JP2004350607A - Telomerase inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a telomerase inhibitor and to provide a method for inhibiting telomerase. <P>SOLUTION: There are provided a double-stranded polynucleotide and a polynucleotide exhibiting RNA interference effect on human telomerase reverse transcriptase gene, RNA and polynucleotide forming those, DNA in which the transcript exhibits the RNA interference effect, a vector containing the DNA, a telomerase reverse transcriptase gene inhibitor and a telomerase inhibitor each containing one or more substances thereof, a method for inhibiting a telomerase reverse enzyme comprising using one or more substances thereof and a method for inhibiting the telomerase; and a medicinal composition containing one or more substances thereof, a reagent kit composed of one or more substances thereof and a method for identifying the double-stranded RNA. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【図面の簡単な説明】
【図１】本発明に係る二重鎖ポリヌクレオチドの配列を示す図である。
【図２】ＴＥＲＴ０２、ＴＥＲＴ０７およびＴＥＲＴ０８の３種類の二重鎖ポリヌクレオチドがそれぞれ、ｈＴＥＲＴ遺伝子およびｈＴＲ遺伝子と共に該二重鎖ポリヌクレオチドをトランスフェクションしたＳａｏｓ−２細胞において、テロメレースを阻害したことを示す図である。テロメレース活性は、テロメレース活性測定における内部標準であるスルホローダミンの蛍光強度（バックグランドを減算）に対する、テロメレースにより付加されたテロメア配列の指標であるフルオレセインの蛍光強度（バックグランドを減算）の比（△ＦＬ／△Ｒ）で示した。図中、ＤＮＡ（−）およびＲＮＡ（−）はそれぞれ、ｈＴＥＲＴ遺伝子および二重鎖ポリヌクレオチドを無添加のときのテロメレース活性を示す。また、ＲＮＡ（−）Ｈｅａｔは、二重鎖ポリヌクレオチドを添加せずに試料を加熱してテロメレースの酵素活性を失活させたときのテロメレース活性を示す。スクランブル（ｓｃｒａｍｂｌｅ）は既知の遺伝子と相同性を持たない二重鎖ポリヌクレオチドであり、陰性対象として用いた。
【図３】ＴＥＲＴ０２およびＴＥＲＴ０８の２種類の二重鎖ポリヌクレオチドがそれぞれ、ｈＴＥＲＴ遺伝子およびｈＴＲ遺伝子と共に該二重鎖ポリヌクレオチドをトランスフェクションしたＳａｏｓ−２細胞において、テロメレースを二重鎖ポリヌクレオチドの濃度依存的に阻害したことを示す図である。テロメレース活性は、テロメレース活性測定における内部標準であるスルホローダミンの蛍光強度（バックグランドを減算）に対する、テロメレースにより付加されたテロメア配列の指標であるフルオレセインの蛍光強度（バックグランドを減算）の比（△ＦＬ／△Ｒ）で示した。図中、ＲＮＡ（−）は、ｈＴＥＲＴ遺伝子および二重鎖ポリヌクレオチドを無添加のときのテロメレース活性を示す。また、ＲＮＡ（−）Ｈｅａｔは、二重鎖ポリヌクレオチドを添加せずに試料を加熱してテロメレースの酵素活性を失活させたときのテロメレース活性を示す。スクランブル（ｓｃｒａｍｂｌｅ）は既知の遺伝子と相同性を持たない二重鎖ポリヌクレオチドであり、陰性対象として用いた。[0001]
[Industrial application fields]
The present invention relates to double-stranded polynucleotides and polynucleotides that exhibit telomerase inhibitory activity, and RNAs and polynucleotides that form them. Further, the present invention relates to DNA characterized in that the transcript exhibits telomerase inhibitory activity. Furthermore, the present invention relates to a vector containing the DNA. The present invention also relates to an inhibitor of telomerase reverse transcriptase and a method of inhibiting telomerase reverse transcriptase. Furthermore, it relates to a telomerase inhibitor and a telomerase inhibition method. The present invention also relates to a pharmaceutical composition comprising at least one of the double-stranded polynucleotide, the polynucleotide, the DNA, and the vector, a cancer disease preventive and / or therapeutic agent, and a reagent kit. Furthermore, the present invention relates to a method for preventing and / or treating cancer diseases, characterized by using at least one of the double-stranded polynucleotide, the polynucleotide, the DNA and the vector. The present invention also relates to a method for identifying a double-stranded polynucleotide having telomerase inhibitory activity using cells that do not express telomerase reverse transcriptase.
[0002]
[Prior art]
Telomerase is an enzyme that performs an elongation reaction of the telomeric portion of a chromosome, and is known to be involved in cell immortalization. Telomeres are simple repetitive sequences present at the end of chromosomal DNA in eukaryotic cells, and contribute to maintaining chromosome stability. A normal DNA polymerase that controls DNA replication in cell division does not completely replicate to the very end of DNA, and therefore telomere is shortened with cell division. In normal somatic cells, proliferation stops after a certain number of divisions, and cell senescence or cell death occurs. On the other hand, the expression of telomerase is recognized in the majority of germ cells having infinite proliferation and immortalized cancer cells. It is considered that the elongation of telomeres shortened with cell division by telomerase is one of the mechanisms for acquiring infinite proliferation of cells.
[0003]
Human telomerase is a complex comprising human telomerase reverse transcriptase (hereinafter abbreviated as hTERT) and human telomerase RNA (hereinafter abbreviated as hTR). (Non-Patent Document 1).
[0004]
Telomerase first recognizes and binds the telomere sequence by hTR having a complementary sequence of telomere in the telomere extension reaction. Next, the hTR is used as a template, and the telomere is extended by hTERT which is a catalytic subunit. Since telomerase activity was experimentally observed only with hTR and hTERT, these two subunits are considered necessary and sufficient elements for telomerase activity. Moreover, since hTR is widely expressed not only in cancer but also in normal tissues, whereas hTERT is expressed specifically in cancer tissues, it is considered that hTERT is a determinant of telomerase activity.
[0005]
Since telomerase is involved in the immortalization of cancer cells, development of telomerase inhibitors has been studied. For example, an antisense method targeting hTR (Non-Patent Documents 2 and 3), a method using a dominant negative form of hTERT (Non-Patent Document 4), a method using a ribozyme (Non-Patent Document 5), and G- It has been reported that telomerase is inhibited by a method using a Kadraplex DNA interactive compound (Non-patent Document 6). All of these methods have drawbacks and few have been developed as practical drugs.
[0006]
In recent years, as a method for suppressing gene expression using RNA, a method using RNA interference has been developed (Non-patent Documents 7 and 8). In mammals, a short RNA consisting of 20 bp to 25 bp short RNA (so-called sense RNA) consisting of a partial sequence of mRNA of a target gene and RNA (antisense RNA) consisting of a base sequence complementary to the base sequence of the RNA. It has been reported that heavy interfering RNA (short interfering RNA; hereinafter abbreviated as siRNA) effectively degrades the mRNA and inhibits the expression of the gene (Non-patent Document 7). Similar to siRNA, it has been reported that short hairpin RNA (hereinafter abbreviated as shRNA), which is a short double-stranded RNA having a hairpin structure, has an RNA interference effect (Non-patent Document 8). The shRNA has a hairpin-like structure because a sense RNA and an antisense RNA are linked by, for example, a polynucleotide, and the sense RNA-derived portion and the antisense RNA-derived portion form a double strand. RNA interference is a useful method that can specifically inhibit the expression of a target gene at a low concentration and with a high probability as compared with other gene expression inhibition methods such as an antisense method and a ribozyme method.
[0007]
For telomerase, suppression by siRNA has been reported (Non-patent Document 9). However, the siRNA had a weak inhibitory effect on telomerase and its effect was transient.
[0008]
The documents cited in this specification are listed below.
[Non-Patent Document 1] Kelleher C. et al., “TRENDS in Biochemical Sciences”, 2002, Vol. 27, p. 572-579.
[Non-Patent Document 2] Feng J. et al., "Science", 1995, Vol. 269, p. 1236-1241.
[Non-patent Document 3] Kondo S. et al., “Oncogene”, 1998, Vol. 16, p. 3323-3330.
[Non-Patent Document 4] Hahn WC et al., “Nature Medicine”, 1999, Vol. 10, p. 1129-1130.
[Non-Patent Document 5] Yokoyama Y. et al., “Cancer Research”, 1998, Vol. 58, p. 5406-5410.
[Non-Patent Document 6] Sun D. et al., “Journal of Medicinal Chemistry”, 1997, Vol. 40, p. 2113-2116.
[Non-Patent Document 7] Elbashir SM, et al., “Nature”, 2001, Vol. 411, p. 494-498.
[Non-patent Document 8] Paddison PJ et al., “Jeans and Development”, 2002, Vol. 16, p. 948-958.
[Non-patent Document 9] Kosciolek BA, et al., “Molecular Cancer Therapeutics”, 2003, Vol. 2, No. 3, p. 209-216.
[Non-Patent Document 10] Edited by Sambrook et al., “Molecular Cloning, Laboratory Manual”, Second Edition, Cold Spring Harbor Laboratory, 1989.
[Non-patent document 11] Muramatsu, M., “Lab Manual Genetic Engineering”, Maruzen Co., Ltd., 1988.
[Non-Patent Document 12] Ehrlich, "PCR Technology, Principles and Applications of DNA Amplification", Stockton Press, 1989.
[Non-patent Document 13] Ulmer KM, "Science", 1983, Vol. 219, p. 666-671.
[Non-Patent Document 14] Kim N.W. et al., “Science”, 1994, Vol. 266, No. 5193, p. 2011-2015.
[Non-Patent Document 15] Wenz C. et al., "The EMBO Journal", 2001, Vol. 20, No. 13, p. 3526-3534.
[Non-Patent Document 16] Tatematsu K. et al., “Oncogene”, 1996, Vol. 13, No. 10, p. 2265-2274.
[Non-patent Document 17] Nakamura Y. et al., “Clinical Chemistry”, 1999, Vol. 45, No. 10, p. 1718-1724.
[Non-patent Document 18] Maesawa C. et al., “Nucleic Acids Research, 2003, Vol. 31, No. 2, p. E4-4.
[Non-patent Document 19] "Biotechnics", 2002, Vol. 32, No. 5, p. 1154-1156, 1158, 1160.
[0009]
[Problems to be solved by the invention]
An object of the present invention is to provide a telomerase inhibitor and a method of inhibiting telomerase that are very specific and useful for telomerase. Another object of the present invention is to provide a pharmaceutical composition containing the telomerase inhibitor, for example, an agent for preventing and / or treating cancer diseases. Furthermore, an object of the present invention is to provide a method for preventing and / or treating a cancer disease characterized by using the telomerase inhibitor.
[0010]
[Means for solving problems]
The present inventors have intensively studied to solve the above problems, and have found a double-stranded polynucleotide capable of inhibiting telomerase by exhibiting RNA interference with the human telomerase reverse transcriptase gene, and have completed the present invention.
