WO2009020344A2

WO2009020344A2 - Small interfering rnas (sirnas) controlling multiple target genes and method for preparing the same

Info

Publication number: WO2009020344A2
Application number: PCT/KR2008/004563
Authority: WO
Inventors: Dong Ki Lee; Chan Il Chang
Original assignee: Postech Acad Ind Found; Dong Ki Lee; Chan Il Chang
Priority date: 2007-08-06
Filing date: 2008-08-06
Publication date: 2009-02-12
Also published as: WO2009020344A3

Abstract

Disclosed are siRNAs shorter than 19 base pairs or nucleotides in length, multi- silencing siRNA, composed of plural siRNAs, capable of regulating the expression of respective target genes, and therapeutics for the treatment of gene control -related diseases, comprising the multi -silencing siRNA as an active ingredient. The siRNAs with fewer nucleotides can be produced at lower cost and can regulate gene expression at a higher efficiency per weight. Also, the multi -silencing siRNAs prepared from the siRNAs are easy to clinically apply, for example, are easily introduced into cells. Thus, the siRNAs find a broad spectrum of applications in the medical industry, including therapeutics for diseases, such as cancers, viral diseases, etc.

Description

[DESCRIPTION]

[invention Title]

SMALL INTERFERING RNAS (siRNAs) CONTROLLING MULTIPLE TARGET GENES AND METHOD FOR PREPARING THE SAME

[Technical Field]

The present invention relates to a small interfering RNA (siRNA) , shorter than 19 base pairs or nucleotides in length, which can regulate the expression of a target gene, a multi- silencing siRNA, composed of a plurality of siRNAs, for regulating the expression of plural target genes, and a therapeutic agent for the treatment of diseases, comprising the siRNA as an active ingredient .

[Background Art]

Small interfering RNA (siRNA) , also known as short interfering RNA or silencing RNA, is a class of 19-21 nucleotide-long double-stranded RNA molecules that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway, where it guides specific RNAi pathway proteins to the targeted messenger RNA (mRNA) complementary thereto so that they cleave the target, breaking it down into smaller portions that can no longer be translated into protein. Via the RNA interference pathway, siRNA, along with short hairpin RNA (shRNA) , can effectively silence gene expression by inducing specific cleavage of target mRNA (G.J. Hannon, RNA interference. Nature 418 (2002) 244-51) . This gene regulation technique using RNA interference induction is far higher in success rate and efficiency than are other various gene regulating methods . Being recognized as an important tool for gene function and as candidate for therapeutic agents for the treatment of various cancers and viral diseases, siRNAs thus have attracted intensive attention (Y. Dorsett, and T. Tuschl, siRNAs: Applications in Functional Genomics and Potential as Therapeutics. Nat Rev Drug Discov 3 (2004) 318-29) .

Initially, siRNA was designed as a 19-bp RNA core duplex that is followed by a two-nucleotide-3' overhang on each strand (termed the 19+2 structure) by the Tuschl research group in 2001 to mimic the Dicer-cleaved product of a long dsRNA. In addition, the 19+2 structure siRNAs were observed to cause specific gene suppression in mammalian cells, without nonspecific gene repression (S.M. Elbashir, J. Harborth, W. Lendeckel, A. Yalcin, K. Weber, and T. Tuschl, Duplexes of 21- nucleotide RNAs mediate RNA Interference in Cultured Mammalian cells. Nature 411 (2001) 494-8) . Thus, since then, most experiments for RNA interference have been performed with 19+2 structure siRNAs .

Recently, attempts have been made to introduce several variations in the 19+2 structure to achieve better silencing efficiency. The first example of structural variation in the canonical 19+2 siRNA was the development of 27 bp-long siRNAs and 29 bp-long shRNAs . These longer RNA duplexes were found to effectively silence the expression of some target mRNAs which could not be silenced by 19 bp-long RNA duplexes (D.H. Kim, M.A. Behlke, S. D. Rose, M.S. Chang, S. Choi, and J.J. Rossi, Synthetic dsRNA Dicer Substrates Enhance RNAi Potency and Efficacy. Nat Biotechnol 23 (2005) 222-6; D. Siolas, C. Lerner, J. Burchard, W. Ge, P. S. Linsley, P.J. Paddison, G.J. Harmon, and M.A. Cleary, Synthetic shRNAs as potent RNAi triggers. Nat Biotechnol 23 (2005) 227-31) .

Accordingly, the development of various antiviral agents is now focused on the use of these siRNAs. However, siRNA or shRNA, which is designed to target only a part of the viral genome, may induce mutations in target base sequences of viruses, which are ready to mutate, incurring the generation of mutant viruses resistant thereto. Indeed, there are reports on cases of producing such viral mutants (A. T. Das, T. R. Brummelkamp, E.M. Westerhout, M. Vink, M. Madiredjo, R. Bernards, and B. Berkhout, Human Immunodeficiency Virus Type 1 Escapes from RNA Interference-mediated Inhibition. J. Virol. 78 (2004) 2601-5) . Hence, there is an imperative need for siRNA that avoids the drug resistance due to such mutations.

More recently, a multi-target RNA interference (RNAi) technology for simultaneously suppressing multiple target genes from being expressed has been developed as an evolved alternative to such conventional methods (D. Grimm, and M.A. Kay, Combinatorial RNAi: A Winning Strategy for the Race against Evolving Targets? MoI Ther 15 (2007) 878-88) . The multi-target RNAi technology is now used to develop therapeutics for cancers as well as viral diseases. In fact, multi-target RNAi plays inhibitory roles in various carcinogenesis-related pathways to regulate the growth of cancer cells in a synergistic manner. Like this, the fundamental concept regarding control in the intracellular expression of multiple genes through RNAi is called "combinatorial RNAi" , and many studies on combinatorial RNAi are ongoing (D. Grimm, and M.A. Kay, Combinatorial RNAi: A Winning Strategy for the Race Against Evolving Targets? MoI Ther 15 (2007) 878-88) .

To date, most multi-target RNAi has been developed based on short or small hairpin RNA (shRNA) . Achievements of multi- target RNAi using recombinant expression vectors from which shRNA having at least one hairpin structure is transcribed were reported (D. Sun, M. Melegari, S. Sridhar, CE. Rogler, and L. Zhu, MultimiRNA Hairpin Method that Improves Gene Knockdown Efficiency and Provides Linked Multi-gene Knockdown. Biotechnigues 41 (2006) 59-63) . In contrast to shRNA that appears in the transcription stage after introduction into cells, siRNA is directly introduced after the chemical synthesis thereof. Thus, structural modifications of siRNA are required for the development of multi-target siRNA.

