US20210207137A1

US20210207137A1 - Rna-interference-inducing nucleic acid molecule able to penetrate into cells, and use therefor

Info

Publication number: US20210207137A1
Application number: US17/110,640
Authority: US
Inventors: Sun Woo Hong
Original assignee: Olix Pharmaceuticals Inc
Current assignee: Olix Pharmaceuticals Inc
Priority date: 2012-05-22
Filing date: 2020-12-03
Publication date: 2021-07-08
Also published as: US10883105B2; US10125362B2; KR20130130653A; JP6139671B2; EP2853597B1; JP2017093448A; JP2015518721A; CN108148838A; KR101581655B9; KR101581655B1; JP6999590B2; KR20150118061A; DK2853597T3; JP6629712B2; HK1211319A1; US20150111948A1; EP2853597A4; CN104755620B; EP2853597A1; ES2716818T3

Abstract

The present invention relates to a novel, RNAi-inducing nucleic acid molecule having cell penetrating ability and the use thereof, and more particularly, to a novel, RNAi-inducing double-stranded nucleic acid molecule, which has a replacement of the phosphate backbone of at least one nucleotide with phosphorothioate or phosphorodithioate, and has a lipophilic compound conjugated thereto, and thus has high target gene-silencing efficiency while having the ability to penetrate cells without needing a separate intracellular delivery vehicle, and to a method of silencing a target gene using the nucleic acid molecule. The nucleic acid structure according to the present invention has both cholesterol modification and phosphorothioate modification introduced therein, and thus has high gene silencing efficiency while having the ability to penetrate cells without needing a separate intracellular delivery vehicle. Thus, it can be delivered into an actual target area in an amount sufficient for induction of RNAi, and thus can overcome the in vivo delivery problem occurring in the prior art. Therefore, the nucleic acid molecule according to the invention can effectively substitute for conventional siRNA molecules to treat cancer or viral infections.

Description

TECHNICAL FIELD

The present invention relates to a novel, RNA interference (RNAi)-inducing nucleic acid molecule having cell penetrating ability and the use thereof, and more particularly, to a novel, RNAi-inducing double-stranded nucleic acid molecule, which has a replacement of the phosphate backbone of at least one nucleotide with phosphorothioate or phosphorodithioate, and has a lipophilic compound conjugated thereto, and thus has high target gene-silencing efficiency while having the ability to penetrate cells without needing a separate intracellular delivery vehicle, and to a method of silencing a target gene using the nucleic acid molecule.

BACKGROUND ART

RNA interference (RNAi) is a mechanism capable of inhibiting the expression of a gene in a highly specific and efficient manner, in which degradation of the mRNA of a target gene is induced by introducing a double-stranded RNA, which comprises a sense strand having a sequence homologous to the mRNA of the target gene and an antisense strand having a sequence complementary to the mRNA of the target gene, into cells or the like, thereby inhibiting the expression of the target gene.
An siRNA that induces this RNA interference is a short (19-21 bp) double-stranded RNA capable of inhibiting the expression of a target gene in a sequence-specific manner, and is currently receiving attention as a therapeutic agent against various diseases, including cancer difficult to treat, viral infections, and hereditary diseases, thanks to its high efficiency and target specificity. For the development of effective therapeutic agents based on an siRNA, various problems associated with stability, silencing efficiency, immune responses, off-target effects and the like, are required to be solved, and among them, effective in vivo delivery is considered most difficult to achieve. An siRNA cannot pass through the cell membrane, because it is highly negatively charged due to its phosphate backbone structure. In addition, because of its small size, the siRNA is quickly removed from blood, and thus it is difficult to deliver the siRNA in an amount sufficient for inducing RNAi to a target area.
In the case of in vitro delivery, many high-efficiency delivery methods that use cationic lipids and cationic polymers have been developed (Sioud M, Sorensen D R Cationic liposome-mediated delivery of siRNAs in adult mice. Biochem Biophys Res Commun 2003; 312: 1220-1225). However, in most cases, in vivo delivery of siRNAs is difficult to achieve with high efficiency, unlike in vitro delivery, and the efficiency of delivery of siRNAs decreases due to their interactions with various proteins in vivo (Bolcato-Bellemin A L, Bonnet M E, Creusat G, et al. Sticky overhangs enhance siRNA-mediated gene silencing. Proceedings of the National Academy of Sciences of the United States of America 2007; 104: 16050-16055). In addition, siRNAs are highly accumulated in a specific organ such as liver or lung, which is not a diseased area, depending on the composition of delivery vehicles, thus inducing toxicity.
Meanwhile, connective tissue growth factor (CTGF/CCN2) is known as a matricellular protein that plays an important role in the differentiation, growth, migration, ECM production, adhesion and the like of cells. In the case of chronic fibrotic disorders that induce fibrosis in various organs to cause damage to the organs, it was found that CTGF is overexpressed in tissues in which fibrotic disorders occur. Also, the relationship between CTGF and fibrosis in the skin has been relatively well studied. In addition, it was observed that the expression of CTGF in a normal skin was inhibited to the basal level, but temporarily increased when the skin was wounded. On the contrary, in the case of keloid or localized sclerosis, it was observed that the overexpression of CTFG was maintained even after wound healing, and when the expression of CTGF was inhibited using an antisense strand or the like, fibrosis and keloid production were inhibited, suggesting that CTGF plays an important role in fibrosis and keloid production (Sisco M, Kryger Z B, O'Shaughnessy K D, et al. Antisense inhibition of connective tissue growth factor (CTGF/CCN2) mRNA limits hypertrophic scarring without affecting wound healing in vivo. Wound Repair Regen 2008; 16: 661-673. DOI: WRR416 [pii]). Pathologically, it is known that a full-length CTGF molecule is involved in a condition in which the hyperproliferation of connective tissue cells and the excessive deposition of extracellular matrix are present. In addition, it is known that CTGF is also involved in conditions associated with the migration and proliferation of endothelial cells and angiogenesis. Examples of diseases and disorders associated with such conditions include the fibrosis, cancer and related diseases and disorders of the skin and major organs, for example, systemic sclerosis, angiogenesis, atherosclerosis, diabetic nephropathy, and renal hypertension. Also, CTGF is known to be useful for wound healing, connective tissue repair, and bone and cartilage repair. In such terms, CTGF was disclosed as an inducer of bone, tissue or cartilage formation disorders such as osteoporosis, osteoarthritis or osteochondritis, arthritis, skeletal disorder, hypertrophic scar, a burn, hemagiectatic hypertrophy, or sound healing (see, for example, U.S. Pat. No. 5,837,258).
Accordingly, the present inventors have made extensive efforts to provide a novel, RNAi-inducing nucleic acid molecule that can be effectively delivered in vitro and in vivo and has cell-penetrating ability, and as a result, have found that, when the phosphate backbone of at least one nucleotide in an RNAi-inducing double-stranded nucleic acid molecule is substituted with phosphorothioate and a lipophilic compound is conjugated to the nucleic acid molecule, the nucleic acid molecule exhibits high target gene silencing efficiency even in vivo without needing a separate intracellular delivery vehicle and, at the same time, has high cell-penetrating ability, thereby completing the present invention.
The above information disclosed in this Background section is only for enhancement of understanding of the background of the present invention, and therefore it may contain information that does not form the prior art that is already known to a person of ordinary skill in the art.

PRIOR ART LITERATURE

Patent Documents

Patent Document 1: U.S. Pat. No. 5,837,258

Non-Patent Documents

Non-Patent Document 1: Sioud M, Sorensen D R Cationic liposome-mediated delivery of siRNAs in adult mice. Biochem Biophys Res Commun 2003; 312: 1220-1225
Non-Patent Document 2: Bolcato-Bellemin A L, Bonnet M E, Creusat G, et al. Sticky overhangs enhance siRNA-mediated gene silencing. Proceedings of the National Academy of Sciences of the United States of America 2007; 104: 16050-16055
Non-Patent Document 3: Sisco M, Kryger Z B, O'Shaughnessy K D, et al. Antisense inhibition of connective tissue growth factor (CTGF/CCN2) mRNA limits hypertrophic scarring without affecting wound healing in vivo. Wound Repair Regen 2008; 16: 661-673. DOI: WRR416 [pii]

DISCLOSURE OF INVENTION

It is an object of the present invention to provide a novel, RNAi-inducing nucleic acid molecule having cell-penetrating ability, which can be effectively delivered in vitro and in vivo, and the use thereof.
To achieve the above object, the present invention provides an RNAi-inducing double-stranded nucleic acid molecule having cell-penetrating ability, wherein the nucleic acid molecule comprising a first strand comprising a region complementary to a target nucleic acid and a second strand that forms a complementary bond with the first strand; and wherein the phosphate backbone of at least one nucleotide in the nucleic acid molecule was substituted with phosphorothioate or phosphorodithioate, and the nucleic acid molecule has a lipophilic compound conjugated thereto.
The present invention also provides a gene-silencing composition containing the above nucleic acid molecule.
The present invention also provides a method for silencing a target gene in a cell, the method comprising introducing the above nucleic acid molecule into the cell.
The present invention also provides a pharmaceutical composition for treating or preventing a connective tissue growth factor (CTGF)-associated disease or disorder, the composition containing the above nucleic acid molecule that targets a CTGF-encoding mRNA.
The present invention also provides a meth of treating or preventing a connective tissue growth factor (CTGF)-associated disease or disorder comprising administering a pharmaceutical composition containing the above nucleic acid molecule targeting a CTGF-encoding mRNA.
The present invention also provides an RNAi-inducing double-stranded nucleic acid molecule having cell-penetrating ability, the nucleic acid molecule comprising: a first strand comprising a region complementary to a connective tissue growth factor (CTGF)-encoding mRNA; and a second strand that forms a complementary bond with the first strand, wherein the phosphate backbone of 1 to 31 nucleotides in the nucleic acid molecule was substituted with phosphorothioate or phosphorodithioate, and the nucleic acid molecule has a lipophilic compound conjugated thereto and has a pair of nucleic sequences selected from the group consisting of a pair of nucleotide sequences of SEQ ID NOS: 149 and 150, a pair of nucleotide sequences of SEQ ID NOS: 151 and 152, and a pair of nucleotide sequences of SEQ ID NOS: 153 and 154.
Other features and embodiments of the present invention will be more apparent from the following detailed descriptions and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the gene silencing efficiencies of siRNA, asiRNA and lasiRNA structures for 24 sequences that target the CTGFs shown in Tables 1 to 3.