[0011]
That is, the present invention
1. RNA comprising the base sequence set forth in any one of SEQ ID NOs: 1 to 6 in the sequence listing,
2. A polynucleotide (polynucleotide A) obtained by binding one or several nucleotides to the RNA having the base sequence set forth in SEQ ID NO: 1 in the sequence listing at the 3 'end of the RNA, and comprising SEQ ID NO: in the sequence listing A double-stranded polynucleotide obtained by hybridization with a polynucleotide comprising 1 to several nucleotides bound to the RNA having the base sequence described in 2 at the 3 ′ end of the RNA is a human telomerase reverse transcriptase gene A polynucleotide A having an RNA interference effect on
3. A polynucleotide (polynucleotide B) obtained by binding one or several nucleotides to the RNA having the base sequence set forth in SEQ ID NO: 2 in the sequence listing at the 3 ′ end of the RNA, and the sequence number in the sequence listing A double-stranded polynucleotide obtained by hybridizing an RNA comprising the nucleotide sequence of 1 with a polynucleotide obtained by binding one or several nucleotides to the 3 ′ end of the RNA is a human telomerase reverse transcriptase gene A polynucleotide B characterized by having an RNA interference effect on
4). A polynucleotide (polynucleotide C) obtained by binding one or several nucleotides to the RNA having the base sequence set forth in SEQ ID NO: 3 in the sequence listing at the 3 ′ end of the RNA, and comprising SEQ ID NO: in the sequence listing A double-stranded polynucleotide obtained by hybridizing an RNA having the base sequence described in 4 with a polynucleotide obtained by binding one or several nucleotides to the 3 ′ end of the RNA is a human telomerase reverse transcriptase gene A polynucleotide C characterized by having an RNA interference effect on
5). A polynucleotide (polynucleotide D) obtained by binding one or several nucleotides to the RNA having the base sequence set forth in SEQ ID NO: 4 in the sequence listing at the 3 ′ end of the RNA, and comprising SEQ ID NO: in the sequence listing A double-stranded polynucleotide obtained by hybridization with a polynucleotide comprising 1 to several nucleotides bonded to the RNA having the base sequence described in 3 at the 3 ′ end of the RNA is a human telomerase reverse transcriptase gene A polynucleotide D characterized by having an RNA interference effect on
6). A polynucleotide (polynucleotide E) obtained by binding one or several nucleotides to the RNA having the base sequence set forth in SEQ ID NO: 5 in the sequence listing at the 3 'end of the RNA, and comprising SEQ ID NO: in the sequence listing A double-stranded polynucleotide obtained by hybridizing an RNA comprising the nucleotide sequence of claim 6 with a polynucleotide obtained by binding one or several nucleotides to the 3 ′ end of the RNA is a human telomerase reverse transcriptase gene A polynucleotide E characterized by having an RNA interference effect on
7). A polynucleotide (polynucleotide F) obtained by binding one or several nucleotides to the RNA having the base sequence set forth in SEQ ID NO: 6 in the sequence listing at the 3 'end of the RNA, and comprising SEQ ID NO: in the sequence listing A double-stranded polynucleotide obtained by hybridizing an RNA having the base sequence of 5 to a polynucleotide obtained by binding one or several nucleotides to the 3 ′ end of the RNA is a human telomerase reverse transcriptase gene A polynucleotide F characterized by having an RNA interference effect on
8). A polynucleotide obtained by binding two deoxythymidylates to the RNA having the base sequence described in any one of SEQ ID NOS: 1 to 6 in the sequence listing at the 3 ′ end of the RNA
9. Any one double-stranded polynucleotide selected from the following group, wherein the double-stranded polynucleotide has an RNA interference effect on a human telomerase reverse transcriptase gene;
(I) a polynucleotide formed by binding one or several nucleotides to the RNA having the base sequence set forth in SEQ ID NO: 1 in the sequence listing at the 3 'end of the RNA and the base sequence set forth in SEQ ID NO: 2 A double-stranded polynucleotide comprising a polynucleotide formed by binding one or several nucleotides to the RNA at the 3 ′ end of the RNA;
(Ii) consisting of a polynucleotide obtained by binding one or several nucleotides to the RNA comprising the base sequence shown in SEQ ID NO: 3 in the sequence listing at the 3 ′ end of the RNA and the base sequence shown in SEQ ID NO: 4 A double-stranded polynucleotide comprising a polynucleotide formed by binding one or several nucleotides to the RNA at the 3 ′ end of the RNA;
and
(Iii) consisting of a polynucleotide obtained by binding one or several nucleotides to the RNA consisting of the base sequence shown in SEQ ID NO: 5 in the sequence listing at the 3 ′ end of the RNA and the base sequence shown in SEQ ID NO: 6 A double-stranded polynucleotide comprising a polynucleotide formed by binding one or several nucleotides to the RNA at the 3 ′ end of the RNA;
10. Any one double-stranded polynucleotide selected from the group below;
(I) a polynucleotide obtained by binding two deoxythymidylic acids to the RNA having the base sequence set forth in SEQ ID NO: 1 in the sequence listing and the 3 'end of the RNA and the RNA consisting of the base sequence set forth in SEQ ID NO: 2 A double-stranded polynucleotide comprising a polynucleotide obtained by binding two deoxythymidylic acids to the 3 ′ end of the RNA; (ii) an RNA comprising the base sequence set forth in SEQ ID NO: 3 in the sequence listing; A duplex consisting of a polynucleotide formed by binding two deoxythymidylic acids at the ends and a polynucleotide formed by binding two deoxythymidylic acids at the 3 ′ end of the RNA to the RNA consisting of the base sequence set forth in SEQ ID NO: 4. Strand polynucleotide,
and
(Iii) An RNA comprising a nucleotide sequence set forth in SEQ ID NO: 5 in the sequence listing and an RNA comprising a nucleotide sequence set forth in SEQ ID NO: 6 and a polynucleotide obtained by binding two deoxythymidylic acids to the 3 ′ end of the RNA. A double-stranded polynucleotide comprising a polynucleotide obtained by binding two deoxythymidylic acids to the 3 ′ end of the RNA;
11. Any one polynucleotide selected from the group below, wherein the polynucleotide has an RNA interference effect on a human telomerase reverse transcriptase gene;
(I) an RNA comprising the base sequence described in SEQ ID NO: 1 in the sequence listing, a polynucleotide forming a loop structure and a polynucleotide (polynucleotide G) formed by binding an RNA comprising the base sequence described in SEQ ID NO: 2 The 3 ′ end of the RNA comprising the base sequence set forth in SEQ ID NO: 1 of the Sequence Listing is linked to the 5 ′ end of the polynucleotide forming the loop structure, and further the 3 ′ end of the polynucleotide forming the loop structure And a polynucleotide G in which the 5 ′ end of RNA consisting of the nucleotide sequence set forth in SEQ ID NO: 2 is bound,
(Ii) an RNA comprising the base sequence described in SEQ ID NO: 1 in the sequence listing, a polynucleotide forming a loop structure, and a polynucleotide (polynucleotide H) formed by binding an RNA comprising the base sequence described in SEQ ID NO: 2 The 5 ′ end of the polynucleotide that forms the loop structure by binding the 5 ′ end of the RNA consisting of the base sequence shown in SEQ ID NO: 1 of the Sequence Listing to the 3 ′ end of the polynucleotide that forms the loop structure A polynucleotide H bound to the 3 ′ end of RNA consisting of the nucleotide sequence set forth in SEQ ID NO: 2,
(Iii) a polynucleotide (polynucleotide I) obtained by binding an RNA comprising the base sequence set forth in SEQ ID NO: 3 in the sequence listing, a polynucleotide forming a loop structure, and an RNA comprising the base sequence set forth in SEQ ID NO: 4 The 3 ′ end of the polynucleotide that forms the loop structure is formed by binding the 3 ′ end of the RNA consisting of the base sequence shown in SEQ ID NO: 3 of the Sequence Listing to the 5 ′ end of the polynucleotide that forms the loop structure. A polynucleotide I bound to the 5 ′ end of RNA consisting of the nucleotide sequence set forth in SEQ ID NO: 4,
(Iv) an RNA comprising the base sequence described in SEQ ID NO: 3 in the sequence listing, a polynucleotide forming a loop structure, and a polynucleotide (polynucleotide J) formed by binding an RNA comprising the base sequence described in SEQ ID NO: 4 The 5 ′ end of the RNA consisting of the base sequence shown in SEQ ID NO: 3 in the sequence listing and the 3 ′ end of the polynucleotide forming the loop structure are combined, and the 5 ′ end of the polynucleotide forming the loop structure. And polynucleotide J in which the 3 ′ end of RNA consisting of the nucleotide sequence set forth in SEQ ID NO: 4 is bound,
(V) a polynucleotide (polynucleotide K) formed by binding an RNA comprising the base sequence set forth in SEQ ID NO: 5 in the sequence listing, a polynucleotide forming a loop structure, and an RNA comprising the base sequence set forth in SEQ ID NO: 6 And the 3 ′ end of the polynucleotide that forms the loop structure by binding the 3 ′ end of the RNA consisting of the base sequence shown in SEQ ID NO: 5 of the sequence listing to the 5 ′ end of the polynucleotide that forms the loop structure. A polynucleotide K bound to the 5 ′ end of RNA consisting of the nucleotide sequence set forth in SEQ ID NO: 6,
and
(Vi) an RNA composed of the base sequence shown in SEQ ID NO: 5 in the sequence listing, a polynucleotide forming a loop structure, and a polynucleotide (polynucleotide L) formed by binding an RNA consisting of the base sequence shown in SEQ ID NO: 6 The 5 ′ end of the polynucleotide that forms the loop structure is formed by binding the 5 ′ end of the RNA consisting of the base sequence shown in SEQ ID NO: 5 of the Sequence Listing to the 3 ′ end of the polynucleotide that forms the loop structure. And a polynucleotide L bound to the 3 ′ end of RNA consisting of the base sequence set forth in SEQ ID NO: 6,
12 Any one polynucleotide selected from the group below, wherein the polynucleotide has an RNA interference effect on a human telomerase reverse transcriptase gene;
(I) a polynucleotide formed by binding of RNA consisting of the base sequence shown in SEQ ID NO: 1 in the sequence listing, polynucleotide consisting of 4 to 23 nucleotides, and RNA consisting of the base sequence shown in SEQ ID NO: 2 Nucleotide M), the 3 ′ end of RNA consisting of the base sequence set forth in SEQ ID NO: 1 in the Sequence Listing is linked to the 5 ′ end of a polynucleotide consisting of 4 to 23 nucleotides, and further 4 to A polynucleotide M, wherein the 3 ′ end of a polynucleotide consisting of 23 nucleotides and the 5 ′ end of an RNA consisting of the base sequence set forth in SEQ ID NO: 2 are linked;
(Ii) a polynucleotide formed by binding of RNA consisting of the base sequence shown in SEQ ID NO: 1 in the sequence listing, polynucleotide consisting of 4 to 23 nucleotides, and RNA consisting of the base sequence shown in SEQ ID NO: 2 (polynucleotide) Nucleotide 5), the 5 ′ end of RNA consisting of the base sequence set forth in SEQ ID NO: 1 in the sequence listing and the 3 ′ end of a polynucleotide consisting of 4 to 23 nucleotides, A polynucleotide N in which the 5 ′ end of a polynucleotide consisting of 23 nucleotides and the 3 ′ end of an RNA consisting of the base sequence set forth in SEQ ID NO: 2 are linked;
(Iii) A polynucleotide comprising a combination of RNA comprising the nucleotide sequence set forth in SEQ ID NO: 3 in the sequence listing, polynucleotide comprising 4 to 23 nucleotides, and RNA comprising the nucleotide sequence set forth in SEQ ID NO: 4 Nucleotide 3), the 3 ′ end of RNA consisting of the base sequence set forth in SEQ ID NO: 3 in the sequence listing is linked to the 5 ′ end of a polynucleotide consisting of 4 to 23 nucleotides, and further 4 to A polynucleotide O in which the 3 ′ end of a polynucleotide consisting of 23 nucleotides and the 5 ′ end of an RNA consisting of the base sequence set forth in SEQ ID NO: 4 are linked;
(Iv) a polynucleotide formed by binding of RNA consisting of the base sequence shown in SEQ ID NO: 3 in the sequence listing, polynucleotide consisting of 4 to 23 nucleotides, and RNA consisting of the base sequence shown in SEQ ID NO: 4 Nucleotide P), wherein the 5 ′ end of the RNA comprising the base sequence set forth in SEQ ID NO: 3 in the sequence listing and the 3 ′ end of the polynucleotide comprising 4 to 23 nucleotides are combined, and further 4 to A polynucleotide P in which the 5 ′ end of a polynucleotide consisting of 23 nucleotides and the 3 ′ end of an RNA consisting of the base sequence set forth in SEQ ID NO: 4 are linked;
