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OLIGOMERIC NUCLEIC ACID MOLECULE ACTIVATING ATOH1 GENE AND USE THEREOF

20220313721 · 2022-10-06

    Inventors

    Cpc classification

    International classification

    Abstract

    The present invention relates to oligomeric nucleic acid molecules and uses thereof for treating hearing loss. The present invention relates to small activating nucleic acid molecules for treating hearing loss. The small activating nucleic acid molecules of the present invention can be double-stranded or single-stranded RNA molecules targeting the promoter region of an ATOH1 gene comprising a first nucleic acid strand and a second nucleic acid strand. The double-stranded RNA molecule targeting the promoter region of the ATOH1 gene comprises two nucleic acid strands of 16 to 35 nucleotides in length, wherein one nucleic acid strand has at least 75% homology or complementarity to a target selected from the promoter region of the ATOH1 gene. The present invention also relates to pharmaceutical compositions comprising the small activating nucleic acid molecules and optionally, a pharmaceutically acceptable carrier, and methods for upregulating the expression of the ATOH1 gene and ATOH1 protein in a cell and treating diseases or conditions, related to insufficient or decreased expression of the ATOH1 gene by using the small activating nucleic acid molecules, or the pharmaceutical composition.

    Claims

    1. A small activating nucleic acid molecule, comprising a first nucleic acid strand and a second nucleic acid strand, wherein the first nucleic acid strand has at least 75% homology or complementarity to a continuous sequence of 16 to 35 nucleotides in length in positions −578 to −386 (SEQ ID NO: 2), positions −339 to −291 (SEQ ID NO: 3), positions −162 to −86 (SEQ ID NO: 4) or positions −55 to −34 (SEQ ID NO: 5) upstream of the transcription start site in a promoter region of an ATOH1 gene, wherein the first nucleic acid strand and the second nucleic acid strand complementarily form a double-stranded nucleic acid structure.

    2. The small activating nucleic acid molecule of claim 1, wherein the first nucleic acid strand and the second nucleic acid strand are present on two different nucleic acid strands.

    3. The small activating nucleic acid molecule of claim 1, wherein the first nucleic acid strand and the second nucleic acid strand are present on the same nucleic acid strand, and the small activating nucleic acid molecule is a hairpin single-stranded nucleic acid molecule, wherein the first nucleic acid strand and the second nucleic acid strand comprise complementary regions forming the double-stranded nucleic acid structure.

    4. (canceled)

    5. The small activating nucleic acid molecule of claim 2, wherein both strands of the small activating nucleic acid molecule have overhangs of of 2 or 3 nucleotides in length at the 3′ terminus.

    6. The small activating nucleic acid molecule of claim 1, wherein the first nucleic acid strand and the second nucleic acid strand independently have 16 to 35 nucleotides in length.

    7. The small activating nucleic acid molecule of claim 1, wherein one strand of the small activating nucleic acid molecule comprises a nucleic acid sequence having at least 75% homology or complementarity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 280-416, or consists of a nucleic acid sequence having at least 75% homology or complementarity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 280-416.

    8. The small activating nucleic acid molecule of claim 1, wherein the first nucleic acid strand has at least 75% homology to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-142, and the second nucleic acid strand has at least 75% homology to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 143-279.

    9. The small activating nucleic acid molecule of claim 1, wherein said first and second strand is selected from the group consisting of: SEQ ID NO: 6 and SEQ ID NO: 143; SEQ ID NO: 7 and SEQ ID NO: 144; SEQ ID NO: 8 and SEQ ID NO: 145; SEQ ID NO: 9 and SEQ ID NO: 146; SEQ ID NO: 10 and SEQ ID NO: 147; SEQ ID NO: 11 and SEQ ID NO: 148; SEQ ID NO: 12 and SEQ ID NO: 149; SEQ ID NO: 13 and SEQ ID NO: 150; SEQ ID NO: 14 and SEQ ID NO: 151; SEQ ID NO: 15 and SEQ ID NO: 152; SEQ ID NO: 16 and SEQ ID NO: 153; SEQ ID NO: 17 and SEQ ID NO: 154; SEQ ID NO: 18 and SEQ ID NO: 155; SEQ ID NO: 19 and SEQ ID NO: 156; SEQ ID NO: 20 and SEQ ID NO: 157; SEQ ID NO: 21 and SEQ ID NO: 158; SEQ ID NO: 22 and SEQ ID NO: 159; SEQ ID NO: 23 and SEQ ID NO: 160; SEQ ID NO: 24 and SEQ ID NO: 161; SEQ ID NO: 25 and SEQ ID NO: 162; SEQ ID NO: 26 and SEQ ID NO: 163; SEQ ID NO: 27 and SEQ ID NO: 164; SEQ ID NO: 28 and SEQ ID NO: 165; SEQ ID NO: 29 and SEQ ID NO: 166; SEQ ID NO: 30 and SEQ ID NO: 167; SEQ ID NO: 31 and SEQ ID NO: 168; SEQ ID NO: 32 and SEQ ID NO: 169; SEQ ID NO: 33 and SEQ ID NO: 170; SEQ ID NO: 34 and SEQ ID NO: 171; SEQ ID NO: 35 and SEQ ID NO: 172; SEQ ID NO: 36 and SEQ ID NO: 173; SEQ ID NO: 37 and SEQ ID NO: 174; SEQ ID NO: 38 and SEQ ID NO: 175; SEQ ID NO: 39 and SEQ ID NO: 176; SEQ ID NO: 40 and SEQ ID NO: 177; SEQ ID NO: 41 and SEQ ID NO: 178; SEQ ID NO: 42 and SEQ ID NO: 179; SEQ ID NO: 43 and SEQ ID NO: 180; SEQ ID NO: 44 and SEQ ID NO: 181; SEQ ID NO: 45 and SEQ ID NO: 182; SEQ ID NO: 46 and SEQ ID NO: 183; SEQ ID NO: 47 and SEQ ID NO: 184; SEQ ID NO: 48 and SEQ ID NO: 185; SEQ ID NO: 49 and SEQ ID NO: 186; SEQ ID NO: 50 and SEQ ID NO: 187; SEQ ID NO: 51 and SEQ ID NO: 188; SEQ ID NO: 52 and SEQ ID NO: 189; SEQ ID NO: 53 and SEQ ID NO: 190; SEQ ID NO: 54 and SEQ ID NO: 191; SEQ ID NO: 55 and SEQ ID NO: 192; SEQ ID NO: 56 and SEQ ID NO: 193; SEQ ID NO: 57 and SEQ ID NO: 194; SEQ ID NO: 58 and SEQ ID NO: 195; SEQ ID NO: 59 and SEQ ID NO: 196; SEQ ID NO: 60 and SEQ ID NO: 197; SEQ ID NO: 61 and SEQ ID NO: 198; SEQ ID NO: 62 and SEQ ID NO: 199; SEQ ID NO: 63 and SEQ ID NO: 200; SEQ ID NO: 64 and SEQ ID NO: 201; SEQ ID NO: 65 and SEQ ID NO: 202; SEQ ID NO: 66 and SEQ ID NO: 203; SEQ ID NO: 67 and SEQ ID NO: 204; SEQ ID NO: 68 and SEQ ID NO: 205; SEQ ID NO: 69 and SEQ ID NO: 206; SEQ ID NO: 70 and SEQ ID NO: 207; SEQ ID NO: 71 and SEQ ID NO: 208; SEQ ID NO: 72 and SEQ ID NO: 209; SEQ ID NO: 73 and SEQ ID NO: 210; SEQ ID NO: 74 and SEQ ID NO: 211; SEQ ID NO: 75 and SEQ ID NO: 212; SEQ ID NO: 76 and SEQ ID NO: 213; SEQ ID NO: 77 and SEQ ID NO: 214; SEQ ID NO: 78 and SEQ ID NO: 215; SEQ ID NO: 79 and SEQ ID NO: 216; SEQ ID NO: 80 and SEQ ID NO: 217; SEQ ID NO: 81 and SEQ ID NO: 218; SEQ ID NO: 82 and SEQ ID NO: 219; SEQ ID NO: 83 and SEQ ID NO: 220; SEQ ID NO: 84 and SEQ ID NO: 221; SEQ ID NO: 85 and SEQ ID NO: 222; SEQ ID NO: 86 and SEQ ID NO: 223; SEQ ID NO: 87 and SEQ ID NO: 224; SEQ ID NO: 88 and SEQ ID NO: 225; SEQ ID NO: 89 and SEQ ID NO: 226; SEQ ID NO: 90 and SEQ ID NO: 227; SEQ ID NO: 91 and SEQ ID NO: 228; SEQ ID NO: 92 and SEQ ID NO: 229; SEQ ID NO: 93 and SEQ ID NO: 230; SEQ ID NO: 94 and SEQ ID NO: 231; SEQ ID NO: 95 and SEQ ID NO: 232; SEQ ID NO: 96 and SEQ ID NO: 233; SEQ ID NO: 97 and SEQ ID NO: 234; SEQ ID NO: 98 and SEQ ID NO: 235; SEQ ID NO: 99 and SEQ ID NO: 236; SEQ ID NO: 100 and SEQ ID NO: 237; SEQ ID NO: 101 and SEQ ID NO: 238; SEQ ID NO: 102 and SEQ ID NO: 239; SEQ ID NO: 103 and SEQ ID NO: 240; SEQ ID NO: 104 and SEQ ID NO: 241; SEQ ID NO: 105 and SEQ ID NO: 242; SEQ ID NO: 106 and SEQ ID NO: 243; SEQ ID NO: 107 and SEQ ID NO: 244; SEQ ID NO: 108 and SEQ ID NO: 245; SEQ ID NO: 109 and SEQ ID NO: 246; SEQ ID NO: 110 and SEQ ID NO: 247; SEQ ID NO: 111 and SEQ ID NO: 248; SEQ ID NO: 112 and SEQ ID NO: 249; SEQ ID NO: 113 and SEQ ID NO: 250; SEQ ID NO: 114 and SEQ ID NO: 251; SEQ ID NO: 115 and SEQ ID NO: 252; SEQ ID NO: 116 and SEQ ID NO: 253; SEQ ID NO: 117 and SEQ ID NO: 254; SEQ ID NO: 118 and SEQ ID NO: 255; SEQ ID NO: 119 and SEQ ID NO: 256; SEQ ID NO: 120 and SEQ ID NO: 257; SEQ ID NO: 121 and SEQ ID NO: 258; SEQ ID NO: 122 and SEQ ID NO: 259; SEQ ID NO: 123 and SEQ ID NO: 260; SEQ ID NO: 124 and SEQ ID NO: 261; SEQ ID NO: 125 and SEQ ID NO: 262; SEQ ID NO: 126 and SEQ ID NO: 263; SEQ ID NO: 127 and SEQ ID NO: 264; SEQ ID NO: 128 and SEQ ID NO: 265; SEQ ID NO: 129 and SEQ ID NO: 266; SEQ ID NO: 130 and SEQ ID NO: 267; SEQ ID NO: 131 and SEQ ID NO: 268; SEQ ID NO: 132 and SEQ ID NO: 269; SEQ ID NO: 133 and SEQ ID NO: 270; SEQ ID NO: 134 and SEQ ID NO: 271; SEQ ID NO: 135 and SEQ ID NO: 272; SEQ ID NO: 136 and SEQ ID NO: 273; SEQ ID NO: 137 and SEQ ID NO: 274; SEQ ID NO: 138 and SEQ ID NO: 275; SEQ ID NO: 139 and SEQ ID NO: 276; SEQ ID NO: 140 and SEQ ID NO: 277; SEQ ID NO: 141 and SEQ ID NO: 278; and SEQ ID NO: 142 and SEQ ID NO: 279.

