ASYMMETRIC siRNA FOR INHIBITING EXPRESSION OF MALE PATTERN HAIR LOSS TARGET GENE

Abstract

The present invention relates to an asymmetric siRNA which inhibits an expression of male pattern hair loss target genes and a use thereof and, more particularly, to an asymmetric siRNA which inhibits an expression of 3-oxo-5-alpha-steroid-4-dehydrogenase 1 (SRD5A1) gene, 3-oxo-5-alpha-steroid-4-dehydrogenase 2 (SRD5A2) gene or androgen receptor (AR) gene, and a composition for prevention or treatment of hair loss comprising the asymmetric siRNA.

Claims

1. siRNA specifically binding to mRNA of a 3-oxo-5-alpha-steroid 4-dehydrogenase 1 (SRD5A1)-encoding gene having SEQ ID NOs: 669, 670 or 671, mRNA of a 3-oxo-5-alpha-steroid 4-dehydrogenase 2 (SRD5A2)-encoding gene having SEQ ID NO: 672, or mRNA of an androgen receptor (AR)-encoding gene having SEQ ID NO: 673 and comprising a sense strand having a length of 15 nt to 17 nt and an antisense strand complementary to the sense strand and having a length of 19 nt or more, wherein a 3-terminus of the sense strand and a 5-terminus of the antisense strand form a blunt end.

2. The siRNA according to claim 1, wherein the siRNA comprises a sense strand having one selected from SEQ ID NOS: 5, 6, 15, 18, 40, 48, 49, 59, 62, 69, 77, 86, 205, 208, 228, 231, 232, 233, 237, 238, 239, 240, 242, 248, 249, 259, 260, 262, 265, 283, 284, 285, 291, 292, 300, 471, 477, 498, 500, 502, 503, 505, 506, 507, 509, 510, 515, 517, 518, 521, 524, 534, 538, 539, and 546 and an antisense strand complementary to the sense strand.

3. The siRNA according to claim 2, wherein the siRNA comprises a sense strand having one selected from the group consisting of SEQ ID NOS: 48, 49, 69, 86, 231, 259, 260, 262, 498, 500, 506, 509, 510, 518, 538, 539, and 546 and an antisense strand complementary to the sense strand.

4. The siRNA according to claim 1, wherein the antisense strand has a length of 19 nt to 24 nt.

5. The siRNA according to claim 1, wherein the antisense strand is selected from the group consisting of SEQ ID NOS: 105, 106, 115, 118, 140, 148, 149, 159, 162, 169, 177, 186, 317, 320, 340, 343, 344, 345, 349, 350, 351, 352, 354, 360, 361, 371, 372, 374, 377, 395, 396, 397, 403, 404, 412, 589, 595, 616, 618, 620, 621, 623, 624, 625, 627, 628, 633, 635, 636, 639, 642, 652, 656, 657, and 664.

6. The siRNA according to claim 5, wherein the antisense strand is selected from the group consisting of SEQ ID NOS: 148, 149, 169, 186, 343, 371, 372, 374, 616, 618, 624, 627, 628, 636, 656, 657, and 664.

7. The siRNA according to claim 1, wherein the sense strand or antisense strand of the siRNA comprises at least one chemical modification.

8. The siRNA according to claim 7, wherein the chemical modification comprises at least one selected from the group consisting of: a modification in which an OH group at a 2 carbon position of a sugar structure in a nucleotide is substituted with CH.sub.3 (methyl), OCH.sub.3 (methoxy), NH.sub.2, F (fluorine), O-2-methoxyethyl-O-propyl, O-2-methylthioethyl, O-3-aminopropyl, or O-3-dimethylaminopropyl; a modification in which oxygen in a sugar structure in a nucleotide is substituted with sulfur; a modification of a nucleotide bond to a phosphorothioate, boranophosphate or methyl phosphonate; a modification to peptide nucleic acid (PNA), locked nucleic acid (LNA), or unlocked nucleic acid (UNA); and cholesterol or cell-penetrating peptide binding.

9. The siRNA according to claim 7, wherein the chemical modification is substitution of an OH group at a 2 carbon position of a sugar structure in a nucleotide with CH.sub.3 (methyl), modification of a nucleotide bond to a phosphorothioate, or cholesterol binding.

10. The siRNA according to claim 9, wherein the chemical modification comprises at least one selected from the group consisting of: a modification in which the OH group at the 2 carbon position of a sugar structure in the 5- or 3-terminus nucleotide of the sense strand is substituted with CH.sub.3 (methyl); a modification in which the OH group at the 2 carbon position of a sugar structure in two or more nucleotides of the sense strand or the antisense strand is substituted with CH.sub.3 (methyl); a modification of 25% or more of bonds between nucleotides in the sense or antisense strand to phosphorothioate; and cholesterol binding at the 3-terminus of the sense strand.

11. A composition for preventing or treating hair loss, the composition comprising the siRNA according to claim 1.

Description

DESCRIPTION OF DRAWINGS

[0017] FIGS. 1A and 1B illustrate results showing the gene inhibitory efficiency of asiRNA against 100 sequences targeting SRD5A1. HuH-7 cells were transfected with 0.3 nM asiRNA targeting each nucleotide sequence, and after 24 hours, the expression level of SRD5A1 mRNA was measured using qRT-PCR, and the graphs show the mean and SD of two repeated experiments.

[0018] FIG. 2 illustrates results showing the gene inhibitory efficiency of asiRNA against 12 sequences targeting SRD5A1. HuH-7 cells were transfected with 0.3 nM, 1 nM, or 10 nM asiRNA targeting each nucleotide sequence, and after 48 hours, the expression level of the SRD5A1 protein was measured by western blotting.

[0019] FIG. 3 illustrates results showing the gene inhibitory efficiency of 12 kinds of cp-asiRNAs targeting SRD5A1 and having various chemical modifications added thereto, through two repeated experiments. HuH-7 cells were incubated with 1 M or 3 M cp-asiRNA targeting each nucleotide sequence, and after 48 hours, the expression level of the SRD5A1 protein was measured by western blotting.

[0020] FIG. 4 illustrates results showing the gene inhibitory efficiency of 2 kinds of cp-asiRNA targeting SRD5A1 and having various chemical modifications added thereto. HuH-7 cells were incubated with 0.3 M, 1 M, or 3 M cp-asiRNA targeting each nucleotide sequence, and after 24 hours, the expression level of SRD5A1 mRNA was measured using real-time PCR, and the graph shows the mean and SD of four repeated experiments.

[0021] FIGS. 5A and 5B illustrate results showing the gene inhibitory efficiency of asiRNA against 112 sequences targeting SRD5A2. HuH-7 cells were transfected with 0.3 nM asiRNA targeting each nucleotide sequence, and after 24 hours, the expression level of SRD5A2 mRNA was measured using qRT-PCR, and the graphs show the mean and SD of two repeated experiments.

[0022] FIG. 6A illustrates results showing the gene inhibitory efficiency of asiRNA against 23 sequences targeting SRD5A2, and FIG. 6B illustrates results showing the gene inhibitory efficiency of asiRNA against 6 sequences and 4 sequences, which target SRD5A2. HuH-7 cells were transfected with 3 nM or 10 nM of asiRNA targeting each nucleotide sequence, and after 48 hours, the expression level of the SRD5A2 protein was measured by western blotting.

[0023] FIG. 7 illustrates results showing the gene inhibitory efficiency of 12 kinds of cp-asiRNA targeting SRD5A2 and having various chemical modifications added thereto, through two repeated experiments. HuH-7 cells were incubated with 1 M of cp-asiRNA targeting each nucleotide sequence, and after 48 hours, the expression level of the SRD5A2 protein was measured by western blotting.

[0024] FIG. 8 illustrates results showing the gene inhibitory efficiency of 2 selected kinds of cp-asisRD5A2. HuH-7 cells were incubated with cp-asiRNA targeting each nucleotide sequence at a concentration ranging from 1.95 nM to 1,000 nM, and after 24 hours, the expression level of SRD5A2 mRNA was measured by RT-PCR.

[0025] FIG. 9 illustrates results showing the gene inhibitory efficiency of 2 selected kinds of cp-asiSRD5A2. HuH-7 cells were incubated with 0.1 M, 0.3 M, 1 M, or 3 M of cp-asiRNA targeting each nucleotide sequence, and after 48 hours, the expression level of the SRD5A2 protein was measured by western blotting.

[0026] FIGS. 10A and 10B illustrate results showing the gene inhibitory efficiency of asiRNA against 118 sequences targeting AR. A549 cells were transfected with 0.3 nM of asiRNA targeting each nucleotide sequence, and after 24 hours, the expression level of AR mRNA was measured through qRT-PCR, the graphs showing the mean and SD of two repeated experiments.

[0027] FIG. 11 illustrates results showing the inhibitory efficiency of asiRNA against 20 sequences targeting AR at a protein level. A549 cells were transfected with 0.3 nM asiRNA targeting each nucleotide sequence, and after 48 hours, the expression level of the AR protein was measured by western blotting, and experiments were repeated three times.

[0028] FIG. 12 illustrates results showing the gene inhibitory efficiency of asiRNA against 9 sequences targeting AR. A549 cells were transfected with 0.1 nM asiRNA targeting each nucleotide sequence, and after 48 hours, the expression levels of AR mRNA and the AR protein were measured by qRT-PCR and western blotting, respectively.

[0029] FIG. 13 illustrates results showing the gene inhibitory efficiency of 9 kinds of cp-asiRNA targeting an AR and having various chemical modifications added thereto. A549 cells were incubated with 1 M or 3 M of cp-asiRNA targeting each nucleotide sequence, and after 48 hours, the expression level of AR mRNA was measured through real-time PCR, and the graph shows the mean and SD of four repeated experiments.

[0030] FIG. 14 illustrates results showing the gene inhibitory efficiency of 9 kinds of cp-asiRNA targeting an AR and having various chemical modifications added thereto. A549 cells were incubated with 1 M of cp-asiRNA targeting each nucleotide sequence, and after 48 hours, the expression level of the AR protein was measured using a western blotting assay.

DETAILED DESCRIPTION AND EXEMPLARY EMBODIMENTS

[0031] Unless otherwise defined, all technical and scientific terms as used herein have the same meanings as those commonly understood by one of ordinary skill in the art to which the present invention pertains. Generally, the nomenclature used herein is well known and commonly used in the art.

[0032] Accordingly, in one aspect, the present invention relates to siRNA specifically binding to mRNA of a 3-oxo-5-alpha-steroid 4-dehydrogenase 1 (SRD5A1)-encoding gene having SEQ ID NO: 678, mRNA of a 3-oxo-5-alpha-steroid 4-dehydrogenase 2 (SRD5A2)-encoding gene having SEQ ID NO: 679, or mRNA of an androgen receptor (AR)-encoding gene having SEQ ID NO: 680 and comprising a sense strand having a length of 15-17 nt and an antisense strand complementary to the sense strand and having a length of 19 nt or more, wherein the 3-terminus of the sense strand and the 5-terminus of the antisense strand form a blunt end.

[0033] The SRD5A1-encoding gene, which is a target gene for male pattern hair loss, for example, androgenetic alopecia, has mRNA Accession Number: NM_001047.3, NM_001324322.1, or NM_001324323.1, which respectively include sequences with SEQ ID: 669, SEQ ID NO: 670, and SEQ ID NO: 671. The SRD5A2-encoding gene has mRNA Accession Number: NM_000348.3 and includes a sequence having SEQ ID NO: 672. The AR-encoding gene has mRNA Accession Number: NM_001011645.2 and includes a sequence having SEQ ID NO: 673.

[0034] In the present invention, siRNA is a concept including all substances having a general RNA interference (RNAi) action. RNAi is an intracellular mechanism for gene regulation that was first found in Caenorhabditis elegans in 1998, and as for the mechanism action, it is known that the antisense strand of a double-stranded RNA introduced into a cell complementarily binds to mRNA of a target gene to thereby induce the degradation of the target gene. In this regard, small interfering RNA (siRNA) is one of the methods of inhibiting gene expression in vitro. siRNAs of 19-21 bp in length are theoretically capable of performing selective inhibition against almost all genes, and thus can be developed as therapeutic agents for various gene-related diseases such as cancer, viral infection, and the like, and is the new candidate drug development technology that has recently drawn the most attention. The first attempt to perform in vivo treatment using siRNA in mammals was in mid-2003, and since then, there have been numerous reports of in vivo treatment thanks to many attempts for application studies.

[0035] However, contrary to the possibility of in vivo treatment, side effects and disadvantages of siRNA have continually been reported. To develop an RNAi-based therapeutic agent, challenges such as: 1) the absence of an effective delivery system; 2) the off-target effect; 3) the induction of immune responses; and 4) intracellular RNAi mechanism saturation need to be overcome. Although siRNAs are an effective method of directly regulating target gene expression, it is difficult to develop a therapeutic agent using such siRNAs due to the above-described problems. With regard thereto, the applicant of the present invention has developed an asymmetric shorter duplex siRNA (asiRNA) structure-related technology (WO2009/078685). asiRNA is an asymmetric RNAi-inducing structure having a shorter double helix length than the 19+2 structure of existing siRNAs. asiRNA is a technology that has overcome known problems with the existing siRNA structure technology, such as the off-target effect, RNAi mechanism saturation, immune responses by TLR3, and the like, and accordingly is used for the development of a new RNAi drug with minimal side effects.

[0036] Based on this, the present invention provides asymmetric siRNA including a sense strand having a length of 15-17 nt and an antisense strand complementary to the sense strand and having a length of 19 nt or more, and thus the siRNA according to the present invention may stably maintain high delivery efficiency without incurring problems such as the off-target effect, RNAi mechanism saturation, immune responses by TLR3, and the like, and may inhibit the expression of a 5-reductase type 1 target gene, a 5-reductase type 2 target gene, and an androgen receptor target gene.

[0037] In the present invention, the term sense strand refers to a polynucleotide having the same nucleic acid sequence as that of the SRD5A1-, SRD5A2-, or AR-encoding gene, and has a length of 15-17 nt. In one embodiment, the sense strand may have a length of 15 nt, 16 nt, or 17 nt.