(V) a polynucleotide formed by binding of RNA consisting of the base sequence shown in SEQ ID NO: 5 in the sequence listing, polynucleotide consisting of 4 to 23 nucleotides, and RNA consisting of the base sequence shown in SEQ ID NO: 6 Nucleotide 3), the 3 ′ end of RNA consisting of the base sequence set forth in SEQ ID NO: 5 in the sequence listing and the 5 ′ end of a polynucleotide consisting of 4 to 23 nucleotides, A polynucleotide Q in which the 3 ′ end of a polynucleotide consisting of 23 nucleotides and the 5 ′ end of an RNA consisting of the base sequence set forth in SEQ ID NO: 6 are linked;
and
(Vi) a polynucleotide formed by binding of RNA consisting of the base sequence set forth in SEQ ID NO: 5 in the sequence listing, polynucleotide consisting of 4 to 23 nucleotides, and RNA consisting of the base sequence set forth in SEQ ID NO: 6 Nucleotide R), the 5 ′ end of the RNA consisting of the base sequence set forth in SEQ ID NO: 5 of the Sequence Listing is linked to the 3 ′ end of a polynucleotide consisting of 4 to 23 nucleotides, and further 4 to A polynucleotide R in which the 5 ′ end of a polynucleotide consisting of 23 nucleotides and the 3 ′ end of an RNA consisting of the base sequence set forth in SEQ ID NO: 6 are linked;
13. DNA comprising the base sequence described in any one of SEQ ID NOs: 7 to 12 in the sequence listing;
14 Any one DNA selected from the following group, wherein the transcript has an RNA interference effect on a human telomerase reverse transcriptase gene;
(I) DNA (DNA1) obtained by binding DNA consisting of the base sequence shown in SEQ ID NO: 7 of the sequence listing, DNA consisting of 4 to 23 nucleotides, and DNA consisting of the base sequence shown in SEQ ID NO: 8 The 3 ′ end of the DNA consisting of the base sequence shown in SEQ ID NO: 7 in the sequence listing and the 5 ′ end of the DNA consisting of 4 to 23 nucleotides are combined, and further from 4 to 23 nucleotides. DNA 1 in which the 3 ′ end of the DNA and the 5 ′ end of the DNA comprising the base sequence set forth in SEQ ID NO: 8 are bound,
(Ii) DNA (DNA2) obtained by binding DNA consisting of the base sequence set forth in SEQ ID NO: 7 in the sequence listing, DNA consisting of 4 to 23 nucleotides, and DNA consisting of the base sequence set forth in SEQ ID NO: 8 And the 5 ′ end of the DNA consisting of the base sequence shown in SEQ ID NO: 7 of the Sequence Listing is linked to the 3 ′ end of the DNA consisting of 4 to 23 nucleotides, and further from 4 to 23 nucleotides. DNA 2 in which the 5 ′ end of the DNA and the 3 ′ end of the DNA consisting of the base sequence set forth in SEQ ID NO: 8 are bound,
(Iii) DNA (DNA3) obtained by binding DNA consisting of the base sequence shown in SEQ ID NO: 9 in the sequence listing, DNA consisting of 4 to 23 nucleotides, and DNA consisting of the base sequence shown in SEQ ID NO: 10 And the 3 ′ end of the DNA consisting of the base sequence shown in SEQ ID NO: 9 of the Sequence Listing is linked to the 5 ′ end of the DNA consisting of 4 to 23 nucleotides, and further comprising 4 to 23 nucleotides. DNA 3 in which the 3 ′ end of the DNA and the 5 ′ end of the DNA comprising the base sequence set forth in SEQ ID NO: 10 are bound,
(Iv) DNA consisting of the base sequence set forth in SEQ ID NO: 9 in the sequence listing, DNA consisting of 4 to 23 nucleotides and DNA consisting of the base sequence set forth in SEQ ID NO: 10 (DNA4) And the 5 ′ end of the DNA consisting of the base sequence shown in SEQ ID NO: 9 of the Sequence Listing is linked to the 3 ′ end of the DNA consisting of 4 to 23 nucleotides, and further from 4 to 23 nucleotides. DNA 4 in which the 5 ′ end of the DNA and the 3 ′ end of the DNA consisting of the base sequence set forth in SEQ ID NO: 10 are bound,
(V) DNA (DNA5) obtained by binding DNA consisting of the base sequence shown in SEQ ID NO: 11 of the sequence listing, DNA consisting of 4 to 23 nucleotides, and DNA consisting of the base sequence shown in SEQ ID NO: 12 The 3 ′ end of the DNA consisting of the base sequence shown in SEQ ID NO: 11 of the sequence listing and the 5 ′ end of the DNA consisting of 4 to 23 nucleotides are combined, and further from 4 to 23 nucleotides. DNA 5 in which the 3 ′ end of the DNA and the 5 ′ end of the DNA comprising the base sequence set forth in SEQ ID NO: 12 are bound,
and
(Vi) DNA consisting of the base sequence set forth in SEQ ID NO: 11 in the sequence listing, DNA consisting of 4 to 23 nucleotides, and DNA consisting of the base sequence set forth in SEQ ID NO: 12 (DNA6) And the 5 ′ end of the DNA consisting of the base sequence set forth in SEQ ID NO: 11 of the Sequence Listing is linked to the 3 ′ end of the DNA consisting of 4 to 23 nucleotides, and further comprising 4 to 23 nucleotides. DNA 6 in which the 5 ′ end of the DNA and the 3 ′ end of the DNA consisting of the base sequence set forth in SEQ ID NO: 12 are bound,
15. 13. Or a vector containing 14 DNAs,
16. 9 above. Or 10. 10. A double-stranded polynucleotide according to any of the above, Or 12. Any one of the polynucleotides of the above 13. Or 14. Any one of the above DNAs, and 15. A telomerase reverse transcriptase gene expression inhibitor comprising at least one of the vectors of:
17. 9 above. Or 10. 10. A double-stranded polynucleotide according to any of the above, Or 12. Any one of the polynucleotides of the above 13. Or 14. Any one of the above DNAs, and 15. A telomerase inhibitor comprising at least one of the vectors of
18. 9 above. Or 10. 10. A double-stranded polynucleotide according to any of the above, Or 12. Any one of the polynucleotides of the above 13. Or 14. Any one of the above DNAs, and 15. A method for inhibiting expression of a telomerase reverse transcriptase gene, comprising using at least one of the vectors of
19. 9 above. Or 10. 10. A double-stranded polynucleotide according to any of the above, Or 12. Any one of the polynucleotides of the above 13. Or 14. Any one of the above DNAs, and 15. A method of inhibiting telomerase, comprising using at least one of the vectors of:
20. 9 above. Or 10. 10. A double-stranded polynucleotide according to any of the above, Or 12. Any one of the polynucleotides of the above 13. Or 14. Any one of the above DNAs, and 15. A pharmaceutical composition comprising an effective amount of at least one of the vectors of
21. 9 above. Or 10. 10. A double-stranded polynucleotide according to any of the above, Or 12. Any one of the polynucleotides of the above 13. Or 14. Any one of the above DNAs, and 15. A preventive and / or therapeutic agent for cancer diseases, comprising an effective amount of at least one of the vectors;
22. 9 above. Or 10. 10. A double-stranded polynucleotide according to any of the above, Or 12. Any one of the polynucleotides of the above 13. Or 14. Any one of the above DNAs, and 15. A method for preventing and / or treating a cancer disease, characterized by using at least one of the vectors
23. 9 above. Or 10. 10. A double-stranded polynucleotide according to any of the above, Or 12. Any one of the polynucleotides of the above 13. Or 14. Any one of the above DNAs, and 15. A reagent kit comprising at least one of the vectors of
24. A method for identifying a double-stranded polynucleotide having a telomerase inhibitory action, wherein a cell that does not express a human telomerase reverse transcriptase gene or a cell that does not express a human telomerase reverse transcriptase gene and a human telomerase RNA gene is used, The cell is transfected with a double-stranded polynucleotide as a test substance together with a human telomerase reverse transcriptase gene expression vector and a human telomerase RNA gene expression vector, and then the telomerase activity is measured and the double-stranded polynucleotide is transfected. An identification method comprising selecting a double-stranded polynucleotide having reduced telomerase activity compared to when not
25. 24. The cell that does not express a human telomerase reverse transcriptase gene or a cell that does not express a human telomerase reverse transcriptase gene and a human telomerase RNA gene is a human osteosarcoma-derived Saos-2 cell. Identification method,
26. 24. The transfection is a lipofection method. Or 25. Identification method,
27. 26. The lipofection method is a lipofection method using cationic liposomes. Identification method,
Consists of.
[0012]
DETAILED DESCRIPTION OF THE INVENTION
In the present invention, a polynucleotide useful for inhibiting telomerase is provided. The polynucleotide in the present specification means a linear polymer in which more than 2 nucleotides are linked by a 3 ′, 5′-phosphodiester bond, and two or more linked by phosphodiester bonds. A linear nucleic acid fragment consisting of about 10 or more nucleotides is also included.
[0013]
One embodiment of the polynucleotide according to the present invention is RNA consisting of a partial sequence of hTERT mRNA and RNA consisting of a base sequence complementary to the base sequence of the RNA. As RNA which concerns on this invention, Preferably RNA which consists of a base sequence as described in any one of sequence number 1 to 6 of a sequence table is mentioned.
[0014]
Each RNA according to the present invention preferably has a nucleotide consisting of one or several base sequences called an overhang sequence bound to the 3 ′ end of the RNA. The overhang sequence has an effect of protecting RNA from nucleases as one of its actions. The overhang sequence is not particularly limited as long as it does not inhibit the RNA interference effect of the RNA, preferably any one consisting of 1 to 10, more preferably 1 to 4, and even more preferably 2 nucleotides. Can also be used. Specifically, sequences such as a sequence composed of deoxythymidylic acid (for example, TT), a sequence composed of uridylic acid (for example, UU), and a sequence in which any nucleotide is bound following deoxythymidylic acid (for example, TN) can be exemplified. More preferably, a sequence consisting of two deoxythymidylic acids is used as an overhang sequence because it can be synthesized inexpensively and has a higher nuclease resistance. The overhang sequence is bound to the ribose 3 ′ hydroxyl site at the 3 ′ end of the RNA according to the present invention by a diester bond.
[0015]
A polynucleotide obtained by binding an overhang sequence consisting of one to several nucleotides to the 3 ′ end of each RNA according to the present invention is also included in the scope of the present invention. As such a polynucleotide, for example, an RNA comprising the base sequence set forth in any one of SEQ ID NOs: 1 to 6 in the sequence listing, an overhang sequence comprising 1 to several nucleotides at the 3 ′ end of the RNA, preferably Includes a polynucleotide obtained by binding a sequence composed of two deoxythymidylic acids.
[0016]
One of the characteristics of these polynucleotides is that one or several nucleotides are bound to the 3 ′ end of the RNA to RNA comprising the polynucleotide and a complementary base sequence of RNA contained in the polynucleotide. The double-stranded polynucleotide obtained by hybridizing with the resulting polynucleotide has an RNA interference effect on the human telomerase reverse transcriptase gene.
[0017]
For example, a polynucleotide obtained by binding one or several nucleotides to the RNA having the base sequence set forth in SEQ ID NO: 1 in the sequence listing at the 3 ′ end of the RNA, and the base set forth in SEQ ID NO: 2 in the sequence listing A double-stranded polynucleotide obtained by hybridizing an RNA consisting of a sequence to a polynucleotide obtained by binding one or several nucleotides to the 3 ′ end of the RNA, Inhibited in a dose-dependent manner. That is, it is considered that this double-stranded polynucleotide exhibited an RNA interference effect on the hTERT gene and inhibited telomerase by inhibiting the expression of the gene. Inhibition of telomerase in the present invention means partial or complete loss of telomerase function.
[0018]
Similarly, a polynucleotide obtained by binding one or several nucleotides to the RNA having the base sequence set forth in SEQ ID NO: 3 in the sequence listing at the 3 ′ end of the RNA, and the sequence described in SEQ ID NO: 4 in the sequence listing. A double-stranded polynucleotide obtained by hybridizing an RNA consisting of a base sequence to a polynucleotide obtained by binding one or several nucleotides to the 3 ′ end of the RNA is also used for the human telomerase reverse transcriptase gene. Showed an RNA interference effect and inhibited telomerase. A polynucleotide obtained by binding one or several nucleotides to the RNA having the base sequence set forth in SEQ ID NO: 5 in the sequence listing at the 3 'end of the RNA; and the base sequence set forth in SEQ ID NO: 6 in the sequence list A double-stranded polynucleotide obtained by hybridizing an RNA obtained by hybridization with a polynucleotide obtained by binding one or several nucleotides to the 3 ′ end of the RNA is also RNA for human telomerase reverse transcriptase gene. Shows interference effect and inhibits telomerase.