    10. (canceled)

    11. The small activating nucleic acid molecule of claim 1, wherein the small activating nucleic acid molecule comprises at least one chemical modification selected from the group consisting of: (1) a modification of a phosphodiester bond connecting nucleotides in the nucleotide sequence of the small activating nucleic acid molecule; (2) a modification of 2′-OH of a ribose in the nucleotide sequence of the small activating nucleic acid molecule; (3) a modification of a base in the nucleotide sequence of the small activating nucleic acid molecule; and (4) at least one nucleotide in the nucleotide sequence of the small activating nucleic acid molecule being a locked nucleic acid.

    12. The small activating nucleic acid molecule of claim 11, wherein the at least one chemical modification is one or more modifications selected from the group consisting of: 2′-fluoro modification, 2′-oxymethyl modification, 2′-oxyethylidene methoxy modification, 2,4′-dinitrophenol modification, locked nucleic acid (LNA), 2′-amino modification, 2′-deoxy modification, 5′-bromouracil modification, 5′-iodouracil modification, N-methyluracil modification, 2,6-diaminopurine modification, phosphorothioate modification, and boranophosphate modification.

    13. The small activating nucleic acid molecule of claim 1, wherein the small activating nucleic acid molecule activates or upregulates the expression of the ATOH 1 gene by at least 10%.

    14. A nucleic acid encoding the small activating nucleic acid molecule of claim 1.

    15. (canceled)

    16. The small activating nucleic acid molecule of claim 1, wherein the first nucleic acid strand has at least 95% homology to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-142, and the second nucleic acid strand has at least 95% homology to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 143-279.

    17. A composition comprising the small activating nucleic acid molecule of claim 1, and a pharmaceutically acceptable carrier.

    18. (canceled)

    19. (canceled)

    20. The small activating nucleic acid molecule of claim 8, wherein all the nucleotides are natural non-chemically modified nucleotides.

    21-29. (canceled)

    30. A method for activating or upregulating the expression of an ATOH1 gene in a cell, comprising administering the small activating nucleic acid molecule of claim 1 to the cell.

    31-34. (canceled)

    35. The method of claim 30, wherein the cell is present in a human body.

    36. (canceled)

    37. (canceled)

    38. A method for treating a disease or condition related to insufficient or decreased expression of the ATOH1 protein in an individual, comprising administering the small activating nucleic acid molecule of claim 1 to the individual.

    39. The method of claim 38, wherein the disease or condition related to insufficient or decreased expression of the ATOH1 protein comprises hearing loss and tumor.

    40. (canceled)

    41. The method of claim 38, wherein the individual is a human.

    42-46. (canceled)

    Description

    BRIEF DESCRIPTION OF THE DRAWINGS

    [0048] FIG. 1 shows changes in the expression of ATOH1 mRNA mediated by ATOH1 saRNA. HEK293T cells were transfected by 403 saRNAs targeting the ATOH1 promoter and mRNA expression of ATOH1 was analyzed 72 h later using QuantiGene 2.0. Shown in the drawing are the changes in ATOH1 mRNA expression relative to control treatment (mock) ranked from −637 to −19 according to the saRNA target positions in the ATOH1 promoter.

    [0049] FIG. 2 shows the hotspot region of functional saRNA in the ATOH1 promoter. HEK293T cells were transfected by 403 saRNAs targeting ATOH1 promoter and mRNA expression of ATOH1 was analyzed 72 h later using QuantiGene 2.0. Shown in the drawing are changes in ATOH1 mRNA expression relative to control treatment (mock) ranked from −637 to -19 according to the target positions of the saRNA in the ATOH1 promoter. The dotted line in FIG. 2 is the limit of 0.5 (log.sub.2) fold increase. The dotted frames show hotspot regions (H1-H4) for functional saRNAs.

    [0050] FIG. 3 shows the screening results for two-step RT-PCR validated by QuantiGene in HEK293T cells. HEK293T cells were transfected with saRNA at a final concentration of 10 nM for 72 h. RNA was extracted using an Qiagen Rneasy kit and qPCR amplification was performed with a 7500 FAST real-time PCR system after reverse transcription. An HPRT1 gene was amplified as internal control. The relative mRNA expression values of ATOH1 after treating cells with a single saRNA are shown. Mock, dsCon2, and RAG9-871i are represented as blank transfection, sequence-independent double-stranded RNA transfection, and small interference RNA control transfection, respectively.

    [0051] FIG. 4 shows the screening results for two-step RT-PCR validated by Quantigene in NKEK cells. Normal human epidermal keratinocytes (NHEK) cells were transfected with saRNA at a final concentration of 10 nM for 72 h. RNA was extracted using a Qiagen Rneasy kit and qPCR amplification was performed with a 7500 FAST real-time PCR system after reverse transcription. An HPRT1 gene was amplified as internal control. The relative mRNA expression values of ATOH1 after treatment of cells with a single saRNA are shown. Mock, dsCon2, and RAGS-871i are represented as blank transfection, sequence-independent double-stranded RNA transfection, and small interference RNA control transfection, respectively.

    [0052] FIG. 5 shows ATOH1 saRNA activating the expression of ATOH1 protein. HEK293T cells were transfected with saRNA at a final concentration of 10 nM for 72 h. After collecting the cells and extracting total cell protein, ATOH1 protein expression was detected by Western Blot and α/β-tubulin was used as an internal control. The relative ATOH1 protein expression values after cell treatment with a single saRNA are shown. Mock, dsCon2, and RAGS-871i are represented as blank transfection, sequence-independent double-stranded RNA transfection, and small interference RNA control transfection, respectively.

    DETAILED DESCRIPTION

    [0053] In the present invention, the related terms are defined as follows.

    [0054] The term “complementarity” as used herein refers to the capability of forming base pairs between two oligonucleotide strands. The base pairs are generally formed through hydrogen bonds between nucleotides in the antiparallel oligonucleotide strands. The bases of the complementary oligonucleotide strands can be paired in the Watson-Crick manner (such as A to T, A to U, and C to G) or in any other manner allowing the formation of a duplex (such as Hoogsteen or reverse Hoogsteen base pairing).

    [0055] Complementarity comprises complete complementarity and incomplete complementarity. “Complete complementarity” or “100% complementarity” means that each nucleotide from the first oligonucleotide strand can form a hydrogen bond with a nucleotide at a corresponding position in the second oligonucleotide strand in the double-stranded region of the double-stranded oligonucleotide molecule without “mispairing”. “Incomplete complementarity” means that not all the nucleotide units of the two strands are bonded with each other by hydrogen bonds. For example, for two oligonucleotide strands each of 20 nucleotides in length in the double-stranded region, if only two base pairs in this double-stranded region can be formed through hydrogen bonds, the oligonucleotide strands have a complementarity of 10%. In the same example, if 18 base pairs in this double-stranded region can be formed through hydrogen bonds, the oligonucleotide strands have a complementarity of 90%. Substantial complementarity refers to at least about 75%, about 79%, about 80%, about 85%, about 90%, about 95%, about 99% or about 100% complementarity.

    [0056] The term “oligonucleotide” as used herein refers to polymers of nucleotides, and includes, but is not limited to, single-stranded or double-stranded molecules of DNA, RNA, or DNA/RNA hybrid, oligonucleotide strands containing regularly and irregularly alternating deoxyribosyl portions and ribosyl portions, as well as modified and naturally or unnaturally existing frameworks for such oligonucleotides. The oligonucleotide for activating target gene transcription described herein is a small activating nucleic acid molecule.

    [0057] The terms “oligonucleotide strand” and “oligonucleotide sequence” as used herein can be used interchangeably, referring to a generic term for short nucleotide sequences having less than 35 bases (including nucleotides in deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)). In the present invention, an oligonucleotide strand can have any of 16 to 35 nucleotides in length.

    [0058] As used herein, the term “first nucleic acid strand” can be a sense strand or an antisense strand. The sense strand of a small activating RNA refers to a nucleic acid strand contained in a small activating RNA duplex which has identity to the coding strand of the promoter DNA sequence of a target gene, and the antisense strand refers to a nucleic acid strand in a small activating RNA duplex which is complementary to the sense strand.

    [0059] As used herein, the term “second nucleic acid strand” can also be a sense strand or an antisense strand. If the first oligonucleotide strand is a sense strand, the second oligonucleotide strand is an antisense strand; if the first oligonucleotide strand is an antisense strand, the second oligonucleotide strand is a sense strand.

    [0060] The term “gene” as used herein refers to all nucleotide sequences required to encode a polypeptide chain or to transcribe a functional RNA. “Gene” can be an endogenous or fully or partially recombinant gene for a host cell (for example, because an exogenous oligonucleotide and a coding sequence for encoding a promoter are introduced into a host cell, or a heterogeneous promoter adjacent to an endogenous encoding sequence is introduced into a host cell). For example, the term “gene” comprises a nucleic acid sequence consisting of exons and introns. Protein-encoding sequences are, for example, sequences contained within exons in an open reading frame between an initiation codon and a termination codon, and as used herein, “gene” can comprise such as a gene regulatory sequence, such as a promoter, an enhancer, and all other sequences known in the art for controlling the transcription, expression or activity of another gene, no matter whether the gene comprises a coding sequence or a non-coding sequence. In one case, for example, “gene” can be used to describe a functional nucleic acid comprising a regulatory sequence such as a promoter or an enhancer. The expression of a recombinant gene can be controlled by one or more types of heterogeneous regulatory sequences.