[0038] The inventors of the present application selected, as target genes, 5-reductase type 1, 5-reductase type 2, and an androgen receptor, which play a major role in inhibiting the synthesis of proteins required for hair follicle growth in male pattern hair loss and inducing hair loss by reducing the dermal papilla. As a result of screening 100 or more siRNAs targeting each target gene and selecting siRNAs with excellent inhibitory efficiency from among the same, it was confirmed that siRNA comprising a sense strand having one selected from SEQ ID NOS: 5, 6, 15, 18, 40, 48, 49, 59, 62, 69, 77, 86, 205, 208, 228, 231, 232, 233, 237, 238, 239, 240, 242, 248, 249, 259, 260, 262, 265, 283, 284, 285, 291, 292, 300, 471, 477, 498, 500, 502, 503, 505, 506, 507, 509, 510, 515, 517, 518, 521, 524, 534, 538, 539, and 546 and an antisense strand complementary to the sense strand, effectively reduced the expression of mRNA of the SRD5A1-, SRD5A2-, or AR-encoding gene.

[0039] Specifically, siRNA comprising a sense strand having SEQ ID NO: 5, 6, 15, 18, 40, 48, 49, 59, 62, 69, 77, or 86 and an antisense strand complementary to the sense strand, may reduce the expression of mRNA of the SRD5A1-encoding gene, siRNA comprising a sense strand having SEQ ID NO: 205, 208, 228, 231, 232, 233, 237, 238, 239, 240, 242, 248, 249, 259, 260, 262, 265, 283, 284, 285, 291, 292, or 300 and an antisense strand complementary to the sense strand, may reduce the expression of mRNA of the SRD5A2-encoding gene, and siRNA comprising a sense strand having SEQ ID NO: 471, 477, 498, 500, 502, 503, 505, 506, 507, 509, 510, 515, 517, 518, 521, 524, 534, 538, 539, or 546 and an antisense strand complementary to the sense strand, may reduce the expression of mRNA of the AR-encoding gene.

[0040] Specifically, it was confirmed that siRNA comprising a sense strand having one selected from the group consisting of SEQ ID NOS: 48, 49, 69, 86, 231, 259, 260, 262, 498, 500, 506, 509, 510, 518, 538, 539, and 546 and an antisense strand complementary to the sense strand also effectively inhibited the expression of the SRD5A1 protein, the SRD5A2 protein, or the AR protein.

[0041] The 3-terminus of the sense strand and the 5-terminus of the antisense strand form a blunt end. For example, the 5-terminus of the antisense strand may include, for example, an overhang of 1 nt, 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, or 8 nt.

[0042] In the present invention, the antisense strand is a polynucleotide complementary to the target gene and has a length of 19 nt or more, for example, 20 nt or more, 21 nt or more, 22 nt or more, 23 nt or more, 24 nt or more, 25 nt or more, 26 nt or more, 27 nt or more, 29 nt or more, 30 nt or more, or 31 nt or more. In one embodiment, the antisense strand may have a length between 19 nt and 24 nt, for example, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, or 24 nt. The antisense strand may have a sequence partially complementary to the sense strand in consideration of the asymmetric structure.

[0043] The antisense strand may, for example, be selected from the group consisting of SEQ ID NOS: 105, 106, 115, 118, 140, 148, 149, 159, 162, 169, 177, 186, 317, 320, 340, 343, 344, 345, 349, 350, 351, 352, 354, 360, 361, 371, 372, 374, 377, 395, 396, 397, 403, 404, 412, 589, 595, 616, 618, 620, 621, 623, 624, 625, 627, 628, 633, 635, 636, 639, 642, 652, 656, 657, and 664.

[0044] Specifically, it was confirmed that siRNA comprising an antisense strand selected from the group consisting of SEQ ID NOS: 148, 149, 169, 186, 343, 371, 372, 374, 616, 618, 624, 627, 628, 636, 656, 657, and 664 also effectively inhibited the expression of the SRD5A1 protein, the SRD5A2 protein, or the AR protein.

[0045] In some embodiments, the sense strand or antisense strand of the siRNA may include one or more chemical modifications.

[0046] General siRNAs are unable to penetrate through the cell membrane due to reasons such as high negative charge, high molecular weight, and the like, and are rapidly degraded and eliminated in the blood, making it difficult to deliver an amount sufficient for RNAi induction to an actual target site. Currently, in the case of in vitro delivery, numerous high-efficiency delivery methods using cationic lipids and cationic polymers have been developed, but in vivo delivery of siRNA as efficient as in vitro delivery thereof is difficult, and siRNA delivery efficiency is reduced by interactions between various proteins present in the living body.

[0047] Therefore, the inventors of the present application developed cell penetrating asiRNA (cp-asiRNA) having self-transfer ability that enables effective intracellular delivery without a separate delivery vehicle by introducing a chemical modification into an asymmetric siRNA structure.

[0048] The chemical modification in the sense strand or the antisense strand may comprise, for example, at least one selected from the group consisting of:

[0049] a modification in which an OH group at the 2 carbon position of a sugar structure in a nucleotide is substituted with CH.sub.3 (methyl), OCH.sub.3 (methoxy), NH.sub.2, F (fluorine), O-2-methoxyethyl-O-propyl, O-2-methylthioethyl, O-3-aminopropyl, or O-3-dimethylaminopropyl;

[0050] a modification in which oxygen in a sugar structure in a nucleotide is substituted with sulfur;

[0051] a modification of a nucleotide bond to a phosphorothioate, boranophosphate or methyl phosphonate;

[0052] a modification to peptide nucleic acid (PNA), locked nucleic acid (LNA), or unlocked nucleic acid (UNA); and

[0053] cholesterol or cell-penetrating peptide binding.

[0054] In one embodiment, the chemical modification in the sense or antisense strand may be substitution of an OH group at the 2 carbon position of a sugar structure in a nucleotide with CH.sub.3 (methyl), modification of a nucleotide bond into phosphorothioate, or cholesterol binding. This may enhance the in vivo stability of siRNA.

[0055] When the OH group at the 2 carbon position of a sugar structure is substituted with CH.sub.3 (methyl) or when the nucleotide bond is modified into a phosphorothioate, resistance to nucleases may be increased, and binding to the cell membrane via cholesterol binding may facilitate the intracellular delivery of siRNA.

[0056] In particular, the chemical modification may include at least one modification selected from the group consisting of: a modification in which an OH group at the 2 carbon position of a sugar structure in the 5- or 3-terminus nucleotide of the sense strand is substituted with CH.sub.3 (methyl); a modification in which an OH group at the 2 carbon position of a sugar structure in two or more nucleotides of the sense strand or the antisense strand is substituted with CH.sub.3 (methyl); a modification of 25% or more of nucleotides bonds in the sense or antisense strand to phosphorothioate; and cholesterol binding at the 3-terminus of the sense strand.

[0057] With regard to the modification in which an OH group at the 2 carbon position of a sugar structure in a nucleotide is substituted with CH.sub.3 (methyl), the OH group at the 2 carbon position of the sugar structure in a nucleotide positioned at the 5-terminus of the sense strand may be substituted with CH.sub.3 (methyl). In addition, a 2-O-methylated nucleoside, in which an OH group at the 2 carbon position of a sugar structure is substituted with CH.sub.3 (methyl), may be continuously or discontinuously included in a 5-terminus to 3-terminus direction of the sense strand. 2-O-methylated nucleosides and unmodified nucleosides may be alternately included in the sense strand. 2, 3, 4, 5, 6, 7, or 8 consecutive 2-O-methylated nucleosides and unmodified nucleosides may be alternately included in the sense strand. For example, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, 2 to 8, or 8 2-O-methylated nucleosides may be present in the sense strand.

[0058] 2-O-methylated nucleosides may be continuously or discontinuously included in a 5-terminus to 3-terminus of the antisense strand. 2-O-methylated nucleosides and unmodified nucleotides may be alternately included in the antisense strand. 2, 3, 4, 5, 6, 7, or 8 consecutive 2-O-methylated nucleosides and unmodified nucleosides may be alternately included in the antisense strand. For example, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or 2-7 2-O-methylated nucleosides may be present in the antisense strand.

[0059] With regard to the modification of a nucleotide bond to a phosphorothioate, at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of bonds between ribonucleotides in the sense strand may be modified into phosphorothioate. In some embodiments, all (100%) of the bonds between ribonucleotides in the sense strand may be modified into phosphorothioate.

[0060] At least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the bonds between ribonucleotides in the antisense strand may be modified into phosphorothioate. In some embodiments, a total (100%) of the bonds between ribonucleotides in the antisense strand may be modified into phosphorothioate.

[0061] In another aspect, the present invention relates to a composition for the prevention or treatment of hair loss, which comprises the siRNA.

[0062] The term treatment as used herein means reducing the symptoms of hair loss or the severity of hair loss in a subject to which the composition is administered or preventing the same from being aggravated, and in some cases may include the progression of hair growth. The term prevention as used herein means preventing or delaying the initiation of hair loss, or reducing the possibility of developing hair loss.

[0063] The composition may further be prepared including one or more pharmaceutically acceptable carriers, in addition to the siRNA as an active ingredient. The pharmaceutically acceptable carrier has to be compatible with the active ingredient of the present invention, and may be one selected from physiological saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of two or more of these components. If necessary, the composition may include other general additives such as an antioxidant, a buffer, a bacteriostatic agent, and the like. In addition, the composition may be formulated into an injectable preparation such as an aqueous solution, a suspension, an emulsion, or the like by further adding a diluent, a dispersing agent, a surfactant, a binder, and a lubricant. In particular, the composition may be formulated into a lyophilized preparation. The lyophilized preparation may be formulated using a method commonly used in the art to which the present invention pertains, and a stabilizer for lyophilization may also be added.

[0064] An administration method of the composition may be determined by one of ordinary skill in the art on the basis of general symptoms of patients and the severity of diseases. In addition, the composition may be formulated into various forms such as powders, tablets, capsules, liquids, injections, ointments, syrups, and the like, and may also be provided in a unit dosage or multiple dosage container, for example, sealed ampoules and vials, and the like.

[0065] The composition may be administered orally or parenterally. The administration route of the composition according to the present invention may be, but is not limited to, for example, oral administration, intravenous administration, intramuscular administration, intraarterial administration, intramedullary administration, intradural administration, intracardiac administration, transdermal administration, subcutaneous administration, intraperitoneal administration, intestinal administration, sublingual administration, or topical administration. The dosage of the composition according to the present invention varies depending on the body weight, age, gender, and health condition of a patient, diet, administration time, administration method, excretion rate, severity of disease, or the like, and may be easily determined by those of ordinary skill in the art. In addition, for clinical administration, the composition of the present invention may be formulated into a suitable form using known techniques.

[0066] In another aspect of the present invention, there is provided a method of preventing or treating hair loss, comprising administering the siRNA to a subject.

[0067] In another aspect, the present invention relates to a use of the siRNA for preventing or treating hair loss.

[0068] In another aspect, the present invention relates to a use of the siRNA for preparing a drug for the prevention or treatment of hair loss.

[0069] Configurations included in the prevention or treatment method according to the present invention are the same as those included in the aforementioned embodiments, and thus the foregoing description may be equally applied to the prevention or treatment method.

[0070] Hereinafter, the present invention will be described in further detail with reference to the following examples. It will be obvious to those of ordinary skill in the art that these examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention.

[Example 1] Screening for 100 Kinds of RNAi-Inducing Double-Stranded Nucleic Acid Molecules Targeting SRD5A1

[0071] To obtain high-efficiency RNAi-inducing double-stranded nucleic acid molecules targeting SRD5A1, the target sequence of the SRD5A1 gene was selected and then asiRNA was designed. The asiRNA structure is different from that of generally known siRNAs, and thus when the nucleotide sequences of asiRNA are designed using a general siRNA design program, it may be somewhat difficult to design an optimized asiRNA. Therefore, asiRNA was constructed by the following method. An NCBI db search was used to obtain information on the SRD5A1 gene (mRNA Accession Number: NM_001047.3, NM_001324322.1, NM_001324323.1), which is the target gene pertaining to male pattern hair loss (androgenetic hair loss). For subsequent animal experiments, nucleotide sequences were secured in consideration of the nucleotide sequence homology with mice, and then 100 kinds of asiRNA were designed according to a design method such as the exclusion of sequences having a GC content of 30-62% and 4 or more G or C consecutive bases, and then synthesized by OliX Inc. (Korea). The synthesized sense and antisense strand RNA oligonucleotides were annealed at 95 C. for 2 minutes through incubation at 37 C. for 1 hour, and the asiRNA annealed by 10% polyacrylamide gel electrophoresis (PAGE) was confirmed using a UV transilluminator.