The Guo research group suggested the use of a RNA nano- structure, so-called Phi29 RNA, based on a bacteriophage introducing multiple siRNAs into cells (A. Khaled, S. Guo, F. Li, and P. Guo, Controllable Self-assembly of Nanoparticles for Specific Delivery of Multiple Therapeutic Molecules to Cancer Cells using RNA Nanotechnology. Nano Lett 5 (2005)

1797-808) . However, the RNA structure is too large in size to easily allow the chemical synthesis thereof, and there is difficulty in its development into actual therapeutics. In addition, the Tuschl group reported a partially complementary siRNA composed of a sense and an antisense strand which can target and regulate respective different genes simultaneously

(M. Hossbach, J. Gruber, M. Osborn, K. Weber, and T. Tuschl,

Gene Silencing with siRNA Duplexes Composed of Target mRNA- complementary and Partially Palindromic or Partially Complementary Single-stranded siRNAs . RNA Biol 3 (2006) 82-9) . The partially complementary siRNA duplexes also suffer from the disadvantage of being low in efficiency of regulation and showing structural instability. In previous studies of the present inventors, various structurally modified siRNAs were chemically synthesized and assayed for RNAi according to structural modifications (C. I. Chang, S. W. Hong, S. Kim, and D. K. Lee, A Structure-activity Relationship Study of siRNAs with Structural Variations. Biochem Biophys Res Commun 359 (2007) 997-1003) .

Leading to the present invention, intensive and thorough research into anticancer or anti-viral agents using various siRNAs resulted in the finding that the expression of target genes can be regulated by not only individual siRNAs ranging in length from 9 to 19 base pairs, but also multi-silencing siRNAs, composed of such individual siRNAs, whether they may be partially single-stranded, and that nano-structures comprising dual, triple or quadruple silencing siRNA can be widely used as therapeutic agents for the treatment of various diseases .

[Disclosure] [Technical Problem]

It is an object to provide an siRNA shorter than 19 base pairs or nucleotides in length, which can regulate the expression of a target gene, a multi-silencing siRNA for regulating the expression of a plurality of target genes, in which a plurality of siRNAs are combined via connection therebetween, and a therapeutic agent for the treatment of diseases such as cancer and viral diseases, comprising the siRNA or the multi-silencing siRNA as an active ingredient.

[Technical Solution] In order to accomplish the object, a small interfering RNA (siRNA) , shorter than 19 base pairs in length, for regulating expression of a target gene thereof is provided.

Preferably, the siRNA may range in length from 15 to less than 19 base pairs . Preferably, the siRNA may be at least 9 base pairs long. Also, the siRNA may be partially single-stranded. Preferably the siRNA is used to prepare a multi- silencing siRNA for regulating the expression of a plurality of target genes . In addition, a therapeutic for treating a disease is provided .

The type of disease includes cancer and viral diseases .

Also provided is a multi-silencing siRNA for regulating expression of plural target genes, comprising a plurality of individual siRNAs with a connection therebetween.

In the multi-silencing siRNA, the connection is achieved in such a manner that 3 '-end of an antisense strand of one individual siRNA is joined to a 5' -end of a sense strand of another individual siRNA. In the multi-silencing siRNA, the multi-silencing siRNA is a dual silencing siRNA consisting of two individual siRNAs which regulate expression of respective target genes .

In the multi-silencing siRNA, the multi-silencing siRNA is a triple silencing siRNA consisting of three individual siRNAs which regulate expression of respective target genes .

In the multi-silencing siRNA, the multi-silencing siRNA is a quadruple silencing siRNA consisting of four individual siRNAs which regulate expression of respective target genes .

Preferably, the multi-silencing siRNA may further comprise an aptamer.

In the multi-silencing siRNA, the at least one of the individual siRNAs may be single stranded.

In the multi-silencing siRNA, at least one of the individual siRNAs may be 15 to 19 base pairs long when it is double stranded, or 15 to 19 nucleotides long when it is single stranded .

In the multi-silencing siRNA, the individual siRNAs are at least 9 base pairs long when it is double stranded or at least 9 nucleotides long when it is single stranded.

Also, a therapeutic for treatment of a disease, comprising the multi-silencing of one of claims 8 to 16 as an active ingredient is provided.

In the therapeutic, the disease includes cancer or viral diseases .

A detailed description will be given of the present invention, below.

The present invention pertains to a small interfering RNA (siRNA) shorter than 19 base pairs (bp) in length, capable of regulating the expression of a target gene.

In an embodiment of this aspect, the siRNA preferably ranges in length from 15 to less than 19 base pairs . No matter how small it may be, the siRNA according to the present invention must be at least 9 base pairs long in order to ensure the regulation of gene expression. In addition, the siRNA may partially single stranded. In accordance with another aspect thereof, the present invention pertains to a multi-silencing siRNA, composed of a combination of different individual siRNAs, capable of regulating the expression of different corresponding target genes simultaneously. In an embodiment of this aspect, the individual siRNAs may be of double-stranded structure or of single-stranded structure with the proviso that at least one of them is double stranded .

Each of the individual siRNAs preferably ranges in length from 15 to less than 19 base pairs for double strand or from 15 to less than 19 nucleotides for single strand. No matter how small it may be, each strand of the individual siRNAs must be at least 9 nucleotides long in order to ensure the regulation of gene expression.

According to a concrete embodiment of this aspect, the multi-silencing siRNA is a dual silencing siRNA (dsiRNA) , composed of a combination of two different independent siRNA segments, capable of regulating the expression of two different target genes .

In the dsiRNA, each of the individual siRNAs may preferably be 15 ~ 19 base pairs or nucleotides long. However small it may be, the base sequence of each of the siRNA segments according to the present invention must be at least 11 base pairs or nucleotides long in order to ensure the regulation of gene expression. In addition, the siRNA segments of the multi-silencing siRNA may independently be of single stranded structure or double stranded structure with the proviso that at least one of them is double stranded.

