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Patent Application Titled "Reic-Expressing Adenovirus Vector" Published Online

June 16, 2014



By a News Reporter-Staff News Editor at Cancer Gene Therapy Week -- According to news reporting originating from Washington, D.C., by NewsRx journalists, a patent application by the inventors Kumon, Hiromi (Okayama, JP); Huh, Namho (Okayama, JP); Sakaguchi, Masakiyo (Okayama, JP); Watanabe, Masami (Okayama, JP), filed on May 25, 2012, was made available online on June 5, 2014 (see also Momotaro-Gene Inc.).

The assignee for this patent application is Momotaro-Gene Inc.

Reporters obtained the following quote from the background information supplied by the inventors: "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)."

In addition to obtaining background information on this patent application, NewsRx editors also obtained the inventors' summary information for this patent application: "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, © a polynucleotide comprising a nucleotide sequence ranging from the 1.sup.st nucleotide to any single nucleotide from the 117.sup.th nucleotide to the 234.sup.th 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 1.sup.st nucleotide to any single nucleotide from the 117.sup.th nucleotide to the 234.sup.th 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)."

For more information, see this patent application: Kumon, Hiromi; Huh, Namho; Sakaguchi, Masakiyo; Watanabe, Masami. Reic-Expressing Adenovirus Vector. Filed May 25, 2012 and posted June 5, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=2237&p=45&f=G&l=50&d=PG01&S1=20140529.PD.&OS=PD/20140529&RS=PD/20140529

Keywords for this news article include: Biotechnology, Oncology, Telomerase, DNA Research, Bioengineering, Carrier Proteins, Momotaro-Gene Inc., Ribonucleoproteins, Cancer Gene Therapy.

Our reports deliver fact-based news of research and discoveries from around the world. Copyright 2014, NewsRx LLC


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Source: Cancer Gene Therapy Week


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