TABLE-US-00001 TABLE1 100KindsofasiRNAnucleotidesequencestargeting 3-oxo-5-alpha-steroid4-dehydrogenase1 100kinds Sequence(5-3) No. Name S AS 1 asiSRD5A11 GCAGAUACUUGAGCCA UGGCUCAAGUAUCUG CUUUGC (SEQIDNO:1) (SEQIDNO:101) 2 asiSRD5A12 AAGCAGAUACUUGAGC GCUCAAGUAUCUGCU UUGCAA (SEQIDNO:2) (SEQIDNO:102) 3 asiSRD5A13 CAAAGCAGAUACUUGA UCAAGUAUCUGCUUU GCAAAU (SEQIDNO:3) (SEQIDNO:103) 4 asiSRD5A14 UGCAAAGCAGAUACUU AAGUAUCUGCUUUGC AAAUAG (SEQIDNO:4) (SEQIDNO:104) 5 asiSRD5A15 GUGCAGUGUAUGCUGA UCAGCAUACACUGCA CAAUGG (SEQIDNO:5) (SEQIDNO:105) 6 asiSRD5A16 UUGUGCAGUGUAUGCU AGCAUACACUGCACA AUGGCU (SEQIDNO:6) (SEQIDNO:106) 7 asiSRD5A17 CAUUGUGCAGUGUAUG CAUACACUGCACAAU GGCUCA (SEQIDNO:7) (SEQIDNO:107) 8 asiSRD5A18 UUUUGGCUUGUGGUUA UAACCACAAGCCAAA ACCUAU (SEQIDNO:8) (SEQIDNO:108) 9 asiSRD5A19 CGGGCAUGUUGAUAAA UUUAUCAACAUGCCC GUUAAC (SEQIDNO:9) (SEQIDNO:109) 10 asiSRD5A110 AUAUCCUAAGGAAUCU AGAUUCCUUAGGAUA UGAUCU (SEQIDNO:10) (SEQIDNO:110) 11 asiSRD5A111 AUCUCAGAAAACCAGG CCUGGUUUUCUGAGA UUCCUU (SEQIDNO:11) (SEQIDNO:111) 12 asiSRD5A112 GAAUCUCAGAAAACCA UGGUUUUCUGAGAUU CCUUAG (SEQIDNO:12) (SEQIDNO:112) 13 asiSRD5A113 AGGAAUCUCAGAAAAC GUUUUCUGAGAUUCC UUAGGA (SEQIDNO:13) (SEQIDNO:113) 14 asiSRD5A114 CUGGAUACAAAAUACC GGUAUUUUGUAUCCA GUAUCU (SEQIDNO:14) (SEQIDNO:114) 15 asiSRD5A115 UACUGGAUACAAAAUA UAUUUUGUAUCCAGU AUCUCC (SEQIDNO:15) (SEQIDNO:115) 16 asiSRD5A116 GAUACUGGAUACAAAA UUUUGUAUCCAGUAU CUCCUG (SEQIDNO:16) (SEQIDNO:116) 17 asiSRD5A117 GAGAUACUGGAUACAA UUGUAUCCAGUAUCU CCUGGU (SEQIDNO:17) (SEQIDNO:117) 18 asiSRD5A118 AGGAGAUACUGGAUAC GUAUCCAGUAUCUCC UGGUUU (SEQIDNO:18) (SEQIDNO:118) 19 asiSRD5A119 CCAGGAGAUACUGGAU AUCCAGUAUCUCCUG GUUUUC (SEQIDNO:19) (SEQIDNO:119) 20 asiSRD5A120 AAUACCAAGGGGAGGC GCCUCCCCUUGGUAU UUUGUA (SEQIDNO:20) (SEQIDNO:120) 21 asiSRD5A121 AAAAUACCAAGGGGAG CUCCCCUUGGUAUUU UGUAUC (SEQIDNO:21) (SEQIDNO:121) 22 asiSRD5A122 GAGGCUUAUUUGAAUA UAUUCAAAUAAGCCU CCCCUU (SEQIDNO:22) (SEQIDNO:122) 23 asiSRD5A123 CAGCCAACUAUUUUGG CCAAAAUAGUUGGCU GCAGUU (SEQIDNO:23) (SEQIDNO:123) 24 asiSRD5A124 UGCAGCCAACUAUUUU AAAAUAGUUGGCUGC AGUUAC (SEQIDNO:24) (SEQIDNO:124) 25 asiSRD5A125 ACUGCAGCCAACUAUU AAUAGUUGGCUGCAG UUACGU (SEQIDNO:25) (SEQIDNO:125) 26 asiSRD5A126 UAACUGCAGCCAACUA UAGUUGGCUGCAGUU ACGUAU (SEQIDNO:26) (SEQIDNO:126) 27 asiSRD5A127 CGUAACUGCAGCCAAC GUUGGCUGCAGUUAC GUAUUC (SEQIDNO:27) (SEQIDNO:127) 28 asiSRD5A128 AUGGAGUGGUGUGGCU AGCCACACCACUCCA UGAUUU (SEQIDNO:28) (SEQIDNO:128) 29 asiSRD5A129 UCAUGGAGUGGUGUGG CCACACCACUCCAUG AUUUCU (SEQIDNO:29) (SEQIDNO:129) 30 asiSRD5A130 AAUCAUGGAGUGGUGU ACACCACUCCAUGAU UUCUCC (SEQIDNO:30) (SEQIDNO:130) 31 asiSRD5A131 GAAAUCAUGGAGUGGU ACCACUCCAUGAUUU CUCCAA (SEQIDNO:31) (SEQIDNO:131) 32 asiSRD5A132 GAGAAAUCAUGGAGUG CACUCCAUGAUUUCU CCAAAA (SEQIDNO:32) (SEQIDNO:132) 33 asiSRD5A133 CCCUGGCCAGCUGGUC GACCAGCUGGCCAGG GCAUAG (SEQIDNO:33) (SEQIDNO:133) 34 asiSRD5A134 UAUGCCCUGGCCAGCU AGCUGGCCAGGGCAU AGCCAC (SEQIDNO:34) (SEQIDNO:134) 35 asiSRD5A135 GCUAUGCCCUGGCCAG CUGGCCAGGGCAUAG CCACAC (SEQIDNO:35) (SEQIDNO:135) 36 asiSRD5A136 UCAUGAGUGGUACCUC GAGGUACCACUCAUG AUGCUC (SEQIDNO:36) (SEQIDNO:136) 37 asiSRD5A137 CAUCAUGAGUGGUACC GGUACCACUCAUGAU GCUCUU (SEQIDNO:37) (SEQIDNO:137) 38 asiSRD5A138 UCCGGAAAUUUGAAGA UCUUCAAAUUUCCGG AGGUAC (SEQIDNO:38) (SEQIDNO:138) 39 asiSRD5A139 CAGUGUAUGCUGAUGA UCAUCAGCAUACACU GCACAA (SEQIDNO:39) (SEQIDNO:139) 40 asiSRD5A140 GCAUGUUGAUAAACAU AUGUUUAUCAACAUG CCCGUU (SEQIDNO:40) (SEQIDNO:140) 41 asiSRD5A141 GUGGCUAUGCCCUGGC GCCAGGGCAUAGCCA CACCAC (SEQIDNO:41) (SEQIDNO:141) 42 asiSRD5A142 GUGUGGCUAUGCCCUG CAGGGCAUAGCCACA CCACUC (SEQIDNO:42) (SEQIDNO:142) 43 asiSRD5A143 UGGUGUGGCUAUGCCC GGGCAUAGCCACACC ACUCCA (SEQIDNO:43) (SEQIDNO:143) 44 asiSRD5A144 AGUGGUGUGGCUAUGC GCAUAGCCACACCAC UCCAUG (SEQIDNO:44) (SEQIDNO:144) 45 asiSRD5A145 UCUUCACGUUUUGUUU AAACAAAACGUGAAG AAAGCA (SEQIDNO:45) (SEQIDNO:145) 46 asiSRD5A146 GACUUGAGAACCCUUU AAAGGGUUCUCAAGU CAGGCU (SEQIDNO:46) (SEQIDNO:146) 47 asiSRD5A147 CUGUUGGCGUGUACAA UUGUACACGCCAACA GUGGCA (SEQIDNO:47) (SEQIDNO:147) 48 asiSRD5A148 UUAUUUGAAUACGUAA UUACGUAUUCAAAUA AGCCUC (SEQIDNO:48) (SEQIDNO:148) 49 asiSRD5A149 UUCCAAUGGCGCUUCU AGAAGCGCCAUUGGA AAGCUU (SEQIDNO:49) (SEQIDNO:149) 50 asiSRD5A150 AAAGGCAUCUGGACUU AAGUCCAGAUGCCUU UGCCUC (SEQIDNO:50) (SEQIDNO:150) 51 asiSRD5A151 AUCAAUGUGCUCUGGU ACCAGAGCACAUUGA UGGCUC (SEQIDNO:51) (SEQIDNO:151) 52 asiSRD5A152 GAUCACUUUCUGUAAC GUUACAGAAAGUGAU CAUUCU (SEQIDNO:52) (SEQIDNO:152) 53 asiSRD5A153 AUCUUCCUUCUAAUAG CUAUUAGAAGGAAGA UUAGCU (SEQIDNO:53) (SEQIDNO:153) 54 asiSRD5A154 GGCAUUGCUUUGCCUU AAGGCAAAGCAAUGC CAGAUG (SEQIDNO:54) (SEQIDNO:154) 55 asiSRD5A155 UGUACAAUGGCGAUUA UAAUCGCCAUUGUAC ACGCCA (SEQIDNO:55) (SEQIDNO:155) 56 asiSRD5A156 CUUCUCUAUGGACUUU AAAGUCCAUAGAGAA GCGCCA (SEQIDNO:56) (SEQIDNO:156) 57 asiSRD5A157 UUCCAAGGUGAGGCAA UUGCCUCACCUUGGA AGGGCC (SEQIDNO:57) (SEQIDNO:157) 58 asiSRD5A158 UCCAAGGUGAGGCAAA UUUGCCUCACCUUGG AAGGGC (SEQIDNO:58) (SEQIDNO:158) 59 asiSRD5A159 GGUUCAUACGGAGUAA UUACUCCGUAUGAAC CACCAC (SEQIDNO:59) (SEQIDNO:159) 60 asiSRD5A160 AUAGUAGAGAUUGUUG CAACAAUCUCUACUA UAUCCA (SEQIDNO:60) (SEQIDNO:160) 61 asiSRD5A161 UGUUGUCUGUGAAAUU AAUUUCACAGACAAC AAUCUC (SEQIDNO:61) (SEQIDNO:161) 62 asiSRD5A162 UUCAAGCUCUGGGUAA UUACCCAGAGCUUGA AAUUCU (SEQIDNO:62) (SEQIDNO:162) 63 asiSRD5A163 UACCUAAUAAGUACCU AGGUACUUAUUAGGU AGAUUG (SEQIDNO:63) (SEQIDNO:163) 64 asiSRD5A164 AUUGUUGUCUGUGAAA UUUCACAGACAACAA UCUCUA (SEQIDNO:64) (SEQIDNO:164) 65 asiSRD5A165 CAAAAGAGCAUCAUGA UCAUGAUGCUCUUUU GCUCUA (SEQIDNO:65) (SEQIDNO:165) 66 asiSRD5A166 CUAUGGACUUUGUAAA UUUACAAAGUCCAUA GAGAAG (SEQIDNO:66) (SEQIDNO:166) 67 asiSRD5A167 CUGUCUUUGAUGGCAU AUGCCAUCAAAGACA GUUGUA (SEQIDNO:67) (SEQIDNO:167) 68 asiSRD5A168 UCUACCUAAUAAGUAC GUACUUAUUAGGUAG AUUGCA (SEQIDNO:68) (SEQIDNO:168) 69 asiSRD5A169 CUAAUCUUCCUUCUAA UUAGAAGGAAGAUUA GCUAUG (SEQIDNO:69) (SEQIDNO:169) 70 asiSRD5A170 CAUUUUCAGAACAAUA UAUUGUUCUGAAAAU GCCAUC (SEQIDNO:70) (SEQIDNO:170) 71 asiSRD5A171 GAUCUCUUCAAGGUCA UGACCUUGAAGAGAU CACUGU (SEQIDNO:71) (SEQIDNO:171) 72 asiSRD5A172 AGAUUGUUGUCUGUGA UCACAGACAACAAUC UCUACU (SEQIDNO:72) (SEQIDNO:172) 73 asiSRD5A173 AGAGAUUGUUGUCUGU ACAGACAACAAUCUC UACUAU (SEQIDNO:73) (SEQIDNO:173) 74 asiSRD5A174 AGACGAACUCAGUGUA UACACUGAGUUCGUC UGACGA (SEQIDNO:74) (SEQIDNO:174) 75 asiSRD5A175 UCCUCCUGGCCAUGUU AACAUGGCCAGGAGG AUGCAG (SEQIDNO:75) (SEQIDNO:175) 76 asiSRD5A176 CUUAAUUUACCCAUUU AAAUGGGUAAAUUAA GCACCG (SEQIDNO:76) (SEQIDNO:176) 77 asiSRD5A177 UGAUGCGAGGAGGAAA UUUCCUCCUCGCAUC AGAAAU (SEQIDNO:77) (SEQIDNO:177) 78 asiSRD5A178 UGUUCUGUACCUGUAA UUACAGGUACAGAAC AUAAUC (SEQIDNO:78) (SEQIDNO:178) 79 asiSRD5A179 CCUGUAACGGCUAUUU AAAUAGCCGUUACAG GUACAG (SEQIDNO:79) (SEQIDNO:179) 80 asiSRD5A180 CCAUUGUGCAGUGUAU AUACACUGCACAAUG GCUCAA (SEQIDNO:80) (SEQIDNO:180) 81 asiSRD5A181 AACAUCCAUUCAGAUC GAUCUGAAUGGAUGU UUAUCA (SEQIDNO:81) (SEQIDNO:181) 82 asiSRD5A182 UAUCCAAAGUUCAGAA UUCUGAACUUUGGAU ACUCUU (SEQIDNO:82) (SEQIDNO:182) 83 asiSRD5A183 ACCUAAAUACGCUGAA UUCAGCGUAUUUAGG UACUUA (SEQIDNO:83) (SEQIDNO:183) 84 asiSRD5A184 CGCUGAAAUGGAGGUU AACCUCCAUUUCAGC GUAUUU (SEQIDNO:84) (SEQIDNO:184) 85 asiSRD5A185 AAUGGAGGUUGAAUAU AUAUUCAACCUCCAU UUCAGC (SEQIDNO:85) (SEQIDNO:185) 86 asiSRD5A186 AUAUCCUACUGUGUAA UUACACAGUAGGAUA UUCAAC (SEQIDNO:86) (SEQIDNO:186) 87 asiSRD5A187 UAUGAGACUAGACUUU AAAGUCUAGUCUCAU ACACAC (SEQIDNO:87) (SEQIDNO:187) 88 asiSRD5A188 AAUGUCACAAUCCCUU AAGGGAUUGUGACAU UUAUUG (SEQIDNO:88) (SEQIDNO:188) 89 asiSRD5A189 GGUCAACUGCAGUGUU AACACUGCAGUUGAC CUUGAA (SEQIDNO:89) (SEQIDNO:189) 90 asiSRD5A190 GCCAUUGUGCAGUCAU AUGACUGCACAAUGG CUACCC (SEQIDNO:90) (SEQIDNO:190) 91 asiSRD5A191 UGUAAGUGGAGAACUU AAGUUCUCCACUUAC ACACAG (SEQIDNO:91) (SEQIDNO:191) 92 asiSRD5A192 CUCUGCCUGUGUGAGU ACUCACACAGGCAGA GCAGCU (SEQIDNO:92) (SEQIDNO:192) 93 asiSRD5A193 ACCGUGAGCCAUCAAU AUUGAUGGCUCACGG UGAGUG (SEQIDNO:93) (SEQIDNO:193) 94 asiSRD5A194 GGUUUCUCUCUGUCUU AAGACAGAGAGAAAC CAUGUC (SEQIDNO:94) (SEQIDNO:194) 95 asiSRD5A195 UAGUCUAGACCUAGUU AACUAGGUCUAGACU AGAAGA (SEQIDNO:95) (SEQIDNO:195) 96 asiSRD5A196 UAGUGUAAAGAAUGAU AUCAUUCUUUACACU ACAAGG (SEQIDNO:96) (SEQIDNO:196) 97 asiSRD5A197 CUGUACCUGUUAUCAA UUGAUAACAGGUACA GGCUAU (SEQIDNO:97) (SEQIDNO:197) 98 asiSRD5A198 GAAUGCUUCAUGACUU AAGUCAUGAAGCAUU CAACAG (SEQIDNO:98) (SEQIDNO:198) 99 asiSRD5A199 UGCCUUAUCAUCUCAU AUGAGAUGAUAAGGC AAAGCA (SEQIDNO:99) (SEQIDNO:199) 100 asiSRD5A1100 CAUCUCAUCUGGAGUU AACUCCAGAUGA GAUGAUAAG (SEQIDNO:100) (SEQIDNO:200)

[Example 2] Screening for RNAi-Inducing Double-Stranded Nucleic Acid Molecules Targeting SRD5A1

[0072] To confirm gene inhibitory efficiency at the mRNA level, 100 selected kinds of asiRNA were transfected into a HuH-7 cell line at a concentration of 0.3 nM, and qRT-PCR was performed to measure the expression level of SRD5A1 mRNA.