[0019]
The RNA and polynucleotide can be prepared, for example, using a known chemical synthesis method. For example, it can be prepared by a DNA / RNA synthesizer using commercially available RNA synthesis reagents (Proligo, Dharmacon Research, ChemGenes, etc.).
[0020]
In one aspect, the inhibition of telomerase is an RNA comprising a polynucleotide obtained by binding one or several nucleotides to the RNA according to the present invention at the 3 ′ end of the RNA, and a base sequence complementary to the RNA. And a double-stranded polynucleotide comprising a polynucleotide formed by binding one or several nucleotides to the 3 ′ end of the RNA. The double-stranded polynucleotide can be obtained by hybridizing these two polynucleotides. Hybridization can be performed by annealing these two polynucleotides by a conventional method. Annealing can be performed by the method described in the Example of this specification as an example.
[0021]
The double-stranded polynucleotide according to the present invention is, for example, a double-stranded polynucleotide selected from the following group (see FIG. 1): (i) RNA consisting of the base sequence described in SEQ ID NO: 1 in the sequence listing A polynucleotide formed by binding two deoxythymidylic acids to the 3 ′ end of the RNA and an RNA comprising the nucleotide sequence set forth in SEQ ID NO: 2 and two deoxythymidylic acids bound to the 3 ′ end of the RNA A double-stranded polynucleotide comprising nucleotides; and (ii) a polynucleotide obtained by binding two deoxythymidylates to the RNA comprising the base sequence set forth in SEQ ID NO: 3 in the sequence listing at the 3 ′ end of the RNA and SEQ ID NO: A duplex comprising an RNA comprising the base sequence according to 4 and a polynucleotide obtained by binding two deoxythymidylates to the 3 ′ end of the RNA A polynucleotide; and (iii) a polynucleotide obtained by binding two deoxythymidylic acids to the RNA comprising the base sequence set forth in SEQ ID NO: 5 in the Sequence Listing and the 3 ′ end of the RNA, and the base sequence set forth in SEQ ID NO: 6 A double-stranded polynucleotide comprising: a polynucleotide comprising two RNAs bonded with two deoxythymidylic acids at the 3 ′ end of the RNA.
[0022]
It is also considered that the inhibition of telomerase can be achieved in one aspect by a polynucleotide that can form a duplex containing the RNA of the present invention and having a hairpin structure. An RNA according to the present invention, an RNA having a base sequence complementary to the RNA, and a polynucleotide formed by linking these two RNAs and combining the polynucleotides forming a loop structure, Polynucleotides having RNA interference effects are also encompassed within the scope of the present invention. Such a polynucleotide preferably binds to the 3 ′ end of the RNA according to the present invention and the 5 ′ end of the polynucleotide forming the loop structure, and is complementary to the 3 ′ end of the polynucleotide forming the loop structure and the RNA. It is desirable that the polynucleotide be bound to the 5 ′ end of RNA consisting of a typical base sequence. Alternatively, the 5 ′ end of the RNA according to the present invention and the 3 ′ end of the polynucleotide forming the loop structure are combined, and the 5 ′ end of the polynucleotide forming the loop structure and the base sequence complementary to the RNA It may be a polynucleotide in which the 3 ′ end of the RNA is bound. A polynucleotide that forms a loop structure means a polynucleotide that exists between two RNAs and that can link both RNAs and that itself forms a loop structure. It is better to design with reference to the sequence described in Non-Patent Document 8. Preferably, it consists of 4 to 23 nucleotides, more preferably 4 to 8 nucleotides. For example, sequences such as TTCAAGAGA (Ambion or Oligoengine), AACGTT, TTAA, CAAGCTTC and the like can be mentioned. Formation of a duplex having a hairpin structure can be performed by annealing a sense RNA-derived portion and an antisense RNA-derived portion by a conventional method.
[0023]
More specific examples of such polynucleotides include polynucleotides selected from the following group: (i) RNA consisting of the base sequence set forth in SEQ ID NO: 1 in the sequence listing, and a polynucleotide consisting of 4 to 23 nucleotides. A polynucleotide obtained by binding a nucleotide and RNA consisting of the base sequence shown in SEQ ID NO: 2 and comprising the 3 ′ end of RNA consisting of the base sequence shown in SEQ ID NO: 1 in the sequence listing and 4 to 23 nucleotides A 5 'end of a polynucleotide consisting of 4 to 23 nucleotides and a 5' end of an RNA consisting of the base sequence set forth in SEQ ID NO: 2 Nucleotides; (ii) RNA consisting of the base sequence set forth in SEQ ID NO: 1 in the sequence listing, consisting of 4 to 23 nucleotides A polynucleotide obtained by binding a polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 2 and RNA having the nucleotide sequence set forth in SEQ ID NO: 1 in the sequence listing and 4-23 nucleotides. To the 3 ′ end of the polynucleotide consisting of 4 to 23 nucleotides, and to the 3 ′ end of the RNA consisting of the nucleotide sequence of SEQ ID NO: 2 (Iii) An RNA comprising the nucleotide sequence set forth in SEQ ID NO: 3 in the Sequence Listing, a polynucleotide comprising 4 to 23 nucleotides, and an RNA comprising the nucleotide sequence set forth in SEQ ID NO: 4 A polynucleotide comprising 3 ′ end of RNA consisting of the nucleotide sequence set forth in SEQ ID NO: 3 in the sequence listing and 4 to 23 nucleotides. The 5 'end of the polynucleotide comprising rhotide was bound, and the 3' end of the polynucleotide comprising 4 to 23 nucleotides and the 5 'end of the RNA comprising the base sequence described in SEQ ID NO: 4 were bound. (Iv) a polynucleotide formed by binding an RNA comprising the nucleotide sequence set forth in SEQ ID NO: 3 in the sequence listing, a polynucleotide comprising 4 to 23 nucleotides, and an RNA comprising the nucleotide sequence set forth in SEQ ID NO: 4. A 5 'end of an RNA comprising the nucleotide sequence set forth in SEQ ID NO: 3 in the Sequence Listing and a 3' end of a polynucleotide comprising 4 to 23 nucleotides, and further 4 to 23 nucleotides. The 5 ′ end of a polynucleotide comprising the nucleotides of 3 and the 3 ′ end of an RNA comprising the base sequence set forth in SEQ ID NO: 4 were bound. (V) a polynucleotide formed by binding of RNA comprising the nucleotide sequence set forth in SEQ ID NO: 5 in the sequence listing, polynucleotide comprising 4 to 23 nucleotides, and RNA comprising the nucleotide sequence set forth in SEQ ID NO: 6. A 3 'end of an RNA comprising the nucleotide sequence set forth in SEQ ID NO: 5 in the Sequence Listing and a 5' end of a polynucleotide comprising 4 to 23 nucleotides, and further 4 to 23 nucleotides A polynucleotide in which the 3 ′ end of a polynucleotide comprising the nucleotides of the above and the 5 ′ end of an RNA comprising the base sequence set forth in SEQ ID NO: 6 are linked; and (vi) the base sequence set forth in SEQ ID NO: 5 in the sequence listing RNA, a polynucleotide consisting of 4 to 23 nucleotides, and an RNA consisting of the base sequence described in SEQ ID NO: 6 bind A 5 ′ end of RNA consisting of the base sequence set forth in SEQ ID NO: 5 in the sequence listing and a 3 ′ end of a polynucleotide consisting of 4 to 23 nucleotides, and four more Or a polynucleotide in which the 5 ′ end of a polynucleotide comprising 23 nucleotides and the 3 ′ end of an RNA comprising the nucleotide sequence set forth in SEQ ID NO: 6 are linked.
[0024]
The RNA, polynucleotide and double-stranded polynucleotide according to the present invention are not limited to those described above, and have one to several base additions, deletions, substitutions, etc. that do not impair their functions. , Other general modifications may be used. Means for introducing base addition, deletion, substitution, etc. are known per se, and are carried out, for example, according to the methods described in the literature (Non-patent Documents 10, 11 and 12) or by modifying those methods. It is possible to use Ulmer's technology (Non-patent Document 13).
[0025]
Inhibition of telomerase can be achieved by using at least one of the double-stranded polynucleotide and polynucleotide according to the present invention. For example, it can be carried out by introducing them into cells. The introduction into the cell can be performed by a known technique, but transfection by the lipofection method is preferable from the viewpoint of introduction efficiency, reproducibility, simplicity, and the like. The lipid vesicles called liposomes used for lipofection are preferably cationic. Examples of the cationic liposome include Lipofectamine, Lipofectamine 2000 (Invitrogen), and Oligofectamine (Invitrogen). Alternatively, commercially available reagents such as TransMessenger (QIAGEN), siRNA transfection kit jetSI (Ambion), and Gene Silencer siRNA Transfection Reagent (Gene Therapy Systems) are commercially available. You can also.
[0026]
Inhibition of telomerase can also be achieved by transcribing a polynucleotide or RNA according to the present invention from DNA in a cell. That is, a DNA whose transcript shows inhibition of telomerase is also included in the present invention. Here, the transcript means RNA produced by transcription of the DNA of the present invention present in the coding strand in a cell. The DNA may be any DNA that encodes the polynucleotide or RNA according to the present invention, and is designed so that the transcription start point (so-called +1 site) is a purine base (G or A). It may be used as a double-stranded DNA comprising the present DNA and its complementary strand, or only the complementary strand of the present DNA can be used as a template DNA.
[0027]
Intracellular transcription of the polynucleotide or RNA according to the present invention is performed by inserting the DNA according to the present invention into a vector and transfecting the obtained vector into the cell by a conventional method (Non-patent Document 10 etc.). Can be achieved.
[0028]
Vector DNA is appropriately selected depending on the type of cells to be introduced. The vector DNA may be one in which a part of the DNA other than the part necessary for growth is missing in addition to the one extracted from naturally occurring ones. For example, vectors derived from chromosomes, episomes and viruses such as bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosome elements, such as baculovirus, papovavirus, SV40, vaccinia virus, adenovirus, Examples include vectors derived from viruses such as fowlpox virus, pseudorabies virus and retrovirus, and vectors in combination thereof, such as vectors derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. A recombinant vector is composed of a gene sequence carrying a target gene sequence and information on replication and control, such as a promoter, a ribosome binding site, a terminator, a signal sequence, an enhancer, etc., and these are combined by a method known per se. Make it. A method known per se can be applied as a method for incorporating a target gene sequence into vector DNA. For example, a method is used in which an appropriate restriction enzyme is selected and treated to cleave DNA at a specific site, and then mixed with DNA used as a similarly treated vector and religated by ligase. Alternatively, a desired recombinant vector can also be obtained by ligating a suitable linker to the gene sequence of interest and inserting it into a multicloning site of a vector suitable for the purpose.