FIG. 2 depicts fluorescence micrographs showing the increase in intracellular uptake efficiency of lasiRNA by cholesterol modification.

FIG. 3 shows the structures of cholesterol- and PS-modified lasiRNAs according to the present invention. Underline: OMe modification; *: PS modification, Chol: cholesterol; Cy3: Cy3.

FIG. 4 depicts fluorescence micrographs showing the increase in intracellular uptake efficiency of chol-lasiRNA by phosphorothioate (PS) modification.

FIG. 5 is a graphic diagram showing a comparison between the gene silencing effects of chol-lasiRNAs according to variation of numbers in phosphorothioate (PS) modifications. Each graph represents the mean±SD of three repeated experiments.

FIG. 6 shows the structure of a chol-lasiRNA-PS7 that targets MyD88. Underline: OMe modification, *: PS modification, and Chol: cholesterol.

FIG. 7 is a graphic diagram showing a comparison between the gene silencing efficiencies of a variety of cell-penetrating lasiRNAs (cp-lasiRNAs). The parenthesized CTGF or MyD88 represents a gene that is targeted by cp-lasiRNAs.

FIG. 8 is a graphic diagram showing the gene silencing efficiencies of the inventive nucleic acid molecules having various lipophilic compound modifications, that is, hydrophobic modifications.

FIG. 9 is a graphic diagram showing the gene silencing efficiency of the inventive nucleic acid molecule according to the lengths of the antisense strand.

FIG. 10 shows the structure of a PS2 modification.

FIG. 11 is a graphic diagram showing the gene silencing efficiencies of the inventive nucleic acid molecules having phosphate backbone modifications.

FIG. 12 is a graphic diagram showing the in vivo target gene silencing efficiencies of the nucleic acid molecules according to the present invention.

FIG. 13 is a graphic diagram showing the in vivo target gene silencing efficiency of the inventive nucleic acid molecule as a function of the concentration thereof.

FIG. 14 is a graphic diagram showing the target gene silencing efficiency of the inventive nucleic acid molecule as a function of duration.