[0073] The HuH-7 cell line was cultured in Dulbecco's Modified Eagle's Medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS, Gibco) and 100 units/ml of penicillin 100 g/ml of streptomycin. HuH-7 cells were seeded in a 96-well plate at a density of 510.sup.3 cells/well, and a reverse transfection experiment was conducted using asiRNA (0.3 nM, OliX Inc.) and Lipofectamine 2000 (1 l/ml, Invitrogen Inc.) in Opti-MEM (a total volume of 100 l) in accordance with Invitrogen's protocol. After 24 hours, RNA purification and cDNA synthesis were performed in accordance with a basic protocol provided by TOYOBO SuperPrep, the expression level of the SRD5A1 gene was examined with a SRD5A1 TaqMan probe (Hs00602694_mH) using a Bio-Rad CFX-4000 machine, and the results are illustrated in FIGS. 1A and 1B.

[0074] From the results of screening the 100 kinds of asiRNA, the 12 top-ranked asiRNAs (in Table 1, No. 5, 6, 15, 18, 40, 48, 49, 59, 62, 77, and 86) were selected, followed by treatment with 0.3 nM, 1 nM, 3 nM, or 10 nM of asiRNA and western blotting to analyze the protein expression inhibitory effects thereof. HuH-7 cells were seeded in a 6-well plate at a density of 2.510.sup.3 cells/well, and then a reverse transfection experiment was conducted using asiRNA and Lipofectamine 2000 (1 l/ml, Invitrogen Inc.) in Opti-MEM (a total volume of 2 ml) in accordance with Invitrogen's protocol. After 48 hours, the cells were lysed using a mammalian protein extraction buffer (GE healthcare), and then proteins were quantified using a Bradford assay. 10 g of the protein of each sample was electrophoresed using 12% SDS-PAGE at 80 V for 20 minutes and at 120 V for 1 hour, and then transferred onto a PVDE membrane (Bio-Rad) at 300 mA for 1 hour and 20 minutes. After transfer, the membrane was blocked in 5% skim milk for 1 hour and allowed to react with SRD5A1 antibody (ABcam, ab110123) at a ratio of 1:2000 for 12 hours. The next day, the resulting membrane was allowed to react with anti-Goat HRP (Santa Cruz) at a ratio of 1:10000 for 1 hour, and then the expression levels of the SRD5A1 protein were compared with each other using ChemiDoc (Bio-Rad). From the results of FIG. 2, 4 asiRNAs (in Table 1, No. 48, 49, 69, and 86) capable of inhibiting SRD5A1 protein expression were selected.

[Example 3] 16 Kinds of Cp-asiRNA Targeting SRD5A1 Gene and Having Self Cell-Penetrating Ability

[0075] SRD5A1 cp-asiRNAs (a total of 16 strands) were designed by applying three modification patterns to 4 kinds (in Table 1, No. 48, 49, 69, and 86) of asiRNA targeting SRD5A1 according to the number and position of 2OMe (methyl), phosphorothioate bonds (PS), and cholesterol, and then synthesized by OliX Inc. (Korea). cp-asiRNA enhances endocytosis efficiency and stability and thus may penetrate through the cell membrane with high efficiency without the aid of a delivery vehicle to thereby inhibit target gene expression. The synthesized sense and antisense strand RNA oligonucleotides were annealed at 95 C. for 2 minutes through incubation at 37 C. for 1 hour, and cp-asiRNAs annealed by 10% polyacrylamide gel electrophoresis (PAGE) were confirmed by a UV transilluminator.

TABLE-US-00002 TABLE2 16strandsofcp-asiRNAnucleotidesequences targetingSRD5A1 No. Name Sequence(5-->3) 1 SRD5A1cp- mUUmAUmUUmGAmAUmACmGU*mA*A* asiRNAS cholesterol 48 2 SRD5A1cp- UUACGUAUUCAAAUmAmAG*C*C*U*C asiRNAAS 48(2,4) 3 SRD5A1cp- UUACGUAUUCAAAUmAmAmG*mC*C*U*C asiRNAAS 48(4,4) 4 SRD5A1cp- UUACGUAUUCAAAUmAmAmG*mC*mC*mU*mC asiRNAAS 48(7,4) 5 SRD5A1cp- mUUmCCmAAmUGmGCmGCmUU*mC*U* asiRNAS cholesterol 49 6 SRD5A1cp- AGAAGCGCCAUUGGmAmAA*G*C*U*U asiRNAAS 49(2,4) 7 SRD5A1cp- AGAAGCGCCAUUGGmAmAmA*mG*C*U*U asiRNAAS 49(4,4) 8 SRD5A1cp- AGAAGCGCCAUUGGmAmAmA*mG*mC*mU*mU asiRNAAS 49(7,4) 9 SRD5A1cp- mCUmAAmUCmUUmCCmUUmCU*mA*A* asiRNAS cholesterol 69 10 SRD5A1cp- UUAGAAGGAAGAUUmAmGC*U*A*U*G asiRNAAS 69(2,4) 11 SRD5A1cp- UUAGAAGGAAGAUUmAmGmC*mU*A*U*G asiRNAAS 69(4,4) 12 SRD5A1cp- UUAGAAGGAAGAUUmAmGmC*mU*mA*mU*mG asiRNAAS 69(7,4) 13 SRD5A1cp- mAUmAUmCCmUAmCUmGUmGU*mA*A* asiRNAS cholesterol 86 14 SRD5A1cp- UUACACAGUAGGAUmAmUU*C*A*A*C asiRNAAS 86(2,4) 15 SRD5A1cp- UUACACAGUAGGAUmAmUmU*mC*A*A*C asiRNAAS 86(4,4) 16 SRD5A1cp- UUACACAGUAGGAUmAmUmU*mC*mA*mA*mC asiRNAAS 86(7,4) m: 2-O-Methyl/RNA *: phosphorothioated bond

[Example 4] Screening for Cp-asiRNA Targeting SRD5A1 Gene and Having Self Cell-Penetrating Ability

[0076] The inhibitory effects of the 12 kinds of cp-asiRNA shown in Table 2 against SRD5A1 expression were examined. A HuH-7 cell line was incubated with 1 M or 3 M of 12 kinds of cp-asiRNA in Opti-MEM media for 24 hours, and then the media were replaced with Dulbecco's Modified Eagle's Medium (Gibco) containing 10% fetal bovine serum (Gibco) and 100 units/ml penicillin 100 g/ml streptomycin, and 24 hours after media replacement, SRD5A1 expression was examined at the protein level. As illustrated in FIG. 3, as the result of repeatedly conducting two experiments, it was confirmed that SRD5A1 cp-asiRNA #49(2,4) and #86(7,4) exhibited gene inhibitory efficiency of 50% or higher.

[0077] The inhibitory effects of the two selected kinds of cp-asiRNA against SRD5A1 expression were examined in a HuH-7 cell line. The HuH-7 cell line was incubated with 0.3 M, 1 M, or 3 M of each of the two kinds of cp-asiRNA in Opti-MEM media for 24 hours, and then SRD5A1 expression was examined at the mRNA level. As the result of repeatedly conducting four experiments, it was confirmed that SRD5A1 cp-asiRNA #49(2,4) and #86(7,4) exhibited gene inhibitory efficiency of 50% or higher at a concentration of 1 M or higher (see FIG. 4).

[Example 5] Screening for 112 Kinds of RNAi-Inducing Double-Stranded Nucleic Acid Molecules Targeting SRD5A2

[0078] To obtain high-efficiency RNAi-inducing double-stranded nucleic acid molecules targeting SRD5A2, the target sequence of the SRD5A2 gene was selected and then asiRNA was designed. The asiRNA structure is different from that of generally known siRNAs, and thus when the nucleotide sequences of asiRNA are designed using a general siRNA design program, it may be somewhat difficult to design an optimized asiRNA. Therefore, asiRNA was constructed by the following method. An NCBI db search was used to obtain information on the SRD5A2 gene (mRNA Accession Number: NM_000348.3) which is thought to target male pattern hair loss (androgenetic hair loss). For subsequent animal experiments, nucleotide sequences with at least 80% homology to that of mice were secured, and then 100 kinds of asiRNA were designed according to a design method such as the exclusion of sequences having a GC content of 30-62% and 4 or more G or C consecutive bases, and then synthesized by OliX Inc. (Korea). The synthesized sense and antisense strand RNA oligonucleotides were annealed at 95 C. for 2 minutes through incubation at 37 C. for 1 hour, and the asiRNA annealed by 10% polyacrylamide gel electrophoresis (PAGE) was confirmed using a UV transilluminator.