[0029]
More specifically, the DNA that becomes the coding strand of the polynucleotide according to the present invention is, for example, DNA selected from the following group, and the transcript has an RNA interference effect on the human telomerase reverse transcriptase gene. (I) DNA consisting of the base sequence shown in SEQ ID NO: 7 in the sequence listing, DNA consisting of 4 to 23 nucleotides, and DNA consisting of the base sequence shown in SEQ ID NO: 8 DNA which is bound, 3 'end of DNA consisting of the base sequence described in SEQ ID NO: 7 in the sequence listing and 5' end of DNA consisting of 4 to 23 nucleotides, and further 4 Or a DNA in which the 3 ′ end of DNA consisting of 23 nucleotides and the 5 ′ end of DNA consisting of the base sequence set forth in SEQ ID NO: 8 are linked; (ii) Sequence Listing DNA comprising the nucleotide sequence set forth in SEQ ID NO: 7, DNA comprising 4 to 23 nucleotides, and DNA comprising the nucleotide sequence set forth in SEQ ID NO: 8, wherein The 5 ′ end of the DNA consisting of the base sequence described in 7 is linked to the 3 ′ end of the DNA consisting of 4 to 23 nucleotides, and the 5 ′ end of the DNA consisting of 4 to 23 nucleotides and the sequence DNA bound to the 3 ′ end of DNA consisting of the base sequence shown in No. 8; (iii) DNA consisting of the base sequence shown in SEQ ID No. 9 of the sequence listing, DNA consisting of 4 to 23 nucleotides and sequence DNA comprising a DNA comprising the base sequence described in No. 10 and having no 3 ′ ends and 4 DNAs comprising the base sequence shown in SEQ ID No. 9 in the sequence listing It binds to the 5 ′ end of DNA consisting of 23 nucleotides, and further binds to the 3 ′ end of DNA consisting of 4 to 23 nucleotides and the 5 ′ end of DNA consisting of the base sequence described in SEQ ID NO: 10. (Iv) DNA consisting of a DNA consisting of the base sequence set forth in SEQ ID NO: 9 in the sequence listing, a DNA consisting of 4 to 23 nucleotides, and a DNA consisting of the base sequence set forth in SEQ ID NO: 10 And the 5 ′ end of the DNA consisting of the base sequence shown in SEQ ID NO: 9 of the Sequence Listing is linked to the 3 ′ end of the DNA consisting of 4 to 23 nucleotides, and further from 4 to 23 nucleotides. DNA in which the 5 ′ end of the DNA and the 3 ′ end of the DNA consisting of the base sequence set forth in SEQ ID NO: 10 are linked; (v) the base sequence set forth in SEQ ID NO: 11 in the sequence listing A DNA comprising 4 to 23 nucleotides of DNA and a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 12, wherein the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 11 in the sequence listing A DNA comprising a 3 'end and a 5' end of DNA consisting of 4 to 23 nucleotides, and further comprising a 3 'end of DNA consisting of 4 to 23 nucleotides and the nucleotide sequence set forth in SEQ ID NO: 12. And (vi) DNA consisting of the base sequence set forth in SEQ ID NO: 11 in the sequence listing, DNA consisting of 4 to 23 nucleotides, and the base sequence set forth in SEQ ID NO: 12. A DNA obtained by binding DNA, the 5 ′ end of the DNA having the base sequence set forth in SEQ ID NO: 11 in the sequence listing and 4 to 23 nucleosides. Becomes' combines the terminal further four to 23 of DNA 5 consisting of the nucleotide 3 'of the DNA from de third DNA consisting terminus nucleotide sequence set forth in SEQ ID NO: 12' terminus is bound DNA.
[0030]
When the DNA serving as the coding strand of the polynucleotide according to the present invention is incorporated into a vector of a type that is transcribed by RNA polymerase III, such as U6 or H1, 5 bases at the 3 ′ end on the side to be transcribed in order to terminate transcription It is preferable to bind poly (T) of 6 bases. Furthermore, depending on the vector, it is preferable to use a further base sequence, for example, a base sequence such as GGAA, after poly (T).
[0031]
Inhibition of telomerase using DNA that is the coding strand of the polynucleotide according to the present invention can be achieved by introducing the DNA into cells. About introduction | transduction of this DNA to the cell, it can carry out with a pSHAG-1 vector system with reference to a previous report (nonpatent literature 8), for example. Alternatively, pSilencer series (Ambion), pSUPER (OligoEngine), retrovirus pSUPER-Retro (OligoEngine), and the like can be used, but are not limited thereto.
[0032]
As a DNA encoding the RNA according to the present invention, for example, a DNA having the base sequence described in any one of SEQ ID NOs: 7 to 12 in the sequence listing can be preferably exemplified.
[0033]
Inhibition of telomerase using DNA encoding RNA according to the present invention is carried out by dividing a vector into which the DNA is inserted and a vector into which a DNA encoding an RNA having a base sequence complementary to the base sequence of the RNA is inserted. We believe that this can be achieved by two RNAs that are transcribed in the cell by transfection into the cell. Alternatively, a similar effect can be obtained by using a vector in which a DNA encoding the RNA according to the present invention and a DNA encoding an RNA comprising a base sequence complementary to the base sequence of the RNA are inserted.
[0034]
Thus, the above DNA and a vector containing the DNA can also be used for inhibition of telomerase, and these are also included in the scope of the present invention.
[0035]
Since the double-stranded polynucleotide and polynucleotide according to the present invention can destroy hTERT mRNA by RNA interference, in the present invention, at least one of the above-mentioned double-stranded polynucleotide, polynucleotide, DNA and vector is used. It is possible to provide a telomerase reverse transcriptase gene expression inhibitor comprising using a telomerase reverse transcriptase gene expression inhibitor and at least one of them.
[0036]
In the present invention, a telomerase inhibitor comprising at least one of the above double-stranded polynucleotide, polynucleotide, DNA and vector, and a telomerase inhibition method comprising using at least one of them Can be provided.
[0037]
Furthermore, the double-stranded polynucleotide, polynucleotide, DNA and vector according to the present invention are medicaments based on inhibiting telomerase, preferably antitumor agents, more preferably active ingredients of medicaments for the treatment of malignant tumors. Useful as. Confirmation of the telomerase inhibitory action includes, for example, a trap method (TRAP method: Non-Patent Document 14), a direct telomerase assay (Direct Telomerase assay: Non-Patent Document 15), a stretch PCR assay (Stretch PCR assay: Non-Patent Document 16), and hybridization. It can be performed by a method such as a protection assay (Hybridization protection assay: Non-patent document 17), a TRE assay (Non-patent document 18), a rapid direct telomerase assay (Non-patent document 19), or the like. Alternatively, it can be confirmed according to the method of Paddyson et al. (Non-Patent Document 8) or the method specifically described in the examples of the present specification.
[0038]
The medicament according to the present invention may be a medicament containing an effective amount of at least one of the above-mentioned double-stranded polynucleotide, polynucleotide, DNA and vector according to the present invention. It is preferable to produce a pharmaceutical composition using the above pharmaceutical carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, and mixtures thereof.
[0039]
The pharmaceutical composition according to the present invention can be used as an agent for preventing and / or treating a disease based on the action of telomerase. Moreover, it can use for the prevention method and / or treatment method of the said disease. Examples of diseases based on the action of telomerase include cancer diseases.
[0040]
Since the expression of telomerase is confirmed in the majority of tumors regardless of the type of tumor, the pharmaceutical composition according to the present invention is considered to be effective in inhibiting the growth of various types of cancer cells. Therefore, when the pharmaceutical composition according to the present invention is used for cancer diseases, the type of tumor to be treated is not particularly limited, and can be applied to either solid tumors or non-solid tumors. The type of solid tumor or non-solid tumor is not particularly limited, and can be applied to all types of tumors expressing telomerase. For example, stomach cancer, esophageal cancer, colon cancer, small intestine cancer, duodenal cancer, lung cancer, liver cancer, gallbladder cancer, pancreatic cancer, kidney cancer, bladder cancer, oral cancer, bone cancer, skin cancer, breast cancer, uterine cancer, prostate cancer, Examples include solid tumors such as brain tumors and neuroblastomas, and non-solid tumors such as leukemias and malignant lymphomas, but the application target of the pharmaceutical composition according to the present invention is not limited to these diseases.
[0041]
On the other hand, telomerase is hardly expressed in normal somatic cells except germ cells. Furthermore, since siRNA and shRNA act only on mRNA, it is considered that genomic genes are not affected at all. From these, it is considered that the medicament and the pharmaceutical composition according to the present invention have few side effects, and are also useful from this viewpoint.
[0042]
The required dose range is not particularly limited, but the effectiveness of the double-stranded polynucleotide, polynucleotide, DNA and vector according to the present invention, dosage form, type of disease, nature of the subject (weight, age, medical condition and other) It is desirable to select as appropriate based on the use of medicine, etc.) and the judgment of the doctor in charge. Specifically, an appropriate dose is preferably in the range of, for example, 0.1 μg to 100 μg per kg of the subject's body weight. However, these dose modifications can be made using general routine experimentation for optimization well known in the art. The above dose can be divided into 1 to 4 times per day, and may be administered intermittently at a rate of once every several days or weeks.
[0043]
What is necessary is just to select a prescription suitable for a dosage form, and what is necessary is just to use this prescription well-known to those skilled in the art. Also, when formulated, these may be used alone or in combination with other compounds or medicaments required for treatment. For example, you may mix | blend the active ingredient of the other telomerase inhibitor or the medicine for antitumors.
[0044]
The double-stranded polynucleotide, polynucleotide, DNA, vector and pharmaceutical composition according to the present invention can be preferably used as a gene therapy agent. In this case, either a non-viral transfection method in which at least one of the double-stranded polynucleotide, polynucleotide and DNA according to the present invention is directly administered by injection or a transfection method using a viral vector is applied. can do.
[0045]
In the non-viral transfection method, in addition to a method in which at least one of the double-stranded polynucleotide, polynucleotide and DNA according to the present invention is directly administered by injection, these are applied to phospholipid vesicles such as liposomes. A method of encapsulating and administering the liposomes is recommended. As the liposome, it is more preferable to use a cationic liposome.
[0046]
In the transfection method using a viral vector, the vector to be used for transfection by incorporating the double-stranded polynucleotide, polynucleotide, or DNA according to the present invention is preferably a retroviral vector, an adenoviral vector, an adeno-associated virus. Examples include vectors, DNA virus vectors such as vaccinia virus vectors, and RNA virus vectors. Efficient administration is possible by using these viral vectors. Furthermore, also in the transfection method using a viral vector, a method of encapsulating the vector in a liposome and administering the liposome is recommended. As the liposome, it is more preferable to use a cationic liposome.
[0047]
The administration form can be selected from systemic administration or local administration. In this case, an appropriate dosage form is selected according to the disease, symptoms and the like. For example, in addition to normal intravenous administration and intraarterial administration, administration can also be performed subcutaneously, intradermally, intramuscularly, or the like. When used for cancer diseases, it is preferable to administer directly to the tumor.
[0048]
The pharmaceutical form can be selected according to the administration form, and when used as a gene therapy agent, it is generally preferable to prepare it as an injection, a drip infusion, or a liposome preparation. In addition, inclusions such as cyclodextrins, solutions, suspensions, fat emulsions, powders, ointments, creams, transdermal absorption agents, transmucosal absorption pills, tablets, capsules, granules, fine granules Preparation of suppositories, suppositories, inhalants, eye drops, ear drops and the like is also possible. However, the pharmaceutical form according to the present invention is not limited thereto.
[0049]
In formulation, an appropriate formulation additive can be used according to the form, and formulation can be made according to a conventional method.
[0050]
Liposomeization, for example, to a solution in which phospholipid is dissolved in an organic solvent (such as chloroform), after adding a solution in which the target substance is dissolved in the solvent, the solvent is distilled off, and a phosphate buffer is added thereto, After shaking, sonication and centrifugation, the supernatant can be filtered and collected.
[0051]
For cyclodextrin inclusion, for example, a solution in which cyclodextrin is heated and dissolved in water or the like is added to a solution in which the target substance is dissolved in a solvent, and then cooled and the deposited precipitate is filtered and sterilized and dried. Can be performed. At this time, the cyclodextrin used may be appropriately selected from cyclodextrins (α, β, γ types) having different pore diameters according to the size of the substance.
[0052]
Solutions for injection can be prepared using a carrier comprising a salt solution, a glucose solution, or a mixture of saline and glucose solution.
[0053]
The suspension can be produced using water, sugars such as sucrose, sorbitol and fructose, glycols such as polyethylene glycol, and oils.
[0054]
For fat emulsification, for example, the desired substance, oil components (vegetable oils such as soybean oil, sesame oil, olive oil, etc., MCT, etc.), emulsifiers (phospholipids, etc.), etc. are mixed and heated to form a solution, and then the required amount of water And emulsifying and homogenizing using an emulsifier (homogenizer, such as a high-pressure jet type or an ultrasonic type). It can also be lyophilized. When emulsifying fat, an emulsification aid may be added. Examples of the emulsification aid include glycerin and saccharides (eg, glucose, sorbitol, fructose, etc.).