    [0061] The term “target gene” as used herein can refer to nucleic acid sequences naturally present in organisms, transgenes, viral or bacterial sequences, can be chromosomes or extrachromosomal genes, and/or can be transiently or stably transfected or incorporated into cells and/or chromatins thereof. The target gene can be a protein-encoding gene or a non-protein-encoding gene (such as a microRNA gene and a long non-coding RNA gene). The target gene generally contains a promoter sequence, and the positive regulation for the target gene can be achieved by designing a small activating nucleic acid molecule having sequence identity (also called homology) to the promoter sequence, characterized as the up-regulation of expression of the target gene. “Sequence of a target gene promoter” refers to a non-coding sequence of the target gene, and the reference of the sequence of a target gene promoter in the phrase “complementary to the sequence of a target gene promoter” of the present invention refers to a coding strand of the sequence, also known as a non-template strand, i.e., a nucleic acid sequence having the same sequence as the coding sequence of the gene. “Target” or “target sequence” refers to a sequence fragment in the sequence of a target gene promoter which is homologous or complementary with a sense oligonucleotide strand or an antisense oligonucleotide strand of a small activating nucleic acid molecule.

    [0062] As used herein, the terms “sense strand” and “sense nucleic acid strand” can be used interchangeably, and the sense oligonucleotide strand of a small activating nucleic acid molecule refers to the first nucleic acid strand having identity to the coding strand of the sequence of a target gene promoter in a small activating nucleic acid molecule duplex.

    [0063] As used herein, the terms “antisense strand” and “antisense nucleic acid strand” can be used interchangeably, and the antisense oligonucleotide strand of a small activating nucleic acid molecule refers to the second nucleic acid strand complementary with the sense oligonucleotide strand in a small activating nucleic acid molecule duplex.

    [0064] The term “coding strand” as used herein refers to a DNA strand in the target gene which cannot be used for transcription, and the nucleotide sequence of this strand is the same as that of an RNA produced from transcription (in the RNA, T in DNA is replaced by U). The coding strand of the double-stranded DNA sequence of the target gene promoter described herein refers to a promoter sequence on the same DNA strand as the DNA coding strand of the target gene.

    [0065] The term “template strand” as used herein refers to the other strand complementary with the coding strand in the double-stranded DNA of the target gene, i.e., the strand that, as a template, can be transcribed into RNA, and this strand is complementary with the transcribed RNA (A to U and G to C). In the process of transcription, RNA polymerase binds to the template strand, moves along the 3′.fwdarw.5′ direction of the template strand, and catalyzes the synthesis of the RNA along the 5′.fwdarw.3′ direction. The template strand of the double-stranded DNA sequence of the target gene promoter described herein refers to a promoter sequence on the same DNA strand as the DNA template strand of the target gene.

    [0066] The term “promoter” as used herein refers to a sequence which plays a regulatory role for the transcription of a protein-coding or RNA-coding nucleic acid sequence by associating with them spatially. Generally, a eukaryotic gene promoter contains 100 to 5000 base pairs, although this length range is not intended to limit the term “promoter” as used herein. Although the promoter sequence is generally located at the 5′ terminus of a protein-coding or RNA-coding sequence, the promoter sequence can also exist in exon and intron sequences.

    [0067] The term “transcription start site” as used herein refers to a nucleotide marking the transcription start on the template strand of a gene. The transcription start site can appear on the template strand of the promoter region. A gene can have more than one transcription start site.

    [0068] The term “identity” or “homology” as used herein means that one oligonucleotide strand (a sense or an antisense strand) of a small activating RNA has similarity to a coding strand or a template strand in a region of the promoter sequence of a target gene. As used herein, the “identity” or “homology” can be at least about 75%, about 79%, about 80%, about 85%, about 90%, about 95%, about 99% or about 100%.

    [0069] The term “overhang” as used herein refers to non-base-paired nucleotides at the terminus (5′ or 3′) of an oligonucleotide strand, which is formed by one strand extending out of the other strand in a double-stranded oligonucleotide. A single-stranded region extending out of the 3′ terminus and/or 5′ terminus of a duplex is referred to as an overhang.

    [0070] As used herein, the terms “gene activation”, “activating gene expression”, “gene up-regulation” and “up-regulating gene expression” can be used interchangeably, and mean an increase in transcription, translation, expression or activity of a certain nucleic acid as determined by measuring the transcriptional level, mRNA level, protein level, enzymatic activity, methylation state, chromatin state or configuration, translation level or the activity or state in a cell or biological system of a gene. These activities or states can be determined directly or indirectly. In addition, “gene activation”, “activating gene expression”, “gene up-regulation” or “up-regulating gene expression” refers to an increase in activity associated with a nucleic acid sequence, regardless of the mechanism of such activation. For example, the nucleic acid sequence plays a regulatory role as a regulatory sequence, the nucleic acid sequence is transcribed into RNA and the RNA is translated into a protein, thereby increasing the expression of the protein.

    [0071] As used herein, the terms “small activating RNA”, “saRNA”, and “small activating nucleic acid molecule” can be used interchangeably, and refer to a nucleic acid molecule that can up-regulate target gene expression and can be composed of the first nucleic acid fragment (antisense nucleic acid strand, also referred to as antisense oligonucleotide strand) containing a nucleotide sequence having sequence identity or homology with the non-coding nucleic acid sequence (e.g., a promoter and an enhancer) of a target gene and the second nucleic acid fragment (sense nucleic acid strand, also referred to as sense oligonucleotide strand) containing a nucleotide sequence complementary with the first nucleic acid fragment, wherein the first nucleic acid fragment and the second nucleic acid fragment form a duplex. A small activating nucleic acid molecule can also be composed of a synthesized or vector-expressed single-stranded RNA molecule that can form a hairpin structure by two complementary regions within the molecule, wherein the first region comprises a nucleotide sequence having sequence identity to the target sequence of a promoter of a gene, and the second region comprises a nucleotide sequence which is complementary to the first region. The length of the duplex region of a small activating nucleic acid molecule is typically about 10 to about 50, about 12 to about 48, about 14 to about 46, about 16 to about 44, about 18 to about 42, about 20 to about 40, about 22 to about 38, about 24 to about 36, about 26 to about 34, and about 28 to about 32 base pairs, and typically about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 base pairs. In addition, the terms “saRNA”, “small activating RNA”, and “small activating nucleic acid molecule” also comprise nucleic acids other than the ribonucleotide, including, but not limited to, modified nucleotides or analogues.

    [0072] As used herein, the term “hotspot” refers to a gene promoter region of at least 30 bp in length. The gathering of targets of functional small activating nucleic acid molecules appears in these hotspot regions, wherein at least 40% of a small activating nucleic acid molecules targeting these hotspot regions can induce a 1.1-fold or more change in the mRNA expression of a target gene.

    [0073] As used herein, the term “synthesis” refers to a method for synthesis of an oligonucleotide, including any method allowing RNA synthesis, such as chemical synthesis, in vitro transcription, and/or vector-based expression.

    [0074] According to the present invention, the expression of the ATOH1 gene is up-regulated by RNA activation, and a related disease (particularly hearing loss) is treated by increasing the expression of the full-length ATOH1 protein. The ATOH1 gene in the present invention is sometimes also called a target gene.

    [0075] The method for preparing a small activating nucleic acid molecule provided by the present invention comprises sequence design and sequence synthesis.

    [0076] The synthesis of the sequence of a small activating nucleic acid molecule of the present invention can be performed by adopting a chemical synthesis or can be entrusted to a biotechnology company specialized in nucleic acid synthesis.

    [0077] Generally speaking, the chemical synthesis comprises the following four steps: (1) synthesis of oligomeric ribonucleotides, (2) deprotection, (3) purification and isolation; and (4) desalination and annealing.

    [0078] For example, the specific steps for chemically synthesizing the saRNA described herein are as follows:

    (1) Synthesis of Oligomeric Ribonucleotides

    [0079] Synthesis of 1 μM RNA was set in an automatic DNA/RNA synthesizer (e.g., Applied Biosystems EXPEDITE8909), and the coupling time of each cycle was also set as 10 to 15 min. With a solid phase-bonded 5′-O-p-dimethoxytriphenylmethyl-thymidine substrate as an initiator, one base was bonded to the solid phase substrate in the first cycle, and then, in the n.sup.th (19≥n≥2) cycle, one base was bonded to the base bonded in the n-1.sup.th cycle. This process was repeated until the synthesis of the whole nucleic acid sequence was completed.

    (2) Deprotection

    [0080] The solid phase substrate bonded with the saRNA was put into a test tube, and 1 mL of a mixed solution of ethanol and ammonium hydroxide (volume ratio: 1:3) was added to the test tube. The test tube was then sealed and placed in an incubator, and the mixture was incubated at 25-70° C. for 2 to 30 h. The solution containing the solid phase substrate bonded with the saRNA was filtered, and the filtrate was collected. The solid phase substrate was rinsed with double distilled water twice (1 mL each time), and the filtrate was collected. The collected eluent was combined and dried under vacuum for 1 to 12 h. Then the solution was added with 1 mL of a solution of tetrabutylammonium fluoride in tetrahydrofuran (1 M), let stand at room temperature for 4 to 12 h, followed by addition of 2 mL of n-butanol. Precipitate was collected to give a single-stranded crude product of saRNA by high-speed centrifugation.

    (3) Purification and Isolation

    [0081] The resulting crude product of saRNA was dissolved in 2 mL of triethylamine acetate solution with a concentration of 1 mol/L, and the solution was separated by a reversed-phase C18 column of high pressure liquid chromatography to give a purified single-stranded product of saRNA.

    (4) Desalination and Annealing

    [0082] Salts were removed by gel filtration (size exclusion chromatography). A single sense oligomeric ribonucleic acid strand and a single antisense oligomeric ribonucleic acid strand were mixed in a 1 to 2 mL of buffer (10 mM Tris, pH=7.5-8.0, 50 mM NaCl) at a molar ratio of 1:1. The solution was heated to 95° C., and was then slowly cooled to room temperature to give a solution containing saRNA.

    [0083] It was discovered in this study that after being introduced into a cell, the aforementioned saRNA could effectively increase the mRNA and protein expression of the full-length ATOH1.