TABLE-US-00003 TABLE3 112strandsofasiRNAnucleotidesequences targeting3-oxo-5-alpha-steroid4- dehydrogenase2 112kinds Sequence(5-3) No. Name S AS 1 asiSRD5A2 GUUCCUGCAGGAGCUG CAGCUCCUGCAGGAACCAGGC 1 (SEQIDNO:201) (SEQIDNO:313) 2 asiSRD5A2 UUCCUGCAGGAGCUGC GCAGCUCCUGCAGGAACCAGG 2 (SEQIDNO:202) (SEQIDNO:314) 3 asiSRD5A2 UCCUGCAGGASCUGCC GGCAGCUCCUGCAGGAACCAG 3 (SEQIDNO:203) (SEQIDNO:315) 4 asiSRD5A2 CCUGCAGGAGCUGCCU AGGCAGCUCCUGCAGGAACCA 4 (SEQIDNO:204) (SEQIDNO:316) 5 asiSRD5A2 CUGCAGGAGCUGCCUU AAGGCAGCUCCUGCAGGAACC 5 (SEQIDNO:205) (SEQIDNO:317) 6 asiSRD5A2 UGCAGGAGCUGCCUUC GAAGGCAGCUCCUGCAGGAAC 6 (SEQIDNO:206) (SEQIDNO:318) 7 asiSRD5A2 GCAGGAGCUGCCUUCC GGAAGGCAGCUCCUGCAGGAA 7 (SEQIDNO:207) (SEQIDNO:319) 8 asiSRD5A2 CAGGAGCUGCCUUCCU AGGAAGGCAGCUCCUGCAGGA 8 (SEQIDNO:208) (SEQIDNO:320) 9 asiSRD5A2 AGGAGCUGCCUUCCUU AAGGAAGGCAGCUCCUGCAGG 9 (SEQIDNO:209) (SEQIDNO:321) 10 asiSRD5A2 ACUUCCACAGGACAUU AAUGUCCUGUGGAAGUAAUGU 10 (SEQIDNO:210) (SEQIDNO:322) 11 asiSRD5A2 CUUCCACAGGACAUUU AAAUGUCCUGUGGAAGUAAUG 11 (SEQIDNO:211) (SEQIDNO:323) 12 asiSRD5A2 AGGUGGCUUGUUUACG CGUAAACAAGCCACCUUGUGG 12 (SEQIDNO:212) (SEQIDNO:324) 13 asiSRD5A2 GGUGGCUUGUUUACGU ACGUAAACAAGCCACCUUGUG 13 (SEQIDNO:213) (SEQIDNO:325) 14 asiSRD5A2 GUGGCUUGUUUACGUA UACGUAAACAAGCCACCUUGU 14 (SEQIDNO:214) (SEQIDNO:326) 15 asiSRD5A2 UGGCUUGUUUACGUAU AUACGUAAACAAGCCACCUUG 15 (SEQIDNO:215) (SEQIDNO:327) 16 asiSRD5A2 GGCUUGUUUACGUAUG CAUACGUAAACAAGCCACCUU 16 (SEQIDNO:216) (SEQIDNO:328) 17 asiSRD5A2 GCUUGUUUACGUAUGU ACAUACGUAAACAAGCCACCU 17 (SEQIDNO:217) (SEQIDNO:329) 18 asiSRD5A2 CUACCUCAAGAUGUUU AAACAUCUUGAGGUAGAACCU 18 (SEQIDNO:218) (SEQIDNO:330) 19 asiSRD5A2 GGUUCCUGCAGGAGCU AGCUCCUGCAGGAACCAGGCG 19 (SEQIDNO:219) (SEQIDNO:331) 20 asiSRD5A2 UACUUCCACAGGACAU AUGUCCUGUGGAAGUAAUGUA 20 (SEQIDNO:220) (SEQIDNO:332) 21 asiSRD5A2 AGACAUACGGUUUAGC GCUAAACCGUAUGUCUGUGUA 21 (SEQIDNO:221) (SEQIDNO:333) 22 asiSRD5A2 CAGACAUACGGUUUAG CUAAACCGUAUGUCUGUGUAC 22 (SEQIDNO:222) (SEQIDNO:334) 23 asiSRD5A2 ACAGACAUACGGUUUA UAAACCGUAUGUCUGUGUACC 23 (SEQIDNO:223) (SEQIDNO:335) 24 asiSRD5A2 CACAGACAUACGGUUU AAACCGUAUGUCUGUGUACCA 24 (SEQIDNO:224) (SEQIDNO:336) 25 asiSRD5A2 GGAUUCCACAAGGUGG CCACCUUGUGGAAUCCUGUAG 25 (SEQIDNO:225) (SEQIDNO:337) 26 asiSRD5A2 GAUUCCACAAGGUGGC GCCACCUUGUGGAAUCCUGUA 26 (SEQIDNO:226) (SEQIDNO:338) 27 asiSRD5A2 AUUCCACAAGGUGGCU AGCCACCUUGUGGAAUCCUGU 27 (SEQIDNO:227) (SEQIDNO:339) 28 asiSRD5A2 UUCCACAAGGUGGCUU AAGCCACCUUGUGGAAUCCUG 28 (SEQIDNO:228) (SEQIDNO:340) 29 asiSRD5A2 UCCACAAGGUGGCUUG CAAGCCACCUUGUGGAAUCCU 29 (SEQIDNO:228) (SEQIDNO:341) 30 asiSRD5A2 CCACAAGGUGGCUUGU ACAAGCCACCUUGUGGAAUCC 30 (SEQIDNO:230) (SEQIDNO:342) 31 asiSRD5A2 CACAAGGUGGCUUGUU AACAAGCCACCUUGUGGAAUC 31 (SEQIDNO:231) (SEQIDNO:343) 32 asiSRD5A2 ACAAGGUGGCUUGUUU AAACAAGCCACCUUGUGGAAU 32 (SEQIDNO:232) (SEQIDNO:344) 33 asiSRD5A2 CAAGGUGGCUUGUUUA UAAACAAGCCACCUUGUGGAA 33 (SEQIDNO:233) (SEQIDNO:345) 34 asiSRD5A2 AAGGUGGCUUGUUUAC GUAAACAAGCCACCUUGUGGA 34 (SEQIDNO:234) (SEQIDNO:346) 35 asiSRD5A2 CUGGAGCCAAUUUCCU AGGAAAUUGGCUCCAGAAACA 35 (SEQIDNO:235) (SEQIDNO:347) 36 asiSRD5A2 UCUGGAGCCAAUUUCC GGAAAUUGGCUCCAGAAACAU 36 (SEQIDNO:236) (SEQIDNO:348) 37 asiSRD5A2 UUCUGGAGCCAAUUUC GAAAUUGGCUCCAGAAACAUA 37 (SEQIDNO:237) (SEQIDNO:349) 38 asiSRD5A2 UUUCUGGAGCCAAUUU AAAUUGGCUCCAGAAACAUAC 38 (SEQIDNO:238) (SEQIDNO:350) 39 asiSRD5A2 GUUUCUGGAGCCAAUU AAUUGGCUCCAGAAACAUACG 39 (SEQIDNO:239) (SEQIDNO:351) 40 asiSRD5A2 UGUUUCUGGAGCCAAU AUUGGCUCCAGAAACAUACGU 40 (SEQIDNO:240) (SEQIDNO:352) 41 asiSRD5A2 AUGUUUCUGGAGCCAA UUGGCUCCAGAAACAUACGUA 41 (SEQIDNO:241) (SEQIDNO:353) 42 asiSRD5A2 UAUGUUUCUGGAGCCA UGGCUCCAGAAACAUACGUAA 42 (SEQIDNO:242) (SEQIDNO:354) 43 asiSRD5A2 GUAUGUUUCUGGAGCC GGCUCCAGAAACAUACGUAAA 43 (SEQIDNO:243) (SEQIDNO:355) 44 asiSRD5A2 GCUCCAGAAACAUACG GCUCCAGAAACAUACGUAAAC 44 (SEQIDNO:244) (SEQIDNO:356) 45 asiSRD5A2 CAUAGGUUCUACCUCA UGAGGUAGAACCUAUGGUCGU 45 (SEQIDNO:245) (SEQIDNO:357) 46 asiSRD5A2 AUAGGUUCUACCUCAA UUGAGGUAGAACCUAUGGUGG 46 (SEQIDNO:246) (SEQIDNO:358) 47 asiSRD5A2 UAGGUUCUACCUCAAG CUUGAGGUAGAACCUAUGGUG 47 (SEQIDNO:247) (SEQIDNO:359) 48 asiSRD5A2 AGGUUCUACCUCAAGA UCUUGAGGUAGAACCUAUGGU 48 (SEQIDNO:248) (SEQIDNO:360) 49 asiSRD5A2 GGUUCUACCUCAAGAU AUCUUGAGGUAGAACCUAUGG 49 (SEQIDNO:249) (SEQIDNO:361) 50 asiSRD5A2 GUUCUACCUCAAGAUG CAUCUUGAGGUAGAACCUAUG 50 (SEQIDNO:250) (SEQIDNO:362) 51 asiSRD5A2 UUCUACCUCAAGAUGU ACAUCUUGAGGUAGAACCUAU 51 (SEQIDNO:251) (SEQIDNO:363) 52 asiSRD5A2 UCUACCUCAAGAUGUU AACAUCUUGAGGUAGAACCUA 52 (SEQIDNO:252) (SEQIDNO:364) 53 asiSRD5A2 CAAAUCUCGGAAAGCC GGCUUUCCGAGAUUUGGGGUA 53 (SEQIDNO:253) (SEQIDNO:365) 54 asiSRD5A2 AAAUCUCGGAAAGCCC GGGCUUUCCGAGAUUUGGGGU 54 (SEQIDNO:254) (SEQIDNO:366) 55 asiSRD5A2 AAUCUCGGAAAGCCCU AGGGCUUUCCGAGAUUUGGGG 55 (SEQIDNO:255) (SEQIDNO:367) 56 asiSRD5A2 GCCCUUAUUCCAUUCA UGAAUGGAAUAAGGGCUUUCC 56 (SEQIDNO:256) (SEQIDNO:368) 57 asiSRD5A2 CCCUUAUUCCAUUCAU AUGAAUGGAAUAAGGGCUUUC 57 (SEQIDNO:257) (SEQIDNO:369) 58 asiSRD5A2 CCUUAUUCCAUUCAUC GAUGAAUGGAAUAAGGGCUUU 58 (SEQIDNO:258) (SEQIDNO:370) 59 asiSRD5A2 CUUAUUCCAUUCAUCU AGAUGAAUGGAAUAAGGGCUU 59 (SEQIDNO:259) (SEQIDNO:371) 60 asiSRD5A2 UUAUUCCAUUCAUCUU AAGAUGAAUGGAAUAAGGGCU 60 (SEQIDNO:260) (SEQIDNO:372) 61 asiSRD5A2 UAUUCCAUUCAUCUUU AAAGAUGAAUGGAAUAAGGGC 61 (SEQIDNO:261) (SEQIDNO:373) 62 asiSRD5A2 AUUCCAUUCAUCUUUU AAAAGAUGAAUGGAAUAAGGG 62 (SEQIDNO:262) (SEQIDNO:374) 63 asiSRD5A2 UUCCAUUCAUCUUUUA UAAAAGAUGAAUGGAAUAAGG 63 (SEQIDNO:263) (SEQIDNO:375) 64 asiSRD5A2 UCCAUUCAUCUUUUAA UUAAAAGAUGAAUGGAAUAAG 64 (SEQIDNO:264) (SEQIDNO:376) 65 asiSRD5A2 UCUCACUUUGUUUCCU AGGAAACAAAGUGAGAAAAAU 65 (SEQIDNO:265) (SEQIDNO:377) 66 asiSRD5A2 UUCUCACUUUGUUUCC GGAAACAAAGUGAGAAAAAUG 66 (SEQIDNO:266) (SEQIDNO:378) 67 asiSRD5A2 UUUCUCACUUUGUUUC GAAACAAAGUGAGAAAAAUGC 67 (SEQIDNO:267) (SEQIDNO:379) 68 asiSRD5A2 UUUUCUCACUUUGUUU AAACAAAGUGAGAAAAAUGCA 68 (SEQIDNO:268) (SEQIDNO:380) 69 asiSRD5A2 UUUUUCUCACUUUGUU AACAAAGUGAGAAAAAUGCAA 69 (SEQIDNO:269) (SEQIDNO:381) 70 asiSRD5A2 AUUUUUCUCACUUUGU ACAAAGUGAGAAAAAUGCAAA 70 (SEQIDNO:270) (SEQIDNO:382) 71 asiSRD5A2 UGGCAGGCAGCGCCAC GUGGCGCUGCCUGCCAGCACU 71 (SEQIDNO:271) (SEQIDNO:363) 72 asiSRD5A2 CUGGCAGGCAGCGCCA UGGCGCUGCCUGCCAGCACUG 72 (SEQIDNO:272) (SEQIDNO:384) 73 asiSRD5A2 GGCAGGCAGCGCCACU AGUGGCGCUGCCUGCCAGCAC 73 (SEQIDNO:273) (SEQIDNO:385) 74 asiSRD5A2 UGCCAGCCCGCGCCGC GCGGCGCGGGCUGGCAGGCGG 74 (SEQIDNO:274) (SEQIDNO:386) 75 asiSRD5A2 UUACUUCCACAGGACA UGUCCUGUGGAAGUAAUGUAG 75 (SEQIDNO:275) (SEQIDNO:387) 76 asiSRD5A2 GUGGAAGUAAUGUAGG CCUACAUUACUUCCACAGGAC 76 (SEQIDNO:276) (SEQIDNO:388) 77 asiSRD5A2 CCCUGAUGGGUGGUAC GUACCACCCAUCAGGGUAUUC 77 (SEQIDNO:277) (SEQIDNO:389) 78 asiSRD5A2 CCUGAUGGGUGGUACA UGUACCACCCAUCAGGGUAUU 78 (SEQIDNO:278) (SEQIDNO:390) 79 asiSRD5A2 CUGAUGGGUGGUACAC GUGUACCACCCAUCAGGGUAU 79 (SEQIDNO:279) (SEQIDNO:391) 80 asiSRD5A2 UGAUGGGUGGUACACA UGUGUACCACCCAUCAGGGUA 80 (SEQIDNO:280) (SEQIDNO:392) 81 asiSRD5A2 GAUGGGUGGUACACAG CUGUGUACCACCCAUCAGGGU 81 (SEQIDNO:281) (SEQIDNO:393) 82 asiSRD5A2 AUGGGUGGUACACAGA UCUGUGUACCACCCAUCAGGG 82 (SEQIDNO:282) (SEQIDNO:394) 83 asiSRD5A2 UGGGUGGUACACAGAC GUCUGUGUACCACCCAUCAGG 83 (SEQIDNO:283) (SEQIDNO:395) 84 asiSRD5A2 GGGUGGUACACAGACA UGUCUGUGUACCACCCAUCAG 84 (SEQIDNO:284) (SEQIDNO:396) 85 asiSRD5A2 GGUGGUACACAGACAU AUGUCUGUGUACCACCCAUCA 85 (SEQIDNO:285) (SEQIDNO:397) 86 asiSRD5A2 GACAUACGGUUUAGCU AGCUAAACCGUAUGUCUGUGU 86 (SEQIDNO:286) (SEQIDNO:390) 87 asiSRD5A2 CUUGGGUGUCUUCUUA UAAGAAGACACCCAAGCUAAA 87 (SEQIDNO:287) (SEQIDNO:399) 88 asiSRD5A2 GCUUGGGUGUCUUCUU AAGAAGACACCCAAGCUAAAC 88 (SEQIDNO:288) (SEQIDNO:400) 89 asiSRD5A2 AGCUUGGGUGUCUUCU AGAAGACACCCAAGCUAAACC 89 (SEQIDNO:289) (SEQIDNO:401) 90 asiSRD5A2 UAGCUUGGGUGUCUUC GAAGACACCCAAGCUAAACCG 90 (SEQIDNO:290) (SEQIDNO:402) 91 asiSRD5A2 GCCAGCUCAGGAAGCC GGCUUCCUGAGCUGGCGCAAU 91 (SEQIDNO:291) (SEQIDNO:403) 92 asiSRD5A2 CGCCAGCUCAGGAAGC GCUUCCUGAGCUGGCGCAAUA 92 (SEQIDNO:292) (SEQIDNO:404) 93 asiSRD5A2 GCGCCAGCUCAGGAAG CUUCCUGAGCUGGCGCAAUAU 93 (SEQIDNO:293) (SEQIDNO:405) 94 asiSRD5A2 UGGAGCCAAUUUCCUC GAGGAAAUUGGCUCCAGAAAC 94 (SEQIDNO:294) (SEQIDNO:406) 95 asiSRD5A2 CUCACUUUGUUUCCUU AAGGAAACAAAGUGAGAAAAA 95 (SEQIDNO:295) (SEQIDNO:407) 96 asiSRD5A2 CAUUUUUCUCACUUUG CAAAGUGAGAAAAAUGCAAAU 96 (SEQIDNO:296) (SEQIDNO:408) 97 asiSRD5A2 CCAUAGGUUCUACCUC GAGGUAGAACCUAUGGUGGUG 97 (SEQIDNO:297) (SEQIDNO:409) 98 asiSRD5A2 ACCAUAGGUUCUACCU AGGUAGAACCUAUGGUGGUGA 98 (SEQIDNO:298) (SEQIDNO:410) 99 asiSRD5A2 CACCAUAGGUUCUACC GGUAGAACCUAUGGUGGUGAA 99 (SEQIDNO:299) (SEQIDNO:411) 100 asiSRD5A2 CCACCAUAGGUUCUAC GUAGAACCUAUGGUGGUGAAA 100 (SEQIDNO:300) (SEQIDNO:412) 101 asiSRD5A2 ACCACCAUAGGUUCUA UAGAACCUAUGGUGGUGAAAA 101 (SEQIDNO:301) (SEQIDNO:413) 102 asiSRD5A2 CACCACCAUAGGUUCU AGAACCUAUGGUGGUGAAAAG 102 (SEQIDNO:302) (SEQIDNO:414) 103 asiSRD5A2 GGACUACCCCAAAUCU AGAUUUGGGGUAGUCCUCAAA 103 (SEQIDNO:303) (SEQIDNO:415) 104 asiSRD5A2 AGGACUACCCCAAAUC GAUUUGGGGUAGUCCUCAAAC 104 (SEQIDNO:304) (SEQIDNO:416) 105 asiSRD5A2 GAGGACUACCCCAAAU AUUUGGGGUAGUCCUCAAACA 105 (SEQIDNO:305) (SEQIDNO:417) 106 asiSRD5A2 UGAGGACUACCCCAAA UUUGGGGUAGUCCUCAAACAU 106 (SEQIDNO:306) (SEQIDNO:418) 107 asiSRD5A2 UUGAGGACUACCCCAA UUGGGGUAGUCCUCAAACAUC 107 (SEQIDNO:307) (SEQIDNO:419) 108 asiSRD5A2 UUUGAGGACUACCCCA UGGGGUAGUCCUCAAACAUCU 108 (SEQIDNO:308) (SEQIDNO:420) 109 asiSRD5A2 CCAAAUCUCGGAAAGC GCUUUCCGAGAUUUGGGGUAG 109 (SEQIDNO:309) (SEQIDNO:421) 110 asiSRD5A2 AGCCCUUAUUCCAUUC GAAUGGAAUAAGGGCUUUCCG 110 (SEQIDNO:310) (SEQIDNO:422) 111 asiSRD5A2 AAGCCCUUAUUCCAUU AAUGGAAUAAGGGCUUUCCGA 111 (SEQIDNO:311) (SEQIDNO:423) 112 asiSRD5A2 GGCUAUGCCCUGGCCA UGGCCAGGGCAUAGCCGAUCC 112 (SEQIDNO:312) (SEQIDNO:424)