In accordance with the present invention, the siRNA for use in RNAi is preferably selected from among siLamin, composed of a combination of base sequences of SEQ ID NOS . 7 and 8, for targeting the Lamin gene, siTIG3, composed of a combination of base sequences of SEQ ID NOS. 9 and 10, for targeting the TIG3 gene, siDBP, composed of a combination of base sequences of SEQ ID NOS. 11 and 12, for targeting the DBP gene, and siOASIS, composed of a combination of base sequences of SEQ ID NOS. 13 and 14, for targeting the OASIS gene. In addition to these, any gene may be a target gene of the siRNA of the present invention as long as the regulation of its expression is involved in the treatment or prevention of diseases. Further, all of the genes necessary for RNAi may be taken as targets according to the present invention.

Also, the present invention pertains to a multi- silencing RNA for regulating the expression of a plurality of target genes, resulting from the combination of a plurality of different individual siRNAs .

In accordance with an aspect thereof, the present invention provides an siRNA for regulating the expression of the target gene thereof.

In an embodiment of the aspect, the present invention provides siLamin (17) , a combination of two 17 nucleotide-long single strands of respective SEQ ID NOS. 1 and 2, which can regulate the Lamin gene . In another embodiment of the aspect, the present invention provides siTIG3(17), a combination of two 17 nucleotide-long single strands of respective SEQ ID NOS. 3 and 4 , which can regulate the TIG3 gene .

In a further embodiment of the aspect, the present invention provides siTIG3 (15) , a combination of two 15 nucleotide-long single strands of respective SEQ ID NOS. 5 and 6 , which can regulate the TIG3 gene . In accordance with another aspect thereof, the present invention provides a multi (dual) -silencing siRNA for regulating the expression of two different genes simultaneously, resulting from the combination of two individual siRNAs.

In an embodiment of this aspect, one of the two individual siRNAs may preferably be single-stranded to form a partially singles-stranded multi (dual) -silencing siRNA. Both or either of the two individual siRNAs is preferably shorter than 19 base pairs in length for double strand or 19 nucleotides in length for single strand and more preferably ranges in length from 15 to less than 19 base pairs or nucleotides. No matter how small it may be, each strand of the individual siRNAs must be at least 9 nucleotides long in order to ensure the regulation of gene expression. Preferably, the individual siRNAs may be partially single- stranded .

In accordance with the present invention, the individual siRNAs for use in RNAi are preferably selected from among siLamin, composed of a combination of base sequences of SEQ ID NOS. 7 and 8, for targeting the Lamin gene, siTIG3, composed of a combination of base sequences of SEQ ID NOS. 9 and 10, for targeting the TIG3 gene, siDBP, composed of a combination of base sequences of SEQ ID NOS. 11 and 12, for targeting the DBP gene, and siOASIS, composed of a combination of base sequences of SEQ ID NOS. 13 and 14, for targeting the OASIS gene. In addition to these, any gene may be a target gene of the siRNA of the present invention as long as the regulation of its expression is involved in the treatment or prevention of diseases. Further, all of the genes necessary for RNAi may be taken as targets according to the present invention. In an embodiment of the aspect, the present invention provides the dual silencing siRNA dsiLamin (19) -TIG3 (19) , a combination of two 19 bp-long siRNAs, composed of two 38-mer single strands of respective SEQ ID NOS. 15 and 16, which can regulate Lamin and TIG3 genes at the same time. In another embodiment of the aspect, the present invention provides the dual silencing siRNA dsiDBP(19)-

TIG3 (19) , a combination of two 19 bp-long siRNAs, composed of two 38-mer single strands of respective SEQ ID NOS. 17 and 18.

In a further embodiment of the aspect, the present invention provides the dual silencing siRNA dsiLamin (19) - DBP (19), a combination of two 19 bp-long siRNAs, composed of two 38-mer single strands of respective SEQ ID NOS. 19 and 20.

In still a further embodiment of the aspect, the present invention provides the dual silencing siRNA dsiOASIS (19) - TIG3 (19) , a combination of two 19 bp-long siRNAs, composed of two 38-mer single strands of respective SEQ ID NOS. 21 and 22.

In still another embodiment of the aspect, the present invention provides the dual silencing siRNA dsiLamin (3 ' 17) - TIG3(5'17), a combination of two siRNAs each less than 19 base pairs long, composed of two 34-mer single strands of respective SEQ ID NOS. 23 and 24, which can regulate Lamin and TIG3 genes at the same time . In yet a further embodiment of the aspect, the present invention provides the dual silencing siRNA dsiLamin (5' 17) - TIG3(5'15), a combination of two siRNAs each less than 19 base pairs long, composed of two 32-mer single strands of respective SEQ ID NOS. 25 and 26, which can regulate Lamin and TIG3 genes at the same time.

In yet another embodiment of the aspect, the present invention provides the dual silencing siRNA dsiLamin (3' 17) - TIG3(3'15), a combination of two siRNAs each less than 19 base pairs long, composed of two 32-mer single strands of respective SEQ ID NOS. 27 and 28, which can regulate Lamin and TIG3 genes at the same time.

In still yet a further embodiment of the aspect, the present invention provides the dual silencing siRNA dsiLamin (5'15) -TIG3 (5' 15) , a combination of two siRNAs each less than 19 base pairs long, composed of two 30-mer single strands of respective SEQ ID NOS. 29 and 30, which can regulate Lamin and TIG3 genes at the same time.

In still yet another embodiment of the aspect, the present invention provides the dual silencing siRNA dsiLamin (3 '15) -TIG3 (5' 15) , a combination of two siRNAs each less than 19 base pairs long, composed of two 30-mer single strands of respective SEQ ID NOS. 31 and 32, which can regulate Lamin and TIG3 genes at the same time. In additional another embodiment of the aspect, the present invention provides the dual silencing siRNA dsiLamin (M15) -TIG3 (5' 15) , a combination of two siRNAs each less than 19 base pairs long, composed of two 30-mer single strands of respective SEQ ID NOS. 33 and 34, which can regulate Lamin and TIG3 genes at the same time.

In additional a further embodiment of the aspect, the present invention provides the partially single-stranded dual silencing siRNA dsiO .5Lamin(19) -TIG3 (19) , a combination of one single-stranded 19-nt siRNA and one double-stranded 19-bp siRNA, composed of two single strands of respective SEQ ID NOS. 35 and 36, which can regulate Lamin and TIG3 genes at the same time.

In additional yet a further embodiment of the aspect, the present invention provides the partially single-stranded dual silencing siRNA dsiLamin(19) -0.5TIG3 (19) , a combination of one double-stranded 19-bp siRNA and one single-stranded 19- nt siRNA, composed of two single strands of respective SEQ ID NOS. 37 and 38, which can regulate Lamin and TIG3 genes at the same time.