[0055]
About powders, pills, capsules, tablets, granules, fine granules, drops, suppositories, inhalants, eye drops, ear drops, ointments, creams, transdermal absorption agents, transmucosal absorption agents, etc. Can also be prepared by commonly used methods.
[0056]
The present invention further provides a method for identifying a double-stranded polynucleotide that inhibits telomerase. The identification method according to the present invention uses a cell that does not express the hTERT gene but expresses the hTR gene (SEQ ID NO: 16), or a cell that does not express both the hTERT gene and the hTR gene. One feature is that a double-stranded polynucleotide as a test substance is transfected together with an hTERT gene expression vector and an hTR gene expression vector, and then telomerase activity is measured. Examples of cells that do not express the hTERT gene but express the hTR gene include human osteosarcoma-derived Saos-2 cells, but the cells that can be used in the identification method according to the present invention are limited to this. There is no. When cells that do not express the hTERT gene but express the hTR gene are used, the hTR gene expression vector need not be introduced, but preferably the hTR gene expression vector is also introduced. The introduction ratio of the hTERT gene expression vector to the hTR gene expression vector is preferably 1: 0.01 to 1:10. The introduction ratio varies depending on the cell to be used, and a more appropriate introduction ratio can be determined by experiment and used. Transfection can be performed by a conventional transfection method (Non-patent Document 10, etc.), but preferably by lipofection. Lipofection can be performed by using a commercially available reagent such as lipofectin, lipofectamine, cellfectin, DMRIE-C or superfect, but it is preferable to use cationic liposomes. Examples of the cationic liposome include lipofectamine, lipofectamine 2000, and oligofectamine.
[0057]
Duplicate test substance with hTERT gene expression vector and hTR gene expression vector in cells that do not express hTERT gene but express hTR gene, or cells that do not express both hTERT gene and hTR gene In such a measurement system constructed by transfection of a strand polynucleotide, only the cell into which the hTERT gene has been introduced exhibits telomerase activity, and it is very likely that the double-stranded polynucleotide has also been introduced into the cell. Therefore, the effect of the double-stranded polynucleotide can be evaluated without much influence of transfection efficiency. The hTR gene and hTERT gene expression vectors may be prepared by inserting hTERT cDNA (SEQ ID NO: 13) and hTR cDNA (SEQ ID NO: 16) into a commonly used mammalian gene expression vector, respectively.
[0058]
A double-stranded polynucleotide as a test substance is selected based on the nucleotide sequence of the hTERT gene, a region containing a sequence specific to RNA having an RNA interference effect, designed from the sequence of the region, and known nucleotide synthesis It can be produced by a method. The sequence of the hTERT gene is the sequence described in SEQ ID NO: 13 in the sequence listing. Sequences unique to RNA having RNA interference effects are already widely known. For example, in the region starting with AA or CA, the GC content is 30% to 70%, preferably 50%. Further, a sequence in which the bias of each base or the sequence in which G or C is continuous is as small as possible is preferable.
[0059]
In the identification method according to the present invention, a desired double-stranded polynucleotide is obtained by selecting a double-stranded polynucleotide having a reduced telomerase activity compared to when the double-stranded polynucleotide is not transfected. can do. The telomerase activity is measured, for example, by the trap method (Non-patent document 14), direct telomerase assay (Non-patent document 15), stretch PCR assay (Non-patent document 16), hybridization protection assay (Non-patent document 17), TRE assay ( Non-patent document 18), rapid direct telomerase assay (non-patent document 19) and the like.
[0060]
Any of the double-stranded polynucleotides, polynucleotides, DNAs, and vectors according to the present invention can be used alone as a reagent. For example, it can be used as a reagent for inhibiting telomerase. The reagent is useful for basic research on the mechanism of cell proliferation and aging involving telomerase, diseases based on the action of telomerase, and the like. When these are reagents, they may contain substances such as buffers, salts, stabilizers, and / or preservatives. In formulating, a known formulating means corresponding to each property may be introduced. The present invention also provides a reagent kit comprising one or more containers filled with one or more of these. These reagents and reagent kits can be used for diagnosis of diseases based on the action of telomerase. For example, if cell death is observed when cancer cells are collected from a cancer patient, cultured and repeatedly treated with a kit containing a double-stranded polynucleotide, the telomerase inhibitor is determined to be an effective patient. Can do. They can also be used as positive controls in the identification method according to the invention.
[0061]
【Example】
EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples.
[0062]
[Example 1]
The double-stranded polynucleotide was designed based on the base sequence of the hTERT gene (SEQ ID NO: 13). First, the base sequence of the sense RNA was designed by selecting a sequence having as few biases as possible from the portion starting with AA or CA in the base sequence of the gene and having a sequence in which G or C is continuous. Moreover, antisense RNA which consists of a base sequence complementary to the base sequence of sense RNA was designed. Then, for the purpose of protecting the double-stranded polynucleotide from nuclease, a sense polynucleotide and an antisense polynucleotide in which an overhang sequence composed of two deoxythymidylates is bound to the 3 ′ ends of the sense RNA and the antisense RNA, respectively. Nucleotides were designed. Both the designed sense and antisense polynucleotides were synthesized by an RNA synthesizer.
[0063]
A double-stranded polynucleotide was prepared by annealing a sense polynucleotide and an antisense polynucleotide. First, each sample was diluted to 50 μM (50 pmol / μl), and then 30 μl of each sample was mixed with 15 μl of 5 × annealing buffer (500 mM potassium acetate, 150 mM HEPES-KOH pH 7.4, 10 mM magnesium acetate) (total amount 75 μl). Subsequently, the mixed solution was heated at 90 ° C. for 1 minute, and allowed to stand at 37 ° C. for 60 minutes after centrifugation. By this operation, the polynucleotide formed a double strand, and the final concentration was 20 μM (20 pmol / μl).
[0064]
An example of the prepared double-stranded polynucleotide is shown in FIG. TERT02 is a double-stranded polynucleotide comprising a polynucleotide in which two deoxythymidylic acids are bound to each 3 ′ end of two RNAs comprising the respective base sequences described in SEQ ID NOs: 1 and 2. TERT07 is a double-stranded polynucleotide comprising a polynucleotide in which two deoxythymidylic acids are bound to each 3 ′ end of two RNAs comprising the respective base sequences described in SEQ ID NOs: 3 and 4. TERT08 is a double-stranded polynucleotide consisting of a polynucleotide in which two deoxythymidylic acids are bound to each 3 ′ end of two RNAs consisting of the respective base sequences described in SEQ ID NOs: 5 and 6. RNAs comprising the nucleotide sequences set forth in SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5 are the 1042-1060th, 1789-1807th and 1812-1830th portions of the hTERT cDNA sequence (SEQ ID NO: 13), respectively. RNA designed from sequence. The RNA consisting of the base sequences shown in SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 is an RNA consisting of a base sequence complementary to the base sequences shown in SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5, respectively. is there.
[0065]
[Example 2]
In order to select a double-stranded polynucleotide exhibiting a telomerase inhibitory action from the double-stranded polynucleotide prepared in Example 1, the human osteosarcoma-derived Saos expressing only the hTR gene but not the hTERT gene -2 cells were used to transfect a double-stranded polynucleotide together with an hTERT gene expression vector and an hTR gene expression vector, and a measurement system for measuring telomerase activity was constructed. Using this measurement system, a double-stranded polynucleotide exhibiting a telomerase inhibitory action was selected.
[0066]
First, Saos-2 cells were 2 × 10 ⁵ The cell / ml was prepared, seeded at 0.5 ml per well in a 24-well plate, and cultured at 37 ° C. overnight (about 12 hours). The duplex polynucleotide along with hTERT gene expression vector and hTR gene expression vector was then mixed with 2.5 μl per well of Lipofectamine 2000 (Invitrogen) in 0.1 ml of serum-free medium to transfect the cells did. Cells were trypsinized 24 hours after transfection and collected by washing once with phosphate buffered saline (PBS). As a negative target, instead of the double-stranded polynucleotide, a scrambled double-stranded polynucleotide having no homology with a known gene (3 ′ of each of the two RNAs comprising the respective base sequences described in SEQ ID NOs: 14 and 15) The cells were treated in the same manner using a double-stranded polynucleotide comprising a polynucleotide having two deoxythymidylic acids bound to the ends.
[0067]
Telomerase activity in the collected cells was measured using TRAPze XL Kit (Oncor) according to the instruction manual of the kit. First, the cells were suspended in 120 μl of 1 × CHAPS lysis buffer attached to the kit, incubated on ice for 20 minutes, centrifuged at 12,000 rpm for 20 minutes at 4 ° C., and the supernatant was collected. After quantifying the protein concentration in the supernatant, it was added to the reaction solution at 200 ng per reaction, and an oligo DNA extension reaction by telomerase was carried out at 30 ° C. for 30 minutes. Then, PCR reaction was performed with the primer pair contained in the reaction solution, the telomerase reaction product was amplified, the ratio with the internal standard was calculated, and the intensity of telomerase activity was measured. Detection was carried out by measuring the fluorescence intensity of fluorescein (index of telomerase reaction product) labeled with the primer and sulforhodamine (index of internal standard) using a spectrofluorometer.
[0068]
The above study was performed using 0.5 μg / well hTERT gene expression vector, 0.54 μg / well hTR gene expression vector, and 20 pmol / well or 2 pmol / well double-stranded polynucleotide. Among the strand polynucleotides, TERT02, TERT07 and TERT08 showed a telomerase inhibitory effect (FIG. 2).
[0069]
Furthermore, for TERT02 and TERT08, a 0.125 μg / well hTERT gene expression vector and a 0.75 μg / well hTR gene expression vector were used, and the double-stranded polynucleotide concentration ranged from 20 pmol / well to 0.2 pmol / well. In the same manner, it was revealed that telomerase was inhibited depending on the double-stranded polynucleotide concentration (FIG. 3).
[0070]
On the other hand, in the sample in which the enzyme activity of telomerase was deactivated by heating, no telomerase activity was detected even without adding double-stranded polynucleotide. It was confirmed that this was reflected (FIGS. 2 and 3).
[0071]
【The invention's effect】
According to the present invention, inhibition of telomerase can be achieved by an RNA interference effect on human telomerase reverse transcriptase gene.
[0072]
The present invention can specifically inhibit the expression of the human telomerase reverse transcriptase gene at a low concentration and with a high probability as compared with other gene expression inhibition methods such as the antisense method and the ribozyme method, thereby inhibiting telomerase. Can do. Moreover, since the double-stranded polynucleotide and the polynucleotide according to the present invention are short-chain polynucleotides, they can be synthesized easily and inexpensively.
[0073]
According to the present invention, it is possible to prevent and / or treat diseases based on the action of telomerase. Since telomerase is expressed in most cancer cells regardless of the type of cancer and is hardly expressed in normal somatic cells other than germ cells, for example, the present invention prevents and / or treats various types of cancer diseases. It is considered effective.
[0074]
As described above, the present invention is useful for basic research on the mechanism of cell proliferation and senescence involving telomerase and diseases based on the action of telomerase. Furthermore, it is very useful for the prevention and / or treatment of diseases based on the action of telomerase, such as cancer diseases.
[0075]
[Sequence Listing Free Text]
SEQ ID NO: 1: RNA designed based on the sequence of human telomerase reverse transcriptase gene (SEQ ID NO: 13).
SEQ ID NO: 2: Designed RNA consisting of a base sequence complementary to the base sequence described in SEQ ID NO: 1.
SEQ ID NO: 3: RNA designed based on the sequence of human telomerase reverse transcriptase gene (SEQ ID NO: 13)
SEQ ID NO: 4: Designed RNA consisting of a base sequence complementary to the base sequence described in SEQ ID NO: 3.
SEQ ID NO: 5: RNA designed based on the sequence of human telomerase reverse transcriptase gene (SEQ ID NO: 13)
SEQ ID NO: 6: Designed RNA consisting of a base sequence complementary to the base sequence described in SEQ ID NO: 5.