[Example 6] Screening for RNAi-Inducing Double-Stranded Nucleic Acid Molecules Targeting SRD5A2

[0079] To confirm gene inhibitory efficiency at the mRNA level, 112 selected kinds of asiRNA were transfected into a HuH-7 cell line at a concentration of 0.3 nM, and qRT-PCR was performed to measure the expression level of SRD5A1 mRNA. The HuH-7 cell line was cultured in Dulbecco's Modified Eagle's Medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS, Gibco) and 100 units/ml of penicillin 100 g/ml of streptomycin. HuH-7 cells were seeded in a 24-well plate at a density of 510.sup.4 cells/well, and a reverse transfection experiment was conducted using asiRNA (0.3 nM, OliX Inc.) and Lipofectamine 2000 (1 l/ml, Invitrogen Inc.) in Opti-MEM (a total volume of 500 l) in accordance with Invitrogen's protocol. After 24 hours, total RNA was extracted using TRIzol (TaKaPa), and then cDNA was synthesized using a high-capacity cDNA reverse transcription kit (Applied Biosystems), and the expression level of the SRD5A2 gene was examined using power SYBR green PCR master Mix (Applied Biosystems), the following primers, and a StepOne real-time PCR system (see FIGS. 5A and 5B).

[0080] The nucleotide sequences of the primers used in the experiment are shown in Table 4 below.

TABLE-US-00004 TABLE4 Primernucleotidesequences Name Sequence(5-3) size Human Forward GAGTCAACGGATTTGGTCGT 186 GAPDH (SEQIDNO:425) Reverse GACAAGCTTCCCGTTCTCAG (SEQIDNO:426) Human Forward TGAACCTGGGTGGCTTATGA 242 SRD5A2 (SEQIDNO:427) Reverse GAAAGGAAAGTTGCTTGGG (SEQIDNO:428)

[0081] From the results of screening 112 asiRNAs, the 23 top-ranked asiRNAs (in Table 3, No. 5, 8, 28, 31, 32, 33, 37, 38, 39, 40, 42, 48, 49, 59, 60, 62, 65, 83, 84, 85, 91, 92, and 100) were selected, and western blotting was performed at a concentration of 10 nM. HuH-7 cells were seeded in a 6-well plate at a density of 2.510.sup.3 cells/well, and then a reverse transfection experiment was conducted using asiRNA and Lipofectamine 2000 (1 l/ml, Invitrogen Inc.) in Opti-MEM (a total volume of 2 ml) in accordance with Invitrogen's protocol. After 48 hours, the cells were lysed using a mammalian protein extraction buffer (GE healthcare), and then proteins were quantified using a Bradford assay. 10 g of the protein of each sample was electrophoresed using 12% SDS-PAGE at 80 V for 20 minutes and at 120 V for 1 hour, and then transferred onto a PVDE membrane (Bio-Rad) at 300 mA for 1 hour and 20 minutes. After transfer, the membrane was blocked in 5% skim milk for 1 hour and then allowed to react with SRD5A2 antibody (ABcam, ab124877) at a ratio of 1:2000 for 12 hours. The next day, the resulting membrane was allowed to react with anti-Rabbit HRP (Santa Cruz) at a ratio of 1:10000 for 1 hour, and then the expression levels of the SRD5A2 protein were compared with each other using ChemiDoc (Bio-Rad). In the present experiment, 4 asiRNAs (in Table 3, No. 31, 59, 60, and 62) capable of inhibiting SRD5A2 protein expression by about 50% or higher were selected (see FIGS. 6A and 6B).

[Example 7] 16 Kinds of Cp-asiRNA Targeting SRD5A2 Gene and Having Self Cell-Penetrating Ability

[0082] SRD5A2 cp-asiRNAs (total 16 strands) were designed by applying three modification patterns to 4 kinds (in Table 3, No. 31, 59, 60, and 62) of asiRNA targeting SRD5A2 according to the number and position of 2OMe (methyl), phosphorothioate bonds (PS), and cholesterol, and then synthesized by OliX Inc. (Korea). cp-asiRNA enhances endocytosis efficiency and stability and thus may penetrate through the cell membrane with high efficiency without the aid of a delivery vehicle to thereby inhibit the expression of the target gene. The synthesized sense and antisense strand RNA oligonucleotides were annealed at 95 C. for 2 minutes through incubation at 37 C. for 1 hour, and cp-asiRNAs annealed by 10% polyacrylamide gel electrophoresis (PAGE) were confirmed using a UV transilluminator.

TABLE-US-00005 TABLE5 16strandsofcp-asiRNAnucleotidesequences targetingSRD5A2 No. Name Sequence(5-->3) 1 SRD5A2cp- mCAmCAmAGmGUmGCmCUmU*G*mU*U asiRNA cholesterol S31 2 SRD5A2cp- AACAAGCCACCUUGmUmGG*A*A*U*C asiRNA AS31(2,4) 3 SRD5A2cp- AACAAGCCACCUUGmUmSmG*mA*A*U*C asiRNA AS31(4,4) 4 SRD5A2cp- AACAAGCCACCUUGmUmGmG*mA*mA*mU*mC asiRNA AS31(7,4) 5 SRD5A2cp- mCUmUAmUUmCCmAUMUCmA*U*mC*U asiRNA cholesterol S59 6 SRD5A2cp- AGAUGAAUGGAAUAmAmGG*G*C*U*U asiRNA AS59(2,4) 7 SRD5A2cp- AGAUGAAUGGAAUAmAmGmG*mG*C*U*U asiRNA AS59(4,4) 8 SRD5A2cp- AGAUGAAUGGAAUAmAmGmG*mG*mC*mU*mU asiRNA AS59(7,4) 9 SRD5A2cp- mUUmAUmUCmCAmUUmCAmU*C*mU*U asiRNA cholesterol S60 10 SRD5A2cp- AAGAUGAAUGGAAUmAmAG*G*G*C*U asiRNA AS60(2,4) 11 SRD5A2cp- AAGAUGAAUGGAAUmAmAmG*mG*G*C*U asiRNA AS60(4,4) 12 SRD5A2cp- AAGAUGAAUGGAAUmAmAmG*mG*mG*mC*mU asiRNA AS60(7,4) 13 SRD5A2cp- mAUmUCmCAmUUmCAmUC*mU*U*mU*U asiRNA cholesterol S62 14 SRD5A2cp- AAAAGAUGAAUGGAmAmUA*A*G*G*G asiRNA AS62(2,4) 15 SRD5A2cp- AAAAGAUGAAUGGAmAmUmA*mA*G*G*G asiRNA AS62(4,4) 16 SRD5A2cp- AAAAGAUGAAUGGAmAmUmA*mA*mG*mG*mG asiRNA AS62(7,4) m: 2-O-Methyl/RNA *: phosphorothioated bond

[Example 8] Screening for Cp-asiRNA Targeting SRD5A2 Gene and Having Self Cell-Penetrating Ability

[0083] The inhibitory effects of the 12 kinds of cp-asiRNA shown in Table 5 against SRD5A2 expression were examined. A HuH-7 cell line was incubated with 1 M or 3 M of 12 kinds of cp-asiRNA in Opti-MEM media for 24 hours, and then the media were replaced with Dulbecco's Modified Eagle's Medium (Gibco) containing 10% fetal bovine serum (Gibco) and 100 units/ml penicillin 100 g/ml streptomycin, and 24 hours after media replacement, SRD5A2 expression was examined at the protein level. As the result of repeatedly conducting two experiments, it was confirmed that SRD5A2 cp-asiRNA #59(4,4) and #62(4,4) exhibited gene inhibitory efficiency of 50% or higher (see FIG. 7).

[Example 9] Confirmation of Inhibitory Efficiency of 2 Selected Kinds of Cp-asiRNA Against Target Gene SRD5A2 Expression

[0084] The inhibitory effects of the two above-selected kinds of cp-asiRNA against SRD5A2 expression were examined in a HuH-7 cell line. The HuH-7 cell line was incubated in Opti-MEM media with 1.95 nM, 3.9 nM, 7.8 nM, 15.6 nM, 31.3 nM, 62.5 nM, 125 nM, 250 nM, 500 nM, or 1,000 nM of each of the two kinds of cp-asiRNA for 24 hours, and then SRD5A2 expression was examined at the mRNA level. As the result of repeatedly conducting three experiments, it was confirmed that SRD5A2 cp-asiRNA #59(4,4) and #62(4,4) had IC.sub.50 values of 22.37 nM and 27.18 nM, respectively (see FIG. 8).

[0085] The inhibitory effects of the two above-selected kinds of cp-asiRNA against SRD5A2 expression were examined in a HuH-7 cell line. The HuH-7 cell line was incubated with 0.1 M, 0.3 M, 1 M, or 3 M of the two kinds of cp-asiRNA in Opti-MEM media for 24 hours, and then the media were replaced with Dulbecco's Modified Eagle's Medium (Gibco) containing 10% fetal bovine serum (Gibco) and 100 units/ml penicillin 100 g/ml streptomycin, and 24 hours after media replacement, SRD5A2 expression was examined at the protein level. As the result of repeatedly conducting two experiments, it was confirmed that, as the treatment concentrations of cp-asiSRD5A2 #59(4,4) and #62(4,4) increased, the protein expression of the target gene SRD5A2 was reduced (see FIG. 9).

[Example 10] Screening for 118 Kinds of RNAi-Inducing Double-Stranded Nucleic Acid Molecules Targeting AR

[0086] To obtain high-efficiency RNAi-inducing double-stranded nucleic acid molecules targeting AR, the target sequence of the AR gene was selected and then asiRNA was designed. The asiRNA structure is different from that of generally known siRNAs, and thus when the nucleotide sequences of asiRNA are designed using a general siRNA design program, it may be somewhat difficult to design an optimized asiRNA. Therefore, asiRNA was constructed by the following method. An NCBI db search was used to obtain information on the AR gene (mRNA Accession Number: NM_001011645.2), which is the target gene pertaining to male pattern hair loss (androgenetic hair loss). For subsequent animal experiments, nucleotide sequences with at least 80% homology to that of mice were secured, and then 100 asiRNAs were designed according to a design method such as the exclusion of sequences having a GC content of 30-62% and 4 or more G or C consecutive bases, and then synthesized by OliX Inc. (Korea). The synthesized sense and antisense strand RNA oligonucleotides were annealed at 95 C. for 2 minutes through incubation at 37 C. for 1 hour, and the asiRNA annealed by 10% polyacrylamide gel electrophoresis (PAGE) was confirmed using a UV transilluminator.