BEST MODE FOR CARRYING OUT THE INVENTION

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Generally, the nomenclature used herein are well known and commonly used in the art.
The definition of main terms used in the detailed description of the invention is as follows.
As used herein, the term “RNAi” (RNA interference) refers to a mechanism by which a double-stranded RNA (dsRNA) consisting of a strand having a sequence complementary to the mRNA of a target gene and a strand having a sequence complementary thereto is introduced into cells or the like to induce the degradation of the mRNA of the target gene to thereby inhibit the expression of the target gene.
As used herein, the term “siRNA” (small interfering RNA) refers to a short double-stranded RNA (dsRNA) that mediates efficient gene silencing in a sequence-specific manner.
As used herein, the term “antisense strand” refers to a polynucleotide that is substantially or 100% complementary to a target nucleic acid of interest. For example, an antisense strand may be complementary, in whole or in part, to a molecule of mRNA (messenger RNA), an RNA sequence that is not mRNA (e.g., microRNA, piwiRNA, tRNA, rRNA and hnRNA) or a sequence of DNA that is either coding or non-coding. The terms “antisense strand” and “guide strand” are used interchangeably herein.
The term “sense strand” refers to a polynucleotide that has the same nucleotide sequence, in whole or in part, as a target nucleic acid, in which the polynucleotide is identical, in whole or in part, a molecule of mRNA (messenger RNA), an RNA sequence that is not mRNA (e.g., microRNA, piwiRNA, tRNA, rRNA and hnRNA) or a sequence of DNA that is either coding or non-coding.
As used herein, the term “gene” is intended to have the broadest meaning, and the gene can encode a structural protein or a regulatory protein. Herein, the regulatory protein includes a transcriptional factor, a heat shock proteins, or a protein that is involved in DNA/RNA replication, transcription and/or translation. Also, the target gene whose expression is to be inhibited is resident in a viral genome which has integrated into the animal gene or may be present as an extrachromosomal element. For example, the target gene may be a gene on an HIV genome. In this case, the genetic construct is useful in inactivating translation of the HIV gene in a mammalian cell.
In one aspect, the present invention is directed to an RNAi-inducing double-stranded nucleic acid molecule having cell-penetrating ability, the nucleic acid molecule comprising a first strand comprising a region complementary to a target nucleic acid; and a second strand that forms a complementary bond with the first strand, wherein the phosphate backbone of at least one nucleotide in the nucleic acid molecule was substituted with phosphorothioate or phosphorodithioate, and the nucleic acid molecule has a lipophilic compound conjugated thereto.
Herein, the first strand corresponds to an antisense strand of siRNA, and the second strand corresponds to a sense strand of siRNA.
In the present invention, the first strand in the RNAi-inducing double-stranded nucleic acid molecule may have a length of 16 to 121 nt, and preferably 24-121 nt. The first strand comprises a region complementary to the target nucleic acid, and the region complementary to the target nucleic acid may have a length of 16 to 31 nt, 19 to 31 nt, or 19 to 21 nt. In addition, the second strand may have a length of 13 to 25 nt, 13 to 21 nt, or 16 to 21 nt.
In the present invention, preferably, the RNAi-inducing double-stranded nucleic acid molecule may comprise a first strand, which is 24-121 nt in length and comprises a region complementary to a target nucleic acid, and a second strand which is 13-21 nt in length and comprises a region that binds complementarily to the region of the first strand, which is complementary to the target nucleic acid.
In an example of the present invention, the nucleic acid molecule having the above-described structure was constructed to have each of 24 sequences targeting CTGF, and as a result, it was found that the constructed nucleic acid molecules generally had high gene silencing efficiencies compared to conventional siRNAs. In the present invention, an RNAi-inducing double-stranded nucleic acid molecule having a long single-stranded region that does not form a complementary bond with the second strand, that is, an siRNA having a long antisense strand, has been named as “lasiRNA”.
The lasiRNA is a novel, asymmetrical RNAi-inducing structure that has a short double-strand length and high gene silencing efficiency, compared to conventional siRNA. In addition, due to the function of the antisense strand having a long overhang structure, the lasiRNA has increased gene silencing efficiency compared to siRNA or asiRNA, and thus will substitute for conventional structures to develop therapeutic agents. In addition, it is characterized in that in that it has a long overhang length compared to other structures, and maintains high activity even when the overhang is modified in various ways. By virtue of these characteristics, relatively many chemical modifications can be freely introduced into the lasiRNA, and thus various functions can be added to the lasiRNA.
In the present invention, the region of the first strand, which is complementary to the target nucleic acid, is preferably 19-21 nt in length. Thus, the first strand comprises a single-stranded region which does not bind to the second strand. Preferably, the first strand may further comprise, in the single-stranded region, a nucleic acid oligonucleotide selected from the group consisting of antisense DNA, antisense RNA, ribozyme and DNAzyme.
In the present invention, the single-stranded region of the first strand, which does not bind complementarily to the second strand, can be linked directly or by a linker to the region that binds complementarily to the second strand. Herein, the linker may be a chemical linker. Examples of the chemical linker include, but are not limited to, a nucleic acid moiety, PNA (a PNA moiety), a peptide moiety, a disulfide bond or a polyethylene glycol moiety.
Moreover, in the present invention, the first strand may further comprise, in the single-stranded region, a sequence that is complementary or non-complementary to the target nucleic acid. When the first strand comprises the complementary sequence, the complementary sequence may be located consecutively from the double-stranded region of the nucleic acid molecule of the present invention, that is, the region of siRNA, which is complementary to the target nucleic acid. Alternatively, the complementary sequence may also be located apart from the double-stranded region. Likewise, the sequence that is targeted by siRNA, and the sequence that is targeted by the ribozyme or DNAzyme of the single-stranded region may be located consecutively or located apart from each other. In addition, in the case in which the single-stranded region of the first strand has the sequence of siRNA, which is complementary to the target gene, when the sequence contained in the single-stranded region is antisense DNA or antisense RNA, the sequence may be at least about 70-80%, more preferably at least about 80-90%, and even more preferably at least 95-99% complementary to the sequence of the target gene targeted by the siRNA, and when the single-stranded region is ribozyme or DNAzyme, the sequence of the single-stranded region may be at least about 50-60% complementary to the sequence of the target gene targeted by the siRNA.
In addition, the single-stranded region may be 5-100 nt in length. If the length of the single-stranded region is less than 5 nt, the effect of increasing the efficiency with which gene expression is inhibited will be insignificant, and if the length is more than 100 nt, the efficiency with which an RNA molecule is synthesized will be reduced. Preferably, the single-stranded region may be 9-100 nt in length or 50 nt or less in length. More preferably, the single-stranded region may be 10-15 nt in length.
In the present invention, at least one of the nucleotides of the single-stranded region in the first strand may comprise a bulky base analog. When an extended sequence comprises a bulky base analog such as a deoxyadenosine derivative having a phenyl group, an mRNA strand that binds complementarily to the extended sequence is cleaved at the location of the bulky base analog. Any bulky base analog that induces this cleavage may be used without limitation in the present invention.
In the present invention, in case of a nucleic structure obtained by extending the antisense strand of siRNA in a manner complementary to a target mRNA sequence, it was predicted that the 5′ end of the nucleic structure will function as the RNAi mechanism while the 3′ end of the nucleic structure will function as an antisense mechanism or guide the 5′ end siRNA to the target mRNA. When the sequence of the antisense 3′-end, which is complementary to mRNA, is DNA, it can induce RNase H-dependent mRNA cleavage. In addition, it was predicted that when at least one of the nucleotides of the single-stranded region of the antisense 3′-end comprises a bulky base analog or the single-stranded region binds to mRNA to form a bulge structure, cleavage could be induced. Further, when a nucleic acid molecule comprising the ribozyme or DNAzyme introduced into the single-stranded region of the first strand can induce synergistic cleavage.
Korean Patent Laid-Open Publication No. 10-2009-0065880 discloses an siRNA structure which is an siRNA molecule consisting of a 19-21 nt antisense strand and a 13-16 nt sense strand, in which the 5′ end of the antisense strand is a blunt end. This siRNA structure inhibits gene expression at high efficiency without causing off-target effects by the sense strand of siRNA or inhibiting other RNAi mechanisms. When the structure of the present invention is applied to this siRNA, off-target effects can be minimized while the above-described effect of the nucleic acid oligonucleotide contained in the single-stranded region of the first strand can be obtained. As used herein, the term “off-target effects” refers to any instance in which the sense strand of siRNA causes the unexpected degradation of other mRNAs or the silencing of the corresponding genes, and the antisense strand of siRNA is paired with undesired targets to cause the degradation of other mRNAs or the silencing of the corresponding genes, even though siRNA is originally used to induce the degradation of mRNA having a sequence complementary to the antisense strand so as to obtain the effect of inhibiting the gene expression of the mRNA.
In an example of the present invention, it was shown that, when cholesterol modifications and PS modifications were performed, the cholesterol modifications increased the cell penetrating ability of the lasiRNA, but when a sufficient number of phosphorothioate (PS) modifications were not introduced, the use of cholesterol alone was not sufficient to effectively induce target gene silencing without a separate intracellular delivery system. Herein, it was shown that the introduction of PS modifications increased the cell penetrating ability in proportion to the number of PS modifications introduced, and when the number of PS modifications was too large, the lasiRNA did not induce RNAi-mediated gene silencing. For this reason, the optimum number of PS modifications was established by comparing gene silencing efficiency after incubation with cells. Specifically, the nucleic acid molecule according to the present invention may characterized in that the phosphate backbone of 1 to 48 nucleotides, preferably 1 to 31 nucleotides, more preferably 2 to 17 nucleotides, and even more preferably 4 to 17 or 12 to 17 nucleotides, is substituted with phosphorothioate.
Herein, the phosphate backbone of nucleotides in the first strand of the nucleic acid molecule may be substituted with phosphorothioate, and the phosphate backbones of nucleotides in a region of the first strand, which excludes a region complementary to a target nucleic acid, may be substituted with phosphorothioate. Herein, the phosphate backbone of 1 to 31 nucleotides, preferably 1 to 17 nucleotides, more preferably 2 to 17 nucleotides, and even more preferably 4 to 17 nucleotides or 12 to 17 nucleotides, in the first strand, may be substituted with phosphorothioate. In addition, the phosphate backbone of 1 to 21 nucleotides, preferably 1 to 17 nucleotides, more preferably 2 to 17 nucleotides, and even more preferably 4 to 17 nucleotides or to 17 nucleotides, in the second strand, may be substituted with phosphorothioate.
In another example of the present invention, it could be seen that the use of PS2 (phosphorodithioate) modification as shown in FIG. 10 in place of PS modification resulted in an increase in the gene silencing efficiency of the inventive nucleic acid molecule compared to conventional siRNA structures, even though it showed reduced gene silencing efficiency compared to PS modification. Thus, the nucleic acid molecule according to the present invention may be characterized in that the phosphate backbone of at least one nucleotide is substituted with phosphorodithioate. Preferably, the phosphate backbone of 1 to 48 nucleotides, preferably 1 to 31 nucleotides, more preferably 2 to 17 nucleotides, and even more preferably 4 to 17 or 12 to 17 nucleotides, in the nucleic acid molecule, may be substituted with phosphorodithioate. Herein, the phosphate backbone of 1 to 31 nucleotides, preferably 1 to 17 nucleotides, more preferably 2 to 17 nucleotides, and even more preferably 4 to or 12 to 17 nucleotides, in the first strand, may be substituted with phosphorodithioate. Alternatively, the phosphate backbone of 1 to 17 nucleotides, preferably 2 to 17 nucleotides, and even more preferably 4 to 17 or 12 to 17 nucleotides, in the second strand, may be substituted with phosphorodithioate.
The lipophilic compound that is used in the present invention results in hydrophobic modification, and may be, for example, a lipid, a lipophilic peptide or a lipophilic protein. As the lipid, cholesterol, tocopherol, or a long-chain fatty acid having 10 or more carbon atoms such as stearic acid or palmitic acid, may be used, but is not limited thereto. In addition, the lipophilic compound such as cholesterol may be conjugated to the 5′ or 3′ end of the first or second strand of the nucleic acid molecule, but is not limited thereto.
The target nucleic acid, not limited thereto, but might be mRNA (messenger RNA), microRNA, piRNA (piwi-interacting RNA), coding DNA sequence or non-coding DNA sequence or the like.
The nucleic acid molecule of the present invention may be a molecule synthesized according to a general method, but is not limited thereto. In other words, in the present invention, the siRNA molecule may be chemically or enzymatically synthesized. The siRNA molecule of the present invention may be derived from naturally occurring genes by standard recombinant techniques. In this case, the siRNA molecule may be substantially complementary at the nucleotide sequence level to at least a portion of mRNA of the target gene, the expression of which is to be modified.
Accordingly, the nucleic acid molecule of the present invention may comprise a chemical modification. The chemical modification may be obtained by replacing the hydroxyl group at position 2′ of ribose of at least one nucleotide, included in the nucleic acid molecule, by any one of a hydrogen atom, a fluorine atom, an —O-alkyl group, an —O-acyl group and an amino group, but is not limited thereto. In order to increase the ability to deliver the nucleic acid molecule, the hydroxyl group may be substituted by any one of —Br, —Cl, —R, —R′OR, —SH, —SR, —N₃and —CN (R=alkyl, aryl, or alkylene). In addition, the chemical modification may be obtained by replacing the phosphate backbone of at least one nucleotide, included in the nucleic acid molecule, by any one of alkylphosphonate form, phosphoroamidate form and boranophosphate form. Further, the chemical modification may be obtained by replacing at least one nucleotide included in the nucleic acid molecule by any one of LNA (locked nucleic acid), UNA (unlocked nucleic acid), morpholino and PNA (peptide nucleic acid). In addition, the chemical modification may be obtained by binding the nucleic acid molecule to one or more selected from the group consisting of lipids, cell penetrating peptides and cell targeting ligands.
In addition, the nucleic acid molecule according to the present invention may be efficiently used for in vitro and in vivo delivery together with various delivery vehicles, such as liposomes, cationic polymers, antibodies, aptamers or nanoparticles, and delivery methods, known to effectively deliver oligonucleotides into cells.
Meanwhile, in an example of the present invention, it was shown that, when the nucleic acid molecule of the present invention, dissolved in a solution such as PBS, was injected without using a separate delivery vehicle, it exhibited a high gene silencing efficiency of 90% or higher in a target area in vivo, suggesting that the nucleic acid molecule of the present invention can be developed directly into an injectable drug without needing a separate formulation process.
Examples of the present invention propose that the RNAi-inducing nucleic acid molecule according to the present invention exhibits a target gene silencing effect. Thus, in another aspect, the present invention is directed to a gene-silencing composition containing an RNAi-inducing nucleic acid molecule. Herein, the nucleic acid molecule may be contained in the form of a nucleic acid complex comprising a cell delivery vehicle bound thereto.
In an example of the present invention, it was found that, when the nucleic acid structure of the present invention was applied to an siRNA targeting the target gene CTGF, the efficiency with which the expression of the target gene is inhibited could be significantly increased, and the cell-penetrating ability thereof could also be maintained for a long period of time. Thus, it will be obvious to those skilled in the art that, even when nucleic acid molecules targeting other target genes are provided according to the present invention, the same results can be obtained.
Meanwhile, the composition for inhibiting gene expression according to the present invention may be provided in the form of a kit for inhibiting gene expression. The kit for inhibiting gene expression may take the form of bottles, tubs, sachets, envelops, tubes, ampoules, and the like, which may be formed in part or in whole from plastic, glass, paper, foil, wax, and the like. The container may be equipped with a fully or partially detachable lid that may initially be part of the container or may be affixed to the container by mechanical, adhesive, or other means. The container may also be equipped with a stopper, allowing access to the contents by a syringe needle. The kit may comprise an exterior package which may include instructions regarding the use of the components.
In still another aspect, the present invention is directed to a method of inhibiting expression of a target gene in a cell using the above RNAi-inducing nucleic acid molecule. That is, the present invention is directed to a method for inhibiting expression of a target gene in a cell, which comprises a step of introducing the above RNAi-inducing nucleic acid molecule into a cell.
In the present invention, the first strand of the RNAi-inducing nucleic acid may be complementary to the mRNA sequence of a target gene.
In the present invention, the target gene may be an endogeneous gene or a transgene.
The nucleic acid molecule according to the present invention is not necessarily limited to a synthetic siRNA and can also advantageously be applied to siRNA or shRNA, which is expressed in cells using an expression vector or the like. In other words, the nucleic acid molecule of the present invention can be expressed in cells to inhibit the expression of the target gene. Thus, in still another aspect, the present invention is directed to a method for inhibiting expression of a target gene in a cell, the method comprising a step of expressing the above RNAi-inducing nucleic acid molecule in the cell.
Meanwhile, the nucleic acid molecule according the present invention can target an mRNA encoding a connective tissue growth factor (CTGF). In an example of the present invention, it was found that the expression of CTGF was inhibited through the introduction of the nucleic acid molecule having the structure according to the present invention into the cell. Thus, in yet another aspect, the present invention is directed to a pharmaceutical composition for treating or preventing a connective tissue growth factor (CTGF)-associated disease or disorder, the composition containing the above nucleic acid molecule that targets a CTGF-encoding mRNA. The present invention also is directed to a method of treating or prevent nq a connective issue growth factor (CTGF)-associated disease or disorder comprising administering a pharmaceutical composition containing the above nucleic acid molecule targeting a CTGF-encoding mRNA.
In addition, the nucleic acid molecule of the present invention can be developed into therapeutic agents against localized diseases, and may be used together with various known cell-specific antibodies, aptamers, ligands or the like, and thus can be developed into therapeutic agents for gene regulation, which exhibit gene silencing effects only in a desired area.
The anticancer composition of the present invention may be provided as a pharmaceutical composition comprising the RNAi-inducing nucleic acid molecule alone or in combination with at least one pharmaceutically acceptable carrier, excipient or diluent. The nucleic acid molecule may be contained in the pharmaceutical composition in a pharmaceutically effective amount according to a disease and the severity thereof, the patient's age, weight, health condition and sex, the route of administration and the period of treatment.
As used herein, the term “pharmaceutically acceptable composition” refers to a composition that is physiologically acceptable and does not cause gastric disorder, allergic reactions such as gastrointestinal disorder or vertigo, or similar reactions, when administered to humans. Examples of said carrier, excipient or diluent may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate and mineral oils.
The pharmaceutical composition may additionally contain fillers, anti-aggregating agents, lubricants, wetting agents, perfumes, emulsifiers and preservatives. Also, the pharmaceutical composition of the present invention may be formulated using a method well known in the art, such that it can provide the rapid, sustained or delayed release of the active ingredient after administration to mammals. The formulation may be in the form of sterile injection solutions, etc.