    [0084] The present invention will be further illustrated with reference to specific examples and drawings below. It should be understood that these examples are merely intended to illustrate the present invention rather than limit the scope of the present invention. In the following examples, study methods without specific conditions were generally in accordance with conventional conditions, such as conditions described in Sambrook, et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or conditions recommended by the manufacturer.

    EXAMPLES

    Example 1: Design and Synthesis of Small Activating Nucleic Acid Molecule Targeting ATOH1 Promoter

    [0085] To screen functional saRNAs capable of activating ATOH1 gene expression, a ATOH1 promoter sequence with a length of 637 bps (−637 bp to −1 bp; SEQ ID NO:1) was used as a template and an saRNA target with a lengths of 19 nucleotides were obtained by moving a base each time. Target sequences were filtered and the criteria for retaining target sequences were: (1) GC content between 30% and 80%, (2) with less than 5 continuous identical nucleotides, (3) with 3 or less dinucleotide repeat sequences; and (4) with 3 or less trinucleotide repeat sequences. There were 402 target sequences remaining after filtration and these entered the screening process as candidates. In addition, one target at the position −1407 bp of the ATOH1 promoter was selected as a candidate target sequence, and the 403 target sequences were selected as candidates to enter screening process.

    [0086] Each sense strand and each antisense strand in the double-stranded small activating RNAs used in the study were 21 nucleotides in length. The 19 nucleotides in the 5′ region of the first nucleic acid strand, e.g., the sense strand of the double-stranded saRNA, had 100% identity to the target sequence of the promoter, and the 3′ terminus of the first nucleic acid strand contained a TT sequence. The 19 nucleotides in the 5′ region of the second nucleic acid strand were fully complementary with the first ribonucleic acid strand sequence, and the 3′ terminus of the second nucleic acid strand contained a TT sequence. The aforementioned two strands of the double-stranded saRNA were mixed at a molar ratio of 1:1, and after annealing, and a double-stranded saRNA was formed. The sequence of the ATOH1 promoter corresponds to position 1 to position 637 from 5′ to 3′ of SEQ ID No: 1 in the sequence listing:

    TABLE-US-00001 −637 aaatgcttgg gcatcagaag tgggagctgt gatcctagct tgggggcagc −587 acagggtagg cggccttctc tctgctttga gtggcttctg ggcgcctggc −537 gggtccagaa tcgcccagag ccgcccgcgg tcgtgcacat ctgacccgag −487 tcagcttggg caccagccga gagccggctc cgcaccgctc ccgcacccca −437 gccgccgggg tggtgacaca caccggagtc gaattacagc cctgcaatta −387 acatatgaat ctgacgaatt taaaagaagg aaaaaaaaaa aaaaacctga −337 gcaggcttgg gagtcctctg cacacaagaa cttttctcgg ggtgtaaaaa −287 ctctttgatt ggctgctcgc acgcgcctgc ccgcgccctc cattggctga −237 gaagacacgc gaccggcgcg aggagggggt tgggagagga gcggggggag −187 actgagtggc gcgtgccgct ttttaaaggg gcgcagcgcc ttcagcaacc −137 ggagaagcat agttgcacgc gacctggtgt gtgatctccg agtgggtggg  −87 ggagggtcga ggagggaaaa aaaaataaga cgttgcagaa gagacccgga  −37 aagggccttt tttttggttg agctggtgtc ccagtgc

    Example 2: High-throughput Screening of saRNAs Targeting ATOH1 Promoter

    (1) Cell Culture and Transfection

    [0087] Normal Human Epidermal Keratinocytes (NHEK) and human renal embryonic cells (HEK293T) were cultured in DMEM media (Gibco). All the media contained 10% bovine calf serum (Sigma-Aldrich) and 1% penicillin/streptomycin (Gibco). The cells were cultured at 37 ° C. under 5% CO2. According to the instructions provided by the manufacturer, RNAiMax (Invitrogen, Carlsbad, Calif.) was used to transfect small activating RNAs at a concentration of 10 nM (unless otherwise specified).

    (2) QuantiGene 2.0 Assay

    [0088] Cells were plated into a 96-well plate at 4000 cells/well and transfected with oligonucleotide duplexes, and 72 h after transfection, the mRNA levels of target genes were quantitatively determined with a QuantiGene 2.0 kit (AffyMetrix, product numbers were QGS-1000, ATOH1 Assay ID: SA-6000952, and HPRT Assay ID: SA-10030). QuantiGene 2.0 assay is based on hybridization technology, wherein mRNA levels were directly quantified with gene-specific probes. The experimental procedure was briefly described as follows: a lysis solution was added to lyse the transfected cells, and the cell lysates were added into a capture well plate coated with probe for ATOH1 and HPRT1 (housekeeping gene) for hybridizing overnight at 55° C. In order to enhance hybridization signal, hybridizations with 2.0 PreAMP Probe, 2.0 AMP Probe and 2.0 Label Probe (provided by Quantigene 2.0 kit) were conducted sequentially in 100 μL of a corresponding buffer solution (provided by Quantigene 2.0 kit). All the hybridizations were conducted with shaking at 50 to 55° C. for 1 h. After the last wash step, 2.0 Substrate was added to the solution and incubated at room temperature for 5 min. Optical signals were detected with an Infinite 200 PRO plate reader (Tecan, Switzerland).

    (3) Screening of Functional saRNAs

    [0089] In order to obtain functional saRNAs capable of activating transcription of ATOH1, HEK293T cells were transfected with the above 403 saRNAs at a concentration of 10 nM, and light signals were analyzed 72 h later by the method described in QuantiGene. As shown in Table 1, 32 (7.9%) and 105 (26.1%) saRNAs showed high and mild activating activities, and 266 (66.0%) saRNAs had no influence on the expression of ATOH1. The maximum activation was 5.88 fold. These saRNAs with activating activity are referred to as functional saRNA.

    TABLE-US-00002 TABLE 1 High-throughput screening results of ATOH1 log.sub.2 value (fold) Number of saRNA activity of changes in ATOH1 saRNAs Percentage High activation ≥1.00 (2.00)~≤2.557 (5.883) 32  7.9% Mild activation ≥0.14 (1.10)~<1.00 (2.00)   105 26.1% No activating <0.14 (1.10) 266 66.0% effect Sum 403  100%

    [0090] FIG. 1 further shows the activity distribution of the ATOH1 saRNAs from high activation to high inhibition.