TABLE-US-00006 TABLE6 118strandsofasiRNAnucleotidesequences targetingandrogenreceptor Sequence(5-3) No. Name S(16mer) As(21mer) 1 asiAR1 GAGAUGAAGCUUCUGG CCAGAAGCUUCAUCUCCACAG (SEQIDNO:429) (SEQIDNO:547) 2 asiAR2 GGAGAUGAAGCUUCUG CAGAAGCUUCAUCUCCACAGA (SEQIDNO:430) (SEQIDNO:548) 3 asiAR3 GUGGAGAUGAAGCUUC GAAGCUUCAUCUCCACAGAUC (SEQIDNO:431) (SEQIDNO:549) 4 asiAR4 UGUGGAGAUGAAGCUU AAGCUUCAUCUCCACAGAUCA (SEQIDNO:432) (SEQIDNO:550) 5 asiAR5 UCUGUGGAGAUGAAGC GCUUCAUCUCCACAGAUCAGG (SEQIDNO:433) (SEQIDNO:551) 6 asiAR6 UGAUCUGUGGAGAUGA UCAUCUCCACAGAUCAGGCAG (SEQIDNO:434) (SEQIDNO:552) 7 asiAR7 CUGAUCUGUGGAGAUG CAUCUCCACAGAUCAGGCAGG (SEQIDNO:435) (SEQIDNO:553) 8 asiAR8 AAGACCUGCCUGAUCU AGAUCAGGCAGGUCUUCUGGG (SEQIDNO:436) (SEQIDNO:554) 9 asiAR9 UUUCCACCCCAGAAGA UCUUCUGGGGUGGAAAGUAAU (SEQIDNO:437) (SEQIDNO:555) 10 asiAR10 ACUUUCCACCCCAGAA UUCUGGGGUGGAAAGUAAUAG (SEQIDNO:438) (SEQIDNO:556) 11 asiAR11 AAGGGAAACAGAAGUA UACUUCUGUUUCCCUUCAGCG (SEQIDNO:439) (SEQIDNO:557) 12 asiAR12 GAAGGGAAACAGAAGU ACUUCUGUUUCCCUUCAGCGG (SEQIDNO:440) (SEQIDNO:558) 13 asiAR13 CUGAAGGGAAACAGAA UUCUGUUUCCCUUCAGCGGCU (SEQIDNO:441) (SEQIDNO:559) 14 asiAR14 CAAAAGAGCCGCUGAA UUCAGCGGCUCUUUUGAAGAA (SEQIDNO:442) (SEQIDNO:560) 15 asiAR15 UCAAAAGAGCCGCUGA UCAGCGGCUCUUUUGAAGAAG (SEQIDNO:443) (SEQIDNO:561) 16 asiAR16 CUUCAAAAGAGCCGCU AGCGGCUCUUUUGAAGAAGAC (SEQIDNO:444) (SEQIDNO:562) 17 asiSR17 CUUCUUCAAAAGAGCC GGCUCUUUUGAAGAAGACCUU (SEQIDNO:445) (SEQIDNO:563) 18 asiAR18 UCUUCUUCAAAAGAGC GCUCUUUUGAAGAAGACCUUG (SEQIDNO:446) (SEQIDNO:564) 19 asiAR19 AGGUCUUCUUCAAAAG CUUUUGAAGAAGACCUUGCAG (SEQIDNO:447) (SEQIDNO:565) 20 asiAR20 AACCAGGGAUGACUCU AGAGUCAUCCCUGCUUCAUAA (SEQIDNO:448) (SEQIDNO:566) 21 asiAR21 UGAAGCAGGGAUGACU AGUCAUCCCUGCUUCAUAACA (SEQIDNO:449) (SEQIDNO:567) 22 asiAR22 UUAUGAAGCAGGGAUG CAUCCCUGCUUCAUAACAUUU (SEQIDNO:450) (SEQIDNO:568) 23 asiAR23 UGUUAUGAAGCAGGGA UCCCUGCUUCAUAACAUUUCC (SEQIDNO:451) (SEQIDNO:569) 24 asiAR24 AUGUUAUGAAGCAGGG CCCUGCUUCAUAACAUUUCCG (SEQIDNO:452) (SEQIDNO:570) 25 asiAR25 GCUAUGAAUGUCAGCC GGCUGACAUUCAUAGCCUUCA (SEQIDNO:453) (SEQIDNO:571) 26 asiAR26 GGCUAUGAAUGUCAGC GCUGACAUUCAUAGCCUUCAA (SEQIDNO:454) (SEQIDNO:572) 27 asiAR27 GAAGGCUAUGAAUGUC GACAUUCAUAGCCUUCAAUGU (SEQIDNO:455) (SEQIDNO:573) 28 asiAR28 UUGAAGGCUAUGAAUG CAUUCAUAGCCUUCAAUGUGU (SEQIDNO:456) (SEQIDNO:574) 29 asiAR29 GAAGCCAUUGAGCCAG CUGGCUCAAUGGCUUCCAGGA (SEQIDNO:457) (SEQIDNO:575) 30 asiAR30 CUGGCUUCCGCAACUU AAGUUGCGGAAGCCAGGCAAG (SEQIDNO:458) (SEQIDNO:576) 31 asiAR31 UGCCUGGCUUCCGCAA UUGCGGAAGCCAGGCAAGGCC (SEQIDNO:459) (SEQIDNO:577) 32 asiAR32 AGUGGGCCAAGGCCUU AAGGCCUUGGCCCACUUGACC (SEQIDNO:460) (SEQIDNO:578) 33 asiAR33 CCAGGAUGCUCUACUU AAGUAGAGCAUCCUGGAGUUG (SEQIDNO:481) (5E0IDNO:579) 34 asiAR34 UCCAGGAUGCUCUACU AGUAGAGCAUCCUGGAGUUGA (SEQIDNO:462) (SEQIDNO:580) 35 asiAR35 AACUCCAGGAUGCUCU AGAGCAUCCUGGAGUUGACAU (SEQIDNO:463) (SEQIDNO:581) 36 asiAR36 UACCGCAUGCACAAGU ACUUGUGCAUGCGGUACUCAU (SEQIDNO:464) (SEQIDNO:582) 37 asiAR37 AGUACCGCAUGCACAA UUGUGCAUGCGGUACUCAUUG (SEQIDNO:465) (SEQIDNO:583) 38 asiAR38 CAAUGAGUACCGCAUG CAUGCGGUACUCAUUGAAAAC (SEQIDNO:466) (SEQIDNO:594) 39 asiAR39 UCAAUGAGUACCGCAU AUGCGGUACUCAUUGAAAACC (SEQIDNO:467) (SEQIDNO:585) 40 asiAR40 UUCAAUGAGUACCGCA UGCGGUACUCAUUGAAAACCA (SEQIDNO:468) (SEQIDNO:586) 41 asiAR41 UUGGAUGGCUCCAAAU AUUUGGAGCCAUCCAAACUCU (SEQIDNO:469) (SEQIDNO:587) 42 asiAR42 AGUUUGGAUGGCUCCA UGGAGCCAUCCAAACUCCUGA (SEQIDNO:470) (SEQIDNO:588) 43 asiAR43 AGAGUUUGGAUGGCUC GAGCCAUCCAAACUCUUGAGA (SEQIDNO:471) (SEQIDNO:589) 44 asiAR44 UCAAGGAACUCGAUCG CGAUCGAGUUCCUUGAUGUAG (SEQIDNO:472) (SEQIDNO:590) 45 asiAR45 CAUCAAGGAACUCGAU AUCGAGUUCCUUGAUGUAGUU (SEQIDNO:473) (SEQIDNO:591) 46 asiAR46 CUACAUCAAGGAACUC GAGUUCCUUGAUGUAGUUCAU (SEQIDNO:474) (SEQIDNO:592) 47 asiAR47 GAACUACAUCAAGGAA UUCCUUGAUGUAGUUCAUUCG (SEQIDNO:475) (SEQIDNO:593) 48 asiAR48 CUUCGAAUGAACUACA UGUAGUUCAUUCGAAGUUCAU (SEQIDNO:476) (SEQIDNO:594) 49 asiAR49 UGAACUUCGAAUGAAC GUUCAUUCGAAGUUCAUCAAA (SEQIDNO:477) (SEQIDNO:595) 50 asiAR50 UGAUGAACUUCGAAUG CAUUCGAAGUUCAUCAAAGAA (SEQIDNO:478) (SEQIDNO:596) 51 asiAR51 GGGCUGAAAAAUCAAA UUUGAUUUUUCAGCCCAUCCA (SEQIDNO:479) (SEQIDNO:597) 52 asiAR52 GAUGGGCUGAAAAAUC GAUUUUUCAGCCCAUCCACUG (SEQIDNO:480) (SEQIDNO:598) 53 asiAR53 UAUUCCAGUGGAUGGG CCCAUCCACUGGAAUAAUGCU (SEQIDNO:481) (SEQIDNO:599) 54 asiAR54 CAUUAUUCCAGUGGAU AUCCACUGGAAUAAUGCUGAA (SEQIDNO:482) (SEQIDNO:600) 55 asiAR55 AGCAUUAUUCCAGUGG CCACUGGAAUAAUGCUGAAGA (SEQIDNO:483) (SEQIDNO:601) 56 asiAR56 UUCAGCAUUAUUCCAG CUGGAAUAAUGCUGAAGAGAG (SEQIDNO:484) (SEQIDNO:602) 57 asiAR57 CUCUUCAGCAUUAUUC GAAUAAUGCUGAAGAGAGCAG (SEQIDNO:485) (SEQIDNO:603) 58 asiAR58 CUGCUCUCUUCAGCAU AUGCUGAAGAGAGCAGUGCUU (SEQIDNO:486) (SEQIDNO:604) 59 asiAR59 AAGCACUGCUCUCUUC GAAGAGAGCAGUGCUUUCAUG (SEQIDNO:487) (SEQIDNO:605) 60 asiAR60 GAAAGCACUGCUCUCU AGAGAGCAGUGCUUUCAUGCA (SEQIDNO:488) (SEQIDNO:606) 61 asiAR61 CAUGAAAGCACUGCUC GAGCAGUGCUUUCAUGCACAG (SEQIDNO:489) (SEQIDNO:607) 62 asiAR62 GUGCAUGAAAGCACUG CAGUGCUUUCAUGCACAGGAA (SEQIDNO:490) (SEQIDNO:608) 63 asiAR63 UUCCUGUGCAUGAAAG CUUUCAUGCACAGGAAUUCCU (SEQIDNO:491) (SEQIDNO:609) 64 asiAR64 GAAUUCCUGUGCAUGA UCAUGCACAGGAAUUCCUGGG (SEQIDNO:492) (SEQIDNO:610) 65 asiAR65 AGGAAUUCCUGUGCAU AUGCACAGGAAUUCCUGGGGG (SEQIDNO:493) (SEQIDNO:611) 66 asiAR66 UCACCAAGCUCCUGGA UCCAGGAGCUUGGUGAGCUGG (SEQIDNO:494) (SEQIDNO:612) 67 asiAR67 ACCAGCUCACCAAGCU AGCUUGGUGAGCUGGUAGAAG (SEQIDNO:495) (SEQIDNO:613) 68 asiAR68 CUACCAGCUCACCAAG CUUGGUGAGCUGGUAGAAGCG (SEQIDNO:496) (SEQIDNO:614) 69 asiAR69 ACCUGCUAAUCAAGUC GACUUGAUUAGCAGGUCAAAA (SEQIDNO:497) (SEQIDNO:615) 70 asiAR70 GACCUGCUAAUCAAGU ACUUGAUUAGCAGGUCAAAAG (SEQIDNO:498) (SEQIDNO:616) 71 asiAR71 UUUGACCUGCUAAUCA UGAUUAGCAGGUCAAAAGUGA (SEQIDNO:499) (SEQIDNO:617) 72 asiAR72 CUUUUGACCUGCUAAU AUUAGCAGGUCAAAAGUGAAC (SEQIDNO:500) (SEQIDNO:618) 73 asiAR73 UCACUUUUGACCUGCU AGCAGGUCAAAAGUGAACUGA (SEQIDNO:501) (SEQIDNO:619) 74 asiAR74 UUCACUUUUGACCUGC GCAGGUCAAAAGUGAACUGAU (SEQIDNO:502) (SEQIDNO:620) 75 asiAR75 CAGUUCACUUUUGACC GGUCAAAAGUGAACUGAUGCA (SEQIDNO:503) (SEQIDNO:621) 76 asiAR76 CAUCAGUUCACUUUUG CAAAAGUGAACUGAUGCAGCU (SEQIDNO:504) (SEQIDNO:622) 77 asiAR77 CUGCAUCAGUUCACUU AAGUGAACUGAUGCAGCUCUC (SEQIDNO:505) (SEQIDNO:623) 78 asiAR78 GCUGCAUCAGUUCACU AGUGAACUGAUGCAGCUCUCU (SEQIDNO:506) (SEQIDNO:624) 79 asiAR79 CCAUCUAUUUCCACAC GUGUGGAAAUAGAUGGGCUUG (SEQIDNO:507) (SEQIDNO:625) 80 asiAR80 CCCAUCUAUUUCCACA UGUGGAAAUAGAUGGGCUUGA (SEQIDNO:508) (SEQIDNO:626) 81 asiAR81 AGCCCAUCUAUUUCCA UGGAAAUAGAUGGGCUUGACU (SEQIDNO:509) (SEQIDNO:627) 82 asiAR82 UCAAGCCCAUCUAUUU AAAUAGAUGGGCUUGACUUUC (SEQIDNO:510) (SEQIDNO:628) 83 asiAR83 GGAAAGUCAAGCCCAU AUGGGCUUGACUUUCCCAGAA (SEQIDNO:511) (SEQIDNO:629) 84 asiAR84 CUGGGAAAGUCAAGCC GGCUUGACUUUCCCAGAAAGG (SEQIDNO:512) (SEQIDNO:630) 85 asiAR85 UUUCUGGGAAAGUCAA UUGACUUUCCCAGAAAGGAUC (SEQIDNO:513) (SEQIDNO:631) 86 asiAR86 UCCUUUCUGGGAAAGU ACUUUCCCAGAAAGGAUCUUG (SEQIDNO:514) (SEQIDNO:632) 87 asiAR87 CCAAGAUCCUUUCUGG CCAGAAAGGAUCUUGGGCACU (SEQIDNO:515) (SEQIDNO:633) 88 asiAR88 UGCCCAAGAUCCUUUC GAAAGGAUCUUGGGCACUUGC (SEQIDNO:516) (SEQIDNO:634) 89 asiAR89 AAGUGCCCAAGAUCCU AGGAUCUUGGGCACUUGCACA (SEQIDNO:517) (SEQIDNO:635) 90 asiAR90 UGCAAGUGCCCAAGAU AUCUUGGGCACUUGCACAGAG (SEQIDNO:518) (SEQIDNO:636) 91 asiAR91 UCUCUGUGCAAGUGCC GGCACUUGCACAGAGAUGAUC (SEQIDNO:519) (SEQIDNO:637) 92 asiAR92 UCAUCUCUGUGCAAGU ACUUGCACAGAGAUGAUCUCU (SEQIDNO:520) (SEQIDNO:636) 93 asiAR93 AGAUCAUCUCUGUGCA UGCACAGAGAUGAUCUCUGCC (SEQIDNO:521) (SEQIDNO:639) 94 asiAR94 CAGAGAUCAUCUCUGU ACAGAGAUGAUCUCUGCCAUC (SEQIDNO:522) (SEQIDNO:640) 95 asiAR95 CACUGGCACUAAAAAA UUUUUUAGUGCCAGUGAACAU (SEQIDNO:523) (SEQIDNO:641) 96 asiAR96 UCACUGGCACUAAAAA UUUUUAGUGCCAGUGAACAUA (SEQIDNO:524) (SEQIDNO:642) 97 asiAR97 GUUCACUGGCACUAAA UUUAGUGCCAGUGAACAUACA (SEQIDNO:525) (SEQIDNO:643) 98 asiAR98 UAUGUUCACUGGCACU AGUGCCAGUGAACAUACAUAA (SEQIDNO:526) (SEQIDNO:644) 99 asiAR99 UGUAUGUUCACUGGCA UGCCAGUGAACAUACAUAAAA (SEQIDNO:527) (SEQIDNO:645) 100 asiAR100 UAUGUAUGUUCACUGG CCAGUGAACAUACAUAAAAAU (SEQIDNO:528) (SEQIDNO:646) 101 asiAR101 GGGUAGUUGCUGAGGU ACCUCAGCAACUACCCAAAGG (SEQIDNO:529) (SEQIDNO:647) 102 asiAR102 UGGGUAGUUGCUGAGG CCUCAGCAACUACCCAAAGGA (SEQIDNO:530) (SEQIDNO:648) 103 asiAR103 CUUUGGGUAGUUGCUG CAGCAACUACCCAAAGGACAG (SEQIDNO:531) (SEQIDNO:649) 104 asiAR104 CCUUUGGGUAGUUGCU AGCAACUACCCAAAGGACAGA (SEQIDNO:532) (SEQIDNO:650) 105 asiAR105 CCACCAUCCACAUGAU AUCAUGUGGAUGGUGGACAUA (SEQIDNO:533) (SEQIDNO:651) 106 asiAR106 CAUUAGUGCCUCUUUG CAAAGAGGCACUAAUGCUUGC (SEQIDNO:534) (SEQIDNO:652) 107 asiAR107 GCAUUAGUGCCUCUUU AAAGAGGCACUAAUGCUUGCU (SEQIDNO:535) (SEQIDNO:653) 108 asiAR108 AGCAUUAGUGCCUCUU AAGAGGCACUAAUGCUUGCUC (SEQIDNO:536) (SEQIDNO:654) 109 asiAR109 AAGCAUUAGUGCCUCU AGAGGCACUAAUGCUUGCUCC (SEQIDNO:537) (SRIIDNO:655) 110 asiAR110 GCCCAUGUUAGCUUAU AUAAGCUAACAUGGGCACUAG (SEQIDNO:538) (SEQIDNO:656) 111 asiAR111 GAAACUUGUUUGUUGG CCAACAAACAAGUUUCUGCCA (SEQIDNO:539) (SEQIDNO:657) 112 asiAR112 GCAGAAACUUGUUUGU ACAAACAAGUUUCUGCCAUUU (SEQIDNO:540) (SEQIDNO:658) 113 asiAR113 AUGGCAGAAACUUGUU AACAAGUUUCUGCCAUUUUUA (SEQIDNO:541) (SEQIDNO:659) 114 asiAR114 AAUGGCAGAAACUUGU ACAAGUUUCUGCCAUUUUUAA (SEQIDNO:542) (SEQIDNO:660) 115 asiAR115 GGAAUCUUUUGUUGCU AGCAACAAAAGAUUCCAAGAU (SEQIDNO:543) (SEQIDNO:661) 116 asiAR116 UGGAAUCUUUUGUUGC GCAACAAAAGAUUCCAAGAUU (SEQIDNO:544) (SEQIDNO:662) 117 asiAR117 UAGUGUUCUGUUCUCU AGAGAACAGAACACUAGCGCU (SEQIDNO:545) (SEQIDNO:663) 118 asiAR118 CUAGUGUUCUGUUCUC GAGAACAGAACACUAGCGCUU (SEQIDNO:546) (SEQIDNO:664)