In additional still another embodiment of the aspect, the present invention provides the partially single-stranded dual silencing siRNA dsiO .5Lamin (3' 17)-TIG3(5' 15), a combination of one single-stranded 17-nt siRNA and one double- stranded 15-bp siRNA, composed of two single strands of respective SEQ ID NOS. 39 and 40, which can regulate Lamin and TIG3 genes at the same time . In additional still a further embodiment of the aspect, the present invention provides the partially single-stranded dual silencing siRNA dsiDBP (19) -0.5TIG3 (19) , a combination of one double-stranded 19-bp siRNA and one single-stranded 19-nt siRNA, composed of two single strands of respective SEQ ID NOS. 41 and 42, which can regulate DBP and TIG3 genes at the same time . In additional yet still a further embodiment of the aspect, the present invention provides the partially single- stranded dual silencing siRNA dsiO .5DBP (19) -TIG3 (19) , a combination of one single-stranded 19-nt siRNA and one double- stranded 19-bp siRNA, composed of two single strands of respective SEQ ID NOS. 43 and 44, which can regulate DBP and TIG3 genes at the same time.

In accordance with a further aspect thereof, the present invention provides a multi (triple) -silencing siRNA for regulating the expression of three different genes simultaneously, resulting from the combination of three individual siRNAs.

In an embodiment of this aspect, at least one of the individual siRNAs is preferably shorter than 19 base pairs or nucleotides in length and more preferably ranges in length from 15 to less than 19 base pairs or nucleotides. No matter how small it may be, each strand of the individual siRNAs must be at least 9 nucleotides long in order to ensure the regulation of gene expression. Preferably, the individual siRNAs may be partially single-stranded. In accordance with the present invention, the individual siRNAs for use in RNAi are preferably selected from among siLamin, composed of a combination of base sequences of SEQ ID NOS. 7 and 8, for targeting the Lamin gene, siTIG3, composed of a combination of base sequences of SEQ ID NOS. 9 and 10, for targeting the TIG3 gene, siDBP, composed of a combination of base sequences of SEQ ID NOS. 11 and 12, for targeting the DBP gene, and siOASIS, composed of a combination of base sequences of SEQ ID NOS. 13 and 14, for targeting the OASIS gene.

In addition to these, any gene may be a target gene of the siRNA of the present invention as long as the regulation of its expression is involved in the treatment or prevention of diseases. Further, all of the genes necessary for RNAi may be taken as targets according to the present invention.

In an embodiment of the aspect, the present invention provides the triple silencing siRNA tsiLamin(19) -DBP (19) - TIG3(17) which can regulate Lamin, DBP and TIG3 genes at the same time, as shown in FIG. 19 (a) .

In another embodiment of the aspect, the present invention provides the triple silencing siRNA tsiLamin(19) - DBP (19) -TIG3 (15) as shown in FIG. 19 (b) . In a further embodiment of the aspect, the present invention provides the triple silencing siRNA tsiLamin(17) - DBP (19) -TIG3 (17) which can regulate Lamin, DBP and TIG3 genes at the same time, as shown in FIG. 20 (a) .

In still a further embodiment of the aspect, the present invention provides the triple silencing siRNA, tsiLamin(17) - DBP (19) -TIG3 (15) , as shown in FIG. 20 (b) .

In yet another embodiment of the aspect, the present invention provides the partially single-stranded triple silencing siRNA tsiLamin (19) -DBP (19) -0.5TIG3 (19) which can regulate Lamin, DBP and TIG3 genes at the same time, as shown in FIG. 21 (a) . In yet a further embodiment of the aspect, the present invention provides the partially single-stranded triple silencing siRNA, tsiLamin (19) -DBP (19) -0.5TIG3 (17) , as shown in

FIG. 21 (b) .

In yet still another embodiment of the aspect, the present invention provides the partially single-stranded, triple silencing siRNA, tsiO .5Lamin (17) -DBP (19) -TIG3 (19) which can regulate Lamin, DBP and TIG3 genes at the same time, as shown in FIG. 22.

In yet still a further embodiment of the aspect, the present invention provides the partially single-stranded, triple silencing siRNA, tsiO .5Lamin(17) -DBP (19) -TIG3 (17) which can regulate Lamin, DBP and TIG3 genes at the same time, as shown in FIG. 23 (a) .

In an additional embodiment of the aspect, the present invention provides the partially single-stranded, triple silencing siRNA, tsiO .5Lamin(17) -DBP (19) TIG3 (15) , as shown in FIG. 23 (b) .

In accordance with still a further aspect thereof, the present invention provides a multi (quadruple) -silencing siRNA for regulating the expression of four different genes at the same time .

In an embodiment of this aspect, at least one of the individual siRNAs is preferably shorter than 19 base pairs or nucleotides in length and more preferably ranges in length from 15 to less than 19 base pairs or nucleotides. No matter how small it may be, each strand of the individual siRNAs must be at least 9 nucleotides long in order to ensure the regulation of gene expression. Preferably, the individual siRNAs may be partially single-stranded.

In accordance with the present invention, the individual siRNAs for use in RNAi are preferably selected from among siLamin, composed of a combination of base sequences of SEQ ID NOS. 7 and 8, for targeting the Lamin gene, siTIG3, composed of a combination of base sequences of SEQ ID NOS. 9 and 10, for targeting the TIG3 gene, siDBP, composed of a combination of base sequences of SEQ ID NOS. 11 and 12, for targeting the DBP gene, and siOASIS, composed of a combination of base sequences of SEQ ID NOS. 13 and 14, for targeting the OASIS gene. In addition to these, any gene may be a target gene of the siRNA of the present invention as long as the regulation of its expression is involved in the treatment or prevention of diseases. Further, all of the genes necessary for RNAi may be taken as targets according to the present invention.

In an embodiment of the aspect, the present invention provides the quadruple silencing siRNA qsiDBP (19) -OASIS (19) - TIG3 (17) -Lamin (19) which can regulate Lamin, DBP, TIG3 and OASIS genes at the same time, as shown in FIG. 25 (a) .

In another embodiment of the aspect, the present invention provides the quadruple silencing siRNA qsiDBP (19)- OASIS (19) -TIG3 (15) -Lamin(19) as shown in FIG. 25 (b) .