    TABLE-US-00003 TABLE 2 Active saRNA sequences, active target sequences thereof and resulting changes in mRNA expression of ATOH1 Fold of changes in relative mRNA expression saRNA Active target sequence (5′-3′) Sense sequence (5′-3′) Antisense sequence (5′-3′) of ATOH1 RAG9-578 GCGGCCTTCTCTCTGCTTT GCGGCCUUCUCUCUGCUUUTT AAAGCAGAGAGAAGGCCGCTT 5.88 (SEQ ID NO: 280) (SEQ ID NO: 6) (SEQ ID NO: 143) RAG9-52 CAGAAGAGACCCGGAAAGG CAGAAGAGACCCGGAAAGGTT CCUUUCCGGGUCUCUUCUGTT 4.17 (SEQ ID NO: 281) (SEQ ID NO: 7) (SEQ ID NO: 144) RAG9-139 CCGGAGAAGCATAGTTGCA CCGGAGAAGCAUAGUUGCATT UGCAACUAUGCUUCUCCGGTT 3.92 (SEQ ID NO: 282) (SEQ ID NO: 8) (SEQ ID NO: 145) RAG9-120 CGCGACCTGGTGTGTGATC CGCGACCUGGUGUGUGAUCTT GAUCACACACCAGGUCGCGTT 3.53 (SEQ ID NO: 283) (SEQ ID NO: 9) (SEQ ID NO: 146) RAG9-339 GAGCAGGCTTGGGAGTCCT GAGCAGGCUUGGGAGUCCUTT AGGACUCCCAAGCCUGCUCTT 3.41 (SEQ ID NO: 284) (SEQ ID NO: 10) (SEQ ID NO: 147) RAG9-128 TAGTTGCACGCGACCTGGT UAGUUGCACGCGACCUGGUTT ACCAGGUCGCGUGCAACUATT 3.37 (SEQ ID NO: 285) (SEQ ID NO: 11) (SEQ ID NO: 148) RAG9-536 GGTCCAGAATCGCCCAGAG GGUCCAGAAUCGCCCAGAGTT CUCUGGGCGAUUCUGGACCTT 3.37 (SEQ ID NO: 286) (SEQ ID NO: 12) (SEQ ID NO: 149) RAG9-53 GCAGAAGAGACCCGGAAAG GCAGAAGAGACCCGGAAAGTT CUUUCCGGGUCUCUUCUGCTT 3.21 (SEQ ID NO: 287) (SEQ ID NO: 13) (SEQ ID NO: 150) RAG9-513 CCGCGGTCGTGCACATCTG CCGCGGUCGUGCACAUCUGTT CAGAUGUGCACGACCGCGGTT 3.19 (SEQ ID NO: 288) (SEQ ID NO: 14) (SEQ ID NO: 151) RAG9-538 CGGGTCCAGAATCGCCCAG CGGGUCCAGAAUCGCCCAGTT CUGGGCGAUUCUGGACCCGTT 3.18 (SEQ ID NO: 289) (SEQ ID NO: 15) (SEQ ID NO: 152) RAG9-150 GCCTTCAGCAACCGGAGAA GCCUUCAGCAACCGGAGAATT UUCUCCGGUUGCUGAAGGCTT 2.83 (SEQ ID NO: 290) (SEQ ID NO: 16) (SEQ ID NO: 153) RAG9-418 ACACCGGAGTCGAATTACA ACACCGGAGUCGAAUUACATT UGUAAUUCGACUCCGGUGUTT 2.81 (SEQ ID NO: 291) (SEQ ID NO: 17) (SEQ ID NO: 154) RAG9-309 AACTTTTCTCGGGGTGTAA AACUUUUCUCGGGGUGUAATT UUACACCCCGAGAAAAGUUTT 2.79 (SEQ ID NO: 292) (SEQ ID NO: 18) (SEQ ID NO: 155) RAG9-331 TTGGGAGTCCTCTGCACAC UUGGGAGUCCUCUGCACACTT GUGUGCAGAGGACUCCCAATT 2.79 (SEQ ID NO: 293) (SEQ ID NO: 19) (SEQ ID NO: 156) RAG9-482 TTGGGCACCAGCCGAGAGC UUGGGCACCAGCCGAGAGCTT GCUCUCGGCUGGUGCCCAATT 2.66 (SEQ ID NO: 294) (SEQ ID NO: 20) (SEQ ID NO: 157) RAG9-54 TGCAGAAGAGACCCGGAAA UGCAGAAGAGACCCGGAAATT UUUCCGGGUCUCUUCUGCATT 2.61 (SEQ ID NO: 295) (SEQ ID NO: 21) (SEQ ID NO: 158) RAG9-533 CCAGAATCGCCCAGAGCCG CCAGAAUCGCCCAGAGCCGTT CGGCUCUGGGCGAUUCUGGTT 2.57 (SEQ ID NO: 296) (SEQ ID NO: 22) (SEQ ID NO: 159) RAG9-158 GGCGCAGCGCCTTCAGCAA GGCGCAGCGCCUUCAGCAATT UUGCUGAAGGCGCUGCGCCTT 2.47 (SEQ ID NO: 297) (SEQ ID NO: 23) (SEQ ID NO: 160) RAG9-122 CACGCGACCTGGTGTGTGA CACGCGACCUGGUGUGUGATT UCACACACCAGGUCGCGUGTT 2.26 (SEQ ID NO: 298) (SEQ ID NO: 24) (SEQ ID NO: 161) RAG9-539 GCGGGTCCAGAATCGCCCA GCGGGUCCAGAAUCGCCCATT UGGGCGAUUCUGGACCCGCTT 2.21 (SEQ ID NO: 299) (SEQ ID NO: 25) (SEQ ID NO: 162) RAG9-572 TTCTCTCTGCTTTGAGTGG UUCUCUCUGCUUUGAGUGGTT CCACUCAAAGCAGAGAGAATT 2.21 (SEQ ID NO: 300) (SEQ ID NO: 26) (SEQ ID NO: 163) RAG9-473 AGCCGAGAGCCGGCTCCGC AGCCGAGAGCCGGCUCCGCTT GCGGAGCCGGCUCUCGGCUTT 2.19 (SEQ ID NO: 301) (SEQ ID NO: 27) (SEQ ID NO: 164) RAG9-502 CACATCTGACCCGAGTCAG CACAUCUGACCCGAGUCAGTT CUGACUCGGGUCAGAUGUGTT 2.11 (SEQ ID NO: 302) (SEQ ID NO: 28) (SEQ ID NO: 165) RAG9-472 GCCGAGAGCCGGCTCCGCA GCCGAGAGCCGGCUCCGCATT UGCGGAGCCGGCUCUCGGCTT 2.11 (SEQ ID NO: 303) (SEQ ID NO: 29) (SEQ ID NO: 166) RAG9-484 GCTTGGGCACCAGCCGAGA GCUUGGGCACCAGCCGAGATT UCUCGGCUGGUGCCCAAGCTT 2.10 (SEQ ID NO: 304) (SEQ ID NO: 30) (SEQ ID NO: 167) RAG9-283 TTGATTGGCTGCTCGCACG UUGAUUGGCUGCUCGCACGTT CGUGCGAGCAGCCAAUCAATT 2.09 (SEQ ID NO: 305) (SEQ ID NO: 31) (SEQ ID NO: 168) RAG9-434 GCCGGGGTGGTGACACACA GCCGGGGUGGUGACACACATT UGUGUGUCACCACCCCGGCTT 2.09 (SEQ ID NO: 306) (SEQ ID NO: 32) (SEQ ID NO: 169) RAG9-251 CCTCCATTGGCTGAGAAGA CCUCCAUUGGCUGAGAAGATT UCUUCUCAGCCAAUGGAGGTT 2.09 (SEQ ID NO: 307) (SEQ ID NO: 33) (SEQ ID NO: 170) RAG9-147 TTCAGCAACCGGAGAAGCA UUCAGCAACCGGAGAAGCATT UGCUUCUCCGGUUGCUGAATT 2.07 (SEQ ID NO: 308) (SEQ ID NO: 34) (SEQ ID NO: 171) RAG9-149 CCTTCAGCAACCGGAGAAG CCUUCAGCAACCGGAGAAGTT CUUCUCCGGUUGCUGAAGGTT 2.07 (SEQ ID NO: 309) (SEQ ID NO: 35) (SEQ ID NO: 172) RAG9-337 GCAGGCTTGGGAGTCCTCT GCAGGCUUGGGAGUCCUCUTT AGAGGACUCCCAAGCCUGCTT 2.05 (SEQ ID NO: 310) (SEQ ID NO: 36) (SEQ ID NO: 173) RAG9-338 AGCAGGCTTGGGAGTCCTC AGCAGGCUUGGGAGUCCUCTT GAGGACUCCCAAGCCUGCUTT 2.01 (SEQ ID NO: 311) (SEQ ID NO: 37) (SEQ ID NO: 174) RAG9-485 AGCTTGGGCACCAGCCGAG AGCUUGGGCACCAGCCGAGTT CUCGGCUGGUGCCCAAGCUTT 1.98 (SEQ ID NO: 312) (SEQ ID NO: 38) (SEQ ID NO: 175) RAG9-336 CAGGCTTGGGAGTCCTCTG CAGGCUUGGGAGUCCUCUGTT CAGAGGACUCCCAAGCCUGTT 1.92 (SEQ ID NO: 313) (SEQ ID NO: 39) (SEQ ID NO: 176) RAG9-503 GCACATCTGACCCGAGTCA GCACAUCUGACCCGAGUCATT UGACUCGGGUCAGAUGUGCTT 1.90 (SEQ ID NO: 314) (SEQ ID NO: 40) (SEQ ID NO: 177) RAG9-152 GCGCCTTCAGCAACCGGAG GCGCCUUCAGCAACCGGAGTT CUCCGGUUGCUGAAGGCGCTT 1.86 (SEQ ID NO: 315) (SEQ ID NO: 41) (SEQ ID NO: 178) RAG9-116 ACCTGGTGTGTGATCTCCG ACCUGGUGUGUGAUCUCCGTT CGGAGAUCACACACCAGGUTT 1.85 (SEQ ID NO: 316) (SEQ ID NO: 42) (SEQ ID NO: 179) RAG9-408 CGAATTACAGCCCTGCAAT CGAAUUACAGCCCUGCAAUTT AUUGCAGGGCUGUAAUUCGTT 1.84 (SEQ ID NO: 317) (SEQ ID NO: 43) (SEQ ID NO: 180) RAG9-409 TCGAATTACAGCCCTGCAA UCGAAUUACAGCCCUGCAATT UUGCAGGGCUGUAAUUCGATT 1.83 (SEQ ID NO: 318) (SEQ ID NO: 44) (SEQ ID NO: 181) RAG9-317 CACACAAGAACTTTTCTCG CACACAAGAACUUUUCUCGTT CGAGAAAAGUUCUUGUGUGTT 1.81 (SEQ ID NO: 319) (SEQ ID NO: 45) (SEQ ID NO: 182) RAG9-330 TGGGAGTCCTCTGCACACA UGGGAGUCCUCUGCACACATT UGUGUGCAGAGGACUCCCATT 1.80 (SEQ ID NO: 320) (SEQ ID NO: 46) (SEQ ID NO: 183) RAG9-616 GGGAGCTGTGATCCTAGCT GGGAGCUGUGAUCCUAGCUTT AGCUAGGAUCACAGCUCCCTT 1.75 (SEQ ID NO: 321) (SEQ ID NO: 47) (SEQ ID NO: 184) RAG9-151 CGCCTTCAGCAACCGGAGA CGCCUUCAGCAACCGGAGATT UCUCCGGUUGCUGAAGGCGTT 1.69 (SEQ ID NO: 322) (SEQ ID NO: 48) (SEQ ID NO: 185) RAG9-615 GGAGCTGTGATCCTAGCTT GGAGCUGUGAUCCUAGCUUTT AAGCUAGGAUCACAGCUCCTT 1.68 (SEQ ID NO: 323) (SEQ ID NO: 49) (SEQ ID NO: 186) RAG9-412 GAGTCGAATTACAGCCCTG GAGUCGAAUUACAGCCCUGTT