in a 6-cm collagen coated Petri dish to 100% confluency. AdenoX-REIC-TSC plasmid (5 μg) digested with Pac I restriction enzyme was transfected using TransIT-293. 24 hours later, medium exchange was performed. After confirmation of cell degradation on day 13, cells were collected. A cell pellet was suspended uniformly in medium, freezing and thawing were repeated 3 times, and then the supernatant obtained by centrifugation was determined to be each primary viral solution.
(ii) Preparation of Secondary Virus
293 cells that had been cultured in the 6-cm collagen coated Petri dish to 70% to 100% confluency were infected with the primary viral solution. Upon viral infection, DMEM containing 5% FBS, 2 mML (final concentration)-glutamine, and a 1% penicillin-streptomycin solution was used. After confirmation of the degradation of the 293 cells, cells were collected, freezing and thawing were repeated 3 times, and then the supernatant obtained by centrifugation was determined to be a secondary virus.
(iii) Preparation of Tertiary Virus
293 cells that had been cultured in a T-75 collagen coated flask to 70% to 100% confluency were infected with the secondary virus. After confirmation of the complete degradation of the 293 cells, the culture solution containing the cells was recovered. After ultrasonication, the supernatant obtained by centrifugation was determined to be a tertiary virus. The resultant was aliquoted at 1 mL/vial, rapidly frozen with dry ice, and then stored at −80° C.
3. Method for Preparation of Purified Recombinant Adenovirus
(i) Scale-Up Preparation
293 cells were cultured in a multilayer cell culture flask until confluent. The tertiary virus titer was confirmed using an Adeno-X (trademark)-Rapid Titer Kit, and then the 293 cells were infected therewith under optimum conditions. After confirmation of the complete degradation of the 293 cells, the cells were collected, and then subjected to freezing and thawing. The supernatant obtained by centrifugation of the resultant was determined to be a viral solution.
(ii) Purification by Cesium Chloride Density-Gradient Centrifugation Method
A cesium chloride solution and the viral solution obtained in (i) above were layered using an ultracentrifugation tube, and then the resultant was subjected to centrifugation at 4° C. and 25,000 rpm for 2 hours. The virus bands formed were collected. A cesium chloride solution was further added, ultracentrifugal separation was performed at 4° C. and 35,000 rpm for 3 hours, and then the virus bands formed were collected. The viral solution was collected by performing ultracentrifugation twice and then dialyzed against 10% glycerol-containing PBS(−) using a dialysis cassette. The solution collected by dialysis was aliquoted (0.5 mL each), and then cryopreserved at −80° C.
4. Quality Inspection of Purified Recombinant Adenovirus
(i) Titer Measurement (TCID50 Method)
The purified viral solution was diluted 1×106-fold with medium and subjected to an 11-step serial dilution (three-fold each) on a collagen coated 96-well plate. The 293 cell suspension (50 μl) of the same quantity was added, and then cells were cultured. Control cells were cultured by adding only a 298 cell suspension and medium. On day 14, the end point of cell degradation was determined by observation under a microscope with the naked eye, and then 50% cell degradation end point (TCID50) was calculated using the following Kaber formula (Table 1). The TCID50 value calculated by this method was well consistent with “pfu”, 1 TCID50=1 pfu was employed.

TCID50=(dilution ratio for the 1st line)×(serial dilution ratio)Σ−0.5  Kaber formula

Where the sum of (the number of wells with cell degradation)/(the number of specimens) at Σ=each dilution step








TABLE 1







Results of the measurement of titer












Virus
Plate #
Titer (pfu/mL)
