EXAMPLES

Hereinafter, the present invention will be described in further detail with reference to examples. It will be obvious to a person having ordinary skill in the art that these examples are illustrative purposes only and are not to be construed to limit the scope of the present invention. In addition, it will be apparent to those skilled in that art that various modifications and variations can be made without departing from the technical scope of the present invention based on this illustration.

Example 1: Screening of RNAi-Inducing Double Stranded Nucleic Acid Molecules Targeting CTGF

Before introduction of various chemical modifications for effective self-delivery structures, in order to secure highly efficient, RNAi-inducing double-stranded nucleic acid molecules targeting CTGF, 50 sequences targeting CTGF were designed, followed by screening.
To compare CTGF gene-silencing efficiency between lasiRNA and conventional RNAi-inducing structures, the siRNA, asiRNA and lasiRNA structures having the nucleotide sequences shown in Tables 1 to 3 below were synthesized. Tables 1 to 3 show 24 nucleotide sequences for each of the siRNA, asiRNA and lasiRNA structures that target CTGF (Capital letters: RNA; small letters: DNA). To test the CTGF mRNA silencing effect of each of the structures having the nucleotide sequences, each of the structures was transfected into HaCaT (ATCC) cells at a concentration of 10 nM, and then the expression levels of CTGF mRNA in the cells were measured by real-time PCR.

TABLE 1

	SiRNA
No	NAME	SEQ	Sequence (5′→3′)

No1	SiRNA		1	sense	GCGAGGAGUGGGUGUGUGAtt
		2	antisense	UCCUCGCAGCAUUUCCCGGtt
	asiRNA
	3	sense	AGGAGUGGGUGUGUGA
		4	antisense	UCCUCGCAGCAUUUCCCGGtt
	lasiRNA
	5	sense	AGGAGUGGGUGUGUGA
		6	antisense	UCACACACCCACUCCUCGCAG
				CAUUUCCCGG

No2	siRNA
	7	sense	AGACCUGUGGGAUGGGCAUtt
		8	antisense	CAGGUCUUGGAACAGGCGCtt
	asiRNA	9	sense	CCUGUGGGAUGGGCAU
		10	antisense	CAGGUCUUGGAACAGGCGCtt
	lasiRNA
	11	sense	CCUGUGGGAUGGGCAU
		12	antisense	AUGCCCAUCCCACAGGUCUUG
				GAACAGGCGC

No3	siRNA
	13	sense	ACAGGAAGAUGUACGGAGAtt
		14	antisense	UUCCUGUAGUACAGCGAUUtt
	asiRNA
	15	sense	GGAAGAUGUACGGAGA
		16	antisense	UUCCUGUAGUACAGCGAUUtt
	lasiRNA
	17	sense	GGAAGAUGUACGGAGA
		18	antisense	UCUCCGUACAUCUUCCUGUAG
				UACAGCGAUU

No4	siRNA
	19	sense	GCACCAGCAUGAAGACAUAtt
		20	antisense	UAUGUCUUCAUGCUGGUGCtt
	asiRNA
	21	sense	CCAGCAUGAAGACAUA
		22	antisense	UAUGUCUUCAUGCUGGUGCtt
	lasiRNA
	23	sense	CCAGCAUGAAGACAUA
		24	antisense	UAUGUCUUCAUGCUGGUCCAG
				CCAGAAAGCU

No5	siRNA	25	sense	GAAGACAUACCGAGCUAAAtt
		26	antisense	UUUAGCUCGGUAUGUCUUCtt
	asiRNA	27	sense	GACAUACCGAGCUAAA
		28	antisense	UUUAGCUCGGUAUGUCUUCtt
	lasiRNA	29	sense	GACAUACCGAGCUAAA
		30	antisense	UUUAGCUCGGUAUGUCUUCAU
				GCUGGUGCAG

No6	siRNA	31	sense	GCUAAAUUCUGUGGAGUAUtt
		32	antisense	AUACUCCACAGAAUUUAGCtt
	asiRNA	33	sense	AAAUUCUGUGGAGUAU
		34	antisense	AUACUCCACAGAAUUUAGCtt
	lasiRNA	35	sense	AAAUUCUGUGGAGUAU
		36	antisense	AUACUCCACAGAAUUUAGCUC
				GGUAUGUCUU

No7	SiRNA	37	sense	GCGAGGUCAUGAAGAAGAAtt
		38	antisense	UUGUUCUUCAUGACCUCGCtt
	asiRNA	39	sense	AGGUCAUGAAGAAGAA
		40	antisense	UUGUUCUUCAUGACCUCGCtt
	lasiRNA	41	sense	AGGUCAUGAAGAAGAA
		42	antisense	UUGUUCUUCAUGACCUCGCCGU
				CAGGGCACU

No8	siRNA	43	sense	UGGAAGAGAACAUUAAGAAtt
		44	antisense	UUCUUAAUGUUCUCUUCCAtt
	asiRNA	45	sense	AAGAGAACAUUAAGAA
		46	antisense	UUCUUAAUGUUCUCUUCCAtt
	lasiRNA	47	sense	AAGAGAACAUUAAGAA
		48	antisense	UUCUUAAUGUUCUCUUCCAGG
				UCAGCUUCGC

(Capital letters: RNA; small letters: DNA)

TABLE 2

	SiRNA

No	NAME	Sequence (5′→3′)

No9	SiRNA	(49) sense	CGGCUUACCGACUGGAAGAtt
		(50) antisense	UCUUCCAGUCGGUAAGCCGtt
	asiRNA	(51) sense	CUUACCGACUGGAAGA
		(52) antisense	UCUUCCAGUCGGUAAGCCGtt
	lasiRNA	(53) sense	CUUACCGACUGGAAGA
		(54) antisense	UCUUCCAGUCGGUAAGCCGCG
			AGGGCAGGCC