CAGGGCUGUAAUUCGACUCTT 1.67 (SEQ ID NO: 324) (SEQ ID NO: 50) (SEQ ID NO: 187) RAG9-162 AAGGGGCGCAGCGCCTTCA AAGGGGCGCAGCGCCUUCATT UGAAGGCGCUGCGCCCCUUTT 1.66 (SEQ ID NO: 325) (SEQ ID NO: 51) (SEQ ID NO: 188) RAG9-417 CACCGGAGTCGAATTACAG CACCGGAGUCGAAUUACAGTT CUGUAAUUCGACUCCGGUGTT 1.64 (SEQ ID NO: 326) (SEQ ID NO: 52) (SEQ ID NO: 189) RAG9-324 TCCTCTGCACACAAGAACT UCCUCUGCACACAAGAACUTT AGUUCUUGUGUGCAGAGGATT 1.61 (SEQ ID NO: 327) (SEQ ID NO: 53) (SEQ ID NO: 190) RAG9-455 CACCGCTCCCGCACCCCAG CACCGCUCCCGCACCCCAGTT CUGGGGUGCGGGAGCGGUGTT 1.61 (SEQ ID NO: 328) (SEQ ID NO: 54) (SEQ ID NO: 191) RAG9-325 GTCCTCTGCACACAAGAAC GUCCUCUGCACACAAGAACTT GUUCUUGUGUGCAGAGGACTT 1.61 (SEQ ID NO: 329) (SEQ ID NO: 55) (SEQ ID NO: 192) RAG9-501 ACATCTGACCCGAGTCAGC ACAUCUGACCCGAGUCAGCTT GCUGACUCGGGUCAGAUGUTT 1.61 (SEQ ID NO: 330) (SEQ ID NO: 56) (SEQ ID NO: 193) RAG9-577 CGGCCTTCTCTCTGCTTTG CGGCCUUCUCUCUGCUUUGTT CAAAGCAGAGAGAAGGCCGTT 1.60 (SEQ ID NO: 331) (SEQ ID NO: 57) (SEQ ID NO: 194) RAG9-191 GGAGACTGAGTGGCGCGTG GGAGACUGAGUGGCGCGUGTT CACGCGCCACUCAGUCUCCTT 1.57 (SEQ ID NO: 332) (SEQ ID NO: 58) (SEQ ID NO: 195) RAG9-148 CTTCAGCAACCGGAGAAGC CUUCAGCAACCGGAGAAGCTT GCUUCUCCGGUUGCUGAAGTT 1.56 (SEQ ID NO: 333) (SEQ ID NO: 59) (SEQ ID NO: 196) RAG9-528 ATCGCCCAGAGCCGCCCGC AUCGCCCAGAGCCGCCCGCTT GCGGGCGGCUCUGGGCGAUTT 1.56 (SEQ ID NO: 334) (SEQ ID NO: 60) (SEQ ID NO: 197) RAG9-552 TTCTGGGCGCCTGGCGGGT UUCUGGGCGCCUGGCGGGUTT ACCCGCCAGGCGCCCAGAATT 1.55 (SEQ ID NO: 335) (SEQ ID NO: 61) (SEQ ID NO: 198) RAG9-279 TTGGCTGCTCGCACGCGCC UUGGCUGCUCGCACGCGCCTT GGCGCGUGCGAGCAGCCAATT 1.53 (SEQ ID NO: 336) (SEQ ID NO: 62) (SEQ ID NO: 199) RAG9-483 CTTGGGCACCAGCCGAGAG CUUGGGCACCAGCCGAGAGTT CUCUCGGCUGGUGCCCAAGTT 1.52 (SEQ ID NO: 337) (SEQ ID NO: 63) (SEQ ID NO: 200) RAG9-628 GGCATCAGAAGTGGGAGCT GGCAUCAGAAGUGGGAGCUTT AGCUCCCACUUCUGAUGCCTT 1.52 (SEQ ID NO: 338) (SEQ ID NO: 64) (SEQ ID NO: 201) RAG9-540 GGCGGGTCCAGAATCGCCC GGCGGGUCCAGAAUCGCCCTT GGGCGAUUCUGGACCCGCCTT 1.50 (SEQ ID NO: 339) (SEQ ID NO: 65) (SEQ ID NO: 202) RAG9-626 CATCAGAAGTGGGAGCTGT CAUCAGAAGUGGGAGCUGUTT ACAGCUCCCACUUCUGAUGTT 1.50 (SEQ ID NO: 340) (SEQ ID NO: 66) (SEQ ID NO: 203) RAG9-515 GCCCGCGGTCGTGCACATC GCCCGCGGUCGUGCACAUCTT GAUGUGCACGACCGCGGGCTT 1.49 (SEQ ID NO: 341) (SEQ ID NO: 67) (SEQ ID NO: 204) RAG9-404 TTACAGCCCTGCAATTAAC UUACAGCCCUGCAAUUAACTT GUUAAUUGCAGGGCUGUAATT 1.49 (SEQ ID NO: 342) (SEQ ID NO: 68) (SEQ ID NO: 205) RAG9-55 TTGCAGAAGAGACCCGGAA UUGCAGAAGAGACCCGGAATT UUCCGGGUCUCUUCUGCAATT 1.49 (SEQ ID NO: 343) (SEQ ID NO: 69) (SEQ ID NO: 206) RAG9-161 AGGGGCGCAGCGCCTTCAG AGGGGCGCAGCGCCUUCAGTT CUGAAGGCGCUGCGCCCCUTT 1.48 (SEQ ID NO: 344) (SEQ ID NO: 70) (SEQ ID NO: 207) RAG9-322 CTCTGCACACAAGAACTTT CUCUGCACACAAGAACUUUTT AAAGUUCUUGUGUGCAGAGTT 1.47 (SEQ ID NO: 345) (SEQ ID NO: 71) (SEQ ID NO: 208) RAG9-106 TGATCTCCGAGTGGGTGGG UGAUCUCCGAGUGGGUGGGTT CCCACCCACUCGGAGAUCATT 1.46 (SEQ ID NO: 346) (SEQ ID NO: 72) (SEQ ID NO: 209) RAG9-56 GTTGCAGAAGAGACCCGGA GUUGCAGAAGAGACCCGGATT UCCGGGUCUCUUCUGCAACTT 1.46 (SEQ ID NO: 347) (SEQ ID NO: 73) (SEQ ID NO: 210) RAG9-320 CTGCACACAAGAACTTTTC CUGCACACAAGAACUUUUCTT GAAAAGUUCUUGUGUGCAGTT 1.44 (SEQ ID NO: 348) (SEQ ID NO: 74) (SEQ ID NO: 211) RAG9-491 CGAGTCAGCTTGGGCACCA CGAGUCAGCUUGGGCACCATT UGGUGCCCAAGCUGACUCGTT 1.43 (SEQ ID NO: 349) (SEQ ID NO: 75) (SEQ ID NO: 212) RAG9-488 GTCAGCTTGGGCACCAGCC GUCAGCUUGGGCACCAGCCTT GGCUGGUGCCCAAGCUGACTT 1.43 (SEQ ID NO: 350) (SEQ ID NO: 76) (SEQ ID NO: 213) RAG9-413 GGAGTCGAATTACAGCCCT GGAGUCGAAUUACAGCCCUTT AGGGCUGUAAUUCGACUCCTT 1.42 (SEQ ID NO: 351) (SEQ ID NO: 77) (SEQ ID NO: 214) RAG9-512 CGCGGTCGTGCACATCTGA CGCGGUCGUGCACAUCUGATT UCAGAUGUGCACGACCGCGTT 1.42 (SEQ ID NO: 352) (SEQ ID NO: 78) (SEQ ID NO: 215) RAG9-113 TGGTGTGTGATCTCCGAGT UGGUGUGUGAUCUCCGAGUTT ACUCGGAGAUCACACACCATT 1.42 (SEQ ID NO: 353) (SEQ ID NO: 79) (SEQ ID NO: 216) RAG9-274 TGCTCGCACGCGCCTGCCC UGCUCGCACGCGCCUGCCCTT GGGCAGGCGCGUGCGAGCATT 1.41 (SEQ ID NO: 354) (SEQ ID NO: 80) (SEQ ID NO: 217) RAG9-634 TGCTTGGGCATCAGAAGTG UGCUUGGGCAUCAGAAGUGTT CACUUCUGAUGCCCAAGCATT 1.39 (SEQ ID NO: 355) (SEQ ID NO: 81) (SEQ ID NO: 218) RAG9-558 AGTGGCTTCTGGGCGCCTG AGUGGCUUCUGGGCGCCUGTT CAGGCGCCCAGAAGCCACUTT 1.39 (SEQ ID NO: 356) (SEQ ID NO: 82) (SEQ ID NO: 219) RAG9-326 AGTCCTCTGCACACAAGAA AGUCCUCUGCACACAAGAATT UUCUUGUGUGCAGAGGACUTT 1.38 (SEQ ID NO: 357) (SEQ ID NO: 83) (SEQ ID NO: 220) RAG9-110 TGTGTGATCTCCGAGTGGG UGUGUGAUCUCCGAGUGGGTT CCCACUCGGAGAUCACACATT 1.37 (SEQ ID NO: 358) (SEQ ID NO: 84) (SEQ ID NO: 221) RAG9-340 TGAGCAGGCTTGGGAGTCC UGAGCAGGCUUGGGAGUCCTT GGACUCCCAAGCCUGCUCATT 1.37 (SEQ ID NO: 359) (SEQ ID NO: 85) (SEQ ID NO: 222) RAG9-506 CGTGCACATCTGACCCGAG CGUGCACAUCUGACCCGAGTT CUCGGGUCAGAUGUGCACGTT 1.36 (SEQ ID NO: 360) (SEQ ID NO: 86) (SEQ ID NO: 223) RAG9-507 TCGTGCACATCTGACCCGA UCGUGCACAUCUGACCCGATT UCGGGUCAGAUGUGCACGATT 1.36 (SEQ ID NO: 361) (SEQ ID NO: 87) (SEQ ID NO: 224) RAG9- AGCTTCTGATTGGCTCTTT AGCUUCUGAUUGGCUCUUUTT AAAGAGCCAAUCAGAAGCUTT 1.35 1407 (SEQ ID NO: 362) (SEQ ID NO: 88) (SEQ ID NO: 225) RAG9-508 GTCGTGCACATCTGACCCG GUCGUGCACAUCUGACCCGTT CGGGUCAGAUGUGCACGACTT 1.35 (SEQ ID NO: 363) (SEQ ID NO: 89) (SEQ ID NO: 226) RAG9-588 CACAGGGTAGGCGGCCTTC CACAGGGUAGGCGGCCUUCTT GAAGGCCGCCUACCCUGUGTT 1.35 (SEQ ID NO: 364) (SEQ ID NO: 90) (SEQ ID NO: 227) RAG9-114 CTGGTGTGTGATCTCCGAG CUGGUGUGUGAUCUCCGAGTT CUCGGAGAUCACACACCAGTT 1.34 (SEQ ID NO: 365) (SEQ ID NO: 91) (SEQ ID NO: 228) RAG9-456 GCACCGCTCCCGCACCCCA GCACCGCUCCCGCACCCCATT UGGGGUGCGGGAGCGGUGCTT 1.34 (SEQ ID NO: 366) (SEQ ID NO: 92) (SEQ ID NO: 229) RAG9-312 AAGAACTTTTCTCGGGGTG AAGAACUUUUCUCGGGGUGTT CACCCCGAGAAAAGUUCUUTT 1.34 (SEQ ID NO: 367) (SEQ ID NO: 93) (SEQ ID NO: 230) RAG9-315 CACAAGAACTTTTCTCGGG CACAAGAACUUUUCUCGGGTT CCCGAGAAAAGUUCUUGUGTT 1.33 (SEQ ID NO: 368) (SEQ ID NO: 94) (SEQ ID NO: 231) RAG9-575 GCCTTCTCTCTGCTTTGAG GCCUUCUCUCUGCUUUGAGTT CUCAAAGCAGAGAGAAGGCTT 1.32 (SEQ ID NO: 369) (SEQ ID NO: 95) (SEQ ID NO: 232) RAG9-467 GAGCCGGCTCCGCACCGCT GAGCCGGCUCCGCACCGCUTT AGCGGUGCGGAGCCGGCUCTT 1.32 (SEQ ID NO: 370) (SEQ ID NO: 