Adeno-X-hREIC
1
1.9 × 1010
2.4 × 1010




2
2.9 × 1010










(ii) Confirmation of Structure

293 cells that had been cultured on a collagen coated 24-well plate to 70% to 100% confluency were infected with 1 μL of a viral solution prepared by diluting the purified viral solution 10-fold with PBS. After confirmation of complete cell degradation, cells were collected. Total DNA was extracted and prepared, digested with Xho I restriction enzyme, and then subjected to agarose gel electrophoresis. Expected fragment sizes of 0.6 kbp, 1.44 kbp, 2.46 kbp, 6.1 kbp, 8 kbp, and 14.5 kbp were detected. The insertion of DNA fragments of the target size was confirmed (FIG. 5).
(iii) RCA (Replication Competent Adenovirus) Check (PCR Method)
HeLa cells that had been cultured on a 24-well plate to 70% to 100% confluency were infected with 1 μL of a viral solution prepared by diluting the purified viral solution 10-fold with PBS. Three days later, cells were collected and then used to prepare total DNA. PCR was performed using the extracted DNA as a template and primers for ElA gene detection (sense: 5′-ATGAGACATATTATCTGCCAC-3 (SEQ ID NO: 8) and antisense: 5′-GTAAGTCAATCCCTTCCTGCAC-3′) (SEQ ID NO: 9). As a result, the 240-bp El gene band to be amplified with the use of these primers was not detected in any of the viral samples, confirming that there was no RCA contamination (FIG. 6).
Example 3
Expression of REIC Protein in Cells Using Ad-SGE-CMV-REIC
PC3 cells or HeLa cells were transfected at 10 MOI (multiplicity of infection) in a serum free medium for 2 hours with:

    the recombinant adenovirus vector (Ad-SGE-CMV-REIC) constructed in Example 2; and
    a recombinant adenovirus vector (Ad-CMV-REIC) containing a construct that had been prepared by inserting only the CMV promoter to a site upstream of REIC/Dkk-3 DNA (without inserting any 3×enh). After 12 hours, intracellular expression of the human REIC protein was detected by Western blot.


The results are shown in FIG. 7. As shown in FIG. 7, REIC gene expression was significantly enhanced when Ad-SGE-CMV-REIC was used, compared to the use of Ad-CMV-REIC.
The results indicate that: increased therapeutic effects (anti-tumor effects) can be expected in human cancer therapy with Ad-SGE-CMV-REIC compared to the use of conventional Ad-CMV-REIC, even when the dosages (viral particles) are the same; and adverse reactions can be expected to be effectively reduced to an extent equivalent to that of the use of conventional Ad-CMV-REIC by further decreasing the dosage of the adenovirus vector.
Example 4
Cell Death Induction by Ad-SGE-CMV-REIC
For examination of in vitro cell death induction, cells were plated on a flat-bottom 6-well plate and then cultured for 24 hours. Cells were transfected with Ad-SGE-CMV-REIC, Ad-CMV-REIC, and a recombinant adenovirus vector (Ad-CAG-REIC) containing a construct prepared by inserting only a CAG promoter (the CAG promoter having a structure prepared by ligating a CMV enhancer and a chicken β-actin promoter to a site upstream of REIC/Dkk-3 DNA) without inserting any 3×enh, at 50 MOI (multiplicity of infection) in serum free media for 2 hours, followed by medium exchange with fresh complete media. An adenovirus vector (Ad-LacZ) containing a LacZ gene was used a control. After 48 hours of incubation, a Hoechst33342 stock solution with a concentration of 2 μg/ml was added, and then cells were incubated in dark conditions for 10 minutes. Hoecht33342 is an interchelator dye that can be used when examining the total amount and degree of the condensation of chromatin (Belloc F et al., Cytometry 1994; 17: 59-65, Maciorowski Z et al., Cytometry 1998; 32: 44-50). Cells with highly condensed and segmented nuclei observed using a fluorescent microscope were identified as non-viable (cell death), and then counted in 3 to 5 different visual fields under a microscope.
To detect cells, for which cell death had been confirmed in vivo, a TUNEL (terminal deoxynucleotidyl transferase-mediated UTP end labeling) assay was performed using an In situ Cell Detection Kit (Fluorescein (Roche)). Specifically, tumor tissue was cut, immersed in an OCT compound, and then quickly frozen in liquid nitrogen. Frozen section (10 μm) samples were fixed and washed using methanol for 30 minutes at room temperature, impregnated with PBS containing 0.1% Triton X-100, and then stained with a TUNEL reaction mixture.
FIG. 8 shows cell death induction rate (%) for each type of cells resulting from the use of each recombinant adenovirus. As shown in FIG. 8, a significantly higher cell death induction rate (%) was obtained when Ad-SGE-CMV-REIC was used, compared to other recombinant adenoviruses.
Example 5
Tumor Growth-Suppressing Effects of Ad-SGE-CMV-REIC (No. 1)
2×105 RENCA cells (mouse kidney cancer-derived cells) were subcutaneously injected to the right femur of female BALB/C mice (6 to 8-week-old) (4 mice per group) on day [−7] (7 days before the initiation of treatment). On day 0, tumors were observed in each mouse. PBS containing 5×109 viral particles/100 μL (Ad-SGE-CMV-REIC, Ad-CMV-REIC, or Ad-CAG-REIC) was intratumorally injected on day 0. An adenovirus vector (Ad-LacZ) containing a LacZ gene was used as a control. Also, the same amount of PBS was injected into each negative control mouse. Tumor size was measured once every 2 weeks. Tumor volume was calculated by the formula found by an experiment, “1⁄2×(w1×w2×w2)” (where “w1” denotes the greatest tumor dimension, and “w2” denotes the smallest tumor dimension).
The results are shown FIG. 9. As shown in FIG. 9, the Ad-SGE-CMV-REIC treatment group exhibited significantly lower tumor volumes compared to those in the other 4 groups, and thus were confirmed to have the strongest tumor growth-suppressing effects compared to the 4 other groups.
Furthermore, subcutaneous tumors were excised on day 14 from each treatment group. Photographs showing typical tumors from two mice (from each treatment group) are shown in FIG. 10. As shown in FIG. 10, the Ad-SGE-CMV-REIC treatment group exhibited significantly lower tumor volumes compared to those of the 4 other groups.
Example 6
Tumor Growth-Suppressing Effects of Ad-SGE-CMV-REIC (No. 2)
2×105 RENCA cells (mouse kidney cancer-derived cells) were subcutaneously injected to the right femur of 6 to 8-week-old female BALB/C mice (4 to 5 mice per group) on day [−7] (7 days before the initiation of treatment). Tumors were confirmed in each mouse on day 0. PBS containing 1×109 viral particles/100 μl (Ad-SGE-CMV-REIC, Ad-CMV-REIC, or Ad-CAG-REIC) was intratumorally injected on day 0. An adenovirus vector (Ad-LacZ) containing a LacZ gene was used as a control. Also, the same amount of PBS was injected to each negative control mouse. Tumor size was measured once every 2 weeks. Tumor volume was calculated by the formula found by an experiment, “1⁄2×(w1×w2×w2)” (where “w1” denotes the greatest tumor dimension, and “w2” denotes the smallest tumor dimension).
The results are shown in FIG. 11. As shown in FIG. 11, the Ad-SGE-CMV-REIC treatment group exhibited significantly lower tumor volumes compared to those of the 4 other groups, and thus confirmed to have the strongest tumor growth-suppressing effects compared to the 4 other groups.
INDUSTRIAL APPLICABILITY
The adenovirus vector of the present invention containing a DNA construct that contains REIC/Dkk-3 DNA can be used as a cancer therapeutic drug.
Sequence Listing Free Text