No10	siRNA	(55) sense	GCAUGAAGCCAGAGAGUGAtt
		(56) antisense	UCACUCUCUGGCUUCAUGCtt
	asiRNA	(57) sense	UGAAGCCAGAGAGUGA
		(58) antisense	UCACUCUCUGGCUUCAUGCtt
	lasiRNA	(59) sense	UGAAGCCAGAGAGUGA
		(60) antisense	UCACUCUCUGGCUUCAUGCCC
			AUGUCUCCGU

No11	siRNA	(61) sense	CACCAUAGGUAGAAUGUAA11
		(62) antisense	UUACAUUCUACCUAUGGUGtt
	asiRNA	(63) sense	CAUAGGUAGAAUGUAA
		(64) antisense	UUACAUUCUACCUAUGGUGtt
	lasiRNA	(65) sense	CAUAGGUAGAAUGUAA
		(66) antisense	UUACAUUCUACCUAUGGUGUU
			CAGAAAUUGA

No12	siRNA	(67) sense	CCUGCAGGCUAGAGAAGCAtt
		(68) antisense	UGCUUCUCUAGCCUGCAGGtt
	asiRNA	(69) sense	GCAGGCUAGAGAAGCA
		(70) antisense	UGCUUCUCUAGCCUGCAGGtt
	lasiRNA	(71) sense	GCAGGCUAGAGAAGCA
		(72) antisense	UGCUUCUCUAGCCUGCAGGAG
			GCGUUGUCAU

No13	SiRNA	(73) sense	CCAGAGAGUGAGAGACAUUtt
		(74) antisense	AAUGUCUCUCACUCUCUGGtt
	asiRNA	(75) sense	GAGAGUGAGAGACAUU
		(76) antisense	AAUGUCUCUCACUCUCUGGtt
	lasiRNA	(77) sense	GAGAGUGAGAGACAUU
		(78) antisense	AAUGUCUCUCACUCUCUGGC
			UUCAUGCCAUG

No14	siRNA	(79) sense	GCGAAGCUGACCUGGAAGAtt
		(80) antisense	UCUUCCAGGUCAGCUUCGCtt
	asiRNA	(81) sense	AAGCUGACCUGGAAGA
		(82) antisense	UCUUCCAGGUCAGCUUCGCtt
	lasiRNA	(83) sense	AAGCUGACCUGGAAGA
		(84) antisense	UCUUCCAGGUCAGCUUCGCA
			AGGCCUGACCA

No15	siRNA	(85) sense	CCGGAGACAAUGACAUCUUtt
		(86) antisense	AAGAUGUCAUUGUCUCCGGtt
	asiRNA	(87) sense	GAGACAAUGACAUCUU
		(88) antisense	AAGAUGUCAUUGUCUCCGGtt
	lasiRNA	(89) sense	GAGACAAUGACAUCUU
		(90) antisense	AAGAUGUCAUUGUCUCCGGGA
			CAGUUGUAAU

No16	siRNA	(91) sense	UCUUUGAAUCGCUGUACUAtt
		(92) antisense	UAGUACAGCGAUUCAAAGAtt
	asiRNA	(93) sense	UUGAAUCGCUGUACUA
		(94) antisense	UAGUACAGCGAUUCAAAGAtt
	lasiRNA	(95) sense	UUGAAUCGCUGUACUA
		(96) antisense	UAGUACAGCGAUUCAAAGAUG
			UCAUUGUCUC

(Capital letters: RNA; small letters: DNA)

TABLE 3

		SEQ
	SiRNA	ID
No	NAME	NO	Sequence (5′→3′)

No17	SiRNA	97	sense	UUGCGAAGCUGACCUGGAAtt
		98	antisense	UUCCAGGUCAGCUUCGCAAtt
	asiRNA	99	sense	CGAAGCUGACCUGGAA
		100	antisense	UUCCAGGUCAGCUUCGCAAtt
	lasiRNA	101	sense	CGAAGCUGACCUGGAA
		102	antisense	UUCCAGGUCAGCUUCGCAAGG
				CCUGACCAUG

No18	SiRNA	103	sense	CAACUAUGAUUAGAGCCAAtt
		104	antisense	UUGGCUCUAAUCAUAGUUGtt
	asiRNA	105	sense	CUAUGAUUAGAGCCAA
		106	antisense	UUGGCUCUAAUCAUAGUUGtt
	lasiRNA	107	sense	CUAUGAUUAGAGCCAA
		108	antisense	UUGGCUCUAAUCAUAGUUGG
				GUCUGGGCCAA

No19	siRNA	109	sense	GUACCAGUGCACGUGCCUGtt
		110	antisense	CAGGCACGUGCACUGGUACtt
	asiRNA	111	sense	CCAGUGCACGUGCCUG
		112	antisense	CAGGCACGUGCACUGGUACtt
	lasiRNA	113	sense	CCAGUGCACGUGCCUG
		114	antisense	CAGGCACGUGCACUGGUACUU
				GCAGCUGCUC

No20	siRNA	115	sense	AGUGCAUCCGUACUCCCAAtt
		116	antisense	UUGGGAGUACGGAUGCACUtt
	asiRNA	117	sense	GCAUCCGUACUCCCAA
		118	antisense	UUGGGAGUACGGAUGCACUtt
	lasiRNA	119	sense	GCAUCCGUACUCCCAA
		120	antisense	UUGGGAGUACGGAUGCACUUU
				UUGCCCUUCU

No21	siRNA	121	sense	CAUGAUGUUCAUCAAGACCtt
		122	antisense	GGUCUUGAUGAACAUCAUGtt
	asiRNA	123	sense	GAUGUUCAUCAAGACC
		124	antisense	GGUCUUGAUGAACAUCAUGtt
	lasiRNA	125	sense	GAUGUUCAUCAAGACC
		126	antisense	GGUCUUGAUGAACAUCAUGUU
				CUUCUUCAUG

No22	siRNA	127	sense	CCAUGACCGCCGCCAGUAUtt
		128	antisense	AUACUGGCGGCGGUCAUGGtt
	asiRNA	129	sense	UGACCGCCGCCAGUAU
		130	antisense	AUACUGGCGGCGGUCAUGGtt
	lasiRNA	131	sense	UGACCGCCGCCAGUAU
		132	antisense	AUACUGGCGGCGGUCAUGGUU
				GGCACUGCGG

No23	siRNA	133	sense	GAACAUUAAGAAGGGCAAAtt
		134	antisense	UUUGCCCUUCUUAAUGUUCtt
	asiRNA	135	sense	CAUUAAGAAGGGCAAA
		136	antisense	UUUGCCCUUCUUAAUGUUCtt
	lasiRNA	137	sense	CAUUAAGAAGGGCAAA
		138	antisense	UUUGCCCUUCUUAAUGUUCUC
				UUCCAGGUCA

No24	siRNA	139	sense	GGAAGACACGUUUGGCCCAtt
		140	antisense	UGGGCCAAACGUGUCUUCCtt
	asiRNA	141	sense	AGACACGUUUGGCCCA
		142	antisense	UGGGCCAAACGUGUCUUCCtt
	lasiRNA	143	sense	AGACACGUUUGGCCCA
		144	antisense	UGGGCCAAACGUGUCUUCCAG
				UCGGUAAGCC

(Capital letters: RNA; small letters: DNA)

Specifically, HaCat cells were cultured in Dulbecco's modified Eagle's medium (Gibco) (supplemented with 10% fetal bovine serum (Gibco) and 100 μg/ml penicillin/streptomycin) in a 100 mm Petri dish. Immediately before transfection, 8×10⁴Hacat cells were seeded into a 12-well plate. Meanwhile, each of the siRNA, asiRNA and lasiRNA was diluted in 1×siRNA duplex buffer (Biosesang Co., Ltd.) at a suitable concentration, and incubated at 90° C. for 2 min and at 37° C. for hour. The annealed siRNAs were electrophoresed on 10% polyacrylamide gel, and then stained with EtBr for 5 min, and the bands were visualized by a UV transilluminator. The siRNAs were transfected into the cells according to the manual provided in Lipofectamine 2000 (Invitrogen), and after 24 hours, the mRNA levels in the cells were measured.
Specifically, after transfection, total RNA was extracted using Isol-RNA lysis reagent (SPRIME), and 500 ng of the RNA was used for cDNA synthesis. cDNA was synthesized using a high-capacity cDNA reverse transcription kit (Applied Biosystems) according to the protocol provided in the kit. The synthesized cDNA was diluted, and then subjected to quantitative real-time PCR using a step one real-time PCR system (Applied Biosystems) according to the protocol provided in the system. The target gene was analyzed using a power SYBR green PCR master mix (Applied Biosystems) with gene-specific primers. The nucleotide sequences of the primers used in the experiment are as follows:
GAPDH-forward 5′-GAG TCA ACG GAT TTG GTC GT-3′(SEQ ID NO: 145)
GAPDH-reverse 5′-GAC AAG CTT CCC GTT CTC AG-3′(SEQ ID NO: 146)
CTGF-forward 5′-CAA GGG CCT CTT CTG TGA CT-3′(SEQ ID NO: 147)
CTGF-reverse 5′-ACG TGC ACT GGT ACT TGC AG-3′(SEQ ID NO: 148)
FIG. 1 shows the results of screening of 24 nucleotide sequences. As shown therein, in 14 sequences among a total of 24 nucleotide sequences, lasiRNAs showed increased activity compared to siRNAs (lasiRNAs showed an increase in gene silencing efficiency of 20% or higher compared to siRNA), and in 5 sequences, siRNAs showed high gene silencing efficiency compared to lasiRNAs, suggesting that lasiRNAs generally show high gene silencing efficiency compared to conventional siRNAs.
Particularly, the IC50 of the siRNAs and lasiRNA showing a gene silencing efficiency of 90% or higher was measured, and as a result, it was shown that lasiRNAs having nucleotide sequences of Nos. 9 and 16 had the lowest IC50. Among them, the nucleotide sequence of No. 9 was selected as a final candidate for modification and self-delivery experiments. Information about the nucleotide sequence of No. 9 is shown in Table 4 below.