96) (SEQ ID NO: 233) RAG9-231 ACGCGACCGGCGCGAGGAG ACGCGACCGGCGCGAGGAGTT CUCCUCGCGCCGGUCGCGUTT 1.31 (SEQ ID NO: 371) (SEQ ID NO: 97) (SEQ ID NO: 234) RAG9-637 AAATGCTTGGGCATCAGAA AAAUGCUUGGGCAUCAGAATT UUCUGAUGCCCAAGCAUUUTT 1.31 (SEQ ID NO: 372) (SEQ ID NO: 98) (SEQ ID NO: 235) RAG9-633 GCTTGGGCATCAGAAGTGG GCUUGGGCAUCAGAAGUGGTT CCACUUCUGAUGCCCAAGCTT 1.30 (SEQ ID NO: 373) (SEQ ID NO: 99) (SEQ ID NO: 236) RAG9-519 AGCCGCCCGCGGTCGTGCA AGCCGCCCGCGGUCGUGCATT UGCACGACCGCGGGCGGCUTT 1.30 (SEQ ID NO: 374) (SEQ ID NO: 100) (SEQ ID NO: 237) RAG9-509 GGTCGTGCACATCTGACCC GGUCGUGCACAUCUGACCCTT GGGUCAGAUGUGCACGACCTT 1.29 (SEQ ID NO: 375) (SEQ ID NO: 101) (SEQ ID NO: 238) RAG9-146 TCAGCAACCGGAGAAGCAT UCAGCAACCGGAGAAGCAUTT AUGCUUCUCCGGUUGCUGATT 1.29 (SEQ ID NO: 376) (SEQ ID NO: 102) (SEQ ID NO: 239) RAG9-531 AGAATCGCCCAGAGCCGCC AGAAUCGCCCAGAGCCGCCTT GGCGGCUCUGGGCGAUUCUTT 1.29 (SEQ ID NO: 377) (SEQ ID NO: 103) (SEQ ID NO: 240) RAG9-614 GAGCTGTGATCCTAGCTTG GAGCUGUGAUCCUAGCUUGTT CAAGCUAGGAUCACAGCUCTT 1.29 (SEQ ID NO: 378) (SEQ ID NO: 104) (SEQ ID NO: 241) RAG9-138 CGGAGAAGCATAGTTGCAC CGGAGAAGCAUAGUUGCACTT GUGCAACUAUGCUUCUCCGTT 1.29 (SEQ ID NO: 379) (SEQ ID NO: 105) (SEQ ID NO: 242) RAG9-129 ATAGTTGCACGCGACCTGG AUAGUUGCACGCGACCUGGTT CCAGGUCGCGUGCAACUAUTT 1.28 (SEQ ID NO: 380) (SEQ ID NO: 106) (SEQ ID NO: 243) RAG9-308 ACTTTTCTCGGGGTGTAAA ACUUUUCUCGGGUGUAAATT UUUACACCCCGAGAAAAGUTT 1.28 (SEQ ID NO: 381) (SEQ ID NO: 107) (SEQ ID NO: 244) RAG9-553 CTTCTGGGCGCCTGGCGGG CUUCUGGGCGCCUGGCGGGTT CCCGCCAGGCGCCCAGAAGTT 1.27 (SEQ ID NO: 382) (SEQ ID NO: 108) (SEQ ID NO: 245) RAG9-439 CAGCCGCCGGGGTGGTGAC CAGCCGCCGGGGUGGUGACTT GUCACCACCCCGGCGGCUGTT 1.27 (SEQ ID NO: 383) (SEQ ID NO: 109) (SEQ ID NO: 246) RAG9-613 AGCTGTGATCCTAGCTTGG AGCUGUGAUCCUAGCUUGGTT CCAAGCUAGGAUCACAGCUTT 1.26 (SEQ ID NO: 384) (SEQ ID NO: 110) (SEQ ID NO: 247) RAG9-119 GCGACCTGGTGTGTGATCT GCGACCUGGUGUGUGAUCUTT AGAUCACACACCAGGUCGCTT 1.25 (SEQ ID NO: 385) (SEQ ID NO: 111) (SEQ ID NO: 248) RAG9-590 AGCACAGGGTAGGCGGCCT AGCACAGGGUAGGCGGCCUTT AGGCCGCCUACCCUGUGCUTT 1.25 (SEQ ID NO: 386) (SEQ ID NO: 112) (SEQ ID NO: 249) RAG9-546 GCGCCTGGCGGGTCCAGAA GCGCCUGGCGGGUCCAGAATT UUCUGGACCCGCCAGGCGCTT 1.25 (SEQ ID NO: 387) (SEQ ID NO: 113) (SEQ ID NO: 250) RAG9-627 GCATCAGAAGTGGGAGCTG GCAUCAGAAGUGGGAGCUGTT CAGCUCCCACUUCUGAUGCTT 1.24 (SEQ ID NO: 388) (SEQ ID NO: 114) (SEQ ID NO: 251) RAG9-530 GAATCGCCCAGAGCCGCCC GAAUCGCCCAGAGCCGCCCTT GGGCGGCUCUGGGCGAUUCTT 1.24 (SEQ ID NO: 389) (SEQ ID NO: 115) (SEQ ID NO: 252) RAG9-159 GGGCGCAGCGCCTTCAGCA GGGCGCAGCGCCUUCAGCATT UGCUGAAGGCGCUGCGCCCTT 1.24 (SEQ ID NO: 390) (SEQ ID NO: 116) (SEQ ID NO: 253) RAG9-403 TACAGCCCTGCAATTAACA UACAGCCCUGCAAUUAACATT UGUUAAUUGCAGGGCUGUATT 1.23 (SEQ ID NO: 391) (SEQ ID NO: 117) (SEQ ID NO: 254) RAG9-388 AACATATGAATCTGACGAA AACAUAUGAAUCUGACGAATT UUCGUCAGAUUCAUAUGUUTT 1.22 (SEQ ID NO: 392) (SEQ ID NO: 118) (SEQ ID NO: 255) RAG9-454 ACCGCTCCCGCACCCCAGC ACCGCUCCCGCACCCCAGCTT GCUGGGGUGCGGGAGCGGUTT 1.22 (SEQ ID NO: 393) (SEQ ID NO: 119) (SEQ ID NO: 256) RAG9-189 AGACTGAGTGGCGCGTGCC AGACUGAGUGGCGCGUGCCTT GGCACGCGCCACUCAGUCUTT 1.21 (SEQ ID NO: 394) (SEQ ID NO: 120) (SEQ ID NO: 257) RAG9-131 GCATAGTTGCACGCGACCT GCAUAGUUGCACGCGACCUTT AGGUCGCGUGCAACUAUGCTT 1.20 (SEQ ID NO: 395) (SEQ ID NO: 121) (SEQ ID NO: 258) RAG9-504 TGCACATCTGACCCGAGTC UGCACAUCUGACCCGAGUCTT GACUCGGGUCAGAUGUGCATT 1.19 (SEQ ID NO: 396) (SEQ ID NO: 122) (SEQ ID NO: 259) RAG9-529 AATCGCCCAGAGCCGCCCG AAUCGCCCAGAGCCGCCCGTT CGGGCGGCUCUGGGCGAUUTT 1.19 (SEQ ID NO: 397) (SEQ ID NO: 123) (SEQ ID NO: 260) RAG9-493 CCCGAGTCAGCTTGGGCAC CCCGAGUCAGCUUGGGCACTT GUGCCCAAGCUGACUCGGGTT 1.19 (SEQ ID NO: 398) (SEQ ID NO: 124) (SEQ ID NO: 261) RAG9-481 TGGGCACCAGCCGAGAGCC UGGGCACCAGCCGAGAGCCTT GGCUCUCGGCUGGUGCCCATT 1.18 (SEQ ID NO: 399) (SEQ ID NO: 125) (SEQ ID NO: 262) RAG9-307 CTTTTCTCGGGGTGTAAAA CUUUUCUCGGGGUGUAAAATT UUUUACACCCCGAGAAAAGTT 1.16 (SEQ ID NO: 400) (SEQ ID NO: 126) (SEQ ID NO: 263) RAG9-564 GCTTTGAGTGGCTTCTGGG GCUUUGAGUGGCUUCUGGGTT CCCAGAAGCCACUCAAAGCTT 1.16 (SEQ ID NO: 401) (SEQ ID NO: 127) (SEQ ID NO: 264) RAG9-410 GTCGAATTACAGCCCTGCA GUCGAAUUACAGCCCUGCATT UGCAGGGCUGUAAUUCGACTT 1.15 (SEQ ID NO: 402) (SEQ ID NO: 128) (SEQ ID NO: 265) RAG9-511 GCGGTCGTGCACATCTGAC GCGGUCGUGCACAUCUGACTT GUCAGAUGUGCACGACCGCTT 1.15 (SEQ ID NO: 403) (SEQ ID NO: 129) (SEQ ID NO: 266) RAG9-406 AATTACAGCCCTGCAATTA AAUUACAGCCCUGCAAUUATT UAAUUGCAGGGCUGUAAUUTT 1.15 (SEQ ID NO: 404) (SEQ ID NO: 130) (SEQ ID NO: 267) RAG9-58 ACGTTGCAGAAGAGACCCG ACGUUGCAGAAGAGACCCGTT CGGGUCUCUUCUGCAACGUTT 1.15 (SEQ ID NO: 405) (SEQ ID NO: 131) (SEQ ID NO: 268) RAG9-547 GGCGCCTGGCGGGTCCAGA GGCGCCUGGCGGGUCCAGATT UCUGGACCCGCCAGGCGCCTT 1.15 (SEQ ID NO: 406) (SEQ ID NO: 132) (SEQ ID NO: 269) RAG9-123 GCACGCGACCTGGTGTGTG GCACGCGACCUGGUGUGUGTT CACACACCAGGUCGCGUGCTT 1.14 (SEQ ID NO: 407) (SEQ ID NO: 133) (SEQ ID NO: 270) RAG9-124 TGCACGCGACCTGGTGTGT UGCACGCGACCUGGUGUGUTT ACACACCAGGUCGCGUGCATT 1.14 (SEQ ID NO: 408) (SEQ ID NO: 134) (SEQ ID NO: 271) RAG9-436 CCGCCGGGGTGGTGACACA CCGCCGGGGUGGUGACACATT UGUGUCACCACCCCGGCGGTT 1.14 (SEQ ID NO: 409) (SEQ ID NO: 135) (SEQ ID NO: 272) RAG9-286 TCTTTGATTGGCTGCTCGC UCUUUGAUUGGCUGCUCGCTT GCGAGCAGCCAAUCAAAGATT 1.13 (SEQ ID NO: 410) (SEQ ID NO: 136) (SEQ ID NO: 273) RAG9-420 ACACACCGGAGTCGAATTA ACACACCGGAGUCGAAUUATT UAAUUCGACUCCGGUGUGUTT 1.13 (SEQ ID NO: 411) (SEQ ID NO: 137) (SEQ ID NO: 274) RAG9-636 AATGCTTGGGCATCAGAAG AAUGCUUGGGCAUCAGAAGTT CUUCUGAUGCCCAAGCAUUTT 1.13 (SEQ ID NO: 412) (SEQ ID NO: 138) (SEQ ID NO: 275) RAG9-289 AACTCTTTGATTGGCTGCT AACUCUUUGAUUGGCUGCUTT AGCAGCCAAUCAAAGAGUUTT 1.13 (SEQ ID NO: 413) (SEQ ID NO: 139) (SEQ ID NO: 276) RAG9-537 GGGTCCAGAATCGCCCAGA GGGUCCAGAAUCGCCCAGATT UCUGGGCGAUUCUGGACCCTT 1.12 (SEQ ID NO: 414) (SEQ ID NO: 140) (SEQ ID NO: 277) RAG9-419 CACACCGGAGTCGAATTAC CACACCGGAGUCGAAUUACTT GUAAUUCGACUCCGGUGUGTT 1.12 (SEQ ID NO: 415) (SEQ ID NO: 141) (SEQ ID NO: 278) RAG9-327 GAGTCCTCTGCACACAAGA GAGUCCUCUGCACACAAGATT UCUUGUGUGCAGAGGACUCTT 1.10 (SEQ ID NO: 416) (SEQ ID NO: 142) (SEQ ID NO: 279)