In a further embodiment of the aspect, the present invention provides the quadruple silencing siRNA qsiLamine(17) -TIG3 (15) -OASIS (19) -DBP (19) which can regulate Lamin, DBP, TIG3 and OASIS genes at the same time, as shown in

FIG. 26.

In still a further embodiment of the aspect, the present invention provides the partially single-stranded quadruple silencing siRNA qsiDBP (19) -OASIS (19) -0.5TIG3 (19) -Lamin (19) which can regulate Lamin, DBP, TIG3 and OASIS genes at the same time, as shown in FIG. 27.

In still another embodiment of the aspect, the present invention provides the partially single-stranded quadruple silencing siRNA qsiDBP (19) -OASIS (19) -0.5TIG3 (17) -Lamin (19) which can regulate Lamin, DBP, TIG3 and OASIS genes at the same time, as shown in FIG. 28 (a) .

In yet another embodiment of the aspect, the present invention provides the partially single-stranded quadruple silencing siRNA qsiO .5Lamin(17) -TIG3 (19) -OASIS (19) -DBP (19) , as shown in FIG. 28 (b) .

In yet a further embodiment of the aspect, the present invention provides the partially single-stranded quadruple silencing siRNA qsiDBP (19) -OASIS (19) -TIG3 (15) -0.5Lamin(17) which can regulate Lamin, DBP, TIG3 and OASIS genes at the same time, as shown in FIG. 29 (a) .

In yet still a further embodiment of the aspect, the present invention provides the partially single-stranded quadruple silencing siRNA qsiLamin (17) -0.5TIG3 (15) -OASIS (19) - DBP (19), as shown in FIG. 29 (b) .

In accordance with still a further aspect thereof, the present invention provides the use of the siRNA of the present invention in the treatment of diseases such as cancer and viral diseases .

By regulating the expression of two or more target genes, the multi-silencing siRNA of the present invention is used as an active ingredient of anti-cancer agents or anti- viral agents in addition to being useful in the treatment and prevention of a variety of diseases .

[Description of Drawings]

FIG. 1 shows sequence structures of siRNAs, having a length less than 19 base pairs (bp) , for regulating the respective target genes thereof in accordance with the present invention, along with conventional 19-bp-long siRNAs.

FIG. 2 shows the regulatory activity of the siTIG3 (15) of the present invention against the expression of the target gene thereof . FIG. 3 shows the sequence structure of the dual silencing siRNA dsiLamin (19) -TIG3 (19) according to the present invention, resulting from the combination of two individual siRNAs .

FIG. 4 shows the regulatory activity of the dual silencing siRNA dsiLamin (19) -TIG3 (19) according to the present invention against the expression of respective target genes . FIG. 5 shows the sequence structure of the dual silencing siRNA dsiDBP (19) -TIG3 (19) according to the present invention, along with the individual siRNAs thereof.

FIG. 6 shows the regulatory activity of the dual silencing siRNA dsiDBP (19) -TIG3 (19) according to the present invention against the expression of respective target genes .

FIG. 7 shows the sequence structure of the dual silencing siRNA, dsiLamin (19) -DBP (19) according to the present invention, along with the individual siRNAs thereof. FIG. 8 shows the regulatory activity of the dual silencing siRNA dsiLamin (19) -DBP (19) according to the present invention against the expression of respective target genes .

FIG. 9 shows the sequence structure of the dual silencing siRNA dsiOASIS (19) -TIG3 (19) according to the present invention, along with the individual siRNAs thereof.

FIG. 10 shows the regulatory activity of the dual silencing siRNA dsiOASIS (19) -TIG3 (19) according to the present invention against the expression of respective target genes .

FIG. 11 shows sequence structures of various dual silencing siRNAs according to the present invention, each resulting from the combination of two individual different siRNAs having a length less than 19 base pairs.

FIG. 12 shows the regulatory activity of the dual silencing siRNA according to the present invention against the expression of respective target genes.

FIG. 13 shows sequence structures of partially single- stranded dual silencing siRNAs (Lamin-TIG3) according to the present invention, resulting from the combination of a single strand and a double strand.

FIG. 14 shows the regulatory activity of partially single-stranded dual silencing siRNAs according to the present invention against the expression of respective target genes .

FIG. 15 shows a partially single-stranded dual silencing siRNA (DBP-TIG3) according to the present invention, resulting from the combination of a single strand and a double strand .

FIG. 16 shows the regulatory activity of the partially single-stranded dual silencing siRNA according to the present invention against the expression of respective target genes.

FIG. 17 shows sequence structures of the triple silencing siRNAs tsiLamin-DBP-TIG3 (a) and tsiTIG3-DBP-Lamin (b) according to the present invention, resulting from the combination of three individual different siRNAs.

FIG. 18 shows the regulatory activity of the triple silencing siRNAs tsiLamin-DBP-TIG3 and tsiTIG3-DBP-Lamin according to the present invention against the expression of respective target genes. FIG. 19 shows sequence structures of the triple silencing siRNAs tsiLamin-DBP-TIG3 (17) (a) and tsiLamin-DBP- TIG3(15) (b) according to the present invention, in which one shorter siRNA is combined with two 19 bp-long siRNAs.

FIG. 20 shows sequence structures of the triple silencing siRNAs tsiLamin(17) -DBP-TTG3 (17) (a) and tsiLamin(17) -DBP-TIG3 (15) (b) according to the present invention, in which two shorter siRNAs are combined with one 19 bp-long siRNA.

FIG. 21 shows sequence structures of the triple silencing siRNAs tsiLamin-DBP-0.5TIG3 (a) and tsiLamin-DBP-

0.5TIG3(17) (b) according to the present invention, in which one 19-mer or shorter single-stranded siRNA is combined with two 19 bp-long siRNA.

FIG. 22 shows the sequence structure of the triple silencing siRNA tsiO .5Lamin (17) -DBP-TIG3 according to the present invention, in which one shorter single-stranded siRNA is combined with two 19-bp long siRNAs.

FIG. 23 shows sequence structures of the triple silencing siRNAs tsiLamin(17) -DBP-TIG3 (17) (a) and tsiLamin(17) -DBP-TTG(15) (b) according to the present invention, in which one shorter single-stranded siRNA is combined with two siRNAs at least one of which is shorter than 19 base pairs in length.