TABLE 4

RNAi-inducing double-stranded
nucleic acid molecule

SiRNA

Sequence

No	name	Sequence (5′-> 3′)	listing

No9	siRNA	sense	CGGCUUACCGA	149
			CUGGAAGAtt
		antisense	UCUUCCAGUCG	150
			GUAAGCCGtt

	asiRNA	sense	CUUACCGACUG	151
			GAAGA
		antisense	UCUUCCAGUCG	152
			GUAAGCCGtt

	lasiRNA	sense	CUUACCGACUG	153
			GAAGA
		antisens	UCUUCCAGUCG	154
			GUAAGCCGCGA
			GGGCAGGCC

(Capital letters: RNA; small letters: DNA)

Example 2: Preparation of Nucleic Acid Molecule According to the Present Invention, and Measurement of Intracellular Uptake Efficiency Thereof

2-1: Effect of Cholesterol Modification
In order to examine the effect of cholesterol modification on the delivery of lasiRNA, the 5′ end of the lasiRNA sense strand, that is, the second strand, was labeled with cy3, and then the difference in uptake of lasiRNA between the presence and absence of cholesterol was observed by a fluorescence microscope. Specifically, the cy3-labeled lasiRNA or chol-lasiRNA structure was incubated in HeLa cells at a concentration of 1 μM for 3 hours, and then the degree of intracellular delivery thereof was measured by observation with a fluorescence microscope.
Specifically, HeLa cells (ATCC) were cultured in Dulbecco's modified Eagle's medium (Gibco) (supplemented with 10% fetal bovine serum (Gibco) and 100 μg/ml penicillin/streptomycin) in a 100 mm Petri dish.
Each of cholesterol-modified lasiRNAs was diluted in single-strand Accell siRNA delivery medium (Thermo scientific) at a suitable concentration, and the cholesterol-modified single strand was incubated at 90° C. for 20-30 sec before annealing. The sense strand and the antisense strand were mixed with each other, and then incubated at 90 for 30 sec and at 37° C. for 1 hour, followed by annealing. The resulting siRNAs were electrophoresed on 10% polyacrylamide gel, and then stained with EtBr for 5 min, and the bands were visualized by a UV transilluminator.
For an incubation test, 2×10⁵HeLa cells were seeded into a cover glass-bottom dish (SPL) at 24 hours before treatment with lasiRNA. After removal of culture media from the prepared dish, the cells were washed twice with 2 ml of 1×DPBS. siRNA, diluted in 100 μL of Accell siRNA delivery medium (Thermo scientific) pre-warmed in a water bath at 37° C., was added to and incubated in the cells. After 3 hours, the Accell medium was removed, and the cells were washed twice with 1×DPBS, and then incubated with 1 ug/ml of Hoechst 33343 (Sigma) in Opti-MEM (gibco) at 37° C. for 10 min to stain the nucleus. After removal of the Hoechst, the cells were washed twice with 1×DPBS, and then added to Opti-MEM medium and observed with a fluorescence microscope (Microscope—Olympus IX81, software—MetaMorph).
As can be seen in FIG. 2, when the intracellular uptake efficiency of cholesterol-modified lasiRNA was examined, and as a result, it could be seen that little or no cy3 fluorescence was observed in the cells in the absence of cholesterol, but lasiRNA-chol obtained by conjugating cholesterol to lasiRNA showed very strong fluorescence.
This suggests that the intracellular delivery of the lasiRNA structure was increased by cholesterol modification.
2-2: Effect of PS Modification
Additionally, in order to examine whether the direct introduction of phosphorothioate (PS) modification into lasiRNA increases the uptake efficiency of the lasiRNA, PS modification was introduced into the 3′ overhang of the antisense strand (i.e., first strand) of chol-lasiRNA, and the change in uptake efficiency of the chol-lasiRNA by PS modification was tested. Each of cy3-labelled chol-lasiRNA-PS(N) structures was incubated in HeLa cells at a concentration of 1 μM for 3 hours, and then the degrees of intracellular delivery of the structures was compared by observation with a fluorescence microscope. For an accurate comparison between cell penetrating abilities between the structures, the condition in which chol-lasiRNA-PS0 shows the lowest fluorescence was set, after which the fluorescence intensities of other structures were compared.
Specifically, as shown in FIG. 3, 0, 4, 7, 12 or 17 PS modification(s) were introduced into the 3′ end of the antisense strand of the Chol-lasiRNA structure, which was then incubated in or transfected into HeLa cells. Then, as described in Example 2-1, the difference in delivery efficiency by the number of PS modifications was observed with a fluorescence microscope. In FIG. 3, the underline and the red color represent OMe modification, represents PS modification, Chol represents cholesterol, and Cy3 represents Cy3 fluorescent dye.
As a result, as shown in FIG. 4, in the case of chol-lasiRNA-PS0 having no PS modification, little or no fluorescence was observed in the HeLa cells, and chol-lasiRNA-PS0 showed low uptake efficiency compared to other samples.
In addition, it was observed that fluorescence became brighter as the number of PS modifications in the antisense strand (i.e., first strand) of lasiRNA increased, and among all the samples, chol-lasiRNA-PS12 and chol-lasiRNA-PS17 having 12 and 17 PS modifications, respectively, showed the brightest fluorescence, indicating that the amount of internalized lasiRNA increased with an increase in the number of PS modifications in chol-lasiRNA.

Example 3: Measurement of CTGF-Silencing Efficiency

The results of the internalization experiment carried out using Cy3-labeled lasiRNA in Example 2 indicated that the direct introduction of cholesterol and PS modifications into the lasiRNA structure enables the lasiRNA to be effectively delivered into cells without needing a delivery vehicle or an additional reagent. However, it is known that when various chemical modifications are introduced into siRNA, the activity of the siRNA somewhat decreases, or the activity of siRNA decreases rapidly depending on the kind of modification. Thus, in order to examine the effect of each modification on the activity of lasiRNA, various lasiRNA structures were transfected into HeLa cells, and then a change in the expression of CTGF mRNA in the cells was measured to determine the effect of each of the modifications on the gene silencing efficiency of the lasiRNA.
In order to examine the effect of PS modification on the gene silencing efficiency of lasiRNA, various PS-modified lasiRNA [chol-lasiRNA-PS(N)] structures were transfected into HeLa cells, and then the expression levels of the CTGF gene in the cells were measured. Specifically, each of chol-lasiRNA-PS(N) structures was transfected into HeLa cells at a concentration of 10 nM, and after 48 hours, the expression levels of CTGF mRNA in the cells were measured by real-time PCR.
Subsequently, at 24 hours before the experiment, 2.5×10⁴HeLa cells were seeded into a 24-well plate. Then, each of the lasiRNAs was transfected using Lipofectamine 2000 according to the protocol provided therein. Next, the cells were cultured in a 5% CO₂incubator for 48 hours, and then the expression level of mRNA in the cells was measured according to the method described in Example 1.
As a result, as shown in FIG. 5, the gene silencing efficiency of the lasiRNAs showed a tendency to decrease as the number of PS modifications in the antisense strand increased, and when 12 or more PS modifications were introduced into the antisense strand, a slight decrease in the silencing activity was observed. Also, it was shown that chol-lasiRNA-PS17 having 17 PS modifications introduced into the antisense strand showed significantly low gene silencing efficiency, and thus showed little or no silencing effect on CTGF, suggesting that the number of PS modifications in the antisense strand is preferably 17 or less, and that 17 or more PS modifications are not suitable for self-delivery of the lasiRNA. Each graph in FIG. 5 represents the mean±SD of three repeated experiments.
Additionally, an increase in the number of PS modifications leads to an increase in the self-delivery efficiency of chol-lasiRNA, but has the disadvantage of reducing the silencing activity of the lasiRNA. In order to establish the optimum modification structure enabling silencing to be induced without needing a vehicle, chol-lasiRNA-PS(N) structures having varying numbers of PS modifications were incubated with HeLa cells, and then the CTGF mRNA levels of the cells were measured to compare the gene silencing efficiencies of the structures. Herein, the cells were treated with 0.1 μM, 0.3 μM and 1 μM of each of the lasiRNAs, and chol-lasiRNA-PS7 (FIG. 6; red: OMe modification; *: PS modification; Chol: cholesterol) targeting MyD88 was also used as a control. Specifically, HeLa cells were incubated with CTGF- or MyD88-targeting chol-lasiRNA-PS(N) structures for 48 hours, and then the expression levels of CTGF mRNA in the cells were measured by real-time PCR.
As a result, as can be seen in FIG. 7, chol-lasiRNA-PS4 showed a gene silencing effect of only about 55% even at the highest concentration (1 μM), and chol-lasiRNA-PS7 and chol-lasiRNA-PS12 showed a CTGF silencing effect of about 95% or higher at 1 μM. For a more accurate comparison of gene silencing efficiency, each of the structures was incubated at lower concentrations, and then the CTGF mRNA level was measured, and the results of the measurement indicated that PS12 most efficiently silenced the CTGF gene even at low concentrations. In addition, it was observed that chol-lasiRNA-PS17 had a gene silencing effect of about 50% even when it was incubated at a high concentration (1 μM), like when it was transfected, suggesting that it is required to optimize the number of PS modifications suitable for increasing the delivery and silencing activity, rather than to introduce a too large number of PS modifications. In addition, MyD88-targeting chol-lasiRNA-PS7 showed no CTGF silencing efficiency, indicating that gene silencing by the cp-lasiRNA structures occurs in a sequence-specific manner.