[Example 11] Screening for RNAi-Inducing Double-Stranded Nucleic Acid Molecules Targeting AR

[0087] To confirm gene inhibitory efficiency at the mRNA level, the 118 selected asiRNAs were transfected into an A549 cell line at a concentration of 0.3 nM, and qRT-PCR was performed to measure the expression level of AR mRNA.

[0088] The A549 cell line was cultured in Dulbecco's Modified Eagle's Medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS, Gibco) and 100 units/ml of penicillin 100 g/ml of streptomycin. A549 cells were seeded in a 96-well plate at a density of 510.sup.3 cells/well, and a transfection experiment was conducted using asiRNA (0.3 nM, OliX Pharmaceuticals Inc.) and RNAiMAX (1 l/ml, Invitrogen Inc.) in Opti-MEM (a total volume of 100 l) in accordance with Invitrogen's protocol. After 24 hours, RNA purification and cDNA synthesis were performed in accordance with a basic protocol provided by TOYOBO SuperPrep, the expression level of the AR gene was examined with an AR TaqMan probe (T) using a Bio-Rad CFX-4000 machine. First, 88 kinds of asiRNA from among the 118 kinds of asiRNA were subjected to an asiRNA screening experiment and the 13 top-ranked asiRNAs (in Table 6, No. 43, 49, 67, 70, 72, 74, 75, 77, 78, 79, 81, 82, and 87) were selected on the basis of inhibitory efficacy against the expression of the target gene, and the 13 selected asiRNAs and the 30 remaining asiRNAs (in Table 6, Nos. 88 to 118) were subjected to a secondary asiRNA screening experiment (see FIGS. 10A and 10B).

[0089] The 20 top-ranked asiRNAs (in Table 6, No. 43, 49, 70, 72, 74, 75, 77, 78, 79, 81, 82, 87, 89, 90, 93, 96, 106, 110, 111, and 118) having gone through secondary asiRNA screening were selected on the basis of inhibitory efficacy against the expression of the target gene, and an experiment for confirming the inhibitory effect of the 20 selected asiRNAs against AR expression at the protein level was performed. A549 cells were seeded in a 12-well plate at a density of 510.sup.4 cells/well, and then a transfection experiment was conducted using asiRNA (0.3 nM, OliX Pharmaceuticals Inc.) and RNAiMAX (1 l/ml, Invitrogen Inc.) in Opti-MEM (a total volume of 1 ml) in accordance with Invitrogen's protocol. After 48 hours, the cells were lysed using a mammalian protein extraction buffer (GE healthcare), and then proteins were quantified using a Bradford assay. 20 g of the protein of each sample was electrophoresed using 10% SDS-PAGE at 80 V for 20 minutes and at 120 V for 1 hour, and then transferred onto a PVDE membrane (Bio-Rad) at 300 mA for 1 hour. After transfer, the membrane was blocked in 5% skim milk for 1 hour and allowed to react with AR antibody (ABcam, ab133273) at a ratio of 1:2000 for 12 hours. The next day, the resulting membrane was allowed to react with anti-Rabbit HRP (Santa Cruz) at a ratio of 1:5000 for 1 hour, and then the expression levels of the AR protein were compared with each other using ChemiDoc (Bio-Rad). From the results, the 9 top-ranked asiRNAs (No. 70, 72, 78, 81, 82, 90, 110, 111, and 118) capable of more effectively inhibiting AR protein expression were selected (see FIG. 11).

[0090] The 9 top-ranked asiRNA candidates (in Table 6, No. 70, 72, 78, 81, 82, 90, 110, 111, and 118) having gone through asiRNA screening were selected on the basis of inhibitory efficacy against the expression of the target gene, and an experiment for confirming the inhibitory effects of the 9 selected asiRNA candidates against AR expression at the mRNA and protein levels and a lower concentration (0.1 nM) was conducted. A549 cells were seeded in a 12-well plate at a density of 510.sup.4 cells/well, and a transfection experiment was conducted using asiRNA and RNAiMAX (1 l/ml, Invitrogen Inc.) in Opti-MEM (a total volume of 0.5 ml) in accordance with Invitrogen's protocol. After 48 hours, total RNA was extracted using TRIzol (TaKaPa), and then cDNA was synthesized using a high-capacity cDNA reverse transcription kit (Applied Biosystems), and the expression level of the AR gene was examined using power SYBR green PCR master Mix (Applied Biosystems), the primers shown in Table 7 below, and a StepOne real-time PCR system.

TABLE-US-00007 TABLE7 Primernucleotidesequences Name Sequence(5-3) size Human Forward GAGTCAACGGATTTGGTC 186 GAPDH GT (SEQIDNO:665) Reverse GACAAGCTTCCCGTTCTC AG (SEQIDNO:666) Human Forward GGGGCTAGACTGCTCAAC 191 AR TG (SEQIDNO:667) Reverse GCCAAGTTTTGGCTGAAG AG (SEQIDNO:668)

[0091] In addition, A549 cells were seeded in a 12-well plate at a density of 510.sup.4 cells/well, and a transfection experiment was conducted using asiRNA and RNAiMAX (1 l/ml, Invitrogen Inc.) in Opti-MEM (a total volume of 0.5 ml) in accordance with Invitrogen's protocol. After 48 hours, the cells were lysed using a mammalian protein extraction buffer (GE healthcare), and then proteins were quantified using a Bradford assay. 20 g of the protein of each sample was electrophoresed using 10% SDS-PAGE at 80 V for 20 minutes and at 120 V for 1 hour, and then transferred onto a PVDE membrane (Bio-Rad) at 300 mA for 1 hour. After transfer, the membrane was blocked in 5% skim milk for 1 hour and allowed to react with AR antibody (ABcam, ab133273) at a ratio of 1:2000 for 12 hours. The next day, the resulting membrane was allowed to react with anti-Rabbit HRP (Santa Cruz) at a ratio of 1:5000 for 1 hour, and then the expression levels of the AR protein were compared with each other using ChemiDoc (Bio-Rad). As the result of the experiment for the 9 selected asiRNAs, it was confirmed that asiRNA #72, 78, and 110 exhibited gene inhibitory efficiency of 50% or higher efficiently even at a concentration of 0.1 nM (see FIG. 12).

[Example 12] 9 Kinds of Cp-asiRNA Targeting AR Gene and Having Self Cell-Penetrating Ability

[0092] AR cp-asiRNAs (a total of 9 kinds) were designed by applying three modification patterns to 3 kinds of asiRNA targeting AR according to the number and position of 2OMe (methyl), phosphorothioate bonds (PS), and cholesterol, and then synthesized by Dharmacon. cp-asiRNA enhances endocytosis efficiency and stability, and thus may penetrate through the cell membrane with high efficiency without the aid of a delivery vehicle to thereby inhibit the expression of the target gene. The synthesized sense and antisense strand RNA oligonucleotides were annealed at 95 C. for 2 minutes through incubation at 37 C. for 1 hour, and cp-asiRNAs annealed by 10% polyacrylamide gel electrophoresis (PAGE) were confirmed using a UV transilluminator.

TABLE-US-00008 TABLE8 9kindsofcp-asiRNAnucleotidesequences targetingAR No. Name Sequence(5-3) 1 cp-asiAR72 mCUmUUmUGmACmCUmGCmUAm*A*U*chol S 2 cp-asiAR72 AUUAGCAGGUCAAAmAmGmU*mG*mA*mA*mC AS(7,4) 3 cp-asiAR72 AUUAGCAGGUCAAAmAmGmU*mG*A*A*C AS(4,4) 4 cp-asiAR72 AUUAGCAGGUCAAAmAmGU*G*A*A*C AS(2,4) 5 cp-asiAR78 mGCmUGmCAmUCmAGmUUmCAm*C*U*chol S 6 cp-asiAR78 AGUGAACUGAUGCAmGmCmU*mC*mU*mC*mU AS(7,4) 7 cp-asiAR78 AGUGAACUGAUGCAmGmCmU*mC*U*C*U AS(4,4) 8 cp-asiAR78 AGUGAACUGAUGCAmGmCU*C*U*C*U AS(2,4) 9 cp-asiAR110 mGCmCCmAUmGUmUAmGCmUUm*A*U*chol S 10 cp-asiAR110 AUAAGCUAACAUGGmGmCmA*mC*mU*mA*mG AS(7,4) 11 cp-asiAR110 AUAAGCUAACAUGGmGmemA*mC*U*A*G AS(4,4) 12 cp-asiAR110 AUAAGCUAACAUGGmGmCA*C*U*A*G AS(2,4) m: 2-O-Methyl RNA, *: phosphorothioated bond, chol: cholesterol

[Example 13] Screening for Cp-asiRNA Targeting AR Gene and Having Self Cell-Penetrating Ability

[0093] The inhibitory effects of the 9 kinds of cp-asiRNA shown in Table 8 against AR expression were examined. An A549 cell line was incubated with 1 M or 3 M of each of the 9 cp-asiRNAs in Opti-MEM media for 24 hours, and then the media were replaced with Dulbecco's Modified Eagle's Medium (Gibco) containing 10% fetal bovine serum (Gibco) and 100 units/ml penicillin 100 g/ml streptomycin, and after 24 hours, AR expression was examined at the mRNA level. As the result of repeatedly conducting four experiments, it was confirmed that the 9 kinds of AR cp-asiRNA exhibited gene inhibitory efficiency of 50% at a concentration of 3 M (see FIG. 13).

[0094] Under the same experimental conditions, the inhibitory effects of the 9 kinds of cp-asiRNA against AR expression were examined at the protein level in an A549 cell line. The A549 cell line was incubated with 1 M or 3 M of each of the 9 cp-asiRNAs in Opti-MEM media for 24 hours, and then the media were replaced with Dulbecco's Modified Eagle's Medium (Gibco) containing 10% fetal bovine serum (Gibco) and 100 units/ml penicillin 100 g/ml streptomycin, and after 24 hours, AR expression was examined at the protein level. Among them, cp-asiRNA #72(7,4), #78(7,4) (4,4) (2,4), and #110(7,4) (4,4) exhibited target gene protein expression inhibitory efficiency of 50% or higher at a concentration of 1 M on the basis of the band intensity of a no treatment (NT) sample and a NT sample (see FIG. 14).

[0095] While the present invention has been described in detail with reference to specific embodiments thereof, it will be obvious to those of ordinary skill in the art that these embodiments are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Therefore, the actual scope of the present invention will be defined by the appended claims and equivalents thereof.

INDUSTRIAL APPLICABILITY

[0096] A 5-reductase type 1-encoding gene, a 5-reductase type 2-encoding gene, and an androgen receptor-encoding gene, which play a major role in inhibiting the synthesis of proteins required for hair follicle growth in male pattern hair loss and inducing hair loss by reducing the size of the dermal papilla, were selected as target genes, and asymmetric siRNA with high inhibitory efficiency against each target gene was selected. siRNA according to the present invention exhibits the ability to inhibit the expression of the target gene for 5-reductase type 1, 5-reductase type 2, or an androgen receptor, and thus may be effectively used as an agent for preventing or treating hair loss.

[0097] While the present invention has been particularly shown and described with reference to specific embodiments thereof, it will be obvious to those of ordinary skill in the art that such embodiments are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Therefore, the actual scope of the present invention should be defined by the appended claims and equivalents thereof.

SEQUENCE LIST FREE TEXT

[0098] Electronic files attached.