FIG. 24 shows sequence structures of the quadruple silencing siRNAs qsiDBP-OASIS-TIG3-Lamin (a) and qsiLamin-

TIG3 -OASIS-DBP (b) according to the present invention, resulting from the combination of four individual different 19 bp-long siRNAs .

FIG. 25 shows sequence structures of the quadruple silencing siRNAs qsiDBP-OASIS-TIG3 (17) -Lamin (a) and qsiDBP- OASIS-TIG3 (15) -Lamin (b) , in which one shorter siRNA is combined with three 19 bp-long siRNAs.

FIG. 26 shows the sequence structure of the quadruple silencing siRNA qsiLamin (17) -TIG3 (15) -OASIS-DBP according to the present invention, in which one or more shorter siRNAs are combined with the other 19 bp-long siRNAs.

FIG. 27 shows the sequence structure of the quadruple silencing siRNA qsiDBP-OASIS-0.5TIG3-Lamin according to the present invention, in which one single-stranded siRNA is combined with three 19 bp-long siRNAs.

FIG. 28 shows sequence structures of the quadruple silencing siRNAs qsiDBP-OASIS-0.5TIG3 (17) -Lamin (a) and qsiO.5Lamin( 17) -TIG3 -OASIS-DBP (b) according to the present invention, in which one shorter single-stranded siRNA is combined with three 19 bp-long siRNAs.

FIG. 29 shows sequence structures of the quadruple silencing siRNAs qsiDBP-OASIS-TIG3 (15) -0.5Lamin(17) (a) and qsiLamin(17) 0.5TIG3 (15) -OASIS-DBP (b) according to the present invention, in which one shorter siRNA and one shorter single-stranded siRNA are combined with two 19 bp-long siRNAs.

FIG. 30 schematically shows the structures of aptamer- multi-silencing siRNAs according to the present invention, with a cell-specific aptamer combined to a dual silencing siRNA (dsiRNA) (a) and to triple silencing siRNA (tsiRNA) (b) .

[Best Mode]

A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as limiting the present invention. EXAMPLE 1. Construction of siRNA and Interfering Activity thereof against Gene Expression

By virtue of previous studies, the present inventors found that siRNA having a core duplex less than 19 base pairs (bp) has the possibility of effectively regulating target genes .

A 15 bp-long siTIG3 having the base sequence represented by SEQ ID NO. 5 or 6, was synthesized and transfected at a concentration of 100 nM or 10 nM into HeLa cells with the aid of Lipofectamine 2000. Real-time RT-PCR was performed to quantitatively analyze the mRNA level of the target gene TIG3. Further, the regulation efficiency of siTIG3 was determined in terms of mRNA level and plotted according to transfection concentration and sequence length. As seen in FIG. 2, the 15-bp-long siTIG3 (siTIG3 15 bp) was found to regulate the expression of the target gene at an efficiency similar to that of the 19-bp-long siTIG3 (siTIG3 19 bp) .

EXAMPLE 2: Construction of Dual Silencing siRNA

In the previous study into the actions thereof, the present inventors found that even when an siRNA is joined with a different duplex at the 3' -terminus of the guide strand

(antisense strand) thereof, its ability to regulate gene expression is maintained (CI. Chang, S. W. Hong, S. Kim, and

D. K. Lee, A Structure-activity Relationship Study of siRNAs with Structural Variations. Biochem Biophys Res Commun 359 (2007) 997-1003) . On the basis of this fact, the present inventors made an assumption that a new siRNA constructed by joining two different 19-bp-long siRNAs with each other may maintain gene regulation efficiency similar to that of the individual 19-bp-long siRNAs.

As shown in FIG.3, siLamin and siTIG3, which target Lamin and TIG3 genes, respectively, were combined to construct a dual silencing siRNA of SEQ ID NO. 15 or 16 [dual silencing siRNA Lamin (19) -TIG3 (19)] . In greater detail, the 5' -end of the 19-mer sense strand of siTIG3 was combined to the 3' terminus of the 19-mer antisense strand of siLamin to afford a 38-mer single strand. Separately, the 5' -end of the 19-mer sense strand of siLamin was combined to the 3' terminus of the 19-mer antisense strand of siTIG3 to afford a 38-mer single strand. Thereafter, these two complementary 38-mer single strands were annealed to construct the dual silencing siRNA dsiLamin (19) -TIG3 (19) 38 bp long.

Likewise, if necessary, two or more siRNAs may be combined between antisense and sense strands, sense and sense strands, and/or antisense and antisense strands, followed by annealing the synthesized, at least 19-mer single strands to construct multi-silencing siRNA.

EXAMPLE 3 : Inhibitory Activity of Dual Silencing siRNA against Gene Expression I

In order to examine dual silencing siRNA for inhibitory activity against gene expression, siLamin of SEQ ID NO. 7 or

8, targeting the Lamin gene only, siTIG3 of SEQ ID NO. 9 or

10, targeting the TIG3 gene only, mixtures of the siLamin and the siTIG3, and the dual silencing siRNA dsiLamin(19) -TIG3 (19) of SEQ ID NO. 15 or 16 were transfected at a concentration of

100 nM or 10 nM into HeLa cells with the aid of Lipofectamine

2000 in the same manner as in Example 1.

Afterwards, the mRNA levels of the target genes Lamin and TIG3 were quantitatively analyzed by real-time RT-PCR. Further, the regulation efficiency of each siRNA was determined in terms of mRNA level and plotted according to transfection concentration in FIG. 4.

As seen in FIG. 4, the dual silencing siRNA, dsiLamin(19) -TIG3 (19) , according to the present invention, was observed to have the same inhibitory activity as that of the simple mixture of individual siLamin and siTIG3.

EXAMPLE 4 : Inhibitory Activity of Dual Silencing siRNA against Gene Expression II An examination was made into whether the same inhibitory activity of dual silencing siRNA as was exemplified in Example 3 is achieved for dual silencing siRNAs targeting other genes. To this end, first, various dual silencing siRNAs were constructed by combining sets of two different siRNAs, such as RNA dsiDBP(19) -TIG3 (19) , composed of two single strands of respective SEQ ID NOS. 17 and 18, dsiLamin (19) -DBP (19) , composed of two single strands of respective SEQ ID NOS. 19 and 20, and dsiOASIS (19) -TIG3 (19) , composed of two single strands of respective SEQ ID NOS. 21 and 22 (see FIGS.5, 7 and 9) •

All of the dual silencing siRNAs, as seen in FIGS. 6, 8 and 10, were observed to effectively regulate the expression of respective double target genes although there was a slight difference in regulative efficiency. Hence, when transfected into cells, the dual silencing siRNAs composed of sets of two different 19-bp-long siRNAs showed regulatory activity against gene expression to the same degree as with simple mixtures of the component 19-bp-long siRNAs.