SEQ ID NO: 7: DNA designed based on the sequence of human telomerase reverse transcriptase gene (SEQ ID NO: 13).
SEQ ID NO: 8: designed DNA comprising a base sequence complementary to the base sequence described in SEQ ID NO: 7
SEQ ID NO: 9: DNA designed based on the sequence of human telomerase reverse transcriptase gene (SEQ ID NO: 13)
SEQ ID NO: 10: Designed DNA consisting of a base sequence complementary to the base sequence described in SEQ ID NO: 9
SEQ ID NO: 11: DNA designed based on the sequence of human telomerase reverse transcriptase gene (SEQ ID NO: 13)
SEQ ID NO: 12: designed DNA comprising a base sequence complementary to the base sequence described in SEQ ID NO: 11
SEQ ID NO: 14: Designed RNA consisting of a base sequence that is not homologous to all genes.
SEQ ID NO: 15: designed RNA comprising a base sequence complementary to the base sequence described in SEQ ID NO: 14
[0076]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows the sequence of a double-stranded polynucleotide according to the present invention.
FIG. 2 shows that three types of double-stranded polynucleotides, TERT02, TERT07 and TERT08, inhibited telomerase in Saos-2 cells transfected with the double-stranded polynucleotide together with hTERT gene and hTR gene, respectively. FIG. Telomerase activity is the ratio of the fluorescence intensity of fluorescein (subtracting the background), which is an index of the telomere sequence added by telomerase, to the fluorescence intensity of sulforhodamine, which is an internal standard in telomerase activity measurement (subtracting the background) (Δ FL / ΔR). In the figure, DNA (-) and RNA (-) indicate telomerase activity when no hTERT gene and double-stranded polynucleotide are added, respectively. RNA (-) Heat indicates telomerase activity when a sample is heated to inactivate the enzyme activity of telomerase without adding a double-stranded polynucleotide. A scramble is a double-stranded polynucleotide that has no homology with a known gene and was used as a negative target.
FIG. 3 shows the concentration of telomerase in double-stranded polynucleotides in Saos-2 cells in which two double-stranded polynucleotides, TERT02 and TERT08, were transfected with the double-stranded polynucleotide together with hTERT gene and hTR gene, respectively. It is a figure which shows having inhibited dependently. Telomerase activity is the ratio of the fluorescence intensity of fluorescein (subtracting the background), which is an index of the telomere sequence added by telomerase, to the fluorescence intensity of sulforhodamine, which is an internal standard in telomerase activity measurement (subtracting the background) (Δ FL / ΔR). In the figure, RNA (-) indicates telomerase activity when no hTERT gene and double-stranded polynucleotide are added. RNA (-) Heat indicates telomerase activity when a sample is heated to inactivate the enzyme activity of telomerase without adding a double-stranded polynucleotide. A scramble is a double-stranded polynucleotide that has no homology with a known gene and was used as a negative target.

Claims (27)

RNA comprising the base sequence set forth in any one of SEQ ID NOs: 1 to 6 in the Sequence Listing. A polynucleotide (polynucleotide A) obtained by binding one or several nucleotides to the RNA having the base sequence set forth in SEQ ID NO: 1 in the sequence listing at the 3 'end of the RNA, and comprising SEQ ID NO: in the sequence listing A double-stranded polynucleotide obtained by hybridization with a polynucleotide comprising 1 to several nucleotides bound to the RNA having the base sequence described in 2 at the 3 ′ end of the RNA is a human telomerase reverse transcriptase gene Polynucleotide A, which has an RNA interference effect on A polynucleotide (polynucleotide B) obtained by binding one or several nucleotides to the RNA having the base sequence set forth in SEQ ID NO: 2 in the sequence listing at the 3 ′ end of the RNA, and the sequence number in the sequence listing A double-stranded polynucleotide obtained by hybridizing an RNA comprising the nucleotide sequence of 1 with a polynucleotide obtained by binding one or several nucleotides to the 3 ′ end of the RNA is a human telomerase reverse transcriptase gene Polynucleotide B, which has an RNA interference effect on A polynucleotide (polynucleotide C) obtained by binding one or several nucleotides to the RNA having the base sequence set forth in SEQ ID NO: 3 in the sequence listing at the 3 ′ end of the RNA, and comprising SEQ ID NO: in the sequence listing A double-stranded polynucleotide obtained by hybridizing an RNA having the base sequence described in 4 with a polynucleotide obtained by binding one or several nucleotides to the 3 ′ end of the RNA is a human telomerase reverse transcriptase gene Polynucleotide C, which has an RNA interference effect on A polynucleotide (polynucleotide D) obtained by binding one or several nucleotides to the RNA having the base sequence set forth in SEQ ID NO: 4 in the sequence listing at the 3 ′ end of the RNA, and comprising SEQ ID NO: in the sequence listing A double-stranded polynucleotide obtained by hybridization with a polynucleotide comprising 1 to several nucleotides bonded to the RNA having the base sequence described in 3 at the 3 ′ end of the RNA is a human telomerase reverse transcriptase gene Polynucleotide D characterized by having an RNA interference effect on A polynucleotide (polynucleotide E) obtained by binding one or several nucleotides to the RNA having the base sequence set forth in SEQ ID NO: 5 in the sequence listing at the 3 'end of the RNA, and comprising SEQ ID NO: in the sequence listing A double-stranded polynucleotide obtained by hybridizing an RNA comprising the nucleotide sequence of claim 6 with a polynucleotide obtained by binding one or several nucleotides to the 3 ′ end of the RNA is a human telomerase reverse transcriptase gene Polynucleotide E, which has an RNA interference effect on A polynucleotide (polynucleotide F) obtained by binding one or several nucleotides to the RNA having the base sequence set forth in SEQ ID NO: 6 in the sequence listing at the 3 'end of the RNA, and comprising SEQ ID NO: in the sequence listing A double-stranded polynucleotide obtained by hybridizing an RNA having the base sequence of 5 to a polynucleotide obtained by binding one or several nucleotides to the 3 ′ end of the RNA is a human telomerase reverse transcriptase gene Polynucleotide F, which has an RNA interference effect on A polynucleotide obtained by binding two deoxythymidylates to the RNA having the nucleotide sequence set forth in any one of SEQ ID NOS: 1 to 6 in the sequence listing at the 3 'end of the RNA Any one double-stranded polynucleotide selected from the following group, wherein the double-stranded polynucleotide has an RNA interference effect on a human telomerase reverse transcriptase gene;
(I) a polynucleotide formed by binding one or several nucleotides to the RNA having the base sequence set forth in SEQ ID NO: 1 in the sequence listing at the 3 'end of the RNA and the base sequence set forth in SEQ ID NO: 2 A double-stranded polynucleotide comprising a polynucleotide formed by binding one or several nucleotides to the RNA at the 3 ′ end of the RNA;
(Ii) consisting of a polynucleotide obtained by binding one or several nucleotides to the RNA comprising the base sequence shown in SEQ ID NO: 3 in the sequence listing at the 3 ′ end of the RNA and the base sequence shown in SEQ ID NO: 4 A double-stranded polynucleotide comprising a polynucleotide formed by binding one or several nucleotides to the RNA at the 3 ′ end of the RNA;
And (iii) a polynucleotide obtained by binding one or several nucleotides to the RNA having the base sequence set forth in SEQ ID NO: 5 in the sequence listing at the 3 ′ end of the RNA and the base sequence set forth in SEQ ID NO: 6. A double-stranded polynucleotide comprising a polynucleotide obtained by binding one or several nucleotides to the RNA at the 3 ′ end of the RNA. Any one double-stranded polynucleotide selected from the group below;
(I) a polynucleotide obtained by binding two deoxythymidylic acids to the RNA having the base sequence set forth in SEQ ID NO: 1 in the sequence listing at the 3 'end of the RNA and the RNA consisting of the base sequence set forth in SEQ ID NO: 2 A double-stranded polynucleotide comprising a polynucleotide obtained by binding two deoxythymidylic acids to the 3 ′ end of the RNA;
(Ii) an RNA comprising a nucleotide sequence set forth in SEQ ID NO: 3 in the sequence listing and an RNA comprising a nucleotide sequence set forth in SEQ ID NO: 4 and a polynucleotide obtained by binding two deoxythymidylic acids to the 3 ′ end of the RNA; A double-stranded polynucleotide comprising a polynucleotide obtained by binding two deoxythymidylic acids to the 3 ′ end of the RNA;
And (iii) an RNA comprising a nucleotide sequence set forth in SEQ ID NO: 5 in the sequence listing and a polynucleotide comprising a base sequence set forth in SEQ ID NO: 6 and a polynucleotide obtained by binding two deoxythymidylic acids to the 3 ′ end of the RNA. A double-stranded polynucleotide comprising a polynucleotide obtained by binding two deoxythymidylic acids to the 3 ′ end of the RNA; Any one polynucleotide selected from the group below, wherein the polynucleotide has an RNA interference effect on a human telomerase reverse transcriptase gene;
(I) an RNA comprising the base sequence described in SEQ ID NO: 1 in the sequence listing, a polynucleotide forming a loop structure and a polynucleotide (polynucleotide G) formed by binding an RNA comprising the base sequence described in SEQ ID NO: 2 The 3 ′ end of the RNA comprising the base sequence set forth in SEQ ID NO: 1 of the Sequence Listing is linked to the 5 ′ end of the polynucleotide forming the loop structure, and further the 3 ′ end of the polynucleotide forming the loop structure And a polynucleotide G in which the 5 ′ end of RNA consisting of the nucleotide sequence set forth in SEQ ID NO: 2 is bound,
(Ii) an RNA comprising the base sequence described in SEQ ID NO: 1 in the sequence listing, a polynucleotide forming a loop structure, and a polynucleotide (polynucleotide H) formed by binding an RNA comprising the base sequence described in SEQ ID NO: 2 The 5 ′ end of the polynucleotide that forms the loop structure by binding the 5 ′ end of the RNA consisting of the base sequence shown in SEQ ID NO: 1 of the Sequence Listing to the 3 ′ end of the polynucleotide that forms the loop structure A polynucleotide H bound to the 3 ′ end of RNA consisting of the nucleotide sequence set forth in SEQ ID NO: 2,
(Iii) a polynucleotide (polynucleotide I) obtained by binding an RNA comprising the base sequence set forth in SEQ ID NO: 3 in the sequence listing, a polynucleotide forming a loop structure, and an RNA comprising the base sequence set forth in SEQ ID NO: 4 The 3 ′ end of the polynucleotide that forms the loop structure is formed by binding the 3 ′ end of the RNA consisting of the base sequence shown in SEQ ID NO: 3 of the Sequence Listing to the 5 ′ end of the polynucleotide that forms the loop structure. A polynucleotide I bound to the 5 ′ end of RNA consisting of the nucleotide sequence set forth in SEQ ID NO: 4,
(Iv) an RNA comprising the base sequence described in SEQ ID NO: 3 in the sequence listing, a polynucleotide forming a loop structure, and a polynucleotide (polynucleotide J) formed by binding an RNA comprising the base sequence described in SEQ ID NO: 4 The 5 ′ end of the RNA consisting of the base sequence shown in SEQ ID NO: 3 in the sequence listing and the 3 ′ end of the polynucleotide forming the loop structure are combined, and the 5 ′ end of the polynucleotide forming the loop structure. And polynucleotide J in which the 3 ′ end of RNA consisting of the nucleotide sequence set forth in SEQ ID NO: 4 is bound,
(V) a polynucleotide (polynucleotide K) formed by binding an RNA comprising the base sequence set forth in SEQ ID NO: 5 in the sequence listing, a polynucleotide forming a loop structure, and an RNA comprising the base sequence set forth in SEQ ID NO: 6 And the 3 ′ end of the polynucleotide that forms the loop structure by binding the 3 ′ end of the RNA consisting of the base sequence shown in SEQ ID NO: 5 of the sequence listing to the 5 ′ end of the polynucleotide that forms the loop structure. A polynucleotide K bound to the 5 ′ end of RNA consisting of the nucleotide sequence set forth in SEQ ID NO: 6,