    SEQ ID NOS: 8 and 9, primers


All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

## Claims

1. A DNA construct for expression of REIC/Dkk-3 DNA, which is prepared by ligating the following (i) to (iv) in this order, from the 5′ terminal side:
(i) a CMV promoter;
(ii) the following REIC/Dkk-3 DNA:
(a) DNA comprising the nucleotide sequence shown in SEQ ID NO: 1, or
(b) a polynucleotide consisting of SEQ ID NO: 3 or SEQ ID NO: 4;

(iii) a polyA addition sequence; and
(iv) enhancers prepared by linking an hTERT (Telomerase Reverse Transcriptase) enhancer, an SV40 enhancer, and a CMV enhancer in this order.

2. The DNA construct according to claim 1, wherein the polyA addition sequence is a polyA addition sequence of bovine growth hormone gene.

3. The DNA construct according to claim 1, containing the nucleotide sequence shown in SEQ ID NO: 6, wherein (ii) the REIC/Dkk-3 DNA, (iii) the polyA addition sequence, and (iv) the enhancers prepared by linking the hTERT (Telomerase Reverse Transcriptase) enhancer, the SV40 enhancer, and the CMV enhancer in this order are ligated.

4. The DNA construct according to claim 1, containing the nucleotide sequence shown in SEQ ID NO: 6.

5. An adenovirus vector, containing the DNA construct according to claim 1.

6. A cancer cell death-inducing agent, containing the adenovirus vector according to claim 5.

7. A tumor cell growth-suppressing agent, containing the adenovirus vector according to claim 5.

8. A cancer therapeutic drug, containing the adenovirus vector according to claim 5.