EXAMPLE 5: Construction of Short Dual Silencing siRNA Although effectively regulating gene expression, the 38- bp-long dual silencing siRNAs might be disadvantageous in gene regulation compared with conventional 19- or 27-bp-long siRNAs due to the longer sequences thereof. Thus, an examination was made into whether dual silencing siRNAs of a sequence shorter than 38-bp were able to effectively regulate gene expression. As seen in FIG. 11, the dsiLamin-TIG3 siRNA 38 bp long was truncated to dual silencing siRNAs composed of 17 bp + 17 bp, 17 bp + 15 bp, or 15 bp + 15 bp. For example, the Lamin(3'17) of SEQ ID NO. 23 or 24 was composed of a duplex 17 bp long from the 3' terminus of the antisense strand of the 19-bp-long siLamin. Likewise, the Lamin(5'17) of SEQ ID NO 25 or 26 stands for a duplex 17 bp long from the 5 terminus of the antisense strand of the 19-bp-long siLamin, the

2 Q O Lamin(5'15) of SEQ ID NO. 27 or 28 for a duplex 15 bp long from the 5' terminus of the antisense strand of the 19-bp-long siLamin, the Lamin(3'15) of SEQ ID NO. 31 or 32 for a duplex 15 bp long from the 3' terminus of the antisense strand of the 19-bp-long siLamin, and the Lamin(M15) of SEQ ID NO 33 or 34 for a 15-bp-long duplex corresponding to a middle region of the 19-bp-long siLamin.

In addition, as for the siRNA inhibitory of TIG3 gene expression, it was truncated into duplexes 15 or 17 bp long from the 5' end of the antisense strand thereof. Thus, TIG3(5'17) stands for a duplex 17 bp long from the 5' end of the antisense strand of siTIG3, and TIG3(5'15) for a duplex 15 bp long from the 5' end of the antisense strand of siTIG3. The short dual silencing siRNA truncates were assayed for intracellular inhibitory activity against target gene expression and the results are summarized in FIG. 12.

EXAMPLE 6 : Assay for Regulatory Activity of Short Dual Silencing siRNA against Gene Expression The siRNAs including the truncated dual silencing siRNAs in accordance with the present invention were assayed for regulatory activity against gene expression in the same manner as in Example 1. It was found that truncated siRNA duplexes 15 or 17 bp long from the 5' end of the antisense strand of the intact siRNA could sufficiently decrease the mRNA level of the TIG3 gene. As for the Lamin gene, its mRNA level was greatly decreased by the truncated siRNA duplex 17 bp long from the 3' end of the antisense strand while the truncated siRNA duplex 17 bp long from the 5' end of the antisense strand almost did not inhibit Lamin gene expression (see FIG. 12) . Even when composed of two different siRNA units shorter in sequence than 19-bp, the dual silencing siRNA structures in accordance with the present invention were found to sufficiently inhibit the expression of corresponding target genes in a sequence-dependent manner.

As such, dual silencing siRNA structures shorter than those composed of sets of two 19-bp-long siRNAs may be decreased in nucleotide number to, for example, a 17 bp + 15 bp duplex, that is, a 32-mer structure, without the regression of inhibitory activity against gene expression. Therefore, the medicines comprising the siRNAs of the present invention as active ingredients can be produced at reduced cost.

EXAMPLE 7: Construction of Partially Single-stranded Dual Silencing siRNA

Dual silencing siRNAs having partial single strands were constructed as follows. Previous studies showed that siRNAs can induce RNAi in the form of a single strand, as well as in the form of double strand to inhibit the expression of target genes (J. Martinez, A. Patkaniowska, H. Urlaub, R. Luhrmann, and T. Tuschl, Single-stranded Antisense siRNAs Guide Target RNA Cleavage in RNAi. Cell 110 (2002) 563-74) . In this example, as shown in FIG. 13, heterogeneous dsiRNAs composed of two different siRNA units were constructed so that one of the siRNA units was single stranded with the lack of the sense strand (expressed by the numeral ^x0.5' in FIG.13) . For example, as seen in FIG. 13, dsiO .5Lamin(19) -TIG3 (19) , which is a combination of two sequences of respective SEQ ID NOS. 35 and 36, is a TIG3 siRNA duplex joined with a 19-mer single stranded antisense siLamin. In dsiLamin(19) -0.5TIG3 (19) composed of two sequences of respective SEQ ID NOS. 37 and 39, a Lamin siRNA duplex is followed by a 19-mer single-stranded antisense siTIG3. In dsiO .5Lamin (3' 17) -TIG3 (5' 15) composed of two sequences of respective SEQ ID NOS. 39 and 40, a 17-mer single stranded antisense siLamin is followed by a 15 bp-long siTIG3 duplex in such a manner that the antisense strand of the duplex is joined to the 3' end of the single strand antisense siLamin. In greater detail, dsiO .5Lamin (19) -TIG3 (19) was constructed by joining the sense strand of TIG3 to the 3' end of the antisense strand of Lamin to give a 38-mer single strand and annealing this 38-mer single strand with the antisense strand of TIG3. Likewise, as for dsiO .5Lamin (3' 17) - TIG3(5'15), the 17-mer antisense strand of Lamin was joined to the 15-mer sense strand of TIG3 to give a 32-mer single strand, followed by annealing this strand with the 15-mer antisense strand of TIG3. The other heterogeneous dsiRNA was also constructed in a similar manner.

EXAMPLE 8: Assay of Partially Single-Stranded Dual Silencing RNAs for Regulatory Activity against Gene Expression The partially single-stranded dsiRNAs according to the present invention was assayed for regulatory activity against gene expression in the same manner as described in Example 3.