Example 4: Measurement of Intracellular Uptake Efficiencies Resulting from Modifications with Other Lipophilic Compounds

In order to examine the effects of lipophilic modifications (hydrophobic modifications) other than cholesterol modification, cp-lasiRNA (cell penetrating lasiRNA) structures according to the present invention, which target survivin gene, were prepared using the following sequences. Herein, cp-lasiRNA-1 has cholesterol conjugated thereto, cp-lasiRNA-2 has conjugated thereto tocopherol in place of cholesterol, and cp-lasiRNA-3 has stearic acid conjugated to the 5′ end of the sense strand in place of cholesterol.

	<cp-lasiRNA (survivin) 31mer>
	cp-lasiRNA (survivin) Antisense 31nt:
	(SEQ ID NO: 169)
	5′ UGAAAAUGUUGAUCUCCUUUCCUAAGACATT 3′

	cp-lasiRNA (survivin) Sense:
	(SEQ ID NO: 170)
	5′ GAGAUCAACAUUUUCA*cholesterol. 3′

	Underline: OMe modification;
	*: PS: phosphorothioate bond.

Each of cp-lasiRNA-1, cp-lasiRNA-2 and cp-lasiRNA-3 was incubated in A549 cells (ATCC) at a concentration of 300 mM for 24 hours according to the method described in Example 2, and then the expression levels of survivin mRNA in the cells were measured by real-time PCR. FIG. 8 shows the mean±DS of two repeated experiments for each of the cp-lasiRNA structures.
After transfection, total RNA was extracted using Isol-RNA lysis reagent (SPRIME), and 500 ng of the RNA was used for cDNA synthesis. cDNA was synthesized using a high-capacity cDNA reverse transcription kit (Applied Biosystems) according to the protocol provided therein. The synthesized cDNA was diluted, and then subjected to quantitative real-time PCR using a step one real-time PCR system (Applied Biosystems) according to the protocol provided therein. The target gene was analyzed using a power SYBR green PCR master mix (Applied Biosystems) together with gene-specific primers. The nucleotide sequences of the primers used in the experiment are as follows:

	Survivin
	Forward
	(SEQ ID NO: 172)
	5′-GCA CCA CTT CCA GGG TTT AT-3′

	Reverse
	(SEQ ID NO: 173)
	5′-CTC TGG TGC CAC TTT CAA GA-3′

As a result, as can be seen in FIG. 8, hydrophobic modifications other than cholesterol modification enabled the target gene to be silenced with high efficiency. In addition, stearyl showed high gene silencing efficiency, even though it was conjugated to the 5′ end of the sense strand, suggesting that the nucleic acid molecule according to the present invention can achieve the desired effect even when a lipophilic compound (hydrophobic modification) is conjugated to various positions of the nucleic acid molecule.

Example 5: Examination of Target Gene Silencing Efficiency According to the Length of Antisense Strand

In order to examine the target gene silencing efficiency of the inventive nucleic acid molecule according to the length of the first strand thereof, each of 31-nt antisense and 21-nt antisense strands was combined with a 16-nt second strand (sense strand) to make cp-lasiRNAs, and then A549 cells were treated with each of the cp-lasiRNAs.

	<cp-lasiRNA (survivin) 31mer>
	cp-lasiRNA (survivin) Antisense 31nt:
	(SEQ ID NO: 169)
	5′ UGAAAAUGUUGAUCUCCUUUCCUAAGACATT 3′

	cp-lasiRNA (survivin) Sense:
	(SEQ ID NO: 170)
	5′ GAGAUCAACAUUUUCA*cholesterol. 3′

	<cp-lasiRNA (survivin) 21mer>
	cp-lasiRNA (survivin) Antisense 21nt:
	(SEQ ID NO: 171)
	5′ UGAAAAUGUUGAUCUCCUUUCC 3′

	cp-lasiRNA (survivin) Sense:
	(SEQ ID NO: 170)
	5′ GAGAUCAACAUUUUCA*cholesterol. 3′
	Underline: OMe modification,
	*: PS (phosphorothioate bond)

	<cp-lasiRNA (CTGF) 31mer>
	cp-lasiRNA (CTGF) Antisense 31nt:
	(SEQ ID NO: 174)
	5′ UCUUCCAGUCGGUAAGCCGCGAGGGCAGGCC 3′

	cp-lasiRNA (CTGF) Sense:
	(SEQ ID NO: 175)
	5′ CTTACCGACTGGAAGA*chol. 3′

	<cp-lasiRNA (CTGF) 21mer>
	cp-lasiRNA (CTGF) Antisense 21nt:
	(SEQ ID NO: 176)
	5′ UCUUCCAGUCGGUAAGCCGCG 3′

	cp-lasiRNA (CTGF) Sense:
	(SEQ ID NO: 175)
	5′ CTTACCGACTGGAAGA*chol. 3′
	Underline: OMe modification,
	*: PS (phosphorothioate bond)

Specifically, each of the nucleic acid molecules was transfected into A549 cells (ATCC) according to the method of Example 1 or incubated in A549 cells for 24 hours according to the method of Example 2. Then, the expression levels of target gene mRNA in the cells were measured by real-time PCR. FIG. 9 shows the mean±SD of two repeated experiments for each of the nucleic acid molecules. Specifically, FIG. 9A shows the gene silencing efficiency of a CTGF-targeting cp-lasiRNA having a 21 mer antisense strand; FIG. 9B shows the gene silencing efficiency of a CTGF-targeting cp-lasiRNA having a 31 mer antisense strand; FIG. 9C shows the gene silencing efficiency of a survivin-targeting cp-lasiRNA having a 21 mer antisense strand; and FIG. 9D shows the gene silencing efficiency of a survivin-targeting cp-lasiRNA having a 31 mer antisense strand. The CTGF silencing efficiency was measured using the primers described in Example 1, and the surviving silencing efficiency was measured using the primers described in Example 4.
As shown in FIG. 9, when the CTGF-targeting cp-lasiRNA was transfected or incubated, the target gene silencing efficiency thereof was higher in the case of the 31-nt antisense stand than in the case of the 21-nt antisense strand (FIGS. 9A and 9B). Likewise, when the surviving-targeting cp-lasiRNA was incubated, the target gene silencing efficiency thereof was higher in the case of the 31-nt antisense strand. Thus, it can be seen that the nucleic acid molecule according to the present invention can be designed to have an antisense strand (i.e., first strand) having varying lengths of 19 nt to 31 nt, and can be used to effectively silence a target gene, but it can more efficiently silence a target gene when it has a 31-nt antisense strand, compared to when it has a 21-nt antisense strand.

Example 6: Examination of Effect of PS2 Modification

The effect of the modification of the phosphate backbone of at least one nucleotide in the nucleic acid molecule with phosphorodithioate (PS2; having a structure shown in FIG. 10) in place of phosphorothioate was examined as follows.
Specifically, each of the following cp-lasiRNA (Survivin) and the following cp-lasiRNA (Survivin)-PS2, obtained by introducing PS2 modification in place of PS modification into the cp-lasiRNA (Survivin), was transfected into or incubated with A549 cells for 24 hours according to the method described in Example 1 or 2. Then, the expression levels of the survivin genes in the cells were measured by real-time PCR in the same manner as described in Example 4. Each graph in FIG. 11 represents the mean±SD of two repeated experiments for each of the cp-lasiRNA (Survivin) structures.

	<cp-lasiRNA (survivin)>
	cp-lasiRNA (survivin) Antisense 31nt:
	(SEQ ID NO: 169)
	5′ UGAAAAUGUUGAUCUCCUUUCCUAAGACATT 3′

	cp-lasiRNA (survivin) Sense:
	(SEQ ID NO: 170)
	5′ GAGAUCAACAUUUUCA*cholesterol. 3′

	Underline: OMe modification,
	*: PS (phosphorothioate bond or
	phosphorodithioate bond)

As a result, as can be seen in FIG. 11, an increase in the gene silencing effect by additional sulfur modification (PS2) was not observed, and the cp-lasiRNA structure having PS2 modification showed reduced gene silencing efficiency compared to the conventional cp-lasiRNA.