And (vi) a polynucleotide formed by binding an RNA comprising the base sequence set forth in SEQ ID NO: 5 in the sequence listing, a polynucleotide forming a loop structure, and an RNA comprising the base sequence set forth in SEQ ID NO: 6 (polynucleotide L) The 5 ′ end of the RNA comprising the base sequence set forth in SEQ ID NO: 5 in the Sequence Listing is linked to the 3 ′ end of the polynucleotide forming the loop structure, and further 5 ′ of the polynucleotide forming the loop structure. A polynucleotide L in which an end and the 3 ′ end of RNA consisting of the nucleotide sequence set forth in SEQ ID NO: 6 are linked. Any one polynucleotide selected from the group below, wherein the polynucleotide has an RNA interference effect on a human telomerase reverse transcriptase gene;
(I) a polynucleotide formed by binding of RNA consisting of the base sequence shown in SEQ ID NO: 1 in the sequence listing, polynucleotide consisting of 4 to 23 nucleotides, and RNA consisting of the base sequence shown in SEQ ID NO: 2 Nucleotide M), the 3 ′ end of RNA consisting of the base sequence set forth in SEQ ID NO: 1 in the Sequence Listing is linked to the 5 ′ end of a polynucleotide consisting of 4 to 23 nucleotides, and further 4 to A polynucleotide M, wherein the 3 ′ end of a polynucleotide consisting of 23 nucleotides and the 5 ′ end of an RNA consisting of the base sequence set forth in SEQ ID NO: 2 are linked;
(Ii) a polynucleotide formed by binding of RNA consisting of the base sequence shown in SEQ ID NO: 1 in the sequence listing, polynucleotide consisting of 4 to 23 nucleotides, and RNA consisting of the base sequence shown in SEQ ID NO: 2 (polynucleotide) Nucleotide 5), the 5 ′ end of RNA consisting of the base sequence set forth in SEQ ID NO: 1 in the sequence listing and the 3 ′ end of a polynucleotide consisting of 4 to 23 nucleotides, A polynucleotide N in which the 5 ′ end of a polynucleotide consisting of 23 nucleotides and the 3 ′ end of an RNA consisting of the base sequence set forth in SEQ ID NO: 2 are linked;
(Iii) A polynucleotide comprising a combination of RNA comprising the nucleotide sequence set forth in SEQ ID NO: 3 in the sequence listing, polynucleotide comprising 4 to 23 nucleotides, and RNA comprising the nucleotide sequence set forth in SEQ ID NO: 4 Nucleotide 3), the 3 ′ end of RNA consisting of the base sequence set forth in SEQ ID NO: 3 in the sequence listing is linked to the 5 ′ end of a polynucleotide consisting of 4 to 23 nucleotides, and further 4 to A polynucleotide O in which the 3 ′ end of a polynucleotide consisting of 23 nucleotides and the 5 ′ end of an RNA consisting of the base sequence set forth in SEQ ID NO: 4 are linked;
(Iv) a polynucleotide formed by binding of RNA consisting of the base sequence shown in SEQ ID NO: 3 in the sequence listing, polynucleotide consisting of 4 to 23 nucleotides, and RNA consisting of the base sequence shown in SEQ ID NO: 4 Nucleotide P), wherein the 5 ′ end of the RNA comprising the base sequence set forth in SEQ ID NO: 3 in the sequence listing and the 3 ′ end of the polynucleotide comprising 4 to 23 nucleotides are combined, and further 4 to A polynucleotide P in which the 5 ′ end of a polynucleotide consisting of 23 nucleotides and the 3 ′ end of an RNA consisting of the base sequence set forth in SEQ ID NO: 4 are linked;
(V) a polynucleotide formed by binding of RNA consisting of the base sequence shown in SEQ ID NO: 5 in the sequence listing, polynucleotide consisting of 4 to 23 nucleotides, and RNA consisting of the base sequence shown in SEQ ID NO: 6 Nucleotide 3), the 3 ′ end of RNA consisting of the base sequence set forth in SEQ ID NO: 5 in the sequence listing and the 5 ′ end of a polynucleotide consisting of 4 to 23 nucleotides, A polynucleotide Q in which the 3 ′ end of a polynucleotide consisting of 23 nucleotides and the 5 ′ end of an RNA consisting of the base sequence set forth in SEQ ID NO: 6 are linked;
And (vi) a polynucleotide formed by binding an RNA comprising the base sequence set forth in SEQ ID NO: 5 in the sequence listing, a polynucleotide comprising 4 to 23 nucleotides, and an RNA comprising the base sequence set forth in SEQ ID NO: 6 ( Polynucleotide R), the 5 ′ end of RNA consisting of the base sequence set forth in SEQ ID NO: 5 in the sequence listing and the 3 ′ end of polynucleotide consisting of 4 to 23 nucleotides are bound, and 4 more Or a polynucleotide R in which the 5 ′ end of a polynucleotide comprising 23 nucleotides and the 3 ′ end of an RNA comprising the base sequence set forth in SEQ ID NO: 6 are linked. DNA comprising the base sequence set forth in any one of SEQ ID NOs: 7 to 12 in the Sequence Listing. Any one DNA selected from the following group, wherein the transcript has an RNA interference effect on a human telomerase reverse transcriptase gene;
(I) DNA (DNA1) obtained by binding DNA consisting of the base sequence shown in SEQ ID NO: 7 of the sequence listing, DNA consisting of 4 to 23 nucleotides, and DNA consisting of the base sequence shown in SEQ ID NO: 8 The 3 ′ end of the DNA consisting of the base sequence shown in SEQ ID NO: 7 in the sequence listing and the 5 ′ end of the DNA consisting of 4 to 23 nucleotides are combined, and further from 4 to 23 nucleotides. DNA 1 in which the 3 ′ end of the DNA and the 5 ′ end of the DNA comprising the base sequence set forth in SEQ ID NO: 8 are bound,
(Ii) DNA (DNA2) obtained by binding DNA consisting of the base sequence set forth in SEQ ID NO: 7 in the sequence listing, DNA consisting of 4 to 23 nucleotides, and DNA consisting of the base sequence set forth in SEQ ID NO: 8 And the 5 ′ end of the DNA consisting of the base sequence shown in SEQ ID NO: 7 of the Sequence Listing is linked to the 3 ′ end of the DNA consisting of 4 to 23 nucleotides, and further from 4 to 23 nucleotides. DNA 2 in which the 5 ′ end of the DNA and the 3 ′ end of the DNA consisting of the base sequence set forth in SEQ ID NO: 8 are bound,
(Iii) DNA (DNA3) obtained by binding DNA consisting of the base sequence shown in SEQ ID NO: 9 in the sequence listing, DNA consisting of 4 to 23 nucleotides, and DNA consisting of the base sequence shown in SEQ ID NO: 10 And the 3 ′ end of the DNA consisting of the base sequence shown in SEQ ID NO: 9 of the Sequence Listing is linked to the 5 ′ end of the DNA consisting of 4 to 23 nucleotides, and further comprising 4 to 23 nucleotides. DNA 3 in which the 3 ′ end of the DNA and the 5 ′ end of the DNA comprising the base sequence set forth in SEQ ID NO: 10 are bound,
(Iv) DNA consisting of the base sequence set forth in SEQ ID NO: 9 in the sequence listing, DNA consisting of 4 to 23 nucleotides and DNA consisting of the base sequence set forth in SEQ ID NO: 10 (DNA4) And the 5 ′ end of the DNA consisting of the base sequence shown in SEQ ID NO: 9 of the Sequence Listing is linked to the 3 ′ end of the DNA consisting of 4 to 23 nucleotides, and further from 4 to 23 nucleotides. DNA 4 in which the 5 ′ end of the DNA and the 3 ′ end of the DNA consisting of the base sequence set forth in SEQ ID NO: 10 are bound,
(V) DNA (DNA5) obtained by binding DNA consisting of the base sequence shown in SEQ ID NO: 11 of the sequence listing, DNA consisting of 4 to 23 nucleotides, and DNA consisting of the base sequence shown in SEQ ID NO: 12 The 3 ′ end of the DNA consisting of the base sequence shown in SEQ ID NO: 11 of the sequence listing and the 5 ′ end of the DNA consisting of 4 to 23 nucleotides are combined, and further from 4 to 23 nucleotides. DNA 5 in which the 3 ′ end of the DNA and the 5 ′ end of the DNA comprising the base sequence set forth in SEQ ID NO: 12 are bound,
And (vi) DNA comprising the base sequence set forth in SEQ ID NO: 11 in the sequence listing, DNA comprising 4 to 23 nucleotides, and DNA comprising the base sequence set forth in SEQ ID NO: 12 (DNA6) And the 5 ′ end of the DNA consisting of the base sequence shown in SEQ ID NO: 11 of the Sequence Listing is linked to the 3 ′ end of the DNA consisting of 4 to 23 nucleotides, and further 4 to 23 nucleotides. DNA 6 in which the 5 ′ end of the DNA consisting of is bound to the 3 ′ end of the DNA consisting of the base sequence set forth in SEQ ID NO: 12. A vector comprising the DNA according to claim 13 or 14. The double-stranded polynucleotide according to any one of claims 9 or 10, the polynucleotide according to any one of claims 11 or 12, the DNA according to any one of claims 13 or 14, And a telomerase reverse transcriptase gene expression inhibitor comprising at least one of the vectors according to claim 15. The double-stranded polynucleotide according to any one of claims 9 or 10, the polynucleotide according to any one of claims 11 or 12, the DNA according to any one of claims 13 or 14, And a telomerase inhibitor comprising at least one of the vectors of claim 15. The double-stranded polynucleotide according to any one of claims 9 or 10, the polynucleotide according to any one of claims 11 or 12, the DNA according to any one of claims 13 or 14, And a method for inhibiting the expression of a telomerase reverse transcriptase gene, wherein at least one of the vectors according to claim 15 is used. The double-stranded polynucleotide according to any one of claims 9 or 10, the polynucleotide according to any one of claims 11 or 12, the DNA according to any one of claims 13 or 14, A method for inhibiting telomerase, comprising using at least one of the vectors according to claim 15. The double-stranded polynucleotide according to any one of claims 9 or 10, the polynucleotide according to any one of claims 11 or 12, the DNA according to any one of claims 13 or 14, And a pharmaceutical composition comprising an effective amount of at least one of the vectors of claim 15. The double-stranded polynucleotide according to any one of claims 9 or 10, the polynucleotide according to any one of claims 11 or 12, the DNA according to any one of claims 13 or 14, A preventive and / or therapeutic agent for cancer diseases, comprising an effective amount of at least one of the vectors according to claim 15. The double-stranded polynucleotide according to any one of claims 9 or 10, the polynucleotide according to any one of claims 11 or 12, the DNA according to any one of claims 13 or 14, A method for preventing and / or treating a cancer disease, comprising using at least one of the vectors according to claim 15. The double-stranded polynucleotide according to any one of claims 9 or 10, the polynucleotide according to any one of claims 11 or 12, the DNA according to any one of claims 13 or 14, A reagent kit comprising at least one of the vectors according to claim 15. A method for identifying a double-stranded polynucleotide having a telomerase inhibitory action, wherein a cell that does not express a human telomerase reverse transcriptase gene or a cell that does not express a human telomerase reverse transcriptase gene and a human telomerase RNA gene is used, The cell is transfected with a double-stranded polynucleotide as a test substance together with a human telomerase reverse transcriptase gene expression vector and a human telomerase RNA gene expression vector, and then the telomerase activity is measured and the double-stranded polynucleotide is transfected. An identification method comprising selecting a double-stranded polynucleotide having reduced telomerase activity as compared to when not. The identification method according to claim 24, wherein the cell not expressing the human telomerase reverse transcriptase gene or the cell not expressing the human telomerase reverse transcriptase gene and the human telomerase RNA gene is a human osteosarcoma-derived Saos-2 cell. . The identification method according to claim 24 or 25, wherein the transfection is a lipofection method. The identification method according to claim 26, wherein the lipofection method is a lipofection method using cationic liposomes.

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