As seen in FIG. 14, dsiLamin(19) -0.5TIG3 (19) in which a Lamin siRNA duplex of SEQ ID NO. 37 was followed by the 19-mer single-stranded antisense siTIG3 of SEQ ID NO. 38 was found to effectively regulate the mRNA expression of both Lamin and TIG3 genes. In contrast, the dsiO .5Lamin(19) -TIG3 (19) in which the 19-mer single-stranded antisense siTIG3 of SEQ ID NO. 35 was joined to the Lamin siRNA duplex of SEQ ID NO. 36 regulated the expression of the TIG3 gene only. However, dsiO.5Lamin(3'17) -TIG3 (5'15) in which the 15 bp-long TIG3 siRNA duplex of SEQ ID NO. 40 was joined to the 3' end of the 19-mer single-stranded antisense siLamin of SEQ ID NO. 39 showed effective regulation against the expression of both TIG3 and Lamin genes . Being able to regulate the expression of two different target genes simultaneously even though they consist of as low as 47 nucleotides, much less than those of two 19-bp-long siRNAs (76 nucleotides in total) , therefore, the partially single-stranded dual silencing siRNAs according to the present invention may make a great contribution to decreasing the production costs of medicines thereof.

EXAMPLE 9: Construction of Triple Silencing siRNAs and Assay for Regulatory Activity against Gene Expression

Triple silencing siRNA (tsiRNA) was constructed using a chemical synthesis method in which three different 19 bp-long siRNAs were joined as illustrated in FIG. 17, and then was assayed for regulatory activity against the expression of respective target genes.

In detail, as shown in FIG. 17 (a) , the antisense strand of Lamin was combined to the sense strand of DBP in the direction of 5' to 3' to give a 38-mer single strand. Likewise, combination was made in the direction of 5' to 3' between the antisense strand of DBP and the sense strand of TIG3 and between the antisense strand of TIG3 and the sense strand of Lamin to give two respective 38-mer single strands. The resulting three 38-mer single strands were annealed with one another to form the most stable triple structure tsiLamin- DBP-TIG3.

Also, tsiTIG3-DBP-Lamin was prepared by synthesizing three single strands and annealing them in the same manner as above described .

When transfected into cells, these triple silencing siRNAs were found to show regulatory activity against the expression of the three respective target genes at efficiencies similar to those of a simple mixture of three individual siRNAs (see FIG. 18) . Accordingly, one siRNA structure according to the present invention can effectively regulate the expression of three target genes at the same time. As was described in relation to dsiRNA, the tsiRNA of the present invention was modified to make truncated structures which became small in the length of individual siRNA duplexes, and/or to partially single-stranded triple structures. In the case of the modified structures, they showed effective regulatory activity against the expression of the target genes .

EXAMPLE 10: Construction of Multiple Silencing siRNA Having Aptamer Sequence and Regulatory Activity Thereof against Gene Expression

As seen in FIG. 30, an RNA nanostructure in which an aptamer sequence was substituted for one duplex of the tsiRNA was constructed so as to selectively introduce two or three siRNAs into cells of interest. Also, an assay performed in the same manner as in Example 1 showed that the multi silencing siRNAs effectively regulated the expression of target genes.

[industrial Applicability]

As described hitherto, the present invention provides an siRNA which has a duplex consisting of less than 19 base pairs and a medicine for the treatment of diseases, comprising the same as an active ingredient. Also the present invention provides a multiple silencing siRNA capable of regulating the expression of multiple target genes, a method for preparing the multiple silencing siRNA from individual siRNAs, and medicines for the treatment of diseases, such as cancer, viral diseases, etc.

Being fewer in the number of nucleotides than conventional siRNAs, the siRNAs according to the present invention can be produced at lower cost and can regulate gene expression at a higher efficiency per weight. Also, the tπulti-silencing siRNAs prepared from the siRNAs of the present invention are easy to clinically apply, for example, are easily introduced into cells . Accordingly, the siRNAs of the present invention find a broad spectrum of applications in the medical industry, including therapeutics for diseases, such as cancers, viral diseases, etc.

[Sequence List Text]

Claims

[CLAIMS]

[Claim l]

A small interfering RNA (siRNA) , shorter than 19 base pairs or nucleotides in length, for regulating expression of a target gene thereof .

[Claim 2]

The siRNA according to claim 1, wherein the siRNA ranges in length from 15 to less than 19 base pairs or nucleotides.

[Claim 3]

The siRNA according to claim 1, wherein the siRNA is at least 9 base pairs or nucleotides long.

[Claim 4]

The siRNA according to claim 1, wherein the siRNA is partially single-stranded.

[Claim 5]

The siRNA according to claim 1, wherein the siRNA is used to prepare a multi-silencing siRNA for regulating the expression of a plurality of target genes .

[Claim 6]

A therapeutic for treating a disease . [Claim 7]

The therapeutic according to claim 6, wherein the disease is selected from among cancer and viral diseases .

[Claim 8]

A multi-silencing siRNA for regulating expression of plural target genes, comprising a plurality of individual siRNAs with a connection therebetween.

[Claim 9]

The multi-silencing siRNA according to claim 8, wherein the connection is achieved in such a manner that the 5' -end of an antisense strand of one individual siRNA is joined to a 5'- end of a sense strand of another individual siRNA.

[Claim lθ]

The multi-silencing siRNA according to claim 8, wherein the multi-silencing siRNA is a dual silencing siRNA consisting of two individual siRNAs which regulate expression of respective target genes.

[Claim ll]

The multi-silencing siRNA according to claim 8, wherein the multi-silencing siRNA is a triple silencing siRNA consisting of three individual siRNAs which regulate expression of respective target genes. [Claim 12]

The multi-silencing siRNA according to claim 8, wherein the multi-silencing siRNA is a quadruple silencing siRNA consisting of four individual siRNAs which regulate expression of respective target genes .

[Claim 13]

The multi-silencing siRNA according to claim 8, further comprising an aptamer.

[Claim 14]

The multi-silencing siRNA according to claim 8, wherein the individual siRNAs are single stranded or double stranded with a proviso that at least one of them is double stranded.

[Claim 15]

The multi-silencing siRNA according to any of claims 8 to 14, wherein at least one of the individual siRNAs is 15 to 19 base pairs long when it is double stranded, or 15 to 19 nucleotides long when it is single stranded.

[Claim 16]

The multi-silencing siRNA according to any of claims 8 to 14, wherein at least one of the individual siRNAs is at least 9 base pairs long when it is double stranded or at least 9 nucleotides long when it is single stranded. [Claim 17]

A therapeutic for treatment of a disease, comprising the multi-silencing of one of claims 8 to 16 as an active ingredient .

[Claim 18]

The therapeutic according to claim 17, wherein the disease includes cancer or viral diseases.