Example 7: Measurement of In Vivo Target Gene Silencing Efficiency of Nucleic Acid According to the Present Invention

In the current development of therapeutic agents based on RNAi technology, the development of effective in vivo RNA delivery technology is most difficult. Many delivery technologies developed to date show high in vitro delivery efficiency, but have a problem in that, when these are applied in vivo, the efficiency thereof significantly decreases. Thus, in order to examine whether the nucleic acid molecule according to the present invention has a high gene silencing effect even in vivo, cp-lasiRNA alone was injected into the skin of rats without using a separate delivery vehicle, and the target gene silencing effect thereof was measured.
Specifically, siRNA (CTGF), cp-lasiRNA (CTGF) or cp-lasiRNA (Scrambled) was dissolved in 100 μl of PBS at concentrations shown in FIG. 12, and each of the solutions was injected intradermally into the rat skin, and after 24 hours, the skin tissue was collected, and the expression of the target gene therein was measured. Specifically, SD rats (Orient Bio Inc.) were anesthetized by intraperitoneal injection with Zoletil and Rompun solution, and then the back of the rats was shaved. A circle having a radius of 5 mm was drawn on the shaved skin portion, and then 100 μl of PBS, siRNA or cp-lasiRNA was injected intradermally into the central portion of the circle by an insulin syringe (BD, 31G). After injection, the skin tissue was taken using 8 mm biopsy punch at the indicated date, and the expression of gene therein was analyzed. The nucleic acids used are as follows.

	cp-lasiRNA (CTGF) Antisense Rat:
	(SEQ ID NO: 177)
	5′-UCUUCCAGUCGGUAGGCAGCUAGGGCAGGGC-3′

	cp-lasiRNA (CTGF) Sense Rat:
	(SEQ ID NO: 178)
	5′-CCTACCGACTGGAAGA*choleterol. 3′

	Underline: OMe modification,
	*: PS (phosphorothioate bond)

The followings were used as siRNA:

	siRNA (CTGF) antisense:
	(SEQ ID NO: 179)
	5′-CUGCCUACCGACUGGAAGATT-3′

	siRNA (CTGF) sense:
	(SEQ ID NO: 180)
	5′-CUGCCUACCGACUGGAAGATT-3′

	Underline: OMe modification

Herein, RNA was extracted using an RNeasy fibrous tissue mini kit (Qiagen), and 1 μg of the RNA was used for cDNA synthesis. cDNA was synthesized using a high-capacity cDNA reverse transcription kit (Applied Biosystems) according to the protocol provided therein. The synthesized cDNA was diluted, and then subjected to quantitative real-time PCR using a step one real-time PCR system (Applied Biosystems) according to the protocol provided therein. The target gene was analyzed using a power SYBR green PCR master mix (Applied Biosystems) together with gene-specific primers. The nucleotide sequences of the primers used in the experiment are as follows. Each graph in FIG. 12 represents the mean±SD of five repeated experiments.

	CTGF-Rat
	Forward
	(SEQ ID NO: 181)
	5′-GGC TCG CAT CAT AGT TG-3′

	Reverse
	(SEQ ID NO: 182)
	5′-CGG GAA ATG CTG TGA GGA GT-3′

siRNA (CTGF), cp-lasiRNA (CTGF) or cp-lasiRNA (Scrambled) was dissolved in 100
of PBS at the indicated concentrations, and each of PBS and the solutions was injected intradermally into the rat skin, and after 24 hours, the skin tissue was collected, and the expression of the target gene therein was measured. Each graph in FIG. 12 represents the mean±SD of five repeated experiments.
As a result, as can be seen in FIG. 12, the expression of CTGF in the group treated with cp-lasiRNA (CTGF) decreased by 80-90% or higher compared to that in the group treated with PBS, cp-lasiRNA (scrambled) or siRNA (CTGF), suggesting that cp-lasiRNA can highly efficiently silence the target gene even in vivo.
Additionally, in order to examine the in vivo gene silencing efficiency of Cp-lasiRNA, cp-lasiRNA was injected into rats at a concentration ranging from 100 μg/injection to 0.1 μg/injection in the same manner as described above, and then the expression of the target gene was measured.
As a result, as shown in FIG. 13, cp-lasiRNA (CTGF) showed a target gene silencing efficiency of 70% or higher even at a low concentration of about 0.3 μg/injection, and had an IC50 value of about 0.21 μg/injection. Each graph in FIG. 13 represents the mean±SD of two repeated experiments.
Additionally, cp-lasiRNA (CTGF) was injected in the same manner as described above, and then on day 1, day 2, day 3 and day 6, the tissue was analyzed to measure the expression of the gene. cp-lasiRNA (CTGF) was injected intradermally, after which the tissue was collected at the indicated date, and the expression of CTGF therein was analyzed by real-time PCR.
As a result, as shown in FIG. 14, it was found that cp-lasiRNA (CTGF) silenced the target gene for at least 5 days. Each graph in FIG. 14 represents the mean±SD of two repeated experiments.

INDUSTRIAL APPLICABILITY

As described above, the nucleic acid structure according to the present invention has both cholesterol modification and phosphorothioate modification introduced therein, and thus has high gene silencing efficiency while having the ability to penetrate cells without needing a separate intracellular delivery vehicle. Thus, it can be delivered into an actual target area in an amount sufficient for induction of RNAi, and thus can overcome the in vivo delivery problem occurring in the prior art. Therefore, the nucleic acid molecule according to the present invention can effectively substitute for conventional siRNA molecules to treat cancer or viral infections.
Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims

1. An RNAi-inducing double-stranded nucleic acid molecule having cell-penetrating ability, wherein the nucleic acid molecule comprising a first strand comprising a region complementary to a target nucleic acid and a second strand that forms a complementary bond with the first strand; and wherein the phosphate backbone of at least one nucleotide in the nucleic acid molecule was substituted with phosphorothioate or phosphorodithioate, and the nucleic acid molecule has a lipophilic compound conjugated thereto.

2. The nucleic acid molecule of claim 1, wherein the RNAi-inducing double-stranded nucleic acid molecule comprises a first strand comprising a region complementary to a target nucleic acid, which is 24-121 nt in length, and a second strand which is 13-21 nt in length and comprises a region that binds complementarily to the region of the first strand, which is complementary to the target nucleic acid.

3. The nucleic acid molecule of claim 2, wherein the 5′ end of the first strand is a blunt end.

4. The nucleic acid molecule of claim 2, wherein the region of the first strand, which is complementary to the target nucleic acid, is 19-31 nt in length.

5. The nucleic acid molecule of claim 1, wherein the phosphate backbone(s) of 1 to 48 nucleotides is (are) substituted with phosphorothioate or phosphorodithioate.

6. The nucleic acid molecule of claim 5, wherein the phosphate backbone(s) of 1 to 31 nucleotides in the first strand of the nucleic acid molecule is (are) substituted with phosphorothioate or phosphorodithioate.

7. The nucleic acid molecule of claim 5, wherein the phosphate backbone(s) of 1 to 17 nucleotides in the second strand of the nucleic acid molecule is (are) substituted with phosphorothioate or phosphorodithioate.

8. The nucleic acid molecule of claim 7, wherein the phosphate backbone(s) of nucleotides in a region of the first strand, other than a region complementary to a target nucleic acid, is (are) substituted with phosphorothioate or phosphorodithioate.

9. The nucleic acid molecule of claim 1, wherein the phosphate backbone of at least one nucleotide in the nucleic acid molecule is substituted with phosphorothioate.

10. The nucleic acid molecule of claim 1, wherein the lipophilic compound is selected from the group consisting of a lipid, a lipophilic peptide, and a lipophilic protein.

11. The nucleic acid molecule of claim 10, wherein the lipid is at least one selected from the group consisting of cholesterol, tocopherol, and a long-chain fatty acid having 10 or more carbon atoms.

12. The nucleic acid molecule of claim 1, wherein the lipophilic compound is conjugated to the end of the first or second strand of the nucleic acid molecule.

13. The nucleic acid molecule of claim 1, wherein the target nucleic acid is at least one inducing RNAi selected from the group consisting of mRNA (messenger RNA), microRNA, piRNA (piwi-interacting RNA), a coding DNA sequence and a non-coding DNA sequence.

14. The nucleic acid molecule of claim 1, hydroxyl group at position 2′ of ribose of at least one nucleotide included in the nucleic acid molecule is substituted with at least one selected from the group consisting of a hydrogen atom, a fluorine atom, an —O-alkyl group, an —O-acyl group and an amino group.

15. The nucleic acid molecule of claim 1, wherein the phosphate backbone of at least one nucleotide, included in the nucleic acid molecule, is substituted with at least one selected from the group consisting of alkylphosphonate form, phosphoroamidate form and boranophosphate form.

16. The nucleic acid molecule of claim 1, at least one nucleotide included in the nucleic acid molecule is substituted with at least one selected from the group consisting of LNA (locked nucleic acid), UNA (unlocked nucleic acid), morpholino and PNA (peptide nucleic acid).

17. The nucleic acid molecule of claim 1, at least one of the nucleotides of the single-stranded region in the first strand comprises a bulky base analog.

18. The nucleic acid molecule of claim 1, wherein the target nucleic acid is mRNA encoding a connective tissue growth factor (CTGF), and the RNAi-inducing double-stranded nucleic acid molecule has a pair of nucleic sequences selected from the group consisting of a pair of nucleotide sequences of SEQ ID NOS: 149 and 150, a pair of nucleotide sequences of SEQ ID NOS: 151 and 152, and a pair of nucleotide sequences of SEQ ID NOS: 153 and 154.

19-24. (canceled)