    [0091] It can be clearly seen from the arrangement of activities of the 403 saRNAs according to their positions within the ATOH1 promoter region that the functional saRNAs are gathered together, that is, in certain promoter regions, the activating saRNAs gathered in specific “hotspot” regions (FIG. 2). As shown in FIG. 2, 4 hotspot regions respectively appeared in region (H1) from −578 to −386, region (H2) from −339 to −291, region (H3) from −162 to −86 and region (H4) from −55 to −34 of the promoter, respectively, showing high gathering of the activating saRNAs, and the proportion of functional saRNAs was 41.0%, 51.6%, 45.6%, and 100%, respectively. These results indicate that the functional saRNAs were not randomly distributed within the promoter, but existed in the specific hotspot regions.

    [0092] The sequence of the hotspot H1 (5′ to 3′: −578 to −386) corresponds to SEQ ID NO: 2 in the sequence listing:

    TABLE-US-00004 gcggccttctctctgctttgagtggcttctgggcgcctggcgggtccaga atcgcccagagccgcccgcggtcgtgcacatctgacccgagtcagcttgg gcaccagccgagagccggctccgcaccgctcccgcaccccagccgccggg gtggtgacacacaccggagtcgaattacagccctgcaattaa

    [0093] The sequence of the hotspot H2 (5′ to 3′: −339 to −291) corresponds to SEQ ID NO: 3 in the sequence listing:

    TABLE-US-00005 tgcacacaagaacttttctcggggtgtaa

    [0094] The sequence of the hotspot H3 (5′ to 3′: −162 to −86) corresponds to SEQ ID NO: 4 in the sequence listing:

    TABLE-US-00006 aaggggcgcagcgccttcagcaaccggagaagcatagttgcacgcgacct ggtgtgtgatctccgagtgggtgggg

    [0095] The sequence of the hotspot H4 (5′ to 3′: −55 to −34) corresponds to SEQ ID NO: 5 in the sequence listing:

    TABLE-US-00007 ttgcagaagagacccggaaagg

    Example 3: Validation of High-Throughput Screening Results

    [0096] Normal Human Epidermal Keratinocytes (NHEK) cells and human renal embryonic cells (HEK293T) were seeded into a 6-well plate with 2×10.sup.5 to 3×10.sup.5 cells/well and were reverse transfected with oligonucleotide duplexes. Total cellular RNAs were extracted using an RNeasy Plus Mini kit (Qiagen). The resulting RNA (1 μg) was reverse transcribed into cDNA using a PrimeScript RT kit containing gDNA Eraser (Takara, Shlga, Japan). The resulting cDNA was amplified by qPCR using an ABI 7500 fast real-time PCR system (Applied Biosystems) and SYBR Premix Ex Taq II reagent (Takara, Shlga, Japan). The reaction conditions were: 95° C. for 3 s, 60° C. for 30 s, and 40 cycles. HPRT1 was used as internal control and RAG9-871i as inhibitory dsRNA control. The phase expression value of the ATOH1 gene was calculated by a method of 2.sup.−ΔΔCT. All the primer sequences used are listed in Table 3.

    [0097] In order to calculate the expression value (E.sub.rel) of ATOH1 (target gene) of an saRNA-transfected sample relative to control treatment (mock), the Ct values of the target gene and the internal control genes were substituted into Formula 1 for calculation.


    E.sub.rel=2.sup.(CtTm−CtTs)/2.sup.(CtRm−CtRs)  (Formula 1)

    [0098] wherein CtTm was the Ct value of the target gene from Mock sample; CtTs was the Ct value of the target gene from the saRNA-treated sample; CtRm was the Ct value of the internal control gene from mock-treated sample; CtRs was the Ct value of the internal control gene from the saRNA-treated sample.

    TABLE-US-00008 TABLE 3 Primer sequences for two-step qRT-PCR analysis Primer Sequence No. Sequence (5′-3′) ATOH1 F1 SEQ ID NO: 417 CCCTTCCAGCAAACAGGTGA ATOH1 R1 SEQ ID NO: 418 GAACGACGGGATAACATTGCG HPRT1 F SEQ ID NO: 419 ATGGACAGGACTGAACGTCTT HPRT1 R SEQ ID NO: 420 TCCAGCAGGTCAGCAAAGAA

    [0099] As shown in FIG. 3 and FIG. 4, randomly selected functional saRNAs were verified in HEK293T cells and NHEK cells, and both showed up-regulation of mRNA expression of ATOH1.

    TABLE-US-00009 TABLE 4 Double-stranded RNA sequences as study controls Double- stranded RNA Sequence No. Sequence (5′-3′) dsCon2-sense SEQ ID NO: 421 ACUACUGAGUGACAGUAGATT strand dsCon2- SEQ ID NO: 422 UCUACUGUCACUCAGUAGUTT antisense strand RAG9-871i- SEQ ID NO: 423 GCUCUGCACUUCUCGACUUTT sense strand RAG9-871i- SEQ ID NO: 424 AAGUCGAGAAGUGCAGAGCTT antisense strand

    Example 4. saRNAs Promoting ATOH1 Protein Expression

    [0100] HEK293T cells were plated into a 96-well plate at a concentration of 3×10.sup.3 to 5×10.sup.3 cells/well, cultured overnight, and transfected with 7 randomly selected functional saRNAs. Seventy two hours after transfection, cells were collected and lysed using a cell lysis buffer (1×RIPA buffer, CST) which contained a protease inhibitor. Proteins were quantified by BCA method (Thermo) and separated by polyacrylamide gel electrophoresis and conversion to a 0.45 μm PVDF membrane. Primary Rabbit monoclonal anti-ATOH1 (Abcam) and α/β-tubulin antibodies (Cell Signaling Technology) were used to detect blots and a secondary anti-rabbit IgG, HRP-linked antibody (Cell Signaling Technology) was used. Signals were detected using an Image Lab (BIO-RAD, Chemistry Doctm MP imaging system).

    [0101] As shown in FIG. 5, the 7 randomly selected activating saRNAs were capable of promoting or increasing the expression of ATOH1 protein.

    [0102] Based on these results, a plurality of saRNAs capable of activating the expression of ATOH1 gene were found through high-throughput screening of saRNAs targeting ATOH1 gene promoter. The saRNAs can up-regulate the expression of the ATOH1 gene on mRNA and at the protein expression level, and can be used to treat a disease or symptom caused by a decrease or insufficient expression of the ATOH1 protein, or used in the preparation of a method or drug for treating the disease or symptom.

    REFERENCES

    [0103] 1. Aragaki, M., K. Tsuchiya, R. Okamoto, S. Yoshioka, T. Nakamura, N. Sakamoto, T. Kanai, and M. Watanabe. 2008. ‘Proteasomal degradation of Atoh1 by aberrant Wnt signaling maintains the undifferentiated state of colon cancer’, Biochem Biophys Res Commun, 368: 923-9. [0104] 2. Bermingham, N. A., B. A. Hassan, S. D. Price, M. A. Vollrath, N. Ben-Arie, R. A. Eatock, H. J. Bellen, A. Lysakowski, and H. Y. Zoghbi. 1999. ‘Math1: an essential gene for the generation of inner ear hair cells’, Science, 284: 1837-41. [0105] 3. Gubbels, S. P., D. W. Woessner, J. C. Mitchell, A. J. Ricci, and J. V. Brigande. 2008. ‘Functional auditory hair cells produced in the mammalian cochlea by in utero gene transfer’, Nature, 455: 537-41. [0106] 4. Gurtler, N., and A. K. Lalwani. 2002. ‘Etiology of syndromic and nonsyndromic sensorineural hearing loss’, Otolaryngol Clin North Am, 35: 891-908. [0107] 5. Izumikawa, M., R. Minoda, K. Kawamoto, K. A. Abrashkin, D. L. Swiderski, D. F. Dolan, D. E. Brough, and Y. Raphael. 2005. ‘Auditory hair cell replacement and hearing improvement by Atoh1 gene therapy in deaf mammals’, Nat Med, 11: 271-6. [0108] 6. Kawamoto, K., S. Ishimoto, R. Minoda, D. E. Brough, and Y. Raphael. 2003. ‘Math1 gene transfer generates new cochlear hair cells in mature guinea pigs in vivo’, J Neurosci, 23: 4395-400. [0109] 7. Kazanjian A., Shroyer S. F. 2011. NOTCH Signaling and ATOH1 in Colorectal Cancers. Curr Colorectal Cancer Rep June; 7(2):121-127. [0110] 8. Leow, C. C., P. Polakis, and W. Q. Gao. 2005. ‘A role for Hath1, a bHLH transcription factor, in colon adenocarcinoma’, Ann N Y Acad Sci, 1059: 174-83. [0111] 9. Liberman, M. C. 1990. ‘Quantitative assessment of inner ear pathology following ototoxic drugs or acoustic trauma’, Toxicol Pathol, 18: 138-48. [0112] 10. Richardson, R. T., and P. J. Atkinson. 2015. ‘Atoh1 gene therapy in the cochlea for hair cell regeneration’, Expert Opin Biol Ther, 15: 417-30. [0113] 11. Zheng, J. L., and W. Q. Gao. 2000. ‘Overexpression of Math1 induces robust production of extra hair cells in postnatal rat inner ears’, Nat Neurosci, 